Tyrosine kinase substrate

Matthiew Piel, Dr Sandrine Etienne-Manneville and Dr Johanna Ivaska for taking the time to review my work. I am grateful to be able to discuss this project with such esteemed members of the scientific community.

I would also like to thank Guillaume Montagnac, my PhD director, who gave me this opportunity to discover the world of research, which I had been dreaming about since high school. Thank you for believing in me and encouraging me, even at my lows. It has been a long and arduous road, but I have always been able to count on your help, feedback and support. I would also like to thank Nadia Elkhatib, who supervised my work from the very start, from the bottom of my heart. You have always believed in me, even when I doubted myself. You brought so much humanity and benevolence to this experience. Talking with you about science and more personal stuff was always a highlight and helped me to persevere through the highs and especially the lows. I would also like to thank the rest of the team: Francesco, Nathalie, Ksenia, Sammy, Stéphane, Jean-Philippe. Thank you for your support, advice and all the memorable life discussions and laughter we shared. It was always a pleasure to come work alongside you. Thank you, as well, to the Jaulin team. A special shout-out to Charlotte, Manu and JB of course. Thank you to the other teams on our floor, with a special shout-out to Sara and Julie.

I would like to thank my friends, who supported me throughout this laborious journey. I wouldn't have been able to keep it together without you guys. Thank you to Lucie, Aurélie, Manon, Alice, Marine, Didier, Gemma, Elodie, Camille H, Béatrice, Camille B, Angélique, and so many more! Lastly, I would like to thank my family, I definitely would not have been able to achieve this without your love and support. Thank you to my parents and my sister, Lena, for encouraging me and believing that I could do anything I set my mind to (def not true but at least it worked out in this case!). Thank you for being so comprehensive and reminding me that it's ok to be ok and that I don't need to stress all the time. Merci à mes grands-parents, surtout ma maminette, pour m'avoir écouté me plaindre de tout ce qui n'allait pas pendant ces 4 ans de thèse. Et merci de venir assister à ma soutenance, ça me fait tellement plaisir. Merci aussi à mamie Francine de m'avoir écoutée et soutenue sur ces deux dernières années. Merci à mon oncle, ma tante et mes cousins pour leur soutien et de venir assister à ma soutenance. J'ai d'autant plus la pression maintenant ! Enfin, merci à mon chéri, Bastien. Tu as été le plus gros soutien pendant ces 4 années et ça aurait été beaucoup plus compliqué de réaliser cet exploit sans toi. Je sais que ça n'a pas toujours été facile et qu'il a fallu faire quelques sacrifices, donc merci de les avoir faits. Merci pour ta patience, d'avoir été aussi compréhensif, je sais que ce n'est pas évident. Et bientôt on pourra profiter à fond, voyager et faire tout ce que j'avais égoïstement mis en attente, youyou ! This thesis would not have seen the light of day without all these lovely people in my life. I am so thankful for each and every one of you. It has been a real team effort, so thank you for everything.

I would also like to thank myself for persevering through all these challenges (and there were many). I never thought I would be able to get where I am today but decided to give it a try anyway. I learned so much during these four years, scientifically but also personally. I wish the journey had been a bit less strenuous, but at least I got there eventually and right now, it's what really matters.

Table of figures

Introduction

I. Mesenchymal migration

Cell migration is known to play a pivotal part in many biological processes, both physiological and pathological. For instance, the morphogenetic movements observed during the embryonic development, such as gastrulation or the neural crest development, originate from cells' ability to migrate as a collective. Similarly, single cell migration is required in order to establish a functioning immune system and response such as with wound healing or leucocyte invasion at inflamed sites. However, migration has also been involved in pathological processes such as in inflammatory diseases where an abnormal increase in leucocytes infiltration in targeted tissues leads to the development of diseases such as psoriasis, Crohn's disease or cancer metastasis.

Metastasis occurs when cancer cells escape the primary tumour by breaching through the basement membrane and invading the surrounding tissue. Cancer cells must undergo phenotypic changes to lose part or all of their epithelial characteristics and acquire mobility.

This process is referred to as the epithelial to mesenchymal transition (EMT). Cancer cells can then invade the stroma either as isolated cells or as a cohesive group of collectively migrating cells.

I.1. Different modes of cell migration

Various modes of migration exist and can be interchangeable over time, in response to external stimuli such as changes in ECM properties or molecular pathway activation. However different those types of migration may be, a common thread can be found between them. All require cell polarisation and a capacity to shape or reshape its cytoskeletal architecture, especially its actomyosin network which is key to generating forces necessary for cell migration. In vivo, cell migration is directional and guided by many different types of cues, including, but not limited to, mechanical, chemical, biochemical and electrical cues.

I.1.1. Collective migration

Collective migration occurs when a group of cells move together, with coordinated directionality and speed (Fig. 1). In most cases these cells form a cohesive structure or cluster and are able to communicate and coordinate their movement through inter-cellular adhesion structures 1 , the actomyosin network or through soluble factors [START_REF] Theveneau | Collective Chemotaxis Requires Contact-Dependent Cell Polarity[END_REF] . Clusters are polarised systems with an overall front and rear type organisation. Leader cells, located at the migration front, both impose the direction and pace of migration to the follower cells [START_REF] Reffay | Orientation and Polarity in Collectively Migrating Cell Structures: Statics and Dynamics[END_REF]4 and collect information regarding the surrounding ECM through integrin interaction. Leader cells generate most of the traction force. 5 Even though leader cells present some features attributed to a front-rear polarity such as lamellipodia 6 and express genes promoting migration [START_REF] Wicki | Tumor invasion in the absence of epithelial-mesenchymal transition: Podoplanin-mediated remodeling of the actin cytoskeleton[END_REF] , they also possess characteristics of an apico-basal polarity 6 .

Although collective migration was for a long time believed to be traction based, similarly to mesenchymal migration, it has recently been shown that clusters can adapt an amoeboid type behaviour [START_REF] Pagès | Cell clusters adopt a collective amoeboid mode of migration in confined non-adhesive environments[END_REF] .

Overall, collective migration allows cells to migrate in a more efficient manner than observed in single cells as they tend to experience less directional changes. It can play a pivotal role during development and can also be observed in pathologies such as cancer metastasis.

I.1.2. Single cell migration

Single cells can adopt two main states when migrating (Fig. 1). They are found to mostly use either a mesenchymal or an amoeboid mode of migration, which they can alternatively use depending on physical and chemical cues. In both theses modes, cells acquire a rear to front organisation I. 1.2.i. Amoeboid migration Amoeboid migration is akin to amoeba movements. Cells present a rounded morphology and undergo extensive cell body deformation due to either the actin cytoskeleton or hydrostatic pressure. Amoeboid migration can be either adhesion dependant or independent. However, in the former, cells develop weaker adhesions sites than those observed in mesenchymal cells [START_REF] Sroka | Phenotype modulation in nonadherent and adherent sublines of Walker carcinosarcoma cells: the role of cell-substratum contacts and microtubules in controlling cell shape, locomotion and cytoskeletal structure[END_REF] .

Cells undergo cycles of expansion and contraction in order to pass through openings in the ECM. Since actin contractions at the rear of the cell propel the cell forward it is considered to be propulsion based. Blebs are a type of cell protrusion that occurs due to changes in hydrostatic pressure across the plasma membrane [START_REF] Boulbitch | Shape instability of a biomembrane driven by a local softening of the underlying actin cortex[END_REF] and are often associated with amoeboid migration.

Blebbs are initially devoid of an actin cortex, yet over time, the actin cortex reforms and can lead to the retraction of the bleb. Amoeboid cells exert forces on the ECM during the formation of blebs or bleb-like structures, either by forming weak adhesions, in a process similar to that observed in mesenchymal cells or through none adhesive contact with the substrate. The latter will allow the cell to exert forces perpendicular to the substrate enabling the cell body to squeeze through extracellular matrix network [START_REF] Sroka | Phenotype modulation in nonadherent and adherent sublines of Walker carcinosarcoma cells: the role of cell-substratum contacts and microtubules in controlling cell shape, locomotion and cytoskeletal structure[END_REF]11 .

This mode of migration requires the cell to be embedded in an ECM or cellular structure. Thus it can only occur in 3D environments.

I.1.2.ii. Mesenchymal migration

Mesenchymal migration is an adhesion based mode of migration which requires the cell to dynamically interact with the ECM through adhesion structures, especially focal adhesions (FAs), in a cyclic manner. Cells form protrusions (Fig. 2) such as lamellipodia (sheet-like) or filopodia (finger-like), driven by actin polymerisation and stabilised by adhesions with the underlying ECM. The coupling of the actomyosin network with these adhesion sites enables cells to exert traction forces which are the driving force of mesenchymal migration. The cell body contraction, powered by the actomyosin cortex 12 , combined with adhesion disassembly at the rear then allows the translocation of the cell body and movement in the direction of migration.

We decided to focus our project around this type of single cell migration which will be delved into more thoroughly in the next part of this chapter. This mode of migration has been extensively studied in planar, 2D substrates. However, in vivo, mesenchymal migration, like most types of migration, typically occurs within the ECM, a 3D environment composed of a complex meshwork of fibres and other associated proteins.

I.2. Adhesion and mesenchymal migration

Cell adhesion is essential in many processes such as tissue homeostasis and cell survival. These structures anchor the cell to its surrounding ECM or to neighbouring cells and allow for communication to be established between those entities. They act as mechanical and biochemical signalling platforms and convey forces between the cell and its surroundings by

The adhesome

FAs are organised structures that can be divided into three spatial and functional layers 13 (Fig. 3). The first layer, closest to the plasma membrane, serves as a signalling layer and contains clusters of integrin, focal adhesion kinase (FAK) and paxillin, the two latter being located on the intracellular side of the cell. Integrins play a central part in FA structure and signalling and their role will be further developed in the following section. Paxillin plays a pivotal role in FA as it bridges the first and second layer by interacting with both FAK, Src and actin binding proteins such as vinculin and talin. When phosphorylated, paxillin can modulate cytoskeletal dynamics by inhibiting RhoA and promoting lamellipodium formation 14 . FAK is a non-receptor tyrosine kinase. When bound to phosphatidylinositol 4,5-bisphosphate (PIP2) membranes, it is capable of activating itself through autophosphorylation. This in turn triggers the recruitment of another kinase, Src, which further phosphorylates and fully activates FAK [START_REF] Tapial Martínez | FAK Structure and Regulation by Membrane Interactions and Force in Focal Adhesions[END_REF] .

The second layer acts as a force transducing layer and comprises talin and vinculin. Talin not only binds to multiple FA components such as integrins, vinculin, FAK and actin, but it also participates in integrin activation. Talin's affinity for integrins is heightened by PIP2 [START_REF] Martel | Conformation, Localization, and Integrin Binding of Talin Depend on Its Interaction with Phosphoinositides[END_REF] . On the other hand, vinculin plays a central role in force transduction as it directly binds actin, recruits cytoskeletal regulators and promotes actin polymerisation. It exists in an active or inactive, auto-inhibited form, respectively at adhesion sites and the plasma membrane. It requires binding to both actin and a FA protein (talin, α-actinin or α-catenin) to be activated 17 . Vinculin's activation enables the recruitment of other proteins to FAs as well as promoting integrin clustering and stabilising FAs.

The third layer, composed of zyxin, vasodilator-stimulated-phosphoprotein (VASP) an actin filament elongator, and α-actinin, regulates actin dynamics. α-actinin is an actin-binding protein that not only contributes to the bundling of actin filaments but also constitutes a multivalent platform through its interaction with both cytoskeletal and regulatory proteins [START_REF] Sjöblom | Alpha-actinin structure and regulation[END_REF] . Zyxin interacts through its LIM domain with many proteins including actin, VASP and α-actinin. It can also shuttle between the nucleus and the cytoplasm and was proposed to modulate gene transcription by binding with transcription factors (TFs) [START_REF] Wang | Zyxin and paxillin proteins: focal adhesion plaque LIM domain proteins go nuclear[END_REF] .

Integrin structure and activation

Integrins play a central part in FA structure and signalling. These heterodimers are transmembrane receptors that link the ECM or counter-receptors in neighbouring cells and the actin cytoskeleton [START_REF] Huttenlocher | Integrins in Cell Migration[END_REF] . They not only provide the cell with direct anchorage to its environment (ECM or adjacent cells), but also constitute a signalling platform.

Integrins are heterodimers of α and β subunits. In mammalian cells, 18α and 8β subunits are expressed and can form altogether 24 heterodimer combinations with ligand and signalling specificity [START_REF] Hynes | Integrins: Bidirectional, Allosteric Signaling Machines[END_REF] . Owing to this multitude of combinations, integrins are able to specifically recognise a large throng of ligands. For instance, they have been shown to bind ECM proteins such as fibronectin, tenascin and collagens [START_REF] Humphries | Integrin ligands at a glance[END_REF] and surface receptors like E-cadherins [START_REF] Humphries | Integrin ligands at a glance[END_REF] . Each subunit contains a short cytoplasmic tail, a single transmembrane domain and large extracellular domain which accounts for ligand specificity.

Many integrins are not constitutively active. They exist either in a low ligand affinity, close and generally inactive state or in a high ligand affinity, open and activated form [START_REF] Erb | Integrin αIIbβ3 Reconstituted into Lipid Bilayers Is Nonclustered in Its Activated State but Clusters after Fibrinogen Binding[END_REF] (Fig. 4). Integrin activation takes place when changes in interaction occur between the transmembrane and cytoplasmic tail of the α and β subunits. This leads to a conformational switch of the extracellular domain from a bent or closed state to an extended or open one (Fig. 4).

Integrin activation can come about in one of two ways; it either arises from intracellular or extracellular cues, otherwise known as inside-out or outside-in signalling respectively.

Although inside-out signalling has been extensively studied, outside-in signalling still remains quite elusive. Talin and kindlins, for instance, are key regulators of integrin activation via inside-out signalling. They interact with the integrin β tail resulting in integrin β tilting and the ensuing conformational change needed for integrin activation [START_REF] Calderwood | The phosphotyrosine binding-like domain of talin activates integrins[END_REF][START_REF] Montanez | Kindlin-2 controls bidirectional signaling of integrins[END_REF] . Extracellular ligand binding can also allow for integrin activation leading to outside-in signalling [START_REF] Du | Ligands "activate" integrin αIIbβ3 (platelet GPIIb-IIIa)[END_REF] .

Once integrins are activated and bind to an extracellular ligand, they can undergo clustering and form a lateral association of hetero-dimers [START_REF] Hynes | Integrins: Bidirectional, Allosteric Signaling Machines[END_REF] . Clustering is an essential step in FA formation.

It can either be initiated through multivalent ligand binding or through the formation of protein complexes bound to the cytoplasmic tails of integrins that will aggregate integrin heterodimers and stabilise them to form a hetero-oligomer. As these clusters grow, paxillin is recruited, further increasing the size of the adhesion to form nascent adhesions.

FA formation and maturation

Nascent adhesions constitute the first structural step to FA formation. These small (≈0, 25µm)

and short-lived adhesions, localised at the lamellipodium, either disassemble or end up maturing into focal complexes (≈0,5µm) and FA (1-5µm) successively. Their maintenance depends on actin polymerisation, thus they are more prevalent at the lamellipodium where actin polymerisation occurs. However they do not require myosin II activity to persist [START_REF] Choi | Actin and α-actinin orchestrate the assembly and maturation of nascent adhesions in a myosin II motor-independent manner[END_REF][START_REF] Alexandrova | Comparative Dynamics of Retrograde Actin Flow and Focal Adhesions: Formation of Nascent Adhesions Triggers Transition from Fast to Slow Flow[END_REF] . Nascent adhesions contain few proteins, namely talin, paxillin, α-actinin and kindlin2.

Focal complexes are the initial but transient maturation stage. Their molecular composition is very similar to that observed in nascent adhesions. However, they are bigger, more elongated than nascent adhesions and localise at the base of the lamellipodium. Force transmission is essential to adhesion maturation as transition from nascent to focal complexes necessitates both α-actinin and myosin II activity [START_REF] Choi | Actin and α-actinin orchestrate the assembly and maturation of nascent adhesions in a myosin II motor-independent manner[END_REF] .

As the lamellipodium moves forward, they continue to lengthen and widen to become FAs.

Their maturation is force dependant and these forces can either originate from actomyosin contractility or from external cues such as the properties of the ECM as will be discussed later on. In any case, these forces affect FA maturation either by altering the conformation and clustering of proteins or by affecting the molecular organisation of these adhesion sites.

Elongated fibrillar adhesions represent the final stage of maturation. They are generally located along the cell centre and have been shown to be involved in fibronectin (FN) fibrillogenesis [START_REF] Pankov | Integrin Dynamics and Matrix Assembly[END_REF] .

Their formation is tension dependant and they allow force transmission between the cell and the substrate but they lack some molecular components found in FAs such as paxillin and vinculin [START_REF] Chen | Immunoelectron microscopic studies of the sites of cellsubstratum and cell-cell contacts in cultured fibroblasts[END_REF][START_REF]Development of cell surface linkage complexes in cultured fibroblasts[END_REF] . This type of adhesion does not promote migration but enables ECM remodelling [START_REF] Pankov | Integrin Dynamics and Matrix Assembly[END_REF][START_REF] Zhong | Rho-mediated Contractility Exposes a Cryptic Site in Fibronectin and Induces Fibronectin Matrix Assembly[END_REF] .

FA turnover/disassembly

FA disassembly plays a crucial role in cell migration as it promotes cell retraction at the rear and the formation of protrusions at the front through adhesion turnover. Therefore a balance between adhesion formation, maturation and disassembly is required to promote cell motility.

Tensile force plays an important part in FA turnover. FAK is considered a key regulator of adhesion turnover as it can modulate actomyosin contractility. At the leading edge, FAK's association with p190RhoGAP and p190RhoGEF leads to the inactivation of RhoA stimulated contractility [START_REF] Tomar | A FAK-p120RasGAPp190RhoGAP complex regulates polarity in migrating cells[END_REF][START_REF] Lim | PyK2 and FAK connections to p190Rho guanine nucleotide exchange factor regulate RhoA activity, focal adhesion formation, and cell motility[END_REF] and subsequently a decrease in tension. As previously mentioned, adhesion maturation requires tension or else adhesions will be disassemble. At the cell rear, FA disassembly is driven by myosin II dependent stress fibre contraction which breaks weak links between integrins and the ECM [START_REF] Vicente-Manzanares | Regulation of protrusion, adhesion dynamics, and polarity by myosins IIA and IIB in migrating cells[END_REF] . FAK and Src will phosphorylate the myosin light chain kinase (MLCK) through the ERK/MAPK pathway, which will in turn activate myosin II contraction and increase contractility [START_REF] Webb | FAK-Src signalling through paxillin, ERK and MLCK regulates adhesion disassembly[END_REF] .

Microtubules (MTs) play pivotal role in FA disassembly, possibly by providing a lattice to bring "relaxing signals" to adhesion sites through motor proteins such as kinesin-1 [START_REF] Kaverina | Microtubule targeting of substrate contacts promotes their relaxation and dissociation[END_REF][START_REF] Krylyshkina | Modulation of substrate adhesion dynamics via microtubule targeting requires kinesin-1[END_REF] . MTs extend to FAs at the cell rear and dephosphorylate FAK which plays both a role in FA maturation and disassembly [START_REF] De Oliveira | SHP-2 regulates myogenesis by coupling to FAK signaling pathway[END_REF][START_REF] Nagano | ZF21 Protein Regulates Cell Adhesion and Motility[END_REF] . This triggers the recruitment of dynamin 41 , a GTPase, allowing for the internalisation of integrins through the clathrin-mediated endocytosis (CME) pathway [START_REF] Ezratty | Clathrin mediates integrin endocytosis for focal adhesion disassembly in migrating cells[END_REF] .

MTs can also contribute to FA disassembly through the delivery of calpain-2 [START_REF] Nagano | ZF21 protein, a regulator of the disassembly of focal adhesions and cancer metastasis, contains a novel noncanonical pleckstrin homology domain[END_REF] , an intracellular calcium-dependant protease. Calpain-2 has been shown to cleave multiple FA components such as, but not limited to, talin, paxillin, FAK or even α-actinin [START_REF] Franco | Calpain-mediated proteolysis of talin regulates adhesion dynamics[END_REF][START_REF] Chan | Regulation of adhesion dynamics by calpain-mediated proteolysis of focal adhesion kinase (FAK)[END_REF][START_REF] Bhatt | Regulation of focal complex composition and disassembly by the calcium-dependent protease calpain[END_REF] .

I.2.1.ii. Hemidesmosomes

Hemidesmosomes (HDs) are integral to epidermal homeostasis as they maintain skin integrity.

The were long believed to be static structures inhibiting cell migration but have been observe to be dynamic in migrating cells [START_REF] Geuijen | Dynamics of the α6β4 Integrin in Keratinocytes[END_REF] . They are multiproteic complexes that anchor the basal pole of epithelial cells to the underlying basement membrane. Similarly to FAs, they constitute a physical, integrin-mediated link between the ECM and the cytoskeleton, more specifically intermediate filaments (IFs) of keratin.

HDs are composed of basement membrane-connecting proteins such as α6β4 integrins, a transmembrane receptor for laminin [START_REF] Walko | Molecular architecture and function of the hemidesmosome[END_REF] , and IFs such as keratins and plectin-1a (plakin family).

Their formation is calcium dependant and requires the dephosphorylation of integrin β4 to allow for β4-plectin binding.

A crosstalk between FAs and HDs exists, due to their close physical proximity in migrating cells, and modulates cell migration [START_REF] Wang | Hemidesmosomes modulate force generation via focal adhesions[END_REF][START_REF] Pora | Hemidesmosomes and Focal Adhesions Treadmill as Separate but Linked Entities during Keratinocyte Migration[END_REF] . For instance, coupling of HDs and FAs can occur via plectin which affects actomyosin contraction as plectin inhibits RhoA-associated kinase (ROCK)-dependent myosin light chain (MLC) contraction [START_REF] Wang | Hemidesmosomes modulate force generation via focal adhesions[END_REF] . Integrin α6β4, through its binding with laminin, can also activate Rac1 which consequentially inhibits Rho activation and contractility, promoting lamellipodium formation [START_REF] Zahir | Autocrine laminin-5 ligates α6β4 integrin and activates RAC and NFκB to mediate anchorage-independent survival of mammary tumors[END_REF] .

I.2.1.iii. Clathrin coated structures/reticular adhesions

Clathrin coated structures

CME constitutes a key process in cell homeostasis by regulating nutrient internalisation, affecting membrane composition and surface receptor signalling. Canonically, receptors or cargoes are recognised at the plasma membrane by adaptor proteins, such as AP2. These adaptors trigger the recruitment and assembly of a clathrin coat along the cytosolic side of the plasma membrane. This leads to the invagination of the plasma membrane into a clathrin coated pit (CCP) which will eventually bud into the cytosol generating a cargo containing vesical. The content of the vesical will then either be recycled to the cell surface or undergo lysosomal degradation.

Classically, CME was involved in cell adhesion by mediating FA disassembly through the internalisation of integrins. For instance, adaptor proteins such as Disabled-2 (Dab2),

Autosomal recessive hypercholesteremia (ARH) and AP-2 were recruited to adhesion sites prior to FA disassembly [START_REF] Ezratty | Clathrin mediates integrin endocytosis for focal adhesion disassembly in migrating cells[END_REF][START_REF] Chao | Focal adhesion disassembly requires clathrin-dependent endocytosis of integrins[END_REF] . However, an unconventional CME was recently shown to form a new type of cellular adhesion. It has been observed that membrane bending and budding can be mechanically prevented in a process called frustrated endocytosis. In such a case, the CME machinery remains intact and well-functioning but mechanical constraints impede the evolution through canonical endocytosis steps. An example of frustrated endocytosis are a specific type of structure named tubular clathrin/AP2 lattices (TCALs). These TCALs have been discovered

to play a part in 3D migration. Similarly to FAs, TCALs anchor cells to collagen fibres, promoting the formation of protrusions in 3D matrices which allows the cell to migrate through the remodelling of the ECM [START_REF] Elkhatib | Tubular clathrin/AP-2 lattices pinch collagen fibers to support 3D cell migration[END_REF][START_REF] Bresteau | Clathrin-coated structures support 3D directed migration through local force transmission[END_REF] . Clathrin coated plaques constitute another type of frustrated endocytosis structure. They are large, flat and stable clathrin coated structures, enriched in αvβ5 integrins. They have also been acknowledged as adhesion sites as they specifically anchor the cell to the ECM [START_REF] Baschieri | Frustrated endocytosis controls contractility-independent mechanotransduction at clathrin-coated structures[END_REF][START_REF] Lock | Reticular adhesions are a distinct class of cell-matrix adhesions that mediate attachment during mitosis[END_REF] .

Reticular adhesions

Reticular adhesions are multiproteic complexes mediating cellular attachment to the ECM, specifically during mitosis [START_REF] Lock | Reticular adhesions are a distinct class of cell-matrix adhesions that mediate attachment during mitosis[END_REF] . Similarly to clathrin plaques, they are rich in αvβ5 integrins but lack many defining adhesome components, thus distinguishing them from FAs. Furthermore, unlike FAs, they do not require actin filaments and talin to form. It appears that reticular adhesions maintain the cells attached to the substrate during mitosis while also providing a track facilitating cell spreading following cell division.

I.2.1.iv. Podosomes or invadopodia

Podosomes and invadopodia are a specific type of actin-based protrusion involved in matrix degradation in coordination with cell migration [START_REF] Clark | Cortactin is an essential regulator of matrix metalloproteinase secretion and extracellular matrix degradation in invadopodia[END_REF] . Podosomes can be found in monocyte derivatives (osteoclasts [START_REF]Nano-topography sensing by osteoclasts -PMC[END_REF] , macrophages,…) and cancer cells unlike invadopodia which are exclusively observed in cancer cells. Invadopodia are particularly effective at matrix degradation as they tend to be more stable (hours) and penetrate deeper into the ECM compared to podosomes (minutes).

Podosomes and invadopodia possess very similar structures to one another, with an actin-rich core surrounded by actin-binding and signalling proteins. Interestingly, they both share molecular similarities with FAs. Actually, they often form in contact with the ECM after FA dissolution and tend to be located on the ventral side of cells. Their formation can be initiated by a multitude of factors such as growth factor stimulation (transforming growth factor-β (TGFβ) [START_REF] Rottiers | TGFbeta-induced endothelial podosomes mediate basement membrane collagen degradation in arterial vessels[END_REF] and epidermal growth factor (EGF)), integrin signalling (specifically through integrin β1 [START_REF] Destaing | β1A integrin is a master regulator of invadosome organization and function[END_REF] ) or even Src signalling pathways which activates cortactin [START_REF] Thomas | Cellular functions regulated by Src family kinases[END_REF] , an actin-binding protein.

Their maturation is actin dependant and further elongation requires microtubules as they deliver components for the tip and for matrix metalloproteinase (MMP) secretion [START_REF] Schoumacher | Actin, microtubules, and vimentin intermediate filaments cooperate for elongation of invadopodia[END_REF][START_REF] Wiesner | KIF5B and KIF3A/KIF3B kinesins drive MT1-MMP surface exposure, CD44 shedding, and extracellular matrix degradation in primary macrophages[END_REF] . Additionally, they possess proteolytic abilities mediated by cortactin and Tyrosine kinase substrate with four SH3 domains (TKS4) [START_REF] Buschman | The novel adaptor protein Tks4 (SH3PXD2B) is required for functional podosome formation[END_REF] . These two proteins are part of the Src signalling pathway.

I.2.2. Cell-cell adhesion structures

Cell-cell adhesions are crucial for the establishment and maintenance of cell and tissue polarity, specifically the apico-basal polarity, and integrity. Various types of intercellular junctions can be found, namely desmosomes, adherens junctions (AJs), tight junctions and gap junctions (Fig. 5). This section will concentrate on two types of intercellular junctions: desmosomes, adherens junctions.

I.2.2.i. Desmosomes

Desmosomes are primarily located in tissues that go through mechanical strain including cells of the myocardium, skin and bladder. They are a multiprotein complex comprising transmembrane proteins from the cadherin family, specifically desmoglein and desmocollin, and linker proteins from the armadillo family (plakoglobin, plakophillin and desmoplakin).

Desmoglein and desmocollin form heterodimers which mediate intercellular adhesion through the homophilic interaction of their extracellular domain. They also bind plakoglobin and plakophillin through their intracellular domain. In turn, plakoglobin and plakophillin both interact with desmoplakin which binds IFs of keratin, tethering the cytoskeleton to desmosomes.

Desmosome formation is generally believed to be incompatible with cell motility, especially when considering single cell migration. During the EMT process, desmosomes, among other types of intercellular adhesion structures, are disassembled. EMT encompasses the various stages an epithelial, immobile and polarised cell undergoes to become a mesenchymal, motile cell [START_REF] Chen | Epithelial-mesenchymal transition (EMT): A biological process in the development, stem cell differentiation, and tumorigenesis[END_REF] . However, some desmosomal proteins such as plakophilin 2 act as actin regulators through RhoA modulation [START_REF] Godsel | Plakophilin 2 Couples Actomyosin Remodeling to Desmosomal Plaque Assembly via RhoA[END_REF][START_REF] Koetsier | Plakophilin 2 affects cell migration by modulating focal adhesion dynamics and integrin protein expression[END_REF] which can affect cell migration. For instance, plakophilin 2 depleted cells showed an increase in stress fibre formation and an increase in size and stability of FAs, leading to a slowing down of migration speed [START_REF] Godsel | Plakophilin 2 Couples Actomyosin Remodeling to Desmosomal Plaque Assembly via RhoA[END_REF][START_REF] Koetsier | Plakophilin 2 affects cell migration by modulating focal adhesion dynamics and integrin protein expression[END_REF] . Furthermore, desmosomes could also hinder cell migration through the regulation of the hippo pathway which is known to control migration among other cellular processes (detailed later on), and therefore YAP translocation to the nucleus (activation) [START_REF] Uttagomol | Evidence for the Desmosomal Cadherin Desmoglein-3 in Regulating YAP and Phospho-YAP in Keratinocyte Responses to Mechanical Forces[END_REF][START_REF] Chen | The hippo pathway is activated and is a causal mechanism for adipogenesis in arrhythmogenic cardiomyopathy[END_REF] . Desmoglein-3 can sequester the transcriptional activator yes-associated protein (YAP) at the plasma membrane, preventing its activation and translocation to the nucleus [START_REF] Uttagomol | Evidence for the Desmosomal Cadherin Desmoglein-3 in Regulating YAP and Phospho-YAP in Keratinocyte Responses to Mechanical Forces[END_REF] .

