Docteur De L'

R Amaris

Guevara Garcia

Thèse Dirigée

Corinne Albiges-Rizo

Doctorat

Mme Caroline Hill

Ellen Mme

Van Obberberghen-Schilling

M Dominique Bourgeois

Président M Mathieu

Mme Corinne Albiges-Rizo

Mme Catherine Picart

THÈSE Pour obtenir le grade de

Keywords: Alkaline Phosphatase ALK, Activin Like-Kinase BMP, Bone Morphogenetic Protein BMPR, BMP receptor Juvenile Polyposus syndrome FOP, Fibrodysplasia Ossificans Progressiva internal Reflection FRAP, Fluorescence Recovery After Room Temperature PVDF, Polyvinylidene difluoride SDS, sodium dodecyl sulfate PFA, Paraformaldehyde TBS, Tris-Buffer Saline PBS, Phosphate Buffer Saline PLL, Poly-L-Lysine HA, Hyaluronan acid Cell culture

This chapter introduces essential terms, background, and state of the art in studying the BMP receptor and integrin signaling modulation.

The first section (I.A.) will focus on the Bone Morphogenetic Protein (BMP) signaling complexity. It explores the interest to study BMP2. The determinants of BMP2 signaling versatility are discussed.

This section also includes a description of BMP receptors (BMPR), their co-receptors, and, finally, the impact on BMP2 signaling defects on human development and diseases.

A second section (I.B.) will deal with the extracellular matrix's bio-physical properties and its role in cell adhesion. As a growth factor reservoir to control the distribution, activation, and presentation of growth factors to cells, the biomimetic extracellular matrix, is an essential actor to assist cooperation between BMPR and integrins. This section will summarize the crucial players in BMP2 signaling modulation and their importance for the cytoskeleton organization and osteogenic process through cooperation with integrinmediated adhesive structures.

Finally, the last section (I.C.) will address the current knowledge about the BMP2 spatio-temporal signaling in distinct in vivo and cellular models and the manipulation of BMP2 signaling in space and time using biomaterials and the optogenetic approach.

Discrete spatial organization and segregation of BMP2 receptors in β3-integrin containing adhesion sites

Bone Morphogenetic Protein, Receptors, and Signaling.

The Bone Morphogenetic Proteins (BMPs) are secreted cytokines that constitute the largest subdivision of the Transforming Growth Factor-β (TGF-β) superfamily. The TGF β family is composed of 4 subfamilies of a total of 30 structurally related growth factors (Figure 1).

The BMPs were first described for their osteogenic potential in 1995 [START_REF] Urist | Bone: formation by autoinduction[END_REF][START_REF] Wozney | Novel regulators of bone formation: molecular clones and activities[END_REF]. However, BMPs have pivotal roles during embryogenesis [START_REF] Hegarty | BMP2 and GDF5 induce neuronal differentiation through a Smad dependant pathway in a model of human midbrain dopaminergic neurons[END_REF], tissue homeostasis [ M. [START_REF] Chau | Gene Expression Profiling Reveals Similarities between the Spatial Architectures of Postnatal Articular and Growth Plate Cartilage[END_REF], cellular proliferation [START_REF] Ning | Opposing roles and potential antagonistic mechanism between TGF-β and BMP pathways: Implications for cancer progression[END_REF], migration [START_REF] Fiedler | BMP-2, BMP-4, and PDGF-bb stimulate chemotactic migration of primary human mesenchymal progenitor cells[END_REF]Laure Fourel, Valat, Faurobert, Guillot, Reynard, et al., 2016) and differentiation. BMP signaling is widely investigated for vertebral skeletal development and homeostasis [START_REF] Salazar | BMP signalling in skeletal development, disease and -177 -repair[END_REF], but it also has fundamental roles in developing the majority of tissues [START_REF] Wagner | BMPs: from bone to body morphogenetic proteins[END_REF].

The main BMPs involved in skeletal development are BMP2,-4,-5,-6,-7, and their antagonist Noggin and Gremlin (reviewed in [START_REF] Salazar | BMP signalling in skeletal development, disease and -177 -repair[END_REF]). Disruption in BMP pathways or their regulation is increasingly associated with a wide range of human pathologies. Because of their full range of functions and implications, some authors even proposed to call them "Body Morphogenetic

Proteins" [START_REF] Reddi | BMPs: From bone morphogenetic proteins to body morphogenetic proteins[END_REF]. Bone Morphogenetic Protein 2 (BMP2)

BMP2 is the most potent osteoinductive factor known so far: it can initiate the differentiation of mesenchymal stem cells into chondrocytes and osteoblasts in vivo and in vitro [START_REF] Ryoo | Critical molecular switches involved in BMP-2-induced osteogenic differentiation of mesenchymal cells[END_REF], as well as the trans-differentiation of muscle cells into bone cells [START_REF] Asakura | Muscle satellite cells are multipotential stem cells that exhibit myogenic, osteogenic, and adipogenic differentiation[END_REF][START_REF] Katagiri | Bone morphogenetic protein-2 converts the differentiation pathway of C2C12 myoblasts into the osteoblast lineage[END_REF] Because of its osteogenic potential, the clinical use of recombinant human BMP2 was approved in 2002 by the Food and Drug Administration and the European Medicines Agency.BMP/2 It is mainly used in combination with an absorbable collagen sponge to repair open bone fractures in spinal fusion and maxillofacial reconstruction. Alternative carrier materials have also been explored, such as alginate hydrogel or polyelectrolyte films (Figure 2). However, despite its potential benefit to bone repair and regeneration, a high percentage of patients suffered complications, such as hematomas, dysphagia, and a heightened risk of cancer [START_REF] Fu | Effectiveness and harms of recombinant human bone morphogenetic protein-2 in spine fusion: A systematic review and meta-analysis[END_REF][START_REF] Ong | Off-label use of bone morphogenetic proteins in the United States using administrative data[END_REF].

Due to the increasing number of adverse event reports and the growing socio-economic need for bone repair therapies, there is now a great need to understand BMP signaling and related molecules to link to translation medicine and improve their clinic use, not only for bone therapy but also others BMP-related diseases. [START_REF] Kolambkar | Spatiotemporal delivery of bone morphogenetic protein enhances functional repair of segmental bone defects[END_REF]) C) Critical size volumetric bone femoral defect repair after implantation using a polyelectrolyte film coating over an implant loaded with BMP2 [START_REF] Bouyer | Surface delivery of tunable doses of BMP-2 from an adaptable polymeric scaffold induces volumetric bone regeneration[END_REF] a.

BMP receptors (BMPR)

The TGF-β superfamily members signal their binding to a heterotetramer composed of a dimer of two type-I and two type-II receptors (Figure 3). Both types of receptors are made of a relatively short extracellular domain, a single transmembrane region, and an intracellular part containing the serine/threonine kinases. In particular, BMP2/4 binds to type-I receptor: ALK3 (Activin Like Kinase-3

or BMPRIa) or ALK6 (activin like kinase-6 or BMPRIb) [START_REF] Ten Dijke | Characterization of type I receptors for transforming growth factor-β and activin[END_REF][START_REF] Yamashita | Bone morphogenetic protein receptors[END_REF] and to type-II receptors: either BMPRII, ActRIIA [START_REF] Allendorph | Structure of the ternary signaling complex of a TGF-beta superfamily member[END_REF] or ActRIIB [START_REF] Miyazono | BMP receptor signaling: Transcriptional targets, regulation of signals, and signaling cross-talk[END_REF]. The expression of receptors is tissuespecific.

I-15

The type-I BMP receptors are mainly distinguished from the type-II by a shorter intracellular domain and the presence of a conserved domain rich in glycine-serine (GS box) (Figure 3). It is assumed that the Type-I receptor's activation requires binding on the ligand and then recruiting the type-II to the cell surface. The oligomerization of the two types of receptors phosphorylates the multiple serine and threonine residues of the Type-I cytoplasmic GS domain by the type-II receptor. Mutation within the GS domain [START_REF] Wieser | GS domain mutations that constitutively activate TβR-I, the downstream signaling component in the TGF-β receptor complex[END_REF] can elevate kinase activity or impair phosphorylation and signal activity

The type-I receptors have higher affinities to the ligand than the type-II receptors [START_REF] Kirsch | BMP-2 antagonists emerge from alterations in the low-affinity binding epitope for receptor BMPR-II[END_REF].

