Remerciements /Acknowledgments

First, I would like to thank my PhD supervisor: Anne HOUDUSSE for welcoming me in this team, for giving me the opportunity to perform a PhD in her team, for supervising me during these four years, for her encouragements and for all her great advises she provides me with. I am really grateful to Françoise OCHSENBEIN, Christopher TOSELAND, Pierre LEGRAND, Carine TISNE, Dietmar MANSTEIN and Christophe LAMAZE for agreeing to be part of my PhD jury and for accepting to evaluate my thesis. I would also like to thank Patricia BASSEREAU, Patrick ENGLAND and Laure STROCHLIC for agreeing to be part of my PhD comity, for the scientific discussions, for their advises and guidance.

Thanks to our collaborators, Lee SWEENEY, András MALNASI-CSIZMADIA and their teams, for all the scientific discussions and thanks for showing me your confidence. Thanks to Mate GIYMESI for the discussions on blebbistatin derivatives, to Tianming LIN for all these ATPase assays on Myo6 and Xiaoyan LIU for all the bacculo viruses.

Thanks to the structural motility team, present and past members, for training me, providing me with good advises, supporting me during these four years and for all and for all the good time and laughs that we shared together: À Guillaume, pour m'avoir transmis les bases du clonage, de la production et caractérisation de protéines recombinantes lorsque je suis arrivée en stage de M2. Merci aussi pour ta patience, ta bonne humeur et gentillesse (go chicken go !).

À Léonid, merci pour ta bonne humeur, tes conseils et tes digressions philosophiques ! Merci aussi pour toutes les protéines purifiées sans oublier toutes les MST.

À Franck, pour toutes les expériences de biologie cellulaire, pour son implication et pour ta bonne humeur.

À Elena, merci de m'avoir formé à la cristallisation et au SAXS. Merci pour ton aide, ta bonne humeur et tout le temps passer à cloner ! À Carlos, merci de m'avoir enseigné le traitement de données cristallo, merci pour ta gentillesse et tes conseils scientifiques.

À Livia, pour ses conseils en cristallisation et ses numéros de danses.

To Lena and Moutse, my office neighbors, thank for all your advises and discussions, thanks for helping me with Pymol, Coot and Buster! À Amandine, merci pour ta bonne humeur et bien sûr merci pour tous les gâteaux ! À Mona et Emma, mes deux stagiaires, pour leur motivation, leur sérieux et leur bonne humeur.

À Vincent, pour les discussions scientifiques et tous les voyages au synchrotron ! Enfin, à Leila, mon indispensable bras droit ! Merci pour tout ton travail, ta motivation et ta gentillesse.

Thanks to the PX1, PX2 and SWING staff, Aurélien THUREAU, Serena SIRIGU, Pierre MONTAVILLE, Bill SHEPARD, Andrew THOMPSOM, Tatiana ISABET and MARTIN SAVKO for providing me with great advises and helps for collecting and processing my data.

Thanks to La Ligue contre le cancer for funding my work.

Enfin merci à ma famille et à mes amis pour leur confiance, leur patience et tout leur soutien ! Merci de croire en moi !

Abstract

The eukaryotic cell nanomotors are able to perform complex mechanical tasks, necessary for tension generation in precise cellular locations. Vesicles formation and transport in the endocytic and secretory pathways can for example result from it. Among these motors, myosins are able, from the hydrolysis of ATP, to generate force in association with actin filaments. Myosins are divided into many classes involved in a wide range of functions and pathologies. Their dysfunction can lead to many diseases including spasticity, asthma, cardiomyopathy, deafness or many cancers... (Coluccio 2020).

Thus, the thesis project is centered on the one hand (I) on the study of the regulation of myosin VI (Myo6), a nanomotor involved in deafness as well as many cancers [START_REF] Dunn | A Novel Role of Myosin VI in Human Prostate Cancer[END_REF][START_REF] Li | Knockdown of Myosin VI by Lentivirus-Mediated Short Hairpin RNA Suppresses Proliferation of Melanoma[END_REF][START_REF] Ma | [END_REF][START_REF] Ma | [END_REF]Yang 2019;[START_REF] Yang | LncRNA UCA1 Promotes Development of Gastric Cancer via the MiR-145/MYO6 Axis[END_REF]. Myo6 is unique among all members of the myosin superfamily because it moves on actin filaments in the opposite direction to all other myosins. This specificity allows it to perform unique cellular roles that cannot be performed by any other myosin. (II) On the other hand, the thesis project focused on the study of the blebbistatin pocket (Straight et al. 2003), an inhibitor pocket within the myosin Motor domain, in order to explore its potential for the development of specific inhibitors. This thesis therefore contributes to:

(I)

Understanding the processes regulating the activity of Myo6 via an approach combining structural biology, biophysical tests and cell biology.

Such regulation involves the existence of an auto-inhibited state of Myo6 (Spink et al. 2008;[START_REF] Fili | NDP52 Activates Nuclear Myosin VI to Enhance RNA Polymerase II Transcription[END_REF]2020), a state in which the latter consumes little ATP and diffuses freely into the cytosol so that the motor is activated only at the site required for its recruitment. The biophysical study of this state has made it possible to better understand how to stabilize or destabilize it in vitro and has enabled us to obtain a first model of the auto-inhibited state using negative staining electron microscopy. This study also allowed us to better understand the differential role of cellular partners of Myo6 for its activation. Mutants promoting the activation or formation of the auto-inhibited state of Myo6 have been developed based on this study.

Their ability to be recruited by different partners of Myo6 could thus be studied in human melanoma cells (MNT-1), these tests confirm a differential recruitment by the partners and suggest non-specific recruitment of mutants promoting activation.

(II)

Understanding the origin of the specificity of the MPH-220, an allosteric modulator derived from the well-known myosin inhibitor blebbistatin and able to specifically inhibit skeletal Myosin-2 from a crystallographic approach.

This study describes that the specificity of this inhibitor with high potential for the development of new treatment against spasticity is based on a single non-conserved residue in the Blebbistatin allosteric pocket of the other myosins-2 [START_REF] Gyimesi | Single Residue Variation in Skeletal Muscle Myosin Enables Direct and Selective Drug Targeting for Spasticity and Muscle Stiffness[END_REF].

Abbreviations

List of figures

List of tables

General introduction

A. The molecular motors

Molecular motors are key proteins able to generate force upon ATP hydrolysis when associated with the cytoskeleton. Force generated by these motors notably result in cell motility, trafficking, division and muscle contraction; key processes for eukaryotic cell integrity and functions.

Among these molecular motors, we can find the kinesins and the dyneins that generate force when associated to microtubules and the myosins that generate force when associated with actin filaments. Cytoskeleton filaments are dynamic and polarized structures with a minus end (slow growth) and a plus end (fast growth) that constrain nanomotors to exert their force in a specific direction.

Indeed, while most kinesins and myosins move toward the plus end, some move in the opposite direction [START_REF] Endres | A Lever-Arm Rotation Drives Motility of the Minus-End-Directed Kinesin Ncd[END_REF][START_REF] Wells | Myosin VI Is an Actin-Based Motor That Moves Backwards[END_REF], dyneins move toward the minus end of the microtubules (Figure 1). Moreover, the spatial distribution of microtubules and actins filaments differs. Microtubules are large (25 nm diameter) filaments which are anchored in centrosomes at the minus end) and radiate out of the centrosomes at the plus end (Figure 1). So doing, the microtubule network allows cytoplasm organization and cargo transport on the microtubule track from cell periphery to nucleus. In contrast, actin filaments are thin (6 nm diameter) fibers mostly found in the cell cortex where they support specialized structures such as microvilli and can mediate local transport (Figure 1). These nanomotors thus complemented each other, offering the possibility to perform a variety of functions notably offering the possibility of active transport of cargoes at precise location on cytoskeleton tracks upon ATP hydrolysis.

B. The Myosin superfamily

The myosin superfamily is a collection of actin-dependent molecular motors. In human, 40 Myosins sorted according to their motor sequence identity. Myosin gene name correspond to their official HUGO human gene name. Unconventional myosin genes are named "MYO" and conventional myosin (also known as myosin II) genes are named "MYH".

Myosins of class II (Myo2) were historically the first myosins discovered by high salt extraction of a muscle extract by Kühne in 1864 (reviewed in Szent-Györgyi 2004) and nowadays, it is the most diverse class with 15 genes in total in human. Myo2 are thus known as conventional myosins that among myosins, have the unique ability to associate in long filaments able to slide along actin filaments (Figure 3). This ability is notably crucial for muscle contraction and cytokinesis (Alonso-Matilla, Thiyagarajan, and O'Shaughnessy 2019; [START_REF] Powers | The Sliding Filament Theory Since Andrew Huxley: Multiscale and Multidisciplinary Muscle Research[END_REF]. In contrast, the remaining myosin classes, unable to associate into myosin filament assemblies, constitute the unconventional myosins and can associate to membrane for intracellular organization like Myo1 or use their force for transporting cargo along actin filaments like Myo5 (Figure 3).

C. Structural organization of the myosins

Myosins present overall the same organization; they contain (from N-terminus to C-terminus)

a Motor domain that generate force, a Lever arm and a divergent Tail (Figure 4).

C.1. The Motor domain

Myosins are characterized by a conserved Motor domain usually located at the N-terminus (with notably the exception of Myo3, Myo16 and Myo18 that contains an additional N-terminus domain prior to the Motor and Myo1, Myo9 and Myo15 that exhibit an unstructured region prior to the Motor domain). This domain is responsible for ATP hydrolysis and interaction with actin.

Through all the myosin classes, it exhibits conserved structural elements allowing conserved mode of force production among myosins. Indeed, the Motor generates force by undergoing several conformational states along the ATPase cycle that depend both on the nucleotide-and actin-binding.

Since the first atomic description of a myosin Motor, thanks to the X-ray crystallography structure of chicken skeletal Myo2 at 2.8 Å [START_REF] Rayment | Three-Dimensional Structure of Myosin Subfragment-1: A Molecular Motor[END_REF], a large amount of myosin Motor atomic structures have been solved allowing a deTailed description of the different features compositing a myosin Motor (reviewed in Sweeney, Houdusse, and Robert-Paganin 2020; Schröder 2020) (Figure 5). The myosin Motor domain is composed of four major subdomains: the N-terminus, the lower-50

and upper-50 that separate an inner cleft and the converter. These subdomains are linked together by highly conserved connectors: switch-II, relay, strut and SH1-helix (reviewed in Robert-Paganin et al.

2020):

-Upper and lower-50 can mediate actin binding to the myosin Motor through the actin binding loops and the helix-turn-helix of the lower-50. The affinity for actin is increased upon closure of the inner cleft (50 kDa cleft) that separate the two subdomains, assisted by the strut connector [START_REF] Coureux | A Structural State of the Myosin V Motor without Bound Nucleotide[END_REF][START_REF] Coureux | Three Myosin V Structures Delineate Essential Features of Chemo-Mechanical Transduction[END_REF]).

-Switch-II, near the Pi binding site, changes conformation depending on the nucleotide bound and can play the role of a door keeper controlling phosphate release [START_REF] Llinas | How Actin Initiates the Motor Activity of Myosin[END_REF] -Switch-I and P-loop are part of the active site of the myosin Motor.

-As a central β-sheet, the transducer is directly affected by conformational changes at the active site or at the actin-binding interface and is thus critical for structural rearrangement transduction across the Motor.

-Rearrangements are transmitted to the converter through the connectors: SH1-helix and relay.

-The converter, which is the most mobile subdomain can then amplify movement thus drives force generation by the myosin Motor. Following the converter, the neck region is found. It is a helical region containing consensus sequence (IQxxxRGxxxR), i.e., IQ motif (Figure 6). These motifs mediate association of the myosin heavy chain to the light chains, which rigidifies the neck region. The ensemble constitutes the Lever arm, whose structure was solved at 2 Å in 1996 [START_REF] Houdusse | Structure of the Regulatory Domain of Scallop Myosin at 2 A Resolution: Implications for Regulation[END_REF]. This rigidified Lever arm allows force generation by amplifying the structure rearrangement of the Motor domain along the force production cycle (H. Lee Sweeney and Houdusse 2010) (Figure 7). The number of IQ motifs depends on the myosin classes from a short neck region containing only 1 IQ for Myo6 to a long Lever arm containing 6 IQs for Myo5. Light chains are calmodulin (CaM) and CaM related proteins. Generally speaking, the essential light chain (ELC) binds the first IQ (closest to the Motor domain) of the conventional myosins, the regulatory light chain (RLC) binds to the second IQ and the CaMs bind to the Lever arm of unconventional myosins, although it should be noted that native light chain has not be clearly identified for all myosin motors; some unconventional myosin motor were reported to be able to bind ELC or RLC and some atypical light chains were found to bind unconventional myosin as well (reviewed in [START_REF] Heissler | Myosin Light Chains: Teaching Old Dogs New Tricks[END_REF].

C.2. The Lever arm

CaM is a highly conserved protein of ~16 kDa composed of two globular regions (N-lobe and C-lobe) attached together through linker. Each lobe is composed of two EF-hands (helix-turn-helix) able to interact with divalent cations, preferentially Ca 2+ which coordinate the two helices together through interaction with the central loop, preferentially to adopt various conformations (open, i.e., EFhand helices perpendicular, semi-open, i.e., intermediate between open and closed, closed, i.e., EFhand helices anti-parallel, uncoupled, i.e., EF-hand helices separated) notably depending on the cation and the protein bound to it. When a CaM lobe adopt a close conformation, its affinity with the IQ motif became lower. CaM conformational changes can thus be an important element of myosin activity regulation. ELC and RLC are 16-25 kDa proteins that exhibit a structure very similar to CaM with 2 lobes and 4 EF-hands, yet they exhibit several degenerated EF-hands that have partially and completely lost their Ca 2+ binding properties (Houdusse and Cohen 1995;reviewed in Heissler and Sellers 2014) (Figure 8). As for CaM, they can adopt extended or compact conformations that impact their affinity for myosin heavy chain. Additionally, phosphorylation of the RLC has been shown to be a key parameter for regulation of the activity of several conventional myosin [START_REF] Craig | Light-Chain Phosphorylation Controls the Conformation of Vertebrate Non-Muscle and Smooth Muscle Myosin Molecules[END_REF].

Figure 8: Scallop Myo2 Lever arm

Lever arm of scallop Myo2 solved by X-ray crystallography at 2.6 Å (PDB: 1QVI; Gourinath et al. 2003). Neck region of myosin heavy chain in purple, ELC in lila, RLC in burgundy. Mg 2+ ion in green and Ca 2+ ion in pink. One of the four EF-hands of the ELC is highlighted in blue.

C.3. Tail

The Tail is a highly divergent part that sets the different myosin classes apart. It can contain various structural elements such as MyTH4-FERM domain, Lever arm extension or single α-helix and often mediate binding to cell partners and dimerization (Figure 9).

D. Kinetic cycle of myosin motors

Force is generated during the ATPase cycle in which the Motor undergoes various conformations depending on its nucleotide state and actin binding. This motor cycle has been extensively characterized through transient kinetic experiments, while biophysical experiments, including optical trapping, have characterized the force produced by these motors (Sweeney and Houdusse 2010). Moreover, atomic structures of myosins in different states of the cycle have been solved using X-ray crystallography and cryo-electron microscopy (Sweeney, Houdusse, and Robert-Paganin 2020; Schröder 2020) allowing a good understanding of the way myosins transform chemical energy from ATP hydrolysis into mechanical work (Figure 10). Force is produced during the powerstroke of the cycle in which the Motor is bound to the actin filament following ATP hydrolysis. Actin binding induces conformation changes allowing Pi release (Pi release state) [START_REF] Llinas | How Actin Initiates the Motor Activity of Myosin[END_REF] and larger rearrangements resulting in the closure of the inner cleft that increases actin affinity and promote the wide swing of the Lever arm responsible of force generation in the mechanical work (strong ADP state). Finally, ADP is released from the Motor after a transition to populate the so-called Rigor state. The Motor then detaches from actin upon ATP binding which triggers conformational rearrangements of the transducer and opening of the cleft, constituting the post-rigor state of the myosin Motor [START_REF] Coureux | Three Myosin V Structures Delineate Essential Features of Chemo-Mechanical Transduction[END_REF]. During the following recovery stroke, the Motor performs a rapid isomerization that ends up populating the prepowerstroke state in which ATP hydrolysis can be facilitated. This state possesses a low affinity for actin and a primed Lever arm ready to perform its next swing once the Motor will rebind actin to start a new cycle. Chapter 1: Mechanistic and structural basis for tuning of myosin VI activity

Myosins

Introduction

The unconventional myosin of class VI (Myo6) was first identified from F-actin column chromatography on an embryonic drosophila melanogaster extract [START_REF] Kellerman | An Unconventional Myosin Heavy Chain Gene from Drosophila Melanogaster[END_REF]. It was named 95F MHC at the time. Myo6 was then identified through PCR screening on RNA isolated from human and porcine cell extracts (Caco-2BBE, LLC-PK1, human peripheral blood leukocyte and human liver) [START_REF] Bement | Identification and Overlapping Expression of Multiple Unconventional Myosin Genes in Vertebrate Cell Types[END_REF] as well as from organisms from various phylogenetic groups such as urchins, fishes and C. Elegans [START_REF] Terasaki | Identification of Actin-Binding Proteins from Sea Urchin Eggs by F-Actin Affinity Column Chromatography[END_REF][START_REF] Kelleher | Myosin VI Is Required for Asymmetric Segregation of Cellular Components during C. Elegans Spermatogenesis[END_REF][START_REF] Coffin | Myosin VI and VIIa Distribution among Inner Ear Epithelia in Diverse Fishes[END_REF].

Myo6 is ubiquitously expressed and can be found at several cell localizations. It was reported to localize on a variety of cell compartments including: vesicles, membrane ruffles, microvilli, Golgi apparatus [START_REF] Buss | The Localization of Myosin VI at the Golgi Complex and Leading Edge of Fibroblasts and Its Phosphorylation and Recruitment into Membrane Ruffles of A431 Cells after Growth Factor Stimulation[END_REF])and nucleus [START_REF] Vreugde | Nuclear Myosin VI Enhances RNA Polymerase II-Dependent Transcription[END_REF].

By performing in vitro motility assays, [START_REF] Wells | Myosin VI Is an Actin-Based Motor That Moves Backwards[END_REF] demonstrate that as opposed to all other myosin motors, Myo6 moves towards the minus end of the actin filament, which suggests that Myo6 may perform functions that can't be easily compensated by any other myosin. For example, as opposed to Myo5, in cultured rat neurons or in zebrafish in vivo, Tail truncated Myo6 can travel across the actin barrier that filters the proteins and vesicles able to travel across the axon [START_REF] Balasanyan | Structure and Function of an Actin-Based Filter in the Proximal Axon[END_REF]. This is likely related to its unique ability to move towards the minus end of actin filaments as actin is predominantly oriented with their plus end facing the cell body.

A. Hearing loss and deafness

In 1995, Myo6 was identified as a "deafness gene" [START_REF] Avraham | The Mouse Snell's Waltzer Deafness Gene Encodes an Unconventional Myosin Required for Structural Integrity of Inner Ear Hair Cells[END_REF] through genetical mapping of the Snell's Waltzer mice (sv/sv) that suffer from recessive deafness (first described in Deol et [START_REF] Deol | Snell's Waltzer, a New Mutation Affecting Behaviour and the Inner Ear in the Mouse[END_REF].

Immunofluorescence on sections of the mouse organ of Corti shows that Myo6 normally concentrates within the inner and outer hair cells of the sensory epithelium of the cochlea, while in sv/sv mouse, no Myo6 was detected and hairs cells degeneration with hair cell stereocilia fusion was seen after only 3 weeks post-birth [START_REF] Avraham | The Mouse Snell's Waltzer Deafness Gene Encodes an Unconventional Myosin Required for Structural Integrity of Inner Ear Hair Cells[END_REF]. At six weeks of age, they found a complete loss of both inner and outer hair cells within the organ of Corti [START_REF] Avraham | The Mouse Snell's Waltzer Deafness Gene Encodes an Unconventional Myosin Required for Structural Integrity of Inner Ear Hair Cells[END_REF] and formation of giant stereocilia 20 days after birth [START_REF] Self | Role of Myosin VI in the Differentiation of Cochlear Hair Cells[END_REF] resulting in deafness (Figure 11). [START_REF] Melchionda | MYO6, the Human Homologue of the Gene Responsible for Deafness in Snell's Waltzer Mice, Is Mutated in Autosomal Dominant Nonsyndromic Hearing Loss[END_REF] then confirmed that Myo6 dysfunction was also responsible for hearing loss in human. Since then, an ever-growing number of pathogen mutations and genetics anomaly were found in the human and mouse Myo6 gene, causing either dominant or recessive genetic hearing loss (Figure 12). [START_REF] Alkowari | Targeted Sequencing Identifies Novel Variants Involved in Autosomal Recessive Hereditary Hearing Loss in Qatari Families[END_REF][START_REF] Brownstein | Novel Myosin Mutations for Hereditary Hearing Loss Revealed by Targeted Genomic Capture and Massively Parallel Sequencing[END_REF][START_REF] Wu | Clinical and Genetic Study of 12 Chinese Han Families with Nonsyndromic Deafness[END_REF][START_REF] Venugopal | Two Monogenic Disorders Masquerading as One: Severe Congenital Neutropenia with Monocytosis and Non-Syndromic Sensorineural Hearing Loss[END_REF][START_REF] Melchionda | MYO6, the Human Homologue of the Gene Responsible for Deafness in Snell's Waltzer Mice, Is Mutated in Autosomal Dominant Nonsyndromic Hearing Loss[END_REF][START_REF] Ahmed | Mutations of MYO6 Are Associated with Recessive Deafness, DFNB37[END_REF][START_REF] Seki | A Novel Splice Site Mutation of Myosin VI in Mice Leads to Stereociliary Fusion Caused by Disruption of Actin Networks in the Apical Region of Inner Ear Hair Cells[END_REF][START_REF] Sampaio-Silva | Exome Sequencing Identifies a Novel Nonsense Mutation of MYO6 as the Cause of Deafness in a Brazilian Family[END_REF]Kim et al. 2018;[START_REF] Alimardani | Targeted Mutation Analysis of the SLC26A4, MYO6, PJVK and CDH23 Genes in Iranian Patients with AR Nonsyndromic Hearing Loss[END_REF]Wang et al. 2019;[START_REF] Wu | Clinical and Genetic Study of 12 Chinese Han Families with Nonsyndromic Deafness[END_REF][START_REF] Tian | Identification of a Novel MYO6 Mutation Associated with Autosomal Dominant Non-Syndromic Hearing Loss in a Chinese Family by Whole-Exome Sequencing[END_REF][START_REF] Talebi | Next-Generation Sequencing Identifies Three Novel Missense Variants in ILDR1 and MYO6 Genes in an Iranian Family with Hearing Loss with Review of the Literature[END_REF][START_REF] Oka | Clinical Characteristics and In Vitro Analysis of MYO6 Variants Causing Late-Onset Progressive Hearing Loss[END_REF] Fluorescence in situ hybridization (FISH) and sequencing experiments have shown that hereditary cochlear aplasia is linked to additional Myo6 copies in genomes (Sun et al. 2020). In contrast Myo6 haploinsufficiency results in progressive hearing loss [START_REF] Seki | Myosin VI Haploinsufficiency Reduced Hearing Ability in Mice[END_REF]. Mutations resulting in Myo6 severe truncation [START_REF] Sampaio-Silva | Exome Sequencing Identifies a Novel Nonsense Mutation of MYO6 as the Cause of Deafness in a Brazilian Family[END_REF][START_REF] Wu | Clinical and Genetic Study of 12 Chinese Han Families with Nonsyndromic Deafness[END_REF] or aberrant splicing [START_REF] Brownstein | Novel myosin mutations for hereditary hearing loss revealed by targeted genomic capture and massively parallel sequencing[END_REF][START_REF] Seki | A Novel Splice Site Mutation of Myosin VI in Mice Leads to Stereociliary Fusion Caused by Disruption of Actin Networks in the Apical Region of Inner Ear Hair Cells[END_REF] were also found to cause moderate to profound hearing loss or deafness. If most of the point mutations are located either in the Motor domain or in the cargo binding domain, suggesting an impact on the motor activity or proper recruitment through cellular partners binding to the cargo binding domain. Only two of them: the L926Q [START_REF] Brownstein | Novel Myosin Mutations for Hereditary Hearing Loss Revealed by Targeted Genomic Capture and Massively Parallel Sequencing[END_REF]) and the R928C [START_REF] Talebi | Next-Generation Sequencing Identifies Three Novel Missense Variants in ILDR1 and MYO6 Genes in an Iranian Family with Hearing Loss with Review of the Literature[END_REF]) mutations were identified in the rest of the Myo6 molecule, and their precise effect on Myo6 function thus remains to be determined.

A recent study achieves auditory rescue of Myo6 WT/C442Y mice up to 5 months by reintroducing Myo6 through in vivo CriprCas9 assessing the importance of the MYO6 gene in hearing loss and deafness and raising the possibility of a future treatment through gene editing [START_REF] Xue | Gene Editing in a Myo6 Semi-Dominant Mouse Model Rescues Auditory Function[END_REF].

In parallel, a closer look at the Snell's Waltzer mice phenotype reveals symptoms related to Myo6 in addition to hearing loss suggesting the versatility of Myo6 motor:

-Fertility problems for male Snell's Waltzer mice [START_REF] Avraham | The Mouse Snell's Waltzer Deafness Gene Encodes an Unconventional Myosin Required for Structural Integrity of Inner Ear Hair Cells[END_REF][START_REF] Zakrzewski | Diverse Functions of Myosin VI in Spermiogenesis[END_REF] -Hypertrophic cardiomyopathy [START_REF] Hegan | Myosin VI and Cardiomyopathy: Left Ventricular Hypertrophy, Fibrosis, and Both Cardiac and Pulmonary Vascular Endothelial Cell Defects in the Snell's Waltzer Mouse[END_REF] -Renal defects and proteinuria [START_REF] Gotoh | Altered Renal Proximal Tubular Endocytosis and Histology in Mice Lacking Myosin-VI[END_REF]

B. Myo6 unique features

Myo6 exhibits a typical organization for a myosin motor with a Motor domain, a Lever arm and a Tail but unique features set Myo6 apart (Figure 13): Sequence analysis shows good conservation of the N-terminus Motor domain typical of a myosin motor [START_REF] Kellerman | An Unconventional Myosin Heavy Chain Gene from Drosophila Melanogaster[END_REF]. Determination of structures of the Myo6 motor (wild-type or mutants) in six different states of the ATPase cycle allows a deTailed comprehension of the Myo6 cycle making the Myo6 cycle the most well described among myosins (Table 1). This shows that, despite directing its force towards the minus-end of actin filaments and despite the slower ATP binding compared to other myosins, the Motor domain conformational changes along the cycle are very similar with the other myosins [START_REF] Gurel | Cryo-EM Structures Reveal Specialization at the Myosin VI-Actin Interface and a Mechanism of Force Sensitivity[END_REF][START_REF] Ménétrey | The Structure of the Myosin VI Motor Reveals the Mechanism of Directionality Reversal[END_REF]2007;2008;2012) (Figure 14). [START_REF] Llinas | How Actin Initiates the Motor Activity of Myosin[END_REF][START_REF] Gurel | Cryo-EM Structures Reveal Specialization at the Myosin VI-Actin Interface and a Mechanism of Force Sensitivity[END_REF][START_REF] Ménétrey | The Structure of the Myosin VI Motor Reveals the Mechanism of Directionality Reversal[END_REF][START_REF] Pylypenko | Role of Insert-1 of Myosin VI in Modulating Nucleotide Affinity[END_REF]2015;Ménétrey et al. 2007;2008;[START_REF] Blanc | An Intermediate along the Recovery Stroke of Myosin VI Revealed by X-Ray Crystallography and Molecular Dynamics[END_REF][START_REF] Ménétrey | Processive Steps in the Reverse Direction Require Uncoupling of the Lead Head Lever arm of Myosin VI[END_REF] Uniquely, the Myo6 Motor was crystallized in an intermediate state between the post-rigor and the pre-powerstroke characterized by an open switch-II (as for post-rigor) but a relay and a SH1 helix close to the pre-powerstroke conformation and a partially reprimed converter. Structure analysis and molecular dynamics thus offer unique insights on the Myo6 recovery stroke (and probably on the recovery stroke of all myosins). It suggests that the Lever arm repriming is not coupled with switch-II closure (as previously proposed from molecular dynamics), but rather be driven by thermal fluctuations [START_REF] Blanc | An Intermediate along the Recovery Stroke of Myosin VI Revealed by X-Ray Crystallography and Molecular Dynamics[END_REF]. The Myo6 Pi release state is also particularly well characterized. Indeed, a serie of Myo6 crystals soaked with Pi for different times revealed that Myo6 can adopt a state exhibiting a primed converter (as in the pre-powerstroke) but an actin binding interface and an open switch-II allowing Pi to escape [START_REF] Llinas | How Actin Initiates the Motor Activity of Myosin[END_REF]. This state was thus proposed to constitute the Pi [START_REF] Llinas | How Actin Initiates the Motor Activity of Myosin[END_REF], the post rigor (PDB: 2VB6, [START_REF] Ménétrey | The Post-Rigor Structure of Myosin VI and Implications for the Recovery Stroke[END_REF], the pre-transition state (PDB: 5O2L, [START_REF] Blanc | An Intermediate along the Recovery Stroke of Myosin VI Revealed by X-Ray Crystallography and Molecular Dynamics[END_REF]) and the pre-powerstroke (PDB: 4ANJ, [START_REF] Ménétrey | Processive Steps in the Reverse Direction Require Uncoupling of the Lead Head Lever arm of Myosin VI[END_REF] Finally, sequence analysis revealed the presence of a unique 25 amino acid insert within Myo6

Motor domain (residues 316-329 in porcine Myo6) [START_REF] Hasson | Porcine Myosin-VI: Characterization of a New Mammalian Unconventional Myosin[END_REF]. This so-called "Insert-

B.2. Myo6 Lever arm

The Myo6 Lever arm was thought to be particularly short as only 1 IQ motif can be found within the Myo6 neck [START_REF] Kellerman | An Unconventional Myosin Heavy Chain Gene from Drosophila Melanogaster[END_REF], Hasson et Mooseker 1994). Yet , uniquely, a ~50 amino acid insert (the Insert-2 or Ins2) distances the IQ from the Motor domain [START_REF] Hasson | Porcine Myosin-VI: Characterization of a New Mammalian Unconventional Myosin[END_REF] which is unexpected for a myosin motor where the IQ is usually directly connected to the converter.

Interestingly, although it does not contain a canonical CaM binding motif [START_REF] Hasson | Porcine Myosin-VI: Characterization of a New Mammalian Unconventional Myosin[END_REF],

is was discovered to be a Ca 2+ -CaM binding element [START_REF] Bahloul | The Unique Insert in Myosin VI Is a Structural Calcium-Calmodulin Binding Site[END_REF]. (A) Model of Myo5 [START_REF] Coureux | A Structural State of the Myosin V Motor without Bound Nucleotide[END_REF] and Myo6 [START_REF] Ménétrey | The Structure of the Myosin VI Motor Reveals the Mechanism of Directionality Reversal[END_REF] The crystal structure of the Motor domain with Ins2/Ca 2+ -CaM in rigor state confirmed the ability of Ins2 to bind to CaM light chain with four Ca 2+ ions [START_REF] Ménétrey | The Structure of the Myosin VI Motor Reveals the Mechanism of Directionality Reversal[END_REF]. Furthermore, this crystal structure revealed that in rigor state, Ins2-CaM complex interacts with the converter resulting in its reorientation of 120° compared to plus-end myosins allowing force generation toward the minusend of the actin filament [START_REF] Ménétrey | The Structure of the Myosin VI Motor Reveals the Mechanism of Directionality Reversal[END_REF]) (Figure 17). Consistently, motility assays revealed that the removal of Ins2 or its replacement by a conventional IQ motif makes Myo6 a plus-end directed motor further confirming that this unique Insert-2 is at the origin of the reverse directionally of Myo6 [START_REF] Park | The Unique Insert at the End of the Myosin VI Motor Is the Sole Determinant of Directionality[END_REF][START_REF] Bryant | The Power Stroke of Myosin VI and the Basis of Reverse Directionality[END_REF] .

Then, in 2007, a crystal structure of Myo6 was obtained in the pre-powerstroke state (PDB:

2V26, Ménétrey et al. 2007). This structure, which contains the Motor domain, the converter and the first helix of the Insert-2, revealed that Myo6 converter undergoes conformational rearrangements between the pre-powerstroke and the rigor-like state which is unexpected for a myosin motor (Ménétrey et al. 2007) (Figure 18). The first helix of the Insert-2 is reorientated by 90° between the two states. As a consequence, Myo6 is able to perform a particularly large stroke compared to other myosins that would not be possible without rearrangement of the converter (Ménétrey et al. 2007). Myo6 powerstroke is thus quite unique among myosins as the powerstroke does not only rely on Lever arm swing but also the rearrangements within the converter (Figure 19). Finally, a crystal structure of the Myo6 Lever arm containing both the Ins2 and IQ motif was obtained in 2009 (3GN4; [START_REF] Mukherjea | Myosin VI Dimerization Triggers an Unfolding of a Three-Helix Bundle in Order to Extend Its Reach[END_REF]) (Figure 20). Consistently, Ins2 binds to Ca 2+ -CaM while the IQ motif binds to apoCaM (no Ca 2+ bound to it), (3GN4, [START_REF] Mukherjea | Myosin VI Dimerization Triggers an Unfolding of a Three-Helix Bundle in Order to Extend Its Reach[END_REF]). However, titration experiments show that the IQ motif is able to bind to CaM at both low and high calcium concentration but with higher affinity at high calcium concentrations. Moreover, a study by electron microscopy suggests that conformational changes occur for the second CaM upon Ca 2+ binding (Batters et al. 2016).

Ca 2+ was thus proposed as a regulatory element of the Myo6 force production. Rhodamine fluorescence quenching [START_REF] Mukherjea | Myosin VI Dimerization Triggers an Unfolding of a Three-Helix Bundle in Order to Extend Its Reach[END_REF] and NMR [START_REF] Yu | Membrane-Induced Lever arm Expansion Allows Myosin VI to Walk with Large and Variable Step Sizes[END_REF] experiments demonstrated the ability of 3HB to unfold to adopt an extended conformation upon artificial dimerization of Myo6 or DPC micelles addition (respectively), highlighting the putative 3HB role for Myo6 regulation and more specifically as a Lever arm extension. Indeed, [START_REF] Mukherjea | Myosin VI Must Dimerize and Deploy Its Unusual Lever arm in Order to Perform Its Cellular Roles[END_REF]Batters et al. 2016 show the ability of residues 834-875 and 833-856 (respectively) positioned at the N-terminus of Myo6 3HB to bind to a CaM supporting the 3HB potential to act as a Lever arm extension following 3HB unfolding. The precise role and composition of this Lever arm extension remains to be clarified. [START_REF] Mukherjea | Myosin VI Must Dimerize and Deploy Its Unusual Lever arm in Order to Perform Its Cellular Roles[END_REF] proposed a recruitment of a third CaM in EGTA conditions (specific Ca 2+ chelator) would allow to extend a linear Lever arm while Batters et al. 2016 proposed that the 833-856 region would fold back on the CaM bound to the IQ upon Ca 2+ addition (pCa 4). Both propositions result in a rigidification of the 3HB N-terminus upon its unfolding (also see section E.6.b).

Following the 3HB, the sequence corresponds to a single α-helix domain (SAH or medial Tail) (aa917-978). Interestingly, a few apolar residues are found within the helix in particular at its Nterminus and around the residue 955 that do not perfectly follow a (i, i+4) distribution (Figure 22). An NMR study of the SAH domain shows that it corresponds to a stiff α-helix with a persistent length of ~200 Å (PDB: 6OBI, [START_REF] Barnes | Remarkable Rigidity of the Single α-Helical Domain of Myosin-VI As Revealed by NMR Spectroscopy[END_REF]. * marks all the hydrophobic residues found within the SAH domain

3HB917 SAH

The distal Tail (979-1034) has been proposed to constitute a domain of 3 nm of diameter when associated to SAH based on a SAXS study (Spink et al. 2008). On the basis of a Rosetta prediction, Spink et al. 2008 thus proposed that the DT domain would be constituted by three helices interacting with each other's (Figure 23). However, an NMR study of the 998-1071 fragment reveals that alone the DT domain is a succession of helices with high flexibility [START_REF] He | Myosin VI Contains a Compact Structural Motif That Binds to Ubiquitin Chains[END_REF] (Figure 23). Perhaps the DT domain can be stabilized by other elements of the Tail in the context of FLMyo6, thus further studies are required to conclude on the structure and dynamics of this domain. The DT domain was also shown to bind ubiquitin (MIU domain) through its distal amino acids 998-1031 [START_REF] Penengo | Crystal Structure of the Ubiquitin Binding Domains of Rabex-5 Reveals Two Modes of Interaction with Ubiquitin[END_REF]. The CBD can interact with a variety of cargos through different binding sites: two sequence motifs: the RRL motif within the CBD n and the WWY motif within the CBD c have been identified as critical for Myo6 interaction with various proteins, while the WKSKNKKR motif has been proposed to constitute a lipid binding site [START_REF] Spudich | Myosin VI Targeting to Clathrin-Coated Structures and Dimerization Is Mediated by Binding to Disabled-2 and PtdIns(4,5)P2[END_REF]). An ubiquitin binding site (MyUb) has also been identified close to the RRL motif. It exhibits higher affinity for ubiquitin than the MIU [START_REF] He | Myosin VI Contains a Compact Structural Motif That Binds to Ubiquitin Chains[END_REF].

MIU f LI f iso1 QQQAVLEQERRDRELALRIAQSEAELISDEAQADLALRR---------NDGTRPKMTPEQMAKEMSEFLSRGPAVLATKAAAG iso2 QQQAVLEQERRDRELALRIAQSEAELISDEAQADLALR--------------------------------RGPAVLATKAAAG iso3 QQQAVLEQERRDRELALRIAQSEAELISDEAQADLALRRSLDSYPVSKNDGTRPKMTPEQMAKEMSEFLSRGPAVLATKAAAG iso4 QQQAVLEQERRDRELALRIAQSEAELISDEAQADLALRRSLDSYPVSKNDGTRPKMTPEQMAKEMSEFLSRGPAVLATKAAAG iso5 QQQAVLEQERRDRELALRIAQSEAELISDEAQADLALR--------------------------------RGPAVLATKAAAG iso6 QQQAVLEQERRDRELALRIAQSEAELISDEAQADLALRRSLDSYPVSKNDGTRPKMTPEQMAKEMSEFLSRGPAVLATKAAAG f MyUb f f SI f iso1 TKKYDLSKWKYAELRDTINTSCDIELLAACREEFHRRLKVYHAWKSKNKKRNTETEQRAPKSVTDYDFAPFLNNSPQQNPAAQ iso2 TKKYDLSKWKYAELRDTINTSCDIELLAACREEFHRRLKVYHAWKSKNKKRNTETEQRAPKSVTDYDFAPFLNNSPQQNPAAQ iso3 TKKYDLSKWKYAELRDTINTSCDIELLAACREEFHRRLKVYHAWKSKNKKRNTETEQRAPKSVTDYDFAPFLNNSPQQNPAAQ iso4 TKKYDLSKWKYAELRDTINTSCDIELLAACREEFHRRLKVYHAWKSKNKKRNTETEQRAPKSVTDY---------PQQNPAAQ iso5 TKKYDLSKWKYAELRDTINTSCDIELLAACREEFHRRLKVYHAWKSKNKKRNTETEQRAPKSVTDY---------PQQNPAAQ iso6 TKKYDLSKWKYAELRDTINTSCDIELLAACREEFHRRLKVYHAWKSKNKKRNTETEQRAPKSVTDY---------AQQNPAAQ iso1 IPARQREIEMNRQQRFFRIPFIRPADQYKDPQSKKKGWWYAHFDGPWIARQMELHPDKPPILLVAGKDDMEMCELNLEETGLT iso2 IPARQREIEMNRQQRFFRIPFIRPADQYKDPQSKKKGWWYAHFDGPWIARQMELHPDKPPILLVAGKDDMEMCELNLEETGLT iso3 IPARQREIEMNRQQRFFRIPFIRPADQYKDPQSKKKGWWYAHFDGPWIARQMELHPDKPPILLVAGKDDMEMCELNLEETGLT iso4 IPARQREIEMNRQQRFFRIPFIRPADQYKDPQSKKKGWWYAHFDGPWIARQMELHPDKPPILLVAGKDDMEMCELNLEETGLT iso5 IPARQREIEMNRQQRFFRIPFIRPADQYKDPQSKKKGWWYAHFDGPWIARQMELHPDKPPILLVAGKDDMEMCELNLEETGLT iso6 IPARQREIEMNRQQRFFRIPFIRPADQYKDPQSKKKGWWYAHFDGPWIARQMELHPDKPPILLVAGKDDMEMCELNLEETGLT iso1 RKRGAEILPRQFEEIWERCGGIQYLQNAIESRQARPTYATAMLQSLLK
The CBD c TRTK and SKKK motif have been proposed to participate in DNA binding [START_REF] Fili | NDP52 Activates Nuclear Myosin VI to Enhance RNA Polymerase II Transcription[END_REF].

Finally, a clathrin interaction binding site overlaps with the RRL motif and requires the LI insert [START_REF] Biancospino | Clathrin Light Chain A Drives Selective Myosin VI Recruitment to Clathrin-Coated Pits under Membrane Tension[END_REF]. pictured in brown, it contains the CBD n that mediates binding (from the N-terminus to the C-terminus) to ubiquitin through its MyUb domain [START_REF] He | Myosin VI Contains a Compact Structural Motif That Binds to Ubiquitin Chains[END_REF], to several protein partners such as GIPC1 through its RRL motif and to the PiP2 lipid through its WKSKNKKR motif [START_REF] Spudich | Myosin VI Targeting to Clathrin-Coated Structures and Dimerization Is Mediated by Binding to Disabled-2 and PtdIns(4,5)P2[END_REF]. The CBD also contains CBD c that can mediate interactions with partners such as Dab2 and TOM1 through its WWY motif and to DNA through its SKKK and TRTK motifs. CBD n and CBD c can be separated by a small insert (SI) in certain spliced isoforms and a long insert (LI) can be found between the distal Tail and the CBD n in certain spliced isoforms and can mediate interaction with clathrin [START_REF] Biancospino | Clathrin Light Chain A Drives Selective Myosin VI Recruitment to Clathrin-Coated Pits under Membrane Tension[END_REF]. Although no structure containing the whole CBD domain is currently available, structures of several fragments were solved by NMR [START_REF] Yu | Myosin VI Undergoes Cargo-Mediated Dimerization[END_REF][START_REF] Wollscheid | Diverse Functions of Myosin VI Elucidated by an Isoform-Specific α-Helix Domain[END_REF]. The structures available are depicted above the diagram with their corresponding PDB code.

Several NMR structures described portions of the cargo binding domain. Yet, up to date, no structure of the complete cargo-binding domain has been described (Figure 26). Thus, parts of the CBD n structure remains unresolved, probably due to the difficulty in purifying a stable CBD construct [START_REF] Shang | Structure Analyses Reveal a Regulated Oligomerization Mechanism of the PlexinD1/GIPC/Myosin VI Complex[END_REF][START_REF] Yu | Myosin VI Undergoes Cargo-Mediated Dimerization[END_REF][START_REF] Spudich | Myosin VI Targeting to Clathrin-Coated Structures and Dimerization Is Mediated by Binding to Disabled-2 and PtdIns(4,5)P2[END_REF]). Moreover, it is unknown from the literature if CBD n and CBD c interact with each other in a folded domain or if it consists in two independent Myo6 domains.

C. Wide range of cell partners links Myo6 to various functions and diseases

Through the various interaction sites within the CBD and the DT, Myo6 has the ability to interact with a wide range of proteins and even with lipids [START_REF] Spudich | Myosin VI Targeting to Clathrin-Coated Structures and Dimerization Is Mediated by Binding to Disabled-2 and PtdIns(4,5)P2[END_REF]) and DNA [START_REF] Fili | NDP52 Activates Nuclear Myosin VI to Enhance RNA Polymerase II Transcription[END_REF]). O'Loughlin, Masters, and Buss (2018) used proteomics to provide a map of the wide Myo6 interactome (Figure 27). These partners were for several of them already characterized as key in many cellular processes and diseases [START_REF] Qiu | Emerging Views of OPTN (Optineurin) Function in the Autophagic Process Associated with Disease[END_REF][START_REF] Ogbu | The Role of Disabled-2 (Dab2) in Diseases[END_REF][START_REF] Viret | Novel Insights into NDP52 Autophagy Receptor Functioning[END_REF][START_REF] Katoh | Functional Proteomics, Human Genetics and Cancer Biology of GIPC Family Members[END_REF]). This emphasizes the potential of Myo6 as a multifunctional motor.

C.1. WWY partners

C.1.a. Dab2

Dab2 is present on clathrin-coated structures at the plasma membrane and can interact with cell surface receptors as well as many proteins (AP2 [START_REF] Mishra | Disabled-2 Exhibits the Properties of a Cargo-Selective Endocytic Clathrin Adaptor[END_REF], ITSN1 [START_REF] Teckchandani | The Clathrin Adaptor Dab2 Recruits EH Domain Scaffold Proteins to Regulate Integrin Β1 Endocytosis[END_REF], EPS15 [START_REF] Teckchandani | The Clathrin Adaptor Dab2 Recruits EH Domain Scaffold Proteins to Regulate Integrin Β1 Endocytosis[END_REF]) and FCHO2 [START_REF] Mulkearns | FCH Domain Only-2 Organizes Clathrin-Coated Structures and Interacts with Disabled-2 for Low-Density Lipoprotein Receptor Endocytosis[END_REF]). These are involved in clathrin endocytosis and were also found to interact with Myo6 through in situ proximity labelling (O'Loughlin, Masters, and Buss 2018). Dab2 interacts with Myo6 through the WWY motif [START_REF] Inoue | DOC-2/DAB2 Is the Binding Partner of Myosin VI[END_REF])(Table 2) and thus mediates Myo6 recruitment to clathrin coated structures suggesting a role of the complex in the transport of vesicles along the actin cytoskeleton [START_REF] Spudich | Myosin VI Targeting to Clathrin-Coated Structures and Dimerization Is Mediated by Binding to Disabled-2 and PtdIns(4,5)P2[END_REF][START_REF] Morris | Myosin VI Binds to and Localises with Dab2, Potentially Linking Receptor-Mediated Endocytosis and the Actin Cytoskeleton[END_REF]. The crystal structure of Dab2 in complex with the Myo6 CBD c provides a deTailed description of the interaction surfaces showing that Dab2 interacts with Myo6 through two interfaces and confirming the role of the WWY motif [START_REF] Yu | Myosin VI Undergoes Cargo-Mediated Dimerization[END_REF]) (Figure 28) ( also see section G.1.). ). Yet the integrity of the WWY motif is not required for the localization of the Myo6 LI isoform on clathrin coated structures [START_REF] Wollscheid | Diverse Functions of Myosin VI Elucidated by an Isoform-Specific α-Helix Domain[END_REF]. Finally, Dab2

was described as a tumor suppressor (see section D.6.).

C.1.b. LMTK2

Lemur Tyrosine Kinase 2 (LMTK2) is an ubiquitous transmembrane serine/threonine kinase [START_REF] Wang | A Novel Transmembrane Ser/Thr Kinase Complexes with Protein Phosphatase-1 and Inhibitor-2[END_REF] that was found to mostly localize near the Golgi complex [START_REF] Kesavapany | Identification of a Novel, Membrane-Associated Neuronal Kinase, Cyclin-Dependent Kinase 5/P35-Regulated Kinase[END_REF]. It is notably involved in endocytosis trafficking and apoptosis and is linked to diseases such a neurodegenerescence, infertility and cancers [START_REF] Cruz | Unraveling the Function of Lemur Tyrosine Kinase 2 Network[END_REF]. LMTK2 was identified as a Myo6 binding partner through a yeast 2-hybrid screen and a GST-pull-down. This shows that LMTK2 binds Myo6 through its C-terminus cytoplasmic domain. Furthermore, mammalian 2hybrid using Myo6 mutants show that LMTK2 is a WWY motif binder [START_REF] Chibalina | Myosin VI and Its Interacting Protein LMTK2 Regulate Tubule Formation and Transport to the Endocytic Recycling Compartment[END_REF]. They were also found to co-localize and co-precipitate together from HeLa cells. Like Myo6, LMTK2 localizes on Rab5 positive early endosomes [START_REF] Chibalina | Myosin VI and Its Interacting Protein LMTK2 Regulate Tubule Formation and Transport to the Endocytic Recycling Compartment[END_REF]. But as opposed to Myo6, LMTK2 does not seem to play a key role in secretory pathways as SEAP secretion is impaired by Myo6 knockdown but not by LMTK2 knockdown [START_REF] Chibalina | Myosin VI and Its Interacting Protein LMTK2 Regulate Tubule Formation and Transport to the Endocytic Recycling Compartment[END_REF]. Both Myo6 and LMTK2 knockdown result in formation of abnormal large endosome structures, impairment of transferrin receptor recycling in perinuclear recycling vesicles from early endosomes [START_REF] Inoue | BREK/LMTK2 Is a Myosin VI-Binding Protein Involved in Endosomal Membrane Trafficking[END_REF][START_REF] Chibalina | Myosin VI and Its Interacting Protein LMTK2 Regulate Tubule Formation and Transport to the Endocytic Recycling Compartment[END_REF].

C.1.c. TOM1 and TOM1L2

The target of Myb1 (TOM1) and TOM1-like2 (TOM1-L2) proteins are early members of the Endosomal Sorting Complexes Required for Transport (ESCRT), which are required for degradation of ubiquitynilated cargos. TOM1 and TOM1-L2 are able to interact with clathrin [START_REF] Seet | Endofin Recruits Clathrin to Early Endosomes via TOM1[END_REF]Katoh et al. 2006) and ubiquitinilated cargos [START_REF] Yamakami | Tom1, a VHS Domain-Containing Protein, Interacts with Tollip, Ubiquitin, and Clathrin[END_REF]Katoh et al. 2006). TOM1 binds also endofin, mediating clathrin recruitment to endosomes [START_REF] Seet | Endofin Recruits Clathrin to Early Endosomes via TOM1[END_REF].

TOM1 interaction with Tollip results in a loss of the interaction of Tollip with phosphatidylinositol-3phosphate [START_REF] Xiao | Tom1 Modulates Binding of Tollip to Phosphatidylinositol 3-Phosphate via a Coupled Folding and Binding Mechanism[END_REF], so that is is ready to bind cargos [START_REF] Mitra | Ubiquitin Interacts with the Tollip C2 and CUE Domains and Inhibits Binding of Tollip to Phosphoinositides[END_REF]). TOM1 has also been identified as a binding partner of phosphatidylinositol 5 phosphate (PI5P), which is implicated in the endosomal sorting of the epidermal growth factor receptor (EGFR). Its recruitment by TOM1 induces a delay in EGFR degradation and fluid-phase bulk endocytosis. The more PI5P is concentrated in the membrane, the slower is the endosomal maturation [START_REF] Boal | TOM1 Is a PI5P Effector Involved in the Regulation of Endosomal Maturation[END_REF].

TOM1 and TOM1-L2 were found to be Myo6 partners (Table 3) based on pull-down and mammalian 2-hybrid experiments. Use of different truncation and mutagenesis shows that TOM1 interact with the Myo6 WWY site within its last 107 amino acids [START_REF] Tumbarello | Autophagy Receptors Link Myosin VI to Autophagosomes to Mediate Tom1-Dependent Autophagosome Maturation and Fusion with the Lysosome[END_REF]. Recently, the crystal structure of the CBD c and a small helix of TOM1 was solved offering a precise description of the binding interface [START_REF] Hu | Structure of Myosin VI/Tom1 Complex Reveals a Cargo Recognition Mode of Myosin VI for Tethering[END_REF]) (Figure 29). TOM1-L2 was found to colocalize with Myo6 on tubulobulbar complex and APPL1/Rab5 positive endosomes during spermatid maturation and Myo6 depletion results in loss of TOM1-L2 localization on these specialized structures although the function of the Myo6-TOM1-L2 complex is unclear [START_REF] Zakrzewski | Myosin VI Maintains the Actin-Dependent Organization of the Tubulobulbar Complexes Required for Endocytosis during Mouse Spermiogenesis † ‡[END_REF]. Interestingly, TOM1 depletion in RPE cells mimics the Myo6 depletion phenotype on autophagy: it delays autophagosome fusion with lysosomes for their degradation [START_REF] Tumbarello | Autophagy Receptors Link Myosin VI to Autophagosomes to Mediate Tom1-Dependent Autophagosome Maturation and Fusion with the Lysosome[END_REF], suggesting a common role for autophagy regulation, notably through correct addressing of Myo6 to endosomes by TOM1, as TOM1 depletion results in loss of Myo6 localization to endosomes [START_REF] Tumbarello | Autophagy Receptors Link Myosin VI to Autophagosomes to Mediate Tom1-Dependent Autophagosome Maturation and Fusion with the Lysosome[END_REF]. Furthermore, TOM1 silencing, similarly to Myo6 silencing, results

on the accumulation of Rab5 endosomes on nuclear periphery on HeLa cells suggesting that the spatial organization of endosomes depends on TOM1-Myo6 interaction (Master et al. 2017). Moreover, TOM1 play a key role in amyloid-β clearance. In Alzheimer disease, amyloid-β neurotoxic accumulation in dystrophic neurites causes memory loss [START_REF] Gwon | TOM1 Regulates Neuronal Accumulation of Amyloid-β Oligomers by FcγRIIb2 Variant in Alzheimer's Disease[END_REF]. Thereby, TOM1 knockdown from sick mice result in an aggravation of Alzheimer disease but was rescued by increase in TOM1 expression [START_REF] Martini | Amyloid-Beta Impairs TOM1-Mediated IL-1R1 Signaling[END_REF]. Although the importance of the Myo6-TOM1 complex has not been clearly established in this context, Myo6 was found to colocalize with TOM1 in dystrophic neurites suggesting a role of the motor for amyloid-β trafficking [START_REF] Makioka | Immunolocalization of Tom1 in Relation to Protein Degradation Systems in Alzheimer's Disease[END_REF]Feuillette et al. 2010).

C.2. RRL partners

C.2.a. GIPC

The GIPC family consists in an ensemble of highly conserved proteins GIPC1, GIPC2 and GIPC3.

GIPC contributes to the endocytosis process by facilitating the formation of clathrin coated pits and ligand clustering in these pits [START_REF] Hasson | Myosin VI: Two Distinct Roles in Endocytosis[END_REF]. It is also involved in receptor recycling, signaling, angiogenesis and cytokinesis [START_REF] Katoh | Functional Proteomics, Human Genetics and Cancer Biology of GIPC Family Members[END_REF]. Through its PDZ domain, GIPC1 can interact with a wide variety of proteins including transmembrane proteins like TGFβR3 (transforming growth factorβ receptor type III) signaling proteins like RGS19 (regulator of G-protein signaling 19) and even viral proteins like HPV-18 E6 (human papillomavirus type 18 protein E6 [START_REF] Katoh | Functional Proteomics, Human Genetics and Cancer Biology of GIPC Family Members[END_REF]). GIPC1 has also been described as a therapeutical target in cancers (see section D.6.).

Yeast 2-hybrid assays first show that GIPC1 can interact with Myo6 [START_REF] Bunn | Protein Interactions with the Glucose Transporter Binding Protein GLUT1CBP That Provide a Link between GLUT1 and the Cytoskeleton[END_REF]. The crystal structure between CBD n and GIPC1 and GIPC2 confirmed GIPC as a Myo6 partner that binds through the Myo6 RRL motif and provided a deTailed description of the complex as an oligomer [START_REF] Shang | Structure Analyses Reveal a Regulated Oligomerization Mechanism of the PlexinD1/GIPC/Myosin VI Complex[END_REF]) (Figure 30), although this oligomerization capacity is controversial (see section G.1.). Moreover, GIPC1 can also form a ternary complex with Myo6 and LARG, an actin network regulator, thus GIPC1 could play a role in endocytosis and actin organization when associated with Myo6 (O'Loughlin, Masters, and Buss 2018). As GIPC1 is able to localize on uncoated vesicles in Snell's Waltzer Myo6-null mice kidney cells, Myo6 is not required for GIPC1 localization on uncoated vesicles (Naccache, Hasson, and Horowitz 2006), perhaps thanks to direct interaction between APPL1 and GIPC1 PDZ domain. Indeed, transfection of a GIPC1 construct lacking the GIPC1 PDZ domain failed to colocalize with uncoated vesicles (Naccache, Hasson, and Horowitz 2006) and interestingly failed to coprecipitate with Myo6 even if the Myo6 binding domain is present (Naccache, Hasson, and Horowitz 2006). These results suggest that GIPC1 localization on APPL1 positive endosomes/vesicles is required for its binding to Myo6.

C.2.b. Autophagy receptors: Optineurin, NDP52 and TAX1BP1

Optineurin (OPTN, [START_REF] Sahlender | Optineurin Links Myosin VI to the Golgi Complex and Is Involved in Golgi Organization and Exocytosis[END_REF] , nuclear dot protein 52 (NDP52, [START_REF] Morriswood | T6BP and NDP52 Are Myosin VI Binding Partners with Potential Roles in Cytokine Signalling and Cell Adhesion[END_REF], Table 4) and Tax1-binding protein 1 (TAX1BP1, Morriswood et al. 2007, Table 5) were all found to interact with Myo6 using a yeast 2-hybrid screen although affinity with TAXBP1 was later found to be rather low in ITC (~30 µM, [START_REF] Hu | Mechanistic Insights into Recognitions of Ubiquitin and Myosin VI by Autophagy Receptor TAX1BP1[END_REF]. They all contain a zinc finger domain that was shown to mediate interaction with Myo6 in the case of TAX1BP1 through an NMR study [START_REF] Hu | Mechanistic Insights into Recognitions of Ubiquitin and Myosin VI by Autophagy Receptor TAX1BP1[END_REF].

Optineurin (OPTN) has been described as a key protein in Golgi organization and vesicle trafficking [START_REF] Ryan | Optineurin: A Coordinator of Membrane-Associated Cargo Trafficking and Autophagy[END_REF][START_REF] Sahlender | Optineurin Links Myosin VI to the Golgi Complex and Is Involved in Golgi Organization and Exocytosis[END_REF] and OPTN mutations are associated with glaucoma and amyotrophic lateral sclerosis [START_REF] Swarup | Altered Functions and Interactions of Glaucoma-Associated Mutants of Optineurin[END_REF].

Through their interaction with LC3 and ubiquitinylated cargos, these proteins were described as autophagy receptors [START_REF] Qiu | Emerging Views of OPTN (Optineurin) Function in the Autophagic Process Associated with Disease[END_REF]J. White et al. 2022;[START_REF] Viret | Novel Insights into NDP52 Autophagy Receptor Functioning[END_REF] and were found to colocalize with Myo6 on autophagosomes suggesting a collaboration between these proteins in autophagy [START_REF] Tumbarello | Autophagy Receptors Link Myosin VI to Autophagosomes to Mediate Tom1-Dependent Autophagosome Maturation and Fusion with the Lysosome[END_REF]2015).

In addition to a cooperation with Myo6 in autophagy, NDP52 was also shown to interact with Myo6 in the nucleus to modulate RNA polymerase II (RNAPII) transcription [START_REF] Fili | NDP52 Activates Nuclear Myosin VI to Enhance RNA Polymerase II Transcription[END_REF]. NDP52 seems to interact with RNAPII as it partially colocalizes with it in dots and co immunoprecipitates with it (HeLa cells). Moreover, using a HeLaScribe extract, transcription levels have been shown to decrease by ~50% upon NDP52 depletion [START_REF] Fili | NDP52 Activates Nuclear Myosin VI to Enhance RNA Polymerase II Transcription[END_REF].

C.2.c. DOCK7 and the guanine nucleotide exchange factors DOCK180 family

DOCK180 activates Rho GTPases by catalyzing their binding to GTP [START_REF] Côté | Identification of an Evolutionarily Conserved Superfamily of DOCK180-Related Proteins with Guanine Nucleotide Exchange Activity[END_REF].

DOCK7 can be mainly found in nervous system where it regulates Schwann cell migration, myelination [START_REF] Yamauchi | ErbB2 Directly Activates the Exchange Factor Dock7 to Promote Schwann Cell Migration[END_REF]) and neurite outgrowth (Watabe- [START_REF] Watabe-Uchida | The Rac Activator DOCK7 Regulates Neuronal Polarity through Local Phosphorylation of Stathmin/Op18[END_REF]). DOCK7 was first described as a partner of Myo6 in neural cells PC12 [START_REF] Majewski | Dock7: A GEF for Rho-Family GTPases and a Novel Myosin VI-Binding Partner in Neuronal PC12 Cells[END_REF]. It was then further established that this interaction occurs through the RRL motif of Myo6 and is also important for neurite outgrowth [START_REF] Sobczak | Interaction of Myosin VI and Its Binding Partner DOCK7 Plays an Important Role in NGF-Stimulated Protrusion Formation in PC12 Cells[END_REF].

The DOCK180 family has also been involved in cytoskeleton remodeling. Interestingly, DOCK7

has been shown to interact with Rac1 and CDC42, two key regulators of the actin network [START_REF] Zhou | Prenylation and Membrane Localization of Cdc42 Are Essential for Activation by DOCK7[END_REF]). Strikingly, it was proposed that DOCK7 forms a complex with LRCH3 and Myo6 (DISP complex) that could result in a strong remodeling of the septin cytoskeleton as coexpression of LRCH3 and DOCK7 results in a dramatic increase in septin structures in cells (O'Loughlin, Masters, and Buss 2018).

C.3. OFD1

Oral-facial-digital syndrome 1 (OFD1) is specifically localized at centriole and centriolar satellites in cells [START_REF] Magistrati | Myosin VI Regulates Ciliogenesis by Promoting the Turnover of the Centrosomal/Satellite Protein OFD1[END_REF] where it regulates centriole elongation and promotes the recruitment of distal appendage proteins [START_REF] Singla | Ofd1, a Human Disease Gene, Regulates the Length and Distal Structure of Centrioles[END_REF][START_REF] Wang | A Distal Centriolar Protein Network Controls Organelle Maturation and Asymmetry[END_REF]. Its defect results in facial, oral and digital malformations as well as brain malformation and polycystic kidney disease in some cases [START_REF] Ko | Oral-Facial-Digital Syndrome Type 1: A Case Report and Review[END_REF].

Immunofluorescence and proximity ligation assays show that OFD1 colocalizes with Myo6 on centriole constituting a link between Myo6 and the microtubule cytoskeleton. Immunoprecipitation and pull down further confirm that OFD1 and Myo6 can interact together independently of the Myo6 isoform. Pull-down using point mutants and/or truncations demonstrate that the OFD1 coiled coil interacts with the Myo6 Tail; CBD c seems critical for its binding as its truncation results in loss of OFD1 binding although usual WWY motif mutation WLY does not impact its binding. Notably, CBD c seems to need to collaborate with more proximal regions of the Tail for optimal binding [START_REF] Magistrati | Myosin VI Regulates Ciliogenesis by Promoting the Turnover of the Centrosomal/Satellite Protein OFD1[END_REF] which emphasizes the importance to obtain structures with larger Tail fragment to understand how different parts of the Tail can collaborate together for optimal binding of partners.

C.4. Clathrin

Clathrin is a key component of clathrin-mediated endocytosis where it assembles in polyhedral cages to shape vesicular and tubular carriers (Wood and Smith 2021) (Figure 31). Of note, Myo6 cannot interact with clathrin light chain b (CLCb) which may suggest different functions for CLCa and CLCb. Indeed, although both are involved in clathrin mediated endocytosis, CLCa is preferentially involved in cell speading and migration [START_REF] Tsygankova | A Unique Role for Clathrin Light Chain A in Cell Spreading and Migration[END_REF]. CLCb exhibits 40% sequence identity with CLCa and is expressed at various levels depending on the tissues as opposed to CLCa which is ubiquitinously expressed. CLCb overexpression in lung cancer causes aberrant growth factor signaling [START_REF] Chen | Crosstalk between CLCb/Dyn1-Mediated Adaptive Clathrin-Mediated Endocytosis and Epidermal Growth Factor Receptor Signaling Increases Metastasis[END_REF].

C.5. Ubiquitin

Ubiquitin is a highly conserved protein involved in signaling and degradation pathways.

Ubiquitin can form polyubiquitin chains and can covalently bind to proteins and form long ubiquitin The Myo6 MyUb domain interacts with K48, K63, K29 and K11 linked ubiquitin chains, with a clear preference for the latter three. The NMR structure of the complex between the CBD n fragment and K63-diUbiquitin revealed that ubiquitin can interact with Myo6 through two different binding interface within the MyUb, key residue for ubiquitin binding on MyUb domain I1072 (number according to the NI isoform of Myo6) was thus identified through pull-down [START_REF] He | Myosin VI Contains a Compact Structural Motif That Binds to Ubiquitin Chains[END_REF]) (Figure 34). Although no structure is available for the MIU-Ubiquitin complex, MIU was first identified in Rabex (for which the structure of the MIU domain and ubiquitin was solved by NMR) and characterized as a conserved motif found on several proteins. The A1013G mutation was thus identified, which prevents the MIU's interaction to ubiquitin [START_REF] Penengo | Crystal Structure of the Ubiquitin Binding Domains of Rabex-5 Reveals Two Modes of Interaction with Ubiquitin[END_REF][START_REF] He | Myosin VI Contains a Compact Structural Motif That Binds to Ubiquitin Chains[END_REF]. The MIU-binding motif shows no affinity or preference towards ubiquitin linkages; however, in cooperation with the MyUb domain, it seems to specifically support K11-linked ubiquitin binding [START_REF] Penengo | Crystal Structure of the Ubiquitin Binding Domains of Rabex-5 Reveals Two Modes of Interaction with Ubiquitin[END_REF][START_REF] He | Myosin VI Contains a Compact Structural Motif That Binds to Ubiquitin Chains[END_REF]) (Table 6).

C.6. Lipid

Myo6 was proposed to bind to liposomes through its CBD n region as it coprecipitates with liposomes. More precisely, liposomes were proposed to bind through WKSKNKKR, aa 1115-1122, since the WASANNNR mutant disrupts lipid binding [START_REF] Spudich | Myosin VI Targeting to Clathrin-Coated Structures and Dimerization Is Mediated by Binding to Disabled-2 and PtdIns(4,5)P2[END_REF]. Using liposomes of different compositions, it appears that Myo6 binds specifically to liposomes containing phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2 or PIP2) [START_REF] Spudich | Myosin VI Targeting to Clathrin-Coated Structures and Dimerization Is Mediated by Binding to Disabled-2 and PtdIns(4,5)P2[END_REF]) (Figure 35).