Although desmosomes tend to inhibit single cell mesenchymal migration, they have been observed to promote invasion in collective cell migration [START_REF] Beaudry | Loss of the Desmosomal Component Perp Impairs Wound Healing In Vivo[END_REF] . Although loss of desmosomal connexions tend to increase single cell migration, Beaudry et al. [START_REF] Beaudry | Loss of the Desmosomal Component Perp Impairs Wound Healing In Vivo[END_REF] showed it decreased wound healing capabilities possibly by impeding the migration sheet to move efficiently.

I.2.2.ii. Adherens junctions

Similarly to desmosomes, adherens junctions (AJs) also display a cadherin core, yet they present a different subset of these proteins like P-cadherin, N-cadherin, VE-cadherin, and E-cadherin [START_REF] Niessen | Tissue organization by cadherin adhesion molecules: dynamic molecular and cellular mechanisms of morphogenetic regulation[END_REF] , the latter being the most common. These transmembrane proteins can perform both homophilic or heterophilic interactions through their extracellular domains. Through their cytoplasmic tail, they can bind p120 catenin and β-catenin. β-catenin recruits actin regulators, more precisely Rac activators and Rho inhibitors [START_REF] Lampugnani | Endothelial adherens junctions and the actin cytoskeleton: an 'infinity net'?[END_REF] , and associates with α-catenin which links the actin cytoskeleton to AJs. Actin dynamics are not only involved in the establishment of AJs but also their maintenance and maturation [START_REF] Baum | Dynamics of adherens junctions in epithelial establishment, maintenance, and remodeling[END_REF] . For instance, local activation of Rac has been shown to be required for the initial establishment of AJs [START_REF] Baum | Dynamics of adherens junctions in epithelial establishment, maintenance, and remodeling[END_REF] . Additionally, Rac and Cdc42 are both involved in the maturation of AJs through the activation of atypical PKC (αPKC).

AJs constitute another cell-cell adhesion structure dissociated when cells undergo EMT and is accompanied by a decrease in E-cadherin expression [START_REF] Gheldof | Cadherins and epithelial-to-mesenchymal transition[END_REF] . However, cancer cells can hijack this process, creating a hybrid epithelial/mesenchymal phenotype where cell-cell adhesions are maintained and necessary for the coordination of collective migration [START_REF] Klymenko | Cadherin composition and multicellular aggregate invasion in organotypic models of epithelial ovarian cancer intraperitoneal metastasis[END_REF] . N-cadherin, for example, has been shown to increase cancer cell migration and invasion in human breast cancer [START_REF] Hazan | Exogenous expression of N-cadherin in breast cancer cells induces cell migration, invasion, and metastasis[END_REF][START_REF] Nieman | N-cadherin promotes motility in human breast cancer cells regardless of their E-cadherin expression[END_REF] . N-cadherin has also been involved in the remodelling of the actin cytoskeleton.

During wound healing, asymmetric distribution of AJs and N-cadherin in cells close to the wound leads to the polarisation of those cells and the establishment of a posterior to anterior Cdc42 gradient favouring the formation of protrusions at the leading edge and therefore promoting the migration of leader cells [START_REF] Sabatini | Homotypic and Endothelial Cell Adhesions via N-Cadherin Determine Polarity and Regulate Migration of Vascular Smooth Muscle Cells[END_REF] .

I.3. Cytoskeleton dynamics and mesenchymal migration

The cytoskeleton is not only involved in cell migration but also in intracellular trafficking and cell morphology. It also constitutes the driving force of cell migration through its dynamic rearrangement. The cell's cytoskeleton comprises three different types of structure: the actin cytoskeleton, microtubules and intermediate filaments. All three actors contribute to cell migration on an individual level (Fig. 6). However, a crosstalk between the three is essential to coordinate the various steps in mesenchymal migration.

I.3.1. Actin cytoskeleton

The actin cytoskeleton constitutes the primary driving force for cell migration, not only through its connexion with adhesion structures like FAs but also through its ability to generate pushing, pulling and resistance forces.

The main component of the actin cytoskeleton is the actin filament, a polar polymer composed of actin subunits or globular actin (G-actin). Actin filaments are constantly remodelled by regulatory proteins which control nucleation, elongation and disassembly rates. These filaments are also arranged into specific networks and bundles, notably by actin-binding proteins, to adapt to specific tasks. Actin filament dynamics are tightly regulated and finely tuned in space and time to adapt the cytoskeletal architecture to the needs of the cell as will be developed through the lens of cell migration.

As previously mentioned, actin polymerisation is considered the main driving force for cell migration. Actin filaments are polarised with a dynamic, ATP-actin bound barbed end which elongates ten times faster than the more stable, ADP-actin bound pointed end, as will be developed later on. Importantly, actin filaments are consistently oriented with the faster polymerising barbed end oriented toward the plasma membrane thus providing a directional force enabling membrane deformation. Conversely, actin filament disassembly by severing and depolymerisation occurs closer to the pointed ends, enabling the release of monomers to replenish actin pools and providing actin for further polymerisation. Actin cytoskeletal dynamics must be tightly regulated by regulatory proteins to define and restrict the actin networks architecture within certain areas of the cell.

I.3.1.i. Actin filament formation and regulatory proteins

Actin monomers (G-actin) are the building blocks of actin filaments. In vitro, the initial and limiting step for actin filament formation, or nucleation, requires the sequential dimerisation and trimerisation of G-actin. Once trimerised, elongation can occur where G-actin, available from a cytoplasmic pool, can be added in an ATP-dependent manner to form double stranded, polarised filaments with a right-handed helical turn. Elongation always occurs at the barbed end, which binds ATP-actin. ATP is then quickly hydrolysed into ADP along the filament, leading to the release of an inorganic phosphate (Pi). Finally, ADP-actin is eventually dissociated from the pointed end. Association and dissociation of G-actin reaches a state of equilibrium within the filament resembling the movement observed on a treadmill. Thus actin filament turnover has been defined as treadmilling [START_REF] Wegner | Head to tail polymerization of actin[END_REF] .

In cells, spontaneous polymerisation and elongation are inhibited by proteins that bind and sequester G-actin, such as profilin [START_REF] Schlüter | Profilins as regulators of actin dynamics[END_REF] , and capping proteins. Therefore, cells use nucleation and elongation factors to overcome this inhibition.

Actin filament nucleation factors

Three major classes of nucleation factors contribute to the formation of actin filaments: the Arp2/3 complex, formins and tandem-monomer-binding nucleators. Arp2/3 is a multiprotein complex involved in the nucleation of new actin branches along pre-existing actin filaments.

This nucleator plays a fundamental role in the establishment of a branched network at the lamellipodium of migrating cells 81 . Arp2/3's activation depends on nucleation promoting factors (NPFs) like wiscott-Aldrich syndrome protein (WASP) family [START_REF] Suetsugu | Activation of nucleation promoting factors for directional actin filament elongation: allosteric regulation and multimerization on the membrane[END_REF] . Arp2/3 mediated actin polymerisation can be restricted by capping proteins to refine the organisation of the branched network [START_REF] Akin | Capping protein increases the rate of actin-based motility by promoting filament nucleation by the Arp2/3 complex[END_REF][START_REF] Reymann | Nucleation geometry governs ordered actin networks structures[END_REF] .

Formins can bind both actin nuclei and the barbed ends of actin filaments through their FH2 domains, thus promoting both actin nucleation and elongation [START_REF] Courtemanche | Mechanisms of formin-mediated actin assembly and dynamics[END_REF] . Once associated with actin, formins hinder the binding of capping proteins to barbed ends, thus preventing the inhibitory effect of capping proteins on actin elongation.

Lastly, tandem-monomer-binding proteins enable the formation of actin nucleation seeds through their tandem G-actin binding motifs, bringing together actin monomers [START_REF] Weston | Actin nucleators in the nucleus: an emerging theme[END_REF] . Examples of tandem-monomer-binding proteins include adenomatous polyposis coli (APC) and junctionmediating and regulatory protein (JMY), JMY being also shown to act as an Arp2/3 activator [START_REF] Zuchero | p53cofactor JMY is a Multifunctional Actin Nucleation Factor[END_REF] .

Actin elongation factors

As previously mentioned, formins possess an additional capacity for actin filament elongation.

However, another family of proteins, the enabled vasodilator-stimulated phosphoprotein (Ena/VASP) family, is a major contributor to actin filament elongation. Ena/VASP proteins enable the elongation of actin filaments by preventing barbed ends from being capped by capping proteins [START_REF] Pula | Role of Ena/VASP proteins in homeostasis and disease[END_REF] . This anti-capping ability can be increased with the conjoined action of profilin [START_REF] Pula | Role of Ena/VASP proteins in homeostasis and disease[END_REF] .

Profilin, an actin monomer binding protein, prevents spontaneous actin nucleation. However, in association with nucleating factors such as formins and Arp2/3, it can coordinate the creation of specific actin networks by polarising its elongation. For example, it heightens the asymmetric polymerisation of actin filaments by favouring actin monomer binding to the barbed end [START_REF] Pring | Profilin-actin complexes directly elongate actin filaments at the barbed end[END_REF] .

Actin filament disassociation

Actin disassembly can happen spontaneously and plays a crucial role as it replenishes the pool of available G-actin which affects polymerisation rates. Spontaneous depolymerisation rates are very low but can be enhanced with the help of depolymerising agents. In such a case, actin disassembly can occur in one of two ways: either the actin networks are dismantled through severing, generating fragments of filaments, or they are depolymerised in which case G-actin is released into the cytosol.

Actin filament depolymerisation and severing factors

Actin depolymerisation factor (ADF)/cofilin is essential for the fragmentation process in both branched and bundled actin networks but has been more extensively studied in the former. In branched networks, it binds to the filament along its pointed end side as it has a higher affinity for ADP-actin (especially when Pi is lost) and induces conformational changes which weaken the filament and triggers the severing and formation of actin fragments [START_REF] Hawkins | Human actin depolymerizing factor mediates a pH-sensitive destruction of actin filaments[END_REF][START_REF] Mcgough | Cofilin Changes the Twist of F-Actin: Implications for Actin Filament Dynamics and Cellular Function[END_REF][START_REF] Galkin | ADF/cofilin use an intrinsic mode of F-actin instability to disrupt actin filaments[END_REF] . These actin fragments have a higher disassembly rate than that observed in the rest of the actin network and therefore contribute to the renewal of the G-actin pool. Additionally, ADF/cofilin enhances the dissociation of G-actin from the pointed ends of actin filaments.

Gelsolin constitute another severing protein with an additional capacity to bind and cap the barbed ends of actin filaments [START_REF] Ono | Mechanism of depolymerization and severing of actin filaments and its significance in cytoskeletal dynamics[END_REF] . Its activation is calcium-dependent and its dissociation with actin filaments is mediated by PIP2. It is interesting to note that gelsolin possesses the ability to dimerise actin monomers thus enabling the nucleation of actin polymerisation.

Force-induced actin filament disassembly

Myosin II constitutes another contributor to actin disassembly, especially within actin bundles.

Myosin II contraction can bend actin fragments, generating conformational changes which induce fragmentation [START_REF] Vogel | Myosin motors fragment and compact membrane-bound actin filaments[END_REF][START_REF] Murrell | F-actin buckling coordinates contractility and severing in a biomimetic actomyosin cortex[END_REF] . Under high myosin II contraction (high density of myosin II), the bundles will disassemble. Furthermore, modulating myosin II activity has also been observed

to affect actin depolymerisation [START_REF] Vallotton | Simultaneous mapping of filamentous actin flow and turnover in migrating cells by quantitative fluorescent speckle microscopy[END_REF][START_REF] Wilson | Myosin II contributes to cell-scale actin network treadmilling via network disassembly[END_REF] . For example, increasing myosin II contractility using calyculin A, which inhibits the dephosphorylation of myosin II and therefore its inactivation, was shown to augment depolymerisation rates [START_REF] Vallotton | Simultaneous mapping of filamentous actin flow and turnover in migrating cells by quantitative fluorescent speckle microscopy[END_REF] .

I.3.1.ii. Actin network formation and organisation

Actin organisation can vary in geometry and size within the cell (Fig. 7). Each type of organisation serves a purpose and requires close coordination with the rest of the network to fulfil it. A key element in the establishment of specific actin filament architectures are actin crosslinking proteins. These proteins play little to no part in the polymerisation of actin filaments but rather connect already existing filaments to one another, creating complex structures. Crosslinkers can be categories depending on the distance by which they bridge actin filaments. For instance, fascin and fimbrin are considered small crosslinkers as they allow for the tight packing of actin filaments into bundles [START_REF] Courson | Actin Cross-link Assembly and Disassembly Mechanics for α-Actinin and Fascin[END_REF] . Conversely, crosslinkers like α-actinin and filamin impose bigger crosslinking distances enabling the formation of both actin filament

Branched actin

Branched actin serves many purposes such as generating force at the lamellipodium for cell migration or for pathogen propulsion. It also constitutes an important step in endocytosis and exocytosis.

As previously mentioned, Arp2/3 plays a fundamental role in the establishment of a branched network (Fig. 7) at the lamellipodium of migrating cells 81 . Arp2/3 mediated actin nucleation is not only modulated by nuclear promoting factors, but also the availability of G-actin and capping proteins. Together, these factors will refine the organisation of the network and determine its architecture and therefore its mechanical properties. For instance, the mesh size of the actin network will affect the structures rigidity [START_REF] Kawska | How actin network dynamics control the onset of actin-based motility[END_REF] . The denser the actin meshwork is, the more rigid the system becomes. If the network becomes dense and therefore stiff enough, it will generate sufficient force to form membrane protrusions [START_REF] Kawska | How actin network dynamics control the onset of actin-based motility[END_REF] . This phenomenon will be further developed in the following pages.

Parallel and anti-parallel actin bundles

Actin bundling plays a key role in the formation of filopodia, stress fibres and through its interaction with FAs. Actin fibres can be grouped either in a parallel or anti-parallel fashion in regards to their polarity and will serve different purposes accordingly (Fig. 7). For example, anti-parallel bundles are especially important in the development of stress fibres while parallel bundles are found in filopodia.

Two mechanisms that are nonexclusive allow for actin bundle formation. As previously mentioned, Arp2/3 mediated nucleation can generate long filaments if uncapped. These long filaments tend to bundle together through electrostatic interactions but can also be stabilised and maintained by crosslinkers. The second mechanism involved in parallel bundle formation is mediated by elongation factors, such as formins 100 , as previously mentioned. Anti-parallel bundles of actin filaments and myosin II can also form spontaneously 101 . They can generate forces through myosin II contraction. Contraction intensity is a function of the length of the fibre, the number of myosin II proteins associated with the filament and the presence of crosslinkers 101 . Myosin II contraction will either lead to the generation of force or to the disassembly of actin bundles as previously cited.

Crosslinking networks

Networks can also be crosslinked to one another. In other words, proteins can bind two preexisting filaments, bridging actin structures together to form complex organisations (Fig. 7).

Although certain crosslinkers, such as fascin and fimbrin, are specifically found in actin bundles, others, like α-actinin can be found in both types of branched and bundled actin structures [START_REF] Courson | Actin Cross-link Assembly and Disassembly Mechanics for α-Actinin and Fascin[END_REF] . This process can affect the mechanical properties of the actin organisatyion 102 .

I.3.1.iii. Actin cytoskeletal dynamics in the context of cell migration

Retrograde flow at the lamellipodium

As previously mentioned, the lamellipodium contains a branched actin organisation 103 initiated by GTPases Rac and Cdc42, which respectively activate WAVE 104 and N-WASP 105 which in turn trigger Arp2/3-mediated actin polymerisation. This branched network is in close proximity to the plasma membrane and polarised so that actin polymerisation occurs mainly towards the membrane. However, actin polymerisation alone is not sufficient to generate enough force to drive membrane deformation (Fig. 8). Indeed, membrane tension constitutes the main opposing force to membrane deformation 106 . As filaments polymerise, they do not generate enough force to bend the plasma membrane and are pushed towards the rear of the cell in a retrograde movement. Once the actin cytoskeleton is anchored to the substrate via FAs, the ensuing friction slows down the retrograde flow [START_REF] Alexandrova | Comparative Dynamics of Retrograde Actin Flow and Focal Adhesions: Formation of Nascent Adhesions Triggers Transition from Fast to Slow Flow[END_REF] , providing sufficient traction force for actin filaments to surpass membrane tension and push the membrane forward thus forming protrusions. it is important to note that myosin centrality also generates a retrograde flow of actin, especially in the lamella 107 .

Myosin II contraction of stress fibres

Stress fibres are also considered to participate in the generation of traction force through myosin II contraction 108 . As previously mentioned, they are a type of antiparallel actin bundle that are parallel to the direction of migration and found outside of the lamellipodium. Stress fibres constitute a link between the surrounding ECM via maturing FAs and can therefore convey force to and from the matrix.

Transverse arcs are another type of contractile fibre, parallel to the leading edge of the cell, at rear of lamella. They are not linked to the substrate but participate in actin disassembly at the rear of the lamella, contributing to the translocation of the cell body 109 .

Rear retraction and the actin cortex

The actin cortex corresponds to a thin layer of actin filaments and bundles that cover the inner face of the plasma membrane. They are myosin II dependent contractile fibres linked to the membrane through ERM (ezrin, radixin, moesin) proteins. They play a key role in defining the morphology of the cell but also in rear contraction during cell migration. Once FAs at the back of the cell disassemble, relieving the cell from its link to the ECM, the actin cortex contracts following RhoA activation thus allowing for the retraction of the cell's rear.

I.3.2. Microtubules

MTs play a multiple of roles in intracellular trafficking, as signalling hubs by retaining or delivering signalling molecules to certain areas and of course as a structural support for the cell given their size and robustness. They are crucial in cell division as they play a key role in chromosome segregation. As MTs play an important part in the establishment of cell polarity, they provide directionality to the cell during cell migration and additional membrane to the lamellipodium, facilitating protrusion formation 106 .

Microtubule dynamics

MTs can undergo cycles of polymerisation and depolymerisation. The process is similar to actin filament polymerisation as is it also ATP dependent. GTP bound tubulin is added to the plus end and is hydrolysed along the filament, with a Pi being released. This leads to conformational changes within the filament, affecting its stability and ultimately triggering depolymerisation.

Furthermore, the plus end is topped with a cap of GTP bound β-tubulin (GTP-cap) which is associated with stabilising proteins and brings stability to the filament 110 . In the presence of this GTP-cap, polymerisation continues but once gone, the tip becomes unstable the MT starts to depolymerise.

Microtubule organisation

MTs originate from two major sites: the centrosome and the Golgi apparatus. Centrosomes are the primary microtubule organising centre (MTOC), the main organelle where MT nucleation occurs in mammalian cells. It is most often located in front of the nucleus, closer to the cell's leading edge and is important in the establishment of the nucleus/centrosome axis. This axis is parallel to the front to rear organisation of migrating cells. The more stable, minus tips are close to the centrosome while the plus ends radiate throughout the cell, toward the edges 110 . This uniform orientation of MTs optimises transportation of cargoes from one end to the other. It is critical to note that, because of the positioning of the centrosome in front of the nucleus, the MT network is asymmetric with bias toward the leading edge (Fig. 9).

The Golgi apparatus plays an important role in non centrosomal MT polymerisation and organisation 111 . In migrating cells it contributes to the asymmetric distribution of MTs as it also orients them toward the leading edge 112 .

I.3.2.ii. Microtubules' contribution to cell migration

Affecting MT dynamics has been shown to impact cell migration. For instance, in fibroblasts, inhibiting MT polymerisation with nocodazole decreases cell migration speed 113 . Similarly, when fibroblast-like cells were treated with colcemid, a drug inducing MT depolymerisation, migration speed was found to be hindered and migration speed reduced 114,115 .

Kinesin mediated transport to the leading edge

MTs are known to serve as tracks for intracellular transport of cargoes. This active process is rendered possible thanks to kinesins and dyneins, two families of motor proteins. A vast majority of kinesins transport cargoes to the plus end of the MT and thus, through selective binding, can deliver specific cargoes to the leading edge to promote cell migration.

Amongst the components transported to the lamellipodium by kinesins are lipids and vesicles which are destined to be exocytosed 116,117 . As membrane tension is a limiting factor for protrusion formation, bringing extra membrane material allows for the tension to be reduced, thus promoting cell migration. Kinesins also bring mRNAs to the cell front contributing to regulating the translation of proteins involved in actin dynamics. For example, studies have shown that mRNAs encoding actin subunits or actin regulators such as Arp2/3 118 , profilin 119 and β-actin 120 to be conveyed through MTs to the lamellipodium. These motor proteins have also been involved in the recycling of integrins.

As previously mentioned, MTs are physically linked to FA and play an important role in their turnover. Kinesins such as KIF15 further contribute to the process by recruiting clathrin adaptor Dab2 which triggers the internalisation of integrins through CME and ultimately their recycling to the front of the cell 121 . In certain cases, cells have been observed to use their MTs to localise mitochondria, the cells energy powerhouse, to the leading edge 122 . Since actin polymerisation, among other reactions, is ATP dependant, it possibly facilitates the occurrence and rate of certain migration promoting processes.

Dynein-dependent nuclear positioning

Dyneins form multiproteic complexes that enable the delivery of generally large cargo toward the minus tips of MTs 123 . Dynein cargoes include, but are not limited to organelles (e.g. vesicles originating from the endoplasmic reticulum and the Golgi apparatus) or protein complexes.

Dyneins play an important part in nuclear positioning during cellular processes such as mitosis and cell migration 123 . For example, dyneins can generate pulling forces on MTs while anchored at the actin cortex thus pulling on the nucleus 123 .

Force generation at the leading edge

Even though the actin cytoskeleton acts as the main force-generating structure in cell migration, MTs have been shown to contribute, at least in part, to the process. MTs can store energy 124 .

They can also in simplified systems generate force 125 and deform the membrane as observed in lipid vesicles 126 . In neurons, MTs are more directly involved in force driven protrusion formation. For example, mechanisms of MT gliding against one another via kinesin-1 127 have been shown to initiate neurite growth. MTs also involved in pseudopod persistence and therefore contribute to cancer cell invasion, especially plus-end tracking protein (+TIP) SLAIN2 which stabilises MT by inhibiting depolymerisation 128 .

Crosstalk with the actin cytoskeleton

Some MT regulators also possess the ability to bind actin, including but not limited to

I.3.3.ii. Type V IFs : lamins

Lamins are one of the main components of the nuclear envelope, which is a physical barrier separating the nucleoplasm from the cytoplasm (Fig. 10). The nuclear envelop is composed of a double membrane with an inner (INM) and outer (ONM) membrane. Under the INM is the lamina which is a meshwork of both lamins and lamin associated proteins which bridge the INM and chromatin. Lamins can also be found in the nucleoplasm, interacting with chromatin 135,136 .

Structure, assembly and disassembly

There are two subtypes of lamins in mammalian cells: A-type lamins which encompass lamin Cyclin dependent kinase 1 (Cdk1 or cdc2) regulates disassembly of lamin A/C and B form lamina. During mitosis, Cdk1 phosphorylates a conserved site on the N-terminal head of lamins which plays an important role in the association of dimers thus dissociating tetramers 138 .

A
Furthermore, in Drosophila, phosphorylation of lamin C on the Cdk1 site also prevents its interaction with chromatin 139 . Once phosphorylation is removed, lamins can associate and recreate the lamina.

I.3.3.iii. Other IFs and cell migration

IFs reorganise during cell migration. They elongate in an APC dependant manner alongside MTs toward the lamella 140 , reaching and associating with mature FAs in a Plectin1 dependent manner 141,142 , but do not extend to the lamellipodium. Indeed, vimentin IFs in the leading edge were shown to inhibit the formation of the lamellipodium. However, phosphorylated vimentin in the form of small, unit length filaments in the lamellipodium is important for actin stability as vimentin-/-cells were shown to be unable to migrate 143 .

Furthermore, certain modulators of actin and MT dynamics involved in cell migration have been observe to also regulate IF dynamics. For instance, Rac1 and Cdc42 were implicated in the recession of vimentin IF from the lamellipodium 144 .

Some IFs (keratin and plectin 145 ) are involved in cell-cell contacts such as hemidesmosomes, as was previously mentioned, which is known to affect both collective and single cell migration.

II. Mechanotransduction: transforming mechanical cues into biochemical signals

Mechanotransduction corresponds to the conversion of physical and mechanical cues into biological and biochemical signals. In other words, cells are able to sense mechanical stimuli in their environment and respond by undergoing various phenotypic changes.

Mechanotransduction has been shown to regulate many cellular processes such as cell fate, gene transcription, survival, proliferation and migration. In the following section will be discussed the ECM and its contribution to mechanical stimulation, the various mechanosensitive structures in the cells and their signalling pathways.

II.1. Mechanical properties of the ECM

factors and cytokines) found in intercellular spaces of connective tissue, synthesised and remodelled by cells. It plays a fundamental role in the shaping and functioning of tissues and organs. Indeed, the ECM not only provides a structural support for cells but serves an instructional role by supplying biochemical and mechanical cues which form the basis of mechanotransduction. It is involved in many processes such as cell migration, differentiation and survival.

Cells must sense and regulate ECM mechanics to promote homeostasis. When considering cellmatrix interactions three key ECM parameters come to mind: molecular composition, structural organisation and mechanical properties. In the context of this project, our main focus will be on the mechanical properties of the ECM

II.1.1. Defining the mechanical properties of the ECM

The mechanical properties of the ECM can be quantified using mechanical quantities such as stress, strain and rigidity. Stress corresponds to the internal forces that particles exert on each other and is a direct result of forces like tension or compression: the greater the force exerted over a small area, the higher the stress. Strain represents the deformation of a material, and the material's resistance to deformation is measured by its stiffness. A linear relationship between stiffness and stress exists so that control of ECM stress is equivalent to control of ECM stiffness.

In other words, increasing matrix or substrate stiffness corresponds to an increase in stress. The ECM being an elastic materiel, the unit of measure used to quantify its elasticity, or its ability to resist a distorting influence, is Young's modulus in Pascal (Pa). The higher the value of Young's modulus is, the more force needs to be applied to the matrix to deform it and thus the stiffer it is.

The matrices mechanical properties depend mainly on its composition. Elastic fibres, such as elastin and fibrillin, provide for the extensibility and resilience of the tissue 146 while collagen fibres, of which type I and III are the most widespread, impart the stiffness (resistance to deformation) and strength of the structure. ECM composition varies during development and depending on the tissue. For instance, bones, mainly composed of collagen type I (<95% dry weight) are the stiffest organ (1,3-7,8GPa) and brains are the softest (0,1-1kPa) 147 .

II.1.2. ECM stiffness and its influence on cells

Studies have shown that cells can be more or less sensitive to mechanical cues depending on the cell type. However, on stiffer substrates, cells generally tend to spread more, form more mature FAs and stress fibres, and exert higher traction forces through an increase in myosin II dependent contractility 148,149 . It is widely accepted that cell migration presents a bell-curve response to force and rigidity. In other words, cell migration increases with substrate stiffness until reaching a threshold of preferred, cell type specific, rigidity, over which migration speed decreases 150,151 . Interestingly, it was recently proposed that talin could shift this biphasic force/rigidity response and displace the threshold to higher values 152 . Additionally, matrix stiffness has been seen to guide cell migration with a general bias towards stiffer substrates termed as durotaxis 153 , although directed migration towards softer environments has also been witnessed 150 . ECM rigidity has been implicated in many cellular processes such as cell cycle regulation 154 , cell differentiation 155 and cell fate. In the following section we will explore the effect of force and rigidity on the different mechanosensitive structures of the cell

II.2. Mechanosensitive structures

Mechanotransduction relies on the cells ability to both sense and respond to physical stimulation. In order to do so, the cell possesses multiple mechanosensitive structures involved in force transmission and mechanical signalling. Here will be reviewing the main structures implicated in mechanotransduction.

II.2.1. Focal adhesions and integrins

FAs are signalling platforms that connect the ECM to the cytoskeleton. FAs are mechanosensitive structures whose assembly, maturation and turnover is heavily dependent on mechanical force. Although mechanical activation of integrin signalling is a key component to mechanotransduction, this segment will focus mainly on the mechanical regulation of FA dynamics. Integrin signalling will be discussed later on.

FA proteins: a force-driven regulation

Integrin's binding affinity to its ligand has been shown to vary upon application of an external mechanical force. For instance, αvβ3 binding to fibronectin requires a conformational change which can be triggered by mechanical force thus increasing the integrin's affinity for the ECM protein 156 . Internal tensile force generated by actomyosin contractility can also affect bond strength as observed between integrin α5β1 and fibronectin 157 . Additionally, force application was shown to promote integrin clustering and downstream signalling by slowing the integrin diffusion within the plasma membrane.

As mentioned previously, talin plays a crucial role in force transduction 158 (Fig. 11). It not only establishes a direct mechanical link between integrins and actin through its FERM domain 159 , but it also recruits vinculin as its vinculin binding domain (VBD) unfolds, becoming accessible under tensile stress 160 . Vinculin itself has also been identified as a force-bearing molecule.