Whereas three of the five type-II receptors (ActRIIA, ActRIIB, and BMPRII) serve for more than 25 different ligands in a promiscuous manner, type-I receptor recruitment seems more restricted [START_REF] Yadin | Structural insights into BMP receptors: Specificity, activation and inhibition[END_REF]. The BMP-mediated signal transduction differs depending on the type-I receptors involved (D. [START_REF] Chen | Differential Roles for Bone Morphogenetic Protein (BMP) Receptor Type IB and IA in Differentiation and Specification of Mesenchymal Precursor Cells to Osteoblast and Adipocyte Lineages[END_REF][START_REF] Zou | Distinct roles of type I bone morphogenetic protein receptors in the formation and differentiation of cartilage[END_REF]. When both receptor subtypes are present, their binding affinity increases dramatically [START_REF] Rosenzweig | Cloning and characterization of a human type II receptor for bone morphogenetic proteins[END_REF].

ALK3 and ALK6 are closely related and differ from the others type-I receptors; they share 85% homology in their kinase domain and 42% identity in their extracellular domain [START_REF] Ide | Growth Regulation of Human Prostate Cancer Cells by Bone Morphogenetic Protein-2[END_REF][START_REF] Katagiri | Bone morphogenetic proteins[END_REF]. Several studies have shown that ALK3 and ALK6 perform different functions in inducing mesenchymal cell differentiation, as ALK3 induces adipogenic while ALK6 is involved in osteoblastic differentiation (D. [START_REF] Chen | Differential Roles for Bone Morphogenetic Protein (BMP) Receptor Type IB and IA in Differentiation and Specification of Mesenchymal Precursor Cells to Osteoblast and Adipocyte Lineages[END_REF][START_REF] Kamiya | Disruption of BMP Signaling in Osteoblasts Through Type IA Receptor (BMPRIA) Increases Bone Mass*[END_REF]. The deletion of ALK3 in osteoblasts also surprisingly leads to an increase in bone mass. (N. Kamiya et al., 2008). In the case of ALK3, few cytoplasmatic direct interactors are described (Figure 3); at the kinase level, FKP12 (Y. G. [START_REF] Chen | Mechanism of TGFβ receptor inhibition by FKBP12[END_REF] acts as a gatekeeper mechanism, setting a threshold for type-I receptor activation in the absence of ligand [START_REF] Peiffer | Activation of BMP Signaling by FKBP12 Ligands Synergizes with Inhibition of CXCR4 to Accelerate Wound Healing[END_REF] Both BMPRII and ALK3 interact with caveolin-1, an essential component of lipid rafts and clathrinindependent internalization, which results in the inhibition of BMP signaling (Anja Nohe, Keating, Underhill, [START_REF] Nohe | Dynamics and interaction of caveolin-1 isoforms with BMP-receptors[END_REF]. Binding to the BMPs decreases the interaction of these BMP receptors with caveolin. While ALK3 appears to be associated with lipid rafts, ALK6 is instead associated with clathrin-rich regions [START_REF] Hartung | Localization of BMP receptors in distinct plasma membrane domains and its impact on BMP signaling[END_REF][START_REF] Nohe | Dynamics and interaction of caveolin-1 isoforms with BMP-receptors[END_REF]. The BRAM1 (BMP receptorassociated molecule1) and XIAP (X-chromosome-linked inhibitor of apoptosis) interact with the Cterminus of ALK3. [START_REF] Kurozumi | BRAM1, a BMP receptor-associated molecule involved in BMP signalling[END_REF]. XIAP, TAB, and TAK1 act downstream upon BMP4 activation. XIAP participates in the BMP signaling pathway as a positive regulator linking the BMP receptors and TAB1-TAK1 [START_REF] Yamaguchi | XIAP, a cellular member of the inhibitor of apoptosis protein family, links the receptors to TAB1-TAK1 in the BMP signaling pathway[END_REF]. Additionally, the Splicing Factor 3b Subunit 4 (SF3b4) is also able to bind ALK3, thereby inhibiting osteochondral cell differentiation [START_REF] Watanabe | Splicing factor 3b subunit 4 binds BMPR-IA and inhibits osteochondral cell differentiation[END_REF] Type-II BMP receptor binds to the receptor complex with a relatively lower affinity. This low affinity leads to greater flexibility by which BMPs can signal using a more diverse set of cell-surface receptors [START_REF] Heldin | Signaling Receptors for TGF-β Family Members[END_REF]. The kinase domains of type-II receptors are constitutively active and phosphorylate the type-I receptor kinase [START_REF] Salazar | BMP signalling in skeletal development, disease and -177 -repair[END_REF]. BMP2 and BMP4 can bind to ActRIIA, ActRIIB (Activin receptor type-IIA and IIB), and BMPRII with at least 50-to 60-fold lower affinity than the type-I receptor ALK3. Also, binding of BMP2 to all type-II receptors is characterized by much faster kinetic rate constants (kon and koff) compared to binding to type-I receptors [START_REF] Nickel | Specification of BMP Signaling[END_REF].

BMPRII is the only receptor from the TGF-β superfamily that is expressed in two splice variants, the long-form (BMPRII-LF) and the short-form (BMPRII-SF). The long-form has a long cytoplasmic tail of 512 amino acids. The long-form and short-form presented differences in their steady-state levels, the kinetics of degradation, intracellular distribution, and internalization rate [START_REF] Amsalem | Differential regulation of translation and endocytosis of alternatively spliced forms of the type II bone morphogenetic protein (BMP) receptor[END_REF].

The intracytoplasmic tail of the long-form acts as a docking site for several proteins (Figure 3). Most of these proteins seem to be involved in the non-canonical signaling [START_REF] Hassel | Proteins associated with type II bone morphogenetic protein receptor (BMPR-II) and identified by twodimensional gel electrophoresis and mass spectrometry[END_REF], including the endocytic EPS15R, the dynein light chain Tctex-1 [START_REF] Machado | Functional interaction between BMPR-II and Tctex-1, a light chain of dynein, is isoform-specific and disrupted by mutations underlying primary pulmonary -171 -hypertension[END_REF], the kinases cGKI, Trb3, LIMK (Foletta, Lim, Soosairajah, Kelly, Stanley, Shannon, He, Das, Massagué, et al., 2003), which gives an essential function of BMPR in the regulation of cytoskeletal proteins. BMPRII can inhibit LIMK phosphorylation of cofilin through cooperation with Cdc42 small GTPase and regulates BMP-induced changes in actin dynamics (Foletta, Lim, Soosairajah, Kelly, Stanley, Shannon, He, Das, Massagué, et al., 2003;[START_REF] Lee-Hoeflich | Activation of LIMK1 by binding to the BMP receptor , BMPRII , regulates BMP-dependent[END_REF], while the addition of BMP-4 reduced this effect (Foletta, Lim, Soosairajah, Kelly, Stanley, Shannon, He, Das, Massagué, et al., 2003). Nevertheless, an alternative mechanism has also been observed during neuronal differentiation, where LIMK1 binding to BMPRII activates the kinase together with the Cdc42, promoting actin polymerization and dendrite formation [START_REF] Lee-Hoeflich | Activation of LIMK1 by binding to the BMP receptor , BMPRII , regulates BMP-dependent[END_REF].On the other hand, phosphatases are involved in regulating SMAD pathways, like PP2A, which interacts with the BMP receptor complex and dephosphorylates the linker sites at SMAD1 to promote SMAD signaling [START_REF] Bengtsson | PP2A regulates BMP signalling by interacting with BMP receptor complexes and by dephosphorylating both the C-terminus and the linker region of Smad1[END_REF].

The type-II receptors ActRIIA and ActRIIB bind BMPs but also activins, while BMPRII only binds BMPs.

BMPRII and ActRIIB compensated each other functionally in mediating BMP2 signaling and BMP2induced osteoblast differentiation (H. [START_REF] Liu | Functional redundancy of type II BMP receptor and type IIB activin receptor in BMP2-induced osteoblast differentiation[END_REF]. BMPRII availability alleviates receptor-level competition between BMPs and activins, where utilization of ActRIIA and ActRIIB by BMPs comes at the expense of activating ligand [START_REF] Lowery | Loss of BMPR2 leads to high bone mass due to increased osteoblast activity[END_REF].

I-17

b.

BMP receptor oligomerization

The oligomerization pattern of BMP Type-I and type-II receptors is flexible, leading to a great diversity in cell signaling. Several factors influence the binding of BMP receptor partners and, thus, the choice of the pathway activation [START_REF] Ehrlich | Endocytosis and trafficking of BMP receptors: Regulatory mechanisms for finetuning the signaling response in different cellular contexts[END_REF][START_REF] Nickel | Specification of BMP Signaling[END_REF]. The different checkpoints can be listed as i) ligand presentation (discussed in chapter I.B), ii) receptor oligomerization, and iii) receptor localization.