Interestingly, addition of liposomes to Myo6 CBD results in significant changes in the Myo6 circular dichroism profile indicative of conformational changes upon liposome binding (α-helix formation) [START_REF] Spudich | Myosin VI Targeting to Clathrin-Coated Structures and Dimerization Is Mediated by Binding to Disabled-2 and PtdIns(4,5)P2[END_REF]) (Figure 35), highlighting the potential of lipid binding for Myo6 regulation. Moreover, FLMyo6 is able to coprecipitate with liposomes only if calcium is added [START_REF] Spudich | Myosin VI Targeting to Clathrin-Coated Structures and Dimerization Is Mediated by Binding to Disabled-2 and PtdIns(4,5)P2[END_REF]Batters et al., 2016). As calcium does not change the ability of CBD to bind liposomes, this is likely due to Ca 2+ inducing FLMyo6 wider conformational changes that will make the lipid binding site available.

Uniquely, lipids seem also able to bind to the proximal Tail of Myo6 as addition of liposomes to the Myo6 proximal Tail results in significant structural rearrangements suggesting putative interaction between the Myo6 3HB and lipids and confirming the importance of lipid binding for Mo6 regulation [START_REF] Yu | Membrane-Induced Lever arm Expansion Allows Myosin VI to Walk with Large and Variable Step Sizes[END_REF]. (PI (4,5)P2 or PIP2) or phosphatidylinositol (3,4,5)-trisphosphate (PI (3,4,5)P). Right: Circular dichroic spectra of the Myo6 Tail with mixed brain liposomes addition (dashed line) or without (solid line) C.7. DNA DNA was also identified as a partner of Myo6. While it seems to be able to weakly bind to the Myo6 IQ-CBD n , highest affinity was found for CBD c and two sequence motifs were found to be particularly important for its binding as their depletion results in 5 or 7-fold decrease in its affinity for DNA [START_REF] Fili | NDP52 Activates Nuclear Myosin VI to Enhance RNA Polymerase II Transcription[END_REF]) (Table 7, Figure 36). In cells, this binding is particularly important for the ability of Myo6 to regulate RNAPII transcription [START_REF] Fili | NDP52 Activates Nuclear Myosin VI to Enhance RNA Polymerase II Transcription[END_REF] C.8. Fine differences in the interaction surface can modulate partner binding to Myo6

C.8.a. RRL partners interact with the RRL motif in different fashions

The correct interaction surface remains to be properly characterized for many Myo6 partners and may be slightly different for each of them. Even if several partners seem to share the same RRL binding motif, comparison of chemical shifts in Myo6 NMR spectrum upon partner addition further confirm that although OPTN, NDP52 and TAX1BP1 are RRL partners whose binding occurs mainly on the CBD n helix 1071-1122 (Figure 37, helix in green), key binding residues differ [START_REF] Biancospino | Clathrin Light Chain A Drives Selective Myosin VI Recruitment to Clathrin-Coated Pits under Membrane Tension[END_REF]. A mutagenesis analysis through a GSTpull-down assay from HEK293T cells using GST Myo6 CBD n (G1080-R1131) lacking the long insert has

shown that Myo6 point mutation R1116A (i.e. "ARL") and L1118A (i.e. "RRA") strongly impact OPTN and GIPC1 binding but impact only weakly NDP52 and TAX1BP1 binding [START_REF] Wollscheid | Diverse Functions of Myosin VI Elucidated by an Isoform-Specific α-Helix Domain[END_REF].

Further characterization through mammalian two-hybrid from RPE cells shows the relative contribution of R1116A and L1118A for GIPC1 and NDP52: using R1116A, GipC1 binding to Myo6 goes down to ~70% compared to WT while using L1118A, it goes down to ~40%. For NDP52, using R1116A, affinity only goes down to 80-90% while L1118A does not impair the binding at all [START_REF] Arden | Loss of Cargo Binding in the Human Myosin VI Deafness Mutant (R1166X) Leads to Increased Actin Filament Binding[END_REF]).

Yet, these findings are controversial: different conclusions were reported by [START_REF] Fili | NDP52 Activates Nuclear Myosin VI to Enhance RNA Polymerase II Transcription[END_REF] using a FRET assay with Myo6 (814-end). In this case, R1116A has barely any effect on NDP52 binding while L1118A reduced it by 4-fold.

Furthermore, several studies have used the R1117A (i.e., RAL) mutant or the 1116RRL1118 to AAA mutant (i.e., AAA) to study interaction of RRL partners with Myo6. Yet, R1117A disrupts the CBD n structural integrity according to circular dichroism [START_REF] Wollscheid | Diverse Functions of Myosin VI Elucidated by an Isoform-Specific α-Helix Domain[END_REF]. Thus, data obtained using these mutants should be carefully considered as they don't show specifically the importance of interaction of Myo6 with RRL partners but rather point out at the importance of the CBD n as a whole.

C.8.b Long insert regulates partner binding

The long insert also plays a key role in the regulation of partner binding. In situ proximity labelling using either CBD NI or LI shows that NI and LI isoforms shared 39 interactions while LI has 16 specific interaction and NI 47 (O'Loughlin, Masters, and Buss 2018).

Interestingly, the CBD n partner binding is isoform dependent. Indeed, the presence of the long insert between the Myo6 DT and CBD domains drastically reduced NDP52, TAX1BP1, OPTN, K63-diUb and GIPC1 binding to Myo6. Actually, the NMR structure of CBD n (PDB: 2N12, [START_REF] Wollscheid | Diverse Functions of Myosin VI Elucidated by an Isoform-Specific α-Helix Domain[END_REF] reveals that the LI insert can fold on the CBD, masking the RRL site [START_REF] Wollscheid | Diverse Functions of Myosin VI Elucidated by an Isoform-Specific α-Helix Domain[END_REF]) and impairing thus the bindings of several CBD n partners to the Myo6 isoforms containing the long insert (Figure 36). In contrast, the long insert is crucial for clathrin binding as it directly participate to its binding site [START_REF] Biancospino | Clathrin Light Chain A Drives Selective Myosin VI Recruitment to Clathrin-Coated Pits under Membrane Tension[END_REF]. Thus, binding of partners on Myo6 RRL motif and binding of clathrin seem to mutually exclude each other.

Although WWY partners were shown to bind both NI and LI isoforms, some of them seem to show a preference for the LI isoform. Indeed, LMTK2 shows a slight preference for the Myo6 LI isoform over the NI isoform in mammalian 2-hybrid assays [START_REF] Chibalina | Myosin VI and Its Interacting Protein LMTK2 Regulate Tubule Formation and Transport to the Endocytic Recycling Compartment[END_REF]) and Dab2 has a strong preference for the LI isoform in fluorescence resonance energy transfert (FRET) using Myo6 814-end [START_REF] Fili | Competition between Two High-and Low-Affinity Protein-Binding Sites in Myosin VI Controls Its Cellular Function[END_REF]).

Myo6 has a wide range of partners that could cooperate or compete with each other with the potential to link Myo6 to various functions notably in the context of diseases.

D. Myo6, a multifunctional motor in cells

Its unique features and its many cellular partners allow Myo6 to perform a wide range of functions for exocytosis, endocytosis, cell motility, organelle morphology and transcription.

D.1. Myo6 in trafficking/signaling events D.1.a. Exocytic/secretory pathway Once synthesized at the endoplasmic reticulum, the proteins must be transported in tubular/vesicular carriers to the Golgi complex for maturation, they are then sorted for correct delivery to organelles or plasma membrane. By transfecting the protein secretion reporter : secreted form of alkaline phosphatase (SEAP) into immortal fibroblastic cell lines of Snell's Waltzer Myo6-null mice, [START_REF] Warner | Loss of Myosin VI Reduces Secretion and the Size of the Golgi in Fibroblasts from Snell's Waltzer Mice[END_REF] revealed that Golgi secretion was reduced by 40% in the absence of Myo6 suggesting a role for Myo6 in the secretory pathway. Further studies took a close look at the impact of Myo6 on this process and proposed a role for it at different steps of the secretory process.

D.1.a.i Myo6 at the trans-Golgi

Golgi is an important crossroad in the secretory pathway as it allows protein maturation and sorting. Myo6 was found to localize to vesicles in the perinuclear region around the Golgi complex independently of its Motor domain [START_REF] Buss | The Localization of Myosin VI at the Golgi Complex and Leading Edge of Fibroblasts and Its Phosphorylation and Recruitment into Membrane Ruffles of A431 Cells after Growth Factor Stimulation[END_REF][START_REF] Warner | Loss of Myosin VI Reduces Secretion and the Size of the Golgi in Fibroblasts from Snell's Waltzer Mice[END_REF]. On Golgi, Myo6 nicely colocalizes with TGN38 showing a localization at the trans Golgi and trans Golgi network in fluorescence and electron microscopy of NRK cells [START_REF] Warner | Loss of Myosin VI Reduces Secretion and the Size of the Golgi in Fibroblasts from Snell's Waltzer Mice[END_REF][START_REF] Buss | The Localization of Myosin VI at the Golgi Complex and Leading Edge of Fibroblasts and Its Phosphorylation and Recruitment into Membrane Ruffles of A431 Cells after Growth Factor Stimulation[END_REF]. This localization of Myo6 seems mediated by its partner OPTN as OPTN silencing results in loss of Myo6 localization at the Golgi [START_REF] Sahlender | Optineurin Links Myosin VI to the Golgi Complex and Is Involved in Golgi Organization and Exocytosis[END_REF].

Interestingly, using immortal fibroblastic cell lines of Snell's Waltzer mice, it was established that the size of the Golgi was reduced by 40% compared to wild-type while Myo6 overexpression nicely rescues Golgi morphology [START_REF] Warner | Loss of Myosin VI Reduces Secretion and the Size of the Golgi in Fibroblasts from Snell's Waltzer Mice[END_REF]. However, in contrast, a more recent study found that Myo6 results in a ~25% Golgi decompaction using HeLa cells [START_REF] Capmany | MYO1C Stabilizes Actin and Facilitates the Arrival of Transport Carriers at the Golgi Complex[END_REF]. Actually, they pointed out that heterogenous outcomes can be obtained depending on cell types, cell spreading and imaging techniques (2D or 3D) [START_REF] Capmany | MYO1C Stabilizes Actin and Facilitates the Arrival of Transport Carriers at the Golgi Complex[END_REF]. Thus, effect of Myo6 on Golgi morphology remains to clarify [START_REF] Capmany | MYO1C Stabilizes Actin and Facilitates the Arrival of Transport Carriers at the Golgi Complex[END_REF].

D.1.a.ii Cargo sorting in polarized cells

Once mature, the proteins must be sorted prior to their secretion. This is particularly important in polarized cells as cell polarity requires differential addressing of newly synthesized proteins either to the basolateral or apical region of the cell [START_REF] Eaton | Cargo Sorting in the Endocytic Pathway: A Key Regulator of Cell Polarity and Tissue Dynamics[END_REF]. Myo6 NI isoform expression was found to be crucial for basolateral addressing; indeed, pulse-chase experiment shows that overexpression of the Myo6 NI Tail results in missorting of VSVG (basolateral sorted protein used as a reporter). Mutagenesis shows that intact CBD n is required for sorting of VSVG to basolateral domain suggesting of role of a CBD n partners in this process [START_REF] Au | Myosin VI Is Required for Sorting of AP-1B-Dependent Cargo to the Basolateral Domain in Polarized MDCK Cells[END_REF]. The authors suggest that the RRL partner OPTN could be involved as electron microscopy shows colocalization of Myo6 and OPTN on clathrin coated vesicles [START_REF] Au | Myosin VI Is Required for Sorting of AP-1B-Dependent Cargo to the Basolateral Domain in Polarized MDCK Cells[END_REF]).

D.1.a.iii Transport to the plasma membrane

To address potential roles of Myo6 for vesicles transport from the Golgi, the temperature sensitive VSV-G construct was transfected in NRK cells allowing its accumulation in Golgi on precise temperature conditions. Transferring cells at 32°C results in their secretion from the trans Golgi to the plasma membrane. In these conditions, immunostaining clearly shows colocalization of Myo6 with VSV-G vesicles secreted by the Golgi on their path to the plasma membrane suggesting a role of Myo6 in their delivery to the plasma membrane [START_REF] Warner | Loss of Myosin VI Reduces Secretion and the Size of the Golgi in Fibroblasts from Snell's Waltzer Mice[END_REF].

D.1.a.iv Myo6 SI may ensure replenishment of secretory granules by tethering

Vesicles carrying signals must accumulate at the plasma membrane prior to their release at appropriate time (replenishment). In neurosecretory cells, secretory granules are key for diffusion of neuropeptides and hormones upon calcium signaling and Myo6 seems to be involved in their replenishment. Indeed, looking by mass spectroscopy at proteins associated to purified secretory granules, Myo6 was found to interact with the granules in the presence of calcium [START_REF] Tomatis | Myosin VI Small Insert Isoform Maintains Exocytosis by Tethering Secretory Granules to the Cortical Actin[END_REF]. TIRF microscopy on PC12 cells reveals that Myo6 knockdown results in the defect in secretory granule retention near the plasma membrane without affecting their speed or biogenesis suggesting a tethering role for Myo6 [START_REF] Tomatis | Myosin VI Small Insert Isoform Maintains Exocytosis by Tethering Secretory Granules to the Cortical Actin[END_REF]. Interestingly, PC12 cells express both SI and NI isoforms but only Myo6 SI isoform is able to rescue secretory granule exocytosis upon Myo6 knockdown, suggesting a unique role of the small insert in this secretory function. Interestingly, the small insert contains a tyrosine phosphorylation site (DYD) that can be phosphorylated by Src kinase. Addition of a Src kinase inhibitor in PC12 cells results in loss of Myo6 mediated exocytosis revealing a possible role of the phosphorylation of the Myo6 DYD motif for secretory granule exocytosis [START_REF] Tomatis | Myosin VI Small Insert Isoform Maintains Exocytosis by Tethering Secretory Granules to the Cortical Actin[END_REF].

A key partner in this process seems to be the ENA/VASP protein: Mena as relocalization of Mena to mitochondries results in reduction of Myo6 localization to secretory granules and reduction of secretory granules near the plasma membrane (Tomatis et al., 2017).

D.1.a.v Myo6 at the plasma membrane for fusion

Myo6 and OPTN colocalize with secretory vesicles at the plasma membrane. TIRF microscopy reveals that depletion of either OPTN or Myo6 results in less vesicle fusion events at the plasma membrane but does not seem to impact vesicles biogenesis at the trans Golgi [START_REF] Bond | Myosin VI and Its Binding Partner Optineurin Are Involved in Secretory Vesicle Fusion at the Plasma Membrane[END_REF] suggesting a role of Myo6 for vesicle fusion perhaps by tethering Myo6 near the plasma membrane as described in the previous section.

D.1.a.vi Tubular carrier fission

Myo6 was also involved in tubular secretion necessary for maturation of a specialized organelle: Melanosomes. Melanosomes synthesize and store melanin pigments in their lumen and are involved in protein recycling through tubular carriers [START_REF] Dennis | BLOC-1 and BLOC-3 Regulate VAMP7 Cycling to and from Melanosomes via Distinct Tubular Transport Carriers[END_REF]. Finally, once matured, they are secreted to epidermal keratinocytes [START_REF] Delevoye | Melanin Transfer: The Keratinocytes Are More than Gluttons[END_REF][START_REF] Wu | Melanosome Transfer: It Is Best to Give and Receive[END_REF], where they assure photoprotection of the epidermis. In this process, Myo6 was proposed to play an additional role during maturation: fission of tubular carriers [START_REF] Ripoll | Myosin VI and Branched Actin Filaments Mediate Membrane Constriction and Fission of Melanosomal Tubule Carriers[END_REF].

Actually, Myo6 NI colocalizes with melanosomes in punctate at the basis of tubular carrier without the need of the Motor. Interestingly, live cell imaging and EM microscopy reveals that depletion of Myo6 or of its cellular partner OPTN seems to decrease tubular carrier fission. Precisely, tubules can form normally but their basis are larger and fissions event are much rarer suggesting a role of Myo6 for constriction at the tubule basis required for their fission. Interestingly, both Arp2/3 and WASH (branched-actin nucleation factors) depletion also reduce melanosome tubular carrier constriction, as seen with Myo6 depletion [START_REF] Ripoll | Myosin VI and Branched Actin Filaments Mediate Membrane Constriction and Fission of Melanosomal Tubule Carriers[END_REF], highlighting the importance of branched actin for tubular carrier fission. In this context, Myo6 could participate in branched actin network formation or act as an anchor that would exerce a precise force at the basis of tubules for their fission.

D.1.b. Myo6 in endocytosis

Endocytosis allows cells to internalize various particles in vesicles from outside the cells.

Soluble molecules can be internalized through pinocytosis, membrane receptors can be internalized in clathrin coated vesicles and large particles, such as pathogens, are internalized in phagosomes.

Eventually, the endocytosis pathway can also be hijacked by parasites. Internalized vesicles are then addressed to the early endosomes for either recycling or degradation after endosome maturation. This process thus requires accumulation of a substance at the plasma membrane, membrane deformation to form the vesicles, fission of the formed vesicle, transport towards the endosomal compartments and fusion. As for secretion pathways, Myo6 was found to be implicated in many steps of the endocytic process.

D. Bbe cells inhibits this movement assessing a key role of Myo6 either for vesicles formation or for their transport [START_REF] Chen | Myosin VI Mediates the Movement of NHE3 down the Microvillus in Intestinal Epithelial Cells[END_REF].

It is thus clear that Myo6 is required for cargo internalization, although it is undetermined through these experiments whether Myo6 is key for cargo accumulation at the plasma membrane, membrane deformation to form the vesicles or vesicle fission.

Studies of CACO-2 polarized cells further established the importance of the Myo6 LI isoform in the endocytic pathway. Indeed, the Myo6 LI isoform was shown to localize on clathrin coated structures [START_REF] Buss | Myosin VI Isoform Localized to Clathrin-Coated Vesicles with a Role in Clathrin-Mediated Endocytosis[END_REF] where it can directly interact with clathrin both free triskelia and cages through an LI isoform specific insert as described above in section C.4. [START_REF] Wollscheid | Diverse Functions of Myosin VI Elucidated by an Isoform-Specific α-Helix Domain[END_REF][START_REF] Biancospino | Clathrin Light Chain A Drives Selective Myosin VI Recruitment to Clathrin-Coated Pits under Membrane Tension[END_REF]. In NRK cell lines expressing stably Myo6 LI Tail as a dominant negative, transferrin uptake was reduced by ~70%, showing the importance of Myo6 motor for normal transferrin uptake through clathrin dependent endocytosis [START_REF] Buss | Myosin VI Isoform Localized to Clathrin-Coated Vesicles with a Role in Clathrin-Mediated Endocytosis[END_REF] while no effect on transferrin uptake were seen in NRK cells stably expressing the Myo6 NI Tail [START_REF] Buss | Myosin VI Isoform Localized to Clathrin-Coated Vesicles with a Role in Clathrin-Mediated Endocytosis[END_REF].

D.1.b.ii Membrane ruffling

To internalize an element from the outside, membrane needs to deform to ultimately catch it into a vesicle. In the very specific context of Salmonella invasion where the endocytic pathway is hijacked by a parasite to allow its entrance in the cell, Myo6 was found to be required for these membrane deformations. Indeed, Myo6 knockout from HeLa cells results in a 60% decrease of Salmonella invasion, a reduction of membrane ruffling from 65% to 45% and a reduction of PI3P in macropinocytic cups from 70% to 33%. Myo6 is found to localize to Salmonella induced membrane ruffles. Myo6 recruitment to membrane ruffles depends on its phosphorylation on T405 through PAK.

Indeed, the T405A mutant mimicking the dephosphorylated state cannot be recruited while the T405E mutant mimicking the phosphorylation state is strongly recruited to membrane ruffle [START_REF] Brooks | MYO6 Is Targeted by Salmonella Virulence Effectors to Trigger PI3-Kinase Signaling and Pathogen Invasion into Host Cells[END_REF].

These findings highlight the potential of Myo6 for deep membrane reshaping for endocytosis of large elements although there is no clear evidence of membrane reshaping by Myo6 in the context of a normal endocytosis.

D.1.b.iii Myo6 can mediate endocytic vesicles fission

A deTailed study shows that Myo6 binds to CLCa46-61 (see section C.4.) in competition with binding to the actin remodeling protein Hip1R (GST pull-down) [START_REF] Biancospino | Clathrin Light Chain A Drives Selective Myosin VI Recruitment to Clathrin-Coated Pits under Membrane Tension[END_REF]. Lack of Myo6 binding to CLCa (CLC knockdown or CLCa I54D mutant) results in aberrant clathrin-coated pits in Caco-2 cells. An increased number of pits exhibit an elongated shape suggesting that Myo6 binding to clathrin may thus provide the force required for constriction and fission (Figure 38). for proper localization of these signaling endosomes (Masters et al. 2017). Analysis of the content of these perinuclear endosomes notably through the biosensor GFP-PX reveals a change of endosomes composition upon Myo6 depletion. A high density of PI3P and EEA1 was found which indicates that more endosomes have started their maturation process. Interestingly, addition of the microtubule depolymerizer: nocodazole on SiMyo6 HeLa cells results in loss of perinuclear localization, although it does not rescue the wild-type phenotype (i.e., cell periphery localization). Thus the authors proposed that Myo6 would tether signaling endosomes at cell periphery preventing their displacement through microtubule for maturation (Masters et al. 2017).

D.1.b.vii Endocytosis defects may result in infertility in male mice

Defect in Myo6 mediated endocytosis may be at the origin of Snell's Walzer male mice (Myo6 KO) fertility problems. In late phasis of spermatogenesis, in WT mice, MYO6, GIPC1 and TOM1-L2 are recruited to the bulbar region of the apical tubulobulbar complexes (TBCs), a highly specialized endocytic structure that correspond to double-membrane invagination of Sertoli cells to the spermatid stabilized by actin [START_REF] Zakrzewski | Myosin VI Maintains the Actin-Dependent Organization of the Tubulobulbar Complexes Required for Endocytosis during Mouse Spermiogenesis † ‡[END_REF]. It associates to Rab5 and APPL1 positive early endosomes but are not present in EEA1-positive (more mature) sorting endosomes, as previously reported (Master et al. 2017). Myo6 KO changes the overall TBCs orientation, Rab5/APPL1-positive vesicles and EEA1

early endosomes distribution, actin organization and the localization of Arp3 and Cortactin (actin binding proteins) as well as Myo6 adaptors (GipC1, TOM1-L2) distribution [START_REF] Zakrzewski | Myosin VI Maintains the Actin-Dependent Organization of the Tubulobulbar Complexes Required for Endocytosis during Mouse Spermiogenesis † ‡[END_REF] resulting in defects in endocytosis in spermatogenesis probably responsible of infertility issues in Myo6-null mice.

D.2. Degradation pathways

D.2.a. Autophagy

Autophagy is a key degradation pathway that can maintain cell homeostasis and growth on starvation condition (non-selective autophagy) and ensure degradation of large structures tagged with ubiquitin such as pathogens and large protein aggregates (selective autophagy). In this process, autophagosomes fused with early endosomes to form the amphisome (maturation process) prior to its fusion with lysosomes for acidic degradation. Autophagosome membranes contain LC3 that allow their binding to autophagy receptors, able to capture ubiquitinylated cargos.

Actually, Myo6 is key for both selective and non-selective autophagy as shown in HeLa, RPE cells and MEFs and primary cortical neurons derived from Myo6 KO mice. MYO6 together with its partner TAX1BP1 was also found to be able to recognize ubiquitinylated Salmonella typhimurium in infected cells thus playing a role for the autophagy-dependent clearance of pathogens [START_REF] Tumbarello | The Autophagy Receptor TAX1BP1 and the Molecular Motor Myosin VI Are Required for Clearance of Salmonella Typhimurium by Autophagy[END_REF]. Its depletion results in an accumulation of autophagosomes and lack of protein aggregate clearance due to a defect in fusion of autophagosomes with lysosomes (monitoring LC3 lipidation and colocalization between autophagosomes and lysosomes) as well as a delay in the formation of amphisomes [START_REF] Tumbarello | Autophagy Receptors Link Myosin VI to Autophagosomes to Mediate Tom1-Dependent Autophagosome Maturation and Fusion with the Lysosome[END_REF]. In this context, Myo6 may be involved in short movement, and tethering of compartment together for fusion.

D.2.b. Mitophagy

Clearance of damaged mitochondries is essential for cell homeostasis. Damaged mitochondries can be taken in charge by a specialized autophagy process called mitophagy. Parkin is recruited upon membrane potential changes and strongly ubiquitinylates outer membrane proteins including the Myo6 partners OPTN, NDP52 and TAX1BP1 that link damaged mitochondria to autophagosomes through LC3 binding [START_REF] Onishi | Molecular Mechanisms and Physiological Functions of Mitophagy[END_REF]. Shortly after membrane depolarization, mitochondries are encaged in a dense actin network isolating the damaged mitochondria from the rest of the network until their degradation [START_REF] Kruppa | Myosin VI-Dependent Actin Cages Encapsulate Parkin-Positive Damaged Mitochondria[END_REF].

Cells deprived from Myo6 exhibit a large number of autophagosomes containing mitochondries which may suggest delay in autophagosomes fusion with lysosomes as previously described in autophagy processes [START_REF] Tumbarello | Autophagy Receptors Link Myosin VI to Autophagosomes to Mediate Tom1-Dependent Autophagosome Maturation and Fusion with the Lysosome[END_REF]) and a defect in mitochondrial respiration [START_REF] Kruppa | Myosin VI-Dependent Actin Cages Encapsulate Parkin-Positive Damaged Mitochondria[END_REF]. Moreover, upon chemically induced mitophagy, Myo6 was found to translocate from a cortex location to a mitochondria membrane where it colocalizes with the mitophagy marker parkin in HEK293 cells. Interestingly, this translocation is independent of OPTN, NDP52 and TAX1BP1

as their knockdown does not affect Myo6 localization. In contrast, I1072A mutation known to abolish Myo6 MyUb domain binding to ubiquitin [START_REF] He | Myosin VI Contains a Compact Structural Motif That Binds to Ubiquitin Chains[END_REF], results in loss of Myo6 translocation to mitochondria. Thus, ubiquitin chains may be the bridge between Myo6 and mitophagy receptors in this context. Despite the fact that lack of motor activity does not impair Myo6 localization to mitochondries, overexpression of the Myo6 Tail (no motor activity) in HEK293 cells, results in disorganization of the actin cage surrounding the damaged mitochondria and reintroduction of the damaged mitochondria in the healthy mitochondrial network [START_REF] Kruppa | Myosin VI-Dependent Actin Cages Encapsulate Parkin-Positive Damaged Mitochondria[END_REF].

D.3. Structure maintenance and organization

D.3.a. Organelle organization

Myo6 was also found to be key for correct organelle positioning and shaping in neurons.

Through interaction with Syntaphilin (SNPH), Myo6 can recruit and anchor mitochondria in presynaptic region of neurons. Indeed, Myo6 knockdown results in loss of the presynaptic localization of the mitochondries while its overexpression increased it. Moreover, Myo6 colocalizes on mitochondries with the mitochondria anchoring protein SNPH and ability to recruit mitochondrie to presynaptic region was lost upon SNPH knock out (Li et al. 2020). Myo6 seems also essential for endoplasmic reticulum (ER) extension towards the axon as addition of Myo6 inhibitor (TIP) reduces dynamic movement of the ER [START_REF] Deng | Dynamic Remodeling of Ribosomes and Endoplasmic Reticulum in Axon Terminals of Motoneurons[END_REF].

D.3.b. Assembly of Cx43 GAP junctions

Immunohistochemistry on mouse frozen heart sections, immunofluorescence on HeLa cells or NRK cells as well as primary ventricular myocytes from rats show that Myo6 can colocalize with Cx43

(connexin-43) GAP junction plaques [START_REF] Waxse | Myosin VI Facilitates Connexin 43 Gap Junction Accretion[END_REF][START_REF] Piehl | Internalization of Large Double-Membrane Intercellular Vesicles by a Clathrin-Dependent Endocytic Process[END_REF]. Interestingly, if Cx43 is still expressed in similar amount in fibroblast cells lacking Myo6, the GAP junction plaque size is smaller [START_REF] Waxse | Myosin VI Facilitates Connexin 43 Gap Junction Accretion[END_REF]) and intercellular communication across GAP junction is reduced [START_REF] Waxse | Myosin VI Facilitates Connexin 43 Gap Junction Accretion[END_REF]. Photobleaching experiments revealed that even if Cx43 amount is not impaired, fibroblasts lacking Myo6 have more difficulties to integrate free Cx43 to GAP junction plaque [START_REF] Waxse | Myosin VI Facilitates Connexin 43 Gap Junction Accretion[END_REF].

ED.3.c. Cilia/microvilli formation and maintenance

In Myo6-null (deaf) mice cochlea, 20 days after birth, many hair cells have died and the remaining are degenerated with abnormal giant stereocilia containing diffuse actin [START_REF] Self | Role of Myosin VI in the Differentiation of Cochlear Hair Cells[END_REF].

Interestingly, in wild-type Guinea pig, no Myo6 can be detected in inner and outer hair cell stereocilia. Myo6 concentrates instead in the cuticular plates (Figure 39) and vestibular organs [START_REF] Hasson | Unconventional Myosins in Inner-Ear Sensory Epithelia[END_REF].

Taken these data together, [START_REF] Frank | Myosin VI: A Structural Role in Actin Organization Important for Protein and Organelle Localization and Trafficking[END_REF] proposed that Myo6 could keep the actin organization of the cuticular plate together preventing undesired cell components entrance in the stereocilia and maintaining the basis of stereocilia in place.

Myo6 was also found to be critical for cilia/microvilli formation and maintenance in other cell types. Myo6 depletion was also found to impair primary cilium formation (that normally originate from centriole) probably by preventing the centriole protein ODF1 degradation through autophagy upon starvation conditions which essential for primary cilliogenesis [START_REF] Magistrati | Myosin VI Regulates Ciliogenesis by Promoting the Turnover of the Centrosomal/Satellite Protein OFD1[END_REF] for morphogenesis of primary branched dendrites by controlling actin filament extension [START_REF] Yoong | Atypical Myosin Tunes Dendrite Arbor Subdivision[END_REF]. Similarly, in drosophila embryos, Myo6 depletion results in an increase of cell area and a decrease of cell elongation upon development highlighting the importance of Myo6 for cell tissues morphogenesis through actin network organization [START_REF] Dye | Self-Organized Patterning of Cell Morphology via Mechanosensitive Feedback[END_REF]. Actually, Myo6 depletion can even lead to severe epithelial organization defect and death of certain drosophila larvae and embryos [START_REF] Millo | Myosin VI Plays a Role in Cell-Cell Adhesion during Epithelial Morphogenesis[END_REF].

Myo6 accumulates in the actin rich cuticular plate at the basis of the stereocilia

Myo6 has thus been proposed to be part of the Rho-dependent signaling pathway that participate to actin cytoskeleton reorganization in response to mechanical stress. In this pathway, Myo6 seems to act as a force sensor as its association with E-cadherin at cellular junctions increased upon mechanical stress resulting in the activation of RhoA signaling required for maintenance of tissues integrity [START_REF] Acharya | A Mechanosensitive RhoA Pathway That Protects Epithelia against Acute Tensile Stress[END_REF][START_REF] Mangold | The Juxtamembrane Domain of the E-Cadherin Cytoplasmic Tail Contributes to Its Interaction with Myosin VI[END_REF].

D.3.d.ii Migration

Myo6 interaction with cellular junctions was also described in the context of cell migration:

Myo6 depletion was found to reduce cell migration of C2C12 myoblast [START_REF] Karolczak | Involvement of Unconventional Myosin VI in Myoblast Function and Myotube Formation[END_REF] and drosophila melanogaster ovarian border cells [START_REF] Geisbrecht | Myosin VI Is Required for E-Cadherin-Mediated Border Cell Migration[END_REF]. In both cases, this could come from defect in cell adhesions which is essential for cell migration [START_REF] Huttenlocher | Adhesion in Cell Migration[END_REF]. Indeed, in myoblast, Myo6 concentrates in adhesion sites together with talin and vinculin [START_REF] Karolczak | Involvement of Unconventional Myosin VI in Myoblast Function and Myotube Formation[END_REF]. Moreover, Myo6 depletion leads to a lower expression level of proteins involved in cell-cell contacts: E-cadherin and the β-catenin (Armadillo) in Drosophila ovarian brush border [START_REF] Geisbrecht | Myosin VI Is Required for E-Cadherin-Mediated Border Cell Migration[END_REF] and talin, FAK/pFAK, M-cadherin and drebrin in mice myoblast [START_REF] Lehka | Formation of Aberrant Myotubes by Myoblasts Lacking Myosin VI Is Associated with Alterations in the Cytoskeleton Organization, Myoblast Adhesion and Fusion[END_REF]. Actually, Myo6 depletion results in an even wider change in the expression level of several key proteins involved in myoblast differentiation (Pax7, MyoD and myogenin), in the cytoskeleton organization (α-actinin, desmin, myosin heavy chains and γ-actin), and in myoblast membrane fusion (myomaker and myomerger) [START_REF] Lehka | Formation of Aberrant Myotubes by Myoblasts Lacking Myosin VI Is Associated with Alterations in the Cytoskeleton Organization, Myoblast Adhesion and Fusion[END_REF]). Yet in myoblast, Myo6 is not expressed in nuclei [START_REF] Lehka | Formation of Aberrant Myotubes by Myoblasts Lacking Myosin VI Is Associated with Alterations in the Cytoskeleton Organization, Myoblast Adhesion and Fusion[END_REF][START_REF] Karolczak | Involvement of Unconventional Myosin VI in Myoblast Function and Myotube Formation[END_REF]) making a direct effect of Myo6 on transcription level unlikely in this context. Instead, [START_REF] Geisbrecht | Myosin VI Is Required for E-Cadherin-Mediated Border Cell Migration[END_REF] proposed that interaction between cell adhesion proteins and Myo6 helps stabilizing each other at the cell-cell junction level as Myo6 protein level is also reduced in brush border cells upon E-cadherin or Armadillo depletion and interaction between these three proteins is found by immunoprecipitation [START_REF] Geisbrecht | Myosin VI Is Required for E-Cadherin-Mediated Border Cell Migration[END_REF].

Beside its importance for proper cell-cell adhesion, Myo6 was also proposed to interact and support the formation of migratory cell leading edge. Indeed, in A431 cells, upon ruffling stimulation through EGF addition, Myo6 is phosphorylated and goes from a juxtanuclear localization to ruffles location at leading edges within minutes [START_REF] Buss | The Localization of Myosin VI at the Golgi Complex and Leading Edge of Fibroblasts and Its Phosphorylation and Recruitment into Membrane Ruffles of A431 Cells after Growth Factor Stimulation[END_REF]. Myo6 depletion in HeLa or A549 cells through siRNA or crisprCas9 significantly decrease ruffling upon EGF addition [START_REF] Buss | The Localization of Myosin VI at the Golgi Complex and Leading Edge of Fibroblasts and Its Phosphorylation and Recruitment into Membrane Ruffles of A431 Cells after Growth Factor Stimulation[END_REF][START_REF] Puri | Overexpression of Myosin VI in Prostate Cancer Cells Enhances PSA and VEGF Secretion, but Has No Effect on Endocytosis[END_REF]. Interestingly, Myo6 truncated from the Tail can localize to membrane ruffles while, in contrast, neither Myo6 Tail nor Myo6 K157R (rigor mutant strongly bound to actin) were able to localize to ruffles when overexpressed (Masters et al. 2017), assessing the importance of a correctly functioning motor for Myo6 localization to ruffles.

Further evidence suggest that Myo6 may control leading edge ruffling by controlling protein delivery to leading edge, indeed, it was shown that Myo6 or OPTN depletion results in loss of EGFR and RAC delivery to the leading edge of the A549 cells thus preventing ruffling and formation of lamellipodia [START_REF] Buss | The Localization of Myosin VI at the Golgi Complex and Leading Edge of Fibroblasts and Its Phosphorylation and Recruitment into Membrane Ruffles of A431 Cells after Growth Factor Stimulation[END_REF][START_REF] Puri | Overexpression of Myosin VI in Prostate Cancer Cells Enhances PSA and VEGF Secretion, but Has No Effect on Endocytosis[END_REF].

D.4. In Transcription

In addition to its many roles in the cytoplasm, Myo6 is expressed in the nucleus in several cell types: in hippocampal neurons [START_REF] Kneussel | Dynein and Muskelin Control Myosin VI Delivery towards the Neuronal Nucleus[END_REF], in adrenal medulla cells [START_REF] Majewski | Myosin VI in the Nucleus of Neurosecretory PC12 Cells: Stimulation-Dependent Nuclear Translocation and Interaction with Nuclear Proteins[END_REF]2011), in native CD4 T cells [START_REF] Zorca | d'accomplir des tâches mécaniques complexes, nécessaires à la génération de tensions dans des emplacements cellulaires précis. La formation ou le transport de vésicules dans des voies d'endocytose et de sécrétion peuvent par exemple en résulter. Parmi ces moteurs, les myosines sont capables, à partir de l'hydrolyse de l'ATP, de générer de la force en association avec des filaments d'actine. Les myosines se divisent en plusieurs classes impliquées dans un large panel de fonctions et de pathologies[END_REF] and HeLa cells [START_REF] Vreugde | Nuclear Myosin VI Enhances RNA Polymerase II-Dependent Transcription[END_REF][START_REF] Fili | NDP52 Activates Nuclear Myosin VI to Enhance RNA Polymerase II Transcription[END_REF] where it colocalizes with its partner NDP52 [START_REF] Fili | NDP52 Activates Nuclear Myosin VI to Enhance RNA Polymerase II Transcription[END_REF] and RNA polymerase II (RNAPII) [START_REF] Vreugde | Nuclear Myosin VI Enhances RNA Polymerase II-Dependent Transcription[END_REF][START_REF] Fili | NDP52 Activates Nuclear Myosin VI to Enhance RNA Polymerase II Transcription[END_REF]. More precisely Myo6 was found to localize mostly in chromatin free regions and near the nuclear envelope [START_REF] Vreugde | Nuclear Myosin VI Enhances RNA Polymerase II-Dependent Transcription[END_REF][START_REF] Majewski | Myosin VI in the Nucleus of Neurosecretory PC12 Cells: Stimulation-Dependent Nuclear Translocation and Interaction with Nuclear Proteins[END_REF]) and upon cell stimulation (through addition of high external concentration of KCl), Myo6 can translocate to the nucleus where its colocalization with RNAPII and transcription factors SP1, SC35, PML and RNAPII mediated transcription activity are increased [START_REF] Majewski | Myosin VI in the Nucleus of Neurosecretory PC12 Cells: Stimulation-Dependent Nuclear Translocation and Interaction with Nuclear Proteins[END_REF]. Actually, Myo6 depletion even results in loss of normal localization of RNAPII [START_REF] Hari-Gupta | Myosin VI Regulates the Spatial Organisation of Mammalian Transcription Initiation[END_REF]. Furthermore, in fluorescence anisotropy, Myo6 CBD c was found to bind DNA with an affinity of ~0.1 µM compatible with a role of Myo6 in transcription [START_REF] Fili | NDP52 Activates Nuclear Myosin VI to Enhance RNA Polymerase II Transcription[END_REF].

Using an in vitro transcription system (HeLaScribe ® Nuclear extract), it has been shown that transcription level decreases by ~25 % upon Myo6 depletion (using anti-Myo6 antibody) while addition of 1 µM of FLMyo6 partially rescue the transcription level [START_REF] Fili | NDP52 Activates Nuclear Myosin VI to Enhance RNA Polymerase II Transcription[END_REF]. In contrast, addition of CBD results in transcription decrease in a CBD concentration dependent manner suggesting a competition between FLMyo6 already present in the sample and the exogenous CBD. Thus, a key role of the CBD to address Myo6 to its transcription function is revealed. Transcription level increases further by 2-fold when 5 µM of the Myo6 nuclear partner NDP52 is added, assessing the importance of this partner in transcription [START_REF] Fili | NDP52 Activates Nuclear Myosin VI to Enhance RNA Polymerase II Transcription[END_REF].

D.5. Myo6 links microtubule and actin tracks

D.5.a. Myo6 and dynein can collaborate with each other

Dynein coprecipitates with Myo6 from whole brain lysate and both can coprecipitate with the muskelin protein. Interestingly, Myo6 ability to coprecipitate with dynein is significantly reduced upon muskelin knockout suggesting that muskelin may constitute a bridge between the two motors [START_REF] Kneussel | Dynein and Muskelin Control Myosin VI Delivery towards the Neuronal Nucleus[END_REF]. Dynein inhibitor: ciliobrevin D results in ~13% loss of nuclear Myo6 intensity in hippocampal neurons and muskelin knockout results in~22% loss assessing the role of dyneins and muskelin for Myo6 translocation to the nucleus and highlighting a collaboration between the microtubule based motor dynein and the actin based motor Myo6 [START_REF] Kneussel | Dynein and Muskelin Control Myosin VI Delivery towards the Neuronal Nucleus[END_REF].

Such a crosstalk between microtubules and actin filaments through dynein and Myo6 has also been reported in the context of influenza virus infection: this virus moves from the cell periphery to the perinuclear region by switching from actin to microtubule tracks transportation. Single virus tracking shows that the switch from actin to microtubules require Myo6 and dynein motors [START_REF] Zhang | A 'Driver Switchover' Mechanism of Influenza Virus Transport from Microfilaments to Microtubules[END_REF].

D.5.b. Myo6 is involved in centrosomes localization

EM and immunofluorescence microscopy confirmed that Myo6 can localize at the centriole [START_REF] Magistrati | Myosin VI Regulates Ciliogenesis by Promoting the Turnover of the Centrosomal/Satellite Protein OFD1[END_REF]. Moreover, while Myo6 depletion from RPE-1 cells does not seem to impair the centriole's structure, it impairs centrosome localization (centriole moves further away from the plasma membrane). Myo6 depletion also induces accumulation of its partner OFD1 at mother and daughter centrioles even though the overall level of expression of OFD1 is not impaired. Super resolution microscopy reveals that OFD1, which normally concentrates at the tips of the centrioles, adopt a general localization all over the centrioles when Myo6 is depleted. Actually, FRAP reveals that an OFD1

pool stayed immobilized at the centriole surface instead of being translocated at the centriole tips [START_REF] Magistrati | Myosin VI Regulates Ciliogenesis by Promoting the Turnover of the Centrosomal/Satellite Protein OFD1[END_REF]). Thus, Myo6 may act as a regulator of the microtubules cytoskeleton by impacting centrosome location and ODF1 organization.

D.6. Myo6 in cancers

Myo6 seems to have an impact on various signaling pathways related to cancer cells notably transcription, migration, receptor surface expression and autophagy, a key process for cell survival under stress conditions [START_REF] White | The Role for Autophagy in Cancer[END_REF]. Precisely, when interacting with APPL1+ signaling endosomes, Myo6 can enhance EGFR signaling and AKT phosphorylation, a key pathway in cancer progression (Masters et al. 2017;[START_REF] Polo | Keeping EGFR Signaling in Check: Ubiquitin Is the Guardian[END_REF]. Moreover, the impact of Myo6 on signaling protein phosphorylation has been reported in several cancer models. Notably, Myo6 in vitro knockdown decreases ERK1/2 phosphorylation (cell proliferation and cell death modulator) in lung [START_REF] Yu | Lentivirus-Mediated Silencing of Myosin VI Inhibits Proliferation and Cell Cycle Progression in Human Lung Cancer Cells[END_REF] and prostate cancer (Wang et al. 2016). Myo6 has also been proposed to be coupled with nuclear receptor expression: indeed, Myo6 Tail can directly interact with estrogen receptor, moreover, in estrogen receptor positive MCF7 cells, Myo6 Tail expression drastically reduced the expression level of estrogen receptor target PS2 and GREB1. Given the key role of estrogen receptor in breast cancer growth [START_REF] Fili | NDP52 Activates Nuclear Myosin VI to Enhance RNA Polymerase II Transcription[END_REF], this shows the potential of Myo6 in regulation of cancer related signaling pathway.

The key role of Myo6 for tumor development has been shown using several cancer cell models: oral squamous carcinoma [START_REF] Zhang | Knockdown of Myosin 6 Inhibits Proliferation of Oral Squamous Cell Carcinoma Cells[END_REF], ovarian carcinoma [START_REF] Yoshida | Lessons from Border Cell Migration in the Drosophila Ovary: A Role for Myosin VI in Dissemination of Human Ovarian Cancer[END_REF]), lymphoid leukemia [START_REF] Jbireal | The Selective Role of Myosin VI in Lymphoid Leukemia Cell Migration[END_REF], melanoma [START_REF] Li | Knockdown of Myosin VI by Lentivirus-Mediated Short Hairpin RNA Suppresses Proliferation of Melanoma[END_REF], hepatocellular carcinoma [START_REF] Ma | Knockdown of Myosin VI Inhibits Proliferation of Hepatocellular Carcinoma Cells In Vitro[END_REF], lung cancer [START_REF] Yu | Lentivirus-Mediated Silencing of Myosin VI Inhibits Proliferation and Cell Cycle Progression in Human Lung Cancer Cells[END_REF], gastric cancer (Wang et al. 2016), colorectal cancer [START_REF] You | Downregulation of Myosin VI Reduced Cell Growth and Increased Apoptosis in Human Colorectal Cancer[END_REF][START_REF] Wang | Circ_0011385 Knockdown Inhibits Cell Proliferation, Migration and Invasion, Whereas Promotes Cell Apoptosis by Regulating MiR-330-3p/MYO6 Axis in Colorectal Cancer[END_REF]) and prostate cancer [START_REF] Dunn | A Novel Role of Myosin VI in Human Prostate Cancer[END_REF][START_REF] Puri | Overexpression of Myosin VI in Prostate Cancer Cells Enhances PSA and VEGF Secretion, but Has No Effect on Endocytosis[END_REF]Wang et al. 2016). In cell cultures

Myo6 knockdown drastically decreases cell proliferation and increases apoptosis in cell models of colorectal cancer [START_REF] You | Downregulation of Myosin VI Reduced Cell Growth and Increased Apoptosis in Human Colorectal Cancer[END_REF][START_REF] Wang | Circ_0011385 Knockdown Inhibits Cell Proliferation, Migration and Invasion, Whereas Promotes Cell Apoptosis by Regulating MiR-330-3p/MYO6 Axis in Colorectal Cancer[END_REF], oral squamous cell carcinoma [START_REF] Zhang | Knockdown of Myosin 6 Inhibits Proliferation of Oral Squamous Cell Carcinoma Cells[END_REF], melanoma [START_REF] Li | Knockdown of Myosin VI by Lentivirus-Mediated Short Hairpin RNA Suppresses Proliferation of Melanoma[END_REF], hepatocellular carcinoma [START_REF] Ma | Knockdown of Myosin VI Inhibits Proliferation of Hepatocellular Carcinoma Cells In Vitro[END_REF] and gastric cancers [START_REF] Yang | LncRNA UCA1 Promotes Development of Gastric Cancer via the MiR-145/MYO6 Axis[END_REF]. In lymphoid leukemia, in vitro Myo6 knockdown results in the reduction of cell migration [START_REF] Jbireal | The Selective Role of Myosin VI in Lymphoid Leukemia Cell Migration[END_REF]. Myo6 is overexpressed in high-grade ovarian carcinoma. Moreover, in vitro, Myo6 knockdown drastically reduced cells spreading and migration [START_REF] Yoshida | Lessons from Border Cell Migration in the Drosophila Ovary: A Role for Myosin VI in Dissemination of Human Ovarian Cancer[END_REF]. In prostate cancer cells, Myo6 knockdown decreases cell migration, growth [START_REF] Dunn | A Novel Role of Myosin VI in Human Prostate Cancer[END_REF] and proliferation in vitro (Wang et al., 2016) and significantly decreases PSA (biomarker in prostate cancer) and VEGF (growth factor that stimulate vascularization and tumor angiogenesis) secretion [START_REF] Puri | Overexpression of Myosin VI in Prostate Cancer Cells Enhances PSA and VEGF Secretion, but Has No Effect on Endocytosis[END_REF].

Furthermore, it was reported that Myo6 is overexpressed, and the overall survival of patients is affected (Kaplan-Meier plotter) in gastric cancer. In this cancer, in vitro knockdown of Myo6 significantly decreases cell migration and proliferation and downregulates cell cycle activators (Wang et al., 2016). In lung cancer, immunohistochemical staining on human tissues reveals that Myo6 expression is higher in lung cancer tissues than in adjacent normal tissues. Interestingly, the highest level of Myo6 expression was seen in the nymph nodes upon metastasis. In vitro Myo6 knockdown results in a significant reduction of cell proliferation [START_REF] Yu | Lentivirus-Mediated Silencing of Myosin VI Inhibits Proliferation and Cell Cycle Progression in Human Lung Cancer Cells[END_REF].

Developing Myo6 specific inhibitors of the motor function for investigation in cells and development of potential anti-tumor drugs is thus of high interest. Currently, only one Myo6 inhibitor:

has been reported: [TIP: 2,4,6-triiodophenol] [START_REF] Heissler | Kinetic Properties and Small-Molecule Inhibition of Human Myosin-6[END_REF]. However, although it is an interesting tool to study Myo6 functions, the druggability of TIP is quite poor as it has a rather low affinity (µM range). Moreover, its mechanical of action is unknown and as it is a very small molecule, it could result in off-target effects.

Myo6 partners also link Myo6 to cancer; GIPC1 was found to be a promising therapeutical target in cancer, it is notably highly overexpressed in prostate and ovarian cancers and has been associated with proliferation and cell survival [START_REF] Chittenden | Therapeutic Implications of GIPC1 Silencing in Cancer[END_REF][START_REF] Westbrook | CAPG and GIPC1: Breast Cancer Biomarkers for Bone Metastasis Development and Treatment[END_REF]. Peptides preventing GIPC1 interaction with its PDZ partners have been developed as an anti-tumoral treatment.

They result in decrease in proliferation, and increase in apoptosis in pancreas cancer [START_REF] Patra | Chemically Modified Peptides Targeting the PDZ Domain of GIPC as a Therapeutic Approach for Cancer[END_REF][START_REF] Pal | Inhibition of Endoglin-GIPC Interaction Inhibits Pancreatic Cancer Cell Growth[END_REF].

In contrast, DNA fingerprinting of normal and cancerous ovarian cell lines reveals that Dab2 is downregulated in ovarian cancer [START_REF] Mok | Molecular Cloning of Differentially Expressed Genes in Human Epithelial Ovarian Cancer[END_REF] and has the ability to inhibit tumor growth in vitro and in vivo [START_REF] Mok | Molecular Cloning of Differentially Expressed Genes in Human Epithelial Ovarian Cancer[END_REF][START_REF] Sweeney | Kinetic tuning of myosin via a flexible loop adjacent to the nucleotide binding pocket[END_REF]. It was found later that Dab2 expression was decreased in many other cancers including breast cancer [START_REF] Martin | Disabled-2 Downregulation Promotes Epithelial-to-Mesenchymal Transition[END_REF], colorectal cancer [START_REF] Kleeff | Down-Regulation of DOC-2 in Colorectal Cancer Points to Its Role as a Tumor Suppressor in This Malignancy[END_REF], gastric cancer [START_REF] Wang | DAB2 Suppresses Gastric Cancer Migration by Regulating the Wnt/β-Catenin and Hippo-YAP Signaling Pathways[END_REF]) and squamous cell cancers (Wang et al. 2016). In contrast, decreasing Dab2 expression through miRNA has been found to promote cancer invasiveness and proliferation in prostate cancer cell lines (Yang 2019). These findings highlight Dab2 possible roles as a tumor suppressor [START_REF] Ogbu | The Role of Disabled-2 (Dab2) in Diseases[END_REF]. Although the « tumor suppression » mechanisms are not clearly established, Dab2 expression level has been shown to regulate several signaling pathways that may be a reason for its tumor suppressor activity. Dab2 downregulation is notably responsible of activation of the Ras/MAPK or Wnt/β-catenin signaling cascade resulting in EMT promotion [START_REF] Wang | DAB2 Suppresses Gastric Cancer Migration by Regulating the Wnt/β-Catenin and Hippo-YAP Signaling Pathways[END_REF][START_REF] Martin | Disabled-2 Downregulation Promotes Epithelial-to-Mesenchymal Transition[END_REF].

To that extent, understanding Myo6 regulation and how partners can orientate Myo6 towards pro or anti-cancer functions is of particular interest. Typically, in the context of breast cancer (MCF-7 cells), Dab2 expression levels is particularly low and its transfection in MCF-7 cells decrease estrogen receptor transcription level thus tumoregenicity, similarly to Myo6 depletion. This is consistent with the ability of Dab2 to block Myo6 ability to bind DNA (fluorescence anisotropy) and to block its in vitro transcription ability in HeLa cells [START_REF] Fili | Competition between Two High-and Low-Affinity Protein-Binding Sites in Myosin VI Controls Its Cellular Function[END_REF]. [START_REF] Fili | Competition between Two High-and Low-Affinity Protein-Binding Sites in Myosin VI Controls Its Cellular Function[END_REF] thus proposed that the tumor suppressor role of Dab2 would be a down regulation of Myo6 action in the nucleus, in particular an attenuation of estrogen receptor expression.

E. Myo6 versatile mechanical properties

A large range of cellular functions and partners have thus been identified. In the course of characterizing the wide range of functions of Myo6, several mechanical roles have been proposed for Myo6, as an anchor, a transporter or an organizer of the actin network (Table 8). Gathering knowledge on the Myo6 cell functions, it is possible that in complex processes, such as mice spermatogenesis, different Myo6 mechanical roles could collaborate [START_REF] Zakrzewski | Myosin VI Maintains the Actin-Dependent Organization of the Tubulobulbar Complexes Required for Endocytosis during Mouse Spermiogenesis † ‡[END_REF]: first in association with GIPC1, Myo6 could transport APPL1-endosomes towards the cell cortex [START_REF] Aschenbrenner | Myo6 Facilitates the Translocation of Endocytic Vesicles from Cell Peripheries[END_REF], then Myo6 could tether them at the cortex to prevent their maturation (Masters et al. 2017). Finally, Myo6 could participate in organization of the actin network by interacting with LARG, Arp3 and cortactin [START_REF] Noguchi | Myosin VI Stabilizes an Actin Network during Drosophila Spermatid Individualization[END_REF][START_REF] O'loughlin | The MYO6 Interactome Reveals Adaptor Complexes Coordinating Early Endosome and Cytoskeletal Dynamics[END_REF].

However, the mechanical role Myo6 performs in several cellular functions remains to be determined.

Cell function Proposed mechanical role(s) Myo6 partner(s) identified References

Secretory pathway

Localization at Golgi ? OPTN [START_REF] Buss | The Localization of Myosin VI at the Golgi Complex and Leading Edge of Fibroblasts and Its Phosphorylation and Recruitment into Membrane Ruffles of A431 Cells after Growth Factor Stimulation[END_REF][START_REF] Warner | Loss of Myosin VI Reduces Secretion and the Size of the Golgi in Fibroblasts from Snell's Waltzer Mice[END_REF][START_REF] Sahlender | Optineurin Links Myosin VI to the Golgi Complex and Is Involved in Golgi Organization and Exocytosis[END_REF][START_REF] Capmany | MYO1C Stabilizes Actin and Facilitates the Arrival of Transport Carriers at the Golgi Complex[END_REF] Cargo sorting ? OPTN ? [START_REF] Au | Myosin VI Is Required for Sorting of AP-1B-Dependent Cargo to the Basolateral Domain in Polarized MDCK Cells[END_REF] Vesicles transport after their secretion by the Golgi apparatus

Transport ?

GAP junction trafficking

Cx43 addressing to GAP junction plaque ? ?

Waxse et al., 2017

Hair cell maintenance

Stereocilia maintenance Actin organization and/or tethering ? [START_REF] Avraham | The Mouse Snell's Waltzer Deafness Gene Encodes an Unconventional Myosin Required for Structural Integrity of Inner Ear Hair Cells[END_REF][START_REF] Hasson | Unconventional Myosins in Inner-Ear Sensory Epithelia[END_REF][START_REF] Self | Role of Myosin VI in the Differentiation of Cochlear Hair Cells[END_REF][START_REF] Frank | Myosin VI: A Structural Role in Actin Organization Important for Protein and Organelle Localization and Trafficking[END_REF]

Tissues morphogenesis

Cellular junctions function ? ?

Microtubule and actin crosstalk

Myo6-dynein collaboration ? Muskelin ? [START_REF] Kneussel | Dynein and Muskelin Control Myosin VI Delivery towards the Neuronal Nucleus[END_REF][START_REF] Zhang | A 'Driver Switchover' Mechanism of Influenza Virus Transport from Microfilaments to Microtubules[END_REF] Centrosome organization ? OFD1 [START_REF] Magistrati | Myosin VI Regulates Ciliogenesis by Promoting the Turnover of the Centrosomal/Satellite Protein OFD1[END_REF] Table 8: link between mechanical properties and cell functions Thus, how the same motor might adapt to different mechanical properties is of outstanding importance to understand how Myo6 functions in cells. Mechanical properties enabling Myo6 to perform these functions have thus been pursued.

E.1. Actin organizer

As Myo6 has been found to impact the morphology of cells and organelles, the motor was suspected to act as an actin network organizer [START_REF] Frank | Myosin VI: A Structural Role in Actin Organization Important for Protein and Organelle Localization and Trafficking[END_REF].

Myo6 was found to maintain the actin-dependent organization of the tubulobulbar complexes required for endocytosis during mouse spermiogenesis [START_REF] Zakrzewski | Myosin VI Maintains the Actin-Dependent Organization of the Tubulobulbar Complexes Required for Endocytosis during Mouse Spermiogenesis † ‡[END_REF], to participate in actin cage formation during mitophagy [START_REF] Kruppa | Myosin VI-Dependent Actin Cages Encapsulate Parkin-Positive Damaged Mitochondria[END_REF] and to maintain actin network supporting stereocilia in cochlear [START_REF] Frank | Myosin VI: A Structural Role in Actin Organization Important for Protein and Organelle Localization and Trafficking[END_REF]. Profound defects in actin cytoskeleton organization were also found in PC12 cells upon Myo6 depletion [START_REF] Majewski | Myosin VI in PC12 Cells Plays Important Roles in Cell Migration and Proliferation but Not in Catecholamine Secretion[END_REF]. Moreover, Myo6 ability to associate with several actin modulators, including LRCH1 and LRCH3 (O'Loughlin, Masters, and Buss 2018) as well as DOCK7 and LARG [START_REF] Wollscheid | Diverse Functions of Myosin VI Elucidated by an Isoform-Specific α-Helix Domain[END_REF][START_REF] O'loughlin | The MYO6 Interactome Reveals Adaptor Complexes Coordinating Early Endosome and Cytoskeletal Dynamics[END_REF] further supports its possible role as an actin organizer.

However, it should be noted that actin organization is not the only way Myo6 could undergo membrane reshaping, indeed, using a fluid supported bilayer lipid model, it was found that Myo6 on its own induces lipid surface remodeling pointed out to possible membrane reshaping through direct contact between Myo6 and lipid instead of indirect contact through actin cytoskeleton [START_REF] Rogez | Reconstitution Reveals How Myosin-VI Self-Organises to Generate a Dynamic Mechanism of Membrane Sculpting[END_REF]).

E.2. Myo6 can dimerize at high concentration to perform transport and anchoring functions

To take several steps on actin without detaching, which is necessary for performing a transporter function, Myo6 need to be at least a dimer so that one head remains attached to actin while the other is detached. Actually, even for anchoring function, having two myosin heads working together is more efficient (Sweeney and Houdusse 2007).

But surprisingly, gel filtration and sucrose density centrifugation show that recombinant full length Myo6 as well as Myo6 from cell extracts are monomeric [START_REF] Lister | A Monomeric Myosin VI with a Large Working Stroke[END_REF]). Actually, Myo6 remains monomeric even after addition of the zero-length cross linker as an attempt to stabilize weak dimers [START_REF] Lister | A Monomeric Myosin VI with a Large Working Stroke[END_REF]). Strikingly, despite being monomeric in vitro, Myo6 were found to be a dimer in cells: Myo6 dimerization study through homoFRET in ARPE-19 cells shows that, when bound to clathrin coated vesicles, a high FRET signal is recorded, as FRET required specific orientation between donor and acceptor, FRET signal increase is unlikely to be due to high monomer concentration in random orientation, rather these results pointed out a possible dimerization of the motor on these vesicles [START_REF] Altman | Precise Positioning of Myosin VI on Endocytic Vesicles in Vivo[END_REF].

Further investigation demonstrates that actually, at high concentration Myo6 can dimerize even in vitro. High concentration can be reached in vitro by Myo6 saturation on actin dense network or Flag Myo6 saturation on an anti-flag column, the Myo6 is then released using ATP or flag peptide (respectively). Rotary shadowing electron microscopy shows that after these treatments 17% of Myo6 is stably dimerized for a few hours [START_REF] Park | Full-Length Myosin VI Dimerizes and Moves Processively along Actin Filaments upon Monomer Clustering[END_REF]. These dimers are able to move processively for ~1.1 µm runs [START_REF] Park | Full-Length Myosin VI Dimerizes and Moves Processively along Actin Filaments upon Monomer Clustering[END_REF]. The authors thus proposed that Myo6 dimerization can be reached and regulated by partner binding. Partners would address Myo6 to clusters creating a high local Myo6 concentration allowing dimerization or even oligomerization. Indeed, formation of such large Myo6partner clusters were reported by several groups (Dos Santos et al. 2022;[START_REF] Karolczak | Involvement of Unconventional Myosin VI in Myoblast Function and Myotube Formation[END_REF][START_REF] Shang | Structure Analyses Reveal a Regulated Oligomerization Mechanism of the PlexinD1/GIPC/Myosin VI Complex[END_REF]. Moreover, Myo6 dimerization ( Spudich et al. 2007;[START_REF] Phichith | Cargo Binding Induces Dimerization of Myosin VI[END_REF][START_REF] Yu | Myosin VI Undergoes Cargo-Mediated Dimerization[END_REF][START_REF] Fili | Competition between Two High-and Low-Affinity Protein-Binding Sites in Myosin VI Controls Its Cellular Function[END_REF] or oligomerization [START_REF] Shang | Structure Analyses Reveal a Regulated Oligomerization Mechanism of the PlexinD1/GIPC/Myosin VI Complex[END_REF] upon partners binding has been reported by several teams.

Overall, Myo6 seems to be indeed able to function as a dimer in cells, but because of the difficulty to form a stable dimer for in vitro experiments, an artificial Myo6 dimer has been extensively used to investigate Myo6 dimer mechanical properties. Artificial dimerization is reached by the addition of a leucine zipper (Lumb, Carr, and Kim 1994)in place of the cargo binding domain usually at aa 992. Using single molecule motility assays, comparison of the artificially dimerized Myo6 to FLMyo6 formed though actin saturation shows that both constructs are processive and take steps of identical size but it should be noted that run length drops from 1.1 to 0.3 µm when the artificially dimerized Myo6 is used [START_REF] Park | Full-Length Myosin VI Dimerizes and Moves Processively along Actin Filaments upon Monomer Clustering[END_REF]. Caution must thus be raised upon studies of chimeric motors in the absence of structural knowledge of how Myo6 motors dimerize.

E.3. Myo6 travelling along actin

When artificially dimerized through a leucine zipper (introduced at aa 992), Myo6 acts as a slow processive motor able to take several steps on actin filaments without detaching in single molecule motility and optical trapping assays [START_REF] Rock | Myosin VI Is a Processive Motor with a Large Step Size[END_REF]. Intriguingly, Myo6 takes irregular steps of 30-36 nm (Rock et al. 2001) which is unusual compare to the processive motor Myo5 [START_REF] Rief | Myosin-V Stepping Kinetics: A Molecular Model for Processivity[END_REF].

Proper stepping requires specific adaptation of the motor, indeed, a processive motor needs:

-high duty ratio (i.e., the motor must spend most of its kinetic cycle on actin so that the dimer does not detach too easily)

-coordination between the two heads of the dimer (called lead and rear heads) must be established to keep one head interacting with actin when the other is detached.

Kinetic studies show that Myo6 has a poorer affinity for ATP compared to Myo5 but it has a good affinity for ADP making the ADP state, in which Myo6 is bound to actin, the most populated state of the cycle and ADP release the limiting step of the Myo6 cycle (De La Cruz, Ostap, and Sweeney 2001).

These data are consistent with a high duty ratio for Myo6 compatible with its transport function.

Coordination between the lead and rear heads of the motor has been described for Myo5 through a « gating » in which the lead head and the rear head coordinate with each other through intramolecular strain [START_REF] Rosenfeld | A Model of Myosin V Processivity[END_REF]). However, Myo6 cannot gate using the same mechanism as this mechanism relies on the directionality of Myo5 Lever arm. This suggests a unique gating mechanism for Myo6 (Sweeney and Houdusse 2007). For the Myo6 artificial dimer (leucine zipper introduced at aa 992), the ADP release rate is twice as big as the ATPase rate per head while both are similar for the monomer, suggesting indeed a collaboration between the two heads of the dimer [START_REF] Morris | Calcium Functionally Uncouples the Heads of Myosin VI[END_REF]. A mechanism for Myo6 stepping has thus been proposed: the lead head stay bound to ADP.Pi and only weakly binds to actin until the rear head can detach (Robblee, Olivares, and de la [START_REF] Robblee | Mechanism of Nucleotide Binding to Actomyosin VI: Evidence for Allosteric Head-Head Communication[END_REF]). Alternatively, it was proposed that the lead head would be the one strongly attached to actin allowing longer run in an hand-over-hand stepping mechanism [START_REF] Park | Full-Length Myosin VI Dimerizes and Moves Processively along Actin Filaments upon Monomer Clustering[END_REF]Sweeney and Houdusse 2007) (Figure 40).

Performing in vitro motility assay on keratocyte actin network with either artificially dimerized Myo6 or multiple Myo6 monomers linked together through a nanosphere, [START_REF] Sivaramakrishnan | Coupled Myosin VI Motors Facilitate Unidirectional Movement on an F-Actin Network[END_REF] showed that a Myo6 monomer ensemble is able to be processive, indicating that dimerization might not be required for this. Monomer ensemble moves at a speed similar to the dimer and is able to transport cargos on long trajectories of ~10 µm [START_REF] Sivaramakrishnan | Coupled Myosin VI Motors Facilitate Unidirectional Movement on an F-Actin Network[END_REF] perhaps due to the fact that it is more likely to have at least one head remaining attached to the actin track in the context of an ensemble of motor compare to a dimer. Such oligomer assembly may be best suited for long distance trafficking while dimers will be able to transport cargo on ~1 µm distance.

Overall, Myo6 seems indeed well suited for cargo transport function.