Indeed, FRET sensors enabled the measurement of tensile force borne by the protein and found that mechanical tension across vinculin was elevated at the leading edge but decreased in disassembling or sliding FAs 161 . It was proposed that vinculin could contribute to the stabilisation of FAs and enable their maturation.

II.2.2. The actomyosin cytoskeleton

The actin cytoskeleton is pivotal in mechanotransduction as its regulation is sensitive to mechanical cues. Furthermore, it not only provides a means to transduce force to the nucleus via the LINC complex, but it also contributes to force generation through myosin II contractility, further increasing and maintaining the force-related activation of certain factors.

II.2.2.i. Mechanosensitive regulators of the actin cytoskeleton

Many actin binding proteins (ABPs), involved in either actin dynamics or structural organisation of actin filaments, were found to be modulated by mechanical stress. Some ABPs see their affinity for actin increased under stress while others require mechanical activation to bind to filaments.

ABPs force mediated increase in affinity

Among the ABPs whose affinity increases with mechanical tension are formins and Arp2/3.

Formins are involved in actin elongation and were found to play a dual role in response to tension. For instance, upon stretching and in the absence of profilin, formin mDia-mediated elongation of actin filaments was shown to be accelerated 162 . However, tension decreases actin filament elongation by formin Bni1p in the absence of profilin, while it is accelerated when Bni1p and profilin are assocated 163 . It seems that the response to mechanical cues could vary from one formin family to the other.

As was previously mentioned, Arp2/3 is a branched actin nucleator responsible for the formation of the lamellipodium. ATP-bound Arp2/3 is found in recently polymerised branches and closest to the plasma membrane, contrary to ADP-bound Arp2/3 which is found in older branches, closer to the rear of the lamella. It was observed that ATP-bound Arp2/3 is twenty times less sensitive to force than ADP-bound Arp2/3 164 . It was speculated that this would promote the formation of protrusions by branches closest to the plasma membrane while those farthest from it would disassemble, replenishing the G-actin pool.

Force-activated ABPs

Among the force-activated, actin binding proteins are the LIM-domain (LIN-11, Isl-1 and MEC-3) proteins FHL, paxillin and zyxin as their binding to actin in the absence of force has not yet been detected 165 . For instance, both zyxin and paxillin have been involved in stress fibre repair under strain 166 . Furthermore, LIM-protein recruitment has be shown to be mediated by myosin II contraction 167 . As many LIM-proteins can also be recruited to the nucleus and act as co-activators of gene transcription 168 , these proteins could contribute to mechanotransduction as relay factors between the cytoskeleton and the nucleus. For example, four and a half LIM domain 2 (FHL2) was found to be retained to tensed actin filaments while in the absence of tension, it shuttled to the nucleus to regulate gene transcription 165,169 .

II.2.2.ii. Force transmission

Actin filament structure under tension

Actin structure and assembly has been found to be affect by force. Indeed, branched networks present increased filament density and tighter filament packing under force which in turn increases the power and efficiency of protrusion 170 . Furthermore, it has been shown that mechanical stress leads to conformational changes within actin filaments. Under 200 picoNewton (pN) tensile force, actin filaments present an increase in filament length and decrease in twist angle by 20°1 [START_REF] Niessen | Tissue organization by cadherin adhesion molecules: dynamic molecular and cellular mechanisms of morphogenetic regulation[END_REF] . As ABP binding to actin filament can impact filament torsion, as observed with cofilin 172 , it is speculated that tension-mediated torsion could in turn modulate ABP binding.

Perinuclear actin cap

The perinuclear actin cap plays an important role in mechanotransduction as it interacts with the LINC complex and contributes to the deformation of the nucleus under stress. It is situated on the dorsal side of the nucleus and contains contractile fibres that connect the nuclear envelope to focal adhesions 173 . It allows for the cell to shape its nucleus depending on cell morphology. Disruption of the perinuclear actin cap or the LINC complex was shown to impede nuclear deformation 173 . Regulation of this actin cap seems to be mediated by STEF, a Rac1

selective GEF, which colocalises with both nesprin-2 and myosin IIB 174 .

II.2.3. The nucleus

Over the years, many studies have shown the relevance of nuclear mechanotransduction. The organelle alone is sensitive to mechanical loading 175 and can respond to external forces exerted, for example, by the cytoskeleton. These cues trigger changes in nuclear stiffness,morphology and chromatin organisation. For instance, an increase in tensile stress exerted on the nucleus or cell can trigger a stiffening of the nucleus 175 .

II.2.3.i. LINC complex

The LINC complex constitutes the main means of force transmission to the nucleus and regulates many processes such as gene transcription, cytoskeletal organisation, nuclear positioning and morphology. However, it is important to note that force transmission can occur independently of the LINC complex as actin polymerisation around the nucleus area alone was observed to deform the organelle 176 .

LINC complex structure

The LINC complex is embedded in the nuclear envelope and constitutes the main connection between the cytoskeleton and the nucleoskeleton (Fig. 12). This complex contains two types of transmembrane proteins: Klarsicht, ANC-1, Syne homology (KASH) domain proteins and Sad1

and UNC-84 (SUN) domain proteins. KASH domain proteins, also called nesprins, are embedded in the outer-nuclear membrane (ONE) and interact with the cytoskeleton on their cytoplasmic side and with SUN proteins within the perinuclear space 177 (Fig. 12). Many nesprin isoforms can be found, due to alternative splicing. The largest isoforms, giant nesprins (nesprins 1 and 2) mostly bind to actin while nesprin-3 and 4 associate with IFs and MTs respectively 178,179 . SUN proteins pass through the inner-nuclear membrane and interact with the nuclear lamina (lamins and lamin interacting proteins). Multiple SUN isoforms can be found, the most widely expressed being SUN1 and SUN2.

Sensing mechanical force

The LINC complex is known to establish a direct link with the cytoskeleton through nesprins which were shown to be subjected to mechanical loading. Nesprin-2 is able to sense tensile forces from myosin II contraction and cell elongation 180 . Even on isolated nuclei, mechanical loading through nesprin-1 alone is sufficient to trigger a mechanical response and a subsequent increase in nuclear stiffness.

LINC complex: mechanosensitive proteins

LINC complex proteins are mechanosensitive as their expression was shown to vary depending on tensile strain. Nesprin-2 and lamin A levels decreased under low levels of shear stress which was shown to affect the activation of certain transcription factors (TFIID and Stat-1/3/5/6) and to increase cell proliferation and aptotosis 181 . Nesprin-3 expression and localisation was also observed to be sensitive to fluid mechanical shear stress and its disruption affected cell morphology and perinuclear cytoskeleton organisation 182 .

Role in cell migration

The LINC complex plays an important role in cytoskeletal reorganisation, nucleus positioning and cell migration in response to mechanical stimulation. Disrupting the complex was shown to affect cell polarisation during wound healing by impeding nuclear position and thus the formation of the nucleus/centrosome axis 183,184 . Nesprin1/2 and SUN1/2, notably, were identified as creating a physical link between the nucleus and the centrosome during neuronal migration 145 . Nesprin2 was also involved in the establishment of perinuclear cytoskeletal organisation and its depletion affected migration speed of mouse embryonic fibroblasts (MEFs)

and neuronal cells 183 . The LINC complex was also shown to impact adhesion traction force.

Cells depleted for nesprin-1 and under cyclic strain showed an increase in both FA number and cell traction, which ultimately led to a decrease in cell migration 185 .

Role in gene expression

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The LINC complex was shown to participate in the regulation of mechanosensitive genes on a genome-wide scale, following mechanical stimulation. Interestingly, disruption of SUN1L affected different mRNA levels depending on the substrate rigidity cells were cultured on. For instance, in cells cultured on a soft substrate, mRNAs targeting FAs or cytoskeletal proteins and lamin B1 were altered 186 .

LINC complex and lamina: working in tandem

A crosstalk between the LINC complex and the lamina, comprising lamins and lamin associated proteins, exists as both are linked through SUN proteins. A study on isolated nuclei demonstrated that, upon tensile strain applied on nesprin-1, interaction with lamin A/C was reinforced through the phosphorylation of emerin, a lamina associated protein 187 .

II.2.3.ii. Lamina and lamins

As previously mentioned, the lamina constitutes a meshwork of lamin and lamin-associated proteins, situated under the INM in the nucleoplasm (Fig. 12).

The lamina: an elastic meshwork for shock absorption

This meshwork possesses elastic mechanical properties and is believe to serve a purpose in shock absorption 188 within the nucleus, participating in the protection of genomic material.

Lamina composition affects those mechanical properties. For example, lamin A deficient MEFs presented a less stiff, more deformable nucleus and displayed less resistance to mechanical loading 189 . Nuclear stiffness seems to depend on A-type lamins as lamin A and C levels but not B levels impact nuclear deformability 190 . However, it is important to mention that chromatin also participates in nuclear mechanics. While lamin A/C seems to govern nuclear deformation under important nuclear strain over 30% stretch, chromatin seems to direct responses to smaller strains 191,192 .

The lamina: mechanosensitive protein expression and activation

Lamin A/C levels were found to positively correlate with ECM stiffness and tensile strain, promoting either adipogenic or osteogenic differentiation 193 . Futhermore, laminA/C turnover seems to be modulated by matrix stiffness. On soft substrates, cells were shown to present an increase in laminA/C phosphorylation which in turn increased laminA/C turnover, favouring a disassembly of the lamina 194 . Emerin activation is also modulated by mechanical strain. Emerin was found to be phosphorylated under increased mechanical loading, with emerin phospho-mutants cells presenting abnormal cytoskeletal organisation and no lamin A/C recruitment to the LINC complex 175 .

Role in nuclear stiffening and cytoskeletal organisation

Seeing as the LINC complex and the lamina's integrity are tightly associated, it seems fitting that mechanical regulation of lamina associated protein would also play a critical role in nuclear integrity, stiffening and nucleo-cytoskeletal coupling. Indeed, lamin A+/-MEFs exhibited a decrease in nuclear stability and resistance to strain as well as a perturbation in nucleocytoskeletal uncoupling 195,196 , similarly to what was observed upon LINC complex disruption.

II.3. Mechanosignalling : transducing a mechanical force into a biochemical signal II.3.1. Integrin signalling

The many signalling pathways regulated by integrin activation are intertwined, providing a multi-level regulation of anchorage-dependent cell survival, proliferation and migration.

Crucial elements of these mechano-activated signalling pathways will be discussed in the following section.

II.3.1.i. Integrin signalling pathways

A key mediator of force-dependent integrin signalling is receptor-type tyrosine-protein phosphatase alpha (RPTP-α) 197 . Upon mechanical activation, RPTP-α activates Src family kinases (SFKs) through dephosphorylation, which in turn phosphorylate and activate Rho GEFs such as Tiam1. This leads to the activation of Rho GTPases Rac1 and Cdc42 which regulate formin and Arp2/3 activation thus enabling the formation of the lamellipodium 198,199 (Fig. 13).

Another crucial signalling element in integrin signalling is FAK as it also acts as a mechanosensor 200 (Fig. 13). Indeed, FAK activation increases with cellular strain and substrate rigidity 201,202 . Upon integrin activation, it undergoes phosphorylation, lifting its auto-inhibited state, and is recruited to the plasma membrane. Activated FAK, on its own or in conjuncture with SFKs, can then phosphorylate targets, activating and recruiting proteins such as paxillin, 130Cas and Rho signalling through GEFs and GAPs. Increasing p130Cas levels have been correlated with regions of higher traction forces in spreading cells 203 . p130Cas in turn promotes c-Jun NH2-terminal kinase (JNK) pathway signalling, triggering the activation of transcription factors such as the activator protein-1 (AP-1) and FOXO. These transcription factors have been involved in the expression of Bim, a pro-apoptotic factor 204,205 . FAK/SFK also participates in 53 the activation of YAP-TAZ and subsequent inhibition of the Hippo pathway as will be developed further on 206 . Additionally, FAK/SFK can phosphorylate other substrates affecting many pathways such as Ras/ERK and PI3K/AKT, which regulate survival, cell proliferation and migration. For example, increasing integrin clustering by increasing substrate rigidity leads to an augmentation of ERK signalling promoting Rho-generated contractility 207 . PI3K/AKT signalling was also shown to be enhanced with matrix stiffness at focal adhesions 208,209 .

II.3.1.ii. Adhesion dependant cell survival: anoikis

Anoikis is a type of apoptosis mostly triggered by cell detachment, loss of integrin signalling and cell shape. As ECM rigidity can affect all of these parameters, it is not surprising to observe that substrate stiffness also affects anoikis. Cell shape was shown to govern whether cells underwent apoptosis or not, possibly as a decrease in cell spreading lead to a decrease in the cell adhesion surface 210 . ECM rigidity has been shown to affect anoikis through death associated protein kinase 1 (DAPK1) 211 . Activation of DAPK1 is dependent on Src and leads

to its localisation at FAs on stiff substrates where it phosphorylates tropomyosin1.1 and talin, reinforcing integrin signalling activation. On soft matrices, DAPK1 dissociates from FA thus activating anoikis, but upon its depletion.

Anoikis mechanisms

Adhesion-dependent cell death is proposed to use one of two routes: the intrinsic (mitochondria)

and the extrinsic (death receptor) pathways (Fig. 14). In the intrinsic pathway, loss of both integrin β1 engagement and ERK signalling activated Bim 212 and Bad which, activates proapoptotic factor Bax and contributes to the permeabilisation of the mitochondria. Bax activation leads to a conformational change and exposure of its BH3 epitope as well as its translocation

to the mitochondria where it will then activate the caspase cascade leading to apoptosis.

Inhibition of ERK signalling and downregulation of EGFR was shown to inhibit anoikis upon cell detachment 212 . The second extrinsic pathway involves the Fas tumor necrosis factor receptor family. Activation of Fas through FADD binding requires caspase-8 activation and the subsequent activation of the caspase cascade. However, caspase-8 can also mediate the activation of Bax/Bak through Bid, linking both the intrinsic and extrinsic pathways 213 .

Eluding anoikis

Anoikis resistance has been observed in pathologies such as cancer metastasis, where cells must elude apoptosis when travelling through blood and lymphatic vessels to colonise distant organs. to confer anoikis resistance and to increase metastatic abilities of lung and breast cancer cells, possibly through desmosomal junctions 219 . Further studies identified α5β1, a fibronectin receptor, promoted upregulation of pro-survival factor Bcl-2 through Akt signalling 220,221 .

Rigidity sensing seems to be another regulator of anoikis and can be hijacked by cells to elude cell death and promote cell growth in soft environments. It was shown that affecting rigidity sensing by depleting contractility, through topomyosin, myosin IIA or α-actinin, decreased cell death on soft substrates [222][223][224] .

II.3.2. YAP/TAZ

The Hippo pathway's main role is to negatively regulate the activation of YAP/TAZ, two redundant, transcriptional activators (Fig. 15). YAP/TAZ is believed to play a central role in mechanotransduction as it is mechanically regulated and triggers the activation or repression of target genes involved in cell proliferation, survival, and cell fate. Although FAK/Src signalling can modulate YAP/TAZ activation, as mentioned above, many other pathways have been implicated in its regulation.

the cytochrome C and Smac/Diablo from the mitochondria enable the activation, in turn, of Caspase-9 and -3 thus leading to apoptosis. Taken from Q. Zia et al ( 2021).

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II.3.2.i. The Hippo pathway

When inactivated, YAP/TAZ is sequestered in the cytoplasm by 14-3-3, a regulatory protein, promoting YAP/TAZ nuclear export or degradation through the proteasome. This prevents YAP/TAZ from associating with TEAD DNA binding proteins and modulating gene transcription (Fig. 15). YAP-TAZ inactivating phosphorylation is mediated by LATS1/2 which is itself activated by the Ste20 family kinases, including but not limited to mAPK4 and MST1/2.

II.3.2.ii. Mechanical regulation of YAP/TAZ

ECM rigidity plays an important role in YAP/TAZ activation (Fig. 16). On stiff substrates, YAP/TAZ is predominantly nuclear where it enables the activation of mechanically regulated genes, whereas it is mostly cytoplasmic when cells are culture on a soft matrix 225 . Cell shape, particularly cell spreading, alone is sufficient to regulate the activation of YAP/TAZ as reducing cell area using micropatterns led to a cytoplasmic location of the activators 225 . Cytoskeletal tension was identified as an important factor in mechanical activation of YAP/TAZ. Indeed, Rho activation and stress fibre formation were shown to be key elements in nuclear translocation of YAP/TAZ 225 . The Hippo pathway has also been associated with junctional mechanotransduction. There is an inverse correlation between cell tension and cell density, therefore at higher cell densities, the Hippo pathway mediates contact inhibition by inactivating YAP/TAZ, decreasing cell proliferation 226 . 58

II.3.3. Mechanically activated ion channels

Ion channels enable an energetically favourable diffusion of ions depending on their charge.

Mechanically activated ion channels have been proposed to convert mechanical stimuli into electrochemical signals. However, it is important to note that this type of ion channel must meet certain requirements to qualify as mechanical transducers. More specifically, the candidate must be expressed in a mechanosensitive cell and loss of the channel should impede mechanotransduction without affecting cell development 227 . Only a few mechanosensitive ion channels have been identified and will be discussed in this section with a focus on PIEZOs.

Mechanosensitive ion channels include, but are not limited to, MscL (mechanosensitive channel large conductance), MscS (small conductance), transient receptor potential channels (TRP) and the PIEZO family. The first identified channel was MscL, a nonselective ion channel whose diameter widens upon membrane tension, thus opening the channel 228 . It was observed to relieve turgor pressure following a hypo-osmotic shock by enabling the release of ions. Another example is the PIEZO family which is involved in many developmental processes, such as angiogenesis 229 and stem cell differentiation 230 but also in and proliferation 236 . Furthermore, osteoblast adaptation to shear stress was found to be dependent on PIEZO 1, which led to the expression of the RUNX2 transcription factor 237 .

III. Gene regulation via epigenetic modifications

Epigenetics encompass the (heritable) changes that can give rise to specific phenotypes without affecting the DNA sequence. Modifications can occur, in response to both external and internal stimuli, to modulate the expression of certain genes in a time and area specific manner. It has been shown to play a central part in many processes like cell division, differentiation and certain diseases such as cancer.

III.1. Chromatin organisation

The human genome is a very long sequence of nucleotides (3 billion base pairs(bp)) that experience condensation to be contained within a 10m in diameter long organelle: the nucleus.

However, not all sections of the genome need be condensed as some genes must remain accessible to undergo transcription and translation. To finely tune the accessibility of certain genes and regulate their expression, DNA must be skilfully organised in a polymeric complex named chromatin.

III.1.1. Nucleosome structure and condensation

Chromatin can be sectioned into fundamental units called nucleosomes. The nucleosome itself contains nucleosomal DNA, of around 145 to 147bp in length. Nucleosomal DNA possesses a higher curvature than free DNA and is wrapped around the nucleosome core, an octamer of canonical histones containing duplicates of H2A, H2B, H3 and H4 238 . The octamer presents a pseudo-two-fold (dyad) symmetry. Each nucleosome is separated from the other by a DNA linker of about 160 to 240 bp which is connected to the nucleosome through a linker histone called H1 (or H5). Together, the nucleosome and the DNA linker constitute another unit of measure, the chromatosome 239 (Fig. 17). Nucleosomes are distributed along the entire length of the genome, creating a chromatin fibre which is then submitted to different levels of compaction thus affecting gene expression.

III.1.1.i. Nucleosome structure

Each histone contains a central -helical region, composed of 3 -helices (1, 2, 3)

connected by two loops (L1 and L2), which together form the histone fold (1-L1-2-L2-3).

Through this histone fold, histones can associate with one another, in an anti-parallel fashion, forming heterodimers of H2A/H2B and H3/H4 240 (Fig. 18). Due to their head-to-toe organisation, the heterodimers adopt a crescent shape. The convex part of the heterodimer, containing L1, L2 and 1 and being positively charged, becomes the main interaction site with negatively charged DNA. The concave part, composed of 2 and 3 is important for the formation of the octamer. Indeed, two 2 and 3 helices from separate heterodimers will interact with each other to form a four-helix bundle. Two H3/H4 heterodimers will interact through H3's 2 and 3 in a parallel manner, forming a tetramer (Fig. 18). The octamer is then created through the formation of a four-helix bundle between H4 and H2's histone fold. Part of the central -helical region is spread outside the histone fold, forming -helices and coil extensions. The N-terminal and C-terminal regions of histones constitute another set of extensions which account for 28% of the core histones. The C-terminal tail of H2B and H3 interact with nucleosomal DNA while H4's tail is located at the heart of the core and participates in the stabilisation of the complex. Ultimately, nucleosomal DNA is wrapped around the octamer, in a 1.6 left-handed supercoil turn, through its interaction with the different histone folds and terminal extensions (histone tails). Nucleosomal DNA's entry and exit contact points (Fig. 17) are located at the H3 N-terminal tail and the N-helix extension.

III.1.1.ii. Nucleosome stacking: chromatin condensation

The sequential positioning of nucleosomes along the genome generates a 10-nm diameter chromatin fibre which is overall negatively charged. Due to this negative charge, it seems chromatin regions are unable to spontaneously condense due to electrostatic repulsion.

Therefore, chromatin compaction requires the involvement of additional, positively charged factors to adopt a three dimensional (3D) structure.

In vitro studies on purified 10nm chromatin fibres showed the fibres condensation depended on H1 and cation concentration (Fig. 19). At a low cation concentration, the 10nm fibre could condense into a supercoil structure, forming a 30nm-diameter fibre, stabilised through internucleosome interactions 241,242 . Although this chromatin structure was believed to be the basic The more condensed chromatin is, the less accessible the genome becomes. Through regulation of chromatin remodelling, the cell is able to regulate genome accessibility and therefore gene transcription.

III.1.2. Modulating chromatin accessibility

There are two main components to chromatin accessibility: the density and the kinetics of chromatin associated proteins, histones being the most impactful. Interestingly, transcriptionally active regions contain a high density of nucleosomes but those nucleosomes possess a high turnover rate. Various mechanisms regulate chromatin remodelling.

III.1.2.i. Nucleosome dynamics

The nucleosome is a very stable yet dynamic structure. Its composition and conformation can vary, impacting its interaction with DNA and therefore modifying chromatin condensation and Finally at high cation concentration, chromatin adopts a large globular structure which can arise from the condensation of either the 30nm or the 10nm fibre.

Taken from K Maeshima et al. (2019).

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gene accessibility. Nucleosome dynamics can be modulated by post-translational modification (PTMs) which are a type of epigenetic modifications, as well as by changes in histone variant composition and through their interaction with other proteins.

Spontaneous unwrapping

DNA

Post-translational modifications

Depending on the type of PTM and its localisation in the nucleosome, it can differentially affect nucleosome dynamics and therefore chromatin remodelling. For instance, epigenetic modifications located at the dyad increase nucleosome disassembly rates, with a complete disassociation of the octamer, while those located at the DNA entry-exit sites impact unwrapping rates. Histone and DNA PTMs will be further developed in the following pages.

DNA sequence

The DNA sequence can also affect nucleosome unwrapping. Nucleotide content can modify the flexibility of the DNA strand and its affinity for the octamer 246 . Variations in nucleosomal DNA sequence, specifically at the entry-exit site can affect unwrapping rates 247 .

Histones variants

The four canonical histones, above mentioned, are mostly involved in the formation of nucleosomes during replication. However, paralogues of these histones, which slightly differ in amino acid composition to the canonical four, can be used throughout the cell cycle. These variants possess varying affinities for DNA, affecting nucleosome stability, and thus have been shown to participate in modulating chromatin accessibility. They will be rapidly mentioned in the context of euchromatin.

III.1.2.ii. Passive chromatin remodelling -TF and nucleosome passive competition

Transcription factors (TFs) play a central role in chromatin remodelling as they provide binding specificity through the recognition of specific DNA sequences. These factors are unable to associate with nucleosome bound DNA. Indeed, nucleosome depleted regions are found upstream and downstream of the transcription starting sites of active genes, where TFs promote gene transcription 248 .

TFs can passively compete with histones/nucleosomes for DNA binding 248 . This mechanism doesn't require any interaction between histones and TFs. A window of opportunity is created during nucleosome disassembly, allowing TFs to bind to DNA. For instance, passive competition between nucleosomes and TFs from the CCAAT/enhancer binding protein (C/EBP) family, namely C/EBP and , allows for the exclusion of the nucleosomes from promoter regions and access to target genes 249 . This mechanism can solely occur in euchromatin as it contains the only high enough nucleosome turnover rates and density required.

Passive competition can be enhanced through cooperative binding of TFs but is not the sole mechanism used by TFs to regulate chromatin remodelling. They can also affect nucleosome integrity by destabilising the histone linker (H1) through their interaction with the DNA linker.

By affecting nucleosome and DNA binding, this process promotes passive competition.

III.1.2.iii. Active chromatin remodelling

Active chromatin remodelling also participates in determining nucleosome spacing, DNA unwrapping and can affect histone variant composition. However, as its name entails, this the processes above mentioned, to achieve wanted chromatin organisation.

III.1.2.iv. Lamina and heterochromatin maintenance

The lamina plays an important role in heterochromatin maintenance and localisation at the nuclear envelope. For instance, lamins A and C, along with lamin B receptor (LBR) anchor heterochromatin to the nuclear envelope. Depletion of both laminA/C and LBR was shown to relocate heterochromatin away from the nuclear envelope and to affect gene transcription 251 .

Emerin and Lap2β, lamin associated proteins embedded within the INM, recruits HDAC3 to the nuclear envelope, promoting the maintenance of heterochromatin [252][253][254] .

III.1.3. Chromatin organisation and gene transcription

Chromatin organisation is non-uniform across the genome, and it has been divided into two categories, depending on the state of compaction of nucleosomes (Fig. 20). Heterochromatin, a state of highly condensed chromatin, is the most widespread form of genomic organisation and is generally associated with transcriptionally repressed regions. Heterochromatin can be either constitutive or facultative. Euchromatin, on the other hand, presents a state of low condensation with highly open and transcriptionally active regions. This type of organisation distance. Specific modifications promoting gene transcription can be found within euchromatin and will be discussed in the following paragraph.

Characteristics of euchromatin

The most common PTMs found in euchromatin are acetylation, mostly associated with gene transcription, and methylation. Methylation can have varying effects on gene regulation depending on the amino-acid containing the methyl group. For instance, chromatin accessibility and gene activation are associated with acetylation of lysin 27 on histone 3 (H3K27ac) as well as trimethylation of lysins 4 and 36 on histone 3 (H3K4 me3 and H3K36 me3). The specific involvement of each modification in gene transcription will be reviewed in a following pages.

A few histone variants have been associated with euchromatin as they promote gene transcription. H2A.Z, for instance, was shown to recruit RNA polymerase II to promoters thus favouring gene transcription in Saccharomyces cerevisiae 257 . Another histone H2A variant, H2A.B was observed to play an important role not only in transcription regulation but also in mRNA processing by promoting the recognition of splice sites by the spliceosome 258 . H3.3

constitutes another example of euchromatin associated variant found in promoters and gene bodies of actively transcribed genes 259 .

III.1.3.ii. Heterochromatin

As previously mentioned, heterochromatin is a highly condensed chromatin structure with low transcriptional activity. It can be spatially discriminated from euchromatin due to its subnuclear localisation as it is found close to the organelles periphery 260 . Heterochromatin has been divided into two categories: constitutive and facultative heterochromatin.

Constitutive heterochromatin

Constitutive heterochromatin represents the most static regions of this type of structure and is highly conserved throughout an organism's life. Furthermore, genomic regions composing constitutive heterochromatin are the same no matter the cell type. They contain very few genes, mostly repetitive motives 261 . Constitutive heterochromatin, more specifically the centromeric region, has been shown to play an important role in chromosome segregation by recruiting cohesin, a multiprotein complex necessary for the separation of sister chromatids to opposite poles 262 .

Characteristics of constitutive heterochromatin

Heterochromatin formation is tightly regulated and RNA interference (RNAi), a type of epigenetic regulatory layer, has been involved in the establishment and function of this type of structure. Inhibiting RNAi by depleting components of this machinery, increases gene transcription in canonical heterochromatin regions, decreases H3K9 methylation and affects proper chromosomal segregation 263 .

Facultative heterochromatin

Facultative heterochromatin is more dynamic than its constitutive counterpart and plays a key role during development and differentiation. This subtype of heterochromatin can oscillate between a transcriptionally active and inactive state depending on encountered cues. For this reason, genomic regions contained within facultative heterochromatin will fluctuate from one cell to another. The size of these regions can vary from a few gene clusters to an entire chromosome such as with the inactivated X chromosome 264 .

Characteristics of facultative hetero chromatin

Facultative heterochromatin formation is dictated by the Polycomb group (PcG) proteins which are histone modifying complexes comprising polycomb repressive complex 1 (PRC1) and PRC2. While PRC1 is involved in the mono-ubiquitination of histone H2A lysine 119 (H2AK119ub) 265 , PRC2 methylates histone H3 lysin 27 (H3K27) 266 which is a hallmark of facultative heterochromatin. PRC2 target genes include TFs involved in embryonic stem cell development (OCT4, SOX2, NANOG) 267 . Dysregulation of PRC2 has not only been involved

with developmental defects which can lead to embryonic or perinatal death 268,269 but has also been associated with certain types of cancer 270 .

As H3K27 residue can be either methylated or acetylated, it is believed that the competition for those two marks could constitute a switch between euchromatin and heterochromatin.

III.2. Epigenetics -Different levels of regulation

Epigenetic modifications include alterations that do not affect DNA sequence but rather genome accessibility. These modifications can occur on three different levels: DNA, RNA and histones.