Two kinds of BMPR oligomerization have been described: the BMP Induced Signaling Complex (BISC)

and the Preformed Complex (PFC) (Figure 4). In BISC, which is localized in caveolae, BMP ligand binding on a homocomplex (BMPRI/BMPRI) induces the recruitment of a type-II receptor, giving rise to non-canonical pathway activation. In PFC that takes place in a clathrin-coated pit, the BMP ligand bind on an already formed heterodimer (BMPRI/BMPRII) and induces a SMAD-dependent pathway activation. The heterotetrameric BMP receptor complexes are stabilized by interactions between the cytoplasmic domains of the receptors (Anja [START_REF] Nohe | The mode of bone morphogenetic protein (BMP) receptor oligomerization determines different BMP-2 signaling pathways[END_REF].

While BMP receptor and BMP ligand endocytosis has been proposed to promote receptor turnover and regulate BMP signal transduction [START_REF] Hartung | Different routes of bone morphogenic protein (BMP) receptor endocytosis influence BMP signaling[END_REF], it is essential to consider the ability of BMP to be internalized or not. Previous studies have indicated that clathrin/dynamin-dependent endocytosis of BMP type-I receptors is conserved in various models, including C. Elegans, Drosophila, mouse, and human fibroblasts [START_REF] Ehrlich | Endocytosis and trafficking of BMP receptors: Regulatory mechanisms for finetuning the signaling response in different cellular contexts[END_REF]. The level of BMPR expression impacts BMP internalization. ALK3 overexpression induces an increase in BMP2 uptake [START_REF] Alborzinia | Quantitative kinetics analysis of BMP2 uptake into cells and its modulation by BMP antagonists[END_REF], and a higher expression of BMPRII short form at the plasma membrane results in enhanced activation of SMAD signaling, stressing the potential importance of the multilayered regulation of BMPRII expression at the plasma membrane [START_REF] Amsalem | Differential regulation of translation and endocytosis of alternatively spliced forms of the type II bone morphogenetic protein (BMP) receptor[END_REF]. The endocytic machinery can drive specific BMP signaling or attenuate retrograde BMP signaling [START_REF] Gilde | Secondary structure of rhBMP-2 in a protective biopolymeric carrier material[END_REF][START_REF] O'connor-Giles | Satellite signaling at synapses[END_REF] As of today, it remains elusive whether the ligand should remain bound to the receptors and become internalized via the clathrin-mediated pathway or if it would be sufficient to have trafficking of the activated receptors, regardless of ligand internalization. It has been proposed that the nature of the ligand-binding receptor assembly may play a role in cell decision-making [START_REF] Hassel | Initiation of Smad-dependent and Smad-independent signaling via distinct BMP-receptor complexes[END_REF].

Furthermore, this oligomerization can be influenced and modulated by ligand and antagonistic proteins such as Noggin [START_REF] Gilboa | Bone morphogenetic protein receptor complexes on the surface of live cells: a new oligomerization mode for serine/threonine kinase receptors[END_REF]. It is worth noting that the BMPRII short form was proved to be unable to activate non-canonical pathways, while it can activate the SMAD-dependent pathway [START_REF] Amsalem | Differential regulation of translation and endocytosis of alternatively spliced forms of the type II bone morphogenetic protein (BMP) receptor[END_REF]. Thus, the short form cannot be endocytosed while the long-form is endocytosed mostly via clathrin, although some authors have reported endocytosis via caveolae [START_REF] Ehrlich | Endocytosis and trafficking of BMP receptors: Regulatory mechanisms for finetuning the signaling response in different cellular contexts[END_REF][START_REF] Hartung | Localization of BMP receptors in distinct plasma membrane domains and its impact on BMP signaling[END_REF].

Figure 4. Two main modes of BMP2 receptor oligomerization (A) BMP2 binds to the type-I receptor, which then binds to type-II. The complex formed is then internalized in caveosomes and triggers MAPK signaling pathways. (B) BMP2 binds to a preformed complex of the two types of receptors (PFC). The complex is internalized in endosomes and triggers a signaling cascade dependent on SMAD proteins. [START_REF] Sieber | Recent advances in BMP receptor signaling[END_REF].

Before ligand binding, about 30 % of the BMP receptors are found as preformed homo-and heterooligomers at the cell surface. Moreover, the percentage of homo and hetero-oligomers increases upon ligand binding, except for BMPRII homodimers, which exhibit only low affinity to the BMP ligands [START_REF] Gilboa | Bone morphogenetic protein receptor complexes on the surface of live cells: a new oligomerization mode for serine/threonine kinase receptors[END_REF][START_REF] Nohe | The mode of bone morphogenetic protein (BMP) receptor oligomerization determines different BMP-2 signaling pathways[END_REF]. The fact that most dimers are homodimers is probably a phenomenon impeding autoactivation of the signaling without ligand.

c. BMP receptor localization and dynamics.

BMP receptors (BMPR) can either be presented in an intracellular and extracellular forms. The extracellular form binds to the BMP ligands and inhibits their action by impeding their fixation to the membrane receptors [START_REF] Natsume | Interaction between soluble type I receptor for bone morphogenetic protein and bone morphogenetic protein-4[END_REF]. The Intracellular BMPR can be localized at the I-19 membrane bound or free at the cytoplasm. At the membrane level, BMP receptors are present as hetero-or homomeric complexes [START_REF] Gilboa | Bone morphogenetic protein receptor complexes on the surface of live cells: a new oligomerization mode for serine/threonine kinase receptors[END_REF].

Different receptors might have different favorite localization. Indeed, ALK3 can be N-glycosylated, while ALK6 cannot, resulting in greater efficiency of processing and plasma membrane expression of ALK3 [START_REF] Hirschhorn | Differential molecular regulation of processing and membrane expression of Type-I BMP receptors: implications for signaling[END_REF]) (Figure 5). However, the mechanisms allowing BMPR stability on the membrane are not totally understood. BMPR are present at the cell surface as hetero-or homomeric complex [START_REF] Gilboa | Bone morphogenetic protein receptor complexes on the surface of live cells: a new oligomerization mode for serine/threonine kinase receptors[END_REF]. While ALK3 mainly associates with detergent-resistant membrane (DRM) fractions, BMPRII was reported to localize to DRM and non-DRM compartments [START_REF] Hartung | Localization of BMP receptors in distinct plasma membrane domains and its impact on BMP signaling[END_REF]. chicken-anti-myc and Alexa 546-labeled donkey-anti-chicken antibodies for detection of membrane-external receptors (red). Following fixation, samples were permeabiliz ed and labeled with mouse-anti-myc and Alexa 488-labeled goat-anti-mouse antibodies for the detection of internal receptors (green). Micrographs depict single confocal planes of representative cells. Bar 10 µm. Adapted from [START_REF] Hirschhorn | Differential molecular regulation of processing and membrane expression of Type-I BMP receptors: implications for signaling[END_REF] Studies suggest that the location of BMPR relative to their ligands and other receptors is a crucial aspect of receptor signaling. A requisite for SMAD-independent signaling is the presence of ALK3 in cholesterol-rich microdomains regulating lateral mobility of type-I receptors, which has been described to be crucial for regulating non-canonical BMP signaling without affecting canonical signaling function [START_REF] Guzman | SMAD versus Non-SMAD Signaling Is Determined by Lateral Mobility of Bone Morphogenetic Protein (BMP) Receptors[END_REF][START_REF] Hartung | Different routes of bone morphogenic protein (BMP) receptor endocytosis influence BMP signaling[END_REF]. BMP receptor type-I and type-II have distinct lateral mobilities within the plasma membrane [START_REF] Guzman | SMAD versus Non-SMAD Signaling Is Determined by Lateral Mobility of Bone Morphogenetic Protein (BMP) Receptors[END_REF], as shown by singleparticle tracking experiments (Figure 6). ALK3 and ALK6 showed to be highly confined in space, both in the presence or absence of the ligand, whereas BMPRII presented two populations, one freely diffusing and the second confined. While the preformed complex, which triggers the SMADdependent pathway, does not require the BMPRII confinement, the non-SMAD seems highly dependent on the localization of BMPRI in membrane microdomains. Thus, non-SMAD signaling might require more stable complexes, possibly to allow interaction with other protein complexes, e.g., those involved in signaling to the cytoskeleton. However, the different receptor recruitment order might not only exert quantitative effects but could also lead to qualitative signaling differences. As the receptors comprise a kinase activity, different lifetimes of the receptor in the assembly might result in different phosphorylation patterns in the cytoplasmic domains of the activated receptors dependent on the enzymatic properties, i.e., Michaelis-Menten constant ("affinity") and turnover number (conversion rate). A type-I receptor that dissociates fast from the BMP ligand-receptor assembly might not be phosphorylated at all sites compared to a type-I receptor whose dissociation rate is slow. Differences in the receptor recruitment order and ligand-specific residence times of the individual type-I and type-II receptors in the ligand-assembled complex might thus enable a fine-tuning of receptor activation leading to differences in the encoded signals [START_REF] Nickel | Specification of BMP Signaling[END_REF] The first attempts To determine BMPR localization were made by the expression of human influenza hemagglutinin (HA)-tagged BMPRII (B. [START_REF] Marom | FORMATION OF STABLE HOMOMERIC AND TRANSIENT HETEROMERIC BMP RECEPTOR COMPLEXES REGULATES SMAD SIGNALING[END_REF]. However, it remains very challenging for BMPRI because of its low expression level. More recently, the spatial distribution of ALK6 and BMPRII was achieved using high-resolution imaging techniques. Using two-color stimulated depleted emission (STED) microscopy (Figure 7), single BMPRII appears to arrange I-21 sparsely, whereas ALK6 assembles in more massive clusters made of multiple receptors when BMP2