E.4. Load sensitivity and anchoring

Actually, it was shown using optical trap experiments that Myo6 is force sensitive and can switch from a transport function to an anchoring function when force higher than 2 pN is applied to the motor (Altman, Sweeney, and Spudich 2004;[START_REF] Chuan | Robust Mechanosensing and Tension Generation by Myosin VI[END_REF]. When a resisting load is applied to Myo6, the motor becomes a much better ADP binder and a poorer ATP binder. Thus, until the resisting load is released, the strong ADP conformation of the motor is favored and the motor stays more stably attached to the actin filament (Altman, Sweeney, and Spudich 2004), which clearly promotes high capacity for this motor to anchor a cargo/organelle to the actin cytoskeleton. Step size and behavior clearly evolved with load [START_REF] Iwaki | A Programmable DNA Origami Nanospring That Reveals Force-Induced Adjacent Binding of Myosin VI Heads[END_REF]).

Interestingly, using Myo6 constructs truncated and artificially dimerized at residue 1021 either by addition of a portion of a Myo2 dimerization domain or by addition of a flag-tag that will mediate dimerization through binding to oligonucleotides (respectively), [START_REF] Nishikawa | Switch between Large Hand-over-Hand and Small Inchworm-like Steps in Myosin VI[END_REF] and [START_REF] Iwaki | A Programmable DNA Origami Nanospring That Reveals Force-Induced Adjacent Binding of Myosin VI Heads[END_REF] found that Myo6 can change its stepping mode upon load and ADP addition. Indeed, through in vitro motility assays, they monitored the movement on actin of each head of a Myo6 artificial dimer upon increasing load. They found that the Myo6 motor can switch from a non-adjacent binding mode (~36 nm between the two head of the dimer) under low load to an adjacent binding mode (~22 nm [START_REF] Nishikawa | Switch between Large Hand-over-Hand and Small Inchworm-like Steps in Myosin VI[END_REF] or ~10 nm [START_REF] Iwaki | A Programmable DNA Origami Nanospring That Reveals Force-Induced Adjacent Binding of Myosin VI Heads[END_REF] between the two head of the dimer) when load is applied. The non-adjacent stepping mode at low load is typical of a hand-over-hand mechanism associated with Myo6 transport function while the adjacent binding mode is typical of an Inchwormlike stepping behavior and could correspond to a transition between Myo6 transport and anchoring functions (Figure 40).

E.5. Large steps for a short Lever arm

When processive, Myo6 undergoes ~30-36 nm steps towards the minus end of the actin filaments which is surprisingly large considering the length of the Myo6 canonical Lever arm [START_REF] Spudich | The Myosin Swinging Cross-Bridge Model[END_REF]). It should be noted that ~11 nm steps were also observed in the other direction although they are 3.5-fold less frequent [START_REF] Rock | Myosin VI Is a Processive Motor with a Large Step Size[END_REF]Nishikawa et al., 2002).

Myo6 has only one conventional IQ motif in its Lever arm, while Myo5, that similarly performs ~36 nm steps, has 6 IQ [START_REF] Rosenfeld | A Model of Myosin V Processivity[END_REF]). The Ins2 sequence constitutes a second CaM binding site that increases the Myo6 Lever arm length. Moreover, during its powerstroke, the Myo6

Lever arm undergoes a very large swing (180°) compared to Myo5 (70°) (see section B.2.). But only considering the length of the Ins2-IQ motif as the Myo6 Lever arm and applying a 180° swing during the powerstoke, a 12 nm stroke is generated, which is in agreement with what has been measured on optical traps (Ménétrey et al. 2007). However, the way the Myo6 dimer can extend to cover the remaining 24 nm is still unclear and controversial.

It was first tested whether the Myo6 Ins2, unlike conventional IQ motif, would allow more flexibility of the motor allowing to cover the missing 24 nm. But 30-36 nm steps can also be achieved when Ins2 is replaced by one of the IQ motif found in Myo5 [START_REF] Park | The Unique Insert at the End of the Myosin VI Motor Is the Sole Determinant of Directionality[END_REF], thus the Myo6 large steps cannot be explained by the singularity of Ins2 alone and the Tail must play an important role for this. Thereby, a combination of TIRF single motility assays and optical trapping experiments have shown that randomization of the proximal Tail sequence results in shorter steps and reduced velocity of artificially dimerized Myo6 (leucine zipper at aa 992) assessing the importance of the proximal region for Myo6 correct stepping (Spink et al. 2008). However, the way this proximal region would contribute to the Myo6 Lever arm stiffness or to the dimerization of the motor remains unclear and at least two models of the active dimer have been proposed.

E.6. Two dimerization models were proposed to address this question

E.6.a. Distal dimerization

As the addition of the proximal region to the Myo6 S1 fragment does not significantly increase the working stroke of the motor, [START_REF] Rock | A Flexible Domain Is Essential for the Large Step Size and Processivity of Myosin VI[END_REF] proposed that the proximal Tail does not constitute a rigid Lever arm, instead, it may act as a flexible linker that will reach the 36 nm via a diffusive process. [START_REF] Zimmermann | Actin Age Orchestrates Myosin-5 and Myosin-6 Run Lengths[END_REF] show that on ADP-actin filaments, Myo6 binds to specific spots spaced from ~36 nm suggesting that preferential binding sites could be reached by Myo6 through diffusion allowing 36 nm steps, in support to the hypothesis proposed by [START_REF] Rock | A Flexible Domain Is Essential for the Large Step Size and Processivity of Myosin VI[END_REF]. Consistently, Spink et al., (2008) proposed that Myo6 would dimerize through the distal cargo binding domain (at the CBD level)

and thus both the 3HB and the SAH would be part of a flexible Lever arm. They were indeed unable to find a clear indication of Myo6 dimerization upon characterization of the proximal and distal Tails with SEC-MALS. In support of this hypothesis, [START_REF] Yu | Myosin VI Undergoes Cargo-Mediated Dimerization[END_REF] have shown that the Myo6 partner Dab2 is able to dimerize the CBD of Myo6 by X-ray crystallography. In this context, Dab2 constitutes a link that connects the two CBDs together without direct interaction between them (see section C.1.a.) (Figure 41).

CBD does not seem able to dimerize on its own, as addition of the zero-length cross linker in CBD at 12 µM results in no dimerization [START_REF] Spudich | Myosin VI Targeting to Clathrin-Coated Structures and Dimerization Is Mediated by Binding to Disabled-2 and PtdIns(4,5)P2[END_REF]). Based on that, [START_REF] Yu | Myosin VI Undergoes Cargo-Mediated Dimerization[END_REF] proposed a general scheme for the formation of the dimer that would be highly dependent on partner binding :

the so-called cargo-mediated dimerization in which distal dimerization of the Myo6 CBD via partner binding results in a dimer exhibiting flexible Lever arms.

E.6.b. Proximal dimerization

In vitro motility assays using a Myo6 (1-991 and 1-1050) motor without any CBD sequence [START_REF] Park | Full-Length Myosin VI Dimerizes and Moves Processively along Actin Filaments upon Monomer Clustering[END_REF] have shown that Myo6 is still dimeric and is able to make 30-36 nm steps on actin suggesting that a proximal dimerization of Myo6 could occur. Further studies in fact showed that an even more truncated Myo6 (1-940) construct was sufficient to make such big steps [START_REF] Mukherjea | Myosin VI Dimerization Triggers an Unfolding of a Three-Helix Bundle in Order to Extend Its Reach[END_REF]). When Myo6 is truncated up to 917 or 920, processivity of the motor is lost (Parks et al., 2006) assessing the importance of including the 920-940 region for internal dimerization (Table 9). Thereby, replacement of the 922-935 sequence with a perfect single alpha helix sequence (SAHmimic mutant) results in loss of Myo6 proximal dimerization in electron microscopy and loss of processivity in single molecule motility assays [START_REF] Mukherjea | Myosin VI Must Dimerize and Deploy Its Unusual Lever arm in Order to Perform Its Cellular Roles[END_REF]. This further supports the regulatory role of the aa 920-940 region in this process. Proximal dimerization thus results in a rigid Lever arm with three CaM binding motif per motor and a long-constrained dimerization structure that promotes coordination between the two heads and is critical to allow reaching two actin-binding sites distant by ~30 nm for each of the heads of the dimer.

Finally, co sedimentation of FLMyo6 and Myo6 Tail 814-end on actin shows its ability to intrinsically dimerize although the affinity is very low [START_REF] Fili | NDP52 Activates Nuclear Myosin VI to Enhance RNA Polymerase II Transcription[END_REF]) making internal dimerization of Myo6 very unlikely to happen in cells without a regulatory signal that would enhance Myo6 dimerization which could be partner binding (see section G.1.).

To check whether the Myo6 proximal dimerization is important for Myo6 cellular functions, SAHmimic was transfected in fibroblasts derived from Snell's Waltzer mice to investigate on its ability to rescue Myo6-mediated endocytosis following transferrin uptake [START_REF] Mukherjea | Myosin VI Must Dimerize and Deploy Its Unusual Lever arm in Order to Perform Its Cellular Roles[END_REF]. As opposed to FLMyo6, SAHmimic is unable to rescue Myo6 mediated transferrin uptake even when dimerization is forced by the addition of a leucine zipper at residue 994. These findings support the importance of a proper proximal dimerization for Myo6 functions [START_REF] Mukherjea | Myosin VI Must Dimerize and Deploy Its Unusual Lever arm in Order to Perform Its Cellular Roles[END_REF]) and emphasizes the importance of the configuration of the dimeric active motor in order to correctly serve its cellular roles. While the proximal dimerization model is more constrained and exhibit a stiffer Lever arm favouring gating between the heads, it is currently unclear whether the distal dimerization model that exhibits much more flexible Lever arms could be better suited for other Myo6 functions.

In an artificial context, a +end Myo6 mutant, in which the Ins2 and the rest of the Lever arm is replaced by two Myo5 IQ motifs is able to translocate cargo from the basis to the tips of filopodia in which it accumulates, although proximal dimerization cannot occur. This suggests that transport may not require the formation of a proximal dimer but rather leave open the question whether in this context Myo6 serve the role of a transporter as an ensemble of monomeric forms of a vesicle or whether distal dimerization plays a role for this cellular role. In cells, Myo6 localizes in two pools in several cell lines. Indeed, despite its ability to interact with actin, Myo6 does not always colocalize with actin rich structures, rather Myo6 can be seen as either diffuse across the cytosol or in punctate in the cytoplasm in LLC-PK1 (pig proximal tubule cells of the kidney), HeLa, NRK [START_REF] Hasson | Porcine Myosin-VI: Characterization of a New Mammalian Unconventional Myosin[END_REF] and Caco-2BBE (human intestinal epithelial line) [START_REF] Peterson | An in Vitro Model for the Analysis of Intestinal Brush Border Assembly. II. Changes in Expression and Localization of Brush Border Proteins during Cell Contact-Induced Brush Border Assembly in Caco-2BBe Cells[END_REF]. Punctates have been proposed to correspond to the active pool of Myo6 while diffused pool could constitute a Myo6 auto-inhibited pool waiting for its recruitment at appropriate time and place.

Overall, the mechanical role of

When unregulated, Myo6 over-dimerization can have deleterious effects for the cell and may possibly highjack other motor functions. Indeed, when artificially dimerized through an optogenetic or chemical approach, Myo6 was targeted to Rab5-positive early endosomes. Individual tracking of endosomes reveals that inducing Myo6 dimer engagement slows down endosome displacement and makes it irregular probably by competing with microtubule transport. Within minutes, endosomes morphology is altered. Ultimately, the endosomes fused together. In contrast, endosomes are not impaired when additional monomeric Myo6 are recruited [START_REF] Ritt | Engaging Myosin VI Tunes Motility, Morphology and Identity in Endocytosis[END_REF]. Thus, the amount, location and timing of activated dimers/oligomers recruitment must be finely regulated in cells to avoid over recruitment.

F.2. An auto-inhibited state

Regulation of unconventional myosins in cells often requires an auto-inhibited state with:

(1) a low ATPase rate, avoiding ATP consumption when force is not needed

(2) a low affinity for actin so the motor can diffuse freely across the actin network until it reaches the place where and when its recruitment is needed Such a state has already been characterized as a back-folded dimer for Myo5 [START_REF] Liu | Three-Dimensional Structure of the Myosin V Inhibited State by Cryoelectron Tomography[END_REF][START_REF] Thirumurugan | The Cargo-Binding Domain Regulates Structure and Activity of Myosin 5[END_REF] or as a back-folded monomer for Myo10 [START_REF] Umeki | Phospholipid-Dependent Regulation of the Motor Activity of Myosin X[END_REF]) and Myo7 [START_REF] Umeki | The Tail Binds to the Head-Neck Domain, Inhibiting ATPase Activity of Myosin VIIA[END_REF][START_REF] Sakai | Structure and Regulation of the Movement of Human Myosin VIIA[END_REF]. Similarly, to Myo10 and Myo7, Myo6 can adopt a back-folded conformation as shown in SAXS (Spink et al. 2008) and negative staining [START_REF] Song | Nucleotide-Dependent Shape Changes in the Reverse Direction Motor, Myosin VI[END_REF][START_REF] Lister | A Monomeric Myosin VI with a Large Working Stroke[END_REF]) experiments in which the Myo6 Tail was proposed to form intra-molecular interactions in order to form a compact back-folded conformation [START_REF] Song | Nucleotide-Dependent Shape Changes in the Reverse Direction Motor, Myosin VI[END_REF]Spink et al. 2008) (Figure 44). Backfolding of the Tail could then prevent motor activity and recruitment, thus constituting the autoinhibited state of Myo6. The existence of such a back-folded conformation, in which the Myo6 Nterminus and C-terminus are close to each other, has been confirmed in cells where a FRET signal between GFP and RFP can be measured upon transfection with GFP-FLMyo6-RFP [START_REF] Fili | NDP52 Activates Nuclear Myosin VI to Enhance RNA Polymerase II Transcription[END_REF]). It has already been proposed as key for the regulation of Myo6 activity since back-folding impedes Myo6

binding to DNA making it a key element in the regulation of Myo6-mediated transcription [START_REF] Fili | NDP52 Activates Nuclear Myosin VI to Enhance RNA Polymerase II Transcription[END_REF]. Taken together these studies established the ability of Myo6 to fold-back (Spink et al. 2008;[START_REF] Fili | NDP52 Activates Nuclear Myosin VI to Enhance RNA Polymerase II Transcription[END_REF]) and indicated the key importance of the CBD . This C-terminal domain was indeed

shown to be capable of interaction with CaM (Batters et al. 2016), with the IQ-DT fragment or with the MD Ins2 fragment [START_REF] Fili | NDP52 Activates Nuclear Myosin VI to Enhance RNA Polymerase II Transcription[END_REF], which could play a role in the stabilization of the Myo6 offstate.

Yet these studies were performed using Myo6 truncations, thus the key structural elements stabilizing the Myo6 back-folded conformation in the context of the full-length motor remain to be explored. In particular, the contribution of the Motor compared to the contribution of the neck, in the context of a full-length motor, remains to be established. Moreover, the importance of the nucleotide state of the Motor to stabilize the auto-inhibited state has not be clearly explored yet as well as possible contribution of the proximal and of distal Tail. DeTailed understanding of interactions going on in the auto-inhibited state and the way to disrupt them would greatly help dissecting Myo6 activation in cells. concentration [START_REF] Morris | Calcium Functionally Uncouples the Heads of Myosin VI[END_REF].

F.3. Myo6 activation

F.3.b. Phosphorylation

Phosphorylation of myosin motors has been described as a way to regulate the activity of several myosin motors. Phosphorylation of non-muscle and smooth Myo2s regulatory light chain results in important conformational changes, resulting in a shift from the inactive backfolded motor to the unfolded active form [START_REF] Craig | Light-Chain Phosphorylation Controls the Conformation of Vertebrate Non-Muscle and Smooth Muscle Myosin Molecules[END_REF].

Moreover, Myo1 and Myo2 actin-activated ATPase are greatly enhanced by phosphorylation of the motor in the myosin-actin interface [START_REF] Brzeska | Regulation of Class I and Class II Myosins by Heavy Chain Phosphorylation[END_REF]. In Myo6, a threonine was identified at the corresponding location [START_REF] Hasson | Porcine Myosin-VI: Characterization of a New Mammalian Unconventional Myosin[END_REF]) (Thr405 in human) and its phosphorylation has been shown to influence Myo6 localization. In A431, Myo6 motor domain T405 phosphorylation occurs upon EGF stimulation, simultaneously, Myo6 relocates to membrane ruffles [START_REF] Buss | The Localization of Myosin VI at the Golgi Complex and Leading Edge of Fibroblasts and Its Phosphorylation and Recruitment into Membrane Ruffles of A431 Cells after Growth Factor Stimulation[END_REF].

Similarly, Myo6 recruitment to Salmonella induced ruffles required T405 phosphorylation [START_REF] Brooks | MYO6 Is Targeted by Salmonella Virulence Effectors to Trigger PI3-Kinase Signaling and Pathogen Invasion into Host Cells[END_REF]. Phosphorylation can be seen on T405 in vitro using PAK enzyme (known to control actin filament turnover) [START_REF] Buss | The Localization of Myosin VI at the Golgi Complex and Leading Edge of Fibroblasts and Its Phosphorylation and Recruitment into Membrane Ruffles of A431 Cells after Growth Factor Stimulation[END_REF][START_REF] Brooks | MYO6 Is Targeted by Salmonella Virulence Effectors to Trigger PI3-Kinase Signaling and Pathogen Invasion into Host Cells[END_REF]). Yet, effect of T405 phosphorylation on Myo6 mechanical properties is unclear, this phosphorylation does not enhance Myo6 ability to cosediment with F-actin [START_REF] Buss | The Localization of Myosin VI at the Golgi Complex and Leading Edge of Fibroblasts and Its Phosphorylation and Recruitment into Membrane Ruffles of A431 Cells after Growth Factor Stimulation[END_REF]. Moreover, its phosphorylation does not impair Myo6 ATPase rate, either using S1 or HMM fragment and does not impact actin sliding capacity [START_REF] Morris | Calcium Functionally Uncouples the Heads of Myosin VI[END_REF]Sweeney and Houdusse 2007).

But T405 is not the only site phosphorylated by the PAK enzyme, indeed, mass spectroscopy analysis of Myo6 immunoprecipitated from A431 cells revealed two Thr phosphorylation sites in the Myo6 CBD within the TINT motif [START_REF] Sahlender | Optineurin Links Myosin VI to the Golgi Complex and Is Involved in Golgi Organization and Exocytosis[END_REF]. These phosphorylation sites can also be mediated by PAK in vitro. Mammalian two hybrids using either the phosphomimetic EINE mutant or the antiphosphorylation mutant AINA revealed that phosphorylation prevent binding to binding partners OPTN, thus TINT phosphorylation could have a role for addressing Myo6 to correct location by preventing Myo6 binding to specific cellular partners [START_REF] Sahlender | Optineurin Links Myosin VI to the Golgi Complex and Is Involved in Golgi Organization and Exocytosis[END_REF]. Moreover, the TINT motif is present in the CBD. Since the CBD is involved in stabilization of the auto-inhibited state, the TINT phosphorylation could regulate Myo6 opening/back folding equilibrium.

In addition, phosphorylation of the SI isoform specific Tyr phosphorylation site (CBD DYD motif)

was also found to be a key regulator of Myo6 mediated secretory granule exocytosis in PC12 cells although its precise role in this process is unknown [START_REF] Tomatis | Myosin VI Small Insert Isoform Maintains Exocytosis by Tethering Secretory Granules to the Cortical Actin[END_REF].

Overall, Myo6 phosphorylation appears as a key parameter for proper Myo6 localization but it remains unclear if these phosphorylations impact Myo6 auto-inhibition by unfolding the motor, by modulating binding to partners or by regulating force generation. Phosphorylation thus remains an interesting parameter to look at for studying how the role Myo6 plays in cells is finely tuned.

F.3.c. Actin track (concentration, age and composition)

High concentration of actin could constitute a regulation signal as well since ATPase assays show that FLMyo6 ATPase activity increased upon actin addition (Dos Santos et al. 2022). Actin being essentially found near the membranes; this parameter could control the locations where the motor will be activated in cells although it could also depend on the age and composition of the actin tracks:

Actin filaments are dynamic structures in which new ATP-actin monomers bind to the filament plus-end which are in general the closest to the membrane surface. Once the monomer is incorporated in the filament, ATP is hydrolyzed resulting in ADP-actin, which is less stiff and less stable. At the minus end, ADP bound actin form the "old" filament while at the + end, the youngest part of the filament is formed with ATP actin [START_REF] Merino | Structural Transitions of F-Actin upon ATP Hydrolysis at near-Atomic Resolution Revealed by Cryo-EM[END_REF]. Interestingly [START_REF] Zimmermann | Actin Age Orchestrates Myosin-5 and Myosin-6 Run Lengths[END_REF] show through in vitro motility assays that Myo6 undergoes as a single molecule processive motor longer run length on ADP-rich actin filament (while Myo5 binds preferentially to ADP.Pi-rich filaments). Actin + end being usually oriented near the plasma membrane, actin "age" could thus be a parameter that control Myo6 activation on precise location.

Actin track composition has been shown to modulate their association with myosins motors; six actin isoforms can be found in mammalian cells and despite presenting a high sequence identity, non-muscle Myo2A and B associate preferentially with α-actin, non-muscle Myo2C with β-actin and Myo7A with γ-actin, highlighting possible regulation of motor recruitment by actin through actin isoform [START_REF] Müller | Distinct Functional Interactions between Actin Isoforms and Nonsarcomeric Myosins[END_REF]. Moreover, the actin binding tropomyosin was also found to modulate the ability of diverse myosin motors to bind to distinct actin tracks [START_REF] Sckolnick | Tropomyosin Isoforms Bias Actin Track Selection by Vertebrate Myosin Va[END_REF][START_REF] Gateva | Tropomyosin Isoforms Specify Functionally Distinct Actin Filament Populations In Vitro[END_REF]Manstein et al. 2020). Although there is no evidence showing that actin track composition could modulate Myo6 recruitment, as pointed out by [START_REF] De Jonge | The MYO6 interactome: selective motor-cargo complexes for diverse cellular processes[END_REF], it could be an interesting aspect to explore.

F.3.d. Partners

Regulation of the Myo6 auto-inhibition could also directly be driven by its cellular partners.

Using FRET experiments, [START_REF] Fili | NDP52 Activates Nuclear Myosin VI to Enhance RNA Polymerase II Transcription[END_REF]2020) show that partners NDP52 and Dab2 can unfold Myo6 (814-end) and [START_REF] Rai | Multimodal Regulation of Myosin VI Ensemble Transport by Cargo Adaptor Protein GIPC[END_REF] shows that GIPC1 can unfold FLMyo6. Moreover [START_REF] Fili | NDP52 Activates Nuclear Myosin VI to Enhance RNA Polymerase II Transcription[END_REF]2020) demonstrate that partners NDP52, DNA and Dab2 can sequester the CBD and prevent its interaction with Myo6 Tail [START_REF] Fili | NDP52 Activates Nuclear Myosin VI to Enhance RNA Polymerase II Transcription[END_REF]2020) suggesting that once Myo6 unfold, partners could have the ability to stabilize the active form of Myo6.

French, Sosnick, and [START_REF] French | Investigations of Human Myosin VI Targeting Using Optogenetically Controlled Cargo Loading[END_REF] show that artificial recruitment of Myo6 to peroxisomes through Dab2 significantly slow down peroxisome movement on microtubule tracks showing the ability of Dab2 to recruit a competent motor on this cellular compartment. In contrast, single molecule tracking in HeLa cells shows that Myo6 diffuses randomly in the cytosol when partner binding is disabled by mutation within the CBD (Dos Santos et al., 2022). These finding assess the potential of partners for Myo6 activation.

Thanks to the recent findings described above [START_REF] Fili | NDP52 Activates Nuclear Myosin VI to Enhance RNA Polymerase II Transcription[END_REF]2020;Dos Santos et al. 2022;[START_REF] Rai | Multimodal Regulation of Myosin VI Ensemble Transport by Cargo Adaptor Protein GIPC[END_REF], partner binding appears as a key parameter for Myo6 activation. Several of them have been proposed to be able to unfold Myo6. Yet, main evidence of Myo6 unfolding by partners have been assessed with FRET titrations that used the Myo6 Tail 814-end, and not the full length motor. The only study that used a full length motor has been published in 2022 (Rai et al.) and has shown the role of GIPC1 in this process but whether all partners are able to do so in the context of a full-length motor remains to clarify.

G. Modulation of the mechanical properties of the activated Myo6

G.1. Cargo nature

Partners play critical roles in addressing the motor to precise cellular locations [START_REF] Fili | NDP52 Activates Nuclear Myosin VI to Enhance RNA Polymerase II Transcription[END_REF], Tumbarello et al. 2012[START_REF] Sahlender | Optineurin Links Myosin VI to the Golgi Complex and Is Involved in Golgi Organization and Exocytosis[END_REF]. They also have been proposed to trigger the unfolding of the motor to activate it (see section F. 3.d.). But considering the variety of mechanical roles and oligomeric states proposed for Myo6 (see section E), it is of outstanding interest to explore the potential of Myo6 partners to instruct Myo6 for a precise mechanical role, facilitating either anchoring, transporting or organizing actin among other functions (Figure 45). To that aim, understanding which partners can induce Myo6 oligomerization, dimerization or stabilize Myo6 as a monomer following its unfolding is of particular interest. Myo6 partners themselve exhibit various oligomeric states: Dab2 is monomeric while OPTN, NDP52 and GIPC1 are dimeric [START_REF] Phichith | Cargo Binding Induces Dimerization of Myosin VI[END_REF][START_REF] Kim | Structural Basis for Recognition of Autophagic Receptor NDP52 by the Sugar Receptor Galectin-8[END_REF] Single molecule motility assays show that the OPTN dimer is able to make Myo6 processive [START_REF] Phichith | Cargo Binding Induces Dimerization of Myosin VI[END_REF]) and rotary shadowing confirmed that Myo6 is dimeric upon OPTN binding.

The X-ray structure of Dab2 (675-713) with Myo6 CBD c reveals that despite being monomeric, Dab2 can mediate indirect Myo6 dimerization through binding on two distinct binding sites of the CBD c , promoting the formation of a 2:2 complex. ITC using a variety of Myo6 and Dab2 mutants and truncations reveals that the first interaction site results in high affinity (~310 nM) through hydrophobic interactions and a second binding site of lower affinity alone (>13 µM) that, when combined with the first binding site results in increased affinity (50-80 nM) and indirect dimerization [START_REF] Yu | Myosin VI Undergoes Cargo-Mediated Dimerization[END_REF].

These results pointed out to a distal dimerization (Figure 45 Interestingly, when either Dab2 mediated dimer or zippered dimers are observed on a minimal actin cortex, zippered dimer generates actin foci while Dab2 mediated dimers do not impact the overall organization of the actin network (Rai et al. 2021).

As a dimeric partner, we would expect NDP52 to promote distal dimerization as proposed for

OPTN. Yet, a FRET-based assay shows that if CBD dimerization can occur through binding to its dimeric partner NDP52, this is much weaker compared to the dimerization that NDP52 can promote with Myo6 814-end, suggesting a strong contribution of residues within the IQ-DT region for promoting Myo6 dimerization [START_REF] Fili | NDP52 Activates Nuclear Myosin VI to Enhance RNA Polymerase II Transcription[END_REF]. [START_REF] Fili | NDP52 Activates Nuclear Myosin VI to Enhance RNA Polymerase II Transcription[END_REF] proposed thus that both distal and proximal dimerization could occur upon NDP52 binding and that the type of dimer could be modulated by the variation of Myo6 partner concentration.

These comparisons highlight thus the potential of partners to modulate both the nature and the number of Myo6 dimers formed with thus a great potential for fine tuning of Myo6 mechanical properties.

In parallel, using a severely truncated fragment of Myo6 (1052-1096), [START_REF] Shang | Structure Analyses Reveal a Regulated Oligomerization Mechanism of the PlexinD1/GIPC/Myosin VI Complex[END_REF] has proposed that the GIPC1 GH2 domain can interact with the Myo6 CBD n forming oligomers using two opposite interfaces (5V6E). In AUC, the complex seems to oligomerize up to 5:5 complex (reached at 150 µM). They proposed that the two proteins could thus form long chains in an assembly of motors.

In our hands, only very short fragments of Myo6 have this property. This kind of oligomerization could then allow Myo6 to perform a transporter role as a motor ensemble enabling very long processive runs [START_REF] Sivaramakrishnan | Coupled Myosin VI Motors Facilitate Unidirectional Movement on an F-Actin Network[END_REF]). Yet, this result might be an artefact since experiments from our lab demonstrate that longer Myo6 CBD fragments form a 1:1 complex by multi-angle light scattering (MALS). [START_REF] Rai | Multimodal Regulation of Myosin VI Ensemble Transport by Cargo Adaptor Protein GIPC[END_REF] supported in contrast that FLMyo6 and GIPC1 can from a 1:1 non processive complex, although the difference in stoichiometry may be due to the very low concentration of Myo6 used in the motility assays (pM) (Figure 45 and46). Still, they found that GIPC1 was able to greatly enhance Myo6 motor velocity (2.5-fold increase compared to Myo6 alone) and to decrease dwell time in motility assays. In multiple motor motility assays, they even found that GIPC1-Myo6 ensembles display fast, smooth, processive movement on cellular actin networks showing the potential of the complex for transport. They proposed using FRET experiments that GIPC1 binding could result in 3HB opening. They thus proposed that GIPC1 may increase flexibility of the motor by opening 3HB and in so doing increase its velocity. But preliminary data of the Houdusse team (Figure 46) suggests that once unfolded, 3HB can also mediate proximal dimerization possibly orienting the motor towards anchoring functions. Overall, the impact of GIPC1 or Dab2 binding to the Myo6 motor remains to be clarified although it clearly appears that Dab2 and GIPC1 partners have very different impact on the Myo6 motor function in similar experimental set ups (Rai et al. 2021;2022). with the two CBD c through two helices (interface A and interface B) [START_REF] Yu | Myosin VI Undergoes Cargo-Mediated Dimerization[END_REF]). This 2:2 complex between Dab2 and CBD c has been proposed to mediate distal dimerization (although an additional proximal dimerization remained to be explored and has been proposed using rotary shadowing [START_REF] Phichith | Cargo Binding Induces Dimerization of Myosin VI[END_REF]. The complex can mediate sparse processive motility (Rai et al. 2021).

(Center) Crystal structure of GIPC1 GH2 domain through 2 interfaces [START_REF] Shang | Structure Analyses Reveal a Regulated Oligomerization Mechanism of the PlexinD1/GIPC/Myosin VI Complex[END_REF] Herrero). A recent study proposed also a 1:1 complex between FLMyo6 and GIPC1 in biophysical assays [START_REF] Rai | Multimodal Regulation of Myosin VI Ensemble Transport by Cargo Adaptor Protein GIPC[END_REF]) but the assays were carried out at low concentration. In this context, more studies are required to identify whether GIPC1 and FLMyo6 act as a 1:1 complex and whether a proximal dimerization can form after association of GIPC1 to FLMyo6.

(Bottom) Crystal structure of TOM1 436-461 with CBD c , the two proteins interact as a 1:1 complex through an interface similar to one of the two binding sites of Dab2 on CBD c : the so-called interface A. In this context, Myo6

has been proposed to act as a non-processive bridge able for example to reunite endosome (bound to TOM1) and autophagosome (bound to autophagy adaptors NDP52, OPTN or TAX1BP1) for fusion during autophagy (Hu et al. 2019). However, no evidence has been collected to support that such ability of a FLMyo6 monomer or dimer to bind two partners has yet been collected.

G.2. Cargo stiffness

Once unfolded, the stiffness of the cargo bound to Myo6 could also contribute to dictate mechanical properties of the motor to orient it towards a transport or anchoring function [START_REF] Shrivastava | Stiffness of Cargo-Motor Linkage Tunes Myosin VI Motility and Response to Load[END_REF]. Indeed, in an optical trap, attaching artificially dimerized Myo6 (leucine zipper at aa992) to DNA origami using DNA linker of different lengths can mimic Myo6 attachment to either stiff (short DNA linker) or flexible (long DNA linker) cargos. These experiments reveal that under load >2pN, both Myo6 linked to flexible or stiff cargo can take steps on the actin track. However, steps are bigger (28 ± 13 nm) with the flexible cargo, while the time between the steps is smaller and the run length is longer suggesting that a flexible linkage between Myo6 and its cargo would facilitate its transport function.

In contrast, under load, Myo6 associated with a stiff linkage exhibit longer stall events suggesting that load induced anchoring could be facilitated by stiff cargo binding to Myo6 [START_REF] Shrivastava | Stiffness of Cargo-Motor Linkage Tunes Myosin VI Motility and Response to Load[END_REF] reinforcing the importance of cargo in dictating the mechanical role of Myo6. Yet it should be noted that this study was performed using DNA origami to mimic cargo stiffness and not actual Myo6 cargoes.

Thus, to complete this study it would be of great interest to extend it using various Myo6 binding partners.

H. Interest of having several partners interacting at once

Since the Myo6 Tail contains several, distinct sites of interaction with partners, this raises the possibility of having several partners interacting at once with Myo6. Moreover, Myo6 NI isoform colocalizes with both TOM1 and GIPC1 on APPL1-positive signaling endosomes [START_REF] Tumbarello | Autophagy Receptors Link Myosin VI to Autophagosomes to Mediate Tom1-Dependent Autophagosome Maturation and Fusion with the Lysosome[END_REF]. In mice, during spermatid maturation, Myo6 localizes with two partners at once: GipC1 and TOM1-L2 that localizes with it to the bulbar region of the TBCs and associates to Rab5 and APPL1 positive early endosomes [START_REF] Zakrzewski | Myosin VI Maintains the Actin-Dependent Organization of the Tubulobulbar Complexes Required for Endocytosis during Mouse Spermiogenesis † ‡[END_REF]. Although DOCK7 has been reported to bind to the RRL motif and TOM1L2 the WWY motif, a recent study shows that depletion of the WWY motif results in loss of the DOCK7 interaction and loss of the RRL motif results in the loss of TOM1L2 interaction in RPE cells (O'Loughlin, Masters, and Buss 2018), suggesting a possible collaboration between the different partners being required for localization.

Actually, even if the localization is similar, the function pursued by the partners can vary; TOM1 and GIPC1 both colocalize with Myo6 on endosomes but immuno-cytochemistry in RPE cells shows that TOM1 depletion results in a dramatic loss of Myo6 on endosomes while with GipC1 depletion, Myo6 localization loss is only partial [START_REF] Tumbarello | Autophagy Receptors Link Myosin VI to Autophagosomes to Mediate Tom1-Dependent Autophagosome Maturation and Fusion with the Lysosome[END_REF].

Thenceforth, the interest of having several partners collaborating should be pursued. With such collaboration, cellular roles could be smooth transition from one mechanical role to another as proposed in [START_REF] Zakrzewski | Myosin VI Maintains the Actin-Dependent Organization of the Tubulobulbar Complexes Required for Endocytosis during Mouse Spermiogenesis † ‡[END_REF], enhancing the mechanical properties of the motor or assuring redundancy.

Fili et al ( 2017) highlights an important distinction between DNA and NDP52; while both can sequester the CBD prior to its binding to the rest of the Tail, only NDP52 can disrupt the interaction and promote unfolding of Myo6 814-end fragment once its interaction is formed. In this context, they proposed that Myo6 would need first to be opened by its nuclear partner NDP52 prior to DNA binding [START_REF] Fili | NDP52 Activates Nuclear Myosin VI to Enhance RNA Polymerase II Transcription[END_REF]. They then pursue the relevance of this hypothesis in the context of Myo6 transcription function. As described in section D.4., addition of Myo6 or NDP52 to HeLaScribe extract increase the transcription level but strikingly, competition with Myo6 CBD NI or CBD c results in transcription decrease while the use of either the CBD LI, CBD n and CBD RAL mutant does not decrease transcription supporting the fact that a partner is required for the recruitment to this localization. Intriguingly, this suggests a greater role of CBD c than CBD n in transcription whereas NDP52 has been reported to be a CBD n partner suggesting that while NDP52 can enhance transcription, it may not mediate Myo6 interaction with RNAPII. Actually, immunoprecipitation of Myo6 and RNAPII shows using different Myo6 truncations that Myo6 CBD c can nicely interact with RNAPII while CBD n can only very weakly interact with it [START_REF] Fili | NDP52 Activates Nuclear Myosin VI to Enhance RNA Polymerase II Transcription[END_REF]). Overall, it seems that Myo6 unfolding occurs through the RRL binding partners which free the CBD c making its interaction with RNAPII possible through binding of DNA.

Possible collaboration between partners for assuring smooth transition between function was also proposed in prostate cancer LNCaP cells, both GIPC1 and LMTK2 were identified as Myo6 partners [START_REF] Puri | Overexpression of Myosin VI in Prostate Cancer Cells Enhances PSA and VEGF Secretion, but Has No Effect on Endocytosis[END_REF]. Considering the localization of GIPC1 on APPL1 early endosomes and the role of LMTK2 in the delivery of endosomes to multivesicular bodies [START_REF] Chibalina | Myosin VI and Its Interacting Protein LMTK2 Regulate Tubule Formation and Transport to the Endocytic Recycling Compartment[END_REF], the authors proposed that GIPC1 would first recruit Myo6 to early endosomes, then LMTK2 will address Myo6 to the perinuclear recycling compartment [START_REF] Puri | Overexpression of Myosin VI in Prostate Cancer Cells Enhances PSA and VEGF Secretion, but Has No Effect on Endocytosis[END_REF].

In autophagy, Myo6 can form a ternary complex with TOM1 and the autophagy receptors TAXBP1, NDP52 or OPTN according to immunoprecipitation from HEK293T cells [START_REF] Hu | Structure of Myosin VI/Tom1 Complex Reveals a Cargo Recognition Mode of Myosin VI for Tethering[END_REF]. The SiTOM1 phenotype nicely mimics that of Myo6 depletion which results in defects in fusion of autophagosomes with lysosomes and a delay in amphisomes formation [START_REF] Tumbarello | Autophagy Receptors Link Myosin VI to Autophagosomes to Mediate Tom1-Dependent Autophagosome Maturation and Fusion with the Lysosome[END_REF].

Interestingly, NDP52, OPTN, T6BP depletion do not mimic this phenotype. Actually, as opposed to Myo6 depletion, the depletion of autophagy receptors impairs autophagosomes biogenesis. As, the Ubiquitin and Myo6 binding site overlaps on these adaptors, they may first link ubiquitinylated cargo to LC3-autophagosomes and interact afterwards with Myo6 for autophagosomes fusion with lysosomes. The authors thus proposed that Myo6 is recruited to endosomes through TOM1. Then

Myo6 will address the TOM1-positive early endosomes to autophagosomes through autophagy adaptor interaction (NDP52, OPTN, T6BP). Delay in fusion may be the result of an incorrect maturation of the autophagosomes (i.e., lack of TOM1-endosomes docking on autophagosomes).

Finally, in melanocytes (MNT-1), if OPTN seems to be important as a Myo6 associated partner for the constriction of tubular carriers, its depletion does not impair Myo6 localization at melanosomes [START_REF] Ripoll | Myosin VI and Branched Actin Filaments Mediate Membrane Constriction and Fission of Melanosomal Tubule Carriers[END_REF] We thus aimed at providing further insights on the Myo6 auto-inhibited state properties via a structural and functional approach. Specifically, we aimed to explore the importance of the Motor domain nucleotide state and identify the relative contribution of interactions within the Tail or in between the CBD and the Motor to be able to determine precise conditions or pathogenous mutations favoring Myo6 opening and activation. In particular, the potential of partners for differential Myo6 activation has been explored. A particular focus of our research concerned the proximal dimerization.

Indeed, while the potential of partners such as Dab2 to promote distal dimerization has been established, there is no clear evidence of Myo6 proximal dimerization upon partners binding or upon other activation pathways in a physiological context.

Our structural insights have been completed by experiments in cells to assess the relevance of our findings in a physiological context. In particular, we highlight the importance of the Myo6 autoinhibition and dimerization in the context of endocytosis and melanosome maturation.

Results and discussion

A. Myosin VI activation: from the auto-inhibited state to its active forms A.1. Methodology

A.1.a. Identifying the conditions stabilizing either a compact or an open conformation

As the Myo6 auto-inhibited state has been proposed to be a compact back-folded monomer, combination of SEC-MALS (steric exclusion chromatography multi angles light scattering) and SEC-SAXS (steric exclusion chromatography small angles light scattering) were used to determine Myo6 compacity and oligomeric state in several buffer conditions. Indeed, for a given molecular weight, elution time in SEC column is already indicative of the compacity of a protein and allows rather quick screening of buffer conditions for their impact on Myo6 compacity. MALS then allows precise measurement of the molecular weight of the protein allowing reliable estimation of the oligomeric state while SAXS provides information on the shape (Figure 47) and dimension of the protein.

Microscale thermophoresis (MST) then allows in vitro estimation of KD between different Myo6 fragments. with high affinity while addition of four point-mutations in a highly exposed CBD c loop (D1157V, Y1159D, D1161R and Q1163V) was sufficient to lose the interaction (Figure 48). This loop had been reported to mediate Myo6 binding to DNA [START_REF] Fili | NDP52 Activates Nuclear Myosin VI to Enhance RNA Polymerase II Transcription[END_REF]). Based on these data, a constitutively back-folded Myo6 construct was engineered by fusing at the Myo6 N-terminus and C-terminus two subdomains ("Jo" and "In") able to recognize each other and subsequently undergo strong attachment while a covalent bound would be created between them [START_REF] Bonnet | Autocatalytic Association of Proteins by Covalent Bond Formation: A Bio Molecular Welding Toolbox Derived from a Bacterial Adhesin[END_REF])(Jo-Myo6-In construct). But surface residues of the Tail and Motor domain-Lever arm regions are not fully conserved in between mouse and pig which may affect the way Myo6 back-folds (Figure 49 and50). We thus first decided to repeat these experiments using the human FL Myo6 construct. I thus performed a SAXS study that shows that human FL Myo6, overall, behaves the same as pig-mouse Myo6: the protein remains closed in MgADP.VO4 and opens upon nucleotide removal in high salt. SEC-SAXS data collected on buffers containing the Mg.ADP.Pi analog comparing the human and chimera Myo6 compact conformation: no significant changes are found. Yet, gel filtration suggests that human Myo6 back-folded conformation would be more stable than the pig/mouse chimera as 400 mM NaCl was found to be sufficient to fully shift the Myo6 pig-mouse chimera elution peak, while it is not the case for the human Myo6: a second and significant peak corresponding to a compact conformation can be seen at 13.8 mL (although the main peak at 12 mL corresponds to the elongated conformation) (Figure 51). Comparison of pig-mouse chimera and human Myo6 sequence highlights differences in polarity/charge of several surface residues between the two constructs that could be responsible of weaker polar contacts within the chimera enabling easier opening upon salt addition. This suggests that Myo6 adopt a more elongated shape. For the human Myo6 (left panel), upon nucleotide removal in high salt concentration conditions, a small peak can be seen on the SEC profile at ~14mL in addition of the main peak at ~12mL. This suggests that a fraction of the human FLMyo6 remains closed even upon nucleotide removal in high salt concentration conditions.

Additionally, triplicate affinity measurements were performed between the CBD and the Myo6 MD Ins2-IQ-3HB (1-917) in microscale thermophoresis to confirm the preliminary results and get a reliable estimation of the KD. Additionally, we looked at the affinity of the CBD against either MD (1-789), MD Ins2

(1-816) and Ins2-IQ-3HB (1-917) to gain greater insights on the relative influence of all the structural elements proposed to maintain the Myo6 back-folded conformation. working group that was gathering on meetings once a week. The goal of these meetings was to make sure of the quality and the reproducibility of the data the teams had obtained, proposing new experiments when needed, so that the results from our multi-disciplinary approaches would be organized in a manuscript.

A.1.b. Proximal dimerization

Before I joined the research on Myo6's ability to dimerize on its own, SEC-MALS was used to determine the oligomeric state of several Myo6 fragments in the 3HB-SAH area as Mukherjea et al.

(2009) had pointed out that the Myo6 1-940 fragment was sufficient to allow processivity, thus highlighting the existence of this proximal dimerization region. The peptide identified as sufficient to allow proximal dimerization was crystallized and its structure was determined. These studies were performed by Elena Sirkia and Carlos Kikuti, engineers in our lab. Based on this structure, the triple mutant T888D, R892E and V903D was designed to disrupt proximal dimerization (without affecting the stability of the folded 3HB) and its efficiency was confirmed in SEC-MALS.

But all these results were performed on peptides in vitro, so finally, to confirm the relevance of this model of proximal dimerization in a more physiological context, we introduced the mutations T888D, R892E and V903D within the FLMyo6 to check its impact on Myo6 motility using singlemolecule assays through a collaboration with Lee Sweeney's lab. Finally, through this collaboration,

we also checked the impact of these mutations T888D, R892E and V903D on transferrin uptake in HeLa cells.

A.1.c. Recruitment and activation of Myo6 by its partners

Our model of the Myo6 auto-inhibited state suggests that the ability of the protein adaptor to interact with Myo6 could depend on its binding site on the motor. To challenge this hypothesis, we explored the ability of RRL and WWY adaptors to differentially recruit Myo6 and activate it either as an active monomer or dimer.

Pull-down assays were first carried out to explore the ability of the various partners to bind Myo6 in conditions favoring either opening or back-folding in vitro. Then, the ability of partners to recruit Myo6 in cell was explored using MNT-1 cells (human melanoma cells). To design this assay, we took advantage of the work of a former Post-Doctoral researcher in our team: Karl Petersen that had revealed that addition to FLMyo6 NI of a point mutation I1072A previously reported to prevent the interaction between the MIU domain and ubiquitin [START_REF] He | Myosin VI Contains a Compact Structural Motif That Binds to Ubiquitin Chains[END_REF]) results in loss of Myo6 endogenous recruitment to melanosome. We thus decided to fuse the Myo6 adaptor with a melanosome targeting tag (MST, Ishida et al. 2014) enabling their addressing to melanosome and looking at their ability to rescue Myo6 recruitment to melanosome. These experiments were performed by the cell biologist engineer of our team, Franck Mayeux.

Finally, the ability of the Myo6 adaptor to activate Myo6 as either a functional monomer or a dimer was monitored by Tianming Lin from Lee Sweeney's lab via performing actin-based ATPase.

Indeed, ATPase assays was a very clear way to monitor whether binding of adaptors unfolds (releases auto-inhibition) and thus increases Myo6 ATPase rate. In addition, it gives an indirect information on the possibility of gating between the two heads when Myo6 forms a dimer. As described above (see section E), formation of a Myo6 dimer results in an unusual "gating" enabling coordination between the two heads of the Myo6 dimer which results in a 2-fold decrease of Myo6 ATPase rate per head compared to a Myo6 monomer.

These assays were carried out with partners Dab2, TOM1 and GIPC1 and we used their Myo6 binding region to avoid domains that might regulate exposure of the minimal interaction domain for the binding of Myo6 (MIR).

CBD n partner: GIPC1

GIPC1 is a CBD n RRL motif adaptor (section C.2.a.) containing an N-terminus GH1 domain allowing dimerization, a PDZ domain allowing binding to various cellular partners notably APPL1, which mediate its recruitment to APPL1-positive endosomes, finally a GH2 domain that mediates its interaction with Myo6 (Figure 52). Importantly, the GIPC1 52-333 crystal structure reveals the organization of the GIPC1 dimer as a swapped dimer in which GH1 and PDZ interact together (PDB: 5V6B, Shang et al. 2017) (Figure 53).

The GH2 domain folds on the PDZ hindering the Myo6-GIPC1 binding interface. This dimer has thus been proposed to constitute an auto-inhibited state of GIPC1 in which GIPC1 cannot bind to Myo6 [START_REF] Shang | Structure Analyses Reveal a Regulated Oligomerization Mechanism of the PlexinD1/GIPC/Myosin VI Complex[END_REF]. They additionally proposed that binding of a PDZ partner would be required to destabilize the GIPC1 dimer enabling Myo6 binding. Indeed, GIPC1 52-333 was co-crystallized with the PDZ partner plexin D1 and although GIPC1 was not degraded according to SDS page gels of the crystals, the GH2 domain could not be seen in the electron density, the authors thus proposed that GH2 is flexible and possibly more available for Myo6 due to GIPC1 conformational changes occurring upon plexin D1 binding [START_REF] Shang | Structure Analyses Reveal a Regulated Oligomerization Mechanism of the PlexinD1/GIPC/Myosin VI Complex[END_REF]. Moreover, addition of a PDZ partner improves Myo6-GIPC1

binding in pull-down (Naccache, Hasson, and Horowitz 2006).

Due to the existence of this GIPC1 auto-inhibited state and the possible reduction in binding affinity for the FL GIPC1 in its capacity to interact with Myo6, we decided to work with GIPC1 constructs containing only the GH2 domain to ensure Myo6 binding in our assays. The ability of GIPC1 GH2 to bind Myo6 was then confirmed by microscale thermophoresis in our hands (Table 10). Finally, as I1072 is present in CBD n near the GIPC1 binding site, we confirmed with this assay that the mutation I1072A

does not impair GIPC1 binding. Note that the I1072A mutation was extensively used to monitor the Myo6 recruitment on melanosome in MNT-1 cells.

Adaptor Myo6 Tail KD n

Interaction with GipC1 GipC1GH2 255-end YFP CBD 164 ± 51 nM 3

GipC1GH2 255-end YFP CBD I1072A 90 ± 34 nM 2

CBDc partner: TOM1

TOM1 is a CBD c WWY motif adaptor (section C.1.c) containing an N-terminus VHS domain and a GAT domain which mediates interaction with TOLLIP and ubiquitin. Moreover, TOM1 C-terminus can bind clathrin through residues 300-366 [START_REF] Seet | Endofin Recruits Clathrin to Early Endosomes via TOM1[END_REF] and double hybrid assays suggest that TOM1 binds Myo6 within its last 104 amino acids [START_REF] Tumbarello | Autophagy Receptors Link Myosin VI to Autophagosomes to Mediate Tom1-Dependent Autophagosome Maturation and Fusion with the Lysosome[END_REF]) (Figure 54).

VHS domains are found in several proteins that are thought to be key in trafficking events. The VHS domain could facilitate membrane association [START_REF] Wang | The Emerging Role of VHS Domain-Containing Tom1, Tom1L1 and Tom1L2 in Membrane Trafficking[END_REF]. Although the VHS domain structure has been solved at 1.5 Å for TOM1 (1ELK; [START_REF] Misra | Structure of the VHS Domain of Human Tom1 (Target of Myb 1): Insights into Interactions with Proteins and Membranes[END_REF], its exact function is not clear despite the fact that it mediates key interaction(s) in trafficking events. As it does not participate to Myo6 binding, we thus excluded this domain from our study. For cell-based assays, we opted for TOM1 299-end as its interaction with Myo6 was reported in [START_REF] Tumbarello | Autophagy Receptors Link Myosin VI to Autophagosomes to Mediate Tom1-Dependent Autophagosome Maturation and Fusion with the Lysosome[END_REF]. For biophysical and biochemical assays, we first generated a serie of TOM1

C-terminus truncations to confirm their stability and ability to bind Myo6 in vitro (Table 11). I characterized the affinity for Myo6 of all these constructs using microscale thermophoresis and kept Tom1207-492 for our pull-down assays as this construct exhibits the highest affinity for Myo6 and colors nicely in coomasie blue. During the course of my PhD, a crystal structure of the Myo6 CBD c bound to TOM1 revealed that the Myo6 binding motif (MBM) of TOM1 forms a single helix between residues 436 and 461 that able to interact with Myo6 in a 1:1 complex with a KD of ~630 nM (determined through ITC) [START_REF] Hu | Structure of Myosin VI/Tom1 Complex Reveals a Cargo Recognition Mode of Myosin VI for Tethering[END_REF]. For ATPase assays we thus switched for this well characterized TOM1 fragment instead ( YFP TOM1436-461).

CBDc partner: Dab2 Dab2 contains a phosphotyrosine-binding (PTB) domain at its N-terminus that mediates binding to PtdIns(4,5)P( 2) and a proline rich (PRD) C-terminus. As opposed to the p96 isoform, the p67 isoform lacks residues 230-447 that mediates interaction with clathrin (clathrin box) and with αadaptin (DPF motif) in clathrin-coated pits. Both isoforms can interact with Myo6 through the Myo6 interacting region 675-713 (MIR) (Figure 54). For our study, we used the Dab2 650-end construct, which was used in [START_REF] Fili | Competition between Two High-and Low-Affinity Protein-Binding Sites in Myosin VI Controls Its Cellular Function[END_REF] and was a kind gift of the Toseland lab. In our hand, this construct exhibits submicromolar affinity in the same range as the TOM1 construct (Table 12).

Adaptor

Introduction

Myosin motor proteins generate force and/or movement from ATP hydrolysis when associated with actin filaments. Conformational changes in the motor as it progresses from ATP hydrolysis to release of inorganic phosphate and ADP on actin are amplified into large movements via a calmodulin (CaM) or light chain binding region referred to as the "Lever arm" (Fig. 1A). To control the functions of myosin motors in cells, the ATPase activity of the motor and its ability to interact with actin must be regulated both spatially and temporally. Thirteen different classes of myosin motors serve diverse cellular functions in mammalian cells 1 . The regulation of their motor activity is however poorly characterized. A general theme for the control of motor activity is the formation of intra-molecular interactions involving the C-terminal Tail region and the Motor domain of these motors. In the active form of the motor, the Tail region interacts with itself or cellular partners. The best understood is the case of the dimeric myosin II (Myo2) class 2 and myosin V (Myo5) class 3 . In cardiac muscle, impairment in the stabilization of the myosin off-state leads to severe cardiomyopathies 4 . Whether lack of regulation of unconventional myosins can also lead to pathology has not been demonstrated.

Perhaps the most divergent form of regulation is emerging for Class VI (Myo6), VIIa (Myo7a), and X (Myo10) myosins, which all contain regions of extended stable single alpha helices (SAH). Indeed, while they are back-folded monomers in their inactive form 5-8 , these motors can self-associate to form active dimers upon activation 9-12 . How back-folding is stabilized is unknown and the nature of the dimerization following unfolding has only been elucidated for Myo10 10,12 , which forms an anti-parallel coiled-coil immediately following the SAH. The SAH thus extends the Lever arm in the case of Myo10 9,13 . Whether this is also the case for the dimeric Myo6 is debated and requires elucidation of its dimerization region 12,14-18 . The manner in which the motor is dimerized and the composition of its Lever arm greatly influence its function. A distinctive dimerization region in Myo10 allows the dimer to easily reach out for neighboring actin tracks and participate in filopodia formation 13 , unlike the vesicle transporter, Myo5, that makes multiple steps on a single actin track.

As a minus-end directed actin motor, Myo6 performs unique cellular roles (reviewed in 19 ), including endocytic vesicle trafficking and maturation, stereocilia maintenance 20 and melanosome maturation 21 , among many others. For these cellular functions, Myo6 must associate with different binding partners, such as Dab2, GIPC1 and TOM1 in distinct endosomal compartments 22-24 and NDP52 in autophagy and in RNA Polymerase II transcription 25 . Initially characterized as a deafness gene 20 , Myo6 is also overexpressed in aggressive cancers 26,27 and its depletion reduces cell migration and proliferation 26,27 .

Full-length Myo6 (FLMyo6) was characterized as a back-folded monomer in vitro 7,8 , which was confirmed to exist in cells by FLIM (Fluorescence lifetime imaging microscopy) 25 . TOM1 and Dab2 bind Myo6 through its WWY motif on the C-terminus part of its cargo-binding domain (CBD c ; Fig. 1A) while GIPC1 and NDP52 bind the RRL motif on the CBD n (Fig. 1A). FRET studies showing that these partners can unfold constructs lacking the Myo6 Motor domain (MD) led to the proposition that all partners could activate Myo6 upon binding 25,28,29 . However, whether the WWY and RRL motifs are both accessible in the FLMyo6 back-folded state is unknown. Detailed studies of Myo6 recruitment are required to investigate the role of partners in the spatio-temporal regulation of its cellular activity.

The configuration of the Myo6 active state, the nature of its Lever arm and its oligomeric state can make critical differences in the way the force produced by the motor is used 17,30,31 . In fact, Myo6 is well adapted to transport as well as to anchor, depending on the load it is working against, according to single molecule assays 32 . Although the capacity of partners to favor either monomeric, dimeric or oligomeric assemblies has been described 14,25,28,29,31,33-35 , the active configuration required to perform the different cellular roles of Myo6 is unknown. In vitro studies have identified a proximal dimerization region 12,18 , but its role for the cellular function of Myo6 is not established, nor is the structure of this region. Furthermore, it is not known whether the dimerization occurs following partner binding, and whether all partners lead to the same motor configuration, which ultimately will determine the nature of the effective Lever arm and mechanical performance of the motor.

A detailed description of the Myo6 off-state, a structural characterization of the proximal dimerization region, and the role of partners in Myo6 regulation are all essential to understand how Myo6 function is regulated in cells. Here we used structural and functional assays to thoroughly investigate these properties of Myo6. We demonstrate that not all partners can relieve Myo6 autoinhibition since not all binding sites are accessible, and importantly we solved the structure of the proximal dimerization domain and demonstrate its validity.

Results

ADP.Pi bound to the motor strongly stabilizes the off-state conformation of Myo6

Previous biophysical characterizations of the Myo6 back-folded state identified contacts between the Myo6 Lever arm and CBD (Fig. 1A) 25,36 . However, a possible role of the Motor domain in back-folding remains to be clarified. Size Exclusion Chromatography coupled with Multi-Angle Light Scattering (SEC-MALS) and SEC coupled with Small-Angle X-ray Scattering (SEC-SAXS) experiments (Sup Fig. 1A-C; Fig. 1B) indicate that FLMyo6 adopts a compact conformation in the presence of ADP.Pi analogs (Radius of gyration (Rg) = 49.23 ± 0.92 Å) (Fig. 1B-C; Sup Fig. 1C) even at high salt concentration (~425 mM NaCl) (Sup Fig. 1A-B). In contrast, when no nucleotide is present (nucleotide-free (NF) condition), FLMyo6 shifts from a compact to an elongated conformation in a salt concentrationdependent manner (at high salt, Rg = 84.18 ± 4.33 Å, elution 1 mL earlier from SEC-MALS) (Fig. 1B; Sup Fig. 1A-C). Overall, high salt dependency of FLMyo6 opening, combined with the lack of salt dependency in presence of a nucleotide, suggests that the Lever arm and the Tail are held together via electrostatic interactions, while the interactions that keep the Tail back-folded on the Head require the Motor domain to be in a nucleotide-bound state of its cycle (Fig. 1D). At very low salt (10 mM KCl) and in the presence of actin, FLMyo6 consumes ATP ~10 fold slower than the Tail-less construct MD Ins2 (Fig. 1A; Sup Fig. 1D), indicating that the back-folded state is auto-inhibited.

3D reconstruction of the Myo6 off-state

To further characterize the Myo6 off-state, negative staining electron microscopy (EM) of FLMyo6 in ADP.VO4 (ADP.Pi analog) resulted in heterogeneous 2D classes, likely due to the intrinsic flexibility of the protein particles. Previous FLIM demonstrated that fusion of the N-and C-termini to fluorescent proteins is compatible with Myo6 back-folding 25 . Thus, we fused the N-and C-termini of Myo6 to two covalent bonding subdomains of Streptococcus pneumoniae pilus adhesin RrgA: Jo and In 37 (the Jo-Myo6-In construct) to attempt to limit the inherent flexibility of the back-folded monomer. (Bottom) Alignment of Myo6 CBD c domain (aa 1143 to 1262 in Q9UM54-2) from different species. Strictly conserved and similar residues are shown in blue and red, respectively. Stars: residues implicated in binding to the Myo6 Head (Table 1

). (H) CBD c (brown) added to the model pictured in (F) (see Methods).

To show that the fusion does not disrupt the Myo6 back-folding, we confirmed that the Jo-Myo6-In heavy chain could bind to two CaM using SDS-PAGE and that the Jo-Myo6-In behaved as a compact folded protein, even in NF/high salt condition using SEC-MALS and SEC-SAXS (Sup Fig. 2A-C). An actin-activated stopped-flow experiment revealed a low Pi release rate for Jo-Myo6-In compared to FLMyo6 wild-type (WT), indicating that the conformational changes required to release Pi were greatly slowed (Sup Fig. 2D). Finally, negative staining EM images of Jo-Myo6-In in ADP.VO4, low salt were collected (Fig. 1E). The 3D reconstruction of the Myo6 off-state at ~17 Å resolution (Fig. 1F, Sup Movie 1) is consistent in shape and dimensions with SAXS data of FLMyo6 (Fig. 1C,F).

Structural model of the Myo6 off-state

The distinct EM density for the Jo-In fusion clearly defines the position of the N-and C-termini of FLMyo6 and demonstrates how it can lock the off-state. We used available Myo6 crystallographic structures to build a model inside the 3D reconstruction (Fig. 1F, see details in Methods). By defining the orientation of the Lever arm, the model revealed that the flexible joint allowing back-folding must be localized around aa 912-918 prior to the SAH. The ~10 nm long SAH ends up close to the Myo6 Nterminus and Converter, where the rest of the Tail can also participate in stabilizing interactions. Importantly, only the pre-powerstroke structure of the Motor domain (which traps ADP.Pi), not the Rigor (NF) structure leads to good model-to-map agreement (Fig. 1F, Sup Fig. 2E). We challenged this model by measuring affinities between Myo6 CBD 1035-end and Myo6 Head fragments (Table 1, Sup Fig. 3). The CBD binds to the Motor domain with low affinity, and the strongest interaction (KD ~150 nM) was measured for MD Ins2/IQ/3HB . Removal of the IQ-3HB region (MD Ins2 ) reduces the affinity by 2-fold. Last, the interaction between Myo6 CBD and MD Ins2/IQ/3HB drops from KD ~150 nM to ~750 nM upon nucleotide removal (Table 1, Sup Fig. 3). These data indicate an interaction of the CBD with both the Motor domain and the Lever arm and highlight the importance of the nucleotide state for optimal interaction.

To define the CBD region that interacts with the MD Ins2/IQ/3HB , we introduced four missense mutations (D1157V.Y1159D.D1161R.Q1163V: CBD c loop mutant) in a conserved and exposed loop of the CBD (Fig. 1G). These mutations abolished the ability of the CBD to bind to MD Ins2/IQ/3HB , suggesting a key role of the CBD c , and this specific loop, in the interaction (Table 1). When this information is used to dock the CBD c , the Myo6 C-terminus is oriented towards the surface, close to the N-terminus consistent with our Jo-Myo6-In model (Fig. 1H, Methods). 1A). Microscale thermophoresis profiles are presented in Sup Fig. 3.

Head

Auto-inhibition of Myo6 and hearing loss

The back-folded model predicts that a sharp kink occurs at the junction between the 3HB and the SAH (Fig. 1F). The N-terminus region of the SAH (aa 922-935) is thus positioned alongside the 3HB and could participate in the stabilization of the Myo6 off-state via apolar residues found in its atypical sequence (Fig. 2A). The importance of the sequence following the 3HB for back-folding was characterized using the previously published Myo6 (SAHmimic) mutant 17 , in which all apolar residues in the SAH were replaced by charged residues to match the i, i+4 alternance of a "perfect SAH sequence" (Fig. 2A). SEC-SAXS and SEC-MALS experiments indicated that FLMyo6 (SAHmimic) adopts an elongated conformation, even upon addition of an ADP.Pi analog, confirming the importance of the residues 922-935 for stabilization of the Myo6 off-state (Fig. 2B-C and Sup Fig. 4A).

Interestingly, a missense mutation present in this region of the SAH (L926Q) leads to deafness in humans 38 . Positioned away from the Motor domain or from Tail regions involved in recruitment (Fig. 2A), the effect of the mutation on Myo6 function had remained elusive. SEC-MALS (Sup Fig. 4A) and SEC-SAXS experiments (Fig. 2B-C) indicate that the L926Q mutation destabilizes the back-folded state. Both FLMyo6 (L926Q) and FLMyo6 (SAHmimic) mutants display higher ATPase rates (2.86 ± 0.12 and 4.83 ± 0.11 s -1 .Head -1 respectively) than the wild-type (0.65 ± 0.08 s -1 .Head -1 ) (Fig. 2D), which confirms the destabilization of the off-state.

We then investigated the impact of back-folding misregulation in the human pigmented melanoma cell line (MNT-1). Myo6 localizes to dot-like subdomains on the surface of pigmented melanosomes to promote membrane constriction and fission for the release of tubular carriers 21 . MNT-1 cells were transiently co-transfected with plasmids encoding (1) fluorescent components associated with pigmented melanosome iRFP VAMP7 21,39 and mCherry MST 40 , and with (2) either FLMyo6 WT, SAHmimic, L926Q or Jo-Myo6-In, all fused to GFP. All GFP Myo6 constructs localize as dots on melanosomes (Sup Fig. 4C), although at distinct levels (Sup Fig. 4B). The co-distribution of GFP FLMyo6 (SAHmimic) or GFP FLMyo6 (L926Q) with melanosomal components was greater than that of the GFP FLMyo6 (WT) (~1.2 fold increase, p ≤ 0.001, Sup Fig. 4B). However, the co-distribution of GFP Jo-Myo6-In with melanosomes was reduced ~3-fold compared to GFP FLMyo6 (Sup Fig. 4B) and the associated cytosolic and diffuse fluorescent signal was more readily observed (Sup Fig. 4C). Collectively, these data indicate that Myo6 auto-inhibition drastically reduces endogenous recruitment to melanosomes while impairment of Myo6 back-folding can result in over-recruitment. These results highlight the importance of the 3HB-SAH region for Myo6 auto-inhibition since the deafness L926Q mutation is sufficient for over-recruitment of the motor. Thus, destabilization of the off-state can lead to pathology.