III.2.1. DNA methylation

In mammalian cells, most of the genome is methylated (≈70-90%) 271,272 so as to prevent inadequate gene transcription. Interestingly, CpG islands are one of a few structures which mostly remain unmethylated to promote gene transcription 273 . DNA methylation is mainly regulated by two protein families: DNA methyltransferases (DNMTs) and demethylases from the ten eleven translocation (TET) family.

III.2.1.i. DNA methylation -DNMTs

DNA methylation regulation and role in epigenetics

There are three levels of regulation of DNMTs. Firstly, they can be regulated through molecular

Active DNA demethylation

Modifications on the amine residue are carried out by activation-induced deaminase/apolipoprotein B mRNA-editing enzyme complex (AID/APOBEC) 282 . The complex creates a base mismatch by modifying the 5mC into a thymine, thus activating the BER pathway. Modifications on the methyl group involve TET proteins. There are three enzymes to this family: Tet1, Tet2 and Tet3. Their first step is to hydroxylate 5mC, forming 5hmC (Fig. 21). This modified cytosine is then either oxidized by TET proteins to form 5-carboxycytosine 283 , or it is modified on its amine residue by AID/APOBEC to form 5-hydroxymethyluracil 284 to be recognised by the BER pathway protein thymine DNA glycosylase (TDG). TDG is a uracil DNA glycosylase which removes/excises the modified 5mC base 285,286 . The methylated base is subsequently replaced with a naked cytosine.

DNA demethylation regulation and role in epigenetics

DNA demethylation can be regulated through various mechanisms. As TET oxidises 5hmC, it necessitates substrates, namely oxygen, -ketoglutarate and iron, specifically Fe(II), as a cofactor to perform this reaction 287 . The availability of these molecules will affect the reaction rate and therefore regulate DNA demethylation. TET proteins and TDG can also be modulated by RNA interference. For instance, micro-RNA (miRNA) 22 has been shown to negatively regulate TET proteins 288,289 , while miRNA-29 targets both TET and TDG, generally increasing DNA methylation 290 . TET activity can also be modulated through protein-protein interactions with calpains for example, affecting its stability and thus increasing its degradation 291 . Other proteins, like transcription factors NANOG 292 and WT1 293 , enable the recruitment of TET proteins to target genes.

TET proteins are enriched around promoters, especially around CpG islands where H3K4me3 levels are high. This particular affinity could be explained by the CXXC domain which allows Tet1 and Tet3 to bind differentially to modified cytosines (C, 5mC and 5hmC) 294,295 . Posttranslational modifications of histones are transient and constitute a key regulator of chromatin accessibility as they primarily occur on histone tails, an important region in the chromatin folding process. Histones can be modified through addition or removal of specific groups, two of the most abundant and well-studied being acetylation and methylation. Other types of PTMs include, but are not limited to, ubiquitinylation, phosphorylation and SUMOylation. Histones contain many residues available for PTMs, and nucleosomes contain a duplicate of each histone. For this reason, histone tails can present multiple modifications, some residues being available for several types of modifications.

III.2.2. Histone modification

While acetylation occurs only on lysine (K) residues, methyl groups can be transferred to both lysine and arginine (R). Unlike acetylation, which is mostly associated with active transcription, histone methylation presents a dual role in gene transcription. However, it is important to note that a crosstalk can exist between modifications, altering the effect of those modifications on transcription. For instance, acetylation of H3 prior to methylation, following estrogen stimulation, allowed for the activation of target genes 296 . These crosstalks will not be developed in the following section.

III.2.2.i. Histone methylation

Histone methylation is tightly regulated by methyltransferases and demethylases to modulate chromatin accessibility throughout an organism's life. Lysine residues can be mono-, di-or trimethylated unlike arginine residues which can only be mono-and symmetrically or asymmetrically dimethylated. Because of its prominence and its involvement in many important processes, lysine methylation will be the main focus of the following section.

Methyltransferases

Methyltransferases can be divided into two groups. The first group contains proteins with a catalytic SET domain and encompasses most methyltransferases 297 . The second group lacks the SET domain and contains a single methyltransferase, Dot1L 298 . However, both groups present similar mechanisms; a methyl group is transferred from S-adenosyl-L-methionine to the amino group of a lysine residue 297 . Each methyltransferase specifically recognises lysine residues according to their location and their degree of methylation, but interacting partners can modify their target. For instance, although MLL1/2 complex alone regulates the mono-and dimethylation of H3K4, Cps60's association with the complex is required to mediate the trimethylation of the residue 299 .

Demethylases

Methylation was long believed to be irreversible. KDM1A was the first demethylase identified as part of a co-repressor complex targeting H3K4 and involved in gene repression 300,301 .

KDM1A possesses an amine oxidase domain, which can only achieve its reaction on mono-or dimethylated substrates 300 . Further demethylases were then identified over the years, and another type of demethylase catalytic activity was identified: the Jumonji C (JmjC) domain.

JmjC requires two cofactors, Fe(II) and α-ketoglutarate to perform its oxidative catalytic reaction and enables the removal of all three states of methylation through hydroxylation 302 .

Similarly to their enzymatic counterpart, demethylases also present a high substrate specificity in terms of residue and degree of methylation. For instance, KDM7A catalyses demethylation of both mono-and dimethylated H3K9 and H3K27 303 .

Effect of methylation on chromatin condensation

Histone methylation as an active transcription mark: H3K4

Among methylation marks associated with euchromatin is the H3K4me3 epigenetic mark. In mammals, specific patterns of H3K4 methylation have been observed within the genome.

Genes tend to present a methylation gradient: H3K4me3 is located near promoters especially within CpG islands 304 , H3K4me2 is mostly concentrated within the gene body and H3K4me1 at the 3' end of genes 305 as well as in enhancers 306 .

H3K4me3 is mostly associated with transcriptionally active promoters where it was found in the vicinity of other activating marks: H3K9ac and H3K14ac 307 . However, studies have shown that H3K4me3 can also be associated with transcriptionally inactive promoters within regions termed bivalent domains. Bivalent domains constitute a combination of both repressive (H3K27me3) and activating (H3K4me3) epigenetic marks 308 . The balance between these two marks shifts depending on surrounding cues and leads to either the activation or repression of the gene. It is believed that this combination of epigenetic marks leaves promoters poised for activation upon stimulation and regulates the transcription of differentiation-specific genes.

Histone methylation as a repressive transcription mark: H3K9 and H3K27

H3K9me3 is a specific epigenetic mark associated with constitutive heterochromatin and is located in silenced genomic regions. H3K9me3 is central to heterochromatin maintenance and function 309 . Indeed, inhibition of this mark by deregulation of SUV39, a methyltransferase involved in the trimethylation of H3K9, leads to abnormal chromatin condensation and therefore impairs chromosome segregation 310 . Furthermore, H3K9me3 plays a pivotal role in heterochromatin propagation as it binds HP1a which in turn recruits SUV39 which methylates surrounding nucleosomes, thus recruiting more HP1a 311,312 . H3K9me3 are localised at pericentric regions while H3K9me2/1 is associated with silent euchromatin domains 313 .

H3K27me3 is a hallmark of facultative heterochromatin and is associated with gene repression.

This mark is mostly mutually exclusive with two activating methylation marks, namely

H3K36me3 314 and H3K4me3 315 , although they can cohabit in bivalent domains 308 as previously explained. H3K27me1 is mostly enriched at activated genes while H3k27me2 seems be located at inactive enhancers, preventing untimely gene activation. In mammals, the main writers for H3K27me2/3 are the PRC2 complexes. Multiple studies showed that upon depletion of PRC2 subunits, a global reduction of those two marks could be witnessed 268,316 .

III.2.2.ii. Histone acetylation

Many studies have shown histone acetylation's involvement in increasing DNA accessibility by promoting a more open chromatin state. Protein acetylation constitutes a relatively dynamic process as acetyl-lysine possesses a rather short lifetime (≈30-180min), which varies depending on the acetylated site 317 . Histone acetylation is regulated by histone acetyltransferases and deacetylases. Although some of these enzymes also target non-histone proteins, the main focus of this section will be on their histone modifying abilities.

Histone acetyltransferases (HATs)

Many have histone acetyltransferases (HATs) been identified over the years and have been divided into subcategories depending on their catalytic domain. The two predominant families are Gcn5 N-acetyltransferases (GNATs) and Morf, Ybf2, Sas2 and Tip60 (MYST) 318 .

HATs function as part of multiprotein complexes which present a great variety in subunit composition. However, each complex possesses a chromatin-binding domains, including but not limited to bromodomains and chromodomains. These domains recognise specific modifications on histone tails, recruiting the complex to appropriate chromatin locations.

Although each HAT subcategory uses a different acetyl transfer mechanism, which will not be discussed here, many require the association with a cofactor possessing a thioester, which will provide for the transferred acetyl group to the Nζ nitrogen of a histone lysine side chain 319 .

HATs' regulation is mediated by protein-protein interactions through their assembly into multiprotein complexes. Their catalytic activity can also be regulated, in some cases such as with particular MYST members, through auto-acetylation 320 .

Histone deacetylases (HDACs)

Similarly to histone methylation, acetylation is highly reversible. The actyl groups are removed from lysine residues by HDACs, of which there are 18 in humans. These HDACs are divided into two families, the histone deacetylase and Sir2 regulatory family, and are further partitioned Studying substrate specificity has proven to be more complex than with acetyltransferases for multiple reasons such as functional redundancy or the presence of some members in several different complexes

Effect of acetylation on chromatin condensation and gene transcription

Lysine residues are positively charged residues which possess a natural affinity for negatively charged DNA or negatively charged domains of surrounding nucleosomes. Acetylation at specific lysine sites (histone tails and histone folds) can neutralise the positive charge thereby weakening the interaction, thus promoting a more open configuration of chromatin, more favourable to active gene transcription. The density of modified lysine residues amplifies the weakening of the bonds and unwrapping of nucleosomal DNA. Indeed, studies have shown that enrichment in histone acetylation at transcription starting sites correlates with active gene expression 322 . Lysine acetylation can also modify the flexibility of histone tails thereby affecting inter-nucleosome interaction and chromatin compaction. For example, increased, H4K16 acetylation impeded chromatin folding and disrupted nucleosome stacking 323,324 .

Histone acetylation marks, such as histone 3 at lysine 27 (H3K27ac), have been associated with active transcription as it is found within promoters, enhancers 325 and the transcription starting sites of active genes. In enhancers specifically, it is presumed H3K27ac's function could be redundant with other epigenetic modifications since depletion of H3K27ac does not affect considerably overall trancriptome 326 .

III.2.3. Non-coding RNA modifications

A large portion of the genome is transcribed into RNA, but is never fully processed into a protein as it does not undergo translation. This group of RNA sequences lacking protein coding are recognised as non-coding RNAs (ncRNA). There are two families of ncRNAs:

housekeeping ncRNAs and regulatory ncRNAs. Housekeeping ncRNAs are essential for cell viability as they regulate RNA splicing, protein translocation and localisation to specific organelles. They will not be discussed in the following section. On the other hand, regulatory ncRNAs mediate processes such as transcriptional gene silencing, post-transcriptional gene silencing and modulate protein localisation. The participation of ncRNAs in epigenetics will be the focus of this section.

NcRNAs can be separated into two categories: short chain ncRNAs (siRNAs, miRNAs, and piRNAs) and long ncRNA (lncRNAs).

III.2.3.i. Short chain ncRNAs

Short chain ncRNAs can be either double stranded, which is the case for both small interfering RNA (siRNA) and microRNA (miRNA), or single stranded RNA sequences, as observed in the case of PIWI interacting RNA (piRNA). The majority of short chain ncRNAs are associated with gene repression as will be exemplified in the following paragraphs. miRNAs miRNAs originate from a single strand of endogenously translated RNA sequence containing short, inverted repeats, forming a series of incomplete hairpins called primary RNA (pri- miRNA). Processing through Drosha, a ribonuclease, cleaves the pri-mRNA at the base of each hairpin loop, generating single hairpin RNAs (pre-miRNA) which can then be exported out of the nucleus through exportin-5 (Fig. 22). Once in the cytoplasm, pre-miRNA is further processed by Dicer, an endoribonuclease, to form complete, short double-stranded miRNAs.

The miRNA is then loaded to Argonaut proteins, AGO and WAGO, which are an integral part of the RNA-induced silencing complex (RISC). RISC processes the double stranded miRNA into a single strand which is then used to find complementary RNA sequences. The target mRNA will then either be cleaved and undergo degradation or RISC binding will prevent

III.2.3.ii. Long ncRNAs

LncRNAs constitute a large heterogeneous group of ncRNA and many have yet to be attributed a function. They can originate from many different mechanisms such as pseudogenes, copies of mutated coding genes that are no longer coding, they can be transcribed from overlapping genes or arise from intergenic regions 335 .

LncRNAs can modulate gene expression through many mechanisms. Some lncRNAs are templates for miRNA through Drosha and Dicer processing. As they possess similar sites to miRNAs, they can negatively regulate the miRNAs, thereby acting as positive regulators of transcription. One example is lincRNA-ROR which has been shown to target miR-145 in pluripotent ESCs 336 . Another example is lncRNA H19 whose expression negatively correlates with that of miRNA let-7 during muscle cell differentiation 337 . LncRNAs can also target mRNA degradation independently of miRNAs.

More interestingly in the context of epigenetics, lncRNAs are able to bind chromatin remodellers to inhibit gene transcription. For instance, H19 binds to methyl-CpG-binding domain protein 1 (MBD1) which in turn binds to both methylated and unmethylated DNA. The complex then recruits methyltransferases SETDB1 and SUV39H1, increasing H3K9 methylation at target genes 338 . Additionally, HOX transcript antisense RNA (HOTAIR) lncRNA recruits PRC2 to a homeobox gene cluster, maintaining gene repression 339,340 . The transcription of another type of lncRNAs, enhancer RNAs (eRNAs) 341 , has been associated with active enhancer and may play a role in regulating chromatin accessibility. A study performed in mouse skeletal cells showed that upon depletion of eRNAs, chromatin accessibility of the MYOG locus, amongst other loci, was decreased.

III.2.4. Mechanical regulation and epigenetics

Mechanical cues have been shown to impact gene transcription by modulating chromatin organisation and epigenetic modifiers. A study by A. Tajik et al 342 examined chromatin deformation and force-induced transcription using 3D magnetic twisting cytometry to apply local and directional stresses on the cell surface. They observed that strain amplitude and direction affected both chromatin deformation and gene transcription and that this process was reliant on both myosin II contraction and an intact nucleo-cytoskeletal coupling 342,343 . Another paper identified a role of chromatin in preventing DNA damage against mechanical stress 344 .

Upon cyclic stretching, cells underwent a rapid decrease in H3K9me3 levels preventing DNA damage by promoting nuclear softening.

As previously mentioned, lamin A/C, whose expression and turnover is sensitive to tensile strain, plays an important role in heterochromatin maintenance and localisation to the nuclear envelope 251 . HDAC3 was reported to be recruited by Emerin and Lap2β, two lamin associated proteins, to the nuclear envelope under tensile strain, increasing deacetylation and promoting heterochromatin along the nuclear periphery [252][253][254] . Interestingly, an increase in cell spreading and myosin II contractility seems to be sufficient to trigger HDAC3 mediated deacetylation and promote chromatin condensation 345 . Another study revealed the participation of methyltransferase EZH2 in chromatin condensation under dynamic stretching of mesenchymal stem cells (MSCs) 346 . Furthermore, it was shown that this myosin II dependent recruitment of EZH1/2 to the nuclear envelope leads to the replacement of constitutive heterochromatin (H3K9me3) with facultative heterochromatin (H3K27me3) 347 . This remodelling is believed to protect chromatin from mechanical strain as it uncouples heterochromatin from the lamina.

These examples showcase how epigenetics modifications are sensitive to mechanical cues and that this process relies heavily on myosin II contraction and nucleo-cytoskeletal coupling.

IV. Mechanical memoryhow cells retain past information

Mechanotransduction illustrates how cells are able to sense mechanical cues and adapt to their environment, undergoing various phenotypic changes depending on those physical signals. For instance, on softer ECMs, cells generally tend to form fewer and smaller focal adhesions, have a reduced spreading area, are less contractile, exert less forces and migrate at a lower speed than cells cultured on stiffer substrates. However, recent studies have brought to light the fact that cells are not only sensitive to their immediate surroundings but are also impacted by previous environments they encountered.

IV.1. Defining mechanical memory

Interestingly, phenotypic changes attributed to specific physical signals have been observed to persist for some time after the mechanical cues subsided (Fig. 23). This ability to retain information and the effects of past mechanical cues has been named "mechanical memory".

This phenomenon has been involved in many cellular processes such as cell migration, invasion, proliferation or even cell fate (Fig. 23) which will be reviewed in the following section.

IV.1.1. Mechanical memory and cell fate IV.1.1.i. Impact of mechanical memory on fibrosis

Mechanical memory was first brought to light in the context of fibrosis, in an attempt to evaluate the long-lasting effects of mechanical cues on myofibroblast activation 348 . It was observed that, in vitro, myofibroblast activation could be modulated through matrix stiffness, stiff ECMs promoting activation while soft environments reverted the phenotypic markers of activation 349 .

In order to study the effect of mechanical conditioning on myofibroblast differentiation, primary rat lung fibroblasts were mechanically primed, using polydimethylsiloxan (PDMS) 80 gels of varying rigidities, over a defined period of time (three weeks), before being submitted to another environment/PDMS rigidity. As expected, mechanical priming of fibroblasts on a pathologically stiff substrate augmented cell proliferation and differentiation into a myofibroblast phenotype as witnessed by the increase in cell contractility and expression of αsmooth muscle actin (αSMA). Interestingly, this myofibroblast activation was maintained for another two weeks even after cells had been transferred to a soft rigidity. In contrast, priming fibroblasts on a soft PDMS partially shielded them from activation when they were shifted to a stiffer substrate. Thus this paper established the basis for mechanical memory through the observation that the effect of mechanical cues could be stored and maintained over time, affecting cellular processes such as proliferation and differentiation.

Another study presented similar results using rat and human primary adipose-derived stem cells (ASC) 350 . It showed that ASCs exhibited a mechanical memory as their differentiation into a pro-fibrotic phenotype was affected by past mechanical cues, as determined by the priming rigidity. Cells initially cultured on a soft substrate, before being plated on a stiff matrix presented a decrease in cell size, expression of αSMA and ECM secretion as compared to cells that had always been cultured on a stiff substrate.

IV.1.1.ii. Impact of mechanical memory on osteogenic activation

The involvement of mechanical memory in mesenchymal stem cell (MSC) fate was also established, using a photo-tuneable substrate softened by light 351 . Human MSCs, isolated from bone marrow, were mechanically dosed for a period of one to ten days on a stiff substrate before the gel was softened. They were then maintained for an additional one to ten days on the softened gel. On the primary stiff substrate, they observed the expression of pre-osteogenic transcription factor RUNX2 increased the longer the cells were cultured on the stiff priming substrate. Similarly to Balestrini et al 348 , the authors observed that the longer the cells were dosed or primed on a stiff substrate, the higher the expression of RUNX2 was after 3 days on a soft secondary substrate. For instance, cells primed for five days on a stiff substrate had around a three-fold increase in RUNX2 expression after three days on a secondary soft substrate compared to cells primed for only three days on a similarly stiff gel. Interestingly, they found a correlation between the duration of mechanical dosing or mechanical priming and the persistence of mechanical memory. The longer cells were primed on a stiff substrate, the longer high expressions of RUNX2 were maintained on the secondary substrate. This suggests that the storage of mechanical information is not a binary process but rather an incremental one, and a function of the exposure time to these mechanical cues. One could argue that mechanical memory could be either transient or irreversible, depending on the mechanical dosing or priming.

Using alginate-based hydrogels, another study showed that the mechanical properties of cell niches directed human MSC (hMSC) lineage 352 . The hydrogel rigidity was modulated through addition or removal of Ca 2+ , enabling a direct switch in environmental stiffness. Cells that experienced a switch from a soft to a stiff environment showed a delay in osteogenic differentiation. On the contrary, cells that were exposed to a softening of the gel (stiff to soft) maintained a strong osteogenic differentiation before showing a decrease in osteogenic markers. These observations seem to corroborate previous findings regarding the transient nature of mechanical memory. In this context, mechanical memory appeared to simply delay the assimilation of immediate physical cues.

IV.1.2. Mechanical memory and cell adhesion/migration

It has been established that the mechanical properties of the ECM impact cell adhesion, morphology and migration. However, the long term effects of past mechanical cues remained elusive until recently when mechanical memory was approached in the context of collective migration 353 . The team developed a technique whereby they would restrain, for a defined amount of time, a monolayer of cells on a modular polyacrylamide substrate, within a central zone, using a PDMS stencil (Fig. 24). The central zone of a defined stiffness would serve as a priming rigidity for the cells. The PDMS stencil would then be removed at the end of the priming period (here three days), allowing the cells to migrate collectively onto the peripheral regions of the hydrogel, exposing them to another rigidity, where collective migration speed would be studied. They observed that multiple cells lines (MCF10A, A421 and MCF7) were able to set-up a mechanical memory as their migration speed on the secondary substrate was modulated depending on the priming ECM stiffness. For instance, cells that were primed on a soft substrate had a lower migration speed, smaller FAs and a decreased phospho-myosin light chain expression (pMLC) expression on a stiff secondary substrate than cells that had always been exposed to a stiff substrate. Interestingly, they observed that the effects of mechanical priming were lessened when they left cells on the priming stiffness for less than a day. This information further confirms the hypothesis that mechanical memory set-up is time-dependent.

Seeing as they studied collective cell migration, they wondered if the observed effects were due to a signal transmission from the back of the monolayer (still in contact with the priming stiffness) to the migration front or if it could be affected by cell proliferation, which is known to vary depending on ECM rigidity. Inhibiting proliferation and removing the centre part of the gel, containing the primary stiffness, did not impede mechanical memory which disproved these hypothetical alternatives to mechanical memory.

Another study on pancreatic cells showed that mechanical memory also impacted cell spreading and traction force 354 . Again, on a stiff secondary matrix, cells primed on a soft substrate exerted less traction force and had smaller spreading areas than cells primed on a stiff substrate. Similar results were obtained with breast cancer cells lines, which studied the effect of a seven day mechanical priming on invasiveness and osteolytic activity 355,356 . Mechanical memory was shown to affect traction force, cell spreading, invasiveness and integrin signalling activation (pFAK).

IV.2. Identified pathways in mechanical memory

Little is known of the mechanisms involved in the set-up of this mechanical memory in cells.

Some players have been identified, namely YAP/TAZ, RUNX2 and miR-21 which have been suggested to serve as "memory storage pathways", but the upstream regulators have not yet been established. Although the interplay between those factors has not been studies in the context of mechanical memory, miR21 has been observed to positively modulate both RUNX2

and YAP/TAZ activation 357,358 . Mechanical memory establishment is possibly quite complex and multi-levelled. In the following section will be developed the known contribution of each of these pathways to mechanical memory establishment.

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IV.2.1. YAP/TAZ signalling in mechanical memory

Yang et al 351 attempted to identify a mechanical sensor which could modulate mechanical activation in response to immediate physical signals. Seeing as YAP/TAZ activation can be modulated by ECM stiffness, they investigated if it could also be involved in mechanical memory set-up. The authors found that, on the primary substrate, YAP/TAZ nuclear localisation (activation) increased the longer the cells were cultured on the stiff gel. They also found that YAP/TAZ activation on the soft secondary substrate increased with mechanical dosing. The longer cells were primed on a stiff substrate, the more YAP/TAZ localised to the nucleus on the soft secondary substrate and the longer this activation was maintained. Thus a correlation between YAP/TAZ activation and mechanical memory was established. Another study observed equivalent effects of mechanical memory on YAP/TAZ activation with the Suit-2.28 pancreatic cancer cell line 354 .

However, it was not until Nasrollahi et al 353 that a direct link was established between mechanical memory and YAP/TAZ was made. Comparably to the previous study, the authors found that YAP/TAZ localisation was modulated by the priming stiffness. On a stiff secondary substrate, cells that were primed on a soft ECM showed lower nuclear localisation of YAP/TAZ than cells that had always been cultured on a stiff matrix 353,354 . Conversely, on a soft secondary substrate, cells that were conditioned on a stiff ECM showed higher levels of nuclear YAP/TAZ than cells that had always been cultured on a soft matrix. Interestingly, upon YAP/TAZ depletion during the priming period, cells where still able to perform mechanosensing but exhibited no more mechanical memory. Cells on a matching ECM rigidity presented similar migration speeds, no matter the priming rigidity. However, the migration speed was still affected by the secondary substrate. In balance with previous results, they showed that on a stiff secondary substrate, soft primed YAP/TAZ depleted cells showed an increase in FA size and pMLC expression compared to the soft primed control cells. They proposed YAP/TAZ to serve as a storage pathway for mechanical memory, without affecting the cells ability to perform mechanosensing.

IV.2.2. RUNX2 signalling in mechanical memory

Another pathway proposed to participate in mechanical memory is that mediated by the transcription factor RUNX2 355 . Authors of this study conditioned breast cancer cells lines and patient-derived xenograft (PDX) primary cells for seven days on a primary substrate before transferring them to a secondary substrate for up to another seven days or injecting them into mice. Similarly to previous studies, they observed that mechanical memory affected invasion and metastasis. For instance, cells that were primed on a stiff substrate before intra-cardiac injection in mice formed more metastases than cells primed on a soft substrate. They then identified RUNX2 as a potential target in mechanical memory, as will be developed later.

RUNX2 expression is mechanosensitive and its nuclear localisation, upon activating phosphorylation, was found to be affected by mechanical priming. Indeed, on a stiff secondary substrate, RUNX2 was nuclear in cells that were primed on a stiff matrix but cytoplasmic in cells that had always been cultured on a soft ECM. Interestingly, when they over expressed a phosphor-mimetic mutant of RUNX2 in cells primed on a soft substrate, they observed an increase in cell invasion compared to control cells similarly conditioned. Conversely, cells primed on a stiff substrate overexpressing a non-phosphorylatable RUNX2 were less invasive than control cells similarly conditioned. Similar results were obtained when looking at metastasis formation in mice. Injecting soft primed cells overexpressing a constitutively active RUNX2 increased metastasis formation compared to soft primed control cells. On the contrary, injecting stiff primed cells overexpressing a non-phosphorylatable RUNX2 impeded metastasis formation compared to stiff primed control cells. Thus RUNX2 was proposed as a mechanical storage pathway.

IV.2.3. miR-21 signalling in mechanical memory

MiR-21 was shown to act as another regulator for mechanical memory as will be presented in the context of mechanical MSC activation 359 . Mechanical priming of MSCs occurred over the course of three passages (three weeks), cells were then transferred to a secondary substrate for an additional two passages (two weeks). Similarly to what had previously been observed 348 , MSC priming affects mechanical myofibroblast activation and this mechanical memory is maintained up to two passages no matter the physical cues. The authors then proceeded to show that, on stiff substrates, mechanosensitive MRTF-A activated miR-21, which was then translocated to the nucleus where it regulated pro-fibrotic gene expression 360 . MiR-21 levels were affected by the priming stiffness, regardless of the changes in mechanical cues. They observed that disrupting miR-21 levels at the end of the priming period, three days prior to transferring cells to their secondary substrate, erased mechanical memory and thus reset cells ability to respond to immediate physical cues. Interestingly, miR-21 levels were found to be relatively stable over the first few days after switching to another rigidity. Seeing as those levels are also seen to gradually increase over the first five to six days on a stiff environment, it could bring some light as to the time-frame of mechanical memory. Two other studies corroborated these results 352,354 , also observing that modulating miR-21 levels could erase or increase the effects of mechanical memory.

IV.3. Mechanical memory and epigenetic modifications

The fact that cells are able to retain past information implies that the knowledge is stored within the cell and slowly released over time, overcoming immediate signals. How cells are able to bypass immediate physical cues still eludes us. One of the most prominent hypotheses is that past information would be stored in the form of epigenetic modifications. Epigenetic modifications are known to regulate gene expression and to be sensitive to mechanical cues, as previously developed.

IV.3.1. Mechanical memory affects chromatin accessibility

Killaars et al 361 explored this theory using hMSCs cultured on phototunable hydrogels 361 . They first validated that, after a three day culture on a hydrogel, both YAP/TAZ activation and histone acetylation increased with substrate stiffness and that nuclear morphology was also affected by matrix rigidity. Indeed, on stiff substrates, nuclei were less spherical and presented lower chromatin condensation than on soft matrices. Expression levels of HDAC (1, 2 and 3) were also lower in cells cultured on stiff substrates while HAT1 was more expressed. They then sought to investigate how mechanical priming would affect these parameters by priming cells on a given rigidity for up to ten days before inducing gel softening and maintaining cells for an additional ten days. They found that mechanical memory had an effect on nuclear morphology, chromatin condensation and acetylation levels and that the timescale of those effects depended on the duration of the priming period (Fig. 25). For instance, on a soft secondary substrate, hMSCs primed on a stiff gel showed higher levels of histone acetylation and lower overall chromatin condensation than cells cultured on a consistently soft gel. Similarly to what had been previously observed, the intensity of those effects increased and were longer lasting when cells were maintained for a longer amount of time on the priming rigidity. In agreement with

Yang et al 351 , they proposed that the length of the priming period would determine the reversible or irreversible nature of mechanical memory (Fig. 25). It is important to note that the irreversibility of mechanical memory is considered in a defined time frame, ten days in the aforementioned study, and should be taken with precaution in unlimited settings. Seeing as chromatin condensation and acetylation levels are modulated by epigenetic modifications and affect gene expression, Killaars' study supports the idea that mechanical memory could be stored in the form of epigenetics.