was added to the cell culture media. [START_REF] Medda | Challenges in imaging cell surface receptor clusters[END_REF]. Furthermore, there is a lack of information regarding the spatial arrangement of BMPRs at the nanoscale and the localization of the different complexes in distinct cellular compartments. In the absence of BMP2, the two different receptors rarely co-localized (upper white arrowhead), and BMPRII did not cluster (lower arrowhead). When cells were exposed to BMP2, BMPRII was associated with the largest ALK6 assemblies. This different behavior could not be appreciated with confocal microscopy. Image adapted [START_REF] Medda | Challenges in imaging cell surface receptor clusters[END_REF].

BMP2 signaling

After binding to the tetrameric receptor complex, BMP2 signals via at least two different pathways.

On the one hand, pathway restricted SMAD proteins (R-SMADs) are recruited to and phosphorylated by the receptors (SMAD-pathway). On the other hand, BMP receptor complexes modulate several parallel signaling cascades, as described for MAPK (i.e., p38, ERK, and JNK) and NF-κB, Jak/Stat, LIMK, PI3K, mTORC, and RhoGTPases. Such non-canonical signaling cascades increase the number of genes, and subsequent cellular responses, whose expression is regulated by BMPs.

a.

Canonical pathway (SMAD)

After BMPR type-I activation, BMPRI can then phosphorylate the R-SMAD (SMAD1/5/8( 9)) in their SSXS pattern in the C-terminal tail. This modification leads to the activation of these signal transducer proteins. Upon phosphorylation, the SMAD proteins form homomeric or heteromeric triplexes with a common SMAD4 (R. N. Wang et al., 2014), translocate into the nucleus, and subsequently regulate SMAD-complexes target gene transcription through direct binding to DNA, interaction with other DNA-binding proteins and recruitment of transcriptional co-activators or co-repressors (Figure 8). Non-canonical pathway (non-SMAD)

BMPs have been described to elicit a variety of cellular responses, including cytoskeletal rearrangements and migration, some of which are present after a few minutes of stimulation, suggesting that they are independent of transcriptional regulation by SMAD [START_REF] Hiepen | BMP2induced chemotaxis requires PI3K recruitment at the cytocortex[END_REF][START_REF] Voorneveld | The BMP pathway either enhances or inhibits the Wnt pathway depending on the SMAD4 and p53 status in CRC[END_REF]. The responsible pathways are collectively referred to as non-SMAD pathways, i.e., Rho-like GTPase, mitogen-activated protein kinase (MAPK), phosphoinositide 3kinase (PI3K), as well as c-SRC and LIMK1 signaling and many more ( Y.E. Zhang, None, 2017) (Figure 9). In the MAP kinase pathway. IAP (Inhibitor of Apoptosis) is an adaptor protein linking BMPRI and TAB1 (TAK1 Binding Proteins). TAB1 activates TAK1 (transforming growth factor-beta activated kinase). The activation of TAK1 promotes the transcriptional activity of Runx2, Dlx5, and Osx through their phosphorylation by MAPK [START_REF] Wu | TGF-β and BMP signaling in osteoblast, skeletal development, and bone formation, homeostasis and disease[END_REF].TAK1 has also been reported to be an activator of JNK (Janus Kinase), p38, and ERK MAPK signaling pathways. The two pathways described above are not wholly independent nor mutually exclusive, as evidenced by the enhancement of SMAD signaling by MAPK described above. Instead, these pathways cooperate and interact to regulate transcriptional and non-transcriptional aspects of BMP2-induced signaling (Anja [START_REF] Nohe | The mode of bone morphogenetic protein (BMP) receptor oligomerization determines different BMP-2 signaling pathways[END_REF][START_REF] Sieber | Recent advances in BMP receptor signaling[END_REF] c.

Diseases by disruption of BMP2 signaling regulation

Cardiovascular and pulmonary system

BMPRs are key factors maintaining adult cardiovascular homeostasis and function. BMP2 signaling has been identified as an inducer of epithelial-to-mesenchymal transition in the endocardial cushions [START_REF] Délot | BMP signaling is required for septation of the outflow tract of the mammalian heart[END_REF][START_REF] Sugi | Bone morphogenetic protein-2 can mediate myocardial regulation of atrioventricular cushion mesenchymal cell formation in mice[END_REF].

BMP2 homozygous mutants are embryonically lethal, with malformations of the amnion and chorion and the heart's developmental abnormalities [START_REF] Rivera-Feliciano | Bmp2 instructs cardiac progenitors to form the heart-valveinducing field[END_REF]H. Zhang & Bradley, I-25 1996). Furthermore, the conditional deletion of ALK3 in the myocardium results in physiological abnormalities [START_REF] Eblaghie | Evidence that autocrine signaling through Bmpr1a regulates the proliferation, survival and morphogenetic behavior of distal lung epithelial cells[END_REF][START_REF] Sun | Prenatal lung epithelial cell-specific abrogation of Alk3-bone morphogenetic protein signaling causes neonatal respiratory distress by disrupting distal airway formation[END_REF]. ALK3 conditional knock-out (ALK3 CKO) embryos displayed defects in the atrioventricular canal (AVC) endocardial cushion formation in the heart [START_REF] Park | Bone morphogenetic protein receptor 1A signalling is despensable for hematopoietic development but essential for vessel and atrioventricular endocardial cushion formation[END_REF].

Pulmonary Arterial Hypertension (PAH) is a rare cardiovascular disorder characterized by abnormal vascular cell proliferation and the pulmonary artery's constriction. [START_REF] Atkinson | Primary pulmonary hypertension is associated with reduced pulmonary vascular expression of type II bone morphogenetic protein receptor[END_REF][START_REF] Puri | Pulmonary arterial hypertension: current therapeutic strategies[END_REF]. PAH is caused by heterozygous germline mutations of the BMPRII gene in familial and idiopathic cases (70% of patients with HPAH) [START_REF] Lane | Heterozygous germline mutations in BMPR2, encoding a TGF-β receptor, cause familial primary pulmonary hypertension[END_REF]. These mutations target sequences that encode the ligand-binding and kinase domain and the long cytoplasmic tail [START_REF] Machado | Pulmonary Arterial Hypertension: A Current Perspective on Established and Emerging Molecular Genetic Defects[END_REF]. These mutations cause nonsense, missense, or frameshift mutations, leading to the loss of correct BMPRII-function. As described previously, BMPR-II inhibits the ability of LIMK1 to phosphorylate cofilin. A truncated PPH mutant of BMPRII cannot bind and inhibit LIMK1, suggesting that the deregulating of actin dynamics may contribute to the PPH (Foletta, Lim, Soosairajah, Kelly, Stanley, Shannon, He, Das, Massagué, et al., 2003).