Differential binding and activation of FLMyo6 by distinct cellular partners

We next aimed at distinguishing whether partners can bind to FLMyo6 in the back-folded state and if binding depends on the specific binding site. Partners interacting either with the RRL motif (GIPC1 34 ) or the WWY motif (TOM1 35 and Dab2 14 ) (Fig. 1A) were examined as our model suggests that in the FL off-state, the WWY motif of the CBD c is buried and unavailable for binding (Fig. 3A-B). We first looked at the ability of His GIPC1 and His TOM1 to bind FLMyo6 using an anti-His pull-down assay on purified proteins, in conditions promoting either Myo6 back-folding (addition of ADP.VO4) or opening (NF, use of the SAHmimic mutant, or addition of Ca 2+ as previously proposed 33,36 ) (Fig. 3D, Sup Fig. 5). Both TOM1 and GIPC1 were able to retain Myo6 in conditions that favor Myo6 opening. In contrast, upon ADP.VO4 addition, the interaction of Myo6 with GIPC1 is maintained, but the interaction with TOM1 is weakened, suggesting that binding of TOM1 requires Myo6 opening. (A) EM density for the Jo-Myo6-In (grey mesh) obtained by negative staining, as in Fig. 1H and Sup Movie 1. The WWY motif (red spheres) of CBD c is buried. The CBD n fragment (beige) (PDB: 5V6E, 34 ) is positioned in the remaining, uninterpreted part of the density so that the RRL motif (red spheres) on CBD n and the I1072 (blue sphere) that has been proposed to mediate interaction between ubiquitin and Myo6 41 are both exposed. Note that no experimental model exists for 36 missing residues between the CBD n and CBD c (dashed lines). (B) Fitting of CBD c -TOM1 structure (PDB: 6J56 35 ) with CBD c (brown) in the model presented in Fig. 1H, TOM1 (yellow) needs to bind in a poorly exposed area and would clash with SAH (green) and CaM (lilac). (C) Fitting of CBD n (beige)-GIPC1 (light blue) structure (PDB: 5V6E 34 ) as for CBD n alone. GIPC1 binding seems compatible with the back-folded conformation of Myo6 on a highly exposed binding site. (D) Anti-His pull-down assays. His-tagged Myo6 partners TOM1 and GIPC1 against FLMyo6 (WT) or (SAHmimic) revealed using SYPRO 42 (Input and last wash pictured in Sup Fig. 5). Crosses: quantification of retained Myo6 (Image-Lab software, Bio-Rad) followed by stoichiometric normalization based on partner concentration (n=4). + means less than 10% Myo6 retained; +++ means more than 20% Myo6 retained. (E) ATPase rates of FLMyo6 (WT) and FLMyo6 (SAHmimic) with 40 µM F-actin and increasing concentrations of GIPC1,TOM1 or Dab2 (n=6). Purple line: ATPase rate of MD Ins2 at 40 µM actin (n=6) for reference.

Next, we assessed the ability of GIPC1, Dab2 and TOM1 to stimulate the ATPase activity of FLMyo6 (Fig. 3E), and found that GIPC1 increases the Myo6 ATPase rate in a concentration-dependent manner, while addition of Dab2 or TOM1 has little impact. Note that partner affinities for Myo6 YFP CBD are all in the submicromolar range (i.e., sufficient to ensure binding in our ATPase assays) (Sup Fig. 6). Lack of activation by TOM1 and Dab2 must thus be due to inaccessibility of the WWY motif in the backfolded FLMyo6. Interestingly, TOM1 and Dab2 increase the ATPase rate of the FLMyo6 (SAHmimic) mutant, as does GIPC1, which indicates that all partners can bind to and stabilize the unfolded state (Fig. 3E).

In this context, we postulate that the RRL motif required for GIPC1 binding to CBD n (Fig. 1A) must be exposed on the surface of the back-folded Myo6, as opposed to the WWY motif. The CBD n fragment (PDB: 5V6E 34 ) was thus positioned in the unexplained density lying in continuity to CBD c in our Jo-Myo6-In 3D reconstruction (Fig. 3A-C, Sup Movie 1).

Collectively, these results demonstrate that not all partners can induce activation of Myo6. Partners Dab2 and TOM1 require another factor promoting Myo6 opening prior to their binding. In contrast, GIPC1 can directly activate FLMyo6, consistent with a previous study 33 .

Assessing the specific recruitment of Myo6 to native organelles by distinct partners

If some partners can relieve Myo6 from auto-inhibition, we reasoned that artificial targeting of these partners to specific cellular membranes would lead to massive recruitment of Myo6. We thus decided to artificially drive GIPC1, TOM1 and Dab2 to melanosome membranes by fusing them to the melanosome-targeting tag (MST) 40 (Sup Fig. 7) and verifying their ability to recruit either open (SAHmimic and L926Q) or locked (Jo-Myo6-In) Myo6. To do so with an optimized signal to noise measurement, we introduced the point mutation I1072A in our Myo6 constructs since it drastically reduces endogenous recruitment of Myo6 to the melanosomes ( GFP FLMyo6 (I1072A), 3.7-fold reduction (p>0.0001) compared to GFP FLMyo6; Fig. 4A; Sup Fig. 8A-B, 9A), while it is not part of the interface with either GIPC1 (Sup Fig. 6), TOM1 or Dab2. Hence, the I1072A mutation provides an easy way to reduce endogenous recruitment to melanosomes and offers a powerful tool to test the ability of distinct exogenous partners to recruit Myo6.

We transiently transfected MNT-1 cells with plasmids encoding for mCherry MST-GIPC1, mCherry MST-TOM1, or mCherry MST-Dab2 for melanosome targeting. Co-transfection with plasmid encoding for GFP Myo6 (I1072A), GFP Jo-Myo6-In (I1072A), GFP FLMyo6 (SAHmimic.I1072A) or GFP FLMyo6 (L926Q.I1072A) provided a quantitative way to compare the ability of these partners to recruit Myo6 to specific organelles such as the melanosomes in cells (see Methods).

Expression of mCherry MST-GIPC1 resulted in ~90% of Myo6-positive melanosomes for all the Myo6 constructs tested (Fig. 4B,E; Sup Fig. 9B), indicating that exogenous GIPC1 can recruit Myo6 to melanosomes independently of Myo6 being open or closed. In contrast, expression of mCherry MST-TOM1 or mCherry MST-Dab2 did not significantly increase the amount of GFP Jo-Myo6-In (I1072A) positive melanosomes (p=0.5005 and p=0.344 respectively). This confirms the ineffectiveness of WWY partners in recruiting back-folded FLMyo6. Yet, their expression results in a 1.3/1.4-fold increase of melanosomes containing active Myo6 mutants GFP FLMyo6 (SAHmimic.I1072A) and GFP FLMyo6 (L926Q.I1072A) (p=0.003 or lower) (Fig. 4C-E; Sup Fig. 9C-D).

Figure 4 -GIPC1 recruits Myo6 to melanosomes independently of Myo6 closure; Dab2 and TOM1 can only recruit Myo6 after the motor has been primed open. (A) Representative fixed MNT-1 cells co-expressing different GFP Myo6 (I1072A), mCherry MST and iRFP VAMP7 constructs. (B) Representative fixed MNT-1 cells co-expressing different GFP Myo6 (I1072A) constructs with mCherry MST-GIPC1 and iRFP VAMP7. (C) Representative fixed MNT-1 cells co-expressing different GFP Myo6 (I1072A) constructs with mCherry MST-TOM1 and iRFP VAMP7. (D) Representative fixed MNT-1 cells co-expressing different GFP Myo6 (I1072A) constructs with mCherry MST-Dab2 and iRFP VAMP7.

(A-D) Green: Myo6 GFP; Cyan: irRFP VAMP7; Magenta: mCherry MST partner. From left to right: entire cell, 3 channels merged; 8x zoom on boxed region: GFP Myo6 / mCherry MST-partners merged, then individual channels. Scale bars: 10µm. Arrowheads: recruitment of Myo6 on melanosomes. (E) Myo6-positive melanosomes quantification of different GFP Myo6 mutants when different mcherry MST tagged partners are expressed (n=3, total cell number~30). Myo6-positive melanosomes are expressed in percentage and normalized to the total number of VAMP7-positive melanosomes. Cells were fixed 48h posttransfection then imaged and processed for quantification. Data are presented as the mean ± SEM. Significant stars: ***, P < 0.001; **, P < 0.01; *, P < 0.05, n.s., not significant (unpaired t test with Welch's correction), for each GFP Myo6 construct, significance of experiments with partners compared to the control without partner (in black on the graph).

Interestingly, I1072A moderately affects the recruitment of Myo6 mutants impaired in autoinhibition. Compared to GFP FLMyo6 (SAHmimic) and GFP FLMyo6 (L926Q), we observe reductions of 1.4 and 1.7-fold in the co-distribution with melanosome components for GFP FLMyo6 (SAHmimic.I1072A) and GFP FLMyo6 (L926Q.I1072A), respectively (Fig. 4A,4E; Sup Fig. 4B-C), which are interestingly similar to the 1.6-fold reduction in recruitment observed for the CBD alone carrying the mutation I1072A (Sup Fig. 8C-D). We thus conclude that the I1072A mutation must reduce the affinity of the CBD for partner(s) responsible for Myo6 endogenous recruitment to melanosomes. In addition, the drastic reduction in GFP FLMyo6 (I1072A) recruitment evidences the role of endogenous partners to promote Myo6 unfolding and indicates the major role of the I1072 residue in this process.

These results illustrate the importance of the recognition of the inactive state, and the distinct ways signaling factors can trigger association or activation of the back-folded state in a compartment for spatial and timely control of motor activity.

A hinge that dimerizes

While we have demonstrated the key role of the sharp kink (hinge) at the 3HB/SAH junction for Myo6 auto-inhibition (Fig. 2; Sup Fig. 4), previous evidence by single molecule motility assays 12,17 already suggested that this region is key for Myo6 proximal dimerization. Since Myo6 proximal dimerization might be critical for a number of functional properties, we wanted to elucidate the structure of the dimerization region. SEC-MALS with six fragments derived from the 3HB/SAH junction (Sup Fig. 10A, C-E) indicated that a rather conserved region (aa 875-940) can self-dimerize with KD App of ~19 µM obtained from titration (Sup Fig. 10A,C). This minimal region corresponds to the last half of the 3HB (i.e., the 2 nd and 3 rd helix) and the first part of the SAH (Sup Fig. 10A-B). In contrast, no dimerization was observed when peptides included the whole 3HB domain, even when peak concentration of 30 μM was reached for the 834-955 peptide (Sup Fig. 10A,D). This data is consistent with previous findings indicating that proximal dimerization requires unfolding of the 3HB ( 17 , Sup Fig. 11A).

Crystals of the 875-940 peptide diffracted to 2.1 Å resolution (Fig. 5A; Sup Table 1). Clear electron density for all residues from 876 to 937 indicates that they form an extended helix that dimerizes in an anti-parallel manner (Fig. 5B; Sup Fig. 12). This anti-parallel dimerization is stabilized by multiple apolar contacts involving 13 residues from each helix, and six polar contacts involving R892 with D900, and T888 with S906 (via a water molecule) (Fig. 5A; Sup Fig. 10B).

mM TCEP. WT elutes as dimers (32 µM at the peak) and T888D.R892E.V903D mutant elutes as monomers (43 µM at the peak). Measured molecular weight is heterogeneous due to the small size of the peptides through elution (low light scattering signal), but at the peaks it coincides with the expected masses. (E) Model of active FLMyo6 dimer (see Methods). (F)

ATPase rates of FLMyo6 (WT) (green), FLMyo6 (T888D.Q892E.V903D) (grey), FLMyo6 (SAHmimic) (blue) and FLMyo6 (L926Q) (yellow) at 40 µM F-actin and increasing concentrations of GIPC1 (from 0 to 2.5 µM) (n=6). ATPase rates of MD Ins2 and zippered HMM dimer 12,44 with no partner added (n=6) are plotted as purple and red thick lines (respectively) as references for monomeric and dimeric Myo6. (G) Fluorescence intensity of internalized transferrin was measured for each of the conditions after treatment with genistein (minimum 58 cells analyzed per condition). Expression of FLMyo6 (WT) construct restored transferrin uptake to wild-type levels, while the FLMyo6 (T888D.R892E.V903D), fails to rescue the defect. Data shown as violin plot (generated with BoxPlotR 45 ) (p < 0.001, ANOVA; Tukey post-hoc comparisons: *** p < 0.001 vs WT, $ p < 0.05 vs Myo6 KO + FLMyo6 (WT)).

At the center of the interface, the structure highlights how residues T888, R892 and V903 contribute to the dimerization (Fig. 5A; Sup Fig. 10B). Three mutations (T888D.R892E.V903D) were introduced into the 875-940 peptide to assess their ability to disrupt proximal dimerization. Importantly, these residues were chosen on the surface of the 3HB so that the mutations would not disturb the bundle (Fig. 5C). (Note that residue 892 can be a Gln or an Arg depending on the species (Sup Fig. 10B) but both are compatible with the formation of the dimer). SEC-MALS confirms that the T888D.R892E.V903D mutant stays monomeric even up to 43 µM (peak concentration in the SEC-MALS experiment) while the WT counterpart is dimeric in similar conditions (Fig. 5D). In addition, motility assays indicated that no movement could be detected when the triple mutant construct is used in single molecule assays, which contrast to robust motility observed for the FLMyo6 (WT) (run length = 0.74 ± 0.26 μm (N=82)).

Finally, a model of the active dimeric configuration of Myo6 bound to F-actin including this crystal structure was built (Fig. 5E, see Methods). The inter-head distance is indeed compatible with the large (~30 nm) stepping previously reported when Myo6 walks processively 12,44 . Taken together, our results strongly support that proximal dimerization requires the formation of an extended antiparallel coiled coil, which can form following destabilization of the 3HB.

GIPC1 promotes unfolding of the Myo6 monomer and proximal dimerization

We further characterized this proximal dimerization region and investigated the ability of partners to promote proximal dimerization of Myo6 using an actin-based ATPase assay. Such dimerization indeed leads to "gating", i.e. coordination between the two Heads of the dimer that translates into slowing of ATP binding to the lead Head while the rear Head is attached 46 . This results in a 50% drop of ATPase rate per Head when Myo6 is dimerized compared to a monomer. The ATPase rate of zippered HMM (Myo6 truncated at R991 followed by a leucine zipper 47 to create a constitutive dimer) in which gating has been characterized 46 is indeed ~50% that of the monomeric MD Ins2 ATPase rate (Fig. 5F).

Indeed, upon addition of GIPC1, we found a ~50% reduction in the maximal ATPase activity per Head for FLMyo6 (WT) compared to MD Ins2 (Fig. 5F), consistent with GIPC1 promoting proximal dimerization of FLMyo6. In contrast, addition of GIPC1 to the FLMyo6 (SAHmimic) mutant is similar to that measured with MD Ins2 , consistent with a role of GIPC1 in fully freeing the Motor domain from Tail inhibition upon stabilizing an extended, monomeric conformation. Importantly, the FLMyo6 (T888D.Q892E.V903D) exhibits ~2-fold higher maximal ATPase rate upon GIPC1 addition, consistent with loss of gating (Fig. 5F). This additional evidence strongly validates the role of these residues in antiparallel proximal dimerization, and the role of this region in controlling motor mechanical properties. Furthermore, we demonstrate for the first time that proximal dimerization (involving 3HB unfolding) can be triggered upon GIPC1 binding.

The L926Q deafness mutation indirectly impairs proximal dimerization

Interestingly, when we used GIPC1 to activate the FLMyo6 (L926Q) construct (Fig. 5F), the maximal ATPase activity that we found was intermediate between monomeric and dimeric FLMyo6. Since our proximal dimerization structure indicates that the L926Q missense mutation does not impact the anti-parallel coiled-coil region itself (Sup Fig. 11B), and since we found that 3HB unfolding is essential for proximal dimerization, we hypothesized that L926Q impairs Myo6 dimerization by perturbing the unfolding of the 3HB. This was previously reported for the FLMyo6 (SAHmimic) mutant 17 .

To monitor 3HB unfolding, we introduced cysteines at two positions of the 3HB surface (T845 and A880), for tetramethylrhodamine (TMR) labelling (Fig. 5C, Sup Fig. 11A). As previously described 18 , a low TMR fluorescence ratio is found when 3HB is folded (fluorescence quenching due to stacking of the rhodamine rings; MD Ins2/IQ/3HB T845C, A880C). This value increases upon 3HB opening (Sup Fig. 11A), and a high TMR fluorescence ratio indicative of 3HB unfolding has been reported for the Myo6 zippered HMM T845C, A880C 18 . Introducing the L926Q mutation in this zippered construct led to an intermediate fluorescence intensity, indicating limited unfolding of the 3HB for the deafness mutant compared to control (Table 2). This suggests a role for the L926Q mutation in limiting the conformational changes of the 3HB required for dimerization, in addition to its effect in destabilizing the off-state.

Construct

Importance of proximal dimerization in cells

To further demonstrate that proximal dimerization of Myo6 occurs via the anti-parallel coiledcoil seen in our structure, we compared the ability of FLMyo6 (WT) and FLMyo6 (T888D.R892E.V903D) to rescue Myo6-mediated transferrin uptake 17 in HeLa cells whose Myo6 was knocked out for Myo6 by using CRISPR/Cas9 (Sup Fig. 13A). FLMyo6 (WT) and the FLMyo6 (T888D.R892E.V903D) were transiently expressed, and the transferrin internalized during a 10 min pulse was quantified. As summarized in Fig. 5G and Sup Fig. 13B, expression of the T888D.R892E.V903D mutant, unable to form the proximal dimer, profoundly decreases the rate of uptake of endocytic vesicles, providing evidence for the need of proximal dimerization to optimize Myo6 function during endocytosis. Furthermore, this also strengthens the evidence that proximal dimerization occurs via an anti-parallel coiled-coil as depicted in our crystal structure.

Discussion

Despite the significance of controlling where and when myosin motors generate forces and move cargoes in cells, careful investigation of how the function of myosin motors is regulated has only been performed for a few classes of myosin 2,3,48,49 , and most extensively for Myo2. The results of this study highlight the importance of regulated inhibition of the Myo6 motor until it reaches its target in a cell and it is activated. Myo6 must cross actin-rich regions in order to diffuse and reach its binding partners which selectively activate motor activity (Fig. 6A). If the motor was not blocked from interacting with and cycling on actin, Myo6 would bind to actin filaments throughout the cell, retarding diffusion to its target sites at the cell membrane. The fact that the L926Q mutant disrupts the folding and regulation of Myo6 (Fig. 2) and causes deafness in humans 38 attests to the critical need for the regulation of this class of myosin motors.

Our structural and functional studies provide a more precise model to account for the interactions stabilizing Myo6 back-folding (Fig. 1H, Table 1). Among the major differences compared to previous models 7,36 , we show that (1) ADP.Pi bound to the Motor domain is essential to lock Myo6 in its back-folded state (Fig. 1B, Table 1); (2) back-folding involves a specific loop of the CBD c (Table 1), which was previously predicted to be external to the folded complex by Alphafold 50,51 (Sup Fig. 14); and (3) the 3HB/SAH junction acts as a critical hinge to control the equilibrium between on/off states of the motor (Fig. 1G-H, Fig. 2). Earlier studies of the folded monomers 25,28,29,36 focused only on interactions within a full-length construct in the absence of nucleotide or within a Motor-less construct, and thus did not fully represent what is happening with the full-length Myo6 monomer saturated with nucleotide.

Intriguingly, a single amino acid change (L926Q) causes deafness 38 and is in fact sufficient to destabilize the back-folded monomer (Fig. 2). This SAH mutation flanks the hinge region that we identified as essential for the off-state of this motor. To further investigate the impact of Myo6 backfolding in myosin recruitment, we used the FLMyo6 (L926Q) and FLMyo6 (SAHmimic) mutants to probe their impact on Myo6 recruitment on melanosomes. What was observed (Sup Fig. 4B-C) was that both constructs lead to greater recruitment than the FLMyo6 (WT). This is not a gain of function, but rather a loss of regulation as, (1) the normal cellular control over the spatial and temporal recruitment of Myo6 has been lost, and (2) fluorescence quenching assays show that both SAHmimic 17 and L926Q (Table 2) mutations impair proximal dimerization and thus Myo6 function. Deafness due to the L926Q mutation in humans may therefore result from the inability of Myo6 monomers to reach their target sites in hair cells due to loss of folded regulation. 2+ , which can allow TOM1 to bind to Myo6. Such an activation cascade was previously proposed 36 . Once unfolded, Myo6 potentially acts as a monomer, as previously proposed 35 upon TOM1 binding; or it can dimerize 29 through proximal dimerization, as demonstrated in this study with GIPC1 binding; or it dimerizes through distal dimerization upon Dab2 binding 14 , which may lead to proximal dimerization.

We next examined the ability of some of the Myo6 binding partners that recognize different regions of the CBD to induce unfolding and recruitment of Myo6. Folding not only prevents cycling of the motor on actin until the cellular target has been reached, but as shown by our actin-activated ATPase (Fig. 3D), pull-down (Fig. 3E) and recruitment assays on melanosomes (Fig. 4), the folding can also prevent interaction with a subset of cellular partners until unfolding occurs, by either a different class of partners, or potentially by a spike in cellular Ca 2+ concentration 33,36 or PIP2 recognition 43,52 . We thus propose a model of the folded off-state of FLMyo6 in which the GIPC1 binding site is available for binding, while the TOM1/Dab2 site is masked (Fig. 3A-C). Interestingly, this demonstrates that not all partners are equivalent in their potential for binding the auto-inhibited form of the motor and to activate Myo6.

While TOM1 and Dab2 cannot trigger Myo6 initial unfolding, once bound they prevent the formation of the off-state due to their incompatibility with it, as previously proposed 25,28 . Depending on the nature of the partner and its distribution, binding will activate the Myo6 motor and could drive either proximal dimerization (Fig. 5F), distal dimerization 14,15 or maintain an activated, monomeric form 35 . Taken together, these results suggest unique roles for partners not only in Myo6 localization, but also in the control of Myo6 activation and function (Fig. 6B).

Once unfolded, binding to its cargo brings two unfolded Myo6 monomers into close apposition, favoring its dimerization 12,14,15,17,18,25 . The experiments summarized in Fig. 5 provide the previously unknown structure of the proximal dimerization region. We present both in vitro and cellular evidence in support of the structure. This structure reveals the dimerization region to be an anti-parallel coiled-coil, as for Myo10 9,11,13 . Mutations of three of the amino acids that stabilize this coiled-coil structure (T888D.Q892E.V903D) abolish dimer formation in in vitro assays, but with no impact on back-folding of the monomer (Fig. 5F). Furthermore, introduction of this Myo6 triple mutant into cells fails to rescue endocytosis (Fig. 5G), providing evidence for the need of proximal dimerization to optimize this cellular function of Myo6.

Myo7A, Myo10 and Myo6 exist as folded monomers in cells until they are activated and recruited by their partners. Formation of an antiparallel dimer may be the mode of dimerization for the three classes that appear to undergo this folded monomer to dimer transition. The structure of the active form of Myo6 has been a long-debated issue 13,15,17,11 , which is resolved by our structure for the proximal dimerization region. As shown in Fig. 5E, Myo6 is unique in that its antiparallel coiled-coil and Lever arm in the dimer are derived from unfolding of a 3HB, with contribution of the SAH. The resulting Lever arm formed by the CaM binding region and the unfolded 3HB (half of which contributes to the coiled-coil) is sufficiently long to account for the ability of Myo6 to take steps that average ~30nm on actin 12,44 . These findings provide a structural framework that can be applied to understanding how motors are recruited and how partners influence motor functions in cells.

Methods

Constructs cloning, expression and purification

Details on protein cloning and purification can be found in Supplementary Methods.

SEC-SAXS

SAXS data were collected on the SWING beamline at synchrotron SOLEIL (France) 53 in HPLC mode at λ = 1.0332150494700432 Å using a Dectris EIGER-4M detector at a 2 m distance. Protein samples were injected at 0.1 mL/min on Superdex 3.2/300 column pre-equilibrated in 20 mM HEPES; 200 mM NaCl; 2 mM MgCl2; 1 mM NaADP; 1 mM AlF4, 0.1 mM EGTA, 1 mM DTT; or 20 mM HEPES; 400 mM NaCl; 2 mM MgCl2, 0.1 mM EGTA, 1 mM DTT; pH 7.5 prior to data acquisition in the SAXS capillary cell. 150 frames of buffer scattering (before the void volume), then 719 frames of elution sample scattering were collected. Exposure time was 1990 ms/frame. Images were processed using the Foxtrot 3.5.10-3979 53 developed at the SOLEIL synchrotron: buffer averaging, buffer substraction from the corresponding frames at the elution peak, and sample averaging were performed automatically. Further data analysis to obtain Rg, I(0), Dmax and molecular weight estimation was done with PRIMUS from ATSAS suite 54 . Dimensionless Kratky plot ((q.Rg) 2 .I(q)/I(0) versus q.Rg) was generated using Microsoft Excel based on I(0) and Rg values found with PRIMUS. 20 envelopes were generated independently with GASBOR 55 and averaged with DAMAVER 56 .

SEC-MALS

For SEC-MALS analysis, samples were injected in a Superdex 200 10/300 Increase (GE Life Sciences) previously equilibrated in the corresponding buffer, and developed at 0.5 ml/min. Data collection was performed every 0.5 sec with a Treos static light scattering detector, and a t-Rex refractometer (both from Wyatt Technologies). Concentration and molecular mass of each data point were calculated with the software Astra 6. 1.7 (Wyatt Technologies).

Microscale Thermophoresis measurements between Myo6 Tail and Head

Microscale thermophoresis experiments were performed on a Monolith NT.115 system (NanoTemper Technologies) using YFP-fusion proteins. The non-fluorescent protein was first treated with 0.5 mM EGTA (+/-2 mM MgADP; 2 mM Na3VO4 for some experiments); then dialysed against 20 mM Hepes pH 7.5, 50 mM NaCl, 2.5 mM MgCl2, 1 mM TCEP and 0.05% (v/v) Tween-20 (+/-2 mM MgADP; 2 mM Na3VO4 for some experiments). Two-fold dilution series (16 in total) of the non-fluorescent protein (« Head » sample) were performed at 25° in the same buffer. The YFP-fused partner was kept at a constant concentration of 100 nM. The samples were loaded into premium capillaries (Nanotemper Technologies) and heated for 30 sec at 60% laser power. All experimental points were measured twice. The affinity was quantified by analyzing the change in thermophoresis as a function of the concentration of the titrated protein using the NTAnalysis software provided by the manufacturer.

Microscale Thermophoresis measurements with Myo6 partners

Two-fold dilution series (16 in total) of the non-fluorescent protein (Myo6 partner) were performed at 25°C in the MST buffer: 20 mM Bis-Tris pH 6.5, 100 mM KCl, 1 mM DTT and 0.05% (v/v) Tween 20. The YFP-fused partner was kept at a constant concentration of 100 nM. Microscale thermophoresis experiments were then performed in similar conditions as above in 20 mM Bis-Tris pH 6.5, 100 mM KCl, 1 mM DTT and 0.05% (v/v) Tween 20. Capillaries were heated for 30 sec at 50% laser power.

ATPase assays.

ATPase assays were performed as previously described 57 . ATPase rate determined from 2-3 preps with 2-3 independent assays per prep. F-Actin was used at 40 µM and Myo6 at 150 nM with 2.5 µM additional CaM in all our experiments. The experiments were all carried out in 10 mM MOPS pH 7.0; 10 mM KCl; 1 mM DTT; 1 mM MgCl2; 1 mM EGTA.

Pi release experiments

Actin-activated phosphate release from Myo6 constructs was monitored using the 7-diethylamino-3-((((2-maleimidyl)ethyl)amino)carbonyl) coumarin labeled phosphate binding protein (MDCC-PBP) as in 57 . Experiments were conducted under single turnover conditions where the substrate was ratelimiting. Actin-stimulated phosphate release was measured in a double-mixing experiment where Myo6 was mixed with a substoichiometric concentration of ATP, aged for 30 seconds, and subsequently mixed with actin and MDCC-PBP (final reaction conditions: 0.5 μM Myo6, 0.25 μM ATP, 14 μM actin, 5 μM MDCC-PBP). Fluorescence was monitored by excitation at 425 nm, and emitted light was collected through a 455 nm long pass glass filter. Myosin solutions were clarified by centrifugation (550k x gmax, 10 minutes, 4°C) after thawing and buffer exchange.

Anti-His Pull-down assay

FLMyo6 SI (WT) or (SAHmimic) were used alone or mixed with partner GIPC1 (His-rTEV-GIPC1 255-end) or TOM1 (His-TOM1 207-492) in a ratio (1/1) (10µM) and 1 µM of extra Calmodulin was added in a total volume of 20 µL. The input was incubated with 40 µL of Ni 2+ beads from cOmplete column (Roche), which were previously equilibrated either in ADP.VO4 Buffer (10 mM HEPES pH 7.5; 100 mM NaCl; 5 mM NaN3; 1 mM MgCl2; 0.1 mM TCEP; 1 mM NaADP; 1 mM Na3VO4; 0.1 mM EGTA; 4 mM imidazole) or ADP.VO4-CaCl2 Buffer (10 mM HEPES pH 7.5; 100 mM NaCl; 5 mM NaN3, 1 mM MgCl2, 0.1 mM TCEP, 1 mM NaADP; 1 mM Na3VO4; 4 mM imidazole; 1 mM CaCl2) or NF Buffer [10mM HEPES pH 7.5; 100 mM NaCl; 5 mM NaN3; 1 mM MgCl2; 0.1 mM TCEP; 0.1 mM EGTA; 4 mM imidazole]. All steps were performed at 4°C. Beads were washed by centrifugation after 1 hour of gentle agitation. Bound proteins were eluted in 600 mM imidazole in the corresponding buffer.

Electron Microscopy

Purified Jo-Myo6-In at 50 µg/ml in 10 mM HEPES; 80 mM NaCl; 1 mM MgCl2; 0.1 mM TCEP; 0.1 mM ADP; 0.2 mM Na3VO4; 0.1 mM EGTA, pH 7.5 was transferred to Carbon Film 300 mesh copper grids (Electron Microscopy Sciences), then stained with 2% uranyl acetate. A total of 284 images were collected with a 200 kV Tecnai G2 microscope under low dose condition with a 4Kx4K F416 TVIPS camera at 0.213 nm/px and treated with the software CryoSPARC 58 . Following CTF determination, template picking was carried out using an initial set of 100 manually picked particles. The resulting 711,671 particles were submitted to a few rounds of 2D classification from which 93,293 particles were selected. These were used in the ab-initio reconstruction that produced the map at 17 Å resolution (FSC).

Model of the Myo6 off-state

We first positioned the Motor domain-Lever arm (residues 1-917) in the Jo-Myo6-In maps from SAXS and EM. Best model-to-map agreement was obtained with PDB 4ANJ 59 (Motor domain and insert-2/Ca 2+ -CaM with ADP.Pi analog bound, in PPS state). The Lever arm from PDB 3GN4 18 was then superimposed to PDB 4ANJ by using the insert-2/Ca 2+ -CaM region, present in both structures, as reference. In the negative staining reconstruction, Jo-In PDB 5MKC 37 was placed in the distinct density that corresponds to it, as expected, with the N-and C-termini pointing towards the center of the main density body occupied by Myo6. The structure of the C-terminal half of the CBD from PDB 3H8D 14 was placed according to structural and biochemical restrictions as follows: (1) the CBD c C-terminus must be near the N-terminus of the fusion protein In; (2) residues D1157, Y1159, D1161 and Q1163 are in contact with the MD Ins2 ; (3) there is still density to be filled close to the N-terminus of the CBD c , that can be filled by CBD n . (Note that this proposed position is opposite to that currently predicted by Alphafold 50,51 for uniprot entry : Q9UM54, due to lack of data for the intermolecular interactions when that model was built). At last, the NMR structure of the SAH domain (residues 919-998; PDB: 6OBI 60 ) was accommodated in the density. This density is narrow up to residue ~955 and then becomes much larger to account for the rest of the model, in which no distinct subdomain can be identified. Thus, our current model lacks the distal Tail (a compact domain of 3 nm in diameter 7 ) and CBD n , for which there seems to remain enough density to be fitted. Model and figure were prepared with Pymol 61 .

MNT-1 cell transfection

MNT-1 cells were cultured in DMEM supplemented with 20 % FBS, 10 % AIM-V medium, 1 % sodium pyruvate, 1 % nonessential amino acids, and 1 % penicillin-streptomycin. For plasmid transfection, 400 000 MNT-1 cells were transfected using nucleofection (NHEM kit, Lonza) on Amaxa device 2 (program T20) with 1.5 µg of iRFP VAMP7 plasmid; 1 µg of mCherry MST plasmid and 3 µg of GFP FLMyo6 plasmid. After transfection cells were seeded in fluorodish containing 1 mL RPMI medium, then 1 mL of complete MNT-1 medium supplemented by 10 % FBS was added 6 h post-transfection. Medium was changed 1-day post-transfection by complete medium then cells were fixed with 4 % PFA at 48 h posttransfection. Cells were stored in the dark at 4 °C in PBS medium until imaging. Fluorescence intensity of each mCherry MST construct was analyzed to ensure equivalent expression levels between the different partners (Sup Fig. 15).

Super resolution imaging and analysis

Samples were imaged in fluorodish using a 100x/1.4 NA oil immersion objective on an inverted Spinning disk confocal microscope (Inverted Eclipse Ti-E Nikon, Spinning disk CSU-X1, Yokogawa) equipped with a Photometrics sCMOS Prime 95B Camera (1200 x 1200 pixels). Z images series were acquired every 0.2 µm. Images were processed with a Live super Resolution module (Live-SR; Gataca systems) based on structured illumination with optical reassignment technique and online processing leading to a two-time resolution improvement 62 . For the figures, Z maximum projection and a substract background (50 pixels) were applied on SR images using the FIJI software. Analysis was done on raw SR images. Melanin pigments (black spots) were automatically detected in a defined region of interest (ROI) (here, cell outlines that were manually drawn) in BrightField images by creating a MIN-intensity z-projection and considering the lowest values, defined using the 'Find Maxima' function of Image J/Fiji and whose spatial coordinates were recorded. To quantify the percentage of melanosomes containing iRFP VAMP7 / mCherry MST / GFP Myo6 proteins at the membrane, additional ROIs centered around each individual detected pigment were generated whose size was defined (0.350 µm diameter). Then, for each detected brightfield spot, an additional automatic detection in the fluorescent channel(s) of interest was performed by creating a MAX intensity z-projection in the ROI around the pigments and considering the highest values. Detected pigments were considered positive for the marker of interest above a threshold (defined by Triangle's automatic thresholding method, calculated on the MAX intensity projection, or manual thresholding in the case of cells expressing the lowest GFP-Myo6), and the percentage of which was calculated. Pigments that were automatically detected very close to each other (within 4 pixels in XY and 2 pixels in Z) and that had overlapping ROIs were automatically removed and eliminated from the analysis to avoid data duplication. Moreover, automatically detected pigmented that were negative for iRFP VAMP7 and/or mCherry MST fluorescent signal were excluded from the analysis because not considered as pigmented melanosomes (positive for membrane-associated components). For each cell, a percentage of Myo6 positive melanosome was calculated and normalized to the total number pigmented melanosomes (co-positive for pigment and iRFP VAMP7 and/or mCherry MST).

Proximal dimer Crystallization, Data Collection, and Structure Determination

The Myo6 875-940 construct was crystallized by hanging drop vapor diffusion at 290 K by mixing 1 µL of 9.8 mg/mL protein solution with 1 µL of reservoir solution (27% PEG 4000, 10 mM MgCl2 and 0.2 M imidazole / malate, pH 6.0). Crystals grew spontaneously as rods 1 to 7 days after. After additional 3 weeks, they were cryo-cooled in liquid nitrogen in a solution containing 28% PEG 4000, 10 mM MgCl2, 0.2 M imidazole / malate, pH 6.0, and 27% ethylene glycol. One exploitable X-ray dataset was collected at the Proxima 1 beamline (Synchrotron Soleil, Gif-Sur-Yvette) and processed with Autoproc 63 . Diffraction limits after treatment with Staraniso 64 with cut-off of 1.2 I/sI were 2.566 Å in two directions, and 2.077 Å 64 in one direction. Initial structure factors were obtained by molecular replacement with Phaser 65 using a helix comprised of 30 serine residues as search model. Initial sequence attribution was obtained with Phenix AutoBuild 66 , followed by several cycles of iterative edition with Coot 67 and refinement with Buster 68 . Resolution was automatically cut by Buster to 2.2 Å based on model-map cross-correlation. The dimer is defined by one of the 2-fold symmetry axis, with crystal contacts between the N-terminus of one dimer and C-terminus of neighboring dimers (Sup Fig. 12B-C, Sup Movie 2). When the carbons are colored according to B-factors (Fig. 5A), the lowest values are found between residues 885 and 913, suggesting that the dimerization interface is comprised within those boundaries.

Single-molecule assays

Biotin labeled CaM was used to selectively exchange only one of the two myosin VI CaMs in the motor domains of the wild-type or the mutant proteins for the single molecule studies as described before 69 . Potential processive movements of FLMyo6, and FLMyo6 (T888D, R892E, V903D) were observed on 20 µl flowcells using Total Internal Fluorescence Microscope (TIRFM) as described 70 . First, 20 µl of Nethyl maleimide (NEM)-modified myosin (0.5 mg/ml) in Buffer A (25 mM imidazole, pH 7.4, 4 mM MgCl2, 1 mM EGTA, 300 mM KCl, 10 mM DTT) was introduced for 2 min to create the attachment sites for actin filaments. To remove the unbound NEM-modified myosin, the flow chamber was washed with buffer B (25 mM imidazole, pH 7.4, 4 mM MgCl2, 1 mM EGTA, 50 mM KCl, 10 mM DTT) containing 1 mg/ml BSA and incubated for 2 minutes. Then 100 nM actin filaments in buffer B were infused into the flowcell and incubated for 2 min. The flowcell was washed with buffer B and 150 nM of the full-length myosin monomers, FLMyo6 or FLMyo6(T888D, R892E, V903D), in buffer B were introduced into the flow cell without ATP. Under these conditions, the actin filaments were saturated by Myo6 monomers, which allowed the monomers to be in close proximity for dimerization, as previously described 12 . Then, Strepavidin Qdots (100 nM; emission at 655 nm) were infused into the flow cell. The Qdots bound specifically to the exchangeable biotinylated-CaM in the IQ domain of myosin VI. Finally, buffer B containing 2 mM ATP and 1 mg/ml BSA was infused into the flow cell. In this step, Myo6-monomers and unbound Qdots were removed from the flow cell while Myo6 dimers showed processive movement along actin filaments.

Model of the Myo6 proximal dimer

The cryoEM structure of Myo6 bound to actin (PDB: 6BNP 71 ) was used as basis for placing two Myo6 Motor domains in rigor and PPS states (grey) at the desired 31 nm distance (Fig. 5E). On each side, the N-terminus of the Lever arm bound to two light chains (pink, PDB: 3GN4 18 ) was aligned to the corresponding residues in the Converter. The crystallized dimerization domain (blue) was then placed with minimal distance from the two Lever arms, leaving a gap of 4.6 nm from each side. This gap needs to be filled by a stretch of 26 amino acid residues that would make 3.9 nm if in a theoretical helix, or up to 9.9 nm if fully extended. SAH (green) (PDB: 6OBI 60 ) was connected to the C-terminus of the dimerization region via a putative kink. Model and figure were prepared with Pymol 61 .

Bundle Unfolding Assay

As previously described 18 , cysteine residues were introduced to replace T845 and A880 in Myo6-917 and Myo6-991-GCN4 constructs with no reactive cysteines. Control constructs contained one reactive cysteine, T845C. One mg of each protein was labeled with a 10-fold molar excess of TMR 5iodoacetamide (5-TMRIA; Anaspec, San Jose, CA) per cysteine (from a stock concentration of 20 mM in dimethylformamide) at 4°C for 1-3 hr. Unbound rhodamine was removed by gel filtration and overnight dialysis. Absorption spectra were measured in a HP Diode Array Spectrophotometer, and fluorescence spectra were obtained in a PTI QM3 luminescence spectrofluorometer. The excitation and emission spectra were measured at 552 and 575 nm, respectively.

Generation of Myo6 null HeLa cells

The Myo6 gene of HeLa cells (ATCC CCL-2) was inactivated by CRISPR/Cas9 gene editing approach. Briefly, HeLa cells were transfected using a combination of three human Myo6 CRISPR plasmid variants (Santa Cruz Biotech SC-401815) -each driving expression of Cas9, GFP and one of the following human Myo6-specific 20-nucleotide gRNAs (5'-3': taatatcaaagttcgatata, acattctgattgcagtgaatc, ccaagtgtttcctgcagaag). Clones of transfected HeLa cells were selected on the basis of GFP fluorescence, and PCR of isolated DNA using primers flanking the targeted genomic sequences. Loss of Myo6 expression was confirmed by western blot.

Transferrin Endocytosis Assay

Normal and Myo6 null HeLa cells were grown in multi-well tissue culture plates on coverslips coated with rat collagen I (Corning). FLMyo6 (WT) or FLMyo6 (T888D.Q892E.V903D) were tagged with a Cterminal mApple for identification of expressing cells. Transfections were performed using the X-tremeGENE 9 DNA transfection reagent (Sigma-Aldrich) following the manufacturer's instructions. Cells were serum starved, but otherwise maintained in normal growth conditions -at 37˚C with 5% CO2, by incubation in serum-free Dulbecco's modified Eagle's medium for 2.5 h. Serum-free medium was supplemented with genistein (600 µM, Cayman Chemical Company) to inhibit caveolae-mediated uptake of transferrin following Myo6 depletion as previously reported 17 . During the final 10 min of serum starvation, Alexa fluor 488-conjugated transferrin (ThermoFisher) was added to the culture medium at 25 µg/ml. Following serum starvation, plates were placed on ice and washed twice with 10 mM HCl and 150 mM NaCl to remove cell surface-bound transferrin. The cells were fixed with ice-cold 4 % paraformaldehyde for 20 min and stained with rabbit anti-dsRed antibody (Takara Bio) and Alexa fluor 568-conjugated anti-rabbit secondary antibody to identify cells expressing mApple-tagged Myo6. Image acquisition was performed with a Leica Application Suite X software on Leica TSC-8 confocal system using a 40X oil immersion objective lens (n.a. = 1.3). Transferrin uptake was determined using ImageJ software: the total transferrin-conjugated fluorescence intensity from sum slice projections of individual cells was subsequently normalized by cell size. Comparative samples were stained, imaged, and processed simultaneously under identical conditions. Data were subjected to one-way analysis of variance with Tukey post-hoc comparison of individual groups to determine statistical significance.

(B) Dimensionless Kratky plot representation. Dotted lines ((qRg) 2 I(q)/I(0) = 1.104 and qRg = √3) intersection highlight the theoretical maximum of a globular protein. In the presence of ADP.AlF4, FLMyo6 (green) and Jo-Myo6-In (orange) spectra result in a bell-shape curve (with a shoulder in the case of Jo-In) with a maximum close to (√3:1.104), suggesting that both proteins are rather globular and well folded. The shoulder in the Jo-Myo6-In profile is typical of multi-domain proteins consistent with the addition of the Jo-In tag. (C) Normalized SEC-MALS profiles of Jo-Myo6-In in conditions that favor opening (red, NF) or closure (blue, ADPVO4). NF buffer: 20 mM Hepes; 300 mM NaCl; 2 mM CaCl2; 0.1 mM EDTA; 0.5 mM TCEP, pH 7.5. ADP.VO4 buffer: 20 mM Hepes; 80 mM NaCl; 2 mM MgCl2; 1 mM NaADP; 1 mM NaVO4; 0.1 mM EGTA; 0.5 mM TCEP, pH 7.5. (D) Myo6 Pi release rates and amplitudes from stopped-flow experiments. (E) Docking of nucleotide-free Myo6 MD Ins2/IQ (PDB: 2BKI 3 ) and Jo-In (PDB: 5MKC 4 ) in the negative staining map. The structure of the Motor domain devoid of nucleotide doesn't fit the map: (left) if the connection between the N-terminus of Myo6 and the C-terminus of Jo is respected, the Lever arm does not fit in the envelope; (right) if the Lever arm is positioned inside the 3D reconstruction, the distance between the Myo6 N-terminus and the fusion subdomain Jo is prohibitive.

Experimental conditions were similar to those described for Sup Fig 1 . Here, "High salt" means 800 mM NaCl; "low salt", 80 mM. (B) Quantification of Myo6-positive melanosomes for GFP FLMyo6 (L926Q), GFP FLMyo6 (SAHmimic), GFP Jo-Myo6-In, and GFP FLMyo6 (WT) (n=3, total cell number ~30). Significance: ***, P < 0.001 (unpaired t test with Welch's correction). (C) Representative fixed MNT-1 cells co-expressing GFP FLMyo6 (WT), GFP Jo-Myo6-In, GFP FLMyo6 (SAHmimic) or GFP FLMyo6 (L926Q) with mCherry MST and iRFP VAMP7. mCherry MST and iRFP VAMP7 are melanosome-associated components. MNT-1 cells were fixed 48h post-transfection, then imaged and processed for quantification. Green: GFP Myo6; Magenta: mCherry MST; Cyan:

Space group P6522

Cell constants a, b, c, α, β, γ Q1163V) mutant was generated through point mutations addition using reverse PCR on the YFP CBD (WT) construct.

Cloning partner constructs

In order to avoid partner (GIPC1, TOM1 and Dab2) degradation and auto-inhibition as previously reported 17,18 , we used truncations containing the published Myo6-binding domains 18-21 instead of FL constructs.

For microscale thermophoresis assays and his-pull-down assays, His GIPC1 construct was generated using cDNA full length mouse GIPC1 (UNIPROT Q9Z0G0). GIPC1 was incorporated in pProEX-HTb plasmid containing in N-terminus 6XHis-tag and a TEV cleavage sequence. Finally, GIPC1 was truncated in N-terminus at position corresponding to aa D255 through reverse PCR in order to keep only the GH2 domain which is sufficient for mediated interaction with Myo6 18 .

For ATPase assays, we used GIPC1 in fusion with the mNeonGreen tag: GIPC1 DNA sequence encoding for residues 238-end was incorporated in the pET28 plasmid containing in the N-terminus 6XHis-tag and a mNeonGreen tag using homemade Gibson Assembly mix 22 .

For microscale thermophoresis and his-pull-down assays, a non-fluorescent His TOM1 construct was generated using cDNA full length human TOM1 (UNIPROT O60784-1). TOM1 207-end was incorporated into pET14 in-frame with an N-terminus 6XHis-tag and a TEV cleavage sequence using homemade Gibson Assembly mix 22 .

For ATPase assays, we used the TOM1 436-461 peptide described as the minimal sequence required for TOM1 binding to Myo6 21 . DNA sequence coding for aa436-461 was incorporated in pET14 plasmid containing in N-terminus 6XHis-tag, yellow fluorescent protein (YFP) and a TEV cleavage sequence. Dab2 His-650-end (tDab2 17 ) was a kind gift of Christopher Toseland.

To identify the minimal sequence involved in proximal dimerization, several Myo6 truncations were generated from mouse Myo6 cDNA (UNIPROT: E9Q3L1). Constructs encoding for aa 875-940 and 875-955 were cloned with a N-terminal 6xHis-tag into pET14 plasmid. The construct encoding for aa 834-955 was cloned into pET14 with a N-terminal 6xHis-tag followed with a Thrombin cleavage site (coding for LVPRGSH). Constructs encoding for aa 880-940 and 888-940 were cloned with a C-terminal 6xHistag into pET14 by PCR and blunt-end ligation. The 912-end construct was cloned into pProEX-HTb with a N-terminal 6xHis-tag through several rounds of sub cloning. For crystallization assays, the construct encoding for aa 875-940 was generated with a N-terminal 6xHis-tag and a TEV cleavage sequence into pProEX-HTb using homemade Gibson Assembly mix 22 . The point mutations T888D, R892E, V903D were added in the backbone encoding for his-rTEV-875-940, all by Quickchange 23 .

Protein expression and purification

Constructs were expressed in E.coli BL21 (DE3) cells (NEB). Cells were grown in 2xYT media until OD560~0.8, expression was then induced by addition of 200 µM isopropyl β-D-1-thiogalactopyranoside (except for Dab2 expression, where 1 mM isopropyl β-D-1-thiogalactopyranoside was used). Cells were lysed by sonication. For purification, the lysate soluble fraction was loaded on an IMAC column (cOmplete 5mL, Roche for all constructs except YFP CBD (WT and mutant) for which HisTrap-FFcrude 5mL, Cytiva was used instead), and proteins were eluted with 200 mM or 300 mM Imidazole. Purest fractions were identified by SDS-PAGE. If needed, pooled fractions were concentrated using Vivaspin concentrators (Sartorius) up to ~5 mL. Concentrated samples were injected in Superdex 200 or 75 16/600 columns (Cytiva) depending on the molecular weight of the target protein. Purest fractions and the final sample were concentrated by ultrafiltration, and protein concentration was determined using Nanodrop 2000 (ThermoScientist). The final sample containing concentrated protein was flash-frozen in liquid nitrogen and stored at -80°C.

For proteins containing a TEV cleavable His-tag, prior to the gel filtration, His-tag was removed by incubation with homemade rTEV protease overnight in a 1/50 mass ratio. The incubate was passed through the cOmplete His-Tag Purification Column again to remove rTEV and the uncleaved fraction, then concentrated and loaded in a Superdex 75-16/60 gel filtration column. Purification buffers are detailed in Sup Table 2.

Constructs cloning for expression in MNT-1 cells

For expression in MNT-1, FLMyo6 was generated from cDNA of full length human Myo6, no inserts isoform (UNIPROT: Q9UM54-5) with shRNA resistance. DNA was transferred to the pEGFP-C1 vector via a XbaI restriction enzyme site. SAHmimic mutations (Glu922 to Glu935 (EAERLRRIQEEMEK) replaced with alternate acidic and basic residues (EERKRREEEERKKREEE) were introduced by reverse PCR. The L926Q mutation was introduced using Quickchange 23 . Transfer of Jo-Myo6-In from baculovirus vector to P-EGFP-C1 was ordered from GenScript. Myo6 CBD was generated by transferring DNA encoding G1037-end from human Myo6, no inserts isoform (UNIPROT: Q9UM54-5) into pEGFP-C1 plasmid using the XbaI restriction enzyme site. I1072A was introduced in previously cloned constructs (see above) using reverse PCR. Mouse GIPC1 (239-end), human TOM1 (299-end as described in 19 ) and human Dab2 (650-end) were transferred in a modified pmCherry-C1 plasmid containing in N-terminus a melanosome-targeting tag (MST tag, aa 1-139 from Mouse MREG -UNIPROT: Q6NVG5) as described in 24 . The MST tag and mCherry are separated by a GGSGGTGG linker. In the mCherry MST-partners constructs, mCherry and GIPC1, TOM1 or Dab2 sequences are separated by the polylinker multiple cloning site SGLRSRAQASNSLTSK.

A.3. Limits of the study and perspectives

A.3.a. Two kinds of contacts participate in the off-state stability

The study of the Myo6 auto-inhibited state described above allows us to identify important parameters for the maintenance of the Myo6 back-folded conformation. We show that both the NF and Mg.ADP.VO4 bound Motors could adopt a compact conformation at low salt concentration (80 mM NaCl). Yet, in the presence of Mg.ADP.VO4, Myo6 remains compact up to 425 mM NaCl while in the NF condition, Myo6 is more sensitive to salt and opens progressively upon salt addition: thus, more than 80 % of the Myo6 molecules are opened at 425 mM NaCl. Moreover, in microscale thermophoresis, the affinity of the MD ins2/IQ/3HB construct for the CBD drops from 144 ± 61 nM to 726 ± 480 nM upon nucleotide removal.

We thus proposed that two kinds of interactions stabilizes Myo6 in its auto-inhibited state: (1) nucleotide-dependent interactions near the Motor domain that can lock Myo6 in its back-folded conformation in a salt independent manner and ( 2) salt dependent interactions along the rest of the Myo6 molecule that mediates back-folding of Myo6 when no nucleotide is bound (Figure 56) and probably explains why Myo6 is able to fold back even when the Motor is absent as previously reported [START_REF] Fili | Competition between Two High-and Low-Affinity Protein-Binding Sites in Myosin VI Controls Its Cellular Function[END_REF]2017;Dos Santos et al. 2022). Yet, shift towards opening as seen with the FLMyo6 L926Q mutant may not be necessary to enable Myo6 recruitment: Loss of nucleotide-dependent contacts within the auto-inhibited state of Myo6 could be sufficient. Indeed, in our pull-down assays, at 100 mM NaCl, nucleotide removal was sufficient to enhance TOM1 binding in conditions in which Myo6 was found to be compact in SEC-MALS in similar conditions. In this context, the Myo6 NF -high salt state was an interesting tool to characterize the polar interactions that must going on within the Myo6 Tail.

The use of the FLMyo6 D1148V, Y1150D, D1152R and Q1154V mutant remained quite limited for studying Myo6 activation upon partner binding. Indeed, these mutations take place in the CBDc and could strongly impair Myo6 interaction with certain partners. In particular, DNA binding to this mutant is very unlikely as fluorescence anisotropy revealed that depletion of the SKKK motif near residues D1148V, Y1150D, D1152R and Q1154V results in a 7-fold decrease Myo6 affinity for DNA [START_REF] Fili | NDP52 Activates Nuclear Myosin VI to Enhance RNA Polymerase II Transcription[END_REF]. It should be noted that in this context, our results are fully consistent with the results reported by [START_REF] Fili | NDP52 Activates Nuclear Myosin VI to Enhance RNA Polymerase II Transcription[END_REF], that suggest that DNA cannot bind to the Myo6 auto-inhibited state since mutations within the same loop decreases binding between the CBD and DNA (see section C.7.) (Figure 58).

A.3.b. Determination of the atomic structure of the Myo6 auto-inhibited state

Our paper thus brings insights on the way the Myo6 auto-inhibited state might be stabilized, yet atomic description of the auto-inhibited state is still missing to get a clearer understanding of Myo6 regulation by the description of the key contacts that Myo6 maintains in its auto-inhibited state.

Additionally, the structure of Myo6 in its auto-inhibited state will bring deTailed structural information on the Myo6 distal Tail and CBD subdomains that are still currently unreported and that could lead to a greater understanding on the way Myo6 interacts with its partners. Such structure could also lead to the discovery of new allosteric pockets within the back-folded Myo6 structure that could be of great interest to develop Myo6 modulators.

I thus performed crystallization assays in order to solve the structure of the FL Myo6 autoinhibited state. To that aim, I used our Jo-Myo6-In construct. Indeed, crystallization of FL Myo6 had been extensively screened without success by a former PhD student of the team: Vicente Planelles-Herrero. Perhaps the Myo6 Tail still explores several conformations in the crystallization drops resulting in heterogeneity in between the Myo6 and making the formation of crystals disfavored.

In contrast, Jo-Myo6-In is trapped in the back-folded conformation as Jo and In subdomains limit the movement of the Tail with respect for the Motor. Crystallization assays were performed in the presence of Mg.ADP.VO4 and EGTA. Jo-Myo6-In appears to give tiny nuclei in basic conditions whose size slightly improved using seeding (Figure 60). Yet, despite extensive optimization, these crystals remained too small and of poor-quality: no diffraction could be measured at the synchrotron beamline proxima 2.

A.3.c. Structural description of the Myo6 Tail

Determining the structure of the whole Myo6 Tail would be of great interest as it would greatly improve our Myo6 auto-inhibited state model and brings us a clearer understanding of the way certain partners may activate Myo6 or not. Indeed, if small fragments of CBD or DT domains are available, a structure of the whole DT-CBD domain is still missing and it is thus unknown how CBD n and CBD c would be organized with respect to one another, potentially limiting the accessibility of certain binding interfaces. Using a severely truncated fragment of CBD n , [START_REF] Shang | Structure Analyses Reveal a Regulated Oligomerization Mechanism of the PlexinD1/GIPC/Myosin VI Complex[END_REF] proposed the formation of a GIPC1-Myo6 complex with high oligomerization. In contrast, using larger fragments that also include CBD c , a 1:1 Myo6/GIPC1 complex was characterized in our lab (Planelles Herrero 2017; see section G.1). Such structures could also help us understand why addition of the LI insert at the N-terminus of the Myo6 CBD could improve binding affinity of partners, such as Dab2 and LMTK-2 on the CBD Cterminus [START_REF] Fili | Competition between Two High-and Low-Affinity Protein-Binding Sites in Myosin VI Controls Its Cellular Function[END_REF], Chibalina et al. 2007).

A.3.d. Activation

Our study highlights the importance of a proper back-folding equilibrium between on and off states of Myo6 for correct recruitment of Myo6 in cells since mutants biased to favor the open conformation, FLMyo6 L926Q and SAHmimic were over-recruited while Jo-Myo6-In was not recruited to melanosomes. Since FLMyo6 L926Q and SAHmimic interact with partners normally unable to bind to the back-folded Myo6, improper folding could potentially result in misrecruitment resulting in activation of undesired pathway.

But to understand to what extent over-recruitment could be deleterious, a functional assay would be required to look at the effect of Myo6 over-recruitment at a cell compartment. In MNT-1, Myo6 is required for constriction and fission of tubular carriers [START_REF] Ripoll | Myosin VI and Branched Actin Filaments Mediate Membrane Constriction and Fission of Melanosomal Tubule Carriers[END_REF], which is essential for melanosome maturation prior to their delivery to keratinocytes. Additionally, these carriers are involved in protein recycling [START_REF] Dennis | BLOC-1 and BLOC-3 Regulate VAMP7 Cycling to and from Melanosomes via Distinct Tubular Transport Carriers[END_REF]). To complete this study, it would now be interesting to monitor fission events frequency using FLMyo6 L926Q open mutants for example or looking at impact on transferrin uptake.

In our case, such a question is particularly tricky as both the L926Q and SAHmimic mutants impact the SAH N-terminus and, as a consequence, the stabilization of the off-state as well as Myo6 dimerization. It is thus difficult to say if pathogenous effect of the L926Q mutation comes from over/misrecruitment of Myo6 or improper dimerization. Actually, mutating the 3HB-SAH region should always be done carefully as it appears to be a challenging task to destabilize back-folding without destabilizing the proximal dimerization by mutating this region. The deTailed structure of the Myo6 auto-inhibited state would thus be of great interest to identify how the SAH domain folds on the 3HB in the context of the auto-inhibited state and how pathogenous mutations, such as L926Q, could both trigger Myo6 opening and reduce the dynamics of the 3HB domain. On the basis of such a structure, it would then be easier to identify mutations that would target only the activation equilibrium without impairing dimerization.

We have explored the role of binding partners for differential recruitment of Myo6 and this study suggests the ability of RRL partners such as GIPC1 to recruit Myo6 independently of its activation state, while partners such as Dab2 and TOM1 would require assistance to open Myo6 prior to their binding. We propose that for WWY motif partners, local high concentration of Ca 2+ ions could timely and spatially control Myo6 recruitment.

Interestingly, [START_REF] French | Investigations of Human Myosin VI Targeting Using Optogenetically Controlled Cargo Loading[END_REF] noticed that when Dab2 is artificially addressed to the Golgi apparatus, Myo6 recruitment does not correlate with the amount of Dab2, consistent with our results that have shown a lack of FL Myo6 or Jo-Myo6-In recruitment to melanosomes via Dab2.

However, in contrast with our results: when Dab2 is artificially addressed to mitochondria, Dab2 can significantly recruit Myo6 to the mitochondria membrane. It should be noted that if, these three organelles are regulated by different Ca 2+ flux/potential that could be responsible for differential recruitment of Myo6 by Dab2 depending on the compartment where Dab2 is addressed [START_REF] Wuytack | PMR1/SPCA Ca2+ Pumps and the Role of the Golgi Apparatus as a Ca2+ Store[END_REF]. This highlights the fact that the Myo6 recruitment depends on several parameters, and it is also possible that the freeing of the regions that bind Myo6 also depend on the other proteins present on these distinct organelles. To go further in this study, it would thus be of interest to repeat our recruitment assays on different organelles and in particular at the Golgi apparatus membrane.

Impact of other parameters on Myo6 unfolding remains to be explored, notably the role of actin or that of post-traductional modifications such as phosphorylation. Indeed, phosphorylation has already been shown to regulate folding of Myo2 [START_REF] Craig | Light-Chain Phosphorylation Controls the Conformation of Vertebrate Non-Muscle and Smooth Muscle Myosin Molecules[END_REF][START_REF] Brzeska | Regulation of Class I and Class II Myosins by Heavy Chain Phosphorylation[END_REF], and impacts Myo6 ATPase activity (De La Cruz, Ostap, and Sweeney 2001) and its recruitment (Buss et al.1998[START_REF] Brooks | MYO6 Is Targeted by Salmonella Virulence Effectors to Trigger PI3-Kinase Signaling and Pathogen Invasion into Host Cells[END_REF][START_REF] Sahlender | Optineurin Links Myosin VI to the Golgi Complex and Is Involved in Golgi Organization and Exocytosis[END_REF]. We thus could exclude its general requirement for either activation or back-folding. Considering the key role of CBD for the offstate stability, it would be of great interest to investigate the impact of LI and SI inserts on the off-state stability. Recent results already show that the LI insert does not impair the interaction between the Myo6 CBD and the IQ-DT fragment (Dos Santos et al. 2022). It would now be interesting to revisit this finding with Motor containing construct.

A.3.e. Proximal dimerization

A.3.e.i. Mechanical consequence of the proximal dimerization

Our study provided a deTailed atomic structure for the Myo6 dimerization region, in which two Myo6s associate as an antiparallel dimer through residues 875-940. Compatibility of this dimer with 30-36 nm steps (Figure 62) and inability of FLMyo6 T888D, R892E and V903D to rescue transferrin uptake support the physiological relevance of this model. Moreover, the ability of GIPC1 to induce gating of FL Myo6, which is lost for the FLMyo6 T888D, R892E and V903D triple mutant, is a strong argument that GIPC1 induces proximal dimerization.

To develop our understanding of the Myo6 function, closer analysis of the mechanical properties of the proximal dimer would be of great interest. Single motility assays suggest that proximal dimerization may be important for Myo6 processivity as no movement could be detected upon addition of the T888D, R892E and V903D mutations on FLMyo6. Yet, to test the role this dimer may play in cells, looking at the impact of T888D, R892E and V903D on Myo6 ability to stay attached to actin under resisting load would be of particular interest. As deTailed description of either the proximal dimer upon GIPC1 binding (this study) or distal dimer upon Dab2 binding [START_REF] Yu | Myosin VI Undergoes Cargo-Mediated Dimerization[END_REF]) are now available, such a study could now be considered. The cryoEM structure of Myo6 bound to actin (PDB: 6BNP, [START_REF] Gurel | Cryo-EM Structures Reveal Specialization at the Myosin VI-Actin Interface and a Mechanism of Force Sensitivity[END_REF]) was used as a basis for placing two myo6 Motor domains in rigor and PPS states (grey) at the desired 31 nm distance. On each side, the N-terminus of the Lever arm bound to 2 light chains (pink, PDB: 3GN4, [START_REF] Mukherjea | Myosin VI Dimerization Triggers an Unfolding of a Three-Helix Bundle in Order to Extend Its Reach[END_REF]) was aligned to the corresponding residues in the converter. The crystallized dimerization domain (blue) was then arbitrarily placed between the two Lever arms, leaving a gap of 4.6 nm on each side. This gap needs to be filled by a stretch of 26 amino acid residues that would correspond to 3.9 nm if in a theoretical helix, or up to 9.9 nm if fully extended.

The rest of the SAH (green) (PDB: 6OBI, [START_REF] Barnes | Remarkable Rigidity of the Single α-Helical Domain of Myosin-VI As Revealed by NMR Spectroscopy[END_REF] was connected to the C-terminus of the dimerization region via a kink. The model is compatible with parallel dimerization of the cargo-binding domain.

Additionally, to complete our model (Figure 62), closer look should be taken to the R849-E875 region. Tryptophan fluorescence (Batters et al. 2016) and microscale thermophoresis assays [START_REF] Mukherjea | Myosin VI Must Dimerize and Deploy Its Unusual Lever arm in Order to Perform Its Cellular Roles[END_REF] proposed that this fragment could have the ability to bind CaM. For Batters et al. 2016, helix 833-856 could fold on the IQ/CaM while [START_REF] Mukherjea | Myosin VI Must Dimerize and Deploy Its Unusual Lever arm in Order to Perform Its Cellular Roles[END_REF] proposed that an additional CaM could be recruited by the 834-875 region which would rigidify the proximal dimer Lever arm compared to one with only two Cam on each side. In fact, the zippered Myo6 dimer was shown to associate with three CaM per HC via HPLC and gel quantification. In our view, folding of residues R849-E875 on IQ/CaM is unlikely as it would result in a shortening of the distance between the two Myo6 heads and thus such a model is not compatible with the 30-36 nm steps previously measured for the molecule (De La Cruz, Ostap, and Sweeney 2001;[START_REF] Rock | Myosin VI Is a Processive Motor with a Large Step Size[END_REF]) (Figure 63). Batters et al., 2016. This leaves a 51.7 Å gap between residues 856 and 875 if the heads are positioned to allow 31 nm steps. Filling this gap would require that the 856-875 region extends fully (a linker would in fact cover ~ 47 Å). This model is thus unlikely as it does not seem compatible with the stepping previously measured for dimeric Myo6.

Å was left between R849 and the C-terminus of the poly-Ala helix, which corresponds to the typical distance found between two residues within an α-helix. It should be noted that this docking is imprecise since it was roughly assembled without any alteration of the cryoEM and crystallographic model of the Lever arm and the dimerization region, this should be reviewed in the context of a dynamic motor (molecular dynamic) to improve this docking. (B) A third CaM could be recruited on residues 834-875 in our model without generating clashes. Close proximity between CaM and the kink between the end of the proximal dimerization area and the SAH (at residue 940) could suggest that interactions may occur here, although such interaction remained to investigated (C) The 833-856 region is folded on the IQ CaM as proposed by

Exploring putative interaction between CaM and the Myo6 930-950 region may bring insights on the way Myo6 dimerizes. Finally, the crystal structure of Ins2-E875 could provide us with a deTailed description of the Myo6 Lever arm when proximal dimerization through residues 875-940 occurs.

These findings could provide a clearer description of the Lever arm rigidity and help understand the kind of function the proximal dimer could play in cell.

A.3.e.iii. FLGIPC1 -Myo6 stoichiometry

To prevent GIPC1 auto-inhibition, all our studies were performed using the GIPC1 GH2 domain only. Yet, in physiological context, GIPC1 contains a GH1 domain that can mediate GIPC1 dimerization (Figure 64) (see results -section A.1.c). Thus, the impact of GIPC1 auto-inhibition and its ability to prevent or to favour Myo6 binding should be monitored using the FL GIPC1. This should also result in the correct description of the real physiological stoichiometry that can occur upon Myo6/GIPC1 recognition. Indeed, Myo6 has been found to exhibit different mechanical properties depending on whether it is functioning as a dimer or an assembly of cargos (see introduction -section E.3). If FL GIPC1 is not compatible with Myo6 binding, the challenge would be to find a way to destabilize the autoinhibited state of GIPC1 for in vitro experiments without perturbing the Myo6/GIPC1 interactions. The most physiological way would be to try adding GIPC1 PDZ partners such as APPL1. This question remains to be explored in future investigations. The formation of the Myo6 proximal dimer relies on 3HB unfolding [START_REF] Mukherjea | Myosin VI Dimerization Triggers an Unfolding of a Three-Helix Bundle in Order to Extend Its Reach[END_REF]) but once the 3HB unfolding has occurred, Myo6 proximal dimerization is exposed, thus in this context, Myo6 may dimerize without assistance of a particular partner. This raises the question of whether all partners of Myo6 could trigger 3HB unfolding and proximal dimerization. Yet, from our study, whether the proximal dimerization can be triggered only by certain partners or whether all partners could trigger proximal dimerization remains an open question.