Additionally, Heo et al 346 also identified an effect of mechanical memory on chromatin organisation. They studied the effect of mechanical cues on MSC lineage commitment by applying dynamic tensile load to MSCs at 1Hz for up to 6h. The authors observed that dynamic tensile loading affected nuclear rigidity and induced rapid, myosin II-mediated chromatin condensation. The team established the involvement of histone methylation in the process as the rapid chromatin condensation could be prevented by inhibiting the PRC2 complex (involved in H3K27 methylation). They also established that this chromatin condensation was dependent on calcium signalling. However, more importantly in the context of mechanical memory, they observed that the effects of dynamic tensile loading on the nucleus were maintained over time.

MSCs were loaded for three hours, maintained in unloaded culture for two days (first resting period), loaded for a second time for three hours and maintained for an additional two days in unloaded culture (second resting period). They observed chromatin condensation levels to be increased after the second loading compared to the first loading. Furthermore, they found chromatin condensation levels decreased over time during the first resting period but not the YAP/TAZ and AcK stainings. For reversible and irreversible conditions, cells were respectively primed for 1 and 10 days prior to in situ softening. They were then maintained for an additional 5 to 10 days, respectively, before analysis. Taken from A.R. Killaars et al. (2019).

second one. Nuclear deformability presented a similar pattern as nuclei were also shown to be more rigid after the second loading. This study also suggests that mechanical memory could be stored through epigenetic modifications, possibly through histone methylation.

IV.3.2. RUNX2 as a chromatin stabiliser promoting mechanical memory

The exact mechanisms involved in maintaining changes in chromatin condensation, in the context of mechanical memory, still elude us. However, transcription factor RUNX2 has been identified to play a part in sustaining chromatin organisation in mechanical memory 355 . As previously mentioned, they conditioned breast cancer cells lines for seven days on a primary substrate before transferring them to a secondary substrate for up to another seven days. They first performed RNA sequencing on cells that were either primed on a stiff or soft substrate to try and establish genes that were differentially regulated between the two conditions. They identified five candidates, transcription factors among which was RUNX2, which were more highly expressed on stiff substrate. As transcription factors modulate gene expression, they then looked at the effect of mechanical priming on chromatin accessibility using Assay for

Transposase-Accessible Chromatin sequencing (ATAQ seq). They identified a set of "delayedclosing" loci whose accessibility was not immediately switched upon transition from a stiff to a soft environment. "Delayed-closing" loci were found to still be accessible after two days on the soft secondary substrate but were considerably less accessible by day five. Thus, they hypothesised that within these loci could be regulatory sites contributing to the longer lasting effects of mechanical memory. Through motif enrichment analysis of these sequences, they identified that the RUNX consensus binding sequence was enriched at those specific loci. They then depleted RUNX2 and showed that upon transition from a stiff to a soft substrate, chromatin accessibility was altered compared to that observed in control cells. Indeed, within half a day on the secondary soft gel, more genomic loci were closed in shRUNX2 cells compared to control cells. Thus they hypothesised that RUNX2, whose expression is mechanosensitive, could act as a chromatin stabiliser in the context of mechanical memory.

IV.4. Mathematical model for mechanical memory

A mathematical model proposed by Price et al 362,363 , provides insights into the possible mechanisms involved in mechanical memory establishment and persistence. To do so, the authors decided to focus primarily on stiff associated mechanical memory, whereby only stiff substrates would generate the establishment of mechanical memory.

IV.4.1. Kinetics of positive feedback loops

Mechanical memory is based on the maintenance of the effects ofpast mechanical cues. These cues shape the cells phenotype. In this model the relationship between the ECM rigidity and the cells phenotype is defined by the cells mechanoactivation state. Mechanoactivation is a function of stiff-sensitive protein activation: the higher these proteins and their downstream signalling pathways are activated, the higher the mechanoactivation. These proteins include, but are not limited to, cytoskeletal and FA components (vinculin, talin and integrins), transcription factors (RUNX2 and YAP/TAZ) and epigenetic modulators (HDACs, HATs and This in turn affects the concentration of stiff-activated proteins and TFs in the cell which will modulate the mechanoresponse and therefore the mechanical memory of the cell. Taken from Price et al. (2021).

miRNAs). One of the key elements in mechanical memory is the positive feedback these factors can have on transcriptional activity and the epigenetic state that enables the persistence of the information relating to past physical signals (Fig. 26). For instance, cell contractility and FA maturation increases YAP/TAZ signalling which in turn further increases FA stability and cellular contractility 364,365 . They considered the existence of two types of positive feedback loops, a fast-acting and a slow-acting positive feedback loop to account for the timescale of mechanical memory. They considered the slow-acting feedback loop to be driven by epigenetic modifiers, such as miR21 and HDACs/HATs, which act on chromatin remodelling. On the other hand, they considered the fast-acting loop to be determined by signalling pathways which regulate activation of YAP/TAZ and RUNX2. These feedback loops are modulated by the synthesis, lifetime/degradation, nuclear import and export rates of mechanoactivating factors.

IV.4.2. Nonlinear dynamics of positive feedback loops

They propose that these positive feedback loops regulate the state of mechanoactivation of the cell by modulating the cells sensitivity to positive reinforcement (α -Fig. 26). α is a timesensitive parameter which is considered to be the sum of the fast-acting and slow-acting positive feedback loops. In the absence of memory, cellular mechanoactivation is considered low on a soft substrate and high on a stiff ECM. However, in the case of mechanical memory, mechanoactivation is no longer directly correlated to matrix stiffness but rather to the cells mechanical history. They proposed mechanical memory to be influenced by both the kinetics of mechanoactivating factors and their ability to generate a feedback loop, thus enabling the maintenance of high levels of mechanoactivation even after a change in ECM stiffness. When mechanical priming is insufficient (in length or signal intensity), those factors do not provide a sufficient enough positive feedback to maintain high mechanoactivation on a soft substrate.

However, when cells are primed for a sufficient period of time on a stiff substrate, mechanoactivating factors are generated and accumulate, creating a pool, providing a positive feedback thereby maintaining high mechanoactivation on a soft substrate. The authors hypothesised that long priming could lead to a permanent mechanical memory which can be associated with cellular differentiation under mechanical cues.

Objectives of the thesis

Cells are known to sense the mechanical properties of the surrounding ECM and to transform these cues into biochemical and biological responses through a process called mechanotransduction. Studies have shown that the capability to perform mechanosensing is mediated predominantly by integrins, a subset of transmembrane receptors linking the ECM to the cell's cytoskeleton and modulating several signalling pathways. In response to the ECM's stiffness, cells undergo various phenotypic changes: on softer substrates they form fewer and smaller focal adhesions, have a reduced spreading area, exert less forces and present slower migration speeds. Interestingly, these phenotypic changes have been observed to persist for some time after the mechanical cues subsided. This ability to retain information and the effects of past mechanical cues is named "mechanical memory". Little is known of the mechanisms involved in the set-up of this mechanical memory. Some players have been identified, namely RUNX2, YAP/TAZ and MicroRNA-21, which have been suggested to serve as "memory storage pathways", but the upstream and downstream regulators have not yet been established.

Furthermore, recent studies suggest that epigenetic modifications could be central to mechanical memory by enabling the long-term storage of past information.

The general objective of my PhD was to investigate the mechanisms supporting mechanical memory. For this, we set out to address a number of questions:

 Are all cell lines/cell types capable of mechanical memory?

 Is the actomyosin cytoskeleton required to build up a mechanical memory?

 Are epigenetic modifications involved in mechanical memory?

 If so, are there specific players involved in mechanical memory that can be identified?

 Which genes do they target that could serve as effectors of mechanical memory?

Materials and methods

I. Cell biology I.1. Cell lines

Human breast cancer cells HS 578T and MDA-MB-231 were a gift from respectively C.

Lamaze (Institute Curie, Paris, France) and P. Chavrier (Institute Curie, Paris, France) while immortalised mouse osteoblasts were a gift from C. Albiges-Rizo (IAB, Grenoble, France). All three cell lines were cultured in DMEM Glutamax (Gibco) supplemented with 10% (v/v) foetal bovine serum (FBS -Sigma-Aldrich) in 10% CO2 humidified air culture.

I.2. Polyacrylamide gels synthesis

Polyacrylamide (PA) gels were synthesised on glass coverslips (18mm diameter No. 1 -Marienfeld) or glass bottom plates (12-24 1.5P black well plate -IBL). Glass surfaces were first incubated for 15min at room temperature (RT) with 2/3v miliQ H2O and 1/3v 3aminopropyltrimethoxysilane (APTMS -Sigma-Aldtrich) before being washed in miliQ H2O.

The glass surface was then incubated for 30min at RT with glutaraldehyde 0.5% in PBS and subsequently washed with miliQ H2O before being dried with compressed air or let to dry. A 15µl drop of PA solution was deposited either on a rinsed glass surface and flattened using the previously treated coverslip. In glass bottom plaques, a 15µl droplet of PA solution was deposited at the bottom of the well and flattened with a non-treated coverslip. The PA solutions were allowed to polymerise for 40-60min before being hydrated with PBS (Gibco) for 15-20min. The coverslips were then removed and rinsed in PBS. PA gels were then treated to enable cross-linking of an ECM protein to the gel surface. Gels were incubated for 30min under slow agitation with 3/5v 50mM Hepes (Sigma-Aldrich) pH 7.5, 1/5v Sulfo-Sampah (Thermo-Frischer -1mg/ml in 50mM Hepes pH 7.5) and 1/5v N-(3-Dimethylaminopropyl'N'ethylcarbodiimide hydrochloride (EDC -Sigma-Aldrich -10mg/ml in 50mM Hepes pH 7.5).

Gels were subsequently cross-linked under UV light for 10min before being washed with sterile PBS. Lastly the PA gels were incubated O/N at 37°C with either 50 μg/ml of rat tail collagen type I (Gibco) in acetic acid (0.02M -VWR) or 20 μg/ml of fibronectin (Roche) in PBS before being rinsed in PBS.

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PA solution of various rigidities were prepared as shown in Table 1, using acrylamide 40% (BioRad), bisacrylamide 2% (BioRad), ammonium persulphate (APS -Sigma-Aldrich) and Tetramethylethylenediamine (TEMED -Invitrogen). PA solution was vortexed and immediately deposited on the appropriate glass surface. Elasticity of the different gels was controlled by Atomic Force Microscopy 366 . The generation of 80 kPa gels was performed according to a previously published protocol 367 .

I.3. Glass surface coating

Sterilised glass coverslips were incubated for 1h at 37°C with either 50 μg/ml of rat tail collagen type I in acetic acid (0.02M) or 20 μg/ml of fibronectin in PBS before being rinsed in PBS.

I.4. Cell seeding on PA hydrogels

To ensure that cells only adhered to the PA gels and prevent them from adhering to the edges of the wells, a defined number of cells was deposited directly on the gel in an 80µl droplet of medium. Prior to this, the edges of the PA gel was dried using aspiration to avoid the droplet from spilling out of the gel. The number of cells within the droplet varied depending on the cell line and the stiffness of the substrate. On soft hydrogels (0.5kPa and 2kPa), the deposited droplet contained 100 000 osteoblast cells/ml, 125 000 HS 578T cells/ml and 212 500 MDA-MB-231 cells/ml. On stiff hydrogels (80kPa), the deposited droplet contained 75 000 osteoblast cells/ml, 100 000 HS 578T cells/ml and 150 000 MDA-MB-231 cells/ml. Cells were allowed to adhere for one to two hours at 37°C before warm medium was added to the well.

I.5. Antibodies and siRNA

Rabbit polyclonal KDM7a antibody was purchased from Invitrogen (PA5-96987), rabbit trimethyl-histone H3 (lys27) antibody was purchased from Cell Signalling (3674), mouse IgG1 di/tri-methyl-histone H3 (lys9) was purchased from Cell Signalling (5327), rabbit FN antibody was purchased from Sigma (F3648) and alexa fluor 633 phalloidin antibodies were purchased from Molecular Probes (A22284).

siRNA control and SMARTpool siRNA against KDM7a, HR and FN were purchased from Dharmacon.

I.6. Chemicals and drugs

Cells were treated during mechanical priming. They were allowed to adhere for 5h on the PA hydrogel before being treated on the first day of mechanical priming. The drug was washed at the end of the priming period before cells were passaged and transferred to a stiff secondary substrate.

To disrupt the actin cytoskeleton or myosin II contractility, cells were treated respectively with 0,5µM of Cytochalasin D (Sigma-Aldrich) or 10µM of blebbistatin (Sigma-Aldrich). To hypomethylate DNA, cells were treated with 1µM of 5-azacytidine (Sigma-Aldrich). 5-aza is a cytidine analogue which can't be methylated, thus cells were treated daily to ensure that the analogue was incorporated in DNA during cell division. To inhibit HDACs in an attempt to increase histone acetylation cells were treated with 37,25nM of Trichostatin A (Sigma-Aldrich).

To inhibit HATs in an attempt to decrease histone acetylation cells were treated cells with 5µM

of Garcinol (Sigma-Aldrich).

I.7. siRNA transfections

Osteoblasts were transfected with siRNAs (at 30nM) against KDM7a, HR and FN1 using Lipofectamine RNAimax reagent (Invitrogen) according to manufacturer's instructions. In a parallel control experiment, osteoblasts were transfected with a non-targeted control siRNA.

To perform depletions of KDM7a, HR and FN1 in osteoblasts, cells were transfected once as described above and then a second time, 48 hours later, with the same siRNAs. One day (24h) after the second transfection, cells were plated on either a soft (0.5kPa) or stiff (80kPa) Col I coated hydrogels for mechanical priming as previously described, or on TCPs (at 5 000 cells/cm2). Samples were harvested on the TCPs at day 4 for WB to analyse for protein levels.

I.8. Immunofluorescence and epifluorescence microscopy

Col I coated glass coverslip (secondary stiff substrate) and were incubated at 37°C with 5% CO2 for 24h.

Cells were pre-extracted with extraction buffer (PIPES 80 mM, EGTA 5 mM, MgCl2 2 mM. pH 6.8, 1% TritonX100 -Sigma-Aldrich) for 20sec at RT for KDM7a antibodies. After three quick washes with PBS, cells were fixed with 4% paraformaldehyde (PFA -ThermoFischer) for 15min at RT before being washed with PBS. Cells were subsequently incubated with primary antibodies (1:200) for 1h at RT before being washed with PBS. Finally, cells were incubated with secondary antibodies (1:200) and alexa fluor 633 phalloidin for 1h at RT and washed with PBS before mounting with Fluoromount-G with Dapi (Invitrogen).

I.9. Indirect immunofluorescence microscopy and fluorescence quantification

Immunofluorescence images were acquired 24h after cell plating on a secondary stiff substrate (Col I coated glass), on a Leica epifluorescence microscope (Leica Microsystems Ltd., Wetzlar, Germany) through 63x (N.A. 1.40) objective and the microscope was steered by the Leica dedicated LasX software®.

To quantify KDM7a and HR fluorescence intensity in the nucleus, the area of interest was selected manually using DAPI staining. The mean fluorescence intensity was then quantified within the region of interest. For each condition, measurements were made on 50-80 cells. To quantify FN1 intensity in the cell body, the area of interest was selected manually using phalloidin staining. The mean fluorescence intensity was then quantified within the region of interest. For each condition, measurements were made on 50-80 cells in 1 to 7 experiments. All quantifications on immunofluorescence images were done with FIJI after background subtraction.

I.10. Time-lapse microscopy and quantification of migration assays

Time-lapse videos were acquired on a Leica epifluorescence microscope (Leica Microsystems Ltd., Wetzlar, Germany) through 10x (Fluotar N.A. 0.32 PH1) objective. A PeCon chamber i8

was installed on the microscope to perform live imaging at 37°C with 5% CO2. The microscope was steered by the Leica dedicated LasX software®.

Mechanically primed cells were passaged and transferred to a Col I coated 80kPa hydrogel (stiff secondary substrate) where they were allowed to adhere for 5h before cells were imaged in bright field or phase contrast, at a 1 image every 20min for 16h. Cells were manually tracked using the "Manual tracking" plugin of FIJI. Forward Migration Indexes were obtained with the Ibidi "Chemotaxis and Migration Tool" in FIJI. For each condition, 70-100 cells were manually tracked in 2 to 16 experiments.

I.11. Quantification of cell division

Cell division was quantified in osteoblasts using the time-lapse obtained during migration assays (as mentioned above). The number of cells dividing over the course of the 16h timelapse was manually counted and normalised on the total amount of cells found in the first frame of the video. For each condition, cell division was measured from a total of 100-200 cells in 3 experiments.

I.12. RNA sequencing and analysis

Osteoblasts were mechanical primed for three days on either a soft (0,5kPa) or stiff (80kPa) Col I coated PA hydrogel, as previously described, and collected at the end of the mechanical priming. RNA-seq was performed on poly(A) selected RNA extracted from 500 000 cells of mouse osteoblast cell line using the RNeasy Plus Mini kit (Qiagen) following manufacturer's protocol. Poly-(A)-selected, first stranded Illumina libraries were prepared with a modified

TruSeq protocol using dUTP method 368 . Three biological replicates per conditions were prepared. Libraries were size selected using AMPure XP SPRI beads, were amplified by PCR (maximum 16 cycles) and purified with AMPure XP beads and paired-end sequenced (75 bp) on the Illumina NextSeq 500 sequencer.

Sequencing reads were pseudo aligned to the ENSEMBL GRCm38 version of the mouse genome and abundance quantified using Kallisto 369 . Differential expression analysis was done using Sleuth R package 370 . Volcano plots and heatmap were plotted using R packages ggplot2 and pheatmap.

I.13. CUT&Tag and analysis

Osteoblasts were mechanical primed for three days on either a soft (0,5kPa) or stiff (80kPa) Col I coated PA hydrogel, as previously described and cells were collected at the end of the 96 mechanical priming. Cut&Tag experiments were performed using the CUT&Tag-IT TM Assay Kit (Active Motif) in two replicates following manufacturer's instructions. Briefly, 50 000 cells per sample were isolated and bound to Concanavalin A paramagnetic beads, incubated with rabbit anti-mouse H3K27me3 monoclonal antibody (Diagenode) or rabbit anti-mouse KDM7a antibody and then with goat anti-rabbit polyclonal secondary antibody. Subsequently, pA-Tn5 was used to tagment the genomic regions surrounding each bound antibody complex. Proteinase K was added to the reaction to stop tagmentation and solubilize DNA. DNA was then purified using columns provided by the manufacturer and PCR was used for the subsequent library amplification. Each sample was associated with a combination of i7 Indexed Primers and i5

Indexed Primers in the PCR amplification reaction. These Indexed Primers are based on Illumina's Nextera adapters. SPRI beads were used to purify DNA. Libraries quality control was assessed using the DNA High Sensitivity kit on an Agilent Bioanalyzer instrument.

Libraries were quantified by quantitative PCR using the Kapa Library quantification kit and sequenced and sequenced using 37 bp paired-end sequencing on an Illumina NextSeq 500 instrument.

Sequencing reads were aligned to the mouse mm10 version of the mouse genome using Bowtie2 371 with the following parameters: --end-to-end --very-sensitive --no-mixed --nodiscordant --phred33 -I 10 -X 700. Peak calling was realized using the RECOGNICER module of SICER2 372 software keeping duplicates and using the following parameters: -w 4000 -egf 0.97 -f 150 -fdr 0.001.

BigWig files normalized for the depth of sequencing of aligned filtered reads were generated using deeptools 373 . Profile and heatmap of normalized read densities were performed using Deeptools.

I.14. Statistical analysis

Graphics were prepared with the open-source software Prism V8.0 and statistical analyses (multiple t tests) were performed using said software.

Results

I. Mechanical memory is cell type dependent and requires the actomyosin cytoskeleton

Mechanical memory has been defined as the cell's ability to retain information and the effects of past mechanical cues and has been shown to affect many cellular processes such as, but not limited to, differentiation, cell migration and chromatin accessibility.

I.1. Mechanical memory is cell type dependent

We first attempted to study cells' ability to establish a mechanical memory using three cell types: immortalised mouse osteoblasts and two human mammary cancer cell lines, HS 578T

and MDA-MB-231. In order to control the mechanical cues cells were exposed to, we utilised polyacrylamide gels of various rigidities, ranging from 0,5 to 80kPa, coated with collagen type I (Col I) to render them adhesive to the cells. Based on the litterature 147,348,353 , we considered hydrogels with a rigidity inferior to 2kPa to be soft while rigidities over 80kPa were considered stiff. Cells were mechanically primed on either a soft (0,5kPa) or stiff (80kPa) Col I coated hydrogel for a period of three days (Fig. 27A), because Nasrollahi et al 353 reported that this priming period is sufficient for both non-cancerous and cancerous MCF-10A and MCF-7 breast cell lines to establish a mechanical memory. At the end of the priming period, cells were passaged and seeded on a secondary stiff (80kPa) substrate before being imaged by time-lapse microscopy for 24h. The effect of past mechanical cues was assessed on the secondary substrate using migration speed as a readout.

I.1.1. Mechanical memory affects cell migration in a cell type dependent manner

We observed that the migration speed of both osteoblasts and HS 578T cells on the secondary substrate was affected by the priming rigidity (Fig. 27B). Indeed, on a stiff secondary substrate, cells that had been previously cultured on a soft substrate migrated at a lower speed than cells that had always been cultured on a stiff hydrogel. Thus, a three day priming period is sufficient for both osteoblasts and HS 578T cells to set-up a mechanical memory. Interestingly, MDA-MB-231 cells presented similar migration speeds on the secondary substrate, regardless of the priming rigidity (Fig. 27B). Consequently, MDA-MB-231 are unable to establish a mechanical memory after a three day priming period. We proceeded to extend the mechanical priming to a 98 seven day period, suspecting three days might not be sufficient for this cell line to set-up a mechanical memory (Fig. 27C). However, similarly to a three day priming period, MDA-MB-231 cells showed equivalent migration speeds on a stiff secondary substrate whether they were previously primed on a soft or stiff substrate. Although this experiment requires additional replicates, as it was only executed once, it suggests that even a seven day priming period might not be sufficient for MDA-MB-231 cells to set-up a mechanical memory. We questioned whether MDA-MB-231 cells are mechanosensitive and actually adjust their migration velocity as a function of substrate stiffness. We observed that cells that did not undergo any specific priming period migrated at a much lower speed when seeded on a soft hydrogel than on a stiff one, much like osteoblasts and HS 578T cells (data not shown). Thus, it appears that MDA-MB-231 cells are only sensitive to their immediate surroundings unlike osteoblasts and HS 578T which are also influence by past mechanical cues.

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We detected in cells lines capable of mechanical memory, and more noticeably with faster migrating osteoblasts, that on the stiff secondary substrate, soft-primed cells tended to experience difficulties to retract their rear compared to stiff-primed cells (Fig. 27D). These observations seem to hint at the fact that soft-primed cells could be more adhesive than stiffprimed cells, which might explain why their migration speed is reduced on a stiff secondary substrate.

I.1.2. Mechanical memory does not seem to affect cell spreading or nuclear size

Cell spreading and nuclear size are known to vary depending on ECM stiffness. Indeed, we observed that all three cell lines presented a rounded morphology on soft (0,5kPa) hydrogels while they were more spread out on stiff hydrogels (Fig. 28A). This confirmed that all three cell lines are capable of sensing the mechanical properties of their environment and performing mechanotransduction. As mechanical memory has also been shown to affect both cell spreading 356 and nuclear size 361 , we wondered whether similar results could be seen in the two cell lines affected by mechanical priming (osteoblasts and HS 578T cells). All three cell lines 100 were mechanically primed for three days on either a soft (0,5kPa) or stiff (80kPa) Col I coated hydrogel, then cultured for a day on a secondary stiff substrate, a Col I coated glass coverslip, before being fixed and stained for immunofluorescence analysis (Fig. 28B). Glass coverslips were chosen as a secondary substrate over 80kPa hydrogels for technical reasons. A minimum thickness must be maintained for polyacrylamide hydrogels as an excessive lowering of the thickness modifies the hydrogels rigidity. As epifluorescence microscopes at a 60x magnification present a relatively small working distance, it was technically more challenging to work with cells plated on hydrogels than coverslip. Furthermore, it has been speculated that cells are unable to sense variations in rigidity above certain thresholds, the upper threshold often Surprisingly, cell and nuclear size (Fig. 28C and B respectively) were unaffected by mechanical priming in all three cell lines. One could have expected to see an effect of mechanical priming on cell and nuclear size in osteoblasts and HS 578T cells since priming rigidity affects migration speed in both cell lines. It is possible that both cell lines are sensitive to differences in substrate rigidity between the glass coverslip (>GPa) and the stiff hydrogels (80kPa). Therefore, seeding cells on the extremely stiff glass coverslip could potentially hinder differences in cell spreading that would've otherwise been visible on an 80kPa hydrogel. Thus it would be interesting to culture cells on a secondary 80kPa hydrogel and see if comparable results are observed in such conditions. Similarly to what had been observed on cell migration speed, MDA-MB-231 cells presented no effect of mechanical priming on cell and nuclear size on a stiff secondary substrate. Curiously, Syed et al 356 showed that matrix stiffness did impact MDA-MB-231 cell size and spreading rate, using hydrogels with equivalent priming rigidities (1kPa and 100kPa). However, the study highlighted that MDA-MB-231 cells adapted to rigidities so that, for instance, cells mechanically primed on a soft (1kPa) hydrogel were more spread on a soft (1kPa) secondary substrate compared to cells that had previously been cultured in tissue culture plastic (TCP). Similarly, they observed that on a 100kPa secondary substrate, cells that had always been cultured on a 100kPa hydrogel had higher spreading rates then cells that were previously on TCP. Therefore, it would be interesting to culture this cell line on an 80kPa secondary substrate to see if comparable results would be observed.

I.2. Mechanical dosing in osteoblasts

It has been shown that mechanical memory is a time dependent process. For instance, cells need to be exposed to mechanical cues for a sufficient amount of time to set-up mechanical memory 351,353 . Therefore we decreased the mechanical priming period of osteoblasts to two days and observed that it was sufficient for the cell line to establish a mechanical memory (Fig. It was also shown that the length of the mechanical priming periods correlates with the length over which mechanical memory is maintained and the strength of the phenotype. For example, Yung et al 351 showed that hMSCs primed for ten days on a stiff substrate presented a stronger nuclear localisation of YAP/TAZ and RUNX2 than cells primed for seven or one day.

Furthermore, the longer hMSCs were primed (or dosed), the longer the memory was maintained. Thus, we increased the length of mechanical priming, using osteoblasts, to see if it affected mechanical memory. However we were met with technical challenges as osteoblasts tended to form aggregates after three to four days on a soft hydrogel, which would increase in size over time. Although, cells were sparsely seeded on the hydrogels to prevent the formation of aggregates on the first two to three days of mechanical priming on soft substrates, it was insufficient to counteract it on longer priming periods. We anticipated that this could affect mechanical memory if cells lost their contact with the ECM, which is why we chose to passage cells every three days, over four passages (twelve days), culturing them on the same rigidity from one passage to the next (Fig. 29B). At the end of each passage, some cells were collected and transferred to a stiff secondary substrate for imaging. We observed similar trends over the four passages (Fig. 29C). On a stiff (80kPa) secondary substrate, cells that were primed on a 104 soft (0,5kPa or 2kPa) hydrogel tended to show reduced migration speeds compared to cells that had always been cultured on a stiff (80kPa or plastic) substrate. Interestingly, migration speeds did not seem be significantly affected by mechanical dosing. Indeed, migration speeds observed after a three day or twelve day priming period were quite similar for a given priming rigidity although we observed a trend towards lower migration speed with increasing culture time. It is possible that, unlike YAP/TAZ and RUNX2 activation, migration speed rapidly plateaus at a maximum or minimum, and is therefore not as sensitive to mechanical dosing. However, more replicates are required to conclude as the experiment was only performed twice.

I.3. Mechanical memory seems to be independent of protein coating

As previously mentioned, polyacrylamide gels are coated with Col I to increase cell adherence to otherwise non-adherent hydrogels. We wondered whether protein coating affected mechanical memory. To explore this hypothesis, we cultured osteoblasts for three days on either a soft (0,5kPa) or stiff (80kPa) Col I coated hydrogel (Fig. 30A). The cells were then passaged and seeded on a secondary stiff (80kPa) hydrogel, coated with either Col I or fibronectin (FN1).

We observed that on a FN1 coated stiff secondary substrate, soft-primed cells still presented lower migration speeds than stiff-primed cells (Fig. 30B). It seems that mechanical memory is not dependent on protein coating, although further matrix proteins should be tested to confirm these results.

I.4. Mechanical memory requires the actomyosin cytoskeleton

The actin cytoskeleton plays a pivotal role in mechanotransduction, as it participates in force transmission to the nucleus through the LINC complex and also generates forces transmitted to the ECM through focal adhesions. We wondered if targeting the actin cytoskeleton during the mechanical priming would impact the cells ability to acquire a mechanical memory. Osteoblasts were mechanically primed for three days on a soft (0,5kPa) or stiff (80kPa) Col I coated hydrogel and treated on the first day with either blebbistatin (10µM) or cytochalasin D (0.5µM), respectively a myosin II ATPase inhibitor and an actin polymerisation inhibitor (Fig. 31A). The cells were washed before being passaged and transferred to a stiff secondary substrate (80kPa) and imaged by time lapse microscopy.

We observed that blebbistatin and cytochalasin D treatment during the priming period did not seem to affect the migration speed of soft-primed cells (Fig. 31B). On the other hand, stiffprimed treated cells treated with these drugs presented a lower migration speed compared to that observed in stiff-primed control cells. Interestingly, stiff-primed treated cells presented a similar migration speed to that observed in soft-primed control cells. Thus, a functional actomyosin network is necessary to set up a mechanical memory during the priming period.