Gastrointestinal system

The deletion of ALK3 in the foregut endoderm leads to anteriorization of the stomach [START_REF] Maloum | Epithelial BMP signaling is required for proper specification of epithelial cell lineages and gastric endocrine cells[END_REF]. Deletion of ALK3 genes causes aggressive polyposis and numerous congenital anomalies, such as facial dysmorphism [START_REF] Septer | Aggressive juvenile polyposis in children with chromosome 10q23 deletion[END_REF]. For instance, in Juvenile Polyposis Syndrome (JPS), an autosomal dominant inherited disorder, mutations in ALK3 and SMAD4 have been associated with gastrointestinal hamartomatous polyps and risk for gastrointestinal cancers [START_REF] Blatter | Somatic alterations in juvenile polyps from BMPR1A and SMAD 4 mutation carriers[END_REF][START_REF] Carr | Germline mutations in SMAD4 disrupt bone morphogenetic protein signaling[END_REF][START_REF] Lodewijk | Gastrointestinal Polyposis Syndromes[END_REF]. A study of several families with JPS identified nonsense inactivating mutations giving rise to ALK3 receptors, which lack the intracellular kinase domain [START_REF] Howe | Germline mutations of the gene encoding bone morphogenetic protein receptor 1A in juvenile polyposis[END_REF]. Other studies confirmed the overall prevalence of ALK3 mutations in JPS patients, revealing point mutations, as well as large deletions of the gene in approximately 23% of the JPS patients [START_REF] Calva-Cerqueira | The rate of germline mutations and large deletions of SMAD4 and BMPR1A in juvenile polyposis[END_REF]. Missense mutations in ALK3 do not lead to decreased expression of the receptor but rather cause localization to the cytoplasm instead of the plasma membrane [START_REF] Howe | BMPR1A mutations in juvenile polyposis affect cellular localization[END_REF].

Elevated expression of BMP receptors in Gastric Cancer is highly associated with tumor-associated angiogenesis and lymphangiogenesis, which facilitate tumor growth, expansion, and spread.

Principally BMPRII was significantly increased in gastric tumors (Z. [START_REF] Sun | Deregulated bone morphogenetic proteins and their receptors are associated with disease progression of gastric cancer[END_REF]. While overexpression of the constitutively active form of ALK-3 suppressed the proliferation of gastric carcinoma cell lines in vitro and in vivo. Additionally, Alk3 CKO embryos displayed multiple abnormalities in vascular development, including vessel remodeling and maturation, which contributed to severe abdominal hemorrhage [START_REF] Park | Bone morphogenetic protein receptor 1A signalling is despensable for hematopoietic development but essential for vessel and atrioventricular endocardial cushion formation[END_REF]

Musculoskeletal system

Numerous studies have highlighted the importance of BMPs and mainly BMP2/-4 in osteoblastic differentiation. Indeed, the deletion of BMP2 and -4 during development leads to a major defect in the placement of bone tissue; these BMPs are necessary for the transition from the pre-osteoblast (Runx2 +) to the immature osteoblast (Runx2 + Osx +). The deletion of BMP2 in the mesenchyme that forms the limb underlines its essential role in postnatal bone formation and osteogenesis [START_REF] Bandyopadhyay | Genetic Analysis of the Roles of BMP2, BMP4, and BMP7 in Limb Patterning and Skeletogenesis[END_REF].

ALK3 is essential for BMP-SMAD signaling in the craniofacial region. Heterozygosity of ALK3 partially rescues craniosynostosis in caBmpr1a; P0Cre mice. Heterozygosity of other type-I BMPR does not rescue craniosynostosis in mice [START_REF] Pan | BmpR1A is a major type 1 BMP receptor for BMP-Smad signaling during skull development[END_REF]. Disturbed BMPR expression usually leads to bone and cartilage disorders. It is interesting to note that the deletion of ALK3 induces not a decrease but an increase in bone mass via a decrease in osteoclastic activity (Nobuhiro [START_REF] Kamiya | Wnt inhibitors Dkk1 and sost are downstream targets of BMP signaling through the type IA receptor (BMPRIA) in osteoblasts[END_REF]. In addition to their effect on osteoblastic differentiation, BMPs play an important role in osteoblastic function. Indeed, the deletion of ALK3 in the mature osteoblast, as well as the deletion of SMAD-4 and the overexpression of Noggin (an inhibitor of the BMP pathway), induce a decrease in osteoblastic activity [START_REF] Devlin | Skeletal overexpression of noggin results in osteopenia and reduced bone formation[END_REF][START_REF] Lowery | The BMP pathway and its inhibitors in the skeleton[END_REF][START_REF] Mishina | Bone morphogenetic protein type IA receptor signaling regulates postnatal osteoblast function and bone remodeling[END_REF] Accordingly, intramuscular AAV-mediated overexpression of a constitutive form of ALK3 results in increased fiber size, maximal force, and muscle mass [START_REF] Sartori | TGFβ and BMP signaling in skeletal muscle: Potential significance for muscle-related disease[END_REF]. Another group independently showed that over-activation of BMP signaling in muscle by ectopic expression of constitutively active ALK3 prevents muscular atrophy. ALK3 expression in myo-endothelial Myf5+ cells is necessary to support the activity of adipogenic progenitors within the muscle. (P. [START_REF] Huang | Intramuscular adipogenesis is inhibited by myo-endothelial progenitors with functioning Bmpr1a signalling[END_REF].

Cancer

Several studies have demonstrated a secure link between mutations of certain BMP receptors and the progression of specific cancers. ALK3 mutations and JPS led to speculation of the role of BMP in colorectal cancer. Indeed, the BMP pathway is inactivated in the majority of sporadic colorectal cancer cases. [START_REF] Hardwick | Bone morphogenetic protein signalling in colorectal cancer[END_REF][START_REF] Kodach | The Bone Morphogenetic Protein Pathway Is Inactivated in the Majority of Sporadic Colorectal Cancers[END_REF]. Genetic variations in ALK3, ALK6, BMPRII, and BMP2/4 are all associated with the risk of developing colon cancer, with the most high-risk phenotypes conferring a 30% increased risk. Elevated BMP2 levels

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with increased malignancy promote lung tumor growth and stimulate angiogenesis in developing tumors [START_REF] Jiang | A conserved post-transcriptional BMP2 switch in lung cells[END_REF].

As BMP2 has been found to induce the expression of cell cycle regulators, this might be a reason for pathological cellular proliferation. In pancreatic cancer, reduced expression of ALK3 was found to be associated with a poor prognosis [START_REF] Voorneveld | Reduced expression of bone morphogenetic protein receptor IA in pancreatic cancer is associated with a poor prognosis[END_REF].

Disruption of bone morphogenetic protein receptor II in mammary tumors promotes metastases through cell-autonomous and paracrine mediators [START_REF] Owens | Disruption of bone morphogenetic protein receptor 2 (BMPR2) in mammary tumors promotes metastases through cell autonomous and paracrine mediators[END_REF]. More recently, the deletion of the BMP receptor ALK3 was found to impair mammary tumor formation and metastasis [START_REF] Owens | Inhibition of BMP signaling suppresses metastasis in mammary cancer[END_REF]. However, the role of BMP signaling in different cancer types starts to emerge, and studies appear contradictory since BMPs can either promote or suppress tumoral progression.

In contrast, BMP signaling has been shown to decreasing the malignant potential of ovarian cancer.

Epithelial ovarian cancer (EOC) cells have the ability to form multi-cellular aggregates in malignant ascites, which dramatically alters cell signaling, survival, and metastatic potential. It was demonstrated [START_REF] Peart | BMP signalling controls the malignant potential of ascites-derived human epithelial ovarian cancer spheroids via AKT kinase activation[END_REF]that patient ascites-derived EOC cells down-regulate endogenous bone morphogenetic protein (BMP) signaling by decreasing BMP ligand expression when grown in suspension culture to form spheroids. Enforced BMP signaling in these cells via constitutively-active BMP receptor type-I. ALK3(QD) receptor expression causes the formation of smaller, more loosely-aggregated spheroids.

Additionally, ALK3(QD)-expressing spheroids have an increased rate of adhesion and dispersion upon reattachment to the substrate. ALK3 constitutive active expression in ovarian cancer cells reduced tumor implantation on peritoneal surfaces and ascites [START_REF] Shepherd | Constitutive activation of BMP signalling abrogates experimental metastasis of OVCA429 cells via reduced cell adhesion[END_REF]. BMP signaling through SMAD3 facilitates cancer cell invasion. Consistent with increased BMPmediated SMAD2/3 signaling during cancer progression, SMAD1/5, and SMAD 2/3 signaling converge in human cancer specimens [START_REF] Holtzhausen | Novel bone morphogenetic protein signaling through Smad2 and Smad3 to regulate cancer progression and development[END_REF].