Through a kinetic study using FRET-based stopped flow, Dos Santos et al. 2022 monitored the dimerization of the Myo6 814-end construct upon NDP52 binding. Interestingly, they found that the dimerization rate is independent of NDP52 concentration. Moreover, they found that Myo6 can gate upon increasing concentrations of actin using a constant concentration of either the NDP52 or Dab2 partners (Dos Santos et al. 2022). As in this context, the kcat was identical for Myo6 supplemented with Dab2 or NDP52 or Myo6 truncated from the Tail, the authors thus proposed that the partners do not regulate the ATPase level, it mainly promotes exposition of the internal dimerization site normally masked when Myo6 adopts a back-folded conformation [START_REF] Fili | NDP52 Activates Nuclear Myosin VI to Enhance RNA Polymerase II Transcription[END_REF]Dos Santos et al. 2022).

They thus proposed that the role of the Myo6 partner may be to control whether the Myo6 internal dimerization would occur by either promoting unfolding and exposing the dimerization area, or by its inability to do so. The region they considered to allow internal dimerization was suggested to occur arbitrarily after the SAH, and thus was not the same as the one we identified in our study. In fact, we demonstrated that the proximal dimerization requires the opening of the 3HB to form and whether Myo6 unfolding automatically results in a 3HB opening in a physiological context remains to be explored.

It should be noted that in contrast with our study, Dos Santos et al. 2022 found that Dab2 was able to activate FLMyo6 in actin-activated ATPase assays. Perhaps this difference is due to the 5-fold higher salt concentration used in this experiment or to the lower concentration of F-actin. Further experiments are needed to understand the differences. Still, in our hand, the inability of TOM1 and Dab2 to recruit a constitutively closed Myo6 (Jo-Myo6-In) in contrast with the constitutively open constructs (SAHmimic or L926Q mutant) on melanosome strongly support the fact that not all partners have the ability to bind to the Myo6 auto-inhibited state which would be necessary so that they could trigger unfolding. We thus propose that partners must at least be split in two classes: in the first class, that likely includes at least some of the RRL partners, the partner unfolds Myo6 possibly triggering 3HB unfolding. In the second class, that includes the WWY partners and other CBD c partners such as DNA [START_REF] Fili | NDP52 Activates Nuclear Myosin VI to Enhance RNA Polymerase II Transcription[END_REF], their binding site being masked in the off-state, they cannot act to unfold and activate on their own. However, they might act to stabilize the Myo6 active form by preventing Myo6 backfolding. However, the identification of partners triggering/stabilizing the proximal dimerization requires further experiments.

In the other hand, Rai et al. 2021;2022 proposed that the Myo6 partners may influence the Myo6 mechanical role in different ways depending on the partner. Indeed, in their hands, using a similar experimental set up, the addition of Dab2 results in the formation of sparse Myo6 dimers minimizing cortical actin reorganization while that of GIPC1 results in the formation of unfolded Myo6 monomers that could mediate long and fast processive runs when assembled in a network via a DNA scaffold. Note that these experiments have been performed in conditions that may not facilitate dimerization (immobilized motor, very low concentration up to ~50 pM). This likely explains why Rai et al., 2022 could only see a 1:1 complex between GIPC1 and Myo6, while our ATPase results pointed out to Myo6 dimerization upon GIPC1 binding. These findings suggest that Dab2 and GIPC1 may have differential impact on Myo6 mechanical properties once unfolded.

It would thus be of interest to see if all partners could promote proximal dimerization to challenge these models. Yet to answer such question, actin activated ATPase assays as performed in our study show limitations: As Dab2 and TOM1 were not able to activate FL Myo6 WT in our ATPase assays, the impact of Dab2 and TOM1 on the Myo6 mechanical properties and in particular on dimerization is unclear. Indeed, all our constitutively opened mutants impaired proximal dimerization and they thus cannot be used to look at the ability of Dab2 or TOM1 to promote gating. Moreover, the impact of TOM1 and Dab2 on the SAHmimic ATPase activity is very similar despite the fact that these two partners were reported to induce different oligomeric states of the motor: TOM1 and Dab2 were proposed to activate Myo6 as a monomer [START_REF] Hu | Structure of Myosin VI/Tom1 Complex Reveals a Cargo Recognition Mode of Myosin VI for Tethering[END_REF] of a distal dimer [START_REF] Yu | Myosin VI Undergoes Cargo-Mediated Dimerization[END_REF], respectively. Thus, either distal dimerization does not induce gating making distal dimers indistinguishable from active monomers, or our experimental conditions do not allow the formation of proper distal dimerization, perhaps due to the use of the SAHmimic mutant. Possibly also, more studies on TOM1 or Dab2 are required to ascertain whether they can mediate distal dimerization since the results from the crystal structures are insufficient to assess the stoichiometry of the Myo6-partners in the context of the FLMyo6.

Overall, to have a clear understanding of the impact of the binding of partners on Myo6, a direct measurement of the oligomeric state is required in complement to the ATPase assays.

Actually, to overcome this limitation, we first aimed to perform interferometric scattering mass spectrometry (iSCAMS) [START_REF] Young | Quantitative Mass Imaging of Single Biological Macromolecules[END_REF] taking advantage of the fact that our collaborator Lee Sweeney was able to gain access to an iSCAMS system in the beginning of my PhD. ISCAMS is a recently developed technique that can provide a good estimation of the molecular weight of biomolecules and biomolecule complexes. It relies on illumination of molecule in solution with a coherent light resulting in light reflection and light scattering by the biomolecule. Interference between the reflected and scattered light at the detector results in a contrasted signal enabling imaging while a scattered signal is proportional with the particle volume, enabling molecular weight determination [START_REF] Young | Quantitative Mass Imaging of Single Biological Macromolecules[END_REF] (Figure 65). Yet this technique exhibits several limitations. In particular, it is challenging to detect particles below 50 kDa and concentrations must be low enough so that a signal can be detected for single particles near the refractive interface. The detection limit was a first issue to overcome in our experiments as the Myo6 partners we were working with were too small to be detected on their own (GIPC GH2 weights ~12 kDa, TOM1207-492 weights ~33 kDa and Dab2 weights ~17 kDa).

I thus fused these partners to bigger proteins. The Myo6 adaptor constructs cloned in fusion with monomericYFP (27 kDa) [START_REF] Shaner | Improved Monomeric Red, Orange and Yellow Fluorescent Proteins Derived from Discosoma Sp. Red Fluorescent Protein[END_REF], mNeongreen (26.5 kDa) [START_REF] Shaner | A Bright Monomeric Green Fluorescent Protein Derived from Branchiostoma Lanceolatum[END_REF] or MICAL-21-623 (71 kDa) [START_REF] Giridharan | MICAL-Family Proteins: Complex Regulators of the Actin Cytoskeleton[END_REF] were used to try to reach a detectable signal. The purified proteins and experimental protocol were then sent to our collaborator Lee Sweeney where the iSCAMS experiments were carried out by Peter Hook. These assays were carried out using different nucleotides, salt concentrations, Myo6 constructs (promoting either closure or opening). Despite several experiments, the results obtained were unconclusive: the association between Myo6 and its adaptors was only rarely seen, and we were not able to detect more than ~4 % of 2:2 complexes between Myo6 and any tested partners. We propose that iSCAMS was inconclusive for our case perhaps due to the low Myo6 concentration required. Actually, a recent study trying to measure the oligomeric state of Myo7a-M7BP complex faced the same limitation that we did as they could only measure a 1:1 complex in iSCAMS despite the very high affinity of the Myo7a-M7BP complex (~6 nM) [START_REF] Liu | A Binding Protein Regulates Myosin-7a Dimerization and Actin Bundle Assembly[END_REF].

We therefore decided now to switch from iSCAMS to analytical ultracentrifugation (AUC) which allows the use of higher concentration of proteins and can detect small proteins. AUC can provide us on stoichiometry of Myo6 in complex with its various partners using Motor less fragments.

These experiments should give us clearer answer regarding Myo6-partners stoichiometry. We will put these data in perspective with the ATPase assays performed by our collaborator to clarify whether partners can instruct the Myo6 motor of unique mechanical functions.

Finally, additional ATPase assays could be conceived to look at the impact of the WWY partners on proximal dimerization. Perhaps, repeating actin activated ATPase assays in higher salt conditions could be sufficient to enable TOM1 or Dab2 binding. Another way could be the addition of Ca 2+ . Indeed, Ca 2+ addition results in enhanced TOM1 binding to Myo6. However, a complication comes from the fact that Ca 2+ addition must be transient as it was shown to result in loss of coordination between the two heads of a dimer [START_REF] Morris | Calcium Functionally Uncouples the Heads of Myosin VI[END_REF]. Although difficult to implement, such study is important as they would provide us with a more complete characterization of the way partners could provide Myo6 with unique mechanical functions. To complete our study, it would be interesting to explore the conservation of the proximal dimerization among species and investigate whether proximal dimerization can occur outside the mammalian phylogenic group (Figure 66). While the proximal dimerization area is overall conserved, Drosophila exhibits the most divergent sequence among the 8 species compared in Fig XX . Indeed, two insertions can be found within the proximal dimerization area suggesting that if the Myo6 proximal dimerization occurs in Drosophila, some adjustment in the structure are likely occurring compared to the structure we have solved. It is however not possible to say whether this would not favor dimerization or if the adjustments would remain local.

A.3.f. Conservation of the proximal dimerization among species

Additionally, chicken, zebrafish and drosophila Myo6 exhibit two divergent residues compared to the human sequence crystallized: Leu881 is replaced with Ser, His or Ala respectively. If Ser and Ala seems compatible with the structure, a bulk residue such as His could result in steric hindrance and make formation of the dimer destabilized. In addition, Gln896 in the human sequence is replaced with Met or Asp. To understand the impact of these sequence changes on the formation and stability of the proximal dimerization, ATPase assays to monitor gating through different species could be performed.

A.3.g. Endogenous recruitment of Myo6 to melanosome

The Myo6 partner OPTN was proposed to be involved in Myo6 mediated tubular carrier fission during melanosome maturation as OPTN silencing similarly to SiMyo6, decreases fission events without decreasing tubule formation in MNT-1 [START_REF] Ripoll | Myosin VI and Branched Actin Filaments Mediate Membrane Constriction and Fission of Melanosomal Tubule Carriers[END_REF]. However, OPTN silencing does not impact Myo6 localization to melanosome suggesting that if OPTN may be required for Myo6 in melanosomes it may not be required for Myo6 recruitment to melanosomes [START_REF] Ripoll | Myosin VI and Branched Actin Filaments Mediate Membrane Constriction and Fission of Melanosomal Tubule Carriers[END_REF]. In contrast, the I1072A mutation reported in our study drastically reduces Myo6 recruitment to melanosomes. The I1072A mutation has previously been reported to drastically reduce ubiquitin binding to the Myo6 MyUb binding site through pull-down assay, [START_REF] He | Myosin VI Contains a Compact Structural Motif That Binds to Ubiquitin Chains[END_REF]) while keeping the overall MyUb domain fold as showing by NMR [START_REF] He | Myosin VI Contains a Compact Structural Motif That Binds to Ubiquitin Chains[END_REF]. It is thus tempting to speculate that recruitment to melanosomes requires Myo6 interaction with ubiquitinylated cargos. [START_REF] He | Myosin VI Contains a Compact Structural Motif That Binds to Ubiquitin Chains[END_REF] shows that ubiquitin chains exhibiting the highest affinity for Myo6 MyUb binding sites are the K63-linked chains. Considering the importance of residue I1072 for Myo6 recruitment to melanosomes, we decided to check whether K63-diUbiquitin could activate Myo6. I produced the K63-diUbiquitin homemade following the previously published protocol [START_REF] Komander | The Structure of the CYLD USP Domain Explains Its Specificity for Lys63-Linked Polyubiquitin and Reveals a B Box Module[END_REF]. DNA encoding for enzymes required for K63link ubiquitin preparation and Ubiquitin DNA were a kind gift for the Komander's lab. We confirmed the ability of K63-diUbiquitin to bind the Myo6 Tail by microscale thermophoresis (KD ~10 µM) (Table 13).

Table 13: K63-diUbiquitin binding to the Myo6 CBD

Microscale thermophoresis experiment performed in 20 mM Tris pH8.5, 100 mM KCl, 1 mM DTT, 0.05 % tween

We then sent the protein to our collaborator Lee Sweeney to evaluate its ability to activate Myo6 in actin activated ATPase assays but remarkably the K63-diUbiquitin was not able to activate wild-type FLMyo6 in contrast to what was seen for GIPC1 (Figure 67). Actin-activated ATPase rate (s -1 .Head -1 ) of 250 nM FLMyo6 wild-type upon increasing concentration of K63-diUb or GIPC1 GH2 (from 0 to 1 µM) (n=6) on 40 µM F-actin in 10 mM MOPS7, 10 mM KCl, 1 mM DTT, 1 mM MgCl2, 1 mM EGTA Thus, while we cannot exclude that ubiquitinylated cargos could help recruit Myo6 to melanosome, it seems that another partner would be required to activate it, possibly the RRL partner OPTN. Importantly, it should be noted that OPTN can be ubiquitinylated (UbOPTN). Interestingly, He et al. 2016 reported, through a pull-down assay, that Myo6's ability to bind UbOPTN is drastically reduced by the I1072A mutant. This could be the reason why even our constitutively open construct FLMyo6 (SAHmimic.I1072A) or FLMyo6 (L926Q.I1072A) were not able to fully rescue Myo6 recruitment to melanosomes. Indeed, the Myo6 affinity for ubiquitinylated partners present at the surface of melanosomes such as OPTN may still be impaired by the mutant. Overall, while our study is not sufficient to describe how endogenous recruitment of Myo6 to melanosomes occur, it has provided interesting insights and tools for further study on the role of Myo6 in melanosome maturation.

Conclusions

The work presented in this thesis offers new insights on Myo6 regulation.

In our study, we highlight the importance of Myo6 back folding for regulation of its activity in cell. Although some Myo6 back-folding is possible using Motor-less Myo6 construct (or FLMyo6 in nucleotide free state) [START_REF] Fili | NDP52 Activates Nuclear Myosin VI to Enhance RNA Polymerase II Transcription[END_REF]2020;Dos Santos et al. 2022;Batters et al. 2016), our study identifies that the presence of ADP.Pi analog within Myo6 motor greatly enhances the stability of the Myo6 back-folded state since the presence of ADP.Pi analog prevents Myo6 opening in high salt concentration and should thus be sufficient to maintain Myo6 back-folded in a physiological context.

Our study also demonstrates that interactions between the head and the Tail involve a highly conserved loop within the CBD c that has been previously reported to bind DNA, making DNA binding to Myo6 auto-inhibited state unlikely, consistently with what has been proposed previously [START_REF] Fili | NDP52 Activates Nuclear Myosin VI to Enhance RNA Polymerase II Transcription[END_REF]).

As a consequence, not all partners can directly interact with the Myo6 auto-inhibited state to trigger Myo6 recruitment and activation. In particular, the WWY motif partners Dab2 and TOM1 were shown to be unable to bind to the Myo6 auto-inhibited state in in vitro and cell-based assays and considering the fact that the WWY motif is particularly buried in our model, it is tempting to propose that none of the Myo6 WWY partners may be able to trigger unfolding. In this context, a signal triggering unfolding must be required to allow recruitment of these partners offering spatial and temporal control of their binding to Myo6. Indeed, in cells, it has been shown that the ability of Dab2

to recruit Myo6 depends on the cell compartment where it is localized [START_REF] French | Investigations of Human Myosin VI Targeting Using Optogenetically Controlled Cargo Loading[END_REF]. This signal might be for example a Ca 2+ ion concentration variation. Indeed, a recent study has proven the ability of Ca 2+ ion to unfold Myo6 [START_REF] Rai | Multimodal Regulation of Myosin VI Ensemble Transport by Cargo Adaptor Protein GIPC[END_REF] as previously proposed (Batters et al. 2016). In our study we confirm through a pull-down assay that Ca 2+ addition indeed improves Myo6 binding to the WWY partner TOM1, further supporting this hypothesis.

In contrast, we demonstrate that the Myo6 RRL motif partner GIPC1 can efficiently bind to the Myo6 auto-inhibited state, as GIPC1 can recruit both FLMyo6 and Jo-Myo6-In to melanosome. GIPC1 can then trigger unfolding and activation, consistent with a recent study showing GIPC1's ability to unfold FLMyo6 in a FRET assays [START_REF] Rai | Multimodal Regulation of Myosin VI Ensemble Transport by Cargo Adaptor Protein GIPC[END_REF]) and supporting a different role of partners for Myo6 activation.

Although GIPC1 GH2 domain has been proposed either to oligomerize Myo6 [START_REF] Shang | Structure Analyses Reveal a Regulated Oligomerization Mechanism of the PlexinD1/GIPC/Myosin VI Complex[END_REF] or to stabilize an active monomer of Myo6 [START_REF] Rai | Multimodal Regulation of Myosin VI Ensemble Transport by Cargo Adaptor Protein GIPC[END_REF]), here we demonstrate GIPC1's ability to trigger Myo6 gating, reflecting its ability to promote Myo6 self-dimerization, which was further supported by the fact that a triple mutant specifically designed by our team to impair Myo6 proximal dimerization disrupts GIPC1's ability to promote Myo6 gating. Due to its ability to oligomerize the Myo6 Tail in AUC and crystal structure [START_REF] Shang | Structure Analyses Reveal a Regulated Oligomerization Mechanism of the PlexinD1/GIPC/Myosin VI Complex[END_REF], and to speed up Myo6 in in vitro motility assays [START_REF] Rai | Multimodal Regulation of Myosin VI Ensemble Transport by Cargo Adaptor Protein GIPC[END_REF]), the Myo6-GIPC1 complex has been proposed to act as a transporter. Yet, a strong gating between the Myo6 heads might be optimal for anchoring function of the motor (Sweeney and Houdusse 2007), our study opens the possibility that GIPC1 may mediate Myo6 anchoring function against load. Actually, as proposed for NDP52, it is possible that the Myo6 stoichiometry induced by GIPC1 binding may be regulated by local concentration offering ability for Myo6 to work either as a transporter or an anchor through its binding to the same partner. Thus, studying the impact of GIPC1 concentrations on Myo6 mechanical properties and the impact of GIPC1 on Myo6's anchoring ability may be of great interest in the future.

Our data also support the idea that each partner may impact Myo6 mechanical properties in in a different manner: although we could not compare TOM1 and Dab2's impact to GIPC1 on Myo6 wild-type ATPase activity in our ATPase assay, comparison of their ability to activate the constitutively open mutant SAHmimic revealed that even in the context of an open construct TOM1 and Dab2 cannot fully activate Myo6 as opposed to GIPC1, suggesting a differential impact of these partners on Myo6 activity. Although, this point still requires investigation, the idea is further supported by crystal structures showing diverse stoichiometries for Myo6 Tail -partner complexes depending on the partner bound [START_REF] Shang | Structure Analyses Reveal a Regulated Oligomerization Mechanism of the PlexinD1/GIPC/Myosin VI Complex[END_REF][START_REF] Hu | Structure of Myosin VI/Tom1 Complex Reveals a Cargo Recognition Mode of Myosin VI for Tethering[END_REF][START_REF] Biancospino | Clathrin Light Chain A Drives Selective Myosin VI Recruitment to Clathrin-Coated Pits under Membrane Tension[END_REF][START_REF] He | Myosin VI Contains a Compact Structural Motif That Binds to Ubiquitin Chains[END_REF][START_REF] Yu | Myosin VI Undergoes Cargo-Mediated Dimerization[END_REF] and supported by functional assays showing that in similar conditions, Dab2 can dimerize Myo6 and mediate a sparse motility while in contrast, GIPC1 stabilizes a monomer and speeds up the motor in ensemble motility assays (Rai et al. 2021;2022).

Moreover, thanks to their incompatibility with the Myo6 auto-inhibited state, CBD c partners sequester CBD c , preventing its rebinding to the Myo6 head, thus, stabilizing Myo6 active form following unfolding. This is consistent with previous competitive experiments with DNA, Dab2 and NDP52 (Fili et al. 2020;2017). While the GIPC1 binding is compatible with the Myo6 auto-inhibited state, its ability to sequester Myo6 CBD to stabilize an active form may not be as strong as the one of DNA or Dab2 and remains to be investigated. In this context, it may be interesting for the motor to bind several partners at once for optimal functioning or for switching for one mechanical role to another.

Our study of the Myo6 auto-inhibited state provides a clearer definition of the Myo6 backfolded state overall shape and dimensions. Consequently, a hinge between the 3HB and SAH must exist to allow Myo6 to fold back making the SAH N-terminus a critical area for the stabilization of the Myo6 auto-inhibited state. The unusual high occurrence of apolar residues within the N-terminus part of the SAH domain of Myo6 may have a role in the auto-inhibited state stability. Thereby, mutations of apolar residues in this region results in Myo6 opening (SAHmimic [START_REF] Mukherjea | Myosin VI Must Dimerize and Deploy Its Unusual Lever arm in Order to Perform Its Cellular Roles[END_REF] or L926Q deafness mutant [START_REF] Brownstein | Novel Myosin Mutations for Hereditary Hearing Loss Revealed by Targeted Genomic Capture and Massively Parallel Sequencing[END_REF]).

But both the SAHmimic [START_REF] Mukherjea | Myosin VI Must Dimerize and Deploy Its Unusual Lever arm in Order to Perform Its Cellular Roles[END_REF] and L926Q mutations (this study) were shown to also have an impact on the Myo6 proximal dimerization by limiting the 3HB dynamics and unfolding required for proximal dimerization. These findings thus demonstrate the close coordination that must exist between the Myo6 3HB and the SAH domain. It also highlights the crucial role of this region for Myo6 regulation and emphasizes the difficulty to study it, since a particular mutation in this region can impact dimerization as well as opening. A more deTailed description of the interactions existing between the Myo6 3HB and the SAH domain would thus be of great interest. Overall, even if our cellbased assays demonstrated that L926Q and SAHmimic mutations can lead to overrecruitment of the motor, it must not necessarily enhance Myo6 functions as they also make the motor dysfunctional and may instead act as dominant negative which is further supported by the fact that SAHmimic is unable to rescue Myo6 function for Golgi maintenance and transferrin uptake, even when artificially dimerized [START_REF] Mukherjea | Myosin VI Dimerization Triggers an Unfolding of a Three-Helix Bundle in Order to Extend Its Reach[END_REF].

Finally, our study offers a new model for Myo6 self-dimerization. The step size that myosins are able to take on actin has initially been proposed to be proportional to the size of its Lever arm, yet the discovery of Myo6 has challenged this hypothesis since despite its short canonical Lever arm, Myo6

was found able to take large steps on actin (see section E.5). Several models were proposed and debated to explain this step size, involving either a distal dimerization mediated by partners [START_REF] Yu | Myosin VI Undergoes Cargo-Mediated Dimerization[END_REF]) and providing Myo6 with a highly flexible Lever arm or internal dimerization mediated either by SAH (Kim et al. 2010) or 3HB [START_REF] Mukherjea | Myosin VI Dimerization Triggers an Unfolding of a Three-Helix Bundle in Order to Extend Its Reach[END_REF] domains. Here thanks to a crystal structure, our study demonstrates the ability of Myo6 to undergoes anti-parallel dimerization through residues 875-940. Importantly, an atomic model of the Myo6 dimer can thus be built thanks to this information. It is compatible with a distance of 31 nm between the two heads of the Myo6 dimer which explains how the motor can step on actin using ~30 nm steps. This model is further supported by the fact that motility assays demonstrated that a triple mutant (that does not interfere with the 3HB fold but disrupts proximal dimerization renders Myo6 unable to step on actin. Finally, the inability of our triple mutant anti-proximal dimerization to rescue Myo6 mediated transferrin uptake supports the importance of proximal dimerization for endocytosis. There are several isoforms of class II myosin whose heavy chains are encoded by:

• MYH1, MYH2, MYH3, MYH4, MYH7B, MYH8, MYH13, MYH15 for skeletal muscle contraction

• MYH6 and MYH7 for cardiac muscle contraction

• MYH11 for smooth muscle contraction

• MYH9, MYH10, MYH14 for non-muscle cells

• Uncharacterized pseudogene MYH16 A.1. Sarcomeric Myo2
Skeletal (SkMyo2) and cardiac (CardMyo2) muscles Myo2 associate with actin in a contractile unit called the sarcomere (Figure 69). The sarcomere is composed of an alternation of thick myosin filaments and thin actin filaments that give a striated aspect to cardiac and striated muscles. CardMyo2 is specifically found in the heart tissue, and its contraction mediates heartbeat, while SkMyo2 is found in all striated muscles and mediates voluntary contraction. In both cases, contraction of the sarcomere is controlled by the actin-associated proteins troponin and tropomyosin. Moreover, mutations in the α-CardMyo2 Lever arm seem to be involved in an autosomal dominant form of atrial septal defect, one of the most common forms of congenital heart malformations [START_REF] Ching | Mutation in Myosin Heavy Chain 6 Causes Atrial Septal Defect[END_REF]. Mutations in SkMyo2 SH1 can cause abnormal formation of type IIa fibers, and has thus been associated with hereditary inclusion body myopathy that causes proximal muscle weakness and atrophy [START_REF] Martinsson | Autosomal Dominant Myopathy: Missense Mutation (Glu-706 --> Lys) in the Myosin Heavy Chain IIa Gene[END_REF]. A mutation on SkMyo2 Motor domain was reported to cause myositis (immune-mediated muscle inflammation) in horses [START_REF] Finno | A Missense Mutation in MYH1 Is Associated with Susceptibility to Immune-Mediated Myositis in Quarter Horses[END_REF].

A.2. Smooth muscle Myo2

Smooth muscle Myo2 (SmMyo2) enables involuntary smooth muscle contraction without adopting a sarcomere organization as opposed to CardMyo2 and SkMyo2. It participates in the contraction of blood vessels, digestive track and lung muscles. Concerning the regulation of force production, SmMyo2 differs from sarcomeric myosin: in SmMyo2, force production is regulated by phosphorylation of the light chain [START_REF] Craig | Light-Chain Phosphorylation Controls the Conformation of Vertebrate Non-Muscle and Smooth Muscle Myosin Molecules[END_REF]. Mutations of SmMyo2 are involved in circulatory system diseases such as non-syndromic thoracic aortic aneurysm and dissection (TAAD) [START_REF] Pomianowski | The Genetics and Genomics of Thoracic Aortic Disease[END_REF], and urinary and intestinal diseases such as Berdon syndrome [START_REF] Gauthier | A Homozygous Loss-of-Function Variant in MYH11 in a Case with Megacystis-Microcolon-Intestinal Hypoperistalsis Syndrome[END_REF][START_REF] Yetman | Clinical and Echocardiographic Prevalence and Detection of Congenital and Acquired Cardiac Abnormalities in Girls and Women with the Turner Syndrome[END_REF]. Additionally, smooth muscle hypercontractility has been associated with asthma [START_REF] Dowell | Airway Smooth Muscle: A Potential Target for Asthma Therapy[END_REF].

A.3. Non-muscle Myo2

Non-muscle Myo2s (NM2A encoded by MYH9, NM2B encoded by MYH10 and NM2C encoded by MYH14), as muscle Myo2, can associate in filaments to perform their functions. The force produced is used for cytokinesis, cell morphology, cell migration, cell adhesion and signaling (reviewed in Newell-Litwa, Horwitz, and Lamers 2015). Proper functioning of NM2s appears to be vital for organism development. Typically, it has been found that depletion of NM2A in mice results in embryo death within 6.5 days [START_REF] Conti | Defects in Cell Adhesion and the Visceral Endoderm Following Ablation of Nonmuscle Myosin Heavy Chain II-A in Mice[END_REF]). Moreover, a mutation in NM2B results in hearts developing outside of the mice bodies [START_REF] Ma | A Point Mutation in Myh10 Causes Major Defects in Heart Development and Body Wall Closure[END_REF].

Still in mice, the L458R mutation in NM2B leads to cyanosis and respiratory distress due to extracellular matrix rearrangement in their lungs (Kim et al. 2018). Aberrant splicing of NM2C has been identified in myotonic dystrophy type 1 (the most common form of muscular dystrophy), suggesting implication of NM2C in the disease [START_REF] Rinaldi | Aberrant Splicing and Expression of the Non Muscle Myosin Heavy-Chain Gene MYH14 in DM1 Muscle Tissues[END_REF]. NM2A mutations result in a rare platelet disorder known as MYH9 syndrome [START_REF] Kelley | Mutation of MYH9, Encoding Non-Muscle Myosin Heavy Chain A, in May-Hegglin Anomaly[END_REF][START_REF] Heath | Nonmuscle Myosin Heavy Chain IIA Mutations Define a Spectrum of Autosomal Dominant Macrothrombocytopenias: May-Hegglin Anomaly and Fechtner, Sebastian, Epstein, and Alport-like Syndromes[END_REF][START_REF] Kunishima | Identification of Six Novel MYH9 Mutations and Genotype-Phenotype Relationships in Autosomal Dominant Macrothrombocytopenia with Leukocyte Inclusions[END_REF][START_REF] Seri | MYH9-Related Disease: May-Hegglin Anomaly, Sebastian Syndrome, Fechtner Syndrome, and Epstein Syndrome Are Not Distinct Entities but Represent a Variable Expression of a Single Illness[END_REF] due to NM2A aggregation. Such condition can lead to hearing loss, cataracts and nephritis [START_REF] Seri | MYH9-Related Disease: May-Hegglin Anomaly, Sebastian Syndrome, Fechtner Syndrome, and Epstein Syndrome Are Not Distinct Entities but Represent a Variable Expression of a Single Illness[END_REF].

Additionally, because of its role in cytokinesis, NM2C could be required for tumor cell division [START_REF] Jana | A Specific Isoform of Nonmuscle Myosin II-C Is Required for Cytokinesis in a Tumor Cell Line[END_REF]. NM2A has been associated to breast cancer invasiveness as reduced expression of NM2A results in reduced invasion of MCF-7 breast cancer cells [START_REF] Derycke | The Role of Non-Muscle Myosin IIA in Aggregation and Invasion of Human MCF-7 Breast Cancer Cells[END_REF]. In contrast, in squamous cell carcinomas (SCCs), NM2A was found to act as a tumor suppressor [START_REF] Schramek | Direct in Vivo RNAi Screen Unveils Myosin IIa as a Tumor Suppressor of Squamous Cell Carcinomas[END_REF], since the knock-out of NM2A accelerates tumor progression.

Moreover, genetic studies of families suffering from hearing loss associated eight NM2C mutations and one NM2A mutation to non-syndromic hearing loss [START_REF] Donaudy | Nonmuscle Myosin Heavy-Chain Gene MYH14 Is Expressed in Cochlea and Mutated in Patients Affected by Autosomal Dominant Hearing Impairment (DFNA4)[END_REF][START_REF] Kim | Genetic Association of MYH Genes with Hereditary Hearing Loss in Korea[END_REF][START_REF] Kim | Discovery of MYH14 as an Important and Unique Deafness Gene Causing Prelingually Severe Autosomal Dominant Nonsyndromic Hearing Loss[END_REF][START_REF] Choi | A Complex Phenotype of Peripheral Neuropathy, Myopathy, Hoarseness, and Hearing Loss Is Linked to an Autosomal Dominant Mutation in MYH14[END_REF][START_REF] Lalwani | Human Nonsyndromic Hereditary Deafness DFNA17 Is Due to a Mutation in Nonmuscle Myosin MYH9[END_REF]. In mice, NM2A is located in the stereocilia of inner ear hair cells [START_REF] Mhatre | Expression of Myh9 in the Mammalian Cochlea: Localization within the Stereocilia[END_REF], and NM2C is mainly found, distinctly of NM2A, in the organ of Corti, the stria vascularis, and in the cells surrounding the scala media [START_REF] Lalwani | Human Nonsyndromic Hereditary Deafness DFNA17 Is Due to a Mutation in Nonmuscle Myosin MYH9[END_REF], reinforcing the role of NM2A and NM2C in hearing, although their precise functions could be different and remain unclear.

B. Targeting myosin Motors against diseases

Therefore, specific targeting of certain myosin isoforms against diseases appears of particular interest. The potential of myosins as therapeutical targets has already been addressed [START_REF] Trivedi | The Myosin Family of Mechanoenzymes: From Mechanisms to Therapeutic Approaches[END_REF]. However, due to the high similarity of their Motor domains, and to the ubiquitous expression of many myosin classes in cells, specific targeting of one myosin class or of one specific member of a myosin class is particularly challenging. Yet, it is an unavoidable challenge to be overcome for the development of efficient therapies without impairing vital functions such as respiration or heartbeat. To date, several myosin inhibitors and activators exhibiting various specificities have been reported (see section B.2.). The majority are directed toward the Myo2s. In particular, several of them, targeting CardMyo2, are now in clinical trials. The β-CardMyo2 inhibitor Mavacamten has been approved by the FDA in April, 2022, highlighting the potential of myosins as druggable targets (Table 14).

B.1. Five allosteric pockets found in myosin Motor domain Thanks to the structures obtained from myosin-drug co-crystals (Planelles- [START_REF] Herrero | Bases Mécanistiques et Structurales de La Régulation de l'activité Des Myosines[END_REF][START_REF] Sirigu | Highly Selective Inhibition of Myosin Motors Provides the Basis of Potential Therapeutic Application[END_REF]Allingham, Smith, and Rayment 2005;[START_REF] Fedorov | The Mechanism of Pentabromopseudilin Inhibition of Myosin Motor Activity[END_REF][START_REF] Zhou | Structural Basis of Fusarium Myosin I Inhibition by Phenamacril[END_REF], five different allosteric pockets in the myosin Motor domain have been described. Drug interactions are mainly hydrophobic and most likely to happen during the pre-powerstroke state (see General introduction; section D: the pre-powerstroke happens at the end of the recovery stroke). The CK-571 binding pocket is an exception as it exists in an intermediate state along the recovery stroke. In the pre-powerstroke state, myosin has low affinity for actin and does not generate force, thus blocking CK-571 was described as a specific inhibitor of SmMyo2 exhibiting a particularly high affinity (IC50 ~9 nM). The crystal structure reveals that CK-571 binding near the SH1 traps the Motor in an intermediate state of the recovery stroke, thus prevents actin binding and force production [START_REF] Sirigu | Highly Selective Inhibition of Myosin Motors Provides the Basis of Potential Therapeutic Application[END_REF]. Intriguingly, even if all the residues involved in CK-571 binding are conserved in Myo2, only SmMyo2 is inhibited, emphasizing that subtle changes among these allosteric machines can be sufficient for gaining selectivity. This supports the potential of selective myosin Motor inhibition for the development of various treatments. The Halogenated pseudilins were uniquely described as inhibitors of both unconventional and conventional myosins. Halogenated pseudilins were first described in 2009 as myosin inhibitors [START_REF] Fedorov | The Mechanism of Pentabromopseudilin Inhibition of Myosin Motor Activity[END_REF]) able to inhibit myosins of class I, II and V in the micromolar range, but not able to inhibit myosins of class VI and class VII [START_REF] Preller | Inhibition of Myosin ATPase Activity by Halogenated Pseudilins: A Structure-Activity Study[END_REF][START_REF] Chinthalapudi | Mechanism and Specificity of Pentachloropseudilin-Mediated Inhibition of Myosin Motor Activity[END_REF][START_REF] Fedorov | The Mechanism of Pentabromopseudilin Inhibition of Myosin Motor Activity[END_REF]). Crystal structures allow deTailed description of the binding site of halogenated pseudilins near the actin loops (PDB: 2XO8, 2XEL, 2JHR). Although halogenated pseudilins are not specific for one myosin isoform, chlorinated and brominated pseudilins were shown to exhibit preference either for Myo1 or Myo5, respectively [START_REF] Preller | Inhibition of Myosin ATPase Activity by Halogenated Pseudilins: A Structure-Activity Study[END_REF]. These findings assess the potential of halogenated pseudilin derivatives for selective inhibition of myosins in the future, especially considering the fact that actin loops exhibit variability among classes (Robert-Paganin et al. 2020), and this could be exploited for the development of specific inhibitors. The risk of off-target binding should be carefully considered, however, as several non-myosin molecules were reported to be inhibited by pseudilin [START_REF] Kunfermann | Pseudilins: Halogenated, Allosteric Inhibitors of the Non-Mevalonate Pathway Enzyme IspD[END_REF][START_REF] Zhang | A Second Target of the Antimalarial and Antibacterial Agent Fosmidomycin Revealed by Cellular Metabolic Profiling[END_REF].

Few inhibitors

The most well characterized inhibitor is blebbistatin (Straight et al. 2003) (see section C.); a Myo2 specific inhibitor whose potential for development of therapy has already been addressed [START_REF] Rauscher | Targeting Myosin by Blebbistatin Derivatives: Optimization and Pharmacological Potential[END_REF][START_REF] Roman | Recovering Actives in Multi-Antitarget and Target Design of Analogs of the Myosin II Inhibitor Blebbistatin[END_REF].

C. Blebbistatin

Blebbistatin (Figure 72) was first discovered as an inhibitor of furrow ingression through cell division (Straight et al. 2003) and named according to its ability to block cell blebbing (Straight et al. 2003). Blebbistatin has the ability to inhibit Myo2 in a submicromolar/micromolar range (Straight et al. 2003) without blocking RLC phosphorylation by MLCK (myosin light chain kinase) (Straight et al. 2003;Zhang et al. 2017;[START_REF] Wang | Blebbistatin Inhibits the Chemotaxis of Vascular Smooth Muscle Cells by Disrupting the Myosin II-Actin Interaction[END_REF]. Steady-state ATPase assays show that blebbistatin is able to efficiently inhibit SkMyo2 S1 activity both in basal and actin-activated assays [START_REF] Kovács | Mechanism of Blebbistatin Inhibition of Myosin II[END_REF]). It can even inhibit Myo2 coming from the invertebrate organisms Dictyostelium discoideum (DictyMyo2),

Argopecten irradians (ScallopMyo2) (Limouze et al. 2004) and Tarantula [START_REF] Zhao | Blebbistatin Stabilizes the Helical Order of Myosin Filaments by Promoting the Switch 2 Closed State[END_REF].

Blebbistatin contains one asymmetric carbon whose geometry appears to be particularly important for Myo2 inhibition: only the (-)-blebbistatin enantiomer have been reported to inhibit Myo2s: (+)-blebbistatin does not inhibit SmMyo2 activity, while the racemic mix does [START_REF] Wang | Blebbistatin Inhibits the Chemotaxis of Vascular Smooth Muscle Cells by Disrupting the Myosin II-Actin Interaction[END_REF]) and (-)-blebbistatin inhibits specifically skMyo2 over (+)-blebbistatin in steady-state ATPase assays [START_REF] Verhasselt | Discovery of (S)-3'-Hydroxyblebbistatin and (S)-3'-Aminoblebbistatin: Polar Myosin II Inhibitors with Superior Research Tool Properties[END_REF]. SkMyo2, to 4-80 µM for SmMyo2 (Limouze et al. 2004;[START_REF] Wang | Blebbistatin Inhibits the Chemotaxis of Vascular Smooth Muscle Cells by Disrupting the Myosin II-Actin Interaction[END_REF]Zhang et al. 2017;[START_REF] Eddinger | Potent Inhibition of Arterial Smooth Muscle Tonic Contractions by the Selective Myosin II Inhibitor, Blebbistatin[END_REF]. Such a poor inhibition of SmMyo2 is surprising, considering that its sequence is highly similar to the well inhibited NM2A and NM2B (Figure 73). Summary of IC50 measured for Myo2 different isoform in various publications [START_REF] Képiró | Para-Nitroblebbistatin, the Non-Cytotoxic and Photostable Myosin II Inhibitor[END_REF][START_REF] Várkuti | A Highly Soluble, Non-Phototoxic, Non-Fluorescent Blebbistatin Derivative[END_REF][START_REF] Verhasselt | Discovery of (S)-3'-Hydroxyblebbistatin and (S)-3'-Aminoblebbistatin: Polar Myosin II Inhibitors with Superior Research Tool Properties[END_REF]Verhasselt, Stevens, et al. 2017;Limouze et al. 2004;Zhang et al. 2017;[START_REF] Dou | Blebbistatin Specifically Inhibits Actin-Myosin Interaction in Mouse Cardiac Muscle[END_REF][START_REF] Farman | Blebbistatin: Use as Inhibitor of Muscle Contraction[END_REF][START_REF] Wang | Blebbistatin Inhibits the Chemotaxis of Vascular Smooth Muscle Cells by Disrupting the Myosin II-Actin Interaction[END_REF][START_REF] Eddinger | Potent Inhibition of Arterial Smooth Muscle Tonic Contractions by the Selective Myosin II Inhibitor, Blebbistatin[END_REF] Specificity has also been investigated in cells using a crosslinkable version of blebbistatin:

azidoblebbistatin. Azidoblebbistatin exhibits the same inhibitory and solubility properties as blebbistatin, but, under illumination at 310 nm, azidoBlebbistatin forms a covalent bond with its binding pocket. Proteins interacting with azidoblebbistatin in Dd cells treated with 5 µM of drug were identified by cell extract fluorescence and mass spectroscopy analysis after cell illumination at 310 nm.

Myo2 was found to be the main partner of azidoblebbistatin. Additionally, few other, non-myosin proteins were able to interact with azidoblebbistatin, but with much lower affinity (>50 µM vs. 6 µM) [START_REF] Képiró | Para-Nitroblebbistatin, the Non-Cytotoxic and Photostable Myosin II Inhibitor[END_REF].

C.2. Solubility and permeability

Blebbistatin is able to cross cell membranes and to inhibit contraction of the muscle tissue within minutes after administration, with an IC50 that is similar to the one found on isolated Myo2

Motors, showing the good capacity of blebbistatin to cross the plasma membrane and to diffuse in biological tissue [START_REF] Dou | Blebbistatin Specifically Inhibits Actin-Myosin Interaction in Mouse Cardiac Muscle[END_REF].

However, blebbistatin suffers from poor solubility: blebbistatin starts to precipitate over 10 µM in water [START_REF] Kolega | Phototoxicity and Photoinactivation of Blebbistatin in UV and Visible Light[END_REF][START_REF] Sakamoto | Blebbistatin, a Myosin II Inhibitor, Is Photoinactivated by Blue Light[END_REF][START_REF] Képiró | Para-Nitroblebbistatin, the Non-Cytotoxic and Photostable Myosin II Inhibitor[END_REF]). In the presence of DMSO, the limiting concentrations are ~7.5 µM at 0.1 % DMSO and ~80 µM at 10 % DMSO [START_REF] Képiró | Para-Nitroblebbistatin, the Non-Cytotoxic and Photostable Myosin II Inhibitor[END_REF].

Solubility is thus in the range of the IC50, which can be problematic in cells when precipitation can damage the model under study [START_REF] Swift | Properties of Blebbistatin for Cardiac Optical Mapping and Other Imaging Applications[END_REF]. This issue has been overcome by the development of the highly soluble blebbistatin derivative 3'-hydroxyblebbistatin [START_REF] Verhasselt | Discovery of (S)-3'-Hydroxyblebbistatin and (S)-3'-Aminoblebbistatin: Polar Myosin II Inhibitors with Superior Research Tool Properties[END_REF]) and also aminoblebbistatin [START_REF] Várkuti | A Highly Soluble, Non-Phototoxic, Non-Fluorescent Blebbistatin Derivative[END_REF].

2005; [START_REF] Ramamurthy | Kinetic Mechanism of Blebbistatin Inhibition of Nonmuscle Myosin IIb[END_REF]. Actually, illumination of blebbistatin in blue light (425 nm) even results in an increase of NM2B ATPase activity from ~0.17 to ~0.36 s -1 [START_REF] Ramamurthy | Kinetic Mechanism of Blebbistatin Inhibition of Nonmuscle Myosin IIb[END_REF].

C.4.c. Blebbistatin requires careful use

These findings make blebbistatin use and blebbistatin mechanism study particularly challenging. Each illumination wavelength used for experiments need a careful check to avoid inactivation or toxicity. In particular, ATP binding to myosin upon blebbistatin addition cannot be studied by tryptophan fluorescence quenching, as such an experiment requires fluorescence measurement at 295 nm, which will result in blebbistatin degradation within 1 s, compromising the subsequent observations [START_REF] Ramamurthy | Kinetic Mechanism of Blebbistatin Inhibition of Nonmuscle Myosin IIb[END_REF]). It should be noted that illumination at 310 nm has no impact on blebbistatin stability, thus crosslink experiments using azidoblebbistatin at 310 nm still look trustworthy [START_REF] Képiró | Para-Nitroblebbistatin, the Non-Cytotoxic and Photostable Myosin II Inhibitor[END_REF].

Despite all its flaws, with careful choice of illumination wavelength and use of « moderate » concentrations, blebbistatin is not too harmful for cell viability and has already allowed great findings concerning Myo2 functions and related diseases. Myo2 inhibition by blebbistatin was found to be beneficial for various medical indications reviewed in [START_REF] Rauscher | Targeting Myosin by Blebbistatin Derivatives: Optimization and Pharmacological Potential[END_REF] : notably, NM2 inhibition can reduce infiltration of leukemia cells in the central nervous system, methamphetamine seeking and lung carcinoma migration. SmMyo2 inhibition can reduce thrombus formation and blood pressure.

CardMyo2 inhibition can reduce cardiac hypercontractility, and SkMyo2 inhibition can reduce spasticity. It was also suggested that blebbistatin's sensitivity to blue light could make it a photodynamic agent against cancer [START_REF] Mikulich | Blebbistatin, a Myosin Inhibitor, Is Phototoxic to Human Cancer Cells under Exposure to Blue Light[END_REF].

C.5. Blebbistatin degradation mechanism

C.5.a. UV-blue light illumination modifies the spectral properties of blebbistatin

In order to identify the reaction product resulting from UV-blue light illumination, evolution of blebbistatin spectral properties was analyzed. The absorption spectra of blebbistatin shows a peak at 430 nm that decreases after illumination in either blue (450 -490 nm) or UV (365nm) for 1 h (Figure 74). Surprisingly, the profiles differ depending on the wavelength used. If blue light has been used, a double peak appears; if UV light was used, absorbance slightly increases between 300 and 380 nm. In contrast, illumination at 510 -560 or 590 -650 nm results in no modification within the absorbance spectrum consistent with the absence of change in toxicity [START_REF] Kolega | Phototoxicity and Photoinactivation of Blebbistatin in UV and Visible Light[END_REF].

C.5.b. The photoinactivation product is not responsible for toxicity

Interestingly, addition of photodegraded blebbistatin to BAECs does not result in cell death, not even 50 µM for 18 h. Hence, Blebbistatin phototoxicity after illumination may come from a short life intermediate product, such as radicals that might be the same for UV and blue light illumination [START_REF] Kolega | Phototoxicity and Photoinactivation of Blebbistatin in UV and Visible Light[END_REF]). Indeed, ROS liberation was detected after blebbistatin and blue light illumination using dihydrorhodamine 123 (ROS sensor). It should be noted that, in contrast, the Singlet Oxygen Sensor Green was not oxidized, which gives us insights on the degradation mechanism of blebbistatin [START_REF] Mikulich | Blebbistatin, a Myosin Inhibitor, Is Phototoxic to Human Cancer Cells under Exposure to Blue Light[END_REF].

Also of interest, the kinetics of degradation of blebbistatin do not depend only on light exposure, but also on buffer conditions: no degradation was seen on PBS or 100% DMSO after 30minute exposure to 480 nm light, but degradation occurs in water (Verhasselt, Roman, Bracke, et al. 2017). Verhasselt, Roman, Bracke, et al. (2017) proposed that degradation may be promoted by hydroxyl or peroxyl radicals in water that will ultimately result in oxidative degradation of blebbistatin.

C.5.c. Development of photostable derivatives

Finally, deTailed analysis of blebbistatin reaction with blue light using femtosecond transient absorption spectroscopy and chemoselective ROS fluorescent probes results in the proposition of a reactional mechanism [START_REF] Li | Dynamics of Oxygen-Independent Photocleavage of Blebbistatin as a One-Photon Blue or Two-Photon Near-Infrared Light-Gated Hydroxyl Radical Photocage[END_REF] for degradation mediated by blue light (Figure 75). Liberation of the hydroxyl radical during the photo-inactivation of blebbistatin had been previously proposed (Verhasselt, Roman, Bracke, et al. 2017).

In this context an electron withdrawing group could prevent or minimize blebbistatin oxidative degradation. Indeed, para substitution of the blebbistatin D-cycle by an electron-withdrawing group: azido, nitro, chloro or amino, abolishes blebbistatin's sensitivity to blue light (no change in absorbance spectra after 15 minutes of illumination at 480 nm). After Hela cells are treated with para-Nitroblebbistatin or para-Aminoblebbistatin and illuminated at 480nm, cell mortality was similar to the non-treated control, while treatments with blebbistatin and para-Chloroblebbistatin result in stronger mortality, characteristic of blue light phototoxicity [START_REF] Várkuti | A Highly Soluble, Non-Phototoxic, Non-Fluorescent Blebbistatin Derivative[END_REF][START_REF] Képiró | Para-Nitroblebbistatin, the Non-Cytotoxic and Photostable Myosin II Inhibitor[END_REF].

Addition of these electron-withdrawing groups also results in loss of fluorescence at 410 nm, which is much more convenient for fluorescence experiments to avoid interferences [START_REF] Várkuti | A Highly Soluble, Non-Phototoxic, Non-Fluorescent Blebbistatin Derivative[END_REF][START_REF] Képiró | Para-Nitroblebbistatin, the Non-Cytotoxic and Photostable Myosin II Inhibitor[END_REF]. Para-Nitroblebbistatin and para-Aminoblebbistatin also keep good pharmaceutical activity: they can efficiently inhibit SkMyo2 and Dicty Myo2 in steady-state ATPase assays, even if para-Aminoblebbistatin exhibits a higher IC50 against SkMyo2 (0.47 ± 0.06 µM) compared to blebbistatin and para-Nitroblebbistatin (0.11 ± 0.009 µM and 0.1 ± 0.004 µM, respectively) [START_REF] Várkuti | A Highly Soluble, Non-Phototoxic, Non-Fluorescent Blebbistatin Derivative[END_REF][START_REF] Képiró | Para-Nitroblebbistatin, the Non-Cytotoxic and Photostable Myosin II Inhibitor[END_REF].

para-Aminoblebbistatin is 40-fold more soluble than blebbistatin making the former particularly well suited for use in cells despite its higher IC50 [START_REF] Várkuti | A Highly Soluble, Non-Phototoxic, Non-Fluorescent Blebbistatin Derivative[END_REF]. In contrast, the blebbistatin-like solubility of para-Nitroblebbistatin limits its use in cells.

C.6. Blebbistatin inhibition mechanism

To understand how blebbistatin inhibits Myo2 ATPase activity, deTailed studies of its impact on various steps of the Myo2 kinetic cycle have been pursued by several groups.

C.6.a. Blebbistatin does not disturb ATP binding and hydrolysis

The fluorescence of the ATP analog MantATP increases when it binds to Myosin. Experiments with MantATP have consistently shown that Blebbistatin does not compete against the nucleotide, as an increase in fluorescence can be seen both with and without excess of blebbistatin using NM2A (Limouze et al. 2004), NM2B [START_REF] Ramamurthy | Kinetic Mechanism of Blebbistatin Inhibition of Nonmuscle Myosin IIb[END_REF]) and SkMyo2 [START_REF] Kovács | Mechanism of Blebbistatin Inhibition of Myosin II[END_REF]). Moreover, blebbistatin doesn't seem to impact basal [γ-32 P]ATP hydrolysis of NM2B either [START_REF] Ramamurthy | Kinetic Mechanism of Blebbistatin Inhibition of Nonmuscle Myosin IIb[END_REF].

C.6.b. Blebbistatin reduces Pi release

C.6.c. Blebbistatin reduces actin binding

Addition of Blebbistatin to cosedimentation experiments using Myo2 S1 and actin during steady state ATP hydrolysis results in blebbistatin mainly present in the supernatant and a slightly smaller fraction of S1 bound to actin [START_REF] Kovács | Mechanism of Blebbistatin Inhibition of Myosin II[END_REF]. This was further confirmed using pyrene-actin fluorescence quenching, in which a large excess of blebbistatin prevented NM2B to rebind to actin after ATP-induced dissociation [START_REF] Ramamurthy | Kinetic Mechanism of Blebbistatin Inhibition of Nonmuscle Myosin IIb[END_REF].

Thin filaments (actin, tropomyosin and troponin filaments) have the ability to bind to myosin thick filaments in the presence of calcium. Addition of calcium to mixes containing thin and thick filaments results in formation of granulates and lots of thin and thick filaments interacting in negative staining. When a homogenate of Tarantula muscles containing both thin and thick filaments was incubated with 100 µM blebbistatin prior to addition of calcium, neither macroscopic granulates or thin-thick filaments bound to each other were seen on negative staining, as opposed to the controls, suggesting that blebbistatin prevents actin-myosin interaction [START_REF] Zhao | Blebbistatin Stabilizes the Helical Order of Myosin Filaments by Promoting the Switch 2 Closed State[END_REF].

These findings are in line with blebbistatin stabilizing the pre-powerstroke (MgADP.Pi) state, a state characterized by low affinity for actin [START_REF] Ramamurthy | Kinetic Mechanism of Blebbistatin Inhibition of Nonmuscle Myosin IIb[END_REF].

C.6.d. Blebbistatin might slightly reduce ADP release

Recently, a crystal structure of DictyMyo2 bound to blebbistatin and ADP has been proposed

to correspond to the Myo2 ADP release state [START_REF] Ewert | Structural and Computational Insights into a Blebbistatin-Bound Myosin•ADP Complex with Characteristics of an ADP-Release Conformation along the Two-Step Myosin Power Stoke[END_REF], proposing an impact of blebbistatin on ADP release. However, in kinetic studies, the impact of blebbistatin on ADP release seems rather limited compared to its impact on Pi release and actin binding:

Impact of blebbistatin on ADP dissociation from acto-NM2B can be monitored using mant-ADP fluorescence. Addition of blebbistatin excess results in lower ADP dissociation rate (~1.3 s -1 instead of 1.8 s -1 ). But DMSO was found to reduce ADP dissociation rate to ~1.4 s -1 on its own. It is thus unclear if blebbistatin effect on ADP release is significant or not [START_REF] Ramamurthy | Kinetic Mechanism of Blebbistatin Inhibition of Nonmuscle Myosin IIb[END_REF]. Overall, the impact of blebbistatin on ADP release, if significant, remains much subtler than its effect on Pi release and actin binding. This is further supported by the fact that in actin cosedimentation, a large majority of blebbistatin is found in the supernatant (i.e., bound to low actin affinity states of the Myo2 motor) (see section C.6.c.).

C.6.e. Blebbistatin does not promote motor dissociation from actin

No significant effect on Myo2 S1 binding rate and affinity to actin was reported upon addition of blebbistatin in excess for the Myo2 high affinity states: rigor (NF) (Kovács et al. 2004 on SkMyo2 andRamamurthy et al. 2004 on NM2B) or ADP [START_REF] Ramamurthy | Kinetic Mechanism of Blebbistatin Inhibition of Nonmuscle Myosin IIb[END_REF]

C.7. Blebbistatin site

The first structure of Myo2 bound to blebbistatin was solved at 2 Å resolution using X-ray crystallography (Allingham, Smith, and Rayment 2005). The Dictyostelium myosin Motor in the pre- Gln637 and Leu641 (Allingham, Smith, and Rayment 2005). Movement of Leu262 and Tyr634 side chains are required to allow blebbistatin binding in its tight pocket (Allingham, Smith, and Rayment 2005). The specificity for the (-)-blebbistatin can be easily understood on the basis of the structure as the chiral alcohol group is able to form a hydrogen bond with the Leu262 carbonyl and Gly240 amine.

An additional hydrogen bond can be found between the Ser456 amine and the blebbistatin carbonyl (Figure 76). It should be noted that this allosteric pocket might be occupied by many other myosin inhibitors. Indeed, BHC [START_REF] Brawley | Synthesis and Evaluation of 4-Hydroxycoumarin Imines as Inhibitors of Class II Myosins[END_REF], aficamten [START_REF] Chuang | Discovery of Aficamten (CK-274), a Next-Generation Cardiac Myosin Inhibitor for the Treatment of Hypertrophic Cardiomyopathy[END_REF], TIP [START_REF] Heissler | Kinetic Properties and Small-Molecule Inhibition of Human Myosin-6[END_REF] and PPBA [START_REF] Hiratsuka | The Interaction of Phe472 with a Fluorescent Inhibitor Bound to the Complex of Myosin Subfragment-1 with Nucleotide[END_REF] were all proposed to occupy the same binding site, although this needs confirmation with high resolution crystal structures. Key residues for blebbistatin binding are not conserved in several unconventional myosins which leads to the specificity of blebbistatin for Myo2s: in particular, Ser456 is replaced by aromatics residues in myosin I, V, X (Allingham, Smith, and Rayment 2005). In fact, mutation of this residue in SmMyo2 or NM2 into Phe results in severe loss of blebbistatin inhibition (although it alters the normal ATPase activity of NM2B (which increases) and NM2C (which decreases)) (Zhang et al. 2017).

Dicty RFG YLL IS FE CI T V Y Q L NM2A RFG YLL IA FE CI T V Y Q L NM2B RFG YLL IA FE CI T V Y S L NM2C RFG YLL IA FE CI T V Y S L Cardiac RFG YLL IA FE CI T V H N L Smooth RFG YLL IA FE CI T V Y Q L Skeletal RFG YLL IA LE CI T V F N L Myo1 RFG YLL IY FE CI/II C V/A F S L Myo5 RFG YLL IY FE CI A V F S I Myo10 RFG YLL IF FE NI A V F S L Myo15 RFG YLL IY FE CI A V F S L
Mutations Ala454Tyr, Trp, Arg, Glu in NM2A-HMM results as well in loss of blebbistatin inhibition in ATPase assays. Other mutations such as Smooth Ser266Leu (highly conserved in Myo2 and Myo1) and Thr474Ala (highly conserved in Myo2, Cys in Myo1 or Ala in Myo5) did not impair blebbistatin ability to inhibit smooth (Zhang et al. 2017). Blebbistatin has no effect on Drosophila melanogaster cells (Straight et al. 2003) due to Met466 in Drosophila Myo2 instead of Ile455 in DictyMyo2 [START_REF] Heissler | Kinetic Characterization of the Sole Nonmuscle Myosin-2 from the Model Organism Drosophila Melanogaster[END_REF]. Similarly, the Ile455Met mutant in NM2A-HMM results in a reduction of the inhibition (although it is less severe than for Ala454Phe) (Zhang et al. 2017).

However, it these studies are not sufficient to explore the rules of specificity in this site as blebbistatin (and derivatives) exhibit various inhibition among Myo2 (Allingham, Smith, and Rayment 2005). Thus, to understand the specificity, small changes within the pocket and in its close proximity should be further investigated. Especially considering how tight the pocket is, indeed the blebbistatin pocket is only 417.77 Å 3 [START_REF] Roman | Recovering Actives in Multi-Antitarget and Target Design of Analogs of the Myosin II Inhibitor Blebbistatin[END_REF].

Moreover, the blebbistatin pocket is rather buried inside the Myo2 Motor. The structure of blebbistatin in complex with the Myo2 Motor cannot reveal how blebbistatin reaches its binding site and detach from it. As blebbistatin inhibition is reversible upon blebbistatin wash out, blebbistatin must find its way out of the pocket. Roman et al. (2018) proposed a potential tunnel that may enable through branching of an aromatic cycle on C6 and C7 would be a promising way to improve blebbistatin potency (Figure 78). To assess this hypothesis, (Verhasselt, Stevens, et al. 2017) thus synthesized new derivatives with bigger A-ring. However, none of them resulted in gain in inhibition potency and they exhibited worse solubility than blebbistatin.

C.8.a. A-cycle modification

C.8.b. C-ring substitution

There is a very limited room for modification on blebbistatin's C-ring, yet an arginine in close proximity could result in an extra H-bond for a short-to-medium size substitute [START_REF] Roman | Synthesis of C-Ring-Modified Blebbistatin Derivatives and Evaluation of Their Myosin II ATPase Inhibitory Potency[END_REF]) (Figure 80). [START_REF] Roman | Synthesis of C-Ring-Modified Blebbistatin Derivatives and Evaluation of Their Myosin II ATPase Inhibitory Potency[END_REF] thus proposed a serie of 4 derivatives. They however result in severe or complete loss of inhibition. Thus C-ring modification does not seem to be the most promising approach for optimizing the potency of blebbistatin.

C.8.c. D-ring substitution

Addition of small groups in D-ring meta position (cyano, hydroxyl or amino) can still inhibit SkMyo2 but are less efficient than blebbistatin (IC50 10-20-fold higher) while bigger groups: carbamoyl and carboxy results in no inhibition [START_REF] Verhasselt | Discovery of (S)-3'-Hydroxyblebbistatin and (S)-3'-Aminoblebbistatin: Polar Myosin II Inhibitors with Superior Research Tool Properties[END_REF])(Figure 81). Actually, the most successful modification of the blebbistatin scaffold was reached through para substitutions of the D-ring (see section C.5.c.). They resulted in improved stability, solubility and fewer toxicity [START_REF] Várkuti | A Highly Soluble, Non-Phototoxic, Non-Fluorescent Blebbistatin Derivative[END_REF][START_REF] Verhasselt | Discovery of (S)-3'-Hydroxyblebbistatin and (S)-3'-Aminoblebbistatin: Polar Myosin II Inhibitors with Superior Research Tool Properties[END_REF][START_REF] Képiró | Para-Nitroblebbistatin, the Non-Cytotoxic and Photostable Myosin II Inhibitor[END_REF].

Strikingly, from all the derivatives described up to date, only two of them exhibit an improved potency compared to blebbistatin (propenoxyBlebb (S-12) and pNitroBlebb (S-29) exhibit a 2.3 and 2.5-fold potency improvement respectively) (Verhasselt, Roman, Bracke, et al. 2017).

Interestingly, [START_REF] Gyimesi | Improved Inhibitory and Absorption, Distribution, Metabolism, Excretion, and Toxicology (ADMET) Properties of Blebbistatin Derivatives Indicate That Blebbistatin Scaffold Is Ideal for Drug Development Targeting Myosin-2[END_REF] pointed out that, if pNitroBlebb exhibits an improved potency for SkMyo2, this is not true for the other Myo2 isoforms tested [START_REF] Gyimesi | Improved Inhibitory and Absorption, Distribution, Metabolism, Excretion, and Toxicology (ADMET) Properties of Blebbistatin Derivatives Indicate That Blebbistatin Scaffold Is Ideal for Drug Development Targeting Myosin-2[END_REF], resulting in a relative specificity. Although it is not sufficient to call pNitroBlebb a specific inhibitor of SkMyo2, it highlights the potential of development of blebbistatin derivatives with both increased potency and selectivity.

Objectives

To further understand the specificity of the blebbistatin binding pocket among myosins, we started a collaboration with András Málnási Csizmadia's group as they were developing a blebbistatin derivative (MPH-220) that showed good stability, no detectable cytotoxicity and exquisite specificity for fast SkMyo2, characteristics that make it a good drug candidate against spasticity. Indeed, they further demonstrate that MPH-220 is an efficient muscle relaxant with no detectable side effects on the cardiac and respiratory functions in mice [START_REF] Gyimesi | Single Residue Variation in Skeletal Muscle Myosin Enables Direct and Selective Drug Targeting for Spasticity and Muscle Stiffness[END_REF]. MPH-220 differs from blebbistatin in two points: the A-ring was replaced by a methylthiophen moiety, and the D-ring was substituted by a morpholino group in para (Figure 82). Similarly, for blebbistatin, photodegradation was found to promote loss of the hydroxyl group [START_REF] Li | Dynamics of Oxygen-Independent Photocleavage of Blebbistatin as a One-Photon Blue or Two-Photon Near-Infrared Light-Gated Hydroxyl Radical Photocage[END_REF]) (see section C.5.c). Yet, we were surprised as András Málnási Csizmadia's group monitored the Mw of MPH-220 over time by mass spectroscopy and could not detect degradation or toxicity after incubation with human fibroblast for 120 hours. Thus, the compound seemed stable in a physiological context, and light does not seem sufficient to degrade the compound.

We first wondered whether the degradation had occurred prior or after MPH-220 binding to skMyo2. I thus co-crystallized Myo2 with the putative degraded form of MPH-220 produced by our collaborators (as presented in the Figure 83). The best crystal I obtained with the degraded form was diffracting to ~3.7 Å only, and I found no density for the compound, so the procedure was inconclusive.

In parallel, András Málnási Csizmadia's group couldn't detect any inhibition of SkMyo2 ATPase activity Growing crystals on limited light exposure might have been helpful to prevent degradation, as I managed to get one crystal containing the non-degraded form of MPH-220. Yet, it may not be a key parameter on its own, as even by limited light exposure, degradation was seen in certain crystals.

Overall, for discovering what leads to MPH-220 degradation, further investigations were needed.

A.1.c. Dithiothréitol (DTT) in combination with light responsible of MPH-220 degradation

We next looked at the impact of crystallization reagents on the dehydroxylation of MPH-220.

The crystallization condition contained ~35-37% PEG 600, 0.1 M Hepes-NaOH, pH 7.2, and a high concentration of reducing reagent (20 mM DTT).

Dehydroxylation of blebbistatin by light can be easily followed by monitoring light absorbance [START_REF] Kolega | Phototoxicity and Photoinactivation of Blebbistatin in UV and Visible Light[END_REF]. We looked at the ability of different parameters to either slow down or aggravate blebbistatin photodegradation by following absorbance from 300 to 500 nm of different time points under exposure to white light.

With the help of Helena Sirkia, engineer in our team, we monitored the effect of the original SkMyo2 protein buffer, of pH (from 6.5 to 9.8), of DTT and of the usual crystallization additives 3% methanol, 3% ethanol and 3% dichloromethane on blebbistatin stability.

When blebbistatin is degraded upon light exposure the spectral properties of blebbistatin evolve (see section C.4.): the absorption peak at ~400-450 nm decreases, and the absorption peak at 330-350 nm increases.