This may simply reflect the central role of the actin cytoskeleton in mechanotransduction:

without the equipment to sense substrate rigidity during the priming period, cells may adapt by default to a phenotype that is similar to cells primed on soft gels.

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II. Mechanical memory affects the epigenetic landscape II.1. Disrupting the epigenetic landscape impedes stiff-associated mechanical memory

Mechanical memory relies on the retaining of past information. One of the most encouraging hypotheses is that this mechanical information could be stored through epigenetic modifications. Price et al 374 proposed a mathematical model for mechanical memory in which epigenetic modulators play a central role. Furthermore, Killaars et al 361 showed, using hMSCs, that mechanical priming affected both chromatin condensation and acetylation levels. On a soft secondary substrate, stiff-primed cells presented higher acetylation levels and lower chromatin condensation than cells that had always been cultured on a soft substrate. Therefore, we decided to generally perturb widely epigenetic modification dynamics, more specifically DNA methylation and histone acetylation, during the mechanical priming to see if it affected mechanical memory. Osteoblasts were mechanically primed on a soft (0,5kPa) or stiff (80kPa)

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Col I coated hydrogel and treated with either 5-azacytidine (5-aza -1µM), a cytidine analogue preventing DNA methylation, or garcinol (5µM), a histone acetyltransferase (HAT) inhibitor (Fig. 31A). As 5-azacytidine is incorporated in the DNA during mitosis, cells were treated daily for the duration of the mechanical priming, whereas garcinol was only added to the media on the first day of priming. Osteoblasts were also treated with trichostatin A (TSA -37.25nM), a histone deacetylase (HDAC) inhibitor on the first day of mechanical priming although the priming period was shortened to two days, to reduce drug toxicity. The cells were washed before being passaged at the end of the priming period and transferred to a stiff secondary substrate (80kPa) where time lapse microscopy was performed.

We observed that none of the drugs affected migration speed of soft-primed cells which was comparable to that of the soft-primed control cells (Fig. 32A andB). However, both 5-aza and TSA treated stiff-primed cells presented reduced migration speeds compared to that observed in stiff-primed control cells. Similarly to what was observed with actomyosin inhibitors, 5-aza and TSA treated stiff-primed cells showed similar migration speeds to that of the soft-primed control cells. Curiously, garcinol treatment did not affect the migration speed of stiff-primed cells. It is interesting to note that DNA methylation is associated with gene repression while histone acetylation is associated with gene activation. As 5-aza and TSA respectively decrease DNA methylation and increase histone acetylation, both should positively affect gene expression. Therefore, these result could hint at a possible involvement of gene repression in mechanical memory and, more specifically, stiff-associated mechanical memory. In general, the epigenetic landscape seems to play an important part in the establishment of stiff-associated mechanical memory. Another possibility that we cannot exclude at this stage and that will require further investigation is that these drugs may perturb the cell's mechanosensitivity during the priming period.

II.2. Identification of potential targets through RNAseq

ECM rigidity is known to affect gene expression, as previously developed. Consequently, we performed RNAseq on osteoblasts to compare the transcriptome of soft-and stiff-primed cells in an attempt to identify potential targets involved in mechanical memory set-up. Cells were culture for three days on either a soft (0,5kPa) or stiff (80kPa) Col I coated hydrogel and were collected at the end of the priming period for RNA extraction and sequencing (Fig. 33A).

In total, 217 genes were found to be differentially regulated, with an adjusted p-value inferior to 0.05, between osteoblasts cultured on a soft and stiff hydrogels (Fig, 33B andC). Among them, gene ontology (GO) analysis revealed genes involved in ECM composition, FAs and cytoskeletal organisation as well as genes implicated in cell proliferation, survival and cell cycle 109 progression. Interestingly, ECM proteins such as fibronectin (FN1), collagens including but not limited to collagens VIa/IIIa/Va, tenascin C (TNC) and syndecan 2 were found to be mostly up-regulated in cells cultured on a soft substrate. Similarly, integrins such as α2a, α8 and α10

were also found to be up-regulated in cells cultured on a soft hydrogel. Regarding the cell cycle, we detected the up-regulation, in osteoblast cultured on a soft substrate, of three genes (Growth arrest-specific protein 1 (GAS1), nbl1 and Protein yippee-like 4 (Ypel4)) involved in growth arrest, specifically at the G1/S transition. Conversely, we noticed the up-regulation, in osteoblast cultured on a stiff substrate, of two genes (transcription factor E2F4 and G1/Sspecific cyclin-D1 (Ccnd1) which positively regulate the G1/S transition. demethylases were found to be up-regulated in soft-primed osteoblasts, namely KDM7a and hairless (HR). KDM7a is a demethylase with dual-specificity towards H3K9 me2 and H3K27

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We first examined KDM7a expression levels in our three cell lines (osteoblast, HS 578T and MDA-MB-231). Cells were mechanically primed for three days on a soft (0,5kPa) or stiff (80kPa) Col I coated hydrogel before being passaged and transferred to a stiff secondary substrate (Col I coated glass) where cells were fixed and processed for immunofluorescence analysis. We observed, on the stiff secondary substrate, that both soft-primed osteoblasts and HS 578T cells tended to present higher KDM7a intensity levels compared to their stiff-primed counterparts (Fig. 34A andB). However, on a stiff secondary substrate, MDA-MB-231 cells tended to have similar KDM7a intensity levels regardless of the priming rigidity. These experiments need to be replicated to validate our initial observations. Additionally, western blots will be made to further confirm these results. It seems that KDM7a levels are sensitive to mechanical priming in our two cells lines capable of establishing a mechanical memory (osteoblast and HS 578T) but not in MDA-MB-231 which do not exhibit mechanical memory.

This suggests that KDM7a could be involved in mechanical memory set-up. We attempted similar experiments to examine HR intensity levels in mechanically primed cells.

Unfortunately, as of yet, we have not found an antibody that functions in our cell lines.

III.2. KDM7a depletion during mechanical priming impairs mechanical memory

We then tested whether the two demethylases affected mechanical memory in osteoblasts by depleting them, using siRNAs targeting either KDM7a or HR, during mechanical priming. Cells underwent a double depletion at -6 and -4 days, prior to mechanical priming (Fig. 34C), to ascertain depletion during the conditioning period. Additionally, the time frame of depletion was calculated so that the protein of interest would be no longer be depleted, and would therefore be fully expressed, when time-lapse microscopy was performed on the stiff secondary substrate. We reasoned that those proteins could potentially play an important role in cell migration, therefore depleting them during the migration assay could bias our read-out of mechanical memory. Following the depletion, cells were cultured for three days on a soft (0,5kPa) or stiff (80kPa) Col I coated hydrogel before being passaged and transferred to a stiff secondary substrate (80kPa) where time-lapse microscopy was performed. Depletion was verified in classical culture conditions (TCP flasks) by Western Blot (WB), four days after the second depletion, amounting to the end of the priming period (day 0). Due to the limited number of cells we were able to collect on hydrogels, all depletion verifications were performed in TCP conditions. We found that, at day 0, siKDM7a and siHR cells presented a partial depletion

We observed that depleting HR did not affect mechanical priming as migration speed was still impacted by mechanical priming upon depletion (Fig. 34D). Indeed, on a stiff secondary substrate, soft-primed siHR cells presented a similar migration speed to soft-primed siCtrl cells.

Similarly, siCtrl and siHR stiff-primed cells showed equivalent migration speeds. However, we observed that siKDM7a cells presented similar migration speed regardless of the priming rigidity. On a stiff secondary substrate, soft-primed siKDM7a cells were found to present a significant increase in migration speed compared to soft-primed siCtrl, comparable to that observed in stiff-primed siCtrl cells. Stiff-primed siKDM7a cells displayed a slight, but nonsignificant, decrease in migration speed compared to stiff-primed siCtrl. It seems that upon KDM7a depletion, cells are no longer impacted by mechanical priming but only by their immediate surroundings. These results are not dissimilar to what was previously observed in

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MDA-MB-231 cells, which we found to be only sensitive to their immediate environment.

Thus, KDM7a appears to be involved and necessary for mechanical memory set-up.

We then looked at the effect of KDM7a depletion, during mechanical priming, on KDM7a intensity levels on a stiff secondary substrate (Col I coated glass -Fig. 34E)). We observed that siKDM7a cells tended to present similar KDM7a intensity levels, no matter the priming rigidity, and that the intensity levels were comparable to that observed in stiff-primed siCtrl cells.

Although these experiment need to be replicated to validate our results, it is possible that depleting KDM7a during mechanical priming might have prevented an up-regulation of the demethylase in soft-primed cells and hindered the storage of past mechanical information.

Additional WBs comparing KDM7a protein levels at the end of the mechanical priming and on the stiff secondary substrate, in siCtrl and siKDM7a conditions, will be done in an attempt to confirm this hypothesis.

III.3. Mechanical memory modulates H3K27 me2/3 levels

As previously mentioned, KDM7a is a demethylase that targets both H3K27 me2 and H3K9 me2, two heterochromatin marks. Therefore, we proceeded to look at the effect of mechanical priming on H3K27 me3 and H3K9 me2/3 intensity levels in our different cells lines. As KDM7a targets H3K27 me2, we assumed that the demethylase would indirectly affect H3K9 me3 levels.

However, it would be interesting to also look at H3K27 me2 levels. Cells were mechanically primed on a soft (0,5kPa) or stiff (80kPa) Col I coated hydrogel for three days before being passaged and transferred on a stiff secondary substrate (Col I coated glass) where cells were fixed and stained.

Regarding H3K27 me3 staining (Fig. 35 A and B), we observed that both stiff-primed osteoblasts and HS 578T cells tended to present higher fluorescence intensity than their softprimed counterparts. Even though further replicates are required since experiments were only done twice, these results suggest that in our cell lines exhibiting mechanical memory, stiffprimed cells present higher H3K27 me3 than soft-primed cells. Interestingly, MDA-MB-231 cells tended to present similar H3K27 me3 intensity, regardless of mechanical priming. Once more, these experiments need to be replicated to confirm those preliminary observations.

However, these results appear to correlate with our previous observations on KDM7a and its probable role in mechanical memory. Concerning H3K9 methylation, in all three cell lines, soft-and stiff-primed exhibited similar H3K9 me2/3 intensity levels, regardless of mechanical We then proceeded to investigate whether modulating H3K27 methylation levels, during mechanical priming, affected mechanical memory. We chose to work with two PRC2 inhibitors, A395 (4µM) and UNC1999 (2µM), as PRC2 complexes are the main writers for H3K27me2/3. A395 and UNC1999 are selective inhibitors for EZH2-EED-SUZ12 and EZH2/1 respectively and should decrease overall H3K27 me2/3 through inhibition of PRC2. Osteoblasts were mechanically primed on soft (0,5kPa) or stiff (80kPa) Col I coated hydrogels for three days, and treated with either one of the two PRC2 inhibitors on the first day of mechanical priming. At the end of the priming period, cells were washed and passaged before being transferred to a stiff secondary substrate (80kPa) where time-lapse microscopy was performed.

We observed, on a stiff secondary substrate, that PRC2 inhibitors did not seem to affect the migration speed of soft-primed cells which was comparable to that of soft-primed control cells (Fig. 35D). Although soft-primed UNC1999 treated cells presented a slight decrease in migration speed compared to soft-primed control cells, further replicates are needed to confirm whether this tendency is significant or not as the experiment was only performed once.

However, both A395 and UNC1999 treatment affected the migration speed of stiff-primed cells which was reduced compared to that of stiff-primed control cells. Interestingly, A395 and

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UNC1999 stiff-primed cells both exhibited similar migration speeds to that observed in softprimed control cells. It seems that inhibiting PRC2 during mechanical priming impedes stiffassociated mechanical memory set-up. Even though these tests are yet to be done, the efficiency of both PRC2 inhibitors will be evaluated by WB and IF.

III.5. Identification of differentially methylated genes through CUT&Tag

Our previous observations suggest that KDM7a plays a key role in mechanical memory set-up.

Furthermore, we found that an up-regulation of KDM7a in soft-primed cells is accompanied with a decrease in H3K27 me3. Thus, we hypothesised that KDM7a could target genes implicated in mechanical memory. In an attempt to identify downstream KDM7a target genes, we decided to performed CUT&Tag as it requires less material than CHIPseq or CUT&RUN 381 .

As previously mentioned, osteoblasts must be sparsely seeded on the hydrogels to prevent aggregate formation, especially on soft hydrogels. Therefore, performing either CHIPseq or CUT&RUN would have proven even more challenging. In parallel to KDM7a, we performed CUT&Tag on H3K27 me3 as it is both a positive control for this method and has been shown to be affected by KDM7a depletion. Osteoblasts were cultured for three days on a soft (0,5kPa)

or stiff (80kPa) Col I coated hydrogel before being collected at the end of the priming period for analysis.

Unfortunately, we were unable to analyse results obtained with the KDM7a antibody as they were close to the IgG negative control (data not shown) and showed non-specific targeting of the antibody, even though the antibody had been successfully tested in IF 382 . However, it is possible that the antibody specificity was not sufficient or needed further optimisation for this method. It is also likely that the method used was, in fact, not optimal for KDM7a as it has been

shown that the transposase (pA-Tn5) complex used for tagmentation in CUT&Tag is quite bulky and can reduce resolution due to steric effects 382 .

Fortunately, CUT&Tag approved H3K27 me3 antibodies were used and we were able to successfully sequence and compare soft and stiff cultured osteoblasts. It is important to note that each condition was duplicated to ensure reproducibility. However, one of the duplicates for cells cultured on a stiff substrate presented similar specificity to the IgG negative control (data not shown) and could not be using in the analysis. Therefore, this experiment will require to be redone in order to validate the following observations. CUT&Tag results done on H3K27 me3 revealed that cells cultured on a stiff hydrogel presented more H3K27 me3 peaks than cells 118 cultured on a soft substrate (Fig. 36A). Interestingly, few H3K27 me3 peaks were specific to cells cultured on a soft hydrogel and absent in cells cultured on a stiff substrate (Fig. 36Ai). In contrast, many H3K27 me3 peaks were found to be specific to cells cultured on a stiff rigidity (Fig. 36Aiii). When comparing peaks that overlapped between the two conditions, it was generally observed that H3K27 me3 signal tended to be slightly higher in cells cultured on a stiff rigidity (Fig. 36Aii). These observations seem to corroborate what we previously observed in osteoblasts using H3K27 me3 IF staining. Furthermore, they suggest that gene repression could be involved in mechanical memory set-up, as H3K27 me3 is a heterochromatin mark involved with gene repression.

We then compared the RNA sequencing hits, specifically genes downregulated in osteoblasts seeded on a stiff substrate, with hypermethylated genes also detected in stiff-primed osteoblasts through CUT&Tag. We reasoned that this could possibly enable us to further restrict the number of genes of interest potentially involved in mechanical memory set-up. Based on our original RNAseq selection of differentially regulated genes with an adjusted p-value under 0.05 (217 genes in total), we found very little overlap between the RNAseq hits and hypermethylated genes. Therefore, we decided to be less stringent with our RNAseq selection and expanded to differentially regulated genes with a p-value under 0.05 (>1500 hits). Through this second, broader selection, we identified 48 genes that were both downregulated (RNAseq) and hypermethylated (CUT&Tag) in osteoblasts cultured for three days on a stiff substrate. Surprisingly, we detected three genes coding for proteins involved directly or indirectly in integrin signalling: Cdc42 effector protein 5 (Cdc42ep5), Rho-h and Akt3. Cdc42ep5 belongs to the Borg protein family which remains elusive at large. However, Cdc42ep5 can bind Cdc42

and have been shown to promote lamellipodium formation 383 . Rho-h is an atypical Rho family member as it lacks the GTP hydrolysis ability. However, it has been shown to not only participate in the regulation of the actin cytoskeleton but also transcription through its association with Kaiso, a transcription modulator 384 . Akt3 is part of the Ser/Thr kinase Akt family comprising three isoforms (Akt1/2/3). The AKT pathway has been shown to regulate cell survival, proliferation and migration and to be enhanced with matrix stiffness at focal adhesions 208,209 . These results suggest that integrin signalling could be involved in mechanical memory and should be investigated in the context of mechanical memory, as will be developed in the discussion.

IV.1. Mechanical memory affects FN1

We sought to investigate proteins that could be downstream effectors of mechanical memory and could affect cell migration. To do so, we looked at the differentially regulated genes identified through our RNAseq. Intriguingly, RNAseq results suggested that several matrix proteins were up-regulated in osteoblasts primed on a soft hydrogel (Fig. 33B). Furthermore, on a stiff secondary substrate, soft-primed osteoblasts seemed to be more adhesive than their stiff-primed counterparts (Fig. 27D). We reasoned that the decrease in cell adhesion on a softsubstrate could lead to an increase in matrix protein secretion. Our hypothesis is that increased adhesion capacities in soft primed cells may impair migration velocity. Consequently, we selected three hits corresponding to ECM proteins identified in our RNAseq analyses: FN1, Col VI and TNC. Unfortunately, we encountered difficulties with the Col VI and TNC antibodies, especially in our mouse osteoblast cell line and decided, for the time being, to concentrate on FN1. Thus we looked at the effect of mechanical memory on FN1 in all three cell lines. Cells were mechanically primed for three days on a soft (0,5kPa) or stiff (80kPa) Col I coated hydrogel before being passaged and transferred on a stiff secondary substrate (Col I coated glass) where cells were fixed and processed for immunofluorescence analysis.

We observed, in the two cell lines capable of mechanical memory (osteoblast and HS 578T), that on a stiff secondary substrate, soft-primed cells tended to exhibit an increase in FN1 signal compared to stiff-primed cells (Fig. 37A andB). In both cell lines, soft-primed cells presented more FN1 fibrils than stiff-primed cells (Fig. 37A). However, MDA-MB-231 cells tended to present similar, low FN1 intensity levels regardless of the priming rigidity. Very few FN1 fibrils could be detected in this cell line. Although these experiments required additional replicates, it appears that cells sensitive to mechanical priming show variations in FN1 levels depending on the priming rigidity. Up-regulation of FN1 has been shown to facilitate adhesion and cell spreading in softer environments such as 3D cultures 385 . We reasoned that, on soft substrates (0,5kPa), decreased adhesion could lead to an increase in FN1 secretion to facilitate cell attachment and spreading.

It would have been interesting to corroborate these observations with FN1 secretion. As FN1 is an integral part of FBS, this method would have required for cells to be cultured in the absence 121 of serum. However, we found that mechanically priming cells with only 2% serum affected mechanical memory (data not shown). On a stiff secondary substrate, 2% stiff-primed cells showed a decrease in migration speed compared to 10% stiff-primed control cells. Therefore we were unable to look at FN1 secretion.

IV.2. FN1 depletion does not affect mechanical memory

Earlier observations suggest that, in cell lines capable of mechanical memory, soft-priming tends to lead to an increase in FN1 levels (Fig. 37B). We also previously detected that, on a stiff secondary substrate, soft-primed osteoblasts seemed to be more adhesive as they experienced more difficulties to retract their rear (Fig. 27D). Thus we hypothesised that the effect of mechanical memory on cell migration could be caused by an increase in cell adhesion, in soft-primed cells, which would in turn impede migration speed. To test this hypothesis, we depleted FN1 during mechanical priming, reasoning that by hindering the cells ability to upregulate FN1 it would prevent soft-primed cells from becoming more adhesive and would impair mechanical memory. Depletion was validated by WB (data not shown). Cells underwent a double depletion (siRNA) at -6 and -4 days, prior to substrate transfer (Fig. 34C). Depleted cells were cultured for three days on a soft (0,5kPa) or stiff (80kPa) Col I coated hydrogel before being passaged and transferred to a stiff secondary substrate (80kPa) Col I coated hydrogel where time-lapse microscopy was performed.

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We detected that FN1 depletion did not affect mechanical memory set-up (Fig. 37C). Indeed, on a stiff secondary substrate, soft-primed siFN1 cells still presented a significant decrease in migration speed compared to stiff-primed siFN1 cells. Furthermore, soft-primed siFN1 osteoblasts presented similar migration speed to the soft-primed siCtrl cells. Similarly, stiffprimed siFN1 and siCtrl cells exhibited comparable migration speeds. Depleting FN1 during mechanical did not affect mechanical memory, suggesting that FN1 is not involved in mechanical memory set-up. However, as many other ECM proteins (Col VI/III/V, TNC, Syn2)

were detected to be up-regulated in cells cultured on a soft substrate, one can wonder if FN1 depletion could have been compensated by other ECM proteins. Targeting a unique ECM protein might not be enough to conclude on our hypothesis.

IV.3. KDM7a regulates FN1 expression

We previously showed that KDM7a regulates mechanical memory and wondered whether it also impacted FN1 levels. For this reason, we looked at FN1 levels in osteoblasts depleted for KDM7a during the priming period. Cells underwent a double depletion (siRNA) at -6 and -4 days, prior to mechanical priming (Fig. 34C). They were then cultured for three days on a soft (0,5kPa) or stiff (80kPa) Col I coated hydrogel before being passaged and transferred to a stiff secondary substrate (Col I coated glass) to be fixed and stained.

We observed that siKDM7a osteoblasts tended to present similar, low FN1 intensity levels regardless of the priming rigidity (Fig. 37D). Furthermore, siKDM7a cells exhibited decreased FN1 intensity levels compared to siCtrl cells. Although further replicates are required, it appears that KDM7a controls FN1 expression levels. These observations make us wonder whether FN1 could be an effector of mechanical memory regulated by KDM7a.

V. Mechanical priming affects cell proliferation V.1. Mechanical memory affects cell proliferation in osteoblasts

Mechanical memory has been shown to affect cell proliferation 348 . Balestrini et al provided evidence that, whether on a soft or stiff secondary substrate, stiff-primed cells consistently presented an increase in cell number compared to soft-primed cells. For this reason, we looked at the effect of mechanical priming on osteoblast proliferation. Cells were cultured for three days on a soft (0,5kPa) or stiff (0,5kPa) Col I coated hydrogel before being passaged and transferred to a stiff (80kPa) secondary substrate where time-lapse microscopy was performed.

Osteoblasts have a very short cell cycle (12h), therefore we were able to count the number of dividing cells over the 16h time-lapse video and normalise it to the total number of cells observed at the beginning of the video. However, HS 578T and MDA-MB-231 cells present longer cycling periods which prevented the use of this method to look at proliferation in these two cell lines. Staining HS 578T and MDA-MB-231 cells on a stiff-secondary substrate with a proliferation markers such as Ki67 will be preferable to study the effect of mechanical priming on cell proliferation in those cell lines.

We observed that, in osteoblasts, mechanical memory not only impacted migration speed but also affected cell proliferation. Indeed, on a stiff secondary substrate, stiff-primed osteoblasts presented a two-fold increase in proliferation rate compared to soft-primed cells (Fig. 37A).

V.2. Disrupting actomyosin contractility and DNA methylation perturbs the effect of mechanical memory on proliferation

As we had selected migration speed as a readout for mechanical memory, we sought to investigate whether drugs previously tested (namely 5-aza, garcinol and blebbistatine) had similar effects on cell proliferation. Garcinol was found to have no significant effect on mechanical memory (Fig. 32A) while 5-aza and blebbistatine (Fig. 32A and31B) both decreased the migration speed of stiff-primed cells. As previously mentioned, cells were treated daily with 5-aza (1M) for the duration of the mechanical priming, whereas blebbistatin (10M) and garcinol (5M) was only added to the media on the first day of priming. At the end of the priming period, cells were washed and passaged before being transferred to a stiff secondary substrate (80kPa) where time-lapse microscopy was performed.

We observed, on a stiff secondary substrate, that none of the drugs affected the proliferation rates of soft-primed cells (Fig. 38A). On the other hand, both 5-aza and blebbistatine treated stiff-primed cells presented a significant decreased in proliferation rates compared to stiffprimed control cells. In fact, 5-aza and blebbistatine stiff-primed cells exhibited proliferation rates comparable to that observed in soft-primed control cells. However, garcinol treatment do not significantly affect proliferation rates of either soft-or stiff-primed cells compared to their respective control. Furthermore, garcinol treated stiff-primed cells still exhibited a significant increase in cell proliferation compared to garcinol treated soft-primed cells. It appears that the three drugs present similar effects on migration speed and cell proliferation.

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V.3. Is the effect of mechanical memory on cell proliferation due to cell cycle arrest?

Cell size 386 , matrix stiffness 387 and mechanical strain 388 have been shown to affect cell cycle progression, particularly through G1/S transition. Uroz et al 389 also showed that cells subjected to higher mechanical tension were more likely to transition from G1 to S and that they presented shorter G1 and S-G2-M phases. According to the RNAseq performed on osteoblasts (Fig. 33B), cells cultured on a soft hydrogel for three days presented an up-regulation of genes involved in G1/S cell cycle arrest while cells cultured on a stiff substrate presented an up-regulation of genes facilitating G1/S transition. Therefore we wondered whether the decrease in cell proliferation observed in soft-primed cells could be due to an arrest in G1/S. We used a fluorescence ubiquitination-based cell cycle indicator (Fucci), which was stably expressed in osteoblasts, allowing us to monitor cell cycle progression. This reporter relies on the expression of two fusion proteins: a portion of the Gemini protein fused with GFP and a portion of Cdt1 fused with RFP. Cdt1 is expressed during G1 and plays a key role in DNA replication while Gemini is a Cdt1 inhibitor necessary for cell cycle progression. Thus, Fucci cells will express the Cdt1-RFP (red) fusion protein when in the G1 phase and the Gemini-GFP (green) fusion for three days on a soft (0,5kPa) or stiff (80kPa) Col I coated hydrogel before being collected for FACS analysis (Fig. 38C).

Combining two replicates we observed that substrate rigidity did not seem to impact the percentage of cells in G1 or S-G2-M (Fig. 38D andE). Indeed, cells cultured on a soft rigidity presented a comparable percentage of cells in G1 as cells cultured on a stiff substrate. Similar results were observed when looked at the percentage of cells in S-G2-M. It seems that cells cultured on a soft substrate do not exhibit a cell cycle arrest in G1/S although the experiment would require more replicates to conclude. Using propidium iodide, on fixed cells, would also allow us to address this question. Propidium iodide is a fluorescent dye that intercalates with DNA. As the DNA content increases over cell division, propidium iodide fluorescence proportionally increases. These differences in fluorescence enable us to determine the percentage of cells in each phase of the cell cycle. Thus, using this technique would provide a more precise representation of the effect of matrix rigidity on the cell distribution in each cell cycle phase.

Conclusion

Cells are known to sense and respond/adapt to the mechanical properties of their immediate surroundings. However, it has been shown that cells can also maintain the effects of past mechanical cues after those cues have subsided, in a process called mechanical memory.

Intriguingly, I found that not all cell types are capable of establishing a mechanical memory.

Furthermore, in our cells types able to set-up a mechanical memory, I observed that a decrease in priming rigidity reduced the migration speed but did not affect cell or nuclear size.

Mechanical memory establishment also seemed to be independent of ECM protein coating.

I observed that the cytoskeleton, specifically actomyosin, plays an important role in mechanical memory. Indeed, impairing actin polymerisation or myosin II contractility, during mechanical priming, affected stiff-associated mechanical memory. As the actin cytoskeleton plays a central role in force transmission, this may simply reflect the significant part actin plays in mechanotransduction.

Interestingly, I also detected that the epigenetic landscape seemed to play a pivotal part in mechanical memory. Widely perturbing epigenetic modification dynamics, such as DNA methylation and histone acetylation, during mechanical priming affected stiff-associated mechanical memory. These results suggest that gene repression could be involved in mechanical memory and, more specifically, stiff-associated mechanical memory.

Through RNA sequencing, I was able to identify a demethylase differentially regulated depending on substrate rigidity: KDM7a. I found that KDM7a expression was affected by mechanical priming only in our cell types capable of establishing a mechanical memory.

Indeed, soft-primed cells exhibited an increase in KDM7a expression. I then depleted KDM7a during mechanical priming, which tended to prevent the increase in KDM7a expression in softprimed cells. This suggests that KDM7a could be an important regulator of mechanical memory, more specifically soft-associated mechanical memory, by enabling gene expression.

I then looked at the methylation levels of H3K9 and H3K27, the two direct targets of KDM7a.

Although H3K9 me2/3 levels were not affected by the priming rigidity, I found that H3K27 me3 levels tended to decrease with the priming rigidity in our cell lines capable of establishing a mechanical memory. Interestingly, inhibiting H3K27 methylation during mechanical memory affected stiff-associated mechanical memory. These results seem to corroborate the involvement of epigenetic modifications in mechanical memory.

I then looked at RNA sequencing hits, specifically fibronectin (FN), which could potentially be a downstream effector of mechanical memory and could explain the decrease in migration speed in soft-primed cells. I observed that FN expression was increased in soft-primed cells capable of establishing a mechanical memory. Interestingly, depleting FN during mechanical priming did not affect mechanical memory suggesting it is not involved in mechanical memory.

However, depleting KDM7a during mechanical priming did affect FN1 expression. These results suggest that FN1 could be an effector of KDM7a but that FN expression level is not sufficient to mediate the effect of mechanical memory on migration speed.

Finally I also observed an effect of mechanical memory on cell division which was affected by the actin cytoskeleton and the epigenetic landscape. It seems that the decrease in division rate observed in soft-primed cells is not related to an increase in the length of the G1 phase, as suspected. It will be interesting to test whether there is a connection between the two phenotypic consequences of mechanical memory on cell velocity and division rate. For example, it will be crucial to determine if both processes are systematically associated in cells able of mechanical memory. It will be equally important to determine whether the same molecular actors of mechanical memory, for instance KDM7a, are involved in the both consequences on migration velocity and division rate.