Other tissues

Conditional knock-out (KO) of ALK3 and BMPRII results in embryonic lethality [START_REF] Park | Bone morphogenetic protein receptor 1A signalling is despensable for hematopoietic development but essential for vessel and atrioventricular endocardial cushion formation[END_REF], and ActRIIB knock-outs die shortly after birth (R. N. Wang et al., 2014). It has demonstrated the requirement of Alk3 in distinct progenitor cell populations derived from the intermediate mesoderm, like kidney and male gonad in mice (di Giovanni, Alday, Chi, [START_REF] Di Giovanni | Alk3 controls nephron number and androgen production via lineage-specific effects in intermediate mesoderm[END_REF]. ALK3 is required for lens and retinal growth but was not essential for the formation of lens. [START_REF] Zhao | Mutually inductive interactions between the lens and retina require ALK3 functions during mouse embryonic development[END_REF]. Expression of the constitutively active ALK3 receptor ALK3QD, in two independent transgenic lines, caused renal aplasia/severe dysgenesis in less than 10% of mice, and renal medullary cystic dysplasia in more than 50%

BMPR co-receptor and other cell surface protein

To control ligand binding to the receptors and, consequently, activation of signaling, cell expressed transmembrane or membrane-anchored co-receptors that selectively aid in ligand binding to the receptors. Some co-receptors enhance ligand binding, whereas others help to limit ligand binding, and their secreted ectodomains may sequester ligands. Additionally, other co-receptors would alter the BMP receptor organization. BMP binding to BMP receptors can be facilitated (or modulated) by so-called co-receptors that lack an intrinsic signaling motif (Figure 11). It has been suggested that binding of co-receptors may influence the orientation of the receptor complexes upon binding with different ligands, thereby determining the phosphorylation of their downstream substrates.

Moreover, the binding kinetics and the stability of the complex formed between ligand and receptor may influence the potency of the signal transduction by the receptor kinase [START_REF] Mueller | Promiscuity and specificity in BMP receptor activation[END_REF]) a.

Impact of BMP co-receptors on BMP ligand

BMPs interact with proteoglycans, which are present both at the cell surface and within the ECM.

Indeed, proteoglycans decorate both ECM proteins such as collagen, fibronectin, fibrillin and other receptors present at the cell membrane, including MuSK (muscle-specific kinase) and BAMBI (BMP and activin membrane-bound inhibitor) [START_REF] Nickel | TGF-β family co-receptor function and signaling[END_REF]. By interacting with the cell surface and ECM, cell-secreted BMPs have a constrained range of action in the pericellular space.

BAMBI has an extracellular domain comparable to other type-I receptors. BAMBI is a negative regulator of BMP signaling during embryonic development [START_REF] Grotewold | Bambi is coexpressed with Bmp[END_REF][START_REF] Onichtchouk | Silencing of TGF-β signalling by the pseudoreceptor BAMBI[END_REF].

Beta glycan is a single-pass transmembrane proteoglycan binds BMP2 and BMP-4 [START_REF] Bilandzic | Betaglycan: A multifunctional accessory[END_REF][START_REF] Nickel | TGF-β family co-receptor function and signaling[END_REF]. Endoglin is also a single-pass transmembrane protein that acts as an accessory protein, binds activin A, BMP-7, and BMP2 and promotes their signaling via SMAD1, 5, and 8 [START_REF] Coles | Practical Analysis Techniques of Polymer Fillers by Fourier Transform Infrared Spectroscopy (FTIR)[END_REF]. Endoglin binds to BMPs only by associating with the corresponding type-II receptors [START_REF] Nickel | TGF-β family co-receptor function and signaling[END_REF]. Previous studies have shown that low expression of endoglin is associated with increased osteogenic potential, while high endoglin expression is linked with high adipogenic potential (ref).

The repulsive guidance molecule (RGMb, also called DRAGON) is also a Glycosylphosphatidylinositol anchored molecule; it binds BMP2/4 and facilitates signaling via BMP type-II and type-I receptors [START_REF] Samad | DRAGON, a Bone Morphogenetic Protein Co-receptor[END_REF]. The RGMa, RGMb homolog, also acts as a BMP co-receptor and facilitates the selective association of BMP ligands to specific type-II receptors. High RGMa expression chooses for ActRII as the signaling receptor of BMP2/4, whereas low RGMa expression permits the same ligands to be selective for BMPRII as their signaling receptor [START_REF] Xia | Repulsive guidance molecule RGMa alters utilization of bone morphogenetic protein (BMP) type II receptors by BMP2 and BMP4[END_REF].

Neogenin has been recently postulated as a BMP receptor. It has been shown to bind BMP2, -4, -6, and -7 and somehow suppresses intracellular activation induced by BMPs by activating the RhoA pathway [START_REF] Hagihara | Neogenin, a receptor for bone morphogenetic proteins[END_REF].

b. Impact of co-receptors on BMPR organization

In addition to modifying the ligand's affinity, it has been suggested that binding of co-receptors may influence the orientation of the receptor complexes upon binding with different ligands. Moreover, the binding kinetics and the stability of the complex formed between ligand and receptor may influence the potency of the signal transduction by the receptor kinase. BMP signaling diversity is enhanced by cross-talk to other transmembrane receptor pathways such as receptor tyrosine kinase (RTK) [START_REF] Biver | Molecular and Cellular Endocrinology Crosstalk between tyrosine kinase receptors , GSK3 and BMP2 signaling during osteoblastic differentiation of human mesenchymal stem cells[END_REF]Zhou, Lee, Lee, Wei, Lim, Lin, Chien, & Chiu, 2013), c-Kit [START_REF] Hassel | Interaction and functional cooperation between the serine/threonine kinase bone morphogenetic protein type II receptor with the tyrosine kinase stem cell factor receptor[END_REF], orphan receptor (Ror)2 [START_REF] Sammar | Modulation of GDF5/BRI-b signalling through interaction with the tyrosine kinase receptor Ror2[END_REF] or G-protein coupled receptor (GPCR) (G. [START_REF] Jin | Progastrin stimulates colonic cell proliferation via CCK2R-and β-arrestin-dependent suppression of BMP2[END_REF].

Such cross-talk can be in synergy by feed-forward mechanisms (H. Liu et al., 2012) or counteract each other for a negative fine-tuning [START_REF] Little | Bone morphogenetic protein heterodimers assemble heteromeric type I receptor complexes to pattern the dorsoventral axis[END_REF][START_REF] Oshimori | Paracrine TGF-β signaling counterbalances BMP-mediated repression in hair follicle stem cell activation[END_REF]. Cross-talk to other cascades is mediated via either protein-protein interactions or transcriptional feedbacks (C. Liu et al., 2012;[START_REF] Schwappacher | Novel crosstalk to BMP signalling: cGMP-dependent kinase I modulates BMP receptor and Smad activity[END_REF]. Protein-protein cross-talk interactions occur directly on the plasma membrane through interactions between BMP receptors and other transmembrane coreceptors, such as c-Kit [START_REF] Hassel | Interaction and functional cooperation between the serine/threonine kinase bone morphogenetic protein type II receptor with the tyrosine kinase stem cell factor receptor[END_REF],

Endothelial BMPRII activity has been shown to be modulated by VEGFR3 in Pulmonary Arterial Hypertension (PAH) [START_REF] Hwangbo | Modulation of Endothelial Bone Morphogenetic Protein Receptor Type 2 Activity by Vascular Endothelial Growth Factor Receptor 3 in Pulmonary Arterial Hypertension[END_REF].

Neurotrophin receptor TrkC, s implicated in the growth and survival of human cancer tissues. The expression of TrkC suppresses bone morphogenetic protein 2 (BMP2)-induced SMAD1 phosphorylation and transcriptional activation by binds and phosphorylating the BMPRII cytoplasmic domain, disrupting the complex with the BMP type-I receptor and, by consequence, a suppression of BMP signaling in colon cancer cells (W. [START_REF] Jin | TrkC Binds to the Bone Morphogenetic Protein Type II Receptor to Suppress Bone Morphogenetic Protein Signaling[END_REF]. The receptor Ror2 (receptor tyrosine kinase-like orphan receptor 2) forms complexes with ALK6, and activation of ALK6 by GDF-5 leads to its transphosphorylation, which then silences BMP induced SMAD signaling and triggers Erk1/2 MAP kinase signaling leading to chondrogenic differentiation [START_REF] Sammar | Modulation of GDF5/BRI-b signalling through interaction with the tyrosine kinase receptor Ror2[END_REF].

Syndecans have been described to play a role in BMP signaling. It has been shown that the exogenous soluble HS improves BMP2-mediated chondrogenic differentiation as well as heparinase treatment of cell-surface HSPGs, while the up-regulation of syndecan-3 (cell-surface HSPGs) suppresses BMP2-mediated SMAD phosphorylation [START_REF] Fisher | Heparan sulfate proteoglycans including syndecan-3 modulate BMP activity during limb cartilage differentiation[END_REF]. Studies in Drosophila showed that Dally, a cell-surface HSPG, potentiates Dpp signaling and acts as a co-receptor [START_REF] Fujise | Dally regulates Dpp morphogen gradient formation in the Drosophila wing[END_REF].

v-β3 integrin and BMP2 share common effects on osteoblasts [START_REF] Lai | αvβ integrins play an essential role in BMP-2 induction of osteoblast differentiation[END_REF]. BMP2 raises the levels of these integrins on the osteoblast surface. Furthermore, BMP2 receptors co-localize or overlap with v and β3 integrin. The v-β3 integrin receptor can cooperate with BMP receptor activation, promoting downstream SMAD and MAP kinase signaling to lead to the proliferation of endothelial cells (Zhou, Lee, Lee, Wei, Lim, Lin, Chien, & Chiu, 2013). In addition, it has an impact on coupling cell migration and cell differentiation (Laure Fourel, Valat, Faurobert, Guillot, Reynard, et al., 2016).