In our hands, in either SkMyo2 buffer protein or water, when exposed to white light, blebbistatin degradation is rather slow. During the first 3h, absorption at 400-450 nm decreased, but no increase can be seen on the 330-350 nm range. After ~18h, a clear absorbance peak starts to emerge at 330-350nm, in particular in the SkMyo2 buffer (Figure 85). We thus repeated this experiment to compare the effect of pH (from 6.5 to 9.8), of DTT ( 20µM or 5 mM), of 3% methanol, of 3% ethanol and of 3% dichloromethane on the absorption spectrum of blebbistatin after ~18h. All the tested condition resulted in aggravation of blebbistatin degradation compared to the control (Blebbistatin in water). Higher absorbance peaks can be seen between 330 and 350 nm for all the tested reagents (Figure 86). From these data, a neutral pH is the most adapted to avoid hydroxyl loss, but high DTT concentration as used on the crystallization condition can be deleterious as 5 mM DTT results in the biggest absorbance increase at 350 nm. Interestingly, repeating the same experiment in the dark, 5 mM DTT does not promote blebbistatin degradation. Thus, a combination of DTT and light exposure is more likely to be the explanation for the degradation seen in our crystal structure.

This certainly makes co-crystallization of SkMyo2 with blebbistatin derivatives in this crystallization condition more difficult. Indeed, I had found that DTT was essential to reduce the multiplicity of these crystals, and to improve the resolution to which they could diffract. Special care should thus be taken to prevent as much as possible light exposure when using DTT with blebbistatin derivatives. In Brief Ma ´te ´Gyimesi,1,2, * A ´da ´m I. Horva ´th, 1 Demeter Tu ´ro ´s, 1 Sharad Kumar Suthar, 1,3 Ma ´te ´Pe ´nzes, 1 Csilla Kurdi, 1 Louise Canon, 4 Carlos Kikuti, 4 Kathleen M. Ruppel, 5 Darshan V. Trivedi, 5 James A. Spudich, 5 Istva ´n L} orincz, 3Anna A ´. Rauscher, 1,2 Miha ´ly Kova ´cs, 1,6 Endre Pa ´l, 7 Sa ´muel Komoly, 7 Anne Houdusse, 4 and Andra ´s Ma ´lna ´si-Csizmadia 1,6,8, *

INTRODUCTION

Spasticity-characterized by involuntary increased tone of skeletal muscles-is a common sensorimotor disorder in patients with brain and spinal cord injuries after stroke, trauma, cerebral palsy, or in multiple sclerosis and several myopathies of different etiologies [START_REF] Li | Spasticity, motor recovery, and neural plasticity after stroke[END_REF][START_REF] Trompetto | Pathophysiology of spasticity: implications for neurorehabilitation[END_REF]. Spasticity-related physical and mental conditions of patients often permanently disable self-supporting life management and ability to work [START_REF] Martin | Epidemiological, humanistic, and economic burden of illness of lower limb spasticity in adults: a systematic review[END_REF][START_REF] Rychlik | Quality of life and costs of spasticity treatment in German stroke patients[END_REF]. Economic burden for post-stroke patients with spasticity is 4-fold higher than those for patients without spasticity [START_REF] Zorowitz | Poststroke spasticity: sequelae and burden on stroke survivors and caregivers[END_REF], which includes direct costs of medications and hospitalization and indirect costs of caregiver assignments and independent daily-lifefacilitating instruments. Spasticity develops as a consequence of complex rearrangements of supraspinal inputs, which transforms the synaptic regu-lation of motoneurons that drives muscle contraction. The resulting exaggerated stretch reflex is accompanied with disrupted balance in regulation from the premotor cortex and reticular formation of the brainstem, which finally leads to aberrantly increased potentiation of motoneurons and hypercontraction of muscles [START_REF] Enslin | Management of spasticity after traumatic brain injury in children[END_REF][START_REF] Mukherjee | Spasticity mechanisms -for the clinician[END_REF][START_REF] Trompetto | Pathophysiology of spasticity: implications for neurorehabilitation[END_REF].

Current muscle relaxants in medical practice target the central nervous system (e.g., baclofen [GABA B agonist] and tizanidine [a2 agonist]), the neuromuscular junction (botulinum toxins), or the sarcoplasmic reticulum in muscle cells (dantrolene; Figure 1A). Due to their aspecific mechanism of action, centrally acting muscle relaxants have a wide range of neurological and cardiovascular side effects (drowsiness, dizziness, depression, and low blood pressure; [START_REF] Meleger | Muscle relaxants and antispasticity agents[END_REF][START_REF] Smit | Heart conduction problems in a tetraplegic patient caused by a single therapeutic dosage of Baclofen[END_REF] and low levels of efficacy [START_REF] Orsnes | Effect of baclofen on gait in spastic MS patients[END_REF], which often necessitate the application of an intrathecal pump surgically implanted inside the body (Medical Advisory, 2005). Systemic treatment with botulinum toxins is impossible due to their lethal effect on respiratory and cardiac functions, and local treatments are painful, costly, and often ineffective [START_REF] Careta | Report of allergic reaction after application of botulinum toxin[END_REF][START_REF] Pavone | Botulinum toxin treatment for limb spasticity in childhood cerebral palsy[END_REF]. Dantrolene has been associated with severe hepatotoxicity and genotoxicity (NIH, 2012); therefore, its clinical use is now restricted to life-threatening malignant hyperthermia. Surgical treatment of severe spasticity is limited to selective dorsal rhizotomy, when the sensory roots that transfer abnormal information from the muscles to the spinal cord are transected. However, the advantage of dorsal rhizotomy over oral medica-tions is only marginal, because the treatment often leads to paralysis, chronic back pain, sensory dysfunction, inflammation, constipation, and cyst formation in the spinal column (cf. NICE interventional guidance 373).

Besides nervous-system-injury-related spasticity, permanent muscle spasms emerge as the leading source of prolonged pain in non-specific low back pain and chronic musculoskeletal pain disorder, fibromyalgia, altogether affecting more than 10% of global population [START_REF] Marques | Prevalence of fibromyalgia: literature review update[END_REF][START_REF] Vos | Years lived with disability (YLDs) for 1160 sequelae of 289 diseases and injuries 1990-2010: a systematic analysis for the Global Burden of Disease Study 2010[END_REF]. Although treatments of muscle spasm in these syndromes with systemic muscle relaxants are statistically effective, See also Figures S1 andS2.

recommendations for their use in clinics underline precautions due to the reported adverse side effects resulting from non-specific targeting of the nervous system [START_REF] Tofferi | Treatment of fibromyalgia with cyclobenzaprine: A meta-analysis[END_REF][START_REF] Van Tulder | Muscle relaxants for non-specific low back pain[END_REF]. These considerations emphasize the unmet medical need for the development of a new-generation anti-spastic drug, which directly targets the effector protein of muscle contraction while avoiding neurological and cardiovascular side effects. Nervous-system-independent inhibition of muscle contraction in spastic patients could also overcome the need for targeting the otherwise disrupted synaptic balance of neuronal regulation.

Selective inhibition of skeletal muscles, however, raises the challenge of developing a small molecule that distinguishes between the structurally and sequentially highly similar myosin-2 isoforms. The ideal candidate inhibits only skeletal muscle fast isoforms (MyHC IIa,b,x/d), because the slow isoform in skeletal muscles (myosin heavy chain [MyHC I]) expressed from the MYH7 gene is identical to b-cardiac myosin, the most abundant motor in the ventricles of the heart (Coluccio, 2020;Figures 1B and S1).

RESULTS

In order to relax muscle fibers, we aimed to arrest the actomyosin force-generation cycle in the actin-detached weak actinbinding state [START_REF] Taka ´cs | Myosin complexed with ADP and blebbistatin reversibly adopts a conformation resembling the start point of the working stroke[END_REF], when myosin heads are decoupled from actin filaments (Ma ´lna ´si-Csizmadia and Kova ´cs, 2010; Sweeney and Hammers, 2018; Figure 1C). Using rational pharmacological design in a model MyHC IIa myosin structure, we developed and optimized a small-molecule inhibitor (MPH-220) into the myosin-2 class-specific blebbistatin-binding pocket (Allingham et al., 2005;[START_REF] Kova ´cs | Mechanism of blebbistatin inhibition of myosin II[END_REF]Limouze et al., 2004;Straight et al., 2003;Figures 1D and S2). MPH-220 enabled selective inhibition of fast skeletal myosin-2 isoforms (further referred to as skeletal myosins; Figure S1) isolated from rabbit psoas muscle, whereas-even at extremely high concentrations-it did not inhibit the ATPase activity of slow skeletal/b-cardiac myosin (further referred to as cardiac myosin) isolated from left ventricle of porcine heart or smooth muscle myosin-2 (Figure 1E; Table S1) or any non-muscle myosin-2 (NM2) isoforms (Figure 1F; Table S1). Sequence analysis of the inhibitor binding site revealed an important difference between the cardiac and skeletal myosins near the morpholine ring of MPH-220 at a location playing a key role in the myosin forcegeneration pathway (Ma ´lna ´si-Csizmadia and Kova ´cs, 2010). The drug-interacting amino acid is uniquely and invariably leucine in fast skeletal myosin heavy chains (Leu 476 in human MyHC IIa), whereas this position is occupied by phenylalanine in all other myosin-2 isoforms (Figures 1B andS1). We hypothesized that this structural difference can be the basis for the extreme selectivity of MPH-220 toward skeletal myosins. Therefore, we assessed the role of a Phe/Leu exchange through mutational analysis of human non-muscle myosin-2C (NM2C), which is not inhibited by MPH-220 in the wild-type form, possibly due to the presence of Phe 490 in the corresponding position. According to our hypothesis, the expressed NM2C F490L variant became sensitive to MPH-220 (Figure 1F), corroborating that the singleamino-acid change determines the observed selectivity.

Considering the selective inhibitory nature of MPH-220 on myosin samples (Figures 1E and1F) and its potential to become a new-generation anti-spastic drug candidate, we performed ATPase inhibition assays on human myosin samples. Expressed human b-cardiac myosin was essentially unaffected by MPH-220, however, the non-selective myosin-2 inhibitor, blebbistatin, completely blocked its ATPase activity (Figure 1G; Table S1). We further tested MPH-220 on skeletal and cardiac myosin samples isolated from patient-derived biopsies (Figure 1H; Table S1). In line with their myosin-2 isoform composition, skeletal myosin samples from m.vastus lateralis and m.soleus-containing 56% and 28% fast isoforms in human muscles (Figure S1)-were inhibited by 70% and 29%, respectively, confirming the hypothesis that only the fast isoform fraction is targeted by MPH-220. More importantly, b-cardiac myosin isolated from human heart left ventricle was unaffected by MPH-220 (Figure 1H; Table S1). By contrast, blebbistatin reduced the activity of m.vastus lateralis and cardiac myosin samples by more than 90%, confirming that MPH-220 inhibition is proportional to the ratio of fast myosin isoform in different tissues (Figure 1H; Table S1). These results provide the basis for future safe systemic administration of MPH-220 to human patients. Left ventricle human biopsy contains cca. 10% a-cardiac myosin isoform (expressed from the MYH6 gene [Figure S1; [START_REF] Miyata | Myosin heavy chain isoform expression in the failing and nonfailing human heart[END_REF], which also contains Phe in the proximal part of HP-helix Phe 474 ). The lack of inhibition of myosin samples from the biopsies confirms that a-cardiac myosin could not be inhibited by MPH-220. We also note that gene products of MYH1, MYH2, and MYH4 encoding fast skeletal muscle myosin heavy chains IIx/d, IIa, and IIb, respectively, are not expressed in adult human heart in significant amount (England and Loughna, 2013; NCBI Gene Expression database).

In order to further analyze the molecular background of selectivity, we crystalized fast skeletal muscle myosin (isolated from high-quality rabbit m.psoas) in the presence of . The crystal structure confirmed that MPH-220 binds to the blebbistatin-binding cleft and its morpholine ring is in close proximity to Leu 476 . This residue is at the N terminus of the so-called HP-helix of the L50 subdomain [START_REF] Cope | Conservation within the myosin motor domain: implications for structure and function[END_REF], which is preceded by the switch-2 loop and followed by the relay region of the motor. Comparison of the MPH-220bound skeletal and blebbistatin-bound Dictyostelium myosin-2 structures confirms that, although the tricyclic cores of the inhibitors, including the chiral OH, are positioned similarly, Phe 466 of the HP-helix in Dictyostelium myosin-2 comes into steric hindrance with MPH-220 (Figure 2B). Likewise, the position of this Phe in cardiac myosin structure also causes steric hindrance with MPH-220 (Planelles-Herrero et al., 2017;Figure 2C). This steric hindrance based on crystal structures together with the biochemical results strongly support that the unique Leu in the HP-helix of fast skeletal muscle myosin isoforms is responsible for the extreme selectivity of MPH-220. This mechanism for selective inhibition among myosin-2 isoforms is analogous to how blebbistatin is selective for myosin-2s due to the presence of the switch-2 Ser/Ala residue in myosin-2s (Allingham et al., 2005), whereas this position is occupied by Tyr/Phe in myosins from other classes that are uninhibited by blebbistatin. The resolved crystal structure also confirmed that MPH-220 selects and stabilizes the pre-powerstroke state. Overall, the structure validated the in silico approach to design molecules for selective inhibitor development (Figure S3).

We further studied the effect of MPH-220 on muscle force generation in living animals (Figure 3A). In agreement with the ATPase inhibitory properties, the S(À) enantiomer of MPH-220 was drastically more effective than the R(+) enantiomer when animals were treated orally with MPH-220 (Figure S4). Consequently, the force-relaxing effect of the racemic mixture was significantly lower than that of the S(À) enantiomer. Although the racemic mixture caused maximum 40% reduction, the S(À) enantiomer could decrease hindleg force by 70% in a concentra-tion-dependent manner (Figures 3B and3C). The 30% residual isometric force level for S(À)-MPH-220 is in line with the presence of uninhibitable slow skeletal muscle isoforms in rat hindleg muscles. This attribute is of high importance because, unlike centrally acting muscle relaxants, MPH-220 would never cause overdose-related patient immobilization due to the remaining slow myosin-mediated muscle tone. Importantly, the decreased force persisted for more than 10 h (Figure 3C), which indicates optimal properties for pharmacological development. The force-relaxing effect after oral administration was only marginally slower than that after intraperitoneal injection (Figure S4), which indicated optimal absorption properties of MPH-220. High endothelial permeability was further confirmed by Caco-2 assay, which showed that MPH-220 is highly permeable with significantly faster transfer rate in the absorptive direction over the excretive direction (Figure S4). Although skeletal muscle relaxation could be efficiently achieved by MPH-220, cardiac function was not perturbed in the concentration range that was effective in skeletal muscle force relaxation experiments (Figures 3F andS4). More impor-tantly, neither cardiac nor respiratory functions differed significantly from the excipient control during the 10-h experiments, suggesting a reasonable safety range between the effective and toxic doses of . The maximum 25% decrease in heart rate and blood flow parameters is most probably due to the 10-h-long anesthesia without food and drink after an overnight starvation period and not the effect of myosin inhibition (Figure S4). Moreover, the identical direction of the change in heart rate and blood flow parameters solely reflects slowing down the heart during anesthesia, because cardiac myosin inhibition preceding heart failure increases heart rate while drastically lowering blood flow, as shown with the non-selective inhibitor, AmBleb (Figure S4). We also examined MPH-220 distribution in rat tissues in a time-dependent manner and observed a dose-dependent, few-fold accumulation of MPH-220 in skeletal muscle samples over other tissues, including brain, heart, kidney, and blood . Accumulation in skeletal muscle persists after 3 h when MPH-220 is completely eliminated from blood and other tissues, whereas its concentration in target muscles is still in the effective concentration range (10-fold higher than the determined inhibitory constant [IC 50 ]). MPH-220 concentration was only temporarily elevated in the liver, which metabolizes MPH-220. We note that MPH-220 concentration in the diaphragm was much lower compared to other striated muscle samples, which explains the lack of respiratory dysfunctions after MPH-220 treatments. This feature is expected to prove even more favorable in human patients due to the substantially higher ratio of MyHC I slow isoforms in human diaphragm (Figure S1). The pronounced accumulation of MPH-220 in skeletal muscles might be the result of high concentration of fast skeletal myosin isoforms, which bind the inhibitor with high affinity. This hypothesis is supported by the significantly lower accumulation of the R(+) enantiomer, which further explains the 4-fold weaker force relaxation by the R(+) enantiomer (cf. Figure S4A ). The species-specific MyHC distribution (Figure S1) also emphasizes the need for testing these pharmacodynamic properties of MPH-220 in larger animals (dogs and mini pigs), which-unlike rodents-have higher slow isoform proportions, more similar to human tissues.

Direct and efficient targeting of skeletal muscle contraction by MPH-220 raises the possibility of the development of a new-generation, nervous-system-independent, anti-spastic drug. Therefore, we investigated the efficiency of MPH-220 to improve spasticity-related gait disorders in rats with brain injury. We applied the recently developed spastic cerebral palsy animal model to provoke paralysis-coupled spasticity in rats (Figure 4A; [START_REF] Yu | Establishing a rat model of spastic cerebral palsy by targeted ethanol injection[END_REF], and gait disorders were analyzed by non-invasive methods focusing on the motoric functions of rats combined with neural-network-based deep-learning movement analysis (Figure 4B; Video S1). Similarly to human patients with stroke or brain trauma, rats showed individually variable gait patterns with different degrees of palsy in the lower limbs. Thus, we analyzed the effect of MPH-220 treatment individually on each animal and focused on treatment-induced changes in spasticity-related gait components.

Rat-A could not use the right forelimb and the left hindleg due to paralysis-coupled spasticity, which resulted in an almost complete one-sided circling behavior in the open-field box 2 days after the surgery (Video S2). 40 min after MPH-220 treatment, the ratio of left turns increased from 15% to 38%, which further improved to 51% 3 h after the treatment (Figure 4C). Even though rat-A walked significantly less after treatment, it was obvious that a major improvement was achieved: after relaxing spasticity in its left hindleg, rat-A was able to use this leg and make quasi-normal steps (Video S3). Rat-B and C also had paralysis-coupled spasticity in the hindlegs, although to a lower degree than rat-A. The most obvious spasticity-related feature in the open-field test was the random falling and the spontaneous cramping of the right hindleg of rat-C (Video S4). Importantly, the relative frequency of both symptoms decreased drastically after MPH-220 treatment (Figure 4D). Besides these serious gait abnormalities, rat-B and C could not stand up in the cylinder assay before MPH-220 treatment. In agreement with the kinetics observed in the open-field assay, 3 and 5 h after oral MPH-220 treatment, both rats could stand up in the cylinder, touching the wall with both forepaws with the same number of events as their healthy littermates (Figure 4D). Next morning, the positive effects on falling and cramping frequencies and on the ability to stand up in the cylinder disappeared as MPH-220 was excreted from their bodies. Rat-D showed spastic gait disorders only in the right hindleg without signs of severe palsy, and all other legs remained unaffected. This animal could not use the right hindleg normally due to stiff muscles, which resulted in abnormal positioning of all four paws (Figures 4E andS5). The spastic leg was offset from the body axis, which hindered normal progression of the limb during walking and resulted in lopsided gait patterns with tilted body axis (Figures 4F andS5; Video S5). After MPH-220 treatment, the gait of this animal improved significantly as it started to use both hindlegs normally. The improved movement was the consequence of relaxed spasticity in the hindlegs, which enabled the animal to pull its leg into the body axis, thereby making normal walking steps ( Figures 4E,4F, and S5; Video S5). The enhanced gait functions remained persistent for 5 h, when paw positions and body symmetry were close to those of the healthy animals. Similarly to the other experiments, the effect relapsed to the pretreatment state after 21 h. These effects could be reproduced 5 and 12 days after the first treatment, corroborating the specific anti-spastic effect of MPH-220 treatment.

Beside the individual patterns of spastic behavior that could be improved by MPH-220 treatment, deep-learning analysis was used to assess the over-ground locomotion of all animals during 15 min of free movement in the open-field test. We used 28 parameters characterizing the 3D position of the limbs and body axis in three movement-speed regimes (Figure S5) to quantify the locomotor features based on pose-estimating deep-learning algorithms and exposed these to a principal-components analysis (PCA). In the global PCA space-including all three speed regimes-the major gait component (PC1) was significantly different from the healthy range, indicating that pyramidal tract damage-induced spastic walk asymmetry is reflected in the major principal gait component (Figure 5A). Most importantly, MPH-220 treatment significantly decreased PC1 scores, the effect of which was maximal at 3 h and no longer detected after 21 h. Importantly, PC1 scores were significantly different from the untreated level, however, the treated values did not differ significantly from the healthy range, which confirms that MPH-220 ameliorated gait pattern of the treated animals to that of the healthy rats. However, when we analyzed the three speed regimes separately, we found that pyramid tract damage mostly affected PC1 in the slow and medium-speed regimes, whereas PC2 and PC3 were significantly affected in the fast regime (Figures 5B andS5). More importantly, PC1 could be significantly improved by MPH-220 treatment in the slow-and medium-speed regimes, and PC3 was significantly improved in the fast regime. We identified the major parameters that predominantly contributed to PC1 and PC3 in the slow, medium, and fast regimes and found a marked difference in the parameter sets defining PC1 and PC3 (Figures 5C, 5D, andS5). Analysis of these components revealed that, in the slow-and medium-speed regimes, MPH-220 treatment has the largest effect on the volumes of the ellipsoids representing paw positions and on the distances between their centers, both suggesting a much-ordered stepping pattern and straightened body axis (Figure 5E). On the other hand, in the fast regime, MPH-220 has the most pronounced effect on the angles of the ellipsoids, which indicates that the treated rats could keep their legs closer to the body axis similarly to the healthy running pattern. These results further solidify that MPH-220 is capable of treating the spastic component of movement abnormalities after brain injury, exhibiting a promising potential to address the unmet medical need for effective antispastic therapies.

DISCUSSION

Our biochemical and in vivo results demonstrate that MPH-220 selectively inhibits fast skeletal myosin-2 isoforms, which enables effective improvements of spastic gait disorders after brain damage as a consequence of efficient reduction of muscle force. These findings emphasize that nervous-system-injury-derived muscle spasticity can be treated by directly targeting the effector protein of muscle contraction, thereby avoiding adverse neurological and cardiovascular side effects that are characteristic of currently used muscle relaxants. Moreover, as MPH-220 proved a promising candidate for further anti-spastic drug development, we tested its pharmacological properties by investigating its off-target effects on human enzymes, including kinase panels and G-protein-coupled and nuclear hormone receptors (Figure S6). We also demonstrate that MPH-220 is not mutagenic and not cytotoxic (Figure S6), which, together with its high-absorption properties, confirms that MPH-220 is safe and possesses optimal attributes for further drug development as a new-generation anti-spastic drug candidate.

Although we acknowledge that MPH-220 may cause general muscle weakness as observed in our animal disease model, we believe that this effect will be significantly less pronounced in human patients due to the higher ratio of MyHC I slow skeletal muscle myosin isoform. In human skeletal muscles, the presence of 40%-60% MyHC I isoform, which is not inhibited by MPH-220, highlights another important aspect of this type of muscle relaxation as opposed to nervous-systemtargeting medications. Although general weakness of aspecific muscle groups may appear by using both types of muscle relaxants, the MPH-220-based anti-spastic effect is not expected to cause complete immobilization of patients due to the residual muscle tone from the uninhibited slow isoform fraction. Another concern may arise regarding extraocular muscles, which express MYH13 gene product containing Leu required for MPH-220 inhibition (Figure S1). Nevertheless, in adult human extraocular muscles, significant amount of a-cardiac myosin heavy chain is expressed [START_REF] Kjellgren | Myosin heavy chain isoforms in human extraocular muscles[END_REF]; therefore, these muscles will also not be completely immobilized by MPH-220. However, these effects might be investigated during future clinical trials. Moreover, based on the complex pathophysiology of spasticity development [START_REF] Trompetto | Pathophysiology of spasticity: implications for neurorehabilitation[END_REF], we suppose that early treatment with an effective anti-spastic drug will significantly delay the onset and markedly reduce the severity of spastic symptoms after nervous system injuries.

Besides traumatic brain and spinal cord injuries in stroke and cerebral palsy, spasticity is also a common symptom in several other diseases of broad etiology (Table 1). Spasticity is a major component of upper motor neuron lesion syndromes, including multiple sclerosis, amyotrophic lateral sclerosis, spastic paraplegia, multiple system atrophy, and atypical Parkinsonism, and also a characteristic symptom in several skeletal muscle myopathies, such as myotonia congenita, myotonia fluctuans, or certain neurodegenerative diseases. Altogether, spasticity in these syndromes affect nearly 1% of the human population (cca. 55 million spastic patients), and more than 10% of people suffer from often-painful muscle stiffness in low back pain and fibromyalgia [START_REF] Marques | Prevalence of fibromyalgia: literature review update[END_REF][START_REF] Vos | Years lived with disability (YLDs) for 1160 sequelae of 289 diseases and injuries 1990-2010: a systematic analysis for the Global Burden of Disease Study 2010[END_REF]. Consequently, a nervous-system-independent, safe, and effective treatment is a significant unmet need from both clinical and socio-economical perspectives. Therefore, MPH-220 is a promising anti-spastic drug candidate to improve quality of life and may contribute to extended life expectancy [START_REF] Blair | Survival and mortality in cerebral palsy: observations to the sixth decade from a data linkage study of a total population register and National Death Index[END_REF][START_REF] Gillard | The negative impact of spasticity on the health-related quality of life of stroke survivors: a longitudinal cohort study[END_REF][START_REF] Slaman | Can a lifestyle intervention improve physical fitness in adolescents and young adults with spastic cerebral palsy? A randomized controlled trial[END_REF][START_REF] Yi | Emerging issues in cerebral palsy associated with aging: a physiatrist perspective[END_REF] of these patients and would provide new therapeutic protocols for clinicians in these diverse indications.

STAR+METHODS

Detailed methods are provided in the online version of this paper and include the following:

d KEY RESOURCES

RESOURCE AVAILABILITY

Lead Contact

Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Andra ´s Ma ´lna ´si-Csizmadia (malna@elte.hu) Materials Availability MPH-inhibitors generated in this study will be made available on request, but we may require a payment and/or a completed Materials Transfer Agreement as there is potential for commercial application.

Data and Code Availability

The published article includes all datasets generated or analyzed during this study. The accession number for the skeletal myosin bound to MPH-220, Mg.ADP.VO 4 reported in this paper is PDB: 6YSY. The atomic model is available in the PDB (Berman et al., 2003).

EXPERIMENTAL MODEL AND SUBJECT DETAILS

Animals 200-250 g male Wistar rats were obtained from Toxi-Coop Ltd. (Hungary) or Animal Facility, Basic Medical Science Center at Semmelweis University (Hungary). Animals were maintained in standard housing conditions with 12-12-hour light-dark periods and were allowed free access to dry rat food and water. All procedures were conducted in accordance with the ARRIVE guidelines and the guidelines set by the European Communities Council Directive (86/609 EEC) and approved by the Animal Care and Use Committee of the Eo ¨tvo ¨s University (registration number: 48/1/2015) under the permission numbers: PE/EA/142-5/2020, PE/EA/143-7/2020.

Human muscle biopsies

Human muscle biopsy samples were prepared, processed and measured in accordance with the Recommended Standards for Muscle Biopsies by the European Reference Network for Neuromuscular Diseases and the European Commission and approved by the Regional Research Ethics Committee of the Coordination Centre of Clinical Studies of University of Pe ´cs under the permission number: 8340-PTE 2020. M.vastus samples were obtained from female (60%) and male (40%) patients with normal creatine kinase (CK) levels and no or non-specific changes in muscle fibers. M.soleus sample was obtained from a female patient with normal creatine kinase (CK) level and non-specific changes in muscle fibers. 50% of patients had undergone electromyography (EMG) examination before biopsy preparation. Neither of the patients had received medications that would affect muscle protein compositions: obtained treatments include antihypertensive medicines, anti-inflammatory drugs and vitamin products.

METHOD DETAILS

Materials

All chemicals were purchased from Sigma-Aldrich (Germany) if otherwise not stated. Sulphobutyl-ether-b-cyclodextrin (Dexolve TM (SBECD)) was obtained from Cyclolab (Hungary). HPLC-grade acetonitrile, chloroform and water were purchased from VWR (PA, USA). Blebbistatin was purchased from Sellekchem (TX, USA), AmBleb Va ´rkuti et al., 2016 is product of Motorpharma Ltd., and isoflurane was purchased from Rotacher-Medical GmbH (Germany). Ames Microplate Format Mutagenicity Assay kit was purchased from Xenometrix (Switzerland).

Proteins

Rabbit actin was prepared as described previously from rabbit muscle acetone powder (Pel-Freez Biologicals Llc., AR, USA) [START_REF] Spudich | The regulation of rabbit skeletal muscle contraction. I. Biochemical studies of the interaction of the tropomyosintroponin complex with actin and the proteolytic fragments of myosin[END_REF]. Briefly, 5-6 g muscle acetone powder was soaked into ice cold G-actin Buffer (20 ml/g powder; 2 mM Tris pH 7.7, 0.2 mM CaCl2, 0.2 mM ATP, 2 mM DTT) and was stirred on ice for 40 min. After stirring wet acetone powder was filtered using filter paper. 2 mM MgCl2 and 50 mM KCl were added to the filtered solution and further incubated at room temperature for 30 min. KCl was added to the final concentration 0.5 M and was stirred for further 60 min. Solution was centrifuged (40,000 rpm, 90 min, 4 C) and pellet was homogenized in G-buffer and excessively dialyzed against G-buffer. After dialysis, solution was centrifuged (40,000 rpm, 90 min, 4 C) and the supernatant was frozen in liquid nitrogen in 1 mL aliquots. Skeletal muscle myosin subfragment-1 (S1) was prepared from rabbit m.psoas according to an earlier published protocol [START_REF] Margossian | Preparation of myosin and its subfragments from rabbit skeletal muscle[END_REF]) by a-chymotryptic digestion (0.25 mg/ml a-chymotrypsin, 10 min, 25 C; reaction was stopped with 3 mM PMSF). Psoas S1 prep was further purified on MonoS 5/50 GL column to produce high-quality prep for crystallization. Cardiac muscle myosin S1 was isolated from left ventricle of porcine heart following cardiac myofibril preparation protocol [START_REF] Tong | Acceleration of crossbridge kinetics by protein kinase A phosphorylation of cardiac myosin binding protein C modulates cardiac function[END_REF]) by a-chymotryptic digestion (0.05 mg/ml a-chymotrypsin, 10 min, 25 C; reaction was stopped with 3 mM PMSF). Human cardiac muscle myosin subfragment-1 (S1) was prepared from adult human left ventricular tissue samples obtained from ethically consented donors (AnaBios Corporation) according to earlier published protocol [START_REF] Margossian | Preparation of myosin and its subfragments from rabbit skeletal muscle[END_REF]) by a-chymotryptic digestion (0.25 mg/ml a-chymotrypsin, 9 min, 25 C; reaction was stopped with 3 mM PMSF). Smooth muscle myosin S1 was prepared from chicken gizzard [START_REF] Trybus | Biochemical studies of myosin[END_REF] by activated papain digestion [START_REF] Seidel | Fragmentation of gizzard myosin by alpha-chymotrypsin and papain, the effects on ATPase activity, and the interaction with actin[END_REF] (0.2 mg/ml papain, 12 min, 25 C; reaction was stopped with 5 mM sodium iodoacetate). Human skeletal muscle myosin was prepared similarly to rabbit skeletal muscle myosin [START_REF] Margossian | Preparation of myosin and its subfragments from rabbit skeletal muscle[END_REF]. Briefly, biopsies were washed in Rigor Buffer (20 mM MOPS pH 7.4,40 mM KCl, 2 mM MgCl 2 ) and myosin was extracted in Myosin Extraction Buffer (20 mM MOPS pH 7.4,500 mM KCl). Extracted myosin was precipitated in 21-volume ultrapure water with 1 mM EDTA. Myosin was collected in tabletop centrifuge, dissolved in Myosin Extraction Buffer and kept on ice until the measurement. The Sf9 codon optimized sequences for NM2 motor domains (Met 1 -Arg 775 for NM2A, Met 1 -Arg 782 for NM2B (B0) and Met 1 -Arg 799 for NM2C (C0)) fused to an Ala-Ser linker and an artificial a-actinin lever arm: (amino acid sequence: AS-EQTKS-DYLKRANELVQWINDKQASLESRDFGDSIE-SVQSFMNAHKEYKKTEKPPKGQEVSELEAIYNSLQTKLRLIKREPFVAPAGLTPNEIDST-WSALEKAEQEHAEALRIELKRQKKIAVLLQKYNRILKKLENWATTKSVYLGSNETGDSI-TAVQAKLKNLEAFDGECQSLEGQSNSDLLSI-LAQLTELNYNGVPELTERKDTFFAQQW-TGVKSSAETYKNTLLAELERLQKIED) as described earlier for NM2C [START_REF] Heissler | Comparative kinetic and functional characterization of the motor domains of human nonmuscle myosin-2C isoforms[END_REF]) with C-terminal FLAG-tag after an Ala-Leu linker (amino acid sequence: AL-DYKDDDK) sequences were synthesized and cloned into pFastBac1 expression vector. The NM2C-F490L construct was also synthesized by Biomatik Co in pFastBac1 expression vector. The pFastBac1/NM2 constructs were overexpressed in Sf9 cells after bacmid preparation from DH10Bac cells. To reduce proteolytic digestion during expression, Pepstatin-A (0.1 mg/l) was added to Sf9 cells [START_REF] Gotoh | Proteolytic activity and recombinant protein production in virus-infected Sf-9 insect cell cultures supplemented with carboxyl and cysteine protease inhibitors[END_REF]. Expressed human b-cardiac myosin S1 [START_REF] Kawana | Biophysical properties of human b-cardiac myosin with converter mutations that cause hypertrophic cardiomyopathy[END_REF] was prepared as earlier described [START_REF] Sommese | Molecular consequences of the R453C hypertrophic cardiomyopathy mutation on human b-cardiac myosin motor function[END_REF].

ATPase measurement

Steady-state ATPase measurements were carried out in 50 mL volume in a flat bottom 384-well plate (Nunc-Thermo Fischer) using NADH-PK/LDH coupled assay described previously [START_REF] Gyimesi | The mechanism of the reverse recovery step, phosphate release, and actin activation of Dictyostelium myosin II[END_REF]. Briefly, myosin samples were mixed with 2% pyruvate kinase/lactate dehydrogenase (PK/LDH) mixture (Sigma P0294), 1 mM phosphoenol-pyruvate (PEP) and 200 mM NADH at 25 C in the presence of 0.5 mM ATP and 25 mM F-actin in ATPase buffer (10 mM MOPS pH 7.0, 4 mM MgCl 2 , 2 mM b-mercaptoethanol) for 15 minutes. Absorbance was followed at 340 nm wavelength to follow the decrease of NADH in Microplate Spectrophotometer (BioTek Epoch). ATPase activity was calculated from linear regression of the time dependent absorbance data collected at 340 nm. Different concentrations of inhibitors were added to the reaction in 0.5 mL DMSO (1% of total volume). DMSO and actin-controls were measured for each measurement set.

Molecular dynamics simulation

Simulations were carried out using the AMBER16 program suite. The ff14SB force field [START_REF] Maier | ff14SB: improving the accuracy of protein side chain and backbone parameters from ff99SB[END_REF] was used in all subsequent simulations to model protein interactions. The initial structure of the Fast Human Skeletal myosin-2 motor domain was modeled using structural homology with the SWISS-MODEL server [START_REF] Waterhouse | SWISS-MODEL: homology modelling of protein structures and complexes[END_REF] using the Human Cardiac myosin-2 motor domain (PDB: 5N69). ADP.VO 4 was substituted to ADP.PO 4 . Coordinates and partials charges for MPH-220 were refined using antechamber of the AMBERTOOLS package with the AM1 semi-empirical Hamiltonian. Force field parameters for the MPH-220 atoms were adopted from GAFF force field [START_REF] Wang | Development and testing of a general amber force field[END_REF] based on structural similarity following the suggestions of the parmchk2 program. We determined the initial position of MPH-220 by RMSD least-square fitting the model's structure with the blebbistatin-Dyctiostelium motor domain (PDB: 3BZ9) binding pocket. The initial position was then determined by RMSD least-square fitting the tricyclic ring of MPH-220 to blebbistatin's tricyclic ring. The water molecules resolved by crystallography were retained and the model was expanded in an 8-Angstrom clearance dodecahedron box with TIP3P water molecules for explicit solvation and counterions were added to neutralize the system's net charge. The complex was minimized with 2000 steps steepest descent followed by 4000 steps of conjugate gradient method while applying 10 kcal/(mol A ˚2) to all atoms except water molecules. The minimization continued with the same protocol while removing the restraint from all H atoms. The same restraints were applied for the following steps. The complex was heated in 3 100-K 20-ps steps to 300 K under NVT conditions, then subsequently equilibrated to 1 bar in 20 ps under NPT conditions. NPT conditions were applied in all further steps. The system was cooled back to 10 K in the next 20 ps. Restraints from this point were only applied to the protein backbone. The system was heated back to 300 K in 10 ps and the simulation was continued for 1ns. The restraint force was then gradually reduced in 1 ns simulations steps to 5, 1, 0.5 and 0 kcal/(mol A ˚2). The simulation was then allowed to continue and equilibrate under NPT conditions for a further 200 ns to obtain the final structures. The trajectories were sampled every 50 ps during the simulation.

Crystallization and data processing

MyCH IIb-MPH-220 crystals were obtained by vapor diffusion using a protein solution at 10 mg/ml containing 10 mM MOPS pH 7.3; 50 mM NaCl; 3 mM MgCl 2 ; 0.2 mM ATP; 3 mM NaN 3 ; 1 mM DTT; 0.1 mM EGTA; 0.1 mM PMSF; 1 mg/ml leupeptin, 2mM MgADP, 2mM VO 4 , 0.5 mM MPH-220 against a reservoir containing 35% PEG600, 20mM DTT; 100mM HEPES pH 7.0; 5% DMSO at 291K. Optimization of crystallization conditions was performed with trypsin in situ proteolysis. Crystals were frozen then shot in the Prox-ima2 beamline (synchrotron Soleil). X-ray diffraction images were processed with AutoProc (Vonrhein et al., 2011). The crystal belongs to the P2 1 2 1 2 1 space group with one molecule per asymmetric unit. The data collection and refinement statistics are presented in Figure S3.

Structure determination and refinement

Structure of the motor domain was initially solved by molecular replacement using 1QVI (Gourinath et al., 2003) as a search model in Molrep [START_REF] Vagin | Molecular replacement with MOLREP[END_REF], and completed with a second search for the lever arm and its essential light chain from 5N69 (Planelles- [START_REF] Herrero | Bases Mécanistiques et Structurales de La Régulation de l'activité Des Myosines[END_REF] with Phaser (McCoy et al., 2007). Refinement constraints for the ligand were automatically generated with Grade (Bricogne et al., 2017). The final model was obtained after several cycles of building in Coot (Emsley and Cowtan, 2004) and refinement with the latest 2019 release of BUSTER-TNT (Bricogne et al., 2017).

Isometric force measurement

Isometric force of rat left hindleg was measured by a force meter (Supertech Ltd.) after the hindleg was fixed and stimulated according to earlier published protocol [START_REF] Pratt | A stepwise procedure to test contractility and susceptibility to injury for the rodent quadriceps muscle[END_REF]. A non-elastic wire was attached to the ankle of the rat and pulled until prestrained position. Spike (R) software controlled stimulator (BioStim, Supertech Ltd.) was used to trigger the n. femoralis through metal electrodes with 100 Hz, 1 s, 4V, 2.5 minute intervals. Data detection was performed with CED Micro 1401 data acquisition interface. Inhibitors were either dissolved in 200 mL DMSO for intraperitoneal injection or in 1ml 30% SBECD solution (dissolved in GIBCO TM Dulbeccos's Phosphate Buffered Saline (D-PBS)) for oral administration.

Non-invasive measurement of cardiovascular and respiratory functions Cardiovascular and respiratory functions were measured using a non-invasive pulse oximeter (MouseOx Plus, Starr Life Sciences Corp., PA, USA). Blood flow parameters shown on figures corresponds to the measured pulse distention, which is the measure of change in blood vessel volumes in the light path of the sensor, which reflects local blood pressure changes. Rats were anesthetized and the neck region was trimmed using a small animal trimmer to facilitate accurate measurements by size specific collar sensor.

Concentration measurement from tissue samples Animals were treated i.p. or per os with MPH-220, over-anesthetized and tissue samples were collected and frozen at À80 C. Tissue samples were thawed and homogenized in D-PBS, and chloroform was added to the homogenate. Samples were thoroughly shaken, vortexed and sonicated. After sonication, samples were centrifuged and the chloroform layer was collected. Chloroform extraction was repeated two more times. Chloroform was evaporated in a fume-hood. After chloroform drying, water-acetonitrile solution (50%/50%) was added to the samples, which were again vortexed and sonicated again. Samples were centrifuged and supernatants were collected and transferred into ultra-centrifuge vials and ultracentrifuged at 60,000 rpm, 10 C for 60 minutes before HPLC-MS injection. Peak area of compound in HPLC was determined at 400 nm and concentration of compound was determined from the calibration curve. HPLC-MS conditions were the following: Merck Purosphere STAR RP-18 endcapped 250 mm X 4.6 mm column, mobile phase: isocratic 50% H 2 O (0.1% TFA) -50% ACN (0.09% TFA), injection volume: 20 mL HPLC, flow rate: 0.5 ml/min, run time: 25 min, detection at 260, 280, 300, 400 and 450 nm, capillary voltage: 3.5 kV, cone voltage: 30 V.

Spasticity animal model

Targeted brain damage was performed to establish a rat model of spastic cerebral palsy. Briefly, animals were pre-anesthetized with 4% isoflurane, which was reduced to 2% after the fixation of the animal on the stereotactic apparatus. First, a 2 cm-long longitudinal incision between ears was carried out, which was followed by the cleaning of the surface of the skull by hydrogen peroxide to visualize the Bregma point. Injection coordinates were determined based on The Rat Brain in Stereotaxic Coordinates, 6th Edition (Paxinos and Watson, 2007): ML À0.8 mm, AP À10 mm and DV 9.7 mm from the dura. A 25 ml Neuros Syringe (Hamilton, NV, USA) with needle size 22G was used to gently inject 15 ml ethanol for left pyramidal tract lesion. After the injection the syringe was left in place for 10 minutes, followed by its removal from the brain and the sealing of the wound.

Neural network based deep-learning analysis of movements in open-field tests

The over-ground locomotion of the animals was recorded in an 80 by 80 cm wide plastic open-field box using an 8 mm thick transparent Plexiglas as the bottom panel over the course of 15 minutes. The arena was placed on a custom-made wooden frame in order to raise it 1 m above the ground level, while keeping the transparent bottom unobstructed. A Microsoft Kinect V2 for XBOX ONE (2013) RGB-D sensor was secured approx. 80 cm below the Plexiglas facing upward to capture depth information from inside the open-field arena (Figure 4B). The Kinect sensor was connected to a Windows PC (i5-9500F, GeForce GTX 1050 2 GB, 16GB DDR4) placed in a separate room via the official adaptor (Kinect Adaptor for Windows PC) and a USB extension cable. The Kinect for Windows SDK V2.0.1410.19000 API-s were used to control the Kinect sensor in MATLAB v. R2019b with the Image Acquisition Toolbox Support Package for Kinect for Windows Sensor add-on.

An in-house MATLAB program was used to calibrate the spatial position of the device by measuring the relative distance of the four corners of the arena to the sensor to ensure uniform depth information values in the whole arena during experiments. Another inhouse MATLAB program was used for image acquisition by the infrared depth camera (512 3 424 / 30 FPS) and the RGB camera (1920 3 1080 / 30 FPS) simultaneously. Video recordings from the depth camera were stored in .mj2 archives using lossless 16-Bit Motion JPEG 2000 compression, while the RGB camera recordings were stored in MP4 formats to allow easy viewer access. The depth video recordings were subjected to a preprocessing MATLAB script to correct the erroneous depth estimates arising from the time-of-flight depth sensing, such as flying pixels and multipath interference. The modified archives were scaled to preserve intensity values of the region of interest (rat in the arena) and exported as 8-Bit uncompressed AVI videos for further use.

A Resnet-50 deep convolutional neural network was trained to predict distinct body parts (nose, neck, center, right forelimb, left forelimb, right hindlimb, left hindlimb, base of the tail) of the animals on the 8-Bit depth recordings using the DeepLabCut toolbox [START_REF] Mathis | DeepLabCut: markerless pose estimation of userdefined body parts with deep learning[END_REF] in the Google Colaboratory python environment. The network training parameters were identical as described in [START_REF] Nath | Using DeepLabCut for 3D markerless pose estimation across species and behaviors[END_REF] with the provided pre-trained resnet_v1_50 architecture using stochastic gradient descent optimizer, multistep learning rate (0,005 until 10000 epochs, 0.02 until 430.000, 0.002 until 500.000), batch size of 1, train-test split of 0.3, and no early stopping. We used $1000 manually annotated frames for the final training and validation sets (training on 500.000 epochs achieved 1.75 mean pixel error for the training and 7.33 mean pixel error for the validation set). A custom MATLAB script was then used to process the previously modified depth archives (25.000 frames / recording) in conjunction with the error corrected numerical output (X and Y coordinates) of the deep learning algorithm of each labeled body part. The animals were first segmented based on the pixel intensity deviation relative to the background. Each frame was then translated and rotated based on the position and angle of the axis between the center of mass of the animal and the position of its nose to create egocentric recording. This transformation and rotation was then carried out on the body part labels as well. The 3 dimensional position of each limb was calculated by combining the transformed output coordinates of the deep neural net with the depth information of the labeled body parts on each frame of the preprocessed and transformed depth recordings. The three dimensional movement maps were exported to further analyses.

In the final algorithm 95% CI least-squares elliptical fittings were computed on the 3D movement maps of each individual limb of the animals and the gait parameters were calculated (Table S3). Centroid displacement thresholds on moving average function of 100 frames were used to calculate distinct locomotion regimes (1 st Regime: < 0.6 pixel deviation, 2 nd Regime: > 0.6 and < 1.2 pixel deviation, 3 rd Regime: > 1.2 pixel deviation.). The gait parameters were scaled using the Z-score method and subjected to principal component analysis and the results were represented in the 3D PC space together with the 95% CI ellipsoids.

Cylinder assay

Motor functions of spastic rats were tested in an open-top, 21 cm diameter, clear plastic cylinder. Forelimb activity was calculated from number of touches of each forelimb against the wall of the arena. Forelimb touch was defined when the whole palm touched the sidewall of the cylinder. Number of sidewall touches was counted for 3 minutes.

Safety panel measurements

G-protein coupled receptor inhibition and activation was tested in the gpcrMAX panel of DiscoverXâ Profiling service (Eurofins). Caco-2 assays were carried out by Absorption and Permeability Services at Eurofins Discovery. Predictor TM hERG Fluroescence Polarization Assay, SelectScreen Kinase Profiling and SelectScreen Nuclear Receptor Profiling was carried out by Thermo Fisher Scientific. Ames MPF TM (Xenometrix, Switzerland) reverse mutagenicity assay was performed according to the manufacturer's guide on TA98 and TA100 bacterial strains in the absence and presence of phenobarbital/b-naphtoflavone-induced rat liver S9 fraction. Cytotoxicity measurements were performed with CyQUANT TM LDH Cytotoxicity assay (Thermo Fisher) according to the manufacturer's protocols with human adult dermal (GIBCO, Thermo Fisher) and lung (Cell Applications Inc., CA, USA) fibroblast strains.

QUANTIFICATION AND STATISTICAL ANALYSIS

Unless otherwise specified, data are presented as means with standard deviations. Principal component analysis data are presented as box and whishkers representation with min to max whiskers in GraphPad Prism software (Graphpad Software Inc.). ATPase activity analysis, IC 50 and maximal inhibition analysis, single exponential fitting and t tests for significance analysis were performed by GraphPad Prism software (Graphpad Software Inc.). Tissue concentration determination was based on standard curve fitting and area under curve analysis by MassLynx software (Waters Ltd.). Principal component analysis was performed by MATLAB software (MathWorks). We did not apply statistical methods to predetermine sample sizes for animal experiments and kept the used subject numbers at a minimum to conform with ARRIVE and 3R principles. Further details of statistical analysis can be found in the figure legends or in the Method Details section.

Supplemental Figures (legend on next page)

Figure S1. Sequence Analysis of Myosin-2 Isoforms from Different Species, Related to Figure 1 (A) The tables show the amino acid residue at the starting point of the HP-helix in myosin-2 isoforms expressed from different MYH genes in eight vertebrate species and the muscle and non-muscle myosin-2 isoforms in D. melanogaster and C. elegans invertebrate species. The alignments below the tables represent the sequence environment of the relevant Leu (blue L) or Phe (black F) residue, including the switch-2 loop (red) and the N-terminal part of the HP-helix (light orange). These data clearly show that all fast skeletal myosin-2 isoforms in all species contain Leu in the morpholine-interacting position, while Phe is present in all slow type myosin-2 s including both cardiac isoforms at this position. The characteristic difference is even more pronounced when comparing the neighboring residues in the HP-helix, which are practically invariant in all myosin isoforms and species. (B-F) Myosin-2 isoform distribution within different muscle types in different species. Literature-based analysis of myosin-2 isoform composition -determined by SDS-PAGE or immune/enzyme histochemistry -of different muscles in rat (B), dog (C), mini pig (D) and human (E) show significant differences in the proportion of the slow isoform (MyHC I, gray) between the species. While rat muscles contain predominantly fast isoforms (MyHC IIa, IIb, IIx/d, orange), the ratio of fast isoforms in human muscles is between 40%-70%. Distributions in dogs and mini-pigs are in between the rat and human levels. These differences bear important consequences on the expected effect size in different species and may require unique consideration of dosage in non-clinical studies on different species (e.g., rats versus dogs/pigs). The relatively high proportion of slow isoform in human muscles also has important positive consequence on the effect profile of an MPH-220 based anti-spastic drug, because overdosing-related complete muscle tone loss is not expected to occur due to the residual non-inhibited slow myosin contractions. This latter condition confers a huge advantage over the currently used nervous system targeting muscle relaxants, where temporary but complete immobility can occur in case of slight overdose. The large difference in slow isoform proportion can also be seen in the diaphragm of different species (F), which indicates that MPH-220 will be even safer for respiratory functions in humans than in rats. Relative ATPase activities are shown for different MPH-220 derivatives tested during the development of MPH-220. MPH-49 is the morpholine derivative of blebbistatin, which shows selective inhibition; however, its solubility remained blebbistatin-like (less than 50 mM in aqueous solutions). MPH-188 and MPH-248 are morpholine derivatives on different scaffolds of the patented MPH-family. MPH-188 showed drastic improvement in solubility (1 mM in aqeous solutions), but its IC 50 value for skeletal myosin was slightly worse than that of MPH-220. MPH-248 retained skeletal myosin specificity of the pyridine ring containing core (MPH-174), but its total inhibition and IC 50 values remained worse than those of MPH-220. We found that the morpholino group (MPH-49) was superior to homomorpholine (MPH-202), thiomorpholine (MPH-203) or piperazine . The IC 50 of MPH-203 was promising; however, its solubility was even lower than that of MPH-49, suggesting that the optimal ring structure for selective and efficient inhibition is the morpholine ring. We also tried to modify the ring structure of the tricyclic core, but neither modification showed improved properties, confirming that MPH-220 is the optimal structure. Mean ± SD are shown in all ATPase experiments; n = 3-9. was not toxic to either fibroblast strain at any time point or concentration, confirming its safe use in further developments. As one of the most important requirements for a lead compound, mutagenicity was measured by the OECD/FDA approved Ames reverse mutagenicity assay using the two most sensitive bacterial strains for testing frameshift (TA98, G) and base-pair substitution (TA100 H) mutation both in the absence (open circles) and presence (closed circles) of activated rat liver S9 fraction. Relative mutagenicity did not show MPH-220 concentration dependency and, more importantly, experiments on neither strain resulted in any data points above the threshold (gray dotted line). Besides MPH-220 we also tested the mutagenicity of the other two morpholine derivatives of MPH-220 (cf. Figure S2) to elucidate whether the lack of mutagenicity is associated with the morpholine group. Neither MPH-49 nor MPH-188 showed concentration dependent increase in mutagenicity levels and, more importantly, neither showed in data points above the OECD/FDA threshold. Lack of mutagenicity was observed for both TA98 and TA100 S.typhimurium strains both in the absence and presence of liver S9 fraction, suggesting that neither the inhibitors nor their major metabolites are mutagenic, which supports safe development of MPH-220 toward human clinical phases. Yet, strikingly, although the changes in MPH-220 pocket are small, they promote a 3.8 Å difference in the priming of the Lever arm. More precisely, no change was found in the switch-II, the transducer is tilted by 0.8 Å, the relay is shifted by 1.6 Å, the N-terminus subdomain by 1.1 Å, the Converter by 2.5 Å, and finally, the Lever arm is tilted by 3.8 Å (Figure 89). This suggest that MPH-220 could stabilize a state slightly different that the one obtained without drug. Yet whether this finding is due to crystal constraints or whether it is relevant in the context of MPH-220 inhibition and is this change proper to MPH-220 or can be more generally be applied to all blebbistatin derivatives required further investigation. 17). Actually, even AmBlebb, pNitroBlebb and blebbistatin which are known as inhibitors of Myo2s exhibit relative specificity among them (Figure 91). For those three compounds, inhibition of NM2A and NM2B is weaker, in particular for AmBlebb. We also noticed that AmBlebb inhibits DictyMyo2 better that SmMyo2, while pNitroBlebb does the opposite. SkMyo2 is the best inhibited Myo2 of all the myosins and the second best IC50 is always found for CardMyo2. Although these differences are not sufficient for making any of these drugs a specific inhibitor of one Myo2 isoform, understanding the origin of these differences and how we can use them for future development of inhibitors is of particular interest. Thus, our team will continue the collaboration with András Málnási-Csizmadia. Indeed, in the course of the development of MPH-220, they synthesized and characterized the inhibition efficiency of more than 144 molecules. A close look at the difference in their activity for different myosins coupled with co-crystal structures of a selected subset of inhibitors may now lead to a better understanding of the rules of selectivity within the Blebbistatin pocket.

Actually, such knowledge could also be used for the development of specific and potent inhibitors of certain unconventional myosins such as Myo6 for which key residues for blebbistatin binding are rather conserved. Nevertheless, it should be noted that blebbistatin itself is unable to inhibit Myo6, highlighting the fact that looking only at this subset of residues is not sufficient to predict inhibition. Additional parameters should be considered to identify key parameters influencing binding of blebbistatin and binding of its derivatives, and to understand the rules of selectivity and potency within the Blebbistatin pocket.

Moreover, [START_REF] Roman | Recovering Actives in Multi-Antitarget and Target Design of Analogs of the Myosin II Inhibitor Blebbistatin[END_REF] pointed out the current difficulty in predicting the efficiency of blebbistatin derivatives in silico as no correlation could be found between IC50 and docking scores in their study (with or without taking water molecules into account).

B.2. Blebbistatin pocket entrance: a parameter than still requires investigation

An important parameter to be considered is the tightness of the blebbistatin tunnel (see introduction section C.7.). Indeed, the drug must be able of both reaching the blebbistatin binding pocket and stay bound to it to efficiently inhibit a myosin motor.

Looking at surface residues in the outer part of the 50 kDa cleft (putative blebbistatin entrance tunnel), important differences can be found among Myo2s, notably the charge distribution at the surface, which differs from one Myo2 to another:

In particular, in part A (Figure 92), bulky and hydrophobic residues can be found at the entrance of the 50 kDa cleft for CardMyo2 and SkMyo2: Ser272 and Val463 from Dicty are replaced by Leu and Phe, respectively, suggesting more difficult access for small molecules, and in particular polar molecules in SkMyo2 and CardMyo2 across this part of the cleft.

In part B (Figure 92), we noticed the formation of a polar contact between SmGlu422/NM2CGlu441 and SmArg273/NM2CArg293 in SmMyo2 and NM2C that could restrict flexibility of

Conclusions

The work presented in this chapter offers new insights on the potential of Blebbistatin allosteric pocket for the development of specific myosin inhibitors.

Thanks to our collaboration with András Málnási-Csizmadia's lab, we had the opportunity to take part in the characterization of the antispastic drug MPH-220. This Blebbistatin derivative has been initially found in a series of more than 144 Blebbistatin derivatives as a highly specific SkMyo2 inhibitor.

It diverges from Blebbistatin by its A-ring: the Blebbistatin methylphenyl is replaced by a methylthiophen moiety resulting in improved solubility. But the most significant change for specificity was the para substitution of the D-ring with a morpholino group. Indeed, more recently independent development of SkMyo2 inhibitors results in the development of skeletostatins, i.e., blebbistatin derivatives characterized by a morpholino group of the D-ring in para (Radnai et al., 2022). As revealed by the crystallographic structure of the complex between rabbit SkMyo2 and MPH-220, in the allosteric pocket, the morpholino group comes 3.1 Å close to Leu476 at the extremity of the HP-helix which corresponds to a phenylalanine in all other Myo2s. Thus, binding of MPH-220 to any other Myo2 would result in a steric clash between this phenylamine, explaining the striking specificity of MPH-220 for SkMyo2. This explanation was further supported by the fact that introduction of mutation Phe490Leu in NM2C Motor rescues MPH-220 ability to inhibit NM2C.

Through characterization of MPH-220 and SkMyo2, we also gain insights on Blebbistatin photodegradation process, which can be accelerated by the addition of alcohol reagents, or DTT. On this basis, we proposed that MPH-220 degradation can be due to the high concentration of DTT used in the crystallization drops. Yet, this does not seem to be an issue in physiological conditions as András Málnási-Csizmadia's lab confirms the efficiency and non-toxicity of the drug in mice, assess its high drugability.

Finally, this study attests of the potential of the Blebbistatin allosteric pocket for specific targeting of Myo2 motors. Indeed, if only MPH-220 results in strong selectivity for SkMyo2, several Blebbistatin derivatives exhibit a relative selectivity among Myo2s, starting with AmBlebb. Our collaboration with András Málnási-Csizmadia is now continuing to unpack the origin of specificity of a series of Blebbistatin derivatives through co-crystallization assays in CardMyo2, SmMyo2 and SkMyo2.

The Blebbistatin allosteric pocket is rather buried, in the inner part of the 50 kDa cleft of the motor.

Thus, to fully understand what rules blebbistatin derivatives specificity among Myo2, the Blebbistatin tunnel should be looked at as well. We already identified several residues at the entrance of the 50 kDa cleft that greatly differ among Myo2s and could at least in part explain why myosins with very similar Blebbistatin pockets are not equally inhibited by Blebbistatin.

Gathering all this information should help understanding why unconventional myosins which exhibit a rather conserved blebbistatin pocket compared to Myo2, such as Myo6, are not inhibited by Blebbistatin, and that understanding could eventually lead to the development of a new Myo6 inhibitor in the future.

General conclusion

Myosin motors are allosteric machines whose force is essential for key processes in cells. Thus, their malfunctions have been associated with many diseases. Understanding how the force produced by these motors can be modulated is thus of outstanding interest. During my PhD, using a structural approach, I aimed at dissecting how the unique molecular functions of Myo6 can be regulated by Myo6 binding partners, how a pathogenous mutant could perturb this process and how the small allosteric effector MPH-220 can specifically inhibit fast SkMyo2.

Using this structural approach, I managed to solve the structure of the anti-spastic drug MPH-220 bound to the Motor domain of SkMyo2. These X-ray crystallographic data offer an unambiguous electron density for MPH-220 within the blebbistatin pocket. Thus, our structural data, together with the ATPase characterization of the NM2C F/L mutant, clearly demonstrated that the morpholino cycle of MPH-220 is incompatible with the presence of a phenylalanine at the N-terminus of the HP-helix of the myosin motor. Thanks to these findings we were thus able to understand the origin of the exquisite specificity of MPH-220 for SkMyo2. Yet such structural approach can be particularly challenging to put in place, especially in the case of allosteric proteins whose conformational changes can greatly complicate their structural characterization. In particular, the crystallographic approach that I used in an attempt to obtain the structure of Myo6 in its auto-inhibited state remains unsuccessful. If the fusion of the Jo and the In tag with the Myo6 N-and C-terminus (respectively) successfully improved the stability of the auto-inhibited state, the crystals obtained with Jo-Myo6-In remained very small and exhibited no X-ray diffraction at the synchrotron beamline proxima-2 (SOLEIL synchrotron). Overall, it seems that the crystallographic approach may not be the most appropriate in this case. In contrast, negative staining EM gave us a very encouraging result since a first ~17 Å 3D reconstruction of the Jo-Myo6-In was obtained using this technique suggesting that in fact cryoEM might be the most promising approach to explore to determine the structure of the Myo6 auto-inhibited state at high resolution in the future.

To offer more complete insights on myosin activity modulation, the structural work was combined with functional assays, experiments in cells and even in living animals thanks to great collaborations with the laboratories of András Málnási-Csizmadia and Lee Sweeney. In this context structural data offers insights enabling to guide the functional assays while the functional assays provide data to confront/validate the hypothesis proposed based on the structural work. Taken all these data together, we gain insights on myosin auto-inhibition and modulation of motor activity using a highly specific inhibitor.

Auto-inhibition is a key regulation mechanism for myosins in general but a clear description of the auto-inhibited state and ways to relieve Auto-inhibition remains poorly understood in most cases.

Actually, a high-resolution structure of the auto-inhibited state is only available in the case of SmMyo2 (Heissler et al. 2021). Thus, working on Myo6 auto-inhibition was of particular interest for understanding the mechanism of auto-inhibition of myosin motors in general. Previous studies have identified interactions between the CBD and the neck that must mediate Myo6 auto-inhibition (Fili et al. 2017, 2020, Batters et al. 2016) and showed ability of partners to relieve Myo6 auto-inhibition [START_REF] Fili | NDP52 Activates Nuclear Myosin VI to Enhance RNA Polymerase II Transcription[END_REF],2020, Rai et al. 2022). In our study, we addressed the importance of the nucleotide for Myo6 auto-inhibition and demonstrated that in the context of a full-length motor, not all partners are able to activate the motor on their own. Moreover, we highlighted that the deafness mutation L926Q can destabilize the Myo6 auto-inhibited state, making Myo6 accessible for partners that normally cannot relieve Myo6 auto-inhibition such as Dab2 and TOM1. Our work on Myo6 thus highlights the importance of the myosin auto-inhibition for appropriate recruitment of the motor.

Being able to modulate myosin activity with small effectors is also of great interest in order to this pocket as this myosin exhibits rather well conserved residues in the blebbistatin pocket although it interestingly is not inhibited by blebbistatin.