Discussion and perspectives

Recent studies have brought to light the fact that cells are not only sensitive to their immediate surroundings but are also impacted by previous environments they encountered. The maintenance of the effects of past mechanical cues, also known as mechanical memory, has been shown to impact many cellular processes such as, but not limited to, differentiation, cell migration and chromatin accessibility. Although some players have been identified, namely RUNX2, YAP/TAZ and MicroRNA-21, which have been suggested to serve as "memory storage pathways", the upstream and downstream regulators have not yet been established.

Deepening our knowledge of the mechanisms involved in mechanical memory would be interesting not only in the context of development and cell differentiation, but also in our understanding of the development of certain pathologies such as fibrosis or cancer metastasis.

I. What differentiates cells able and those unable to establish a mechanical memory?

We initially wondered whether different cell types were capable of establishing a mechanical memory. Interestingly, out of the three cell types that we tested (osteoblasts, HS 578T and MDA-MB-231 cells) only two showed an effect of priming rigidity on migration speed.

Preliminary data (not shown) on two other cell types, MDA-MB-436 (human adenocarcinoma) and MRC5 (human lung fibroblasts), suggest that these two cell lines are also unable to establish a mechanical memory after a three day priming period.

Price et al 362,363 developed a mathematical model suggesting that mechanical memory depends not only on the duration but also on the intensity of the mechanical cue. Therefore, it could be argued that 80kPa is not stiff enough or that seven days is insufficient for MDA-MB-231 cells to establish a mechanical memory. However, MDA-MB-231 cells were also mechanically primed on classical tissue culture plastic (TCP) with similar results. Furthermore, MDA-MB-231 cells were shown to present YAP activation and translocation to the nucleus on 9.6kPa and 60kPa 390 substrates, suggesting that the intensity of the mechanical cue does not explain why this cell type do not show mechanical memory.

It would be interesting to compare the transcriptome of cell lines able and unable of establishing a mechanical memory to identify potential targets that could explain these differences.

osteoblasts would be a first step to answering this question. If we do identify some potential targets, it could be interesting to deplete or overexpress them and see if it impedes or rescues mechanical memory.

II. KDM7a as a potential regulator of mechanical memory

II.1. Validating KDM7as involvement in mechanical memory

My results show that, in osteoblasts, soft-primed cells increase KDM7a expression, which is also accompanied by a decrease in H3K27 me3. In addition, KDM7a expression is required to establish a mechanical memory in cells primed on soft. It would be interesting to overexpress KDM7a during a stiff mechanical priming and check if it affects migration speed and H3K27 me3 levels as in soft-primed cells. As soft-primed MDA-MB-231 cells do not seem to overexpress KDM7a, it would also be interesting to overexpress the demethylase in these cells to see if it could 'restore" mechanical memory, including its impact on migration speed and heterochromatin levels. The ultimate demonstration of a role of KDM7a in mechanical memory would require to knock down its expression during the priming and restore it during the migration test. Different genetic systems allow for this type of expression modulation in time and I will test them in the near future.

II.2. Identifying KDM7a downstream targets

This study shows that KDM7a affects mechanical memory set-up. I reasoned that, KDM7a being a demethylase overexpressed in soft-primed cells, it could target the demethylation of genes involved in the set-up of soft-associated mechanical memory. In an attempt to answer this question I decided to perform CUT&Tag using an antibody targeting KDM7a on osteoblasts seeded for three days on either a soft or stiff PA hydrogel. This method required the least amount of material/cells compared to CHIP sequencing and CUT&RUN. As our protocol for mechanical priming generated a limited amount of cells, CUT&Tag appeared to be quite an optimal choice. However, as previously mentioned, the results obtained with the KDM7a antibody were similar to that observed with the IgG negative control and, therefore, inconclusive. I suspect that either the antibody lacked specificity or the technique selected was not ideal. It has been recommended to use CUT&RUN for transcription factors as the transposase (pA-Tn5) complex used for tagmentation in CUT&Tag is quite bulky and can reduce resolution due to steric effects 382 . Although CUT&RUN requires more material than CUT&Tag, it might constitute a more viable option to identify KDM7a target genes.

As of yet, I have compared the transcriptome of osteoblasts seeded on either a soft or stiff substrate for three days. This enabled us to identify potential targets differentially expressed depending on substrate rigidity. However, I have not yet verified whether these genes remain differentially expressed after being transferred to a stiff secondary substrate for a day.

Therefore, I intend to perform RNA sequencing on osteoblasts seeded for a day on a stiff secondary substrate with a previous three day mechanical priming. Furthermore, RNA sequencing could also be performed on osteoblasts depleted for KDM7a during mechanical priming. Comparing the differentially expressed genes in control and KDM7a depleted osteoblasts could orient us towards genes whose expression could be modulated by KDM7a.

This might enable us to restrict potential players involved in mechanical memory.

III. A potential role of adhesion molecules in mechanical memory

According to the GO performed on RNA sequencing hits, many of the differentially regulated genes were involved in ECM composition and focal adhesion organisation. Surprisingly, those genes were found to be up-regulated in osteoblast seeded for three days on a soft substrate. I initially assumed that genes relating to adhesion would be up-regulated on a stiff substrate as increased traction forces favour focal adhesion maturation. However, upon seeing the RNA sequencing analysis, I reasoned that cells seeded on a soft substrate have a harder time adhering to the substrate and could attempt to compensate by overexpressing adhesive molecules, thus increasing their adhesiveness.

III.1. Integrins and mechanical memory

I initially investigated the potential role of integrins as it is conventionally accepted that cells are able to sense the mechanical properties of their immediate surroundings through focal 133 adhesions and, more specifically integrins. I suspected that, much like the actin cytoskeleton, targeting integrins would hinder mechanical memory. Thus I attempted to block integrin β1 using the 4B4 and AIIB2 blocking antibodies and both integrins β3 and β5 using cilengitide. I treated our mouse osteoblasts with these inhibitors during mechanical priming and measured migration speed on a stiff secondary substrate. Intriguingly, I observed that treatment with blocking antibodies did not affect the migration speed of osteoblasts nor their capacity to establish a mechanical memory (data not shown). Unfortunately, I had not taken into consideration the fact that these blocking antibodies are specific to human integrins and did not effectively work in mouse cell lines such as our mouse osteoblasts. Thus, the potential involvement of integrins in mechanical memory will require further investigation using blocking antibodies functioning in mice such as integrin β1 mouse blocking antibody MAB1997.

As mentioned above, RNA sequencing analysis revealed the up-regulation, in osteoblasts cultured on a soft substrate, of genes coding for focal adhesion components such as integrins α8, α10 and α2b. Both integrins α8 391 and α10 392 dimerise with the β1 subunit while integrin α2b dimerises with β3 393 . These results suggest there could be an effect of mechanical memory on integrins' activity, particularly of heterodimers involving integrins β1 and β3. Measuring integrin protein levels, by western blot and immunofluorescent staining, on a stiff secondary substrate would enable us to confirm that mechanical memory regulate integrins expression level. Different flavour of integrins could lead to different migration capacities. Thus, integrins' expression level could be an effector of mechanical memory tuning cell migration velocity, the readout we chose to monitor. It will be interesting to overexpress these integrins in stiff-primed cells to check if it leads to a reduction of cell migration speed.

It is important to note that, among those up-regulated integrins, all three integrin heterodimers are receptors for fibronectin [391][392][393] .

III.2. Anoikis resistance and mechanical memory III.2.1. Anoikis resistance through adhesion molecules and mechanical memory

Studies have shown that ECM rigidity affects fibronectin on various levels. For instance, fibronectin assembly has been shown to increase with matrix rigidity 394 and fibronectin affinity and binding to integrin increase upon application of an external mechanical force 156,157 .

Conversely, fibronectin has also been shown to modulate substrate rigidity. For example, the conformational changes undergone by fibronectin during ECM maturing has been shown to affect the mechanical properties of the ECM and to increase the cells response to rigidity 395 .

Investigators observed that cells seeded on a soft (7.5kPa) matrix for 24h presented a reduced integrin β1 staining compared to cells seeded on a stiff (33kPa) substrate. However, as the matrix matured over 72h, cells seeded on a soft substrate presented increasingly elongated β1 containing adhesion structures.

Although fibronectin has been mostly associated with more rigid matrices, Han et al 219 showed that upon cell detachment, lung and breast cancer cells showed an increase in fibronectin expression accompanied by an increase in cell aggregation. Interestingly, they observed that this fibronectin up-regulation conferred anoikis resistance, possibly through an increase in desmosomal junctions. Fibronectin was also shown to increase FAK signalling through αvβ1 binding, promoting cell survival 396 .

I previously showed that fibronectin tended to be overexpressed in soft-primed cells in our cell lines capable of mechanical memory. It is important to note that, in those same cell lines (osteoblasts and HS 578T), cells tended to form aggregates on a soft substrate. These observations suggest that both osteoblasts and HS 578T, upon a reduction in adhesion on soft substrates, could increase their adhesive capacities, by expressing and secreting adhesion molecules like fibronectin, thereby favouring the formation of aggregates. Since cell adhesion molecules, such as fibronectin, have been shown to reduce anoikis and increase cell aggregation upon cell detachment, I wonder whether a similar process could be at play in osteoblasts and HS 578T cells. By increasing the expression and secretion of adhesion-related molecules, osteoblasts and HS 578T could bypass anoikis and incidentally reduce cell migration velocity upon replating cells on the secondary substrate.

To test this hypothesis I could target adhesion molecules overexpressed in soft-primed osteoblasts. This might prove challenging as, according to our RNA sequencing results, many appear to be up-regulated. I will first have to verify the up-regulation of said proteins (e.g. tenascin C, collagens III/V/VI). Once up-regulated proteins have been identified, I will test their effect on mechanical priming by depleting them during mechanical priming. I could also attempt to artificially induce integrin activation in osteoblasts and HS 578T cells, to mimic cell anchorage during soft mechanical priming, in an effort to prevent the increase in adhesion molecules. Integrin activation could be achieved by addition of Mg 2+ /Mn 2+ or stimulatory antibodies, during mechanical priming.

III.2.2. Other regulators of anoikis resistance and mechanical memory

There are many factors, other than adhesion molecules, which can affect anoikis resistance following cell detachment. These include, but are not limited to, signalling pathways, growth factors or long non-coding RNAs 397 . For instance, long non-coding RNA APOC1P1-3 has been shown to promote anoikis survival in MDA-MB-231 cells through inhibition of caspases 3, 8 and 9 and by lifting the inhibition of the pro-survival factor Bcl-2 by Mir-188-3p 398 . TGF-β1

signalling was also shown to promote anoikis resistance in MDA-MB-231 cell lines 399 . As MDA-MB-231 cells do not exhibit mechanical memory nor did they form aggregates on soft substrates, I wonder whether a different anoikis-resistance mechanism could be used to ensure cell survival, independent of cell anchorage and adhesion molecules.

It would be interesting to investigate whether a cell type's response to cell detachment, and the pro-survival factors used by the cell to evade anoikis could affect mechanical memory. I could attempt to identify a cell line whose survival is known to be adhesion independent, such as HEK29 and test its ability to establish a mechanical memory.

IV. Mechanical memory: a central role of the nucleus

It has been established that the nucleus is a mechanosensitive organelle through structures like the LINC complex or the lamina. As both those structures have been shown to affect chromatin remodelling and gene expression, it seems only fitting to investigate their potential role in mechanical memory.

Our study demonstrates that MDA-MB-231 cells appear to only be sensitive to the rigidity of their immediate surroundings, unable to store the effects of past mechanical cues. Furthermore, it seems that mechanical priming does not affect KDM7a expression and H3K9 me2/3 or H3K27 me3 methylation levels in MDA-MB-231 cells. As both the literature and our study strongly suggest that epigenetic modifications serve as a means to store mechanical memory, I 136 wonder whether epigenetic modifications associated with mechanical memory could be impaired in this cell line, affecting its ability to store past mechanical information. Cells have been shown to increase their nuclear rigidity with ECM stiffness 175 . It has been speculated that this modulation in nuclear stiffness protects the genetic material from damage caused by mechanical strain. The lamina, composed of lamins, affects nuclear rigidity and possesses an elastic mechanical property believed to serve an additional purpose in shock absorption 188 . However, lamins are not the only determining factor in nuclear rigidity as heterochromatin also affects the nucleus' mechanical properties 251,346 .

Our preliminary studies suggest that mechanical priming could affect lamin A/C levels in HS 578T but not in MDA-MB-231 cells (Fig. 39). It seems that soft-primed HS 578T cells show a decrease in lamin A/C fluorescence intensity compared to their stiff-primed counterparts. On the other hand, MDA-MD-231 cells appear to present similar lamin A/C fluorescence levels regardless of the priming rigidity. Further immunofluorescence and WB analyses of lamin A/C expression levels in all three cell lines would be required to confirm these preliminary observations. However, these preliminary results suggest that mechanical memory could have an impact on lamin A/C.

Lamins can be present either in monomeric form or as a polymeric network constituting the lamina. Lamin A/C turnover has been shown to be modulated by substrate rigidity 194 . Therefore, it could be possible to observe a change in the quantity of associated or monomeric lamin without overall lamin levels being affected by mechanical memory. Lamina dissociation has also been linked to the cell cycle in Drosophila melanogaster 139 . Mechanical memory having an impact on cell division, it could in turn impact the dynamics of lamina association/dissociation. Therefore, it would be interesting to measure the thickness of lamina through lamin A/C stainings at the nuclear periphery, which could reflect on possible variations in lamin association/dissociation.

Lamins and heterochromatin both affect nuclear rigidity. In our cell lines capable of establishing a mechanical memory, heterochromatin and lamin A/C levels seem to be modulated by priming rigidity. However, MDA-MB-231 cells, which do not exhibit a mechanical memory, seem to present similar levels of heterochromatin and lamin A/C, regardless of the priming rigidity.

These observations suggest there could be a correlation between nuclear rigidity and mechanical memory. Since cell and nuclear shape are affected by ECM rigidity, I reasoned that nuclear deformation could trigger epigenetic modifications, enabling the establishment of a mechanical memory set-up. I also wondered whether low nuclear deformability could hinder the establishment of a mechanical memory. It has been proposed that nuclear shape affects chromatin conformation and accessibility. Nava et al 344 showed that nuclear deformation temporarily affects heterochromatin in an attempt to prevent mechanically induced DNA damage. Therefore it would be interesting to first determine whether mechanical memory affects the nuclear rigidity of osteoblasts and MDA-MB-231 cells using atomic force microscopy (AFM). I could also decrease lamin A/C levels (using siRNAs) in MDA-MB-231 cells during mechanical priming, in an effort to decrease nuclear rigidity and see if it affects migration speed, KDM7a and heterochromatin levels. Conversely, I could try to increase nuclear rigidity by overexpressing lamin A/C in our cell lines capable of establishing a mechanical memory (osteobalsts and HS 578T cells) to see if it affects mechanical memory.

IV.2. The LINC complex: force transmission and gene regulation

The LINC complex physically connects the lamina, and hence the genome, to cytoplasmic cytoskeletal networks. This connection allows for the detection of environmental mechanical cues, possibly altering the regulation of nuclear architecture and genome function. For instance, Alam et al 186 showed that the LINC complex participates in the regulation of mechanosensitive genes on a genome-wide scale, following mechanical stimulation. However, the exact mechanisms by which the LINC complex could modulate gene expression still elude us. It has been suggested that the effect of the LINC complex on gene expression could be mediated through nuclear deformation 186 . As was previously evoked, the nuclear shape has been proposed to mechanically affect chromatin conformation which, in turn, could affect gene accessibility to transcription factors. A study by Gupta et al 400 provides evidence that actindependent nuclear deformation could contribute to gene expression. Alternatively, it has been proposed that direct mechanical force transfer to the lamina, likely via the LINC complex, could affect genome organisation and gene expression 342 . Therefore the LINC complex appeared to be an interesting candidate to investigate in the context of mechanical memory set-up. The object of my thesis was to study the mechanism of mechanical memory, using human mammary cancer cell lines (HS 578T and MDA-MB-231) as well as a non-cancerous cell line of mice osteoblasts. Cells were primed on polyacrylamide (PA) gels, of various rigidity (ranging from 0,5kPA to GPa), for a period of 3 days before being transferred to a PA gel of 80kPa. Cells were maintained on this secondary substrate for a day and various parameters were measured to study mechanical memory. We first established that only two of our cells lines, namely osteoblasts and HS 578T established a mechanical memory. Soft-primed cells displayed lower migration speeds and proliferation rates on a stiff secondary substrate than cells that had 191 been consistently cultured on a stiff substrate. We showed that disrupting the cytoskeleton affected stiff-related mechanical memory, possibly by impeding the transmission of external information to the cell. We then investigated the potential role of epigenetics in storing mechanical memory. Disrupting the epigenetic landscape by targeting overall histone or DNA methylation also affected stiff mechanical memory set up. Thus we performed RNA sequencing (RNAseq) and identified two demethylases, and a set of extracellular matrix (ECM) proteins, such as fibronectin 1 (FN1), that showed higher levels of expression in soft primed cells compared to stiff primed cells. Only one demethylase, KDM7a, seemed to play a role in mechanical memory as depleting it affected the cells ability to retain past information. On a stiff secondary substrate, KDM7a depleted cells displayed similar migration speeds no matter the priming rigidity. Concerning FN1, we hypothesised that an increased expression in softprimed cells could affect FN1 secretion, rendering cells more adhesive and impeding migration.

However, FN1 depletion during the priming period did not affect mechanical memory, thus it is seems unlikely to be involved in mechanical memory. Abstract: Not unlike us, cells have the ability to sense and be affected by rigidity variations in their immediate environment. For instance, we move more slowly on a soft terrain, such as mud, than on a more rigid surface like concrete. Quite similarly, cells migrate much more slowly on a soft environment than on a stiff one. This phenomenon was described as a "mechanical memory". We are attempting to describe the mechanisms by which cells are able to setup a mechanical memory.

As of yet, we have shown that mechanical memory in cells depends on a protein which affects the DNAs architecture. All cells possess the same DNA sequence, but its shape is changed to allow cells to expressed different genes and acquire different characteristics. We identified that KDM7a, a protein involved in these changes in DNA structure is necessary for cells to store information on their mechanical history.

III. 3 .

 3 Mechanical memory modulates H3K27 me2/3 levels ................................................... III.4. Impairing H3K27 methylation during mechanical priming affects mechanical memory 116 III.5. Identification of differentially methylated genes through CUT&Tag ........................... IV. Analysis of potential mechanical memory effectors .......................................................... IV.1. Mechanical memory affects FN1................................................................................... IV.2. FN1 depletion does not affect mechanical memory ...................................................... IV.3. KDM7a regulates FN1 expression................................................................................. V. Mechanical priming affects cell proliferation ........................................................................ V.1. Mechanical memory affects cell proliferation in osteoblasts ........................................ V.2. Disrupting actomyosin contractility and DNA methylation perturbs the effect of mechanical memory on proliferation .......................................................................................... V.3. Is the effect of mechanical memory on cell proliferation due to cell cycle arrest? ....... able and those unable to establish a mechanical memory? ............ II. KDM7a as a potential regulator of mechanical memory ........................................................ II.1. Validating KDM7as involvement in mechanical memory ............................................ II.2. Identifying KDM7a downstream targets ....................................................................... III. A potential role of adhesion molecules in mechanical memory ........................................ III.1. Integrins and mechanical memory ................................................................................. III.2. Anoikis resistance and mechanical memory .................................................................. III.2.1. Anoikis resistance through adhesion molecules and mechanical memory .......... III.2.2. Other regulators of anoikis resistance and mechanical memory ......................... IV. Mechanical memory: a central role of the nucleus ............................................................ IV.1. Mechanical memory and nuclear stiffness: a potential involvement of lamin A/C .......

Figure 1 :

 1 Figure 1: Schematic of the different types of cellular migration. ........................................... 17

Figure 2 : 19 Figure 3 : 21 Figure 4 : 22 Figure 5 : 27 Figure 6 : 30 Figure 7 : 35 Figure 8 : 37 Figure 9 : 39 Figure 10 : 42 Figure 11 : 46 Figure 12 : 49 Figure 13 : 53 Figure 14 : 55 Figure 15 : 57 Figure 16 : 58 Figure 17 : 60 Figure 18 : 61 Figure 19 : 62 Figure 20 : 65 Figure 21 : 70 Figure 22 : 75 Figure 23 : 79 Figure 24 : 82 Figure 25 : 86 Figure 26 : 88 Figure 27 : 99 Figure 28 :Figure 29 :Figure 30 :Figure 31 :Figure 32 :Figure 33 :Figure 34 :Figure 35 :Figure 36 :Figure 37 :Figure 38 :Figure 39 : 5  1  2  3  1  84 

 2193214225276307358379391042114612491353145515571658176018611962206521702275237924822586268827992829303132333435363738395123184 Figure 2: Schematic of the different types of actin architecture commonly observed during mesenchymal cell migration. .................................................................................................... 19 Figure 3: Schematic of the adhesome formed at integrin-mediated, cell matrix adhesions. .. 21 Figure 4: Schematic of the process of integrin activation. ...................................................... 22 Figure 5: Schematic of apicobasolateral polarisation of junctions and the cytoskeleton in epithelial cells. .......................................................................................................................... 27 Figure 6: Organisation of the various cytoskeletal networks during mesenchymal cell migration. ................................................................................................................................. 30 Figure 7: Schematic of the different types of actin filament organisation. ............................. 35 Figure 8: Schematic of the role of the actin cytoskeleton and FA during protrusion formation in mesenchymal migrating cells. .............................................................................................. 37 Figure 9: Schematic of the cytoskeletal organisation of microtubules, actin filaments and intermediate filaments in mesenchymal migrating cells. ......................................................... 39 Figure 10: Schematic of the architecture of lamins in the nucleus. ........................................ 42 Figure 11: Model representing the response of the talin molecular clutch to ECM stiffness and the resulting force transmission. ............................................................................................... 46 Figure 12: Schematic of the LINC complex. .......................................................................... 49 Figure 13: Integrin signalling in mechanotransduction. .......................................................... 53 Figure 14: Schematic of the intrinsic and extrinsic anoikis pathways. ................................... 55 Figure 15: Schematic of the mechanical regulation of YAP/TAZ. ......................................... 57 Figure 16: Regulation of YAP/TAZ activation depending on both mechanical and topological cues. .......................................................................................................................................... 58 Figure 17: Schematic of the chromatosome. ........................................................................... 60 Figure 18: Schematic of histone assembly into a nucleosome. ............................................... 61 Figure 19: Schematic of three different types of chromatin condensation depending on cation concentration. ........................................................................................................................... 62Figure 20: Schematic of the different states of chromatin condensation and accessibility across the genome. .............................................................................................................................. 65 Figure 21: Schematic of the relationship between DNMTs and TETs. .................................. 70

Figure 1 :

 1 Figure 1: Schematic of the different types of cellular migration. Cells can migrate as single cells or collectively, as a cohesive group. They can adopt a traction-based, mesenchymal mode of migration which requires both a strong adhesion to the surrounding ECM and Rac1-dependent actin polymerisation. They can also exhibit a propulsion-based, amoeboid mode of migration which requires low to no adhesion and RhoA-dependent actomyosin contractility at the rear of the cell. Taken from D.L. Pages and E. Dornier et al. (2020).

Figure 2 :

 2 Figure 2: Schematic of the different types of actin architecture commonly observed during mesenchymal cell migration. The zoomed regions depict the following types of structures: i) the actin cortex, ii) stress fibres which are contractile fibres, iii) the lamellipodium and iv) the filopodium. The two former structures are different types of protrusion that depend on actin polymerisation. Taken from L. Blanchoin et al. (2014)

Figure 3 :

 3 Figure 3: Schematic of the adhesome formed at integrin-mediated, cell matrix adhesions. (A) The molecular organisation divided into (B) three spatial and functional layers: the adhesome signalling, the adhesion organisation and the actin regulatory layer. Taken from Pinelopi A. Nikolopoulou (2021)

Figure 4 :

 4 Figure 4: Schematic of the process of integrin activation. Integrin activation through the recruitment of intracellular adaptors, such as talin and kindlin, leads to integrin clustering and focal adhesion assembly in a force dependent manner. Lastly, integrin inactivation and focal adhesion disassembly are driven by tensile forces and microtubules. Taken from Yasmin et al.(2020)

Figure 5 :

 5 Figure 5: Schematic of apicobasolateral polarisation of junctions (adherans, junction, tight junction, desomosome and hemidesmosomes) and the cytoskeleton (actin and intermediate filaments) in epithelial cells. Taken from M. Rübsam et al. (2018).

Figure 6 :

 6 Figure 6: Organisation of the various cytoskeletal networks during mesenchymal cell migration. Cells first establish a front-rear polarity (1) before forming protrusions, notably a lamellipodium(2), through actin polymerisation. The lamellipodium is subsequently stabilised by the formation of new adhesions(3). Finally, the cell contracts (4) and rear retraction(5) enables the translocation of the cell body toward the front of the cell. Taken from S.Seetharaman and C. Etienne-Manneville. (2020)

Figure 7 :

 7 Figure 7: Schematic of the different types of actin filament organisation. (A) Branched actin network are generated by NPFs and Arp2/3. (B) Actin networks are formed by long crosslinkers which organise actin filaments, generated by formins and ENA/VASP, into networks. (C) Straight actin bundles are formed by shorter crosslinkers. (D) Anti-parallel, contractile bundles are linked by myosin II. Taken from L. Blanchoin et al. (2013).

Figure 9 :

 9 Figure 9: Schematic of the cytoskeletal organisation of microtubules, actin filaments and intermediate filaments in mesenchymal migrating cells. Taken from S. Etienne-Manneville (2013)

Figure 10 :

 10 Figure 10: Schematic of the architecture of lamins in the nucleus. The nuclear envelope, a physical barrier separating the nucleoplasm from the cytoplasm, contains the INM, the ON and a meshwork of A-and B-type lamins. Nuclear pores (NPC), constitute the only opening between the cytosol and nucleoplasm. Taken from Vahabikashi et al. (2022).

Figure 11 :

 11 Figure 11: Model representing the response of the talin molecular clutch to ECM stiffness and the resulting force transmission. On a soft ECM, tensile strain is insufficient to induce talin unfolding, preventing recruitment of vinculin thus leading to a weakening or breakage of the bond between integrins and the ligand. Conversely on a stiff ECM, high tensile strain enables the activation of talin and recruitment of vinculin, strengthening the vinculin-dependent clutch and allowing for force transmission to the cell. Taken from Z.Sun et al. (2016).

Figure 12 :

 12 Figure 12: Schematic of the LINC complex. The LINC complex passes through the nuclear envelope, connecting the cytoskeleton to the lamin meshwork, thus enabling force transmission to the nucleus. Taken from S.A.Mosaddad et al. (2021)

Figure 13 :

 13 Figure 13: Integrin signalling in mechanotransduction. Integrin activation, through talinmediated conformational changes, enables integrin clustering which, in turn, initiates robust intracellular integrin signalling. Thus ensues the activation of Rho, Rac and Cdc42 signalling, involved in cytoskeletal organisation, and the activation of several transcription factors,namely MRTF/SRF, TEAD and c-Jun, mediating the expression of pro-migration, antiapoptotic and pro-mitogenic genes. Taken from J.Cooper et al. (2019)

  Many alterations in signalling pathways have been found in cancer cells, notably increased activation of the ERK and PI3K/AKT, survival promoting pathways. For instance, activating mutations of B-RAF, an upstream activator of the MEK/ERK signalling pathway have been 55 found in melanomas. Akt has been observed to be hyper-activated in metastatic cells possessing PTEN phosphatase mutations, an antagonist of the PI3K/AKT pathway 214 .Cell aggregations constitutes another technique developed by malignant cells to promotesurvival in otherwise pro-apoptotic environments. For instance, metastatic breast and prostate carcinoma cells were shown to form multicellular, homotypic aggregates through interactions between T-antigen and galectin-3 215 . Disruption of these interactions led to a decrease of survival and growth of the aggregate. E-cadherin was also found to be upregulated in spheroids and to mediate cell-cell adhesion leading to the upregulation of the ERK pathway 216 and possibly the PI3K/Akt through ErbB4217,218 . Matrix secretion, notably fibronectin, was shown

Figure 14 :

 14 Figure 14: Schematic of the intrinsic and extrinsic anoikis pathways. Cell detachment leads the activation of both pathways. Activation of the extrinsic pathways up-regulates FAS and FasL, inducing the activation of Caspase-8 and subsequent activation of Casapase-7 and -3. Conversely, activation of the intrinsic pathway activates pro-apoptotic Bcl-2 proteins, enabling the permeabilisation of the mitochondria membrane through Bax and Bak. The subsequent release of

Figure 15 :

 15 Figure 15: Schematic of the mechanical regulation of YAP/TAZ. The Hippo pathway negatively regulates YAP/TAZ activation and localisation. YAP/TAZ is sequestered in the cytoplasm through its phosphorylation by MST1/2, MAP4Ks and LATS1/2. Upon mechanical tension mediated by FAs, Adherens junctions, the actin cytoskeleton or the LINC complex,YAP/TAZ inhibition is lifted, enabling its activation and subsequent translocation to the nucleus. In the nucleus, YAP/TAZ activates transcription through its binding with the transcription factor TEAD. Taken fromDasgupta et al. (2019).

Figure 16 :

 16 Figure 16: Regulation of YAP/TAZ activation depending on both mechanical and topological cues Taken from Steve. MBInfo.

Figure 17 :

 17 Figure 17: Schematic of the chromatosome. The chromatosome consists of a core histone octamer (H2A, H2B, H3 and H4) wrapped in DNA (1.65 turn), linker DNA arms and the linker histone (H1). Taken from AM.A Östürk et al. (2020).