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ECM and its impact on BMP signaling

The extracellular matrix (ECM) is the non-cellular component present within all tissues and organs.

Apart from its role as structural support, the ECM provides the biochemical and physical cues to control tissue architecture by driving specific cell programs [START_REF] Mammoto | Mechanical control of tissue and organ development[END_REF]. The ECM appears to play a significant role in BMP's presentation to cells to control signaling [START_REF] Migliorini | Learning from BMPs and their biophysical extracellular matrix microenvironment for biomaterial design[END_REF]. The cellular context depends on the BMPR expression repertoire and the co-receptor involvement to potentiate or inhibit the BMP signaling

The extracellular matrix (ECM)

The ECM is highly dynamic and versatile; although ubiquitously present, its composition and structure vary significantly from tissue to tissue. Its considerable heterogeneity characterizes the ECM in terms of the composition of a dynamic and complex array of several fibrillar proteins and proteoglycans [START_REF] Frantz | The extracellular matrix at a glance[END_REF][START_REF] Hynes | The Extracellular Matrix: Not Just Pretty Fibrils[END_REF]. The architecture and the mechanical and biomechanical properties of ECM depend on its composition, assembly, and functional characteristics based on the type of tissues, age, and physio-pathological conditions [START_REF] Engler | Matrix Elasticity Directs Stem Cell Lineage Specification[END_REF][START_REF] Frantz | The extracellular matrix at a glance[END_REF]. For example, the epithelial tissues are elastic, while the bone tissues must be rigid and resistant. (Figure 12) provides a summarized scheme of ECM and its interaction with the cell. A matrix of fiber proteins and proteoglycans with specific physical characteristics as the fiber strength and pore size. These proteins also influence the biochemical cues and the ability to act as a growth factor reservoir. Adapted from [START_REF] Migliorini | Learning from BMPs and their biophysical extracellular matrix microenvironment for biomaterial design[END_REF] The physical properties of the matrix, such as stiffness, elasticity, porosity, and topography, regulate the anchoring of cells and allow the matrix to play its role in the maintenance and architecture of tissues. The matrix is also a more or less porous structure that determines the diffusion of nutritive elements. These topographical features are essential for the function of many tissues. The cell can also respond and generate forces according to the matrix's rigidity via adhesive receptors, notably integrins, which transform this mechanical signal into a biochemical signal. Thus, the physical properties of the matrix regulate adhesion, migration [START_REF] Engler | Matrix Elasticity Directs Stem Cell Lineage Specification[END_REF] but also can have an impact on growth factor signaling, for example, through shaping concentration gradients or activating growth factor-like TGFβ [START_REF] Discher | Growth Factors, Matrices, and Forces Combine and Control Stem Cells[END_REF][START_REF] Wipff | Myofibroblast contraction activates latent TGF-β1 from the extracellular matrix[END_REF] The matrix, by its biochemical properties, transmits numerous signals to the cell. These signals come from the protein components of the matrix that includes: fibrillar proteins (collagen, fibronectin, vitronectin, and laminin) and proteoglycans (glycosaminoglycan chains) [START_REF] Frantz | The extracellular matrix at a glance[END_REF][START_REF] Hynes | The Extracellular Matrix: Not Just Pretty Fibrils[END_REF], but also from other constituents such as the growth factors which are presented to the cell by the components of the matrix. The matrix thus plays a role as a reservoir of growth factors, including BMPs.

a. ECM as a growth factor reservoir

There are two strategies by which the ECM can present growth factors to the surrounding cells. The first involves making the growth factors insoluble, protecting them from degradation, or concentrates them locally through their binding to the ECM proteins. The second strategy for growth factor presentation by the ECM involves enzymes like metalloproteases, which degrade the ECM's structural components. This action results in ECM remodeling and allows the release of growth factors trapped within an insoluble state.

Several studies have already highlighted many interactions between the growth factors and the matrix [START_REF] Martino | Matrix Enhance Tissue Healing[END_REF], mainly to the fibrillar ECM proteins (Figure 13.A). These fibrillar proteins include multiple independently folded domains whose sequences are highly conserved.

Some of these domains bind adhesion receptors such as integrins, and some other domains bind growth factors to regulate their distribution, activation, and presentation to the cells. (Figure 13. B).

In the case of the BMPs, fibrillar proteins as fibronectin, fibrillin, collagen type-IIA, and IV are known to have specific BMPs and BMP prodomains binding sites as reviewed in [START_REF] Hynes | The Extracellular Matrix: Not Just Pretty Fibrils[END_REF] and, close to them, interaction sites for other cell membrane receptors such as integrins or heparinbinding sites. [START_REF] Martino | Heparin-binding domain of fibrin(ogen) binds growth factors and promotes tissue repair when incorporated within a synthetic matrix[END_REF][START_REF] Martino | Engineering the Growth Factor Microenvironment with Fibronectin Domains to Promote Wound and Bone Tissue Healing[END_REF] . FnII domains found in fibronectin (FN) can bind BMP2/4 through the FN 12-14 domain, which is localized very close to the FN 7-11 domain known to interact with integrins [START_REF] Martino | The 12th-14th type III repeats of fibronectin function as a highly promiscuous growth factor-binding domain[END_REF]. Collagen (COL)-II contains a chordin-like VWC domain, which binds to Transforming Growth Factor β1 (TGF-β1) and BMP2 [START_REF] Garcia Abreu | Chordinlike CR domains and the regulation of evolutionarily conserved extracellular signaling systems[END_REF].

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Structural analyses have also revealed that the N-terminal prodomain of collagen type-IIA binds BMP2 [START_REF] Zhu | Type IIA procollagen containing the cysteine-rich amino propeptide is deposited in the extracellular matrix of prechondrogenic tissue and binds to TGF-β1 and BMP-2[END_REF], whereas the C-terminal part of COL-IV binds BMP(DPP called in Drosophila). The Dpp-binding motif identified in the C-terminal domains of the two collagen IV subunits in Drosophila are highly conserved across phyla, suggesting that this interaction may be significant in other contexts (X. [START_REF] Wang | Type IV collagens regulate BMP signalling in Drosophila[END_REF]. Fibrillins 1, 2, and 3 are critically involved in determining tissue elasticity and stiffness [START_REF] Jensen | Dissecting the fibrillin microfibril: Structural insights into organization and function[END_REF], which can eventually impact BMP diffusion and gradient. Findings from Nistala et al. [START_REF] Nistala | Extracellular microfibrils control osteoblast-supported osteoclastogenesis by restricting TGFβ stimulation of RANKL production[END_REF] identify fibrillins bind via their Nterminal part to BMP prodomain in BMP2, -4, -7, and -10, [START_REF] Robertson | Regulation of the bioavailability of TGF-β and TGF-β-related proteins[END_REF]. The sequestration of BMPs by fibrillin can inhibit their binding to BMP receptors.

It is widely accepted that the ECM is not merely the reservoir for BMPs but is a direct regulator of their biological activities [START_REF] Sedlmeier | Extracellular regulation of BMP signaling: welcome to the matrix[END_REF] by providing the binding sites, regulating their dose and availability, and providing a mechanical signal to the cells. Finally, ECM's dynamic properties are essential not only for BMP sequestration but also for BMP protection from neutralization by soluble inhibitors. The precise regulation of ligand availability at the ECM or the cell surface ensures that BMPs, act locally in a highly controlled manner rather than in free diffusion.

b. BMP signaling as a pathway for matrix remodeling

BMPs and BMP-related molecules participate in matrix remodeling. BMPs promote ECM proteins' production in vitro (Laure Fourel, Valat, Faurobert, Guillot, Reynard, et al., 2016;[START_REF] Yoon | Bmpr1a and Bmpr1b have overlapping functions and are essential for chondrogenesis in vivo[END_REF].