Figure 1 : 15 Figure 2 : 16 Figure 3 :

 1152163 Figure 1: Simplistic view of the organization of the cytoskeleton and molecular motors in cells..….p.15 Figure 2: Unrooted phylogenetic tree of the 40 human myosin genes sorted in classes………………..p.16 Figure 3: Myosin classes can adopt various conformations enabling them for diverse functions…..p.17Figure 4: Diagram of myosin general organization…………………………………………………………………………p.18 Figure 5: Myosin head organization……………………………………………………………………………………….….…p.19 Figure 6: IQ motif among myosins…………………………………………………………………………………………..…….p.20 Figure 7: Lever arm conformational change during the last step of the powerstroke……………….……p.21 Figure 8: Scallop Myo2 Lever arm……………………………………………………………………………….………….………p.22 Figure 9: Myosins general organization………………………………………………………………………………………….p.23 Figure 10: kinetic cycle of a myosin motor………………………………………………………………………..…………….p.24 Figure 11: sensory hair cell degenerescence upon Myo6 loss………………………………….…………………….p.27 Figure 12: deafness/hearing loss mutation reported for Myo6……………………………………….……..……..p.28 Figure 13: Overall organization of the Myo6 motor……………………………………………………………………….p.29 Figure 14: ATPase cycle of Myo6…………………………………………………………………………………………….……..p.31 Figure 15: Myo6 Pi release…………………………………………………………………………………………………….………p.32 Figure 16: Leu310 hinders ATP binding due to the presence of Insert-1………………………………………..p.32 Figure 17: Ins2 reorientates Myo6 converter towards the minus end of actin filament…………….…..p.33 Figure 18: Converter rearrangement between the pre-powerstoke and the rigor-like state reorients Insert-2………………………………………………………………………………………………………………………………………….p.34 Figure 19: Myo6 can undergoes a particularly large stroke due to converter rearrangement………..p.35 Figure 20: Myo6 Lever arm…………………………………………………………………………………………………………….p.36 Figure 21: Myo6 triple helix bundle is stabilized by hydrophobic interactions……………………………….p.36 Figure 22: sequence alignment of SAH domain among species………………………………………………….….p.37 Figure 23: DT domain organization…………………………………………………………………………………………..……p.38 Figure 24: localization of Myo6 isoforms in MDCK cells…………………………………………………………………p.39 Figure 25: sequence alignment of human Myo6 Tail (998-end) isoforms…………………………………….…p.39 Figure 26: Distal Tail and Cargo binding domain, key platforms for Myo6 recruitment………………….p.40 Figure 27: Diagram of Myo6 interactome………………………………………………………………………………….….p.41 Figure 28: Dab2 fragment 675-711 form a 2:2 complex with Myo6 CBD c ……………………….……….……p.42 Figure 29: TOM1 fragment 436-462 form a 1:1 complex with Myo6 CBD c …………………..……………..…p.44 Figure 30: GIPC1 GH2 domain forms long oligomers when bound to the CBD n fragment………………p.46 Figure 31: Clathrin organization…………………………………………………………………………………………………...p.48 Figure 32: CLCa fragment bound to CBD n fragment…………………………………………………………………….…p.49 Figure 33: Ubiquitin chains linkage………………………………………………………………..……………………………..p.50 Figure 34: K63-diUbiquitin bound to the CBD n fragment…………………………………………………………….…p.51 Figure 35: Myo6 CBD binds to PIP2 resulting in conformational changes………………………………………p.52 Figure 36: DNA binding motif……………………………………………………………………………………………………..…p.53 Figure 37: CBD n organization……………………………………………………………………………………………………..….p.54 Figure 38: Increasing number of elongated vesicles suggest the role of Myo6 for fission……………...p.61 Figure 39: sensory ear hair cell organization and Myo6 distribution……………………………………………..p.65 Figure 40: Myo6 behavior switches mode under load…………………………………………………………………...p.75 Figure 41: Distal dimerization…………………………………………………………………………………………..……………p.77 Figure 42: SAH mediated dimerization………………………………………………………………………………………..…p.79 Figure 43: Proximal dimerization………………………………………………………………………………………………..…p.79 Figure 44: Myo6 auto-inhibited state model…………………………………………………………………………….…..p.82 Figure 45: Myo6 oligomeric states instruct this motor for specific, distinct mechanical properties.p.87 Figure 46: Myo6 oligomeric state induced by binding of various partners…………………………………….p.90 Figure 47: Characterization of Myo6 auto-inhibited state using FLMyo6 SI pig-mouse chimera…….p.96

Figure 4 : 18 Figure 5 : 19 Figure 6 : 20 Figure 7 : 21 Figure 8 : 22 Figure 9 : 23 Figure 10 : 24 Figure 11 : 27 Figure 12 : 28 Figure 13 : 29 Figure 14 : 31 Figure 15 : 32 Figure 16 : 32 Figure 17 : 33 Figure 18 : 34 Figure 19 : 35 Figure 20 : 36 Figure 21 : 36 Figure 22 : 37 Figure 23 : 38 Figure 24 : 39 Figure 25 : 39 Figure 26 : 40 Figure 27 : 41 Figure 28 : 42 Figure 29 : 44 Figure 30 : 46 Figure 31 : 48 Figure 32 : 49 Figure 33 : 50 Figure 34 : 51 Figure 35 : 52 Figure 36 : 53 Figure 37 : 54 Figure 38 : 61 Figure 39 : 65 Figure 40 :Figure 41 :Figure 42 :Figure 43 :Figure 44 :Figure 45 :Figure 46 :Figure 47 :

 4185196207218229231024112712281329143115321632173318341935203621362237233824392539264027412842294430463148324933503451355236533754386139654041424344454647 Figure 1: Simplistic view of the organization of the cytoskeleton and molecular motors in cells..….p.15 Figure 2: Unrooted phylogenetic tree of the 40 human myosin genes sorted in classes………………..p.16 Figure 3: Myosin classes can adopt various conformations enabling them for diverse functions…..p.17Figure 4: Diagram of myosin general organization…………………………………………………………………………p.18 Figure 5: Myosin head organization……………………………………………………………………………………….….…p.19 Figure 6: IQ motif among myosins…………………………………………………………………………………………..…….p.20 Figure 7: Lever arm conformational change during the last step of the powerstroke……………….……p.21 Figure 8: Scallop Myo2 Lever arm……………………………………………………………………………….………….………p.22 Figure 9: Myosins general organization………………………………………………………………………………………….p.23 Figure 10: kinetic cycle of a myosin motor………………………………………………………………………..…………….p.24 Figure 11: sensory hair cell degenerescence upon Myo6 loss………………………………….…………………….p.27 Figure 12: deafness/hearing loss mutation reported for Myo6……………………………………….……..……..p.28 Figure 13: Overall organization of the Myo6 motor……………………………………………………………………….p.29 Figure 14: ATPase cycle of Myo6…………………………………………………………………………………………….……..p.31 Figure 15: Myo6 Pi release…………………………………………………………………………………………………….………p.32 Figure 16: Leu310 hinders ATP binding due to the presence of Insert-1………………………………………..p.32 Figure 17: Ins2 reorientates Myo6 converter towards the minus end of actin filament…………….…..p.33 Figure 18: Converter rearrangement between the pre-powerstoke and the rigor-like state reorients Insert-2………………………………………………………………………………………………………………………………………….p.34 Figure 19: Myo6 can undergoes a particularly large stroke due to converter rearrangement………..p.35 Figure 20: Myo6 Lever arm…………………………………………………………………………………………………………….p.36 Figure 21: Myo6 triple helix bundle is stabilized by hydrophobic interactions……………………………….p.36 Figure 22: sequence alignment of SAH domain among species………………………………………………….….p.37 Figure 23: DT domain organization…………………………………………………………………………………………..……p.38 Figure 24: localization of Myo6 isoforms in MDCK cells…………………………………………………………………p.39 Figure 25: sequence alignment of human Myo6 Tail (998-end) isoforms…………………………………….…p.39 Figure 26: Distal Tail and Cargo binding domain, key platforms for Myo6 recruitment………………….p.40 Figure 27: Diagram of Myo6 interactome………………………………………………………………………………….….p.41 Figure 28: Dab2 fragment 675-711 form a 2:2 complex with Myo6 CBD c ……………………….……….……p.42 Figure 29: TOM1 fragment 436-462 form a 1:1 complex with Myo6 CBD c …………………..……………..…p.44 Figure 30: GIPC1 GH2 domain forms long oligomers when bound to the CBD n fragment………………p.46 Figure 31: Clathrin organization…………………………………………………………………………………………………...p.48 Figure 32: CLCa fragment bound to CBD n fragment…………………………………………………………………….…p.49 Figure 33: Ubiquitin chains linkage………………………………………………………………..……………………………..p.50 Figure 34: K63-diUbiquitin bound to the CBD n fragment…………………………………………………………….…p.51 Figure 35: Myo6 CBD binds to PIP2 resulting in conformational changes………………………………………p.52 Figure 36: DNA binding motif……………………………………………………………………………………………………..…p.53 Figure 37: CBD n organization……………………………………………………………………………………………………..….p.54 Figure 38: Increasing number of elongated vesicles suggest the role of Myo6 for fission……………...p.61 Figure 39: sensory ear hair cell organization and Myo6 distribution……………………………………………..p.65 Figure 40: Myo6 behavior switches mode under load…………………………………………………………………...p.75 Figure 41: Distal dimerization…………………………………………………………………………………………..……………p.77 Figure 42: SAH mediated dimerization………………………………………………………………………………………..…p.79 Figure 43: Proximal dimerization………………………………………………………………………………………………..…p.79 Figure 44: Myo6 auto-inhibited state model…………………………………………………………………………….…..p.82 Figure 45: Myo6 oligomeric states instruct this motor for specific, distinct mechanical properties.p.87 Figure 46: Myo6 oligomeric state induced by binding of various partners…………………………………….p.90 Figure 47: Characterization of Myo6 auto-inhibited state using FLMyo6 SI pig-mouse chimera…….p.96

Figure 48 :Figure 49 :Figure 50 :Figure 51 :Figure 52 :Figure 53 :Figure 54 :Figure 55 :Figure 56 :Figure 57 :Figure 58 : 159 Figure 59 :Figure 60 :Figure 61 :Figure 62 :Figure 64 :Figure 65 :Figure 66 : 171 Figure 67 :Figure 68 :Figure 69 :Figure 70 :Figure 71 :Figure 72 :Figure 74 :Figure 82 :Figure 85 :Figure 86 :Figure 90 :Figure 91 :Figure 92 :

 48495051525354555657581595960616264656617167686970717274828586909192 Figure 48: Myo6 sequence alignment………………………………………………………………………………………..….p.98 Figure 49: Surface residues conservation between pig and mouse Myo6………………………………………p.98 Figure 50: Myo6 pig-mouse chimera opens more easily upon salt concentration……………………….…p.99 Figure 51: GIPC1 organization………………………………………………………………………………………………….....p.102 Figure 52: GIPC1 auto-inhibited state prevents binding to Myo6 ………………….………………..…………..p.103 Figure 53: TOM1 organization……………………………………………………………………………………………………..p.104 Figure 54: Dab2 organization………………………………………………………………………………………………….……p.105 Figure 55: Main interactions stabilizing the Myo6 auto-inhibited………………………………………………..p.157 Figure 56: CBD c loop mutant (D1148V, Y1150D, D1152R and Q1154V) has only little impact on Myo6 compacity in vitro……………………………………………………………………………………………………..…………………p.158 Figure 57: DNA binding motifs and CBD c loop mutant (D1148V, Y1150D, D1152R and Q1154V) within CBD c ……………………………………………………………………………………………………………………………………..……..p.159 Figure 58: D1148V, Y1150D, D1152R and Q1154V mutations disrupt Myo6 CBD recruitment to melanosomes………………………………………………………………………………………………………………………………p.159 Figure 59: Jo-Myo6-In crystallization drops without seeding (right) and with seeding (left)………..p.160 Figure 60: Jo-Myo6-In is a multidomain protein……………………………………………………………………….…p.161 Figure 61: Model of the active FL Myo6 proximal dimer………..……………………………………………………p.164 Figure 62: Model of the active FL Myo6 proximal dimer with modelization of the R849-E875 region ………………………………………………………………………………………………………………………………………………......p.165 Figure 64: Model of the GIPC1-Myo6 complex that could either form a single dimer or a large oligomer of dimers………………………………………………………………………………………………………………………..…………...p.167 Figure 65: interferometric scattering mass spectrometry principle……………………………………………..p.170 Figure 66: Sequence alignment of the 851-949 (human gene numbering) amino acids among species. ……………………………………………………………………………………………………………………………………………………p.171 Figure 67: K63-diUbiquitin is unable to increase the Myo6 ATPase rate …………………………………..…p.173 Figure 68: Myo2 subfragments………………………………………………………………………………….……………..…p.178 Figure 69: Overall sarcomere organization………………………………………………………………….……………...p.179 Figure 70: Relative abundance of Myo2 isoforms in human muscles ………………………………………....p.180 Figure 71: Allosteric binding pockets within the myosin motor...................................................... p.183 Figure 72: (-)-Blebbistatin scaffold……………………………………………………………………………………………….p.186 Figure 73: Blebbistatin IC50 for various Myo2 isoforms……………………………………………….……………….p.187 Figure 74: Blebbistatin photodegradation……………………………………………………………………………………p.190 Figure 75: Putative photochemical mechanism for blebbistatin photo-inactivation upon 416 nm illumination ………………………………………………………………………………………………………………………………..p.191 Figure 76: Blebbistatin binding site……………………………………………………………………..…..………………...p.195 Figure 77: Blebbistatin tunnel within the DictyMyo2 pre-powerstroke motor………………………….…p.196 Figure 78: Identification of most promising positions for A-ring substitution………………………………p.197 Figure 79: A-ring derivatives from Verhasselt et al. (2017)…………………………………………………….……p.198 Figure 80: The Arg238 backbone is close to the blebbistatin C-ring and was proposed as a potential way to mediate additional polar contact in the allosteric pocket………………………………………………..p.198 Figure 81: D-ring derivatives of blebbistatin…………………………………………………………………………….…p.199 Figure 82: Comparison between blebbistatin and MPH-220…………………………………………………….…p.200 Figure 83: Degraded MPH-220 within the SkMyo2 motor……………………………………….……………….…p.201 Figure 84: MPH-220 fitted in SkMyo2 blebbistatin allosteric pocket (6YSY)………………………………..p.204 Figure 85: Absorption of 10 µM blebbistatin in water or SkMyo2 buffer…………………………………….p.205 Figure 86: Absorption of 10 µM blebbistatin supplemented with various reagents after 18h in white light………………………………………………………………………………………………………………………………………….….p.206 Figure 87: Example of crystallization drops for SkMyo2 MPH-220 crystals with (right) and without (left) DTT……………………………………………………………………………………………………………………………………………..p.206 Figure 88: Superimposition of SkMyo2 allosteric pocket with MPH-220 (PDB:6YSY) and without MPH-220………………………………………………………………………………………………………………………………………………p.235 Figure 89: Superimposition of SkMyo2 with MPH-220 (PDB: 6YSY) and without MPH-220………….p.235

Figure 1 :

 1 Figure 1: Simplistic view of the organization of the cytoskeleton and molecular motors in cells (Ross, Ali, and Warshaw 2008)Actin filaments (in red) are found at the cell periphery while microtubules (green) radiate from microtubuleorganizing centers (MTOC). In cartoon: myosin Va (dark brown) and myosin VI (light blue) moving towards the plus or minus ends of the actin filaments (respectively). Kinesins (yellow) and dyneins (dark blue) moving towards the plus or minus end of the microtubules (respectively). Contacts between actin filaments and microtubules allowcollaboration. In particular, vesicles (sand) can thus switch from actin-based motion to microtubule-based motion or from microtubule-based motion to actin-based motion thanks to collaboration between the molecular motors.

  myosin genes are expressed and divided in 12 classes of myosins (class I, II, III, V, VI, VII, IX, X, XV, XVI, XVIII, and XIX) (Odronitz and Kollmar 2007; Quintero, Moore, and Yengo 2012) (Figure 2). Most of them are ubiquitously expressed in cells.

Figure 2 :

 2 Figure 2: Unrooted phylogenetic tree of the 40 human myosin genes sorted in classes (Quintero, Moore, and Yengo 2012)

Figure 3 :

 3 Figure 3: Myosin classes can adopt various conformations enabling them for diverse functions (Top) Conventional Myo2 (in blue) associate in thick filaments through their coiled coil Tail and slide along actin filaments (yellow) (Bottom) Unconventional myosins; (right) Myo1 associates with the membrane through its Tail (blue) and interacts with the actin filaments through its Motor domain (grey). So doing Myo1 can participate in cell organization. (left) Myo5 binds cargo (green) and associates as a dimer through its Tail (blue) to take processive steps on the actin track (yellow). Light chains in the neck region are depicted in orange, note that their number vary depending on the myosin isoform.

Figure 4 :

 4 Figure 4: Diagram of myosin general organization Simplified representation of the general organization of a myosin with from N-terminus to C-terminus a Motor domain (grey) (preceded by an N-terminus domain for Myo3, Myo16 and Myo18) a Lever arm (orange) and a divergent Tail (light blue).

Figure 5 :

 5 Figure 5: Myosin head organization (A) Simplified organization of myosin Motor domain and neck. Color code pictured on the figure (B) Motor domain of scallop Myo2 bound to Mg.ADP.VO4 (Pi analog) solved by X-ray crystallography at 2.6 Å (PDB: 1QVI; (Gourinath et al. 2003)). Color code as in (A) (C) close up on scallop Myo2 active site. The key nucleotide binding residues are highlighted as sticks.

Figure 6 :

 6 Figure 6: IQ motif among myosins (Heissler and Sellers 2014) IQ sequence from myosin I, II, V, VI, VII, X, XIV. First position is occupied by a hydrophobic residue, position 2 is occupied by a conserved Gln, position 6 is occupied by a positively charged residue and position 11 is preferentially occupied by a positively charged residue.

Figure 7 :

 7 Figure 7: Lever arm conformational change during the last step of the powerstroke (Robert-Paganin et al. 2020) Comparison between Myo1b in strong-ADP (PDB: 6C1H, Mentes et al. 2018) and rigor state (PDB: 6C1C, Mentes et al. 2018). Motor domain is in grey, the converter is in green and the IQ is in blue. An arrow emphasizes the swing performed by the Lever arm in between strong-ADP and rigor state.

Figure 9 :

 9 Figure 9: Myosins general organization (Li and Zhang 2020) General organigram of the 12 different myosins found in human. Domains are colored depending on their nature with the conserved Motor domain in red; the IQ motifs in dark grey (note that the number of IQ motifs varies depending on the myosin class); coiled coil, single α-helix, Lever arm extension in pink; the unstructured regions in light grey and the structured domains in blue (GTD: globular Tail domain, CBD: cargo binding domain, SH3: SRC homology 3 domain, MyTH4-FERM: myosin Tail homology 4-band 4.1, ezrin, radixin, moesin, GAP: GTPaseactivating protein, ANK domain: ankyrin domain, PH: pleckstrin homology domain).

Figure 10 :

 10 Figure 10: kinetic cycle of a myosin motor (Sweeney and Houdusse 2010) Schematic representation of myosin ATPase cycle. Actin is represented as yellow dots, myosin Motor domain as an oval either white circled in black when it exhibits a low actin affinity or blue when it exhibits a high actin affinity, the converter is representer in green and the Lever arm is represented in light blue and switch position along the ATPase cycle.

  are thus refined machines that generate force, according to a rather conserved mechanism. Yet strong structural divergence in the Tail, the addition of an extra N-terminus domain as well as subtle differences in the Motor domain, notably in the actin loops (Robert-Paganin et al. 2020), instruct the different myosin classes to various functions from muscle contraction (Sweeney and Hammers 2018) to gene expression (Louvet and Percipalle 2009) and drug-associated memory (Briggs et al. 2017). Myosins are also associated with several diseases or disorders like hypertrophic cardiomyopathy, deafness, spasticity and several cancers (Coluccio 2020). This makes the study of myosin motors of particular interest both for the characterization of the many intracellular processes they are involved in as well as for the development of targeted treatments when diseases are linked to myosin malfunction or overexpression.

Figure 11 :

 11 Figure 11: sensory hair cell degenerescence upon Myo6 loss (Self et al. 1999) Scanning electron microscopy of sensory hair cells of wild-type (A, C, E, G) or Snell's Waltzer (Myo6-null) (B, D, F, H) mice cochlea from 1 to 20 days after birth. On the left part of the figure, we can clearly see that the hair cells degenerate to form giant stereocilia in Myo6-null mice as opposed to wild-type mice on the right.

Figure 12 :

 12 Figure 12: deafness/hearing loss mutation reported for Myo6 Myo6 diagram with the Motor domain (MD) in grey, the Insert-2/calmodulin (Ins2/CaM) in purple, the IQ motif/calmodulin (IQ/CaM) in red, the triple helix bundle (3HB) in blue, the single α-helix (SAH) in green, the distal Tail (DT) in orange and the cargo binding domain (CBD) in brown. Deafness mutations are indicated on the fulllength Myo6 diagram. fs stands for frame shift, fs* indicates the position of the translation termination, c. stand for coding DNA, > indicates nucleotide substitution, ins indicates an Insertion, X indicates a stop. Mutations from

Figure 13 :

 13 Figure 13: Overall organization of the Myo6 motor Myo6 organization diagram: From the N-terminus to C-terminus, Myo6 contains a Motor domain (MD) in grey, the Insert-2 (Ins2) in purple bound to a Ca 2+ -CaM in lila, the IQ motif with its associated calmodulin in red, the triple helix bundle (3HB) in blue, the single α-helix (SAH) in green, the distal Tail (DT) in orange and the cargo binding domain (CBD) in brown

  release state of Myo6. The different crystals describe different positions of Pi within the Myo6 Motor depending on the time they were soaked in Pi which traces the putative Pi route to escape the Motor.Moreover, deTailed analysis of the structures together with kinetic studies of Myo6 mutants designed to impair the actin binding or cleft closure suggest that Pi release must occur before the cleft closure and Lever arm swing but after a conformational change occurring due to actin binding, offering deTailed insights on the force generation mechanism of Myo6 and possibly other myosin motors[START_REF] Llinas | How Actin Initiates the Motor Activity of Myosin[END_REF] (Figure15).

Figure 14 :

 14 Figure 14: ATPase cycle of Myo6 Motor domain in blue, converter in green, Lever arm in purple, actin in orange. When not present in the experimental structure, putative position of the converter is depicted as a green circle and the putative position of the Insert-2, at the basis of the Lever arm as a purple dotted line. X-ray structures of the Pi release (PDB: 4PJN,

Figure 15 :

 15 Figure 15: Myo6 Pi release

1 "

 1 is localized near the actin binding element switch-I[START_REF] Ménétrey | The Structure of the Myosin VI Motor Reveals the Mechanism of Directionality Reversal[END_REF] and slows down the Myo6 ATP binding rate compared to other myosins by repositioning Leu310 and creating selective steric hindrance for ATP but not ADP binding[START_REF] Pylypenko | Role of Insert-1 of Myosin VI in Modulating Nucleotide Affinity[END_REF]) (Figure16).

Figure 16 :

 16 Figure 16: Leu310 hinders ATP binding due to the presence of Insert-1 (Pylypenko et al. 2011)Myo6 active site with or without Insert-1 in rigor-like state (ATP modelized in X-ray crystallography structures). In wild-type Leu310 is closer to the ATP binding pocket generating hindrance and slowing-down its binding while upon removal of Insert-1, Leu310 moves out of the pocket resulting in faster ATP binding[START_REF] Pylypenko | Role of Insert-1 of Myosin VI in Modulating Nucleotide Affinity[END_REF]

Figure 17 :

 17 Figure 17: Ins2 reorientates Myo6 converter towards the minus end of actin filament (adapted from Ménétrey et al. 2005).

Figure 18 :

 18 Figure 18: Converter rearrangement between the pre-powerstoke and the rigor-like state reorients Insert-2 (Ménétrey et al. 2007) (A) Comparison of the converter (green) and Insert-2 (purple) position in the rigor like and in the pre-powerstroke state. If the position of the central β-sheet of the converter is rather conserved, the hinges (black) and the helices adopt very different conformations resulting in the repositioning of Insert-2. (B) Superimposition of Myo6 in the rigor-like (black) and pre-powerstroke (colors) states highlights the 90° repositioning of the Insert-2 between the two states.

Figure 19 :

 19 Figure 19: Myo6 can undergoes a particularly large stroke due to converter rearrangement (Spudich and Sivaramakrishnan 2010) Schematic representation of Myo2, Myo5 and Myo6 strokes with the Motor domain in blue, the converter in green and the neck in dark blue bound to light chains in yellow. Myo6 Tail is depicted in orange and actin is depicted in red. Position of the Lever arm in the pre-powerstroke (before the stroke) and the rigor states (after the stroke) are displayed. Lever arm position in the pre-powerstroke is shown in transparency. Myo2 and Myo5 undergoes a ~70° stroke while Myo6 undergoes a ~180° stroke.

Figure 20 :

 20 Figure 20: Myo6 Lever arm[START_REF] Mukherjea | Myosin VI Dimerization Triggers an Unfolding of a Three-Helix Bundle in Order to Extend Its Reach[END_REF] Myo6 Lever arm with Ins2 in purple bound to Ca 2+ -CaM in lila and IQ motif in cyan bound to apoCaM. A triple helix bundle (blue) follows the IQ motif and has been proposed to unfold and act as a Lever arm extension[START_REF] Mukherjea | Myosin VI Dimerization Triggers an Unfolding of a Three-Helix Bundle in Order to Extend Its Reach[END_REF]) by revealing a new CaM binding site[START_REF] Mukherjea | Myosin VI Dimerization Triggers an Unfolding of a Three-Helix Bundle in Order to Extend Its Reach[END_REF] Batters et al. 2016)

Figure 21 :

 21 Figure 21: Myo6 triple helix bundle is stabilized by hydrophobic interactions The NMR structure of the Myo6 3HB (PDB: 2LD3; Yu et al. 2012) is similar to the structure solved in the context of the whole Lever arm (PDB: 3GN4, Mukherjea et al. 2009). The only difference is that the NMR structure proposes a conformation for the loop region that is disordered in the crystal structure. Residues are coloured depending on their charges: residues with positively charged side chains are in blue, residues with negatively charged side chains

Figure 22 :

 22 Figure 22: sequence alignment of SAH domain among speciesResidues are colored depending on their charges: residues with positively charged side chains are in blue, residues with negatively charged side chains are in red, residues with polar uncharged side chains are in purple.

Figure 23 :

 23 Figure 23: DT domain organization (Left) SAXS envelope of a Myo6 fragment containing the SAH and the DT domain. Structure of SAH and DT comes from Rosetta prediction. SAH is in green and DT in orange (Spink et al. 2008). (Right) Top 20 conformers found by NMR for the 998-1071 fragment containing a portion of the DT domain. High variability between the conformers shows a high flexibility of the fragment when purifies alone (PDB: 2N11, Wollscheid et al. 2016)

Figure 24 :

 24 Figure 24: localization of Myo6 isoforms in MDCK cells (adapted from Au et al. 2007) Location of GFP Myo6 in MDCK cells of Myo6 isoforms (NI, SI, LI and SI+LI). Myo6 location is plotted in green (left panels) and actin (stained using rhodamine phalloidin) is plotted in red (middle panels). Merged images of Myo6 and actin are pictured in the right panels. Scale bar: 10 µm.

Figure 26 :

 26 Figure 26: Distal Tail and Cargo binding domain, key platforms for Myo6 recruitment Diagram representing the different domains of the most distal part of the Myo6 Tail. The distal Tail, that contains the ubiquitin binding motif (MIU) (Penengo et al. 2006) is depicted in orange. The cargo binding domain (CBD) is

Figure 27 :

 27 Figure 27: Diagram of Myo6 interactome (O'Loughlin, Masters, and Buss 2018) Diagram of Myo6 interaction network identified by BioID. Myo6 (white circle) is represented in the middle of the diagram. Partners are gathered in groups according to their subcellular locations. GIPC1, LARG, DOCK7 and LRCH3, that were used as baits in secondary BioID screens in O'Loughlin, Masters, and Buss (2018) study, are represented in yellow. Interactions identified by BioID are represented with a solid line. The diagram was completed with the interactions (represented through dashed lines) reported in the literature prior to the O'Loughlin, Masters, and Buss (2018) study.

Figure 28 :

 28 Figure 28: Dab2 fragment 675-711 form a 2:2 complex with Myo6 CBD c Crystal structure of the Dab2 (675-711) -CBD c complex (PDB: 3H8D, Yu et al. 2009). Dab2 interaction with Myo6 involved the WWY motif. Myo6 and Dab2 interacts together through two interfaces thanks to whom Dab2 can mediate indirectly Myo6 CBD c dimerization.

Figure 29 :

 29 Figure 29: TOM1fragment 436-462 form a 1:1 complex with Myo6 CBD c Crystal structure of the TOM1 (436-462 -numbering according to human TOM1 canonical isoform) -CBD c complex (PDB 6J56, Hu et al. 2019). Dab2 interaction with Myo6 involved the WWY motif. Contrary to Dab2, TOM1 interacts with Myo6 CBD c via a unique interface resulting in the formation of a 1:1 complex.

Figure 30 :

 30 Figure 30: GIPC1 GH2 domain forms long oligomers when bound to the CBD n fragment Crystal structure of the GIPC1 -CBD n complex (PDB 5V6E, Shang et al. 2017). GIPC1 and CBD n form oligomers through two interfaces

Figure 31 :

 31 Figure 31: Clathrin organization[START_REF] Halebian | Structure and Assembly of Clathrin Cages[END_REF] Left: Structure of a clathrin triskelion from cryoEM model (PDB: 3IYV,[START_REF] Fotin | Molecular Model for a Complete Clathrin Lattice from Electron Cryomicroscopy[END_REF]. Right: CryoEM map of a clathrin cage with triskelions pictured in red, blue and yellow (PDB: 3IYV,[START_REF] Fotin | Molecular Model for a Complete Clathrin Lattice from Electron Cryomicroscopy[END_REF]

Figure 32 :

 32 Figure 32: CLCa fragment bound to CBD n fragment NMR structure of the CLCa (46-61) -CBD n ( 1050-1131 )complex (PDB 6E5N, Biancospino et al. 2019). Clathrin bound with Myo6 through two helices: the helix G1080-R1131 (green) which is also involved in interaction with partners of Myo6 RRL motif and the last helix of the long insert (blue) making its binding isoform specific.

  chains to address them to degradation pathways. Ubiquitin chains (polyubiquitin) are either linear (M1) (Ubiquitin C-terminus bound to N-terminus of the following ubiquitin) or branched through bonds between the ubiquitin C-terminus and a lysine side chain of the following ubiquitin (K6, K11, K27, K29, K33, K48 or K63) (Akutsu, Dikic, and Bremm 2016; Komander and Rape 2012) (Figure33). Each kind of ubiquitin chain links the ubiquitinylated proteins to a specific fate, K11 and K48 chains mediate protein degradation at proteasome[START_REF] Akutsu | Ubiquitin Chain Diversity at a Glance[END_REF][START_REF] Komander | The Ubiquitin Code[END_REF] while K63 can mediate interaction between two proteins[START_REF] Akutsu | Ubiquitin Chain Diversity at a Glance[END_REF][START_REF] Komander | The Ubiquitin Code[END_REF].

Figure 33 :

 33 Figure 33: Ubiquitin chains linkage Left: X-ray crystal structure of K63-diUbiquitin (PDB: 3H7P, Weeks et al. 2009). Lys63 is colored in purple, all the other lysines able to give rise to another kind of ubiquitin linkage (Lys6, Lys11, Lys27, Lys29, Lys33 and Lys48) are colored in orange. Right: example of ubiquitin linkages extracted from Komander and Rape 2012. Ubiquitins are represented as red spheres that carried a number corresponding to the type of linkage (48 for K48 linkage, 63 for K63 linkage and 11 for K11 linkage)

Figure 34 :

 34 Figure 34: K63-diUbiquitin bound to the CBD n fragmentThe NMR structure of the K63-diUbiquitin / CBD n complex (1080-1122) (PDB 2N13,[START_REF] He | Myosin VI Contains a Compact Structural Motif That Binds to Ubiquitin Chains[END_REF]. Ubiquitin binds through two interfaces that involve two different helices of the CBD n . The Key residue for binding I1072 (blue) is found in one of the two interfaces.

Figure 35 :

 35 Figure 35: Myo6 CBD binds to PIP2 resulting in conformational changes Left: The Myo6 Tail sedimentation with mixed brain liposomes or liposomes specifically containing phosphoinositides (PI), phosphatidylserine (PS), phosphatidylinositol-3-phosphate (PI(3)P), phosphatidylinositol-4-phosphate (PI(4)P), phosphatidylinositol-5-phosphate (PI(5)P), phosphatidylinositol-4,5-bisphosphate

Figure 36 :

 36 Figure 36: DNA binding motif NMR structure of CBD c (PDB: 2KIA, Yu et al. 2009) is pictured in magenta. The TRTK (blue) and SKKK (green) motifs, whose depletion result in 5/7-fold decrease in affinity for DNA thus could be implicated in DNA binding to Myo6.

Figure 37 :

 37 Figure 37: CBD n organization NMR structure of Myo6 CBD n fragment 1050-1137 (PDB: 2N12, Wollscheid et al. 2016) containing the last helix of the long insert and the first two helices of CBD n . Long insert is colored in blue, key helix for CBD n partner binding is colored in green, it contains the RRL motif, colored in red. Note that the long insert folds on the RRL motif, provoking hindrance for binding of RRL partners

Figure 38 :

 38 Figure 38: Increasing number of elongated vesicles suggest the role of Myo6 for fission (Biancospino et al. 2019)

Figure 39 :

 39 Figure 39: sensory ear hair cell organization and Myo6 distribution.

Figure 40 :

 40 Figure 40: Myo6 behavior switches mode under load (Nishikawa et al. 2010; Iwaki et al. 2016) (A) Model of Myo6 stepping mechanism on actin (brown). Myo6 Motor domain is colored in red, its converter is in green and its Lever arm in blue. CaM are colored in purple. Left: hand-over-hand mechanism resulting in 36 nm steps. Right: Inchworm-like mechanism resulting in 10-22 nm steps (Nishikawa et al. 2010). (B) Example of the trajectory of each head of a Myo6 dimer on actin in optical trapping assay coupled with DNA nanospring. Step

Figure 41 :

 41 Figure 41: Distal dimerization (Mukherjea et al. 2009) Myo6 distal dimerization. The Motor domain is colored in grey, the Insert-2 in purple, the IQ in cyan, the folded 3HB in blue, the SAH in green, the DT in orange and the CBD in purple. Actin is colored in yellow. The two Myo6 molecules interact with each other at the level of the CBD.

  Single molecule motility of Myo6 constructs as assessed by fluorescence imaging with one nanometer accuracy (FIONA) (after actin saturation in rigor)[START_REF] Park | Full-Length Myosin VI Dimerizes and Moves Processively along Actin Filaments upon Monomer Clustering[END_REF][START_REF] Mukherjea | Myosin VI Must Dimerize and Deploy Its Unusual Lever arm in Order to Perform Its Cellular Roles[END_REF]; The FIONA method can track the position of a single fluorophore with ~1.5 nm resolutionUsing molecular dynamic on Myo6 907-980 then 907-940,(Kim et al. 2010) proposed a dimerization of the SAH domain based on formation of salt bridges upon low salt condition (Figure42).Yet peptides that include the SAH domain (with residues 920-940) remain monomeric at high concentration(45.6 µM in SEC-MALS: Spink et al. 2008; 1 mM in NMR: Barnes et al. 2019) excluding a role for this region as triggering proximal dimerization.

Figure 42 :

 42 Figure 42: SAH mediated dimerization (Kim et al. 2010) Myo6 dimerized through SAH association thanks to the formation of salt bridges. The Motor domain is colored in grey, the Insert-2 in purple, the IQ in cyan, the unfolded 3HB in blue, the SAH in green, the DT in orange and the CBD in purple. Actin is colored in yellow.

Figure 43 :

 43 Figure 43: proximal dimerization (Mukherjea et al. 2009) Myo6 dimerized in between SAH and 3HB domain upon unfolding of the 3HB. The Motor domain is colored in grey, the Insert-2 in purple, the IQ in red, the unfolded 3HB in blue, the SAH in green, the DT in orange and the CBD in brown. Actin is colored in yellow.

F. 1 .

 1 Myo6 has been well explored with single molecule biophysical studies, indicating the role the motor may fulfill in cells. Although Myo6 can exist as a compact monomer (Lister et al. 2004), it has been demonstrated that the motor is able to dimerize in high concentration conditions (Parks et al. 2006), which can notably be reached when the motor clusters on vesicles (Altman et al. 2007). Characterization of the Myo6 dimer using in vitro motility assays demonstrated that the motor can take processive runs and can switch to an anchoring function under resisting load (Altman, Sweeney, and Spudich 2004; Chuan, Spudich, and Dunn 2011). Uniquely, the steps that Myo6 takes were found to be irregular and larger than expected. If progress has been made in understanding how Myo6 can take such large steps (Ménétrey et al. 2005; Ménétrey et al. 2007), the precise architecture of the Myo6 dimer remains debated and several models are currently coexisting (Mukherjea et al. 2009; 2014; Kim et al. 2010; Yu et al. 2009; Spink et al. 2008). More precise description of the architecture of the Myo6 dimer(s) is thus of high relevance. This could greatly help understanding why this motor takes irregular steps and how load can constrain it to adopt an anchoring function. Indeed, the lack of knowledge on the architectures of Myo6 processive dimer(s) and anchoring dimer(s) greatly limit our ability to specifically study these Myo6 functions, in particular in cells. F. Regulation of the Myo6 active/inactive pool equilibrium by an auto-inhibited state Engaging large amount of active Myo6 is deleterious, control of myo6 activation is necessary

Figure 44 :

 44 Figure 44: Myo6 auto-inhibited state model (Spink et al. 2008) Model of a back-folded Myo6 build based on SAXS study of FLMyo6 in nucleotide free state. The Motor domain, the Ins2 and the IQ are colored in grey, the 3HB in blue, the SAH in green, the DT in orange and the CBD in magenta. In this model, the Tail folds on itself near the neck region

  ; Shang et al. 2017; Dos Santos et al. 2022), ubiquitin can form long oligomeric chains[START_REF] Akutsu | Ubiquitin Chain Diversity at a Glance[END_REF] and clathrin can assemble in trikele or even cages[START_REF] Halebian | Structure and Assembly of Clathrin Cages[END_REF]. This stoechiometry can also influence the way partners will intruct Myo6 for its function, attaching Myo6 monomeric motors together without direct contact between them or inducing direct dimerization between Myo6 molecules.

Figure 45 :

 45 Figure 45: Myo6 oligomeric states instruct this motor for specific, distinct mechanical propertiesMyo6 is represented in different conformations. The motor domain is colored in grey, the Insert-2 in purple, the IQ in red, the 3HB in blue, the SAH in green, the DT in orange and the CBD in brown. Once unfolded, Myo6 was proposed to adapt various conformation to perform its function. Myo6 was proposed to function as a monomeric bridge linking cargoes together[START_REF] Hu | Structure of Myosin VI/Tom1 Complex Reveals a Cargo Recognition Mode of Myosin VI for Tethering[END_REF], as a proximal dimer[START_REF] Mukherjea | Myosin VI Dimerization Triggers an Unfolding of a Three-Helix Bundle in Order to Extend Its Reach[END_REF] 2014), as a distal dimer[START_REF] Yu | Myosin VI Undergoes Cargo-Mediated Dimerization[END_REF] or as an assembly of monomers[START_REF] Shang | Structure Analyses Reveal a Regulated Oligomerization Mechanism of the PlexinD1/GIPC/Myosin VI Complex[END_REF]. Due to their different conformations and stoechiometries each of them could lead to a different function of Myo6. A more deTailed characterization of these models is now required to determined which are relevants in Myo6 functions in cells, note that several of them could exist but would fulfill different mechanical functions.

  In contrast, TOM1 (6J56;[START_REF] Hu | Structure of Myosin VI/Tom1 Complex Reveals a Cargo Recognition Mode of Myosin VI for Tethering[END_REF], clathrin light chain A (6E5N;[START_REF] Wagner | Myosin VI Drives Clathrin-Mediated AMPA Receptor Endocytosis to Facilitate Cerebellar Long-Term Depression[END_REF] were proposed to form a 1:1 complex with Myo6 on the basis of their crystallographic or NMR structure, although these studies did not look at the impact of these partners on the functional motility properties or the impact on possible proximal dimerization. Finally, a FRET study revealed DNA inability to dimerize Myo6 814-end excluding both proximal and distal dimerization upon DNA binding.Hu et al. (2019) thus hypothesized that, as a non processive monomer, Myo6 plays the role of a bridge between between cellular compartments (Figure45and 46), linking for example endosomes (via TOM1) and autophagy receptors through two different binding interfaces.From various oligomeric states to various dimer architectures of Myo6 upon partner binding, all the results obtained on the study of Myo6-partner binding characterization (see above) highlight the unique ability of Myo6 partners to instruct the motor for a specific architecture and oligomeric state, which can result in various mechanical properties thus likely in various cellular mechanistic roles. Actually, this diversity of Myo6-partners complex structures could at least in part explain why one single motor such as Myo6 can fulfill a wide range of cellular functions switching for example from an anchoring function to a transport function depending on the partner bound to the motor.

Figure 46 :

 46 Figure 46: Myo6 oligomeric state induced by binding of various partners (Top) Crystal structure of the 2:2 complex between Myo6 CBD c and Dab2 fragment 675-713, each Dab2 interacts

Figure 47 :

 47 Figure 47: How to read a Kratsky plot (Stanford.edu) Theoretical kratsky plot that can be obtained from SAXS for proteins of different shapes. q is the scattering vector q=4π sin(θ)/λ with λ the radiation wavelength and 2θ the scattering angle.

Figure 48 :

 48 Figure 48: Characterization of Myo6 auto-inhibited state using FLMyo6 SI pig-mouse chimera (A) SEC-MALS profiles of FL Myo6 SI pig mouse chimera in either MgADP.VO4 buffer: 20 mM Hepes; 2 mM MgCl2; 1 mM NaADP; 1 mM NaVO4; 0.1 mM EGTA; 0.5 mM TCEP, 50 mM or 300 mM NaCl, pH 7.5 or NF buffer: 20 mM Hepes; 2 mM MgCl2; 0.1 mM EGTA; 0.5 mM TCEP, 300 mM NaCl, pH 7.5. Myo6 remained monomeric in all buffers

Figure 49 :Figure 50 :

 4950 Figure 49: Myo6 sequence alignment Sequence is colored depending on the domains. * marks the surface residues that differs between the human Myo6 and the pig-mouse Myo6 construct used in the preliminary experiments

Figure 51 :

 51 Figure 51: Myo6 pig-mouse chimera opens more easily upon salt concentration SEC profiles on Superdex 3.2/300 column pre-equilibrated in 20 mM Hepes; 200 mM NaCl; 2 mM MgCl2; 1 mM NaADP; 1 mM AlF4 (Pi analog), 0.1 mM EGTA, 1 mM DTT or in in 20 mM Hepes; 400 mM NaCl; 2 mM MgCl2; 0.1 mM EGTA, 1 mM DTT. FLMyo6 elution peak shifts upon nucleotide removal in high salt concentration conditions.

  the knowledge gathered on Myo6 opened and closed conformations, a combination of SEC-SAXS and SEC-MALS could finally be applied to the characterization of (1) the Myo6 engineered construct designed to be constitutively back-folded (Jo-Myo6-In) and (2) mutants suspected to trigger Myo6 opening. My role in the project was to perform all biochemical steps, in particular for the FL motors and partners in order to eliminate fully the proteolysis fragments that are sometimes present in a preparation. In addition, I took in charge the SEC-SAXS study while Carlos Kikuti, a research engineer in our time took in charge the SEC-MALS experiments. Additionally, Carlos Kikuti, the research engineer in our team pushed forward the characterization of the Jo-Myo6-In in negative staining, thanks to a collaboration with Daniel Levy's team (Institut Curie), to get more deTailed information of the overall shape and organization of the Myo6 back-folded conformation.To complete this structural study, we collaborated with Cédric Delevoye (Institut Curie) and Lee Sweeney's team (University of Florida) to set up functional assays. Lee Sweeney's team performed actin-based ATPase activity measurements on the Myo6 to pursue the impact of back-folding and opening on Myo6 activation while I took in charge the purification optimization of Myo6 and partners constructs needed for these experiments. For the cell-based assays, I participated in analyzing the cellbased assays on melanosomes that were performed by Franck Mayeux, an engineer in our team under the guidance of Cédric Delevoye, a researcher notably specialized in melanosomes. These cell-based assays allowed us to monitor the co-distribution of either constitutively open or back-folded Myo6 in cells with melanosome markers to gain insight on the impact of back-folding dysregulation on Myo6 recruitment in cells. I was also in charge of animating with Anne Houdusse and Lee Sweeney the

Figure 52 :

 52 Figure 52: GIPC1 organization GIPC1 contains a N-terminus GH1 domain allowing dimerization, a PDZ domain allowing binding to various cellular partners notably APPL1 and a GH2 domains able to mediate interaction with Myo6.

Figure 53 :

 53 Figure 53: GIPC1 auto-inhibited state prevents binding to Myo6The GIPC1 dimer (PDB: 5V6B;[START_REF] Shang | Structure Analyses Reveal a Regulated Oligomerization Mechanism of the PlexinD1/GIPC/Myosin VI Complex[END_REF], with one copy in Green and the other one in blue. GH1 of the first dimer is found to interact with the PDZ of the second one resulting in the formation of a dimer.

Figure 54 :

 54 Figure 54: TOM1 organization TOM1 contains a N-terminus VHS domain, a GAT domain allowing binding to various cellular partners notably TOLLIP and a myosin binding motif (MBM) within its C-terminus.

Figure 54 :

 54 Figure 54: Dab2 organization Dab2 contains a N-terminus phosphotyrosine-binding (PTB) domain. The region between residues 230 and 447 is only present in isoform p96 and contains clathrin box and Asp-Pro-Phe (DPF) motifs. Dab2 C-terminus is a proline rich domain (PRD) that notably mediates interaction with Myo6 through the Myo6 interacting region (MIR).

Figure 1 -

 1 Figure 1 -Importance of ADP.Pi for the compact, back-folded Myo6 conformation. (A) Schematic representation of FLMyo6 with the Motor domain (MD, grey), CaM binding sites (Ins2/IQ, purple/red), CaM (lilac/pink), 3-helix bundle (3HB, blue), single alpha helix (SAH, green), distal Tail (DT, orange) and CBD (brown). Residue numbers correspond to human Myo6, Uniprot entry Q9UM54-2. (B) Dimensionless Kratky plot representation from SEC-SAXS. FLMyo6 in the presence of ADP.AlF4 (green) results in a bell-shape spectrum with a maximum close to the intersection of the dashed lines

Figure 2 -

 2 Figure 2 -Role of the proximal Myo6 sequence in the stabilization of the Off-state.

( A )

 A Model of Myo6 opening/back-folding. Back-folding requires the SAH to fold back on the 3HB. The L926 residue (red cross) leads to deafness when mutated into Gln 38 . (Insert) Mutations of the apolar residues at the N-terminus of the SAH to convert it into a « perfect SAH» sequence (SAHmimic) turn Myo6 into a constitutive monomer 17 . (B) (Top) Dimensionless Kratky plot representation from SEC-SAXS. FLMyo6 in NF/high salt is pictured in black. In the presence of ADP.AlF4, FLMyo6 (L926Q) (yellow) and FLMyo6 (SAHmimic) (light blue) spectrums correspond to an elongated shape, as opposed to FLMyo6 WT (green). (Bottom) Rg of FLMyo6 WT, L926Q and SAHmimic determined by SEC-SAXS experiments in the presence of ADP.AlF4 and FLMyo6 in NF/high salt. (C) Rg of FLMyo6 WT, L926Q and SAHmimic determined by SEC-SAXS experiments in the presence of ADP.AlF4 and FLMyo6 in NF/high salt. Rg values were extracted from linear fits of the Guinier plots shown in Sup Fig. 1C using primusqt (ATSAS suite 39 ). (D) Actin-activated ATPase rate of FLMyo6 WT, L926Q, SAHmimic and MD Ins2 (n=6).

Figure 3 -

 3 Figure 3 -GIPC1 can bind to and activate the back-folded form of Myo6, while Dab2 and Tom1 can only bind Myo6 once the motor has been primed open.(A) EM density for the Jo-Myo6-In (grey mesh) obtained by negative staining, as in Fig.1Hand Sup Movie 1. The WWY motif (red spheres) of CBD c is buried. The CBD n fragment (beige) (PDB: 5V6E,34 ) is positioned in the remaining, uninterpreted part of the density so that the RRL motif (red spheres) on CBD n and the I1072 (blue sphere) that has been proposed to mediate interaction between ubiquitin and Myo6 41 are both exposed. Note that no experimental model exists for 36 missing residues between the CBD n and CBD c (dashed lines). (B) Fitting of CBD c -TOM1 structure (PDB: 6J5635 ) with CBD c (brown) in the model presented in Fig.1H, TOM1 (yellow) needs to bind in a poorly exposed area and would clash with SAH (green) and CaM (lilac). (C) Fitting of CBD n (beige)-GIPC1 (light blue) structure (PDB: 5V6E34 ) as for CBD n alone. GIPC1 binding seems compatible with the back-folded conformation of Myo6 on a highly exposed binding site. (D) Anti-His pull-down assays. His-tagged Myo6 partners TOM1 and GIPC1 against FLMyo6 (WT) or (SAHmimic) revealed using SYPRO42 (Input and last wash pictured in Sup Fig.5). Crosses: quantification of retained Myo6 (Image-Lab software, Bio-Rad) followed by stoichiometric normalization based on partner concentration (n=4). + means less than 10% Myo6 retained; +++ means more than 20% Myo6 retained. (E) ATPase rates of FLMyo6 (WT) and FLMyo6 (SAHmimic) with40 µM

Figure 5 -

 5 Figure 5 -Myo6 can form an anti-parallel dimer through residues 875-940 which allow large steps. (A) (Left) X-ray crystallography structure of mouse Myo6 875-940 antiparallel dimer colored according to B-factor from 18.60 Å 2 (dark blue) to 150.81 Å 2 (red). (Right) Key residues for dimer stabilization. Apolar contacts are mediated by residues I878, L881, I885, T888, R892, I895, Q896, Y899, V903, E907, L909, L910, L913 (pictured in green). Dotted blue line: polar contacts. Residues mutated in our triple mutant (T888D.R892E.V903D) are underlined. (B) Close-up of the dimerization interface of Myo6 875-940 (crystallographic dimer) in the electronic density. (C) Triple helix bundle (PDB: 2LD3 43 ) domain. T888, R892, V903, T845C and A880C pictured as sticks are surface residues. (D) SEC-MALS profiles of

Figure 6 -

 6 Figure 6 -Importance of a folded monomer for regulation. (A) When auto-inhibited, Myo6 can diffuse across actin-rich regions without interacting with actin. Once recruited by a partner, Myo6 is activated and starts performing its cellular function. (B) Scheme representing possible activation mechanisms for Myo6. Myo6 MD is pictured in grey, Ins2/CaM in purple, IQ/CaM in red/pink, 3HB in blue, SAH in green, DT in orange and CBD in brown. Partner binding sites are labeled in garnet. The binding site (WWY) for Dab2 and TOM1 is blocked, preventing recruitment of Myo6 without a prior unfolding signal prior to unblock their binding. GIPC1 can bind the accessible RRL motif resulting in Myo6 recruitment and opening. Other signals can act as unfolding factors such as Ca2+ , which can allow TOM1 to bind to Myo6. Such an activation cascade was previously proposed36 . Once unfolded, Myo6 potentially acts as a monomer, as previously proposed35 upon TOM1 binding; or it can dimerize29 through proximal dimerization, as demonstrated in this study with GIPC1

Supplementary Figure 6 -

 6 iRFP VAMP7. From left to right: Entire cell: 3 channels merge and individuals; 8x zoom of the boxed region: merged GFP Myo6 / mCherry MST, individual channels. Scale bars: 10 µm. Affinity of the Myo6 tail for partners by Microscale Thermophoresis. (Top) Dissociation constant (KD) ± KD confidence determined by microscale thermophoresis of Myo6 CBD constructs against Myo6 partners. (Bottom) Integration of thermophoresis profiles against concentration of partners (nM) for one replicate shown as an example. The affinity was quantified by analyzing the change in thermophoresis as a function of the concentration of the titrated protein using the NTAnalysis software provided by the manufacturer. Error bars: standard deviation. Fit: "Kd model" equation from NTAnalysis.(A) Scheme of the Myo6 Tail showing the oligomerization states found for different constructs using SEC-MALS in 10 mM Tris pH 7.5; 50 mM NaCl; 5 mM NaN3; 0.5 mM TCEP. (B) (Top) schematic representation of the dimer crystal structure. Negatively charged residues are circled in red, positively charged residues are circled in blue, hydrophobic residues are circled in green and polar, uncharged residues are circled in purple. Residues involved in apolar contacts are circled in green. Three residues found to be essential for dimerization (T888D, R892E, V903D) are marked with a red cross. (Bottom) Sequence alignment of 851-949 (human protein numbering). Red: conserved residues; blue: conserved in 5 out of 6 species. *: residues inside the dimerization area that are not 100% conserved (S881 and M896). In the structure, M896 faces itself and seems compatible with the crystallized dimer. *: residues mutated in this study to disrupt proximal dimerization. Residues T888 and V903 are well conserved. The crystal structure shows that the replacement of R892 by Q in the human sequence should be compatible with proximal dimerization. (C) Titration of the 875-940 fragment by SEC-MALS in 10 mM Tris pH 7.5; 50 mM NaCl; 5 mM NaN3; 0.5 mM TCEP. Thin lines: light scattering (normalized); thick lines: molecular masses. A KD App of ~19 µM was calculated from a fit of molecular weight against the concentration at the peak from each profile -the affinity is probably underestimated because of the size exclusion resin. (D) Normalized SEC-MALS profiles of the 834-955, 880-940 and 888-940 constructs (concentration at the peak stated on Sup Fig.10A) found to be monomeric in 10 mM Tris pH 7.5; 50 mM NaCl; 5 mM NaN3; 0.5 mM TCEP, unlike the dimeric 875-955 construct (red). (E) Normalized SEC-MALS profile of the 912-end construct (25 µM at the peak) found to be monomeric in 10 mM Tris pH 7.5; 50 mM NaCl; 5 mM NaN3; 0.5 mM TCEP.

Figure 56 :

 56 Figure 56: Main interactions stabilizing the Myo6 auto-inhibited state (see section A.2.)

Figure 57 :

 57 Figure 57: CBD c loop mutant (D1148V, Y1150D, D1152R and Q1154V) has only little impact on Myo6 compacity in vitro SEC profile on Superdex 3.2/300 column pre-equilibrated in 20 mM hepes; 200 mM NaCl; 2 mM MgCl2; 1mM NaADP; 1mM AlF4 (Pi analog), 0.1mM EGTA, 1mM DTT

Figure 58 :

 58 Figure 58: DNA binding motifs and CBD c loop mutant (D1148V, Y1150D, D1152R and Q1154V) within CBD c CBD c NMR structure (PDB: 2KIA, Yu et al. 2009) is pictured in magenta, TRTK and SKKK motif found to be involved in DNA binding are highlighted in blue and green respectively. D1148, Y1150, D1152 and Q1154 are highlighted in red

Figure 59 :

 59 Figure 59: D1148V, Y1150D, D1152R and Q1154V mutations disrupt Myo6 CBD recruitment to melanosomes Representative fixed MNT-1 cells co-expressing different GFP CBD (WT) or GFP CBD (D1148V, Y1150D, D1152R and Q1154V) constructs with control mcherry MST and iRFP VAMP7

Figure 60 :

 60 Figure 60: Jo-Myo6-In crystallization drops without seeding (right) and with seeding (left)

Figure 61 :

 61 Figure 61: Jo-Myo6-In is a multidomain protein (Left) 3D-reconstruction of Jo-Myo6-In construct based on negative staining electron microscopy. Jo-Myo6-In is modeled with the density (see section A.2). Density is less defined for Jo and In domains suggesting flexibility (Right) Dimensionless Kratsky plot representation, dotted lines ((qRg)2I(q)/I(0) = 1.104 and qRg = √3) intersection highlight theoretical maximum of folded proteins. SEC-SAXS was carried out on Superdex 3.2/300 column preequilibrated in 20 mM hepes; 200 mM NaCl; 2 mM MgCl2; 1 mM NaADP; 1 mM AlF4 (Pi analog), 0.1 mM EGTA, 1 mM DTT or in in 20 mM hepes; 400 mM NaCl; 2 mM MgCl2; 0.1 mM EGTA, 1 mM DTT

A. 3 Figure 62 :

 362 Figure 62: Model of the active FL Myo6 proximal dimer (extracted from Canon et al., in preparation, see section A.2.)

Figure 63 :

 63 Figure 63: Model of the active FL Myo6 proximal dimer with modelization of the R849-E875 region (A) docking of a 26 amino acid helix composed of alanine was docked between residues 849 and 875. A distance of ~3.8 Å was left between R849 and the C-terminus of the poly-Ala helix, which corresponds to the typical

Figure 64 :

 64 Figure 64: Model of the GIPC1-Myo6 complex that could either form a single dimer or a large oligomer of dimers Model of Myo6 proximal dimer (light chains not shown). Motor domain in grey, Ins-2 in purple, IQ in red, unfolded 3HB in blue, SAH in light green, DT in orange, CBD in brown. GIPC1 is picture in green. As a dimer, it exhibits two GH2 domain that can bind either to the two cargos binding domain of the same Myo6 proximal dimer (right) or bind to cargo binding domains belonging to two different motors resulting in the formation of large oligomers (right)

Figure 65 :

 65 Figure 65: interferometric scattering mass spectrometry principle (adapted from Young et al. 2018) Characterization of a 12 nM BSA sample in iSCAMS, (A) schematic representation of the iSCAMS experimental approach, (B) Differential interferometric scattering image (scale bar 0.5 µm), (C) Representative image for different oligomeric states (D) Scattering plot of single molecule and corresponding histogram

Figure 66 :

 66 Figure 66: Sequence alignment of the 851-949 (human gene numbering) amino acids among species. Red corresponds to conserved residues, blue to residues conserved in at least 6 out of 8 species. Top: * marks residues inside the dimerization area that are poorly conserved. * marks the residues mutated that are sufficient to disrupt proximal dimerization. Bottom: residues colored depending on their conservation in the crystal structure, residues poorly conserved inside the dimerization interface are plotted in yellow

Figure 67 :

 67 Figure 67: K63-diUbiquitin is unable to increase the Myo6 ATPase rate.

Chapter 2 :

 2 Inhibition of Myosin motor by small molecules binding in the 50 kDa cleft Introduction A. Conventional myosins (Myo2): therapeutical targets Due to the long coiled-coil domain that mediates dimerization of the myosin heavy chain, Myo2s are constitutive hexamers (2 heavy chains and 4 light chains, which comprise 2 essential light chains (ELC) and 2 regulatory light chains (RLC)) (Figure 68). These dimers can then associate and form large fibers to pursue their cellular functions. Historically, several myosin fragments were defined based on proteolysis. Chymotrypsin cleaves myosin in heavy meromyosin (HMM), which contains the Motor domain plus the Lever arm and the beginning of the coiled coil, and light meromyosin (LMM), which contains the remaining Cterminus. HMM can be cleaved by papain in subfragment S1 (Motor domain plus Lever arm) and S2(remaining part of the coiled coil)[START_REF] Lowey | Substructure of the Myosin Molecule. I. Subfragments of Myosin by Enzymic Degradation[END_REF]) (Figure68). S1 is sufficient to produce force and slides actin filaments, although it is monomeric.

Figure 68 :

 68 Figure 68: Myo2 subfragments (Planelles Herrero 2017) Schematic representation of Myo2 with (from right to left) the Motor domain in blue, the Lever arm bound to the essential light chain (ELC) in pink and the regulatory light chain (RLC) in lilac and the long coiled coil Tail in purple. The delimitation of the subfragments 1 (S1) and 2 (S2), the heavy meromyosin (HMM) and the light meromyosin (LMM) are depicted.

Figure 69 :

 69 Figure 69: Overall sarcomere organization (Warshaw, 2016) Overall sarcomere organization with the thick myosin filaments in red and the thin actin filaments in grey. The Z-disc delineates the lateral borders of the sarcomere. During the powerstroke, Myo2 generates force resulting in association of the Myo2 head with the actin filament and the contraction of the sarcomere.

Figure 70 :

 70 Figure 70: Relative abundance of Myo2 isoforms in human muscles (Gyimesi et al. 2020) Myo2 isoform distribution in different human muscle type (literature-based analysis). The slow Myo2 isoform (I) is present in all muscles and represents at least ~30% of the Myo2. Fast Myo2 isoforms are available in various proportion depending on the muscle type.

Figure 71 :

 71 Figure 71: Allosteric binding pockets within the myosin Motor β-CardMyo2 Motor domain plus essential light chain (MDE) from structure PDB: 5N69 (Planelles-Herrero et al. 2017) is displayed with its Lever arm in purple, its converter in green, its SH1 in orange, its relay in yellow, its upper-50 in blue and its lower-50 in light blue. Omecamtiv mercabil (PDB: 5N69; Planelles-Herrero et al. 2017), CK-571 (PDB: 5M05; Sirigu et al. 2016), Blebbistatin (PDB: 1YV3; Alligham et al. 2005), Pentabromopseudilin (PDB: 2JHR; Fedorov et al. 2009) and phenamacril (PDB: 6UI4; Zhou et al. 2020) are represented in sticks in their allosteric binding pocket.

  were also found against unconventional myosins: 2,4,6-triiodophenol (TIP) was proposed as a low affinity inhibitor of unconventional Myo6[START_REF] Heissler | Kinetic Properties and Small-Molecule Inhibition of Human Myosin-6[END_REF] and Myo-Vin1[START_REF] Islam | A Myosin V Inhibitor Based on Privileged Chemical Scaffolds[END_REF]) as a ~6 µM inhibitor of unconventional Myo5. Perhaps the best characterized unconventional myosin inhibitor is Phenamacril. Phenamacril, a fungicide, has been identified as a specific submicromolar inhibitor of unconventional Myo1. Even among Myo1, phenamacril exhibits a certain specificity: Myo1s from plant pathogen Fusarium are efficiently inhibited by phenamacril while human Myo1c isn't[START_REF] Wollenberg | Phenamacril Is a Reversible and Noncompetitive Inhibitor of Fusarium Class I Myosin[END_REF]. The crystal structure of F. graminearum Myo1 in complex with Phenamacril was recently solved at 2.65 Å (PDB: 6UI4;[START_REF] Zhou | Structural Basis of Fusarium Myosin I Inhibition by Phenamacril[END_REF] revealing that the compound binds to the 50 kDa cleft. The authors identified Met375 as a key residue for phenamacril inhibition and selectivity among Myo1s, as the introduction of the methionine in position 375 in Myo1 isoforms that are not usually inhibited by phenamacril renders them inhibited.

Figure 72 :

 72 Figure 72: (-)-Blebbistatin scaffold Red star marks the asymetric carbon

Figure 73 :

 73 Figure 73: Blebbistatin IC50 for various Myo2 isoforms[START_REF] Rauscher | Targeting Myosin by Blebbistatin Derivatives: Optimization and Pharmacological Potential[END_REF]

Figure 74 :

 74 Figure 74: Blebbistatin photodegradation (Kolega 2004)Blebbistatin absorption spectra after 1 h illumination at 365 nm, 450 nm, or not illuminated. Arrows mark absorbance peaks that appear within the spectra after illumination at 450-490 nm and * marks an absorbance peak that decreases following 450-490 or 365 nm illumination. Overall, these data show that light exposure alters the structural integrity of Blebbistatin resulting in a modification of its spectral properties.

Figure 75 :

 75 Figure 75: Putative photochemical mechanism for blebbistatin photo-inactivation upon 416 nm illumination (Li et al. 2018) Water-dependent mechanism consisting in a water-dependent protonation followed by an excited state intramolecular proton transfer (ESIPT). Finally, electron transfer results in liberation of the toxic • OH radical.

Ramamurthy

  et al. (2004) employed a fluorescent phosphate binding protein in a stopped flow apparatus to measure Pi release rates after incubation of NM2B, actin and ATP. They noticed that Blebbistatin could only block Pi release for 1 s. Past that time, normal Pi release rate was recovered, which is explained by the 425 nm wavelength used as excitation for the phosphate binding protein in their experiments. Illumination at that wavelength results in blebbistatin inactivation within 1 s as described in sections C.4. and C.5. Overall, blebbistatin seems to impact the Myo2 Pi burst without interfering with binding of the nucleotide.

  powerstroke state using MgADP. VO4 (MgADP.Pi analog) allowed direct comparison of the structure in the apo and bound state and showed that blebbistatin does not induce any major conformational change within the pre-powerstroke structure (r.m.s.d = 0.407 Å). Blebbistatin lies in the 50 kDa cleft in between the upper-50 and the lower-50, close to the Pi pocket, switch I, switch II and P-loop. It Interacts with the ΦLL motif, HP and HW helix of the lower-50 mainly through apolar contacts: benzyl ring (D-ring) is enclosed by Dicty residues Leu262, Phe466, Glu467 and Val630. Tetrahydropyrrolo ring (C-ring) with Ser456 and Ile471 and methylquinolinone (A and B-rings) with Tyr261, Thr474, Tyr634,

Figure 76 :

 76 Figure 76: Blebbistatin binding site (Top) Blebbistatin in its allosteric pocket in the DictyMyo2 Motor domain, zoom pictures key residues surrounding blebbistatin (PDB:1YV3, Allingham, Smith, and Rayment 2005). Blue dotted lines represent hydrogen bonds.(Bottom) The table highlights the conservation of the myosin residues within the blebbistatin allosteric pocket. In particular, one residue is poorly conserved in switch-2 (sw-II, red) resulting in the loss of the blebbistatin inhibition for unconventionalMyo1, Myo5 and Myo10.

  blebbistatin access to its binding site. Identification of the blebbistatin tunnel using CAVER results in the identification of a tight access tunnel making drug assess and dissociation within the prepowerstroke very constrained by the tunnel residues (Roman et al. 2018) (Figure 77). How blebbistatin enters and dissociates from the Motor thus remains an open question.

Figure 77 :

 77 Figure 77: Blebbistatin tunnel within the DictyMyo2 pre-powerstroke Motor (Roman et al. 2018) Blue spheres correspond to the blebbistatin tunnel detected by CAVER in top and side views. The protein surface is pictured in white and blebbistatin is pictured in cyan. The tunnel presented is very tight which emphasizes the difficulty for Blebbistatin to access its binding pocket in a pre-powerstroke state.

Figure 78 :

 78 Figure 78: Identification of most promising positions for A-ring substitution (Lucas-Lopez et al. 2008)

Figure 79 :

 79 Figure 79: A-ring derivatives from(Verhasselt, Stevens, et al. 2017)

Figure 80 :

 80 Figure 80: The Arg238 backbone is close to the blebbistatin C-ring and was proposed as a potential way to mediate additional polar contact in the allosteric pocket[START_REF] Roman | Synthesis of C-Ring-Modified Blebbistatin Derivatives and Evaluation of Their Myosin II ATPase Inhibitory Potency[END_REF]

Figure 81 :

 81 Figure 81: D-ring derivatives of blebbistatin(Verhasselt, Roman, Bracke, et al. 2017)

Figure 82 :

 82 Figure 82: Comparison between blebbistatin and MPH-220. MPH-220 differs from Blebbistatin by its A-ring (methylthiophen) and by the substitution of its D-ring with a morpholino group

Figure 83 :

 83 Figure 83: Degraded MPH-220 within the SkMyo2 Motor (Left) Degraded MPH-220 fitted in the electron density of SkMyo2-MPH220 crystal structure at 2.1 Å. The red arrow shows the expected position for the hydroxyl function; the purple arrow shows an unexpected water molecule nearby the degradation site which does not appear in the published structures with blebbistatin. (Right) Putative degradation product of MPH-220 where the hydroxyl group is missing.

Figure 84 :

 84 Figure 84: MPH-220 fitted in SkMyo2 blebbistatin allosteric pocket (6YSY) Nice electron density (in blue) can be seen for MPH-2220. All the features of MPH-220 seems present within the density and in particular the hydroxyl group.

Figure 85 :

 85 Figure 85: Absorption of 10 µM blebbistatin in water or SkMyo2 buffer Absorption spectra of blebbistatin in water (top panel) or SkMyo2 buffer (bottom panel). Blebbistatin absorption spectra after 0 to 187min exposure to white light are plotted. The spectra of Blebbistatin after 18h exposure to white light is plotted is inserted at the top left corner of each panel. On this spectrum, a clear absorbance peak (marked by a blue arrows) resulting from blebbistatin degradation can be detected at ~340nm. Degradation seems more severe in SkMyo2 buffer than in water.

Figure 86 :

 86 Figure 86: Absorption of 10 µM blebbistatin supplemented with various reagents after 18h in white light. Absorption spectra of blebbistatin in water supplemented with 20 µM DTT, 5 mM DTT, 3 % Methanol, 3 % Ethanol, 0.025 % Dichloromethane or in SkMyo2 buffer at pH 7.3, 6.5 or 9.8. Spectra of Blebbistatin in water or in SkMyo2 buffer a pH7.3 after 18h are plotted as refence to see which condition(s) could aggravate or slow down Blebbistatin degradation. All conditions tested result in a large peak at 340 nm pointed out to a degradation of the Blebbistatin. Worst conditions seem to be the addition of 5 mM DTT.

Figure 87 :

 87 Figure 87: Example of crystallization drops for SkMyo2 MPH-220 crystals with (right) and without (left) 20 mM DTT DeTailed crystallization protocol can be found in the method of the Gyimesi et al. 2020 paper.

Figure 1 .

 1 Figure 1. MPH-220 Inhibits Skeletal Muscle Myosin with Extreme Selectivity Independently of the Nervous System (A) Current muscle relaxants target the central nervous system or act peripherally, whereas MPH-220 directly inhibits myosin. (B) Human myosin-2 isoforms contain phenylalanine (black F) at the beginning of the HP-helix between the switch-2 loop (red) and the relay region, except for fast skeletal myosin isoforms, where this position is leucine (blue L; cf. Figure S1). (C) MPH-220 was designed to bind into the blebbistatin-binding cavity of the motor domain, to enable inhibition in the actin-detached state. (D) Close-up view of the communication center of the functional regions (actin-binding cleft, switch-2 loop of the active site, and the HP-relay-converter region) in the homology model of MyHC IIa with bound MPH-220 (orange) reveals interaction between the morpholino group and Leu 476 in the HP-helix. (E) Actin-activated ATPase inhibition of three myosin-2 isoforms (cf. Table S1). (F) Actin-activated ATPase inhibition of NM2A, NM2B, NM2C and the NM2C F490L variant. (G) Actin-activated ATPase inhibition of expressed human b-cardiac myosin with MPH-220 and blebbistatin. (H) Actin-activated ATPase inhibition of human muscle myosin samples from biopsies of m. soleus, m. vastus lateralis, and heart ventricle. In contrast to MPH-220, blebbistatin fully inhibited m. vastus lateralis sample and b-cardiac myosin samples. Mean ± SD are shown in all ATPase experiments; n = 3-9. See also Figures S1 and S2.