Figure 18 :

 18 Figure 18: Schematic of histone assembly into a nucleosome. Nucleosomeformation first requires the hetero-oligomerisation of H3 with H4 and H2A with H2B. Two H3/H4 dimers then associate into a tetramer followed by the binding of an H2A/H2B dimer to each H3/H4, thus forming an octamer of histones (histone core). Lastly, the octamer is wrapped by a 146bp DNA segment thus forming a nucleosome. Taken from J.A. Alberts et al. (2002)

Figure 19 :

 19 Figure 19: Schematic of three different types of chromatin condensation depending on cation concentration. In the absence of salt (left panel), chromatin forms a decondensed, 10nm fibre. At low cation concentration (centre panel), chromatin condenses into a supercoil structure forming a 30nm-diameter fibre.

Figure 20 :

 20 Figure 20: Schematic of the different states of chromatin condensation and accessibility across the genome. Unlike closed chromatin, the more dynamic permissive chromatin allows for chromatin remodelling through transcription factors, for instance, into a more open conformation where RNA polymerise II (Pol II) can bind an transcribe target genes. Taken from S.L. Klemm et al. (2019).

  DNA methylation is mediated by a family of DNA methyltransferases (DNMTs -Fig.21) of which there are five: DNMT1/2/3a/3b/4. Of those five, only three (DNMT1, DNMT3a and DNMT3b) catalyse the addition of a methyl group to cytosin-5. All three methylases possess a similar mechanism but differ in their role. DNMT3s have a ubiquitous role, are non-selective and have been involved in de novo methylation274 . DNMT1, on the other hand, maintains DNA methylation patterns during replication. It recognises hemi-methylated DNA and provides a methyl moiety to the symmetrical CpG site 275 .

  interactions. For instance, DNMT3 can form heterotetrameric complexes with a truncated variant DNMT3L, the latter binding unmethylated H3K4 and activating the methyltransferase activity of DNMT3 276 . DNMT1 constitutes another example through its interaction with a specific RNA translated from the C/EBPα gene. This association enables the sequestering of DNMT1, decreasing methylation of the C/EBPα gene and further increasing its translation277 .PTMs represent another level of regulation of DNMT activity. For instance, KDM1, a lysine demethylase, targets and stabilises DNMT1. Depletion of KDM1 leads to an increase in DNMT1 methylation and a decrease in overall DNA methylation278 . Furthermore, phosphorylation of DNMT1 through the PI3K-mTOR pathway, can prevent its hypermethylation and also participate in its stability 279 . DNMT3a activity can also be modulation through phosphorylation by CK2 which inhibits DNA methylation and relocates the methyltransferase to heterochromatin 280 .Lastly, DNMT activity can be modified through alternative splicing, providing isoforms with different activities. For example, DNMT3 isoforms can increase DNA methylation, possibly by acting as an accessory protein for DNMT3a 281 .III.2.1.ii. DNA demethylation -TETsDNA can be actively or passively demethylated. Passive demethylation occurs when methyltransferases are inhibited, notably DNMT1 which is involved in methylation maintenance in dividing cells. There are several mechanisms involved in the active demethylation of DNA. As covalent carbon-carbon bonds are too strong to be severed, methylated cytosine-5 (5mC) nucleotides must undergo modifications on their amine and methyl groups to be replace by non-methylated cytosines. 5mC replacement is dependent on the base excision repair (BER) pathway which corrects DNA damage from oxidation, deamination and alkylation.

Figure 21 :

 21 Figure 21: Schematic of the relationship between DNMTs and TETs. TET family members (Tet1, Tet2 and Tet3) catalyse the replacement of 5mC nucleotides by unmethylated cytosines. TETs antagonise DNMTs which enable the maintenance and de novo methylation of DNA. Taken from F. Lyko (2017).

  into four categories (classes I, II, III, IV) based on sequence homology. HDACs also function as part of multiprotein complexes which present one of two catalytic mechanisms requiring either a zinc cofactor (histone deacetylase family) or NAD+ cofactor (Sir2 family) 321 .

Figure 22 :

 22 Figure 22: Schematic of the processing of small non-coding RNAs. Primary RNA or pri-miRNA are cleaved by endoribouclease Drosha, generating a double stranded precursor miRNA (pre-miRNA). The pre-miRNA is then cleaved by Dicer, generating a mature miRNA which is loaded onto AGO, enabling gene silencing in a sequence specific manner. Taken from K Svortsova et al. (2028).

translation 327 .

 327 Although miRNAs are targets of epigenetic modifications, such as DNA methylation, they are considered epigenetic modulators as they can participate in the regulation of DNMTs and HDACs. For instance, miR-29 targets both DNMT3s as it possesses complementary sequence to their 3'-UTRs and thus reduces overall DNA methylation 328 . miR-140 indirectly modulates histone acetylation levels by negatively regulating HDAC4 through 3'-UTR binding 329 . siRNAs siRNAs are exogenous, fully complementary double stranded ncRNAs derived from virus', transposons, or transgene trigger. They undergo a similar processing as miRNAs. They are shortened into 19-24 nucleotide fragments by Dicer before being processed through RISC. siRNAs are believed to be involved in heterochromatin maintenance as depletion of Dicer and members of RISC leads to a decrease in H3K9me3 and transcriptional de-repression 263 . piRNAs piRNAs are single stranded RNA sequences of 26-31 nucleotides. Primary, newly transcribed piRNAs, are first cleaved by Zucchini (Zuc) and its cofactor Minotaur (Mino), generating a piRNA intermediate. The latter then binds to PIWI proteins, which are part of the Argonaut family. PIWI-associated piRNA intermediate is further cleaved by Zuc or the Papi-dependent trimmer before being methylated, forming a mature PIWI-piRNA complex. PIWI-piRNA complexes have been shown to differentially regulate gene transcription. For instance, in Drosophila, piRNAs activate their own gene transcription in the sub-telomeric region 330 . When PIWI was mutated, euchromatin markers (H3K4me2-me3, H3K9) decreased while heterochromatin markers (H3K9me2-me3, HIP1) increased. Furthermore, an ectopic insertion of piRNA sequences induced a local increase of H3K9me2/3 331 through the recruitment of heterochromatin associated proteins HP1a 332 and Su(var)3-9 333 . In the germ cells of mice models, piRNAs have been involved in the regulation of transposon methylation through DNMT3s 334 .

Figure 23 :

 23 Figure 23: Schematic of a mechanical memory experiment and the biological processes known to be affected by mechanical priming. Cells mechanically conditioned or primed on a stiff substrate (stiff-primed cells) have been shown to maintain phenotypic characteristics associated with a stiff substrate even after being transferred to a soft secondary substrate. Similarly, soft-primed cells have been observed to sustain phenotypic characteristics associated with a soft substrate after being transferred to a stiff secondary substrate. This retaining of past information has been names mechanical memory and has been shown to impact many cellular processes such as adhesion, cell shape, migration/invasion, proliferation and cell fate.

Figure 24 :

 24 Figure 24: Description of the synthesis of a polyacrylamide (PA) substrate of heterogeneous stiffness. PA solutions of different composition are sequentially polymerised to obtain an ECMwith contiguous primary and secondary regions with distinct rigidities. Taken from S.Nasrollahi et al. (2017)

Figure 25 :

 25 Figure 25: Experimental outline of reversible and irreversible mechanical dosing based on

Figure 26 :

 26 Figure 26: Schematic of mechanosensitive positive feedback loops based on the literature and a proposed mathematical model for mechanical memory. In this model, positive reinforcement (α) is influenced by fast-and slow-acting positive feedback loops. The degree of activation of these loops depends on the intensity and magnitude of the mechanical cue.

Figure 27 :

 27 Figure 27: Mechanical memory is cell type dependent. A) Osteoblasts, HS 578T and MDA-MB-231 were mechanically primed on either a soft (0.5kPalight blue) or a stiff (80kPadark blue) Col I coated PA hydrogel for 2 to 7 days before being passaged. They were then transferred to a stiff (80kPa) secondary substrate where they were cultured for 1 day before analysis. B) Average migration speed of osteoblasts, HS 578T and MDA-MB-231 cells after 1 day on a stiff secondary substrate after a 3 day mechanical priming (St3-St1 / So3-St1). Mechanical priming affects the migration speed of osteoblasts and HS 578T cells but not MDA-MB-231 cells. Horizontal square brackets denote statistical significance (p < 0.05). 16 > N > 3 C) Average migration speed of MDA-MB-231 cells after 1 day on a stiff secondary substrate with a previous 3 to 7 day mechanical priming. Cells primed for 3 or 7 days displayed similar a migration speed regardless of the priming rigidity. Horizontal square brackets denote statistical significance (p < 0.05). 3 > N > 1 D) Representative time-lapse sequence of osteoblast migration after one day on a stiff (80kPa) secondary substrate after a 3 day mechanical priming (St3-St1 / So3-St1). Images were taken every 20min. Scale bar, 50µMm. Error bars = SEM. Absence of brackets; non-significant, p < 0.05; *, p < 0.001; ***.

Figure 28 :

 28 Figure 28: Effect of mechanical memory on cell spreading or nuclear size. A) Representative image, in phase contrast microscopy, of osteoblasts and MDA-MB-231 cells after 1 day on either a soft (0.5kPa -So1) or a stiff (80kPa -St1) Col I coated PA hydrogel Both osteoblasts and MDA-MB-231 cells present a rounded morphology on a soft substrate and a more spread-out, spindle-like morphology on a stiff substrate. B) Quantification of the cell area of osteoblast, HS 578T and MDA-MB-231 cells on a stiff secondary substrate after a 3 day mechanical priming (St3-St1 / So3-St1). In all three cell lines, cell area is

  is comparable regardless of the priming rigidity. 5 > N > 2 C) Quantification of the nuclear area of osteoblast, HS 578T and MDA-MB-231 cells on a stiff secondary substrate after a 3 day mechanical priming (St3-St1 / So3-St1). In all three cell lines, nuclear area is similar regardless of the priming rigidity. 4 > N > 2 Scale bar, 100µMm. Error bars = SEM. Absence of brackets; non-significant.

Figure 29 :

 29 Figure 29: Mechanical dosing in osteoblasts. A) Average migration speed of osteoblasts after 1 day on a stiff secondary substrate after a 2 to 3 day mechanical priming. A 2 and 3 day mechanical priming affects the migration speed of osteoblasts. Horizontal square brackets denote statistical significance (p < 0.05). 16 > N > 3 B) Osteoblasts were mechanically primed for 3 days on either a soft (0.5 and 2 kPa Col I coated hydrogel) or a stiff (80kPa Col I coated hydrogel and plastic) substrate before being passaged. Part of the collected cells were transferred to a stiff secondary substrate for 1 day before analysis. The rest of the cells were cultured for an additional 3 days on a substrate of the same stiffness as that used for the previous 3 day mechanical priming (So3-So3 / P2 or St3-St3 / P2), before being passaged. Again the cells were transferred to either a stiff secondary substrate or cultured for an additional 3 days on a similar substrate as that used for previous mechanical priming (So3-So3-So3 / P3 or St3-St3-St3 / P3). This process was repeated over 4 passages. C) Average migration speed of osteoblasts after 1 day on a stiff secondary substrate after a 3 to 12 day mechanical priming. A similar trend is observable within each passage: soft-primed osteoblasts present a decrease in migration speed compared to stiff-primed osteoblasts. N = 2. Error bars = SEM. Absence of brackets; non-significant.

Figure 30 :

 30 Figure 30: Impact of protein coating on mechanical memory. A) Osteoblasts were mechanically primed for 3 days on either a soft (0.5kPa) or a stiff (80kPa) PA hydrogel coated with either Col I or FN1. Cells were passaged at the end of the mechanical priming and transferred to a stiff secondary substrate (80kPa Col I coated PA hydrogel) for 1 day before analysis. B) Average migration speed of osteoblasts after 1 day on a stiff secondary substrate with a previous 3 day mechanical priming (St3-St1 / So3-St1). Mechanical priming affects the migration speed of osteoblasts regardless of the protein coating. Horizontal square brackets denote statistical significance (p < 0.05). N = 3. Error bars = SEM. Absence of brackets; non-significant, p < 0.05; *, p < 0.001; ***.

Figure 31 :

 31 Figure 31: Mechanical memory requires the actin cytoskeleton. A) Osteoblasts were mechanically primed for 3 days on either a soft (0.5kPa) or a stiff (80kPa) Col I coated PA. Cells were treated with inhibitory drugs for the duration of the mechanical priming. Cells were then washed and transferred to a stiff (80kPa) secondary substrate where they were cultured for 1 day before analysis. B) Average migration speed of osteoblasts after 1 day on a stiff secondary substrate with a previous 3 day mechanical priming (St3-St1 / So3-St1) and treatment with either blebbistatin (10µM) or cytochalasin D (0.5µM). Both cytochalasin D and blebbistatin treatments decrease the migration speed of stiff-primed osteoblasts. Horizontal square brackets denote statistical significance (p < 0.05). 5 > N > 2. Error bars = SEM. Absence of brackets; non-significant, p < 0.05; *, p < 0.001; ***.

Figure 32 : 3 > N > 2 .

 3232 Figure 32: Disrupting the epigenetic landscape impedes stiff-associated mechanical memory. A) Average migration speed of osteoblasts after 1 day on a stiff secondary substrate with a previous 3 day mechanical priming (St3-St1 / So3-St1) and treatment with either 5azacytidine (1µM) or garcinol (5µM) during mechanical priming. Garcinol treatment did not affect mechanical memory unlike 5-azacytidine treatment which decreased the migration speed of stiff-primed cells. Horizontal square brackets denote statistical significance (p < 0.05). 5 > N > 2. B) Average migration speed of osteoblasts after 1 day on a stiff secondary substrate with a previous 2 day mechanical priming and trichostatin A (37.25nM) treatment during mechanical priming. Trichostatin A treatment reduced the migration speed of stiffprimed osteoblasts. Horizontal square brackets denote statistical significance (p < 0.05). 3 > N > 2. Error bars = SEM. Absence of brackets; non-significant, p < 0.01; **, p < 0.001; ***.

Figure 33 :

 33 Figure 33: Identification, through RNAseq, of 2 demethylases differentially regulated in osteoblasts depending on substrate rigidity. A) Osteoblasts were mechanically primed for 3 days on either a soft (0.5kPa) or a stiff (80kPa) Col I coated PA hydrogel before being collected for RNAseq. B) Volcano plot of differentially expressed genes identified between

Figure 34 :

 34 Figure 34: Effect of mechanical memory on KDM7a. A) Representative immunofluorescent staining of osteoblast, HS 578T and MDA-MB-231 cells for phalloidin (purple), KDM7a (green) and DAPI (blue), cultured for a day on a stiff secondary substrate (Col I coated glass) after a 3 day mechanical priming (St3-St1 / So3-St1). B) Quantification of the fluorescence intensity of KDM7a stainings in osteoblasts, HS 578T and MDA-MB-231 cultured for a day on a stiff secondary substrate (Col I coated glass) after a 3 day mechanical priming (St3-St1 / So3-St1).

Figure 35 :

 35 Figure 35: H3K27 methylation affects mechanical memory set-up. A) Representative immunofluorescent staining of osteoblast, HS 578T and MDA-MB-231 cells for phalloidin (purple), H3K9 me2/3 (red), H3K27 me3 (green) and DAPI (blue), cultured for a day on a stiff secondary substrate (Col I coated glass) after a 3 day mechanical priming (St3-St1 / So3-St1). B) Quantification of the fluorescence intensity of H3K27 me3 stainings in osteoblasts, HS 578T and MDA-MB-231 cultured for a day on a stiff secondary substrate (Col I coated

Figure 36 :

 36 Figure 36: Substrate rigidity affects H3K27 methylation levels. A) Heatmaps describing the H3K27 me3 CUT&Tag enrichment genome wide in osteoblasts cultured for 3 days on either a soft (0.5kPablue) or stiff (80kPared) Col I coated PA hydrogel. i. Methylation peaks for H3K27 me3 that are specific to osteoblasts cultured on a soft substrate. Very few H3K27 me3 peaks are only found in osteoblasts cultured on a soft substrate.ii. Methylation peaks for H3K27 me3 found in both osteoblasts cultured on a soft and stiff substrate. These overlapping peaks tend to be increased in osteoblasts cultured on a stiff substrate, showing an increase in methylation in these regions. iii. Methylation peaks for H3K27 me3 that are specific to osteoblasts cultured on a soft substrate. Osteoblasts cultured for 3 days on a stiff substrate present an increase in H3K27 me3 peaks undetected in osteoblasts cultured for a similar duration on a soft substrate.

Figure 38 :

 38 Figure 38: Mechanical priming affects cell proliferation. A) Percentage of dividing cells over 1 day on a stiff secondary substrate with a previous 3 day mechanical priming (St3-St1 / So3-St1) and treatment with either 5-azacytidine (1µM), garcinol (5µM) or blebbistatin (10µM) during mechanical priming. Treatment with 5-azacytidine and blebbistatin during mechanical priming decreased cell proliferation in stiff-primed cells whereas garcinol did not significantly affect cell proliferation regardless of the mechanical priming. N = 3. Horizontal square brackets denote statistical significance (p < 0.05). B) Osteoblasts were stably transfected with Fucci cell cycle reporter. Fucci osteoblasts will express the Cdt1-RFP (red) fusion protein in G1 phase and the Gemini-GFP (green) fusion protein during S, G2 and M phases. C) Osteoblasts were mechanically primed for 3 days on either a soft (0.5kPa) or a stiff (80kPa) Col I coated PA hydrogel before being collected and undergoing FACS. D) Percentage of FACSed osteoblasts in G1 (red) and S-G2-M (green) at the end of a 3 day culture on either a soft (So3) or stiff (St3) Col I coated PA hydrogel. Cells presented a similar percentage of cells in G1 and S-G2-M regardless of the substrate rigidity. N= 2. E) Representative FACS data showing the percentage of Fucci osteoblasts in G1 (red) or S-G2-M (green) after a 3 day culture on either a soft (So3) or stiff (St3) Col I coated PA hydrogel.

Figure 39 :

 39 Figure 39: Lamin A/C is affect by mechanical priming in cell lines that establish mechanical memory. Representative immunofluorescent staining of HS 578T and MDA-MB-231 cells for phalloidin (purple), Lamin A/C (red) and DAPI (blue), cultured for a day on a stiff secondary substrate (Col I coated glass) after a 3 day on a soft or stiff secondary substrate (Col I coated glass) after a 3 day mechanical priming (St3-St1 / So3-St1.

  séquençage de l'ARN (RNAseq) et identifié deux déméthylases, ainsi qu'un ensemble de protéines de la matrice extracellulaire (MEC), comme la fibronectine 1 (FN1), qui présentaient des niveaux d'expression plus élevés dans les cellules conditionnées sur un substrat mou que celles conditionnées sur un substrat rigide. Seule une déméthylase, KDM7a, semble jouer un rôle dans la mémoire mécanique, car sa déplétion affecte la capacité des cellules à retenir les informations passées. Sur un substrat secondaire rigide, les cellules dépourvues de KDM7a ont montré des vitesses de migration similaires, quelle que soit la rigidité de conditionnement. En ce qui concerne le FN1, nous avons émis l'hypothèse qu'une expression accrue dans les cellules conditionnées sur un substrat mou pourrait affecter la sécrétion du FN1, rendant les cellules plus adhésives et ralentissant la migration. Cependant, la déplétion de FN1 pendant la période d'amorçage n'a pas affecté la mémoire mécanique, il semble donc peu probable qu'il soit impliqué dans la mémoire mécanique.Cells are known to sense the mechanical properties of the extracellular matrix (ECM) they contact and transform these cues into biochemical and biological responses through a process called mechanotransduction. Studies have shown that the cells capability to perform mechanosensing is mediated predominantly by integrins, a subset of transmembrane receptors linking the ECM to the cell's cytoskeleton and modulating several signalling pathways. In response to the ECM's stiffness cells undergo various phenotypic changes: on softer substrates they form fewer and smaller focal adhesions, have a reduced spreading area, exert less forces and present slower migration speeds. Interestingly, these phenotypic changes have been observed to persist for some time after the mechanical cues subsided. This ability to retain information and the effects of past mechanical cues is named "mechanical memory". Little is known of the mechanisms involved in the set-up of this mechanical memory of cells. Some players have been identified, namely RUNX2, YAP/TAZ and MicroRNA-21, which have been suggested to serve as "memory storage pathways", but the upstream regulators have not yet been established. Deepening our knowledge of this mechanical memory would be especially interesting in regard to cancer and the process of metastasis. The stiffness of certain types of neoplastic tumours, such as in breast and colon cancers, have been identified to be higher than that of neighbouring healthy tissue. Furthermore, these observations have been correlated with an increasing risk of cancer progression and metastasis formation. Mechanical memory could presumably affect the ability of cancer cells to migrate in normal, softer tissues and facilitate the dissemination of those cells once they have left the stiffer primary site. A better understanding of the mechanisms involved in mechanical memory could therefore enable us to develop new therapeutic targets.

Titre:

  Etude des mécanismes supportant la mémoire mécanique cellulaire. Mots clés : Mémoire Mécanique, Epigénétique, Mécanotransduction, Migration Résumé : Tout comme nous, les cellules ont la capacité de sentir et d'être affectées par les variations de rigidité de leur environnement immédiat. Par exemple, nous sommes plus lents sur un terrain mou, comme la boue, que sur une surface rigide comme le béton. De même, les cellules migrent plus lentement dans un environnement mou que dans un environnement rigide. Ce phénomène a été décrit comme la "mémoire mécanique" et nous tentons de décrire les mécanismes de cette mémoire mécanique. Pour l'instant, nous avons montré que la mémoire mécanique des cellules dépend d'une protéine qui affecte l'architecture de l'ADN. Toutes les cellules possèdent la même séquence d'ADN, mais sa forme est modifiée pour permettre aux cellules d'exprimer différents gènes et d'acquérir différentes caractéristiques. Nous avons identifié que KDM7a, une protéine impliquée dans ces changements de structure de l'ADN est nécessaire pour que les cellules puissent stocker des informations sur leur histoire mécanique. Title: Study of the mechanisms supporting cellular mechanical memory. Keywords: Mechanical Memory, Epigenetics, Mechanotransduction, Migration

  has been shown to spontaneously undergo a partial unwrapping from nucleosomes, shaping the nucleosome into a transient state which occurs 2 to 10% of the time 245 . Studies have demonstrated that nucleosomal DNA and histones interact at multiple sites (9-10 pairs of distinct structures). Dissociation starts at the entry-exit point but not all sites immediately disengage, only around three or four of them initially do 238 . This partial unwrapping of DNA is considered a steppingstone to further unwrapping. Indeed, as it reveals DNA binding sites,

proteins can interact with the nucleosomal DNA which both stabilises and increases the unwrapping from the nucleosome 245 . It is important to note that this process is possibly quite transient as partially unwrapped nucleosomal DNA does not seem to be as stable as its fully wrapped counterpart. However, it does appear that, unlike many other processes involved in nucleosome dynamics, this spontaneous unwrapping enables gene accessibility independently of chromatin remodelling factors.

  Gene transcription initiation occurs at promoter sites. Interestingly, about 70% of promoters are associated with CpG islands which can be methylated to regulate gene silencing. Another key modulator of gene expression are enhancers, which activate transcription through the recruitment of TFs to promoters. Although they can be, enhancers do not require to be in close proximity with promoters to regulate transcription. Indeed, it was shown that chromatin looping 256 enabled enhancers to come into physical contact with promoters even from a

	only represents 2 to 3% of the genome 255 . Chromatin remodelling plays a pivotal role in DNA
	packaging and in many other processes such as replication, transcription and chromosome
	segregation.
	III.1.3.i. Euchromatin
	Euchromatin conformation allows for preferential binding of transcriptional machinery due to
	its open configuration. It is mostly found on genes being actively transcribed, on enhancer and
	promoter regions.
	66

Table 1 :

 1 PA composition to obtain varying hydrogel rigidities.

	Stiffness (kPa)	PBS	Acrylamide 40%	Bis-acrylamide 2%	APS	TEMED
	0,5	87%	10.1%	1.5%	1%	0.3%
	2	82.8%	14%	22.2%	1%	0.3%
	80	6.6%	40%	52%	1%	0.3%

  ).

	Soft-priming increases KDM7a expression in osteoblasts and HS 567T cells but not in MDA-MB-
	231 cells. 2 > N > 1. C) Osteoblasts underwent a double depletion, 72h and 24h prior to
	mechanical priming, using control siRNAs and siRNAs targeting KDM7a or HR. Cells were then
	mechanically primed for 3 days on either a soft (0.5kPa) or stiff (80kPa) Col I coated PA hydrogel
	before being transferred for a day on a stiff secondary substrate for analysis. Depletion was
	verified, by WB, 4 days after the second depletion, in cells cultured on TCP. D) Average migration
	speed of osteoblasts, depleted for either KDM7a or HR, after 1 day on a stiff secondary substrate
	with a previous 3 day mechanical priming (St3-St1 / So3-St1). HR depletion did not affect
	mechanical memory while KDM7a depletion increased the migration speed of soft-primed
	osteoblasts. Horizontal square brackets denote statistical significance (p < 0.05). 3 > N > 2. E)
	Quantification of the fluorescence intensity of KDM7a stainings in osteoblasts depleted twice for
	either siCtrl or siKDM7a, cultured for a day on a stiff secondary substrate (Col I coated glass)
	after a 3 day mechanical priming (St3-St1 / So3-St1). Depleting KDM7a during mechanical
	priming the increase in KDM7a expression observed in soft-primed cells. N=2. Error bars =
	SEM. Absence of brackets; non-significant, p < 0.05; *, p < 0.01; **.

IV.2. The LINC complex: force transmission and gene regulation ........................................ Bibliography ...................................................................................................................................... Review on the constraints and frustration in the clathrin dependent endocytosis pathway ...... Resumé de thèse en francais ............................................................................................................. Résumé de thèse en anglais ...............................................................................................................

29A). On a stiff secondary substrate, cells that had been primed for two days on a soft hydrogel presented a decreased migration speed compared to cells that had been continuously cultured on a stiff substrate. Thus, two days are enough for cells to set up a mechanical memory. It will be interesting to test shorter priming periods and determine how it impacts cell velocity on the secondary substrate to better delineate the dynamics of mechanical memory.

Acknowledgements

I would first like to thank the members of my jury, Dr Sara Wickström, Dr Boris Hinz, Dr compared to siCtrl cells (data not shown). We will complete these verifications by looking at the kinetics of depletion over the mechanical priming (days -3 to -1) by WB and through immunofluorescence analyses.

affected by mechanical priming in our cells lines capable of establishing a mechanical memory.

The LINC complex and the lamina's integrity are tightly associated and both play a critical role in nuclear integrity and stiffening. Therefore, I wonder whether the LINC complex could be involved in mechanical memory set-up. Interestingly, our RNA sequencing results showed an up-regulation of Sun2 in osteoblasts seeded on a soft substrate. Sun proteins, through their association with lamins and lamin associated proteins (such as emerin) 175 provide organisational capacity to chromatin. For instance, depletion of Sun1/2 was shown to increase H3K9 me3 marks and inhibit adipogenic gene expression 401 . Verification of this potential overexpression of Sun2 would be first required. If confirmed, I could target Sun2 and other LINC complex proteins like nesprins, using siRNAs, to disrupt the connection between the cytoskeleton and the nuclear envelope and to see if it affects migration speed, KDM7a and heterochromatin levels.

Resumé de thèse en francais

Les cellules sont connues pour détecter les propriétés mécaniques de la matrice extracellulaire (MEC) avec laquelle elles sont en contact et pour transformer ces signaux en réponses biochimiques et biologiques par un processus appelé la mécanotransduction. Des études ont montré que la capacité des cellules à effectuer la mécanotransduction est principalement médiée par les intégrines, un sous-ensemble de récepteurs transmembranaires reliant la MEC au cytosquelette de la cellule et modulant plusieurs voies de signalisation. En réponse à la rigidité de la MEC, les cellules subissent divers changements phénotypiques : sur des substrats plus mous, elles forment des adhésions focales moins nombreuses et plus petites, ont une zone d'étalement réduite, exercent moins de forces et présentent des vitesses de migration plus lentes.

Il est intéressant de noter que ces changements phénotypiques persistent pendant un certain temps après la disparition des signaux mécaniques. Cette capacité à retenir l'information et les effets des signaux mécaniques passés est appelée "mémoire mécanique". Les mécanismes impliqués dans la mise en place de cette mémoire mécanique des cellules sont encore peu connus. Certains acteurs ont été identifiés, à savoir RUNX2, YAP/TAZ et MicroRNA-21, qui serviraient de "voies de stockage de la mémoire", mais les régulateurs en amont n'ont pas encore été établis. L'objet de ma thèse était d'étudier le mécanisme de la mémoire mécanique, en utilisant des lignées cellulaires de cancer mammaire humain (HS 578T et MDA-MB-231) ainsi qu'une lignée cellulaire non cancéreuse d'ostéoblastes de souris. Les cellules ont été amorcées sur des gels de polyacrylamide (PA), de différentes rigidités (allant de 0,5kPA à GPa), pendant une période de 3 jours avant d'être transférées sur un gel de PA de 80kPa. Les cellules ont été maintenues sur ce substrat secondaire pendant une journée et différents paramètres ont été mesurés pour étudier la mémoire mécanique. Nous avons d'abord établi que seules deux de nos lignées cellulaires, à savoir les ostéoblastes et HS 578T ont établi une mémoire mécanique. Les cellules conditionnées sur un substrat mou ont montré des vitesses de migration et des taux de prolifération plus faibles sur un substrat secondaire rigide que les cellules qui avaient été continuellement cultivées sur un substrat rigide. Nous avons montré que la perturbation du cytosquelette affectait la mémoire mécanique liée à un substrat rigide, probablement en empêchant la transmission d'informations externes à la cellule. Nous avons ensuite étudié le rôle potentiel de l'épigénétique dans le stockage de la mémoire mécanique. Perturber le paysage