Figure 2 .

 2 Figure 2. Crystal Structure of MPH-220-Bound Fast Skeletal Muscle Myosin (A) Cartoon representation of the heavy chain (gray) of rabbit skeletal muscle myosin-2 in Mg.ADP.VO 4 (spheres, blue) pre-powerstroke state with the essential light chain bound to the lever arm. MPH-220 binds to the bottom of the actin-binding cleft close to the HP-helix, with its morpholine ring in close proximity of Leu 476 . Blue mesh, electron density corresponding to MPH-220 at sigma 1.0. (B) Comparison of the MPH-220 (orange) binding site in MyHC IIb (gray/beige) and the blebbistatin (yellow) binding site in Dictyostelium myosin-2 (dark gray/ brown; PDB: 1YV3; Allingham et al., 2005). The chiral OH groups of both inhibitors interact with the same residues Gly 248 and Leu 270 (Gly 240 and Leu 262 in Dictyostelium). Mesh, surfaces based on Van-der-Walls radii, calculated with PyMol. (Bottom) Residues involved in compound binding in Ligplot+ v2.2 (Laskowski and Swindells, 2011) representation are shown; U50, upper 50-kDa subdomain; L50, lower 50-kDa subdomain (gray); sw-2, switch-2 loop (red); HP, HPhelix (beige). Hydrogen bonds (up to 3.2 A ˚) are shown as green lines. (C) MPH-220-bound MyHC IIb structure with superimposed cardiac HP-helix illustrates the steric clash between the morpholine ring of MPH-220 and the cardiac HP-helix Phe 473 (red) residue. See also Figure S3.

Figure 3 .

 3 Figure 3. MPH-220 Reduces Skeletal Muscle Force without Cardiovascular Effects (A) Isometric force of rat hindleg was measured while vital functions were detected by a non-invasive pulse oximeter. (B and C) Dose-dependent force reduction after MPH-220 treatment persisted for more than 10 h. (D) Heart rate (red circle) and pulse distention-reflecting local blood flow at the carotid artery-(maroon circle) show the maximal signal deflections during the measurements (60-600 min) on 1-4 animals/dose. (E and F) Cardiovascular and respiratory functions as a function of time after treatment with sulfobutyl-ether-b-cyclodextrin (SBECD) control or MPH-220. Note that permanent oxygen level is maintained by O 2 -supplemented isoflurane anesthesia. (G) Dose-dependent MPH-220 accumulation in rat muscle tissues 60 min after oral treatment. Mean ± SD are shown; n = 2-3. (H and I) Time-dependent MPH-220 accumulation in rat tissues after 35 mg/kg intraperitoneal (i.p.) treatment. Mean ± SD are shown; n = 2-11. See also Figure S4.

Figure 4 .

 4 Figure 4. MPH-220 Efficiently Improves Gait Functions of Spastic Rats after Brain Injury (A) Position of pyramidal tract lesion (red dots). (B) Open-field gait analysis was performed by deep-learning algorithms coupled to sub-millimeter 3D movement detection. Dots represent the determined positions; right and left forelimbs (RF and LF) and right and left hindlegs (RH and LH). (C) Fractional turn orientation of spastic rat before (gray) and after (orange) 15 mg/kg MPH-220 treatment. (D) Falling and spontaneous cramping rates normalized to the total distance traveled (upper) and the number of touches on the cylinder wall (lower) after 20 mg/kg MPH-220 treatment. (E) Paw position distribution of the healthy (upper) and treated (lower) animals before and after 20 mg/kg MPH-220 treatment in slow regime. (F) Averaged distances between the left (upper) and right (lower) fore-and hindlimbs in each frame of the 3D recordings relative to the healthy values. Mean ± SD are shown. See also Figure S5.

Figure 5 .

 5 Figure 5. Principal-Component Analysis of Spastic Rats Reveals Details of Improved Gait Components after MPH-220 Treatment (A and B) Box and whiskers (min to max) representation of global (A) and speed-specific (B) principal-component (PC) analysis of gait parameters of healthy (gray) and operated (colored) animals before (darker) and after (lighter) MPH-220 treatment. (C) Parameter contributions to PC1 in the slow and medium regimes and to PC3 in the fast regimes (cf.Figure S5). (D) Heatmap of major parameters contributing to PC1 (blue-orange) and PC3 (purple-orange; cf.Figure S5). (E) Schematic representation of MPH-220 effects on paw positions in slow and fast regimes. See also Figure S5.

Figure S2 .

 S2 Figure S2. Inhibitory Properties of Different MPH-220 Derivatives, Related to Figure 1

Figure S3 .

 S3 Figure S3. Crystal Structure Refinement Statistics and Comparison of Crystal and Modeled Structures Used for Inhibitor Development, Related to Figure 2

  (A) Data collection and refinement statistics (molecular replacement). Data from one crystal. *Values in parentheses are for highest-resolution shell. (B) Cartoon representation of the crystal structure of MyHC IIb (gray/beige), and the superimposed modeled structure of MyHC IIa from molecular dynamic simulations (yellow/maroon). MPH-220 binding site reveals that the position and orientation of MPH-220 in the crystal structure (orange) and in the modeled structure (green) is similar. Amino acid residue carbons in gray (crystal structure) and maroon (model), and carbons for MPH-220 in orange (crystal structure) and green (model) are shown. (C) Close-up of the MPH-220 binding site reveals that HP-helix position is well predicted; in particular, Leu 476 position is very similar. The alignment also reveals a moderate shift in MPH-220 position between the homology model and the crystal structure; in particular we see slightly different position and orientation of the morpholino group. Still, the homology model predicts well the sub-pocket explored by this cycle; residues Leu 476 , Glu 477 , Cys 480 , Asn 600 and Val 650 interact indeed with the morpholino group in both crystal structure and homology model. These results confirm that the modeled structure that we used for inhibitor development (cf. Figures 1 and S2) is reliable and the results from those experiments are trustworthy. (D) Close-up view of the crystal structure with bound MPH-220 colored according to B-factors from 21.1 A ˚2 (dark blue) to 125.2 A ˚2 (red). ADP, VO 4 and MPH-220 are represented by thicker lines, the sphere represents the Mg 2+ ion.

Figure S5 .

 S5 Figure S5. Gait Analysis of MPH-220-Treated Rats, Related to Figures 4 and 5

(

  A) Paw position distribution of healthy (upper three rows) and treated (lower three rows) animals in the three speed-regimes (slow (less than 0.036 m/s), medium (0.036-0.072 m/s) and high (more than 0.072 m/s)), 1, 3, 5, 9 and 21 hours after the first measurement (healthy control) or before and 1, 3, 5, 9 and 21 hours after 20 mg/kg oral MPH-220 treatment. (B) Averaged distances between the paws in each frame of the 3D recordings in healthy (gray band) and treated (colored dots) animals; green: left fore-and hindlimbs, pink: right fore-and hindlimbs, red: left and right hindlimbs, maroon: left and right forelimbs, purple: left fore-and right hindlimbs, blue: right fore-and left hindlimbs. Schematic representation of distances is shown above the plots. Statistical analyses show that left fore-and hindlimbs are significantly closer in the operated rats than in the healthy animals, however, 1-5 hours after MPH-220 treatment the difference becomes insignificant. The right fore-and hindlimbs are significantly farther in the operated rats before treatment, which becomes non-significant 1 hour after MPH-220 treatment. These together indicate that MPH-220 treatment resulted in a significantly straightened body axis. Data are mean ± SD; n = 2. (C) Principal component (PC) analysis: PC1, PC2 and PC3 scores of healthy (gray) and operated (colored bars) rats in all, slow, medium-speed and fast movement regimes (upper left quartet). PC1 (blue, upper right quartet), PC2 (eggplant, lower left quartet) and PC3 (purple, lower right quartet) scores before (darker) and after (lighter) 20 mg/kg oral MPH-220 treatment in all, slow, medium-speed and fast movement regimes. Boxplots with min-to-max whishkers, n = 4. (D) Parameter contributions to PC1 (upper row), PC2 (middle row) and PC3 (lower row) in the slow (left column), medium-speed (middle column) and fast (right column) movement regimes. Dotted lines represent the threshold for major contributions to PC (3.57%). Boxplots with min-to-max whishkers, n = 4. (E) Explained variance in all, slow, medium-speed and fast movement regimes; PC1, PC2 and PC3 are colored according to panels C-D. (F) The computed gait parameters were scaled (Z-score) and averaged. The columns represent the individual gait parameters (1-28, defined below the heatmap) in the slow (S1-28), medium-speed (M1-28) and fast (F1-28) movement regimes. The separate row represents the average of healthy animals at zero minute, and the other six rows represent the parameter scores beforeand 1, 3, 5,. PC analysis revealed that in the slow regime MPH-220 has the largest effect on X-y axis length and the consequent volumes of the ellipsoids representing paw positions in 3D and certain distances between the centers of ellipsoids (blue brackets) contributing to PC1; in the fast regime MPH-220 has the largest effect on the angles of the ellipsoids (purple brackets) contributing to PC3. Parameter description used in PCA calculations: video recordings were analyzed in three walking speed categories. The numbers encode different parameters of the ellipsoids fitted to data points from 3D position analysis. LF: left front limb, RF: right front limb, LH: left hind limb, RH: right hind limb.

Figure S6 .

 S6 Figure S6. Safety Profiling of MPH-220 Supports Further Drug Development due to Lack of Mutagenicity and Interaction with Off-Target Enzymes, Related to STAR Methods (A) We tested the effects of 5 mM MPH-220 on 186 different human G-protein-coupled-receptors (GPCRs) covering 60 different receptor families including adrenergic, dopamine, P2Y and serotonin receptor families (by DiscoverX-Eurofins). According to the highly selective nature of MPH-220, neither GPCR was inhibited or activated above 30%, which is a standard reference threshold for hit compounds. (B) Beside the gpcrMAX panel, inhibition of 484 kinases was measured in the presence of 5 mM MPH-220. Neither kinase was inhibited by more than 20%, which indicates that MPH-220 does not interfere with the signal transduction pathways in human cells. This Select Screen Kinase Profiling panel (by Thermo Fisher) contains all available wild-type and the most abundant mutant forms of human kinases. (C-E) MPH-220 was also tested on 23 human hormone nuclear receptors (HNR) at 5 mM concentration (by Thermo Fisher). In agreement with the kinase and GPCR results, all investigated receptors remained unaffected by MPH-220 due to the lack of significant binding to any of the HNRs. Mean ± SEM are shown; n = 2 in (A-E). (F) Cytotoxicity was measured with CyQUANT TM LDH Cytotoxicity assay on human adult dermal fibroblast (hDFa, pink) and human adult lung (hLFa, purple) fibroblast cell lines in the presence of 20 mM (open circles) and 50 mM (closed circles) MPH-220 for 48 and 120 hours. MPH-220was not toxic to either fibroblast strain at any time point or concentration, confirming its safe use in further developments. As one of the most important requirements for a lead compound, mutagenicity was measured by the OECD/FDA approved Ames reverse mutagenicity assay using the two most sensitive bacterial strains for testing frameshift (TA98, G) and base-pair substitution (TA100 H) mutation both in the absence (open circles) and presence (closed circles) of activated rat liver S9 fraction. Relative mutagenicity did not show MPH-220 concentration dependency and, more importantly, experiments on neither strain resulted in any data points above the threshold (gray dotted line). Besides MPH-220 we also tested the mutagenicity of the other two morpholine derivatives of MPH-220 (cf. FigureS2) to elucidate whether the lack of mutagenicity is associated with the morpholine group. Neither MPH-49 nor MPH-188 showed concentration dependent increase in mutagenicity levels and, more importantly, neither showed in data points above the OECD/FDA threshold. Lack of mutagenicity was observed for both TA98 and TA100 S.typhimurium strains both in the absence and presence of liver S9 fraction, suggesting that neither the inhibitors nor their major metabolites are mutagenic, which supports safe development of MPH-220 toward human clinical phases.

Figure 88 :

 88 Figure 88: Superimposition of SkMyo2 allosteric pocket with MPH-220 (PDB:6YSY) and without MPH-220. MPH-220 is pictured in orange, SkMyo2 bound to MPH-220 is pictured in grey and SkMyo2 without MPH-220 is pictured in green.

Figure 89 :

 89 Figure 89: Superimposition of SkMyo2 with MPH-220 (PDB: 6YSY) and without MPH-220. MPH-220 is pictured in orange, SkMyo2 bound to MPH-220 is pictured in grey and SkMyo2 without MPH-220 is pictured in green.

1 .

 1 Potential of blebbistatin derivatives for specific myosin targetingAs we show in[START_REF] Gyimesi | Single Residue Variation in Skeletal Muscle Myosin Enables Direct and Selective Drug Targeting for Spasticity and Muscle Stiffness[END_REF], the difference in sequence of only one residue within the blebbistatin allosteric pocket is sufficient to reach high selectivity among different types of Myo2.Thereby selective inhibitors of SkMyo2 (skeletostatins) (Figure90), also obtained by para-substitution of blebbistatin D-ring with a morpholino group (or two other bulk groups) were recently described the same strategy as MPH-220 by another group[START_REF] Radnai | Discovery of Selective Inhibitors for In Vitro and In Vivo Interrogation of Skeletal Myosin II[END_REF]).

Figure 90 :

 90 Figure 90: comparison of skeletostatin and blebbistatin[START_REF] Radnai | Discovery of Selective Inhibitors for In Vitro and In Vivo Interrogation of Skeletal Myosin II[END_REF]

Figure 91 :

 91 Figure 91: Inhibition of different Myo2 subclasses by blebbistatin, paraNitroBlebbistatin and Amino-Blebbistatin (Gyimesi et al. 2021).

  study motor mechanism, provide tools for biology experiments and also to cure diseases. The study of MPH-220 offers a great example of the potential of myosin allosteric modulator to treat diseases and in particular the potential of the blebbistatin allosteric pocket for developing highly specific inhibitors against Myo2. Moreover, during the course of the development of MPH-220, several blebbistatin derivatives exhibiting different selectivities among Myo2 have been identified by András Málnási-Csizmadia's lab. Characterization of a collection of inhibitor-myosin complexes through cocrystallization could offer insights to dissect the origin of the specificity and guide design of specific inhibitors for distinct Myo2. It may even perhaps provide clues to design Myo6 specific inhibitors in

Table 1 :

 1 structure of Myo6 motor in different states of the ATPase cycle…………………………………..….p.30

Table 2 :

 2 Dab2-Myo6 affinity reported in the literature………………………………………………….…..………….p.[START_REF] Yu | Membrane-induced lever arm expansion allows myosin VI to walk with large and variable step sizes[END_REF]

Table 3 :

 3 TOM1-Myo6 affinity reported in the literature…………………………………………………………………p.[START_REF] Spitzer | BoxPlotR: a web tool for generation of box plots[END_REF]

Table 4 :

 4 NDP52-Myo6 affinity reported in the literature………………………………………………….…………...p.47

Table 5 :

 5 TAX1BP1-Myo6 affinity reported in the literature……………………………………………….……….….p.47

Table 6 :

 6 Ubiquitin-Myo6 affinity reported in the literature………………………………………………………..….p.51

Table 7 :

 7 DNA-Myo6 affinity reported in the literature……………………………………………….………….………p.53

Table 8 :

 8 link between mechanical properties and cell functions…………………………………….……….…….p.71

Table 9 :

 9 Myo6 proximal dimerization………………………………………………………………………………..…..……..p.78

Table 10 :

 10 GIPC1 interaction with the Myo6 CBD……………………………………………………….….……………..p.104

Table 11 :

 11 TOM1 interaction with Myo6 CBD……………………………………………………….……….………………p.105

Table 12 :

 12 TOM1 and Dab2 interaction with Myo6 CBD………………………………….….…………………..…….p.106

Table 13 :

 13 K63-diUbiquitin binding to the Myo6 CBD……………………………………………………………………p.173

Table 14 :

 14 clinical trials studies on myosin modulators …………........…………………………………………….p.182

Table 15 :

 15 Data collection and refinement statistics of the crystal structure of SkMyo2 bound to degraded MPH-220……………………………………………………………………………………………………………………..p.202

Table 16 :

 16 Data collection and refinement statistics of the SkMyo2 pre-powerstroke state crystal ……………………………………………………………………………………………………………………………………………………p.234

Table 17 :

 17 Conservation of key residues for blebbistatin binding between conventional myosins and unconventional Myo6………………………………………………………………………………………………………………….p.238

Table 1 :

 1 structure of Myo6 Motor in different states of the ATPase cycle

	Structures from

Table 2 :

 2 Dab2-Myo6 affinity reported in the literature

Table 3 :

 3 TOM1-Myo6 affinity reported in the literature

Table 4 :

 4 NDP52-Myo6 affinity reported in the literature

	Publication	KD	TAX1BP1 construct	Myo6 construct	Technic used
	Hu et al. 2018	93.9 ± 10.8 µM 30.7 ± 1.2 µM 90.0 ± 11.4 µM	725-789 -UBZ1+2-human 754-789 -UBZ2 human 725-753 -UBZ1 -human	R1036-T1135 LI-CBD n human L1073-K1119 short Nter CBD n human	ITC 25°C in 20 mM Tris (pH 7.5), 100 mM NaCl and 1 mM
					DTT

Table 5 :

 5 TAX1BP1-Myo6 affinity reported in the literature

Publication KD Ubiquitin construct Myo6 construct Technic used He et al. 2016

	2.1 ± 0.8 µM 13.5 ± 2.0 µM 2.0 ± 1.0 µM 2.7 ± 0.4 µM	K63-diUb	MyUb MIU-MyUb Iso1 MIU-MyUb Iso2 MIU-MyUb Iso2 A1013G	Fluorescence polarization assay 22°C in 20 mM Tris pH 7.6, 200 mM NaCl, and 5%
	11.4 ± 11.4 µM		MIU-MyUb Iso3	glycerol
	5.2 ± 1.5 µM 3.9 ± 0.1 µM 0.8 ± 0.4 µM 4.9 ± 1.0 µM	K11-diUb	MyUb MIU-MyUb Iso1 MIU-MyUb Iso2 MIU-MyUb Iso2 A1013G
	6.1 ± 1.5 µM		MIU-MyUb Iso3
	20.1 ± 1.1 µM 45.8 ± 5.1 µM 14.5 ± 8.5 µM -44.5 ± 8.5 µM	K48-diUb	MyUb MIU-MyUb Iso1 MIU-MyUb Iso2 MIU-MyUb Iso2 A1013G MIU-MyUb Iso3

Table 6 :

 6 Ubiquitin-Myo6 affinity reported in the literature

  (see section D.4).

	Publication	KD	DNA construct	Myo6 construct	Technic used
	Fili et al. 2017	3.5 ± 0.7 µM 2.5 ± 0.4 µM 1.2 ± 0.35 µM 0.1 ± 0.02 µM	DNA (40 pb)	FLMyo6 (1-1253) IQ-end (814-1253) IQ-DT (814-1060) CBD (1060-1253) CBD n (1060-1120)	Anisotropy fluorescence, 25 °C in 50 mM Tris pH 7.5, 150 mM NaCl and 1
		1.3 ± -0.7 µM		CBD c (1121-1253)	mM DTT
		0.12 ± 0.03 µM 0.5 ± 0.18 µM		CBDc ΔTRKR (1121-1253) CBDc ΔSKKK (1121-1253)
		0.7 ± 0.15 µM

Table 7 :

 7 DNA-Myo6 affinity reported in the literature

(Left) Representative

  electron microscopy images of different morphology of clathrin coated pits. Scale bar:

	EEA1-positive endosomes. Rather Myo6 NI colocalizes with transferrin receptor on Rab5 positive
	endosomes suggesting its recruitment after vesicles uncoating but before fusion with EEA1 early
	endosomes. In this context, Myo6 does not colocalize with Dab2 but colocalize with GIPC1 and the
	overexpression of the dominant negative: Myo6 NI Tail results in delay in transferrin uptake
	(Aschenbrenner, Lee, and Hasson 2003).
	D1.b.vi Myo6 seems to control early endosome localization and maturation
	Myo6 colocalizes in a large majority of APPL1-positive signaling endosomes along the actin
	filaments on the cell cortex (structured illumination microscopy on HeLa cells) (Masters et al. 2017). In
	100 nm. (Right) Bar graph representing the number of each kind of clathrin coated structures found in different contrast, EEA1-positive (more mature) endosomes do not colocalize with Myo6 although they are
	conditions (CTRL: no clathrin transfected, KD: CLCa knockdown, WT: transfection of wild-type CLCa after CLCa surrounded by actin filaments. Upon Myo6 silencing, APPL1-positive endosomes relocalize to the
	knockdown and I54D: transfection of CLCa I54D mutant (unable to bind Myo6) after CLCa knockdown. Accumulation of elongated vesicles when Myo6 is no longer able to bind CLCa (I54D mutant) suggests the nuclear periphery together with an accumulation of actin filaments, assessing the importance of Myo6
	importance of Myo6-clathrin interaction for fission.
	The authors thus proposed that, during vesicle invagination Myo6 will bind to CLCa and
	compete out Huntingtin interacting protein 1-related protein (Hip1R) promoting Hip1R dissociation
	from the cages and recruitment by epsins at the uncoated neck where Hip1R regulates actin filament
	polymerization, then Dab2 may bind Myo6 into an active dimer leading to fission.
	D.1.b.iv Vesicle transport
	Following vesicle fission, Myo6 still seems to have a role to play for transport. Indeed, single
	particle tracking reveals that microsphere speed is slowed down by overexpression of the Myo6 Tail
	(as a dominant negative) when phagocytosis of microspheres are studied in ARPE-19 cells (Hewage
	and Altman 2018). Moreover, a Myo6 mutant directed to the + end of actin filament results in
	redirecting endosomes to the cell cortex creating clusters at filopodia basis assessing the importance
	of myo6 directionality for proper localization and transport of endosomes in cells (Masters and Buss
	2017).
	D.1.b.v Myo6 NI localizes on uncoated vesicles to allow substance entrance
	Even if Myo6 LI was the one specifically found on clathrin coated vesicles, Myo6 NI seems to
	also have its role to play in the endocytic process but in later stages of the process. Indeed, Myo6 NI
	colocalizes with transferrin receptors whose transport is mediated by clathrin-mediated endocytosis.
	Yet, intriguingly, Myo6 NI does not colocalize with clathrin coated structures (like Myo6 LI) or with

Table 9 :

 9 Myo6 proximal dimerization

  Signals enabling Myo6 to switch from a back-folded auto-inhibited state to an unfolded activated one have been explored and several factors have been proposed, some are shared with other unconventional motors and others are unique to Myo6.It has been well established in several myosin motors such as Myo1 and Myo5, as well as Myo6, that the CaM bound to the IQ motif is sensitive to Ca 2+ ions[START_REF] Bahloul | The Unique Insert in Myosin VI Is a Structural Calcium-Calmodulin Binding Site[END_REF][START_REF] Manceva | Calcium Regulation of Calmodulin Binding to and Dissociation from the Myo1c Regulatory Domain[END_REF][START_REF] Krementsov | Myosin V: Regulation by Calcium, Calmodulin, and the Tail Domain[END_REF]. The role of Ca 2+ ions in the destabilization of the backfolded state of Myo6 was explored byBatters et al. (2016) who proposed that the CaM bound on the IQ motif previously identified as Ca 2+ sensitive[START_REF] Bahloul | The Unique Insert in Myosin VI Is a Structural Calcium-Calmodulin Binding Site[END_REF], would regulate the back-folded state. They indeed identified that its interaction with the CBD n depends on Ca 2+ concentration by a pull-interactions between Myo6 and certain partners, it is not clear from these data whether Ca 2+ ions directly increase the affinity between the Myo6 Tail and certain partners or whether they indirectly enhance Myo6 ability to bind partners by relieving auto-inhibition.In any case, high Ca 2+ concentrations can only be transient so that the motor would unfold and undergo recruitment. Low Ca 2+ concentrations are required for the Myo6 motor to function properly: Indeed, direct impact of calcium on Myo6 motor activity has been investigated. At high calcium concentration (pCa 5), Myo6 S1 ATPase rate is significantly reduced compared to low calcium condition (pCa 9). In contrast, artificially dimerized Myo6 (leucine zipper at aa 994) ATPase rate remains unchanged. Yet ADP release and actin filament sliding rate decreases in a calcium concentration dependent manner. The decrease upon reducing Ca 2+ concentrations is even stronger for the artificially dimerized Myo6. The authors thus proposed decoupling of the two heads of the dimer at high Ca 2+

	F.3.a. Ca 2+ ions
	2019) shows through
	immunoprecipitation that addition of 2 mM CaCl2 increased Myo6-clathrin binding. They also pointed
	out that clathrin interacts with Myo6 through CLCa46-61 (Biancospino et al. 2019) which is a flexible and
	unfolded part of the clathrin light chain but folded as an helix when bound to Myo6. As the

down experiment. The interaction is lost when Ca 2+ is added

(Batters et al. 2016)

. Metal shadow electron microscopy suggests even more that upon calcium addition, CaM could detached from the IQ

[START_REF] Song | Nucleotide-Dependent Shape Changes in the Reverse Direction Motor, Myosin VI[END_REF]

supporting previous experiments from Bahloul et al 2004 and the hypothesis that a conformational change in this Calmodulin could trigger unfolding

(Batters et al. 2016)

.

But Fili et al. (2017)

showed by pull-down assays that MD Ins2 can interact with CBD with or without Ca 2+ , suggesting that unfolding regulation upon calcium may not be a relevant mechanism in the context of FLMyo6 since even if CaM CBD N interactions are lost, some remaining contact may keep the folded state together. Yet, in presence of ATP and a 10-fold excess of actin, intramolecular FRET suggests the ability of calcium (pCa 4) to unfold Myo6 when the motor is in a specific condition where it can bind to actin and cycle

[START_REF] Rai | Multimodal Regulation of Myosin VI Ensemble Transport by Cargo Adaptor Protein GIPC[END_REF]

. In this context, Ca 2+ may facilitate Myo6 activation in actin rich regions of the cell but the importance of calcium in on/off regulation thus needs further investigation.

While it is still unclear whether Ca 2+ ions are sufficient to open Myo6, it can modulate the strength of the interaction between Myo6 and its partners, and thus influence the recruitment of Myo6 in cells. Fat blot and cosedimentation with liposomes show that Myo6 interacts much better with lipids at high calcium concentration

(Batters et al. 2016)

. Moreover,

Biancospino et al. (

conformation of the CLCa46-61 has been reported to be reversibly modulated by Ca 2+ ions, they proposed calcium as a key regulator of the Myo6-clathrin interaction

[START_REF] Deluca-Flaherty | Uncoating Protein (Hsc70) Binds a Conformationally Labile Domain of Clathrin Light Chain LCa to Stimulate ATP Hydrolysis[END_REF]

.

Although these studies

(Batters et al. 2016;[START_REF] Biancospino | Clathrin Light Chain A Drives Selective Myosin VI Recruitment to Clathrin-Coated Pits under Membrane Tension[END_REF]

highlighted that Ca 2+ ions can improve the

  and 46). More recently, FRET titration confirmed the ability of Dab2 to dimerize Myo6 814-end fragment[START_REF] Fili | Competition between Two High-and Low-Affinity Protein-Binding Sites in Myosin VI Controls Its Cellular Function[END_REF]) and seems to promote gating of the FLMyo6 upon increasing concentration of actin (DosSantos et al., 2022). It has been further confirmed in gliding filament assays that Dab2 activates Myo6 and makes the motor processive[START_REF] Phichith | Cargo Binding Induces Dimerization of Myosin VI[END_REF][START_REF] French | Investigations of Human Myosin VI Targeting Using Optogenetically Controlled Cargo Loading[END_REF] Rai et al. 2021) similarly to what was seen with OPTN[START_REF] Phichith | Cargo Binding Induces Dimerization of Myosin VI[END_REF]. Electron micrography consistently shows that Dab2 dimerizes

	FLMyo6.

But, even if both OPTN and Dab2 can dimerize, in rotary shadowing only 2% of dimer were seen in Dab2 while 17 % were seen with OPTN in similar conditions

[START_REF] Phichith | Cargo Binding Induces Dimerization of Myosin VI[END_REF]

) which pointed out to the weaker ability of Dab2 to form dimers compared to OPTN. Consistently with low dimerization level using Dab2, single molecule and ensemble molecule motility assays show that Myo6 dimerized by Dab2 behaves differently than the artificially dimerized Myo6 (leucine zippered dimer, i.e., 100% dimers). Although the run lengths are similar, with Dab2, less motile events occurred with bigger pauses between them, showing that Dab2 enables Myo6 for a sparse processive motility.

location and mechanical properties are likely dictated by a collection of successive or simultaneous interactions with cargo proteins, lipids and actin network. Thus, cellular Myo6 works under various load, oligomeric states and concentration conditions, and this makes this motor able to perform a wide range of functions.

  suggesting the existence of an addition unknown Myo6 partner to address Myo6 to melanosomes.

	This chapter explores structural insights of Myo6 mechanic regulation enabling its multifunctionality. Indeed, even if progress has been made in understanding how Myo6 function can be modulated through various signals (partners binding, calcium), mechanism that constricts full-length Myo6 selection, substitution and releases of cargos is not clearly established. Moreover, identification of key parameters able to orientate Myo6 mechanical characteristic towards anchoring, transporter or actin organizer function is still an on-going process: stiffness, the nature of the partner bound or the local Myo6 concentration has been proposed as key parameters. Overall, Myo6 Objectives Considering the growing importance of Myo6 in pathologies such as cancers and deafness,

answering such questions is of particular interest as they are necessary to describe the precise role Myo6 plays in pathogenous processes, which can lead to the development of therapies targeting Myo6 in the future.

Table 10 :

 10 GIPC1 interaction with the Myo6 CBD

	MST done in 20 mM BisTris pH6.5, 100 mM KCl, 1 mM DTT, 0.05% tween or in 20 mM BisTris pH6.5, 100 mM
	KCl, 1 mM DTT, 0.05 % tween

Table 11 : TOM1 interaction with Myo6 CBD

 11

	Tom1	Myo6	KD (nM)	n
	Tom1394-461	yfp Myo61035-fin	878 ± 321	2
	Tom1408-461	yfp Myo61035-fin	4024 ± 1836	1
	Tom1207-492	yfp Myo61035-fin	444 ± 135	2
	Tom1207-461	yfp Myo61035-fin	554 ± 180	2
	MST done in 20 mM BisTris pH6.5, 100 mM KCl, 1 mM DTT, 0.05 % tween or in 20 mM BisTris pH 6.5, 100 mM
	KCl, 1 mM DTT, 0.05 % tween

Table 12 : TOM1 and Dab2 interaction with Myo6 CBD

 12

	Myo6 Tail	KD	n

MST done in 20 mM BisTris pH6.5, 100 mM KCl, 1mM DTT, 0.05 % tween or in 20 mM BisTris pH 6.5, 100 mM KCl, 1 mM DTT, 0.05 % tween

Table 1 -

 1 Main contacts that stabilize the back-folded conformationDissociation constant (KD) ± KD confidence determined by microscale thermophoresis of Myo6 Head constructs against Myo6 Tail constructs (constructs schematized in Fig.

		Tail	Motor state	KD (nM)	n
	MD Ins2/IQ/3HB	YFP CBD	ADP.VO4	144 ± 61	3
	MD Ins2	YFP CBD	ADP.VO4	343 ± 197	3
	MD	YFP CBD	ADP.VO4	3920 ± 1453	3
	Ins2/IQ/3HB	YFP CBD	-	250 ± 86	4
	MD Ins2/IQ/3HB	YFP CBD	NF	726 ± 480	2
	MD Ins2/IQ/3HB	YFP CBDD1157V.Y1159D.D1161R.Q1163V	ADP.VO4	n.b.	3

Fluorescence Ratio without Actin + ATP Fluorescence Ratio with Actin + ATP Molar Ratio of Labeling per

				Myosin Head
	MD Ins2/IQ/3HB T845C	256.1 ± 24.4	243.8 ± 14.5	1.03
	MD Ins2/IQ/3HB T845C, A880C	22.5 ± 5.8	22.6 ± 7.2	2.10
	zippered HMM A880C	238.2 ± 23.7	232.3 ± 30.4	1.11
	zippered HMM T845C, A880C	206.5 ± 19.6	214.3 ± 12.6	2.22
	Zippered HMM T845C, A880C, L926Q	147.53 ± 30.4	164.6 ± 18.7	2.14

Table 2 -

 2 L926Q stabilizes the 3HB.

	Fluorescence observed by TMR labeling of one or two cysteine residues inserted into the three-helix
	bundle of monomers (MD Ins2/IQ/3HB ) and dimers (zippered HMM). Fluorescence was analyzed by a ratio

of the emission values to that of the absorption values for each construct from four independent measurements (n=4). Mean values (±SD) are reported. The molar ratio was calculated by comparing the myosin concentration to the concentration of the incorporated TMR.

Table 1 -Crystallographic structure of the Myo6 dimerization domain -data and refinement statistics.

 1

		29.39 Å, 29.39 Å, 295.65 Å
		90.00°, 90.00°, 120.00°
	Resolution	49.27 Å -2.22 Å
	Data completeness in resolution range	66.9 %
	Rmerge	0.17
	I/s(I)	1.02 at 2.22 Å
	R, Rfree	0.304, 0.343
	Wilson B-factor	39.2 Å 2
	Average B, all atoms	55.0 Å 2
	Total number of atoms	1051
	Unique reflections	2977
	Rfree test set	301 reflections (10.1%)
	Ramachandran outliers	0
	Sidechain outliers	8.8%
	Solvent content*	45.1%
	Supplementary

* Solvent content estimated by Matthews_coef 10

Table 14 :

 14 clinical trials studies on myosin modulators (www.clinicaltrials.gov)

	Clinical trials references on (www.clinicaltrials.gov) and concerning a myosin modulator. Most of them concern
	CardMyo2 and show the potential of myosin modulator to cure diseases

  on NM2B) using transient pyrene fluorescene assay. Lack of impact of blebbistatin on S1 actin dissociation thus suggests no effect of the blebbistatin on the actin bound motor.C.6.f. Blebbistatin must stabilize the pre-powerstroke stateFrom these studies, it thus appears that blebbistatin must stabilize the pre-powerstroke of the Motor in which Pi is not yet released from the Motor and the actin affinity is low to inhibit Myo2 activity.Stabilization of the pre-powerstroke state by blebbistatin was supported by a study in the context of Myo2 thick filaments. The structure of the thick filament depends on the nucleotide state of myosin: MgADP.Pi results in helical order while NF or ADP Motors leads to disordered filaments: this can be clearly identified by electron microscopy and Fourier transform. Treatment with 100 µM blebbistatin for 2h slows down filament loss of order upon nucleotide loss through apyrase addition consistent with the stabilization of the MgADP.Pi state stabilization by blebbistatin. This treatment is also able to reorder a disordered MgADP filament into a helical order filament consistent with the fact that blebbistatin has the ability to promote a pre-powerstroke like state even without Pi addition. It should be noted that blebbistatin addition is not able to reorganize a disordered NF filament onto a helical order one[START_REF] Zhao | Blebbistatin Stabilizes the Helical Order of Myosin Filaments by Promoting the Switch 2 Closed State[END_REF].

Table 15 :

 15 Data collection and refinement statistics of the crystal structure of SkMyo2 bound to

	degraded MPH-220

TABLE d

 d

	RESOURCE AVAILABILITY
	B Lead Contact
	B Materials Availability
	B Data and Code Availability
	d EXPERIMENTAL MODEL AND SUBJECT DETAILS
	B Animals
	B Human muscle biopsies
	d METHOD DETAILS

B Materials B Proteins B ATPase measurement B Molecular dynamics simulation B Crystallization and data processing B Structure determination and refinement

Table 1 .

 1 Prevalence of Diseases with Broad Etiologies in which Muscle Spasticity Is One of the Major Characteristic Symptoms

	Disease	Background	Symptom	Prevalence (per 100,000)	Patient Number	Ratio of Spasticity
	Stroke	blood flow disruption	weakness,	100-600	30 million	38%-46%
		in the brain either by	paralysis,	(Kim et al., 2015;		(Lundstro ¨m
		a blood clot or by	spasticity,	Yi et al., 2020)		et al., 2008; Opheim
		hemorrhage	coordination			et al., 2014; Urban
			and balance			et al., 2010; Wissel
			problems,			et al., 2013; 11-14
			memory problems			million spastic
						patients)
	Multiple	autoimmune disease	fatigue, gait	300-400 in US	2.3 million	50% a (1.5-2 million
	sclerosis (MS)	attacking the myelin	difficulties,	(Wallin et al., 2019)		spastic patients)
		sheaths of nerve fibers	numbness,
			spasticity,
			weakness, vision
			problems
	Cerebral	brain development	muscle stiffness,	150-300	17 million	70%-75% (Novak,
	palsy (CP)	disorders or disruption,	spasticity,	(Kruse et al., 2009;		2014; Pulgar
		due to genetic	ataxia, tremors,	Pulgar et al., 2019;		et al., 2019; 12 to
		mutations, infections,	gait disorders	Van Naarden Braun		13 million spastic
		trauma, or injury		et al., 2016)		patients)
	Amyotrophic	degeneration of	muscle weakness,	4 to 5	14-15,000 (US) b	40% (Meyer
	lateral	cortical motor	atrophy, spasticity	(Xu et al., 2020)		et al., 2019;
	sclerosis (ALS)	neurons, the motor				6,000 spastic
		nuclei of the brainstem,				patients)
		and the anterior horn
		cells of the spinal cord
	Traumatic	blow or jolt to the	paralysis, sensory	1% (up to 15%;	1.7-2 million	5%-19% (Bose
	brain injury (TBI)	head or a head	impairments,	Dewan et al., 2018),	new TBI/year in	et al., 2015;
		injury that disrupts	headaches,	475,000	US (Enslin et al.,	Verplancke et al.,
		normal functions	coordination,	children/year (US;	2020), 68-71	2005; 30-35 million
			spasticity	Enslin et al., 2020)	million	spastic patients)
					worldwide (Dewan
					et al., 2018)
	Multiple system	progressive	slow movement,	2-6 (Bjornsdottir
	atrophy (MSA)	neurodegenerative	muscle stiffness,	et al., 2013)
		disease with alpha-	spasticity,
		synuclein accumulation	incoordination,
		in glia (form of atypical	tremors, bladder
		Parkinsonism)	control problems
	Spastic	defects in transport	progressive gait	3-6
	paraplegia	of proteins, lipids	disorder, spasticity
		through long nerve
		fibers (axons)
	Myotonia	abnormality of	painless spasm,	1 to 2
	congenita	skeletal muscle	difficulty relaxing	(Romitti
	(Becker	membranes	muscles (myotonia),	et al., 2015)
	disease)	resulting in	rigidity, abnormally
		hyperexcitability	enlarged muscles
			(hypertrophy)
	Spinal cord	nerve damage due to,	pain, increased	2-8 (Witiw and
	injury or	e.g., thoracic spine	reflexes in legs that	Fehlings, 2015)
	tumors	herniated discs damage	can cause spasticity,
		the nerves	muscle weakness,
			numbness or tingling

a WHO Atlas multiple sclerosis resources in the world 2008 (http://www.who.int/mental_health/neurology) b NIH amyotrophic lateral sclerosis (ALS) brochure (https://www.ninds.nih.gov/Disorders)

Table 17 :

 17 Conservation of key residues for blebbistatin binding between conventional myosins and unconventional Myo6

Gyimesi et al., 2020, Cell 183, 335-346 October 15, 2020 ª 2020 Elsevier Inc. https://doi.org/10.1016/j.cell.2020.08.050

MTA-ELTE Motor Pharmacology Research Group, Pa ´zma ´ny Pe ´ter se ´ta ´ny 1/c, 1117 Budapest, Hungary

Motorpharma, Ltd., Szila ´gyi Erzse ´bet fasor 27, 1026 Budapest, Hungary

Printnet, Ltd., Kisgo ¨mb utca25-27, 1135 Budapest, Hungary

Structural Motility, Institut Curie, Paris Universite ´Sciences et Lettres, Sorbonne Universite ´, CNRS UMR144, 75005 Paris, France

(legend on next page)

Acknowledgments

The authors greatly acknowledge the Cell and Tissue Imaging (PICT-IBiSA) -Institut Curie, member of the French National Research Infrastructure France-BioImaging (ANR10-INBS-04) and in particular Anne Sophie Macé for her help in the quantification of the results; the CurieCoreTech Mass Spectrometry Proteomics and Recombinant protein; the beam line scientists of PX1 and SWING (SOLEIL synchrotron) for excellent support during data collection; Pierre-Damien Coureux for preliminary experiments in negative staining electron microscopy; Margaret Titus for her help with Jo-Myo6-In cloning and comments on the manuscript; Virginie Ropars for her help in SAXS data optimization, Guillaume Jousset for its advises in biochemistry and Christopher Toseland for accepting to provide us with Dab2 DNA.

ACKNOWLEDGMENTS

We would like to thank Prof. A ´rpa ´d Dobolyi for help in rat operations; Ma ´te ´Winternitz, Noe ´mi Karnok, Kinga Oravecz, and Henrietta Ho ´f for technical assistance; and beam line scientists of PX2A (SOLEIL synchrotron) for excellent support during data collection. We also thank Prof. James R. Sellers (NIH-NHLBI), A ´da ´m Miklo ´si (ELTE), and Csaba Szalontai (ELTE) for detailed discussions and helpful comments on the manuscript and Prof. Matthias Preller (MHH) for helpful discussions regarding MD simulations. This study has been supported by the Hungarian National Research, Development and Innovation Office (NVKP 16-1-2016-0051 to M.G., A.A ´.R., and A.M.-C.) and the Hungarian Ministry of Finance (GINOP-2.1.7-15-2016-02580 to M.G. and A.A ´.R.). L.C. is the recipient of a PhD fellowship from Ligue Contre le Cancer IP/SC-16058. A.H. was supported by grants from CNRS, FRM DCM20181039553, ANR-17-CE11-0029-01, and AFM 21805. The A.H. team is part of the Labex CelTisPhyBio:11-LBX-0038, which is part of the IDEX PSL (ANR-10-IDEX-0001-02 PSL). D.V.T., K.M.R., and J.A.S. are supported by National Institutes of Health grants R01 GM033289 and R01 HL1171138. D.V.T. is supported by an American Heart Association postdoctoral fellowship (17POST33411070).

Funding H.L.S. was supported by National Institutes of Health Grant DC009100. A.H. is supported by grants from CNRS and ANR-19-CE11-0015-02. A.H. and C.D. were supported by grants from ANR-17-CE11-0029-01. The A.H., D.L. and C.D. teams are part of the Labex Cell(n)Scale ANR-11-LBX-0038 and IDEX PSL (ANR-10-IDEX-0001-02-PSL). K.P. is a recipient of a Marie Curie fellowship 797150 MELANCHOR. V.P.-H. and L.C. are recipient of a PhD fellowship from Ligue contre le cancer GB/MA/SC-12630 and IP/SC-16058.

Data availability.

The atomic model of the Myo6 proximal dimer is available on the PDB [START_REF] Berman | Announcing the worldwide Protein Data Bank[END_REF] under the accession code 8ARD. The other data supporting the findings of this study are available from the corresponding author upon request.

Author contributions

A.H. and H.L.S. designed and directed the research. L.C., V.J.P.-H., K.J.P. and C.D. were involved in project management. H.S., X.L., L.H., L.C., T.L., C.K., L.V., A.D., E.F., P.H., V.J.P.-H., D.M. cloned, produced and purified constructs. L.C., T.L., C.K., H.S., L.H., A.D., L.V., P.H., V.J.P.-H., D.M. performed biophysical and biochemical assays. C.K. performed grid optimization for negative staining EM that were collected by ADC. D.L. provided access to the 200kV microscope. C.K. analyzed the negative staining data and performed the 3D reconstruction. H.S. characterized the limits of the proximal dimerization region, crystallized it and C.K. solved the structure. F.M., Y.I.L. and K.J.P. performed the cell-based assays. L.C., C.K., A.H., H.L.S., F.M., Y.I.L., H.S., L.H., A.D., L.V., K.J.P. and V.J.P.-H. analyzed the data. J. S.-C. provided technical help in quantification of the cell-based assays. L.C., A.H., C.D., C.K. and H.L.S. wrote the initial version of the paper, with the help of F.M. and V.J.P.-H. and all authors reviewed it. A.H. and H.L.S. provided fundings.

Competing interests

The authors declare no competing financial interests.

Supplementary information How Myosin VI Traps its Off-State, is Activated and Dimerizes

Louise Canon 1 , Carlos Kikuti 1 , Vicente J. Planelles-Herrero 1 , Tianming Lin 2 , Franck Mayeux 1 , Helena Sirkia 1 , Young il Lee 2 , Leila Heidsieck 1 , Léonid Velikovsky 1 , Amandine David 1 , Xiaoyan Liu 2 , Dihia Moussaoui 1 , Emma Forest 1,3 , Peter Höök 2 , Karl J. Petersen 1 , Aurélie Di Cicco 4 , Julia Sires-Campos 5 , Daniel Lévy 4 , Cédric Delevoye 5 , H. Lee Sweeney 2,* , Anne Houdusse 1,*

Supplementary Figures

Supplementary Figure 1

Supplementary Figure 2 -The Jo-In fusion stabilizes the off-state with no effect on its conformation.

(A) SDS-PAGE profile of Jo-Myo6-In stained with SYPRO 2 . The estimated molecular weight based on SDS-PAGE is given in red. The CaM/FLMyo6 molar ratio was determined from the intensities of the corresponding bands (Table: mean ± standard deviation from 4 replicates). Intensity of Myo6 and CaM bands were estimated on ImageLab software (Bio-Rad). Ratio CaM/FLMyo6 was estimated by calculating (CaM band intensity)/(Myo6 band intensity) and normalized by theoretical molecular weight to get a molar ratio. MD Ins2 was used as a control as it binds 1 CaM per Myo6 3 . Note that, on SDS-PAGE, purified Jo-Myo6-In-Flag appears as two distinct bands, one at its expected molecular weight 176 kDa, and another band around 250 kDa . On SEC, the faster band was found to elute slightly earlier, in a broader peak merged to the one corresponding to the slower band. LC-MS concluded that both bands correspond to Jo-Myo6-In with 68.5% sequence coverage. Exactly the same peptides were found in both bands, and the Jo and In peptides containing the expected covalent Lys-Asn bond could not be found. Therefore, we assume that the slower band corresponds to the protein whose covalent Lys-Asn bond is correctly formed (="locked"), whereas the faster band is composed by folded, but not locked, protein (="closed").

Supplementary Figure 3 -Affinity of the Myo6 CBD for different Myo6 Head or Neck constructs.

Microscale thermophoresis profiles and fits corresponding to the data exposed in Table 1.

Supplementary Figure 4 -Role of the proximal Myo6 sequence in the stabilization of the off-state.

(A) Normalized SEC-MALS profiles of FLMyo6 (SAHmimic) (blue) and FLMyo6 (L926Q) (yellow) mutants, plus FLMyo6 (WT) in conditions that favor opening (black) or closure (green), as references.

Supplementary Figure 5 -SDS-PAGE stained with colloidal blue corresponding to Anti-His pull-down in Fig. 3D.

Input (I), flow through (FT), last wash (W) and elution (E) fractions corresponding to anti-His pull-down pictured in Fig. 3D.

Supplementary Figure 7 -Localization of MST-partners at melanosomal membrane in MNT-1 cells.

Representative MNT-1 cells co-expressing mCherry MST-partners and iRFP VAMP7. MNT-1 were fixed 48h post-transfection then imaged. In order: mCherry MST-GIPC1, mCherry MST-TOM1, mCherry MST-Dab2. Magenta: mCherry MST-partners; Cyan: iRFP VAMP7. From left to right: entire cell (3 channels merged, individual channels); zoom 8x boxed region ( mCherry MST-partners / iRFP VAMP7 merged, individual channels). Results show colocalization of mCherry MST-partners all around the iRFP VAMP7 melanosomes.

Supplementary Figure 8 -I1072 is key for Myo6 specific recruitment on melanosomes.

(A) Representative fixed MNT-1 cells co-expressing GFP Myo6 +/-I1072A mutation with mcherry MST and iRFP VAMP7. mCherry MST and iRFP VAMP7 are melanosome-associated components. MNT-1 cells were fixed 48h post-transfection then imaged and processed for quantification. Note that the control FLMyo6 (WT) is the same as in Sup

Supplementary Figure 10 -Characterization of the dimerization of proximal region by MALS.

The 875-940 fragment is sufficient for dimerization. Importantly, a peptide missing the first 5 aa (aa 880-940) failed to dimerize even when its peak concentration was higher than 70 µM, which emphasised the key role of residues 875-880 for the stability of the dimerization. Additionally, in line with a recent study 5 , no dimerization was found via the SAH, unlike previously proposed by molecular dynamics simulations 6 . Indeed, no dimerization was observed for the Tail fragment aa 912-end, (containing the whole SAH), even when its peak concentration was 25 µM.

Supplementary Figure 11 -Proximal dimerization is regulated by 3HB unfolding which is impaired by the pathogenic L926Q deafness mutation. (A)

Supplementary Figure 13 -Endocytosis is impaired if Myo6 cannot dimerize through its proximal region

Supplementary Figure 14 -Alphafold 8,9 predicts a back-folded FLMyo6 with an exposed CBD c

Alphafold modelisation of FLMyo6 (UNIPROT: Q9UM54) was manually docked within our 3D reconstruction of Jo-Myo6-In (grey mesh). CBD c is found outside of the electron density, with residues D1157, Y1159, D1161, Q1163 highly exposed (red circle), inconsistently with our experimental data.

Supplementary Figure 15 -Fluorescence of MST-partners localized on melanosomes, normalized to the VAMP7 signal.

Quantification of the fluorescence signal of mCherry MST-partners on melanosomes in MNT-1 cells expressing different Myo6 constructs. Global intensities were calculated by using the macro described in Methods, normalized to the iRFP VAMP7 signal (total melanosomal surface area). (For constructs including a rTEV cleavage site, homemade rTEV was added 1:50 m/m, and a second passage through IMAC was performed before SEC)

Supplementary Table 2 -Purification buffers used for constructs expressed in E. Coli

Supplementary Movies

Supplementary Movie 1 3D view of the Myo6 off-state docked in the negative staining reconstruction (from Fig 3A).

Supplementary Movie 2

Representation of the crystal packing of the dimerization Myo6 fragment 875-937 as calculated with PyMol. Each crystallographic dimer is represented in a different color.

Supplementary Material and Methods

Cloning, expression and purification from SF9/baculovirus system.

The full-length wild-type Myo6 (FLMyo6) was generated using human Myo6 cDNA splice form without the large insertion (Q9UM54-2 in UNIPROT). The small insert was removed through sub cloning to obtain a FLMyo6 construct without any spliced insert (corresponding to isoform Q9UM54-5). The FLMyo6 (no inserts) construct was then used in all in vitro experiments requiring a full-length construct except for the Anti-His pull-down experiment, for which the small insert isoform was used. The deafness mutant (L926Q) and triple mutant (T888D.Q892E.V903D -R892 in mouse corresponds to Q892 in human, see Sup Fig. 10B) in the anti-parallel dimerization region were produced from FLMyo6 with no inserts by reverse PCR. As previously described 11 , a mutant FLMyo6 (SAHmimic) was made where the residues from Glu922 to Glu935 (EAERLRRIQEEMEK) were replaced with alternate acidic and basic residues (EERKRREEEERKKREEE) to match the (i, i+4) phasing observed in the predicted Myo6 SAH domain.

For microscale thermophoresis and ATPase assays, previously described constructs were used: MD (1-789) 12 , MD Ins2 (2-816) 3 , MD Ins2/IQ/3HB (1-917) 13 . The Myo6 zippered dimer was created by truncation at R991 followed by a leucine zipper (PDB: GCN4 14 ) as previously described 15 .

For the bundle unfolding experiments, a monomeric "cys-lite" construct was made by C-terminal truncation at amino acid Q919 and introduction of C321S, C362S, and C611A. To this construct, either a T845C mutation alone, or the combination of T845C and A880C mutations was introduced for rhodamine labeling, as previously described 13 . A dimeric "cys-lite" construct was made by introduction of the C321S, C362S, C611A mutations in the Myo6 zippered dimer 15 . Into this construct, either a T845C mutation alone, or the combination of T845C and A880C mutations were introduced for rhodamine labeling, as previously described 13 , with or without the addition of a deafness-causing mutation (L926Q). Each of these constructs had a Flag tag (GDYKDDDDK) at its N-terminal end to facilitate purification. Expression in baculovirus system and purification were performed as previously described 16 . Jo and In-Flag sequences 4 were synthesized (Eurofins genomics) and fused to Myo6 N-terminus (linker Gly-Ser) and C-terminus (linker Gly), in pVL1392 for expression in Sf9 cells. Purification was achieved using the published protocol for FLMyo6 16 except that, for EM studies, purification was performed by replacing ATP with ADP.VO4 in the lysis buffer. For increased purity, a SEC step was performed using a Superdex 200 Increase column (Cytiva) developed in 10 mM Hepes; 80 mM NaCl; 5mM NaN3; 1 mM MgCl2; 0.1 mM TCEP; 0.1 mM ADP; 0.2 mM VO4; 0.1 mM EGTA; pH 7.5.

Constructs cloning, expression and purification from Escherichia coli

Cloning Myo6 constructs Ins2/IQ/3HB was generated using our human FLMyo6 NI construct (see previous section), DNA sequence encoding for aa783-917 was transferred into pPROX-HTB plasmid containing in N-terminus 6XHis tag and a TEV cleavage sequence (coding for ENLYFQG). Myo6 YFP CBD was generated through several round of subclonings from cDNA mouse Myo6 (E9Q3L1 in UNIPROT). Myo6 was incorporated in pET14 plasmid containing in N-terminus 6XHis-tag, yellow fluorescent protein (YFP) and a TEV cleavage sequence. Finally, Myo6 was truncated in N-terminus at position corresponding to aa M1032 through reverse PCR. The YFP CBD (D1157V.Y1159D.D1161R.

C.3. Fluorescence

Blebbistatin is fluorescent (emission peak at 410 nm), which can be an advantage for monitoring the drug, but can also be damaging, notably for cell imaging at this wavelength. Yet, the very bright fluorescence found in cell imaging mostly comes from blebbistatin precipitate rather than the soluble part, thus fluorescence will not be an issue as far as the solubility of the blebbistatin derivative is high enough to avoid precipitation. This is supported by the fact that 3'hydroxyblebbistatin does not cause undesired fluorescence signal in cell imaging [START_REF] Verhasselt | Discovery of (S)-3'-Hydroxyblebbistatin and (S)-3'-Aminoblebbistatin: Polar Myosin II Inhibitors with Superior Research Tool Properties[END_REF].

C.4. Blebbistatin stability and toxicity

C.4.a. Blebbistatin is cytotoxic under blue light and UV

When bovine aortic endothelial cells (BAECs) are supplemented with blebbistatin and illuminated at 450-490 nm or 365 nm, cell death overcomes within hours, and proportionally to the blebbistatin concentration (0-20 µM), even after very short illumination time (1 s). Similarly, 10 -200 µM blebbistatin was found to be phototoxic following illumination with blue light (390 -470 nm) to various cancer cell lines: FEMX-1, LNCaP, Du145, U87 and F11-hTERT [START_REF] Mikulich | Blebbistatin, a Myosin Inhibitor, Is Phototoxic to Human Cancer Cells under Exposure to Blue Light[END_REF]. In contrast, illumination of BAECs supplemented with blebbistatin had no toxic effect at 510 -560 nm or 590 -650 nm: cells continue to migrate and remain alive with a correct shape, even after 12h. These data suggest that if 20 µM blebbistatin is not toxic on its own, it becomes toxic after illumination at 450 -490 nm or 365 nm [START_REF] Kolega | Phototoxicity and Photoinactivation of Blebbistatin in UV and Visible Light[END_REF]).

Yet, it should also be noted that even without blue or UV illumination, blebbistatin can result in cell death if it is used at high concentrations and/or for long times: over 50 µM blebbistatin, significant toxicity can be seen after 24 h in F11-hTERT, FEMX-I, Du145 and LNCaP [START_REF] Mikulich | Blebbistatin, a Myosin Inhibitor, Is Phototoxic to Human Cancer Cells under Exposure to Blue Light[END_REF]. Moreover, 4 different pancreatic adenocarcinoma cell lines [START_REF] Duxbury | Inhibition of Pancreatic Adenocarcinoma Cellular Invasiveness by Blebbistatin: A Novel Myosin II Inhibitor[END_REF] remain viable up to 200 µM blebbistatin, but 50% cell death is reached using 400 µM.

C.4.b. Blebbistatin is inactivated by blue light and UV

Interestingly, once illuminated by UV, blebbistatin is no longer able to inhibit NM2A/B ATPase activity, or to disrupt actin filament sliding and actin network organisation in cells, suggesting that photodegradation induces loss of blebbistatin pharmaceutical activity [START_REF] Kolega | Phototoxicity and Photoinactivation of Blebbistatin in UV and Visible Light[END_REF][START_REF] Sakamoto | Blebbistatin, a Myosin II Inhibitor, Is Photoinactivated by Blue Light[END_REF] by the putative degraded form. It should be noted that, as the hydroxyl group mediates two key polar contacts between blebbistatin and Myo2 (see section C.7.), it is not surprising that loss of the hydroxyl group leads to loss of inhibitory activity. Therefore, the degradation as we observed is more likely to happen once the drug is bound to Myo2.

Interestingly, additional density likely corresponding to a water molecule was found nearby the degradation site (marked by a purple arrow on the Figure 83). This density is not seen in any To understand the origin of the degradation in SkMyo2 MPH-220 crystals, we thus hypothesized that:

-MPH-220 could be degraded by radiation damage due to X-ray -MPH-220 could react with one or some of the ingredients in the crystallization solution -MPH-220 degradation could be caused by exposure to light, as is the case for blebbistatin

A.1.a. Radiation damage

We contacted the beamline scientist of proxima-2 (Synchrotron SOLEIL) William Shepard to discuss with him the possibility of radiation damage of MPH-220. As the overall radiation dose at proxima-2 is way below the Garman limit (30 MGy), and as sulphur is much more prone to radiation damage than a hydroxyl group, radiation damage did not seem likely in this case.

To confirm this hypothesis, I prepared new crystals of SkMyo2 with MPH-220 with limited exposure to light. The newly obtained crystals were then shot at proxima-2 beamline (Synchrotron SOLEIL) using different radiation doses (~1.9MGy and ~0.4MGy) for each one. Two crystals diffracting at 2.1 Å and 2.5 Å were obtained, yet all datasets corresponded to the degraded form of MPH-220 even at the low dose of ~0.4MGy, making the hypothesis of MPH-220 degradation through radiation damage very unlikely.

A.1.b. SkMyo2 -non degraded MPH-220 structure

Finally, a third crystal, diffracting to 3.2 Å, was obtained. This time the crystal contained the complete MPH-220 at both ~1.9MGy and ~0.4Mgy (Figure 84). Data were analyzed and published on Gyimesi et al. 2020 (PDB: 6YSY).

Overall, this does not seem to be a major issue in the physiological context. As mentioned above, no MPH-220 mediated toxicity was found on cultured fibroblasts (see section A.1). Additionally, MPH-220 gives good results on rat muscle relaxation [START_REF] Gyimesi | Single Residue Variation in Skeletal Muscle Myosin Enables Direct and Selective Drug Targeting for Spasticity and Muscle Stiffness[END_REF], see section A.2.).

A.2. Article describing MPH-220 specificity and potential for spasticity treatment

We thus published an article in October 2020 describing MPH-220 specificity and its potential for

DECLARATION OF INTERESTS

The authors declare the following competing interests: employment, A.M.-C. and M.G. are owners of Motorpharma, Ltd. and A.A ´.R. and M.G. are parttime employed by Motorpharma, Ltd.; related patents, PCT/EP2017/051829, WO/2017/129782, HU1800129A2, PCT/HU2019/050017, WO/2019/ 202346A2, and WO/2019/202346A3; J.A.S. is a cofounder and member of the scientific advisory boards of Cytokinetics and MyoKardia, biotechnology companies developing small molecules that target the sarcomere for the treatment of various muscle diseases; K.M.R. is on the scientific advisory board at MyoKardia; and J.A.S., D.V.T., and K.M.R. are cofounders of Kainomyx Inc., a biotechnology company focused on developing small molecules to target tropical diseases.

SUPPORTING CITATIONS

The following references appear in the Supplemental Information: [START_REF] Billington | Characterization of three full-length human nonmuscle myosin II paralogs[END_REF][START_REF] Va ´rkuti | A highly soluble, non-phototoxic, non-fluorescent blebbistatin derivative[END_REF][START_REF] Kova ´cs | Functional divergence of human cytoplasmic myosin II: kinetic characterization of the non-muscle IIA isoform[END_REF]; [START_REF] Wang | Kinetic mechanism of non-muscle myosin IIB: functional adaptations for tension generation and maintenance[END_REF] All three inhibitors were administered to rats in different concentration ranges and cardiovascular (heart rate, blood flow) and respiratory functions (breath rate, oxygen saturation) were monitored. 16 mg/kg AmBleb killed the animals within 15 minutes due to heart failure (G), while neither 13 mg/kg MPH-188 (H) nor 18 mg/kg MPH-220 (I) had any significant effect on cardiovascular and respiratory functions. Note that we measured MPH-220 effect on longer timescales (parallel with force measurements). (J) We repeated the experiments several times and found that MPH-220 does not affect cardiac functions to a greater extent than the solvent excipient during the long anesthetic period (cf. Figures 2E and2F). Data are mean ± SD, n = 1-4. (K) hERG potassium channel inhibition measurement up to 5 mM MPH-220. We measured hERG inhibition up to 5 mM, because the concentrations of MPH-220 in cardiac tissue even upon treatment with high doses (up to 130 mg/kg) were in this concentration range (3-7 mM). At this concentration range we did not observe inhibition of hERG potassium channels and we did not observe any concentration dependence of inhibition on cardiomyocytes, which would otherwise potentiate a drug candidate for QT prolongation leading to life-threatening arrhythmias [START_REF] Pollard | An introduction to QT interval prolongation and non-clinical approaches to assessing and reducing risk[END_REF]. Mean ± SEM are shown; n = 2.

A.3. MPH-220 may induce rearrangements within the Myo2 Motor

Finally, we aimed at comparing SkMyo2 with or without MPH-220 bound to characterize the rearrangements required for MPH-220 binding. I managed to obtain the structure of SkMyo2 in the pre-powerstroke state at 3.2 Å in the same crystallization condition (Table 16).

SkMyo2 ADP-VO4 Wavelength 0.98010 A Resolution range 71.7 -3.215 (3.33 -3.215 the Upper-50 and hinder entrance of small molecules, in particular rather apolar molecules. In contrast, CardMyo2 and SkMyo2 exhibit a Gly instead of a Glu, leaving more space for ligand entrance. Yet, deTailed understanding of the way blebbistatin can enter its pocket and of the relative importance of these surface residues remains to be obtained. To that aim, molecular simulations of Myo2s pre-powerstroke dynamics and mutagenesis assays could greatly help understanding how blebbistatin and its derivatives manage to enter in the site and how residue changes within the entrance of the pocket can impact blebbistatin and blebbistatin derivatives inhibition. Coupled with a deTailed analysis of blebbistatin pocket bound to derivatives exhibiting different specificities (see section B.1.), this knowledge could lead to a greater understanding of the criteria that determines the specificity of a compounds in such a tight pocket. Perhaps helping the development of future specific inhibitors against myosins.

Abstract

ABSTRACT

The eukaryotic cell nanomotors are able to perform complex mechanical tasks, necessary for tension generation in precise cellular locations. Vesicles formation and transport in the endocytic and secretory pathways can for example result from it. Among these motors, myosins are able, from the hydrolysis of ATP, to generate force in association with actin filaments. Myosins are divided into many classes involved in a wide range of functions and pathologies. Their dysfunction can lead to many diseases including spasticity, asthma, cardiomyopathy, deafness or many cancers... (Coluccio 2020).

Thus, the thesis project is centered on the one hand (I) on the study of the regulation of myosin VI (Myo6), a nanomotor involved in deafness as well as many cancers [START_REF] Dunn | A Novel Role of Myosin VI in Human Prostate Cancer[END_REF][START_REF] Li | Knockdown of Myosin VI by Lentivirus-Mediated Short Hairpin RNA Suppresses Proliferation of Melanoma[END_REF][START_REF] Ma | Knockdown of Myosin VI Inhibits Proliferation of Hepatocellular Carcinoma Cells In Vitro[END_REF][START_REF] Ma | [END_REF]Yang 2019;[START_REF] Yang | LncRNA UCA1 Promotes Development of Gastric Cancer via the MiR-145/MYO6 Axis[END_REF]. Myo6 is unique among all members of the myosin superfamily because it moves on actin filaments in the opposite direction to all other myosins. This specificity allows it to perform unique cellular roles that cannot be performed by any other myosin.

(II) On the other hand, the thesis project focused on the study of the blebbistatin pocket (Straight et al. 2003), an inhibitor pocket within the myosin motor domain, in order to explore its potential for the development of specific inhibitors. This thesis therefore contributes to: (I)

Understanding the processes regulating the activity of Myo6 via an approach combining structural biology, biophysical tests and cell biology. Such regulation involves the existence of an auto-inhibited state of Myo6 (Spink et al. 2008;[START_REF] Fili | NDP52 Activates Nuclear Myosin VI to Enhance RNA Polymerase II Transcription[END_REF]2020), a state in which the latter consumes little ATP and diffuses freely into the cytosol so that the motor is activated only at the site required for its recruitment. The biophysical study of this state has made it possible to better understand how to stabilize or destabilize it in vitro and has enabled us to obtain a first model of the auto-inhibited state using negative staining electron microscopy. This study also allowed us to better understand the differential role of cellular partners of Myo6 for its activation. Mutants promoting the activation or formation of the auto-inhibited state of Myo6 have been developed based on this study. Their ability to be recruited by different partners of Myo6 could thus be studied in human melanoma cells (MNT-1), these tests confirm a differential recruitment by the partners and suggest non-specific recruitment of mutants promoting activation.

(II)

Understanding the origin of the specificity of the MPH-220, an allosteric modulator derived from the well-known myosin inhibitor blebbistatin and able to specifically inhibit skeletal Myosin-2 from a crystallographic approach. This study describes that the specificity of this inhibitor with high potential for the development of new treatment against spasticity is based on a single non-conserved residue in the Blebbistatin allosteric pocket of the other myosins-2 [START_REF] Gyimesi | Single Residue Variation in Skeletal Muscle Myosin Enables Direct and Selective Drug Targeting for Spasticity and Muscle Stiffness[END_REF]). KEYWORDS myosin VI; auto-inhibition; deafness; myosin II; MPH-220; spasticity; blebbistatin; structural biology