Analysis of ALK3 CKO and ALK6 -/-embryos reveals an essential role of BMP signaling in the production and remodeling of cartilage ECM proteins in vivo, which likely impact the cartilage-tobone transition through a defect of chondrocyte proliferation and survival [START_REF] Yoon | Bmpr1a and Bmpr1b have overlapping functions and are essential for chondrogenesis in vivo[END_REF]. The ECM may be affected not only by the absence of BMPR but also by their mutations. Haupt et al. [START_REF] Haupt | Dynamic DNA Methylation and Histone Modifications Contribute to Lentiviral Transgene Silencing in Murine Embryonic Carcinoma Cells[END_REF] showed that fibroblasts mimicking the Fibrodysplasia Ossificans Progressiva (FOP) pathology, a mutation in ALK2 (R206H/+), have an altered collagen content and organization within the fibroproliferative tissue. Additionally, BMPRII-deficient cells express genes indicative of altered biophysical properties, including up-regulation of ECM proteins such as fibrillin-1 and integrins [START_REF] Hiepen | BMPR2 acts as a gatekeeper to protect endothelial cells from increased TGFβ responses and altered cell mechanics[END_REF].

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Furthermore, the mammalian tolloid (TLD) family of metalloproteinases (BMP-1/TLD) is essential for tissue patterning and ECM assembly. The BMP-1/TLD family is preserved in species ranging from Drosophila to humans, and their importance is highlighted by the lethal embryonic phenotype of BMP-1/Tll1 homozygous null mice, which display heart malformations and abnormal procollagen processing. BMP-1/TLD proteases are involved in the biosynthetic processing of a wide range of ECM precursors, the crosslinking enzymes lysyl oxidase (LOX), and LOX-like, laminin basement membrane proteins, and small leucine-rich proteoglycans (SLRPG) osteoglycin and probiglycan [START_REF] Muir | Metalloproteinases in Drosophila to humans that are central players in developmental processes[END_REF][START_REF] Goff | BMP-1/tolloid-like proteinases synchronize matrix assembly with growth factor activation to promote morphogenesis and tissue remodeling[END_REF]. However, it is worth noting that cleavage of collagen by BMP-1 induces the production of endotrophin known to be highly inflammatory and to be present in fibrosis and tumoral tissue [START_REF] Heumüller | C-terminal proteolysis of the collagen VI α3 chain by BMP-1 and proprotein convertase(s) releases endotrophin in fragments of different sizes[END_REF]. BMP-1 and its enhancer PCPE-1 have been shown to bind to FN (G. [START_REF] Huang | Fibronectin binds and enhances the activity of bone morphogenetic protein 1[END_REF]. More recent results indicate that collagen fibrillogenesis depends on the FN matrix, at least in part since FN fibrils provide binding sites for BMP-1 to facilitate procollagen processing [START_REF] Saunders | Fibronectin matrix as a scaffold for procollagen proteinase binding and collagen processing[END_REF]. These binding sites and matrix processing supports that the activity, bioavailability, and diffusion of extracellular BMP signaling agonists and antagonists are further regulated by ECM components [START_REF] Umulis | The extracellular regulation of bone morphogenetic protein signaling[END_REF].

c. Biomimetic Extracellular Matrix

Overall, biomaterial engineering explores the concept that ECM proteins bind and present growth factors as a solid-phase ligand. Many studies provided evidence that local application of BMP2 in its immobilized form is suitable and most efficient to trigger the SMAD pathway, which is essential for osteogenic differentiation of bone precursor cells. In the design of materials, traditionally, the immobilization of the BMPs (and related molecules) on material surfaces may be performed by different methodologies: via adsorption or physical entrapment [START_REF] Migliorini | Tuning cellular responses to BMP-2 with material surfaces[END_REF], which allows a slow-release and internalization of the molecule, a second strategy is an immobilization through biotin-streptavidin, or by chemical linker as covalent bonding, which leads to a sustained presentation of BMP2.

In particular, Picart team has developed a polyelectrolyte multilayer film (Poly-l-lysine/Hyaluronan) that allows the presentation of the BMP2 at the basal side of the cells, i.e., where they adhere. The stiffness of the film can be modulated by the chemical crosslinking of the polyelectrolyte constituting the films. When slightly crosslinked, these soft films do not allow good cell adhesion and spreading, but BMP2 can rescue this solely when it is presented in a matrix-bound manner and not when it is added in solution (T Crouzier, Fourel, Boudou, Albiges-Rizo, & Picart, 2011) (Figure 14). Interestingly, BMP2 can also be printed onto the layer-by-layer biopolymeric films to form a long-range gradient while remaining bioactive. Such BMP gradients can be used to spatially-control cell differentiation [START_REF] Almodóvar | Spatial patterning of BMP-2 and BMP-7 on biopolymeric films and the guidance of muscle cell fate[END_REF].

Figure 14. The matrix-bound presentation of BMP2 allows the presentation of BMP2 at the ventral side of the cells and the spatial confinement of BMP receptor complexes. When BMP2 is physically bound to the film, it is spatially confined, and its diffusion is restricted. Besides, the occupancy rate of BMP2 receptors is enhanced with a possible formation homo-and heterodimeric receptor complexes and ligand/receptor binding is not limited by diffusion. Furthermore, due to the proximity of growth factor receptors and adhesion receptors, cross -talk between these two types of receptors is possible. B) Sustained effect of matrix-bound BMP2 on cell morphology. Cell morphology is observed 16 h after plating the cells. Actin and nucleus staining of C2C12 cells revealed that for soft (PLL/HA) films, sBMP2 did not induce any noticeable effect on cell spreading, but bBMP2 induced a striking increase in cell spreading. The scale bar is 20 µm. Adapted from [START_REF] Crouzier | Presentation of BMP-2 from a soft biopolymeric film unveils its activity on cell adhesion and migration[END_REF] I-37

Adhesion Structures and Mechanobiology

The cells sense the applied forces or the rigidity of the matrix at the adhesion sites via the integrins

a. Integrins

Integrins are transmembrane glycoproteins and the primary receptors for cell adhesion to the extracellular matrix. They have a crucial role in many processes: embryonic development, tissue repair, immune response, and cancers (Albiges-Rizo, Destaing, Fourcade, [START_REF] Albiges-Rizo | Actin machinery and mechanosensitivity in invadopodia, podosomes and focal adhesions[END_REF][START_REF] Roca-Cusachs | Finding the weakest link -exploring integrinmediated mechanical molecular pathways[END_REF].

At the structural level, integrins form heterodimers composed of an  unit and a  unit-linked noncovalently. The two subunits are transmembrane proteins containing a large extracellular domain and a short cytoplasmic domain [START_REF] Arnaout | INTEGRIN STRUCTURE, ALLOSTERY, AND BIDIRECTIONAL SIGNALING[END_REF]. Eighteen α chains and eight β chains are known, a total of 24 combinations between these  and  chains, each of them having an affinity for ECM molecules. [START_REF] Humphries | Integrin ligands at a glance g rin Li and s at a Glance RSK and at g a[END_REF][START_REF] Hynes | Integrins: bidirectional, allosteric signaling machines[END_REF] (Figure 15). et al., 2002) The heterodimers are more or less specific. For instance, the integrin αvβ3 recognizes many ligands such as fibronectin, vitronectin, and laminin. The integrins expression depends on cell type, and their activation depends on the nature of the matrix components.

Integrins, as their name suggests, integrate the cell into its microenvironment and provide two-way communication between the cell and the biochemical and physical signals of the matrix and growth factors in the environment. Integrins bind their ECM ligands and get activated. This activation is characterized by the priming of integrin or the transition between two conformational states with different affinities for the ligand and the propagation of intracellular signals induced by ligand binding [START_REF] Humphries | Proteomic Analysis of Integrin-Associated Complexes Identifies RCC2 as a Dual Regulator of Rac1 and Arf6[END_REF]. The two directions of integrin-dependent signaling have different biological consequences. During "inside-out" signaling, an intracellular activator, such as talin, binds

to the cytoplasmic domain of the β subunit and induces conformational changes leading to increased affinity for extracellular ligands.

Integrins also function as traditional receptors by transmitting information from the extracellular medium via "outside-in" signaling. Integrin binding to its extracellular ligands changes its conformation, and like many of its multivalent ligands, the latter induces the regrouping or "clustering" of integrins. Combining these two elements induces intracellular signaling, controlling cell polarity, cytoskeleton structure, gene expression, cell survival, and proliferation. Although conceptually separating the "outside-in" and "inside-in" signals, these two processes are most often interconnected, and one generally does not go without the other (Figure 16).

Figure 16 Integrin structure and bidirectional integrin signaling. While outside-in signaling, Integrins binds to the ECM matrix and transmits the information into the cell. Inside out-signaling controls adhesion strength and enables sufficiently strong interactions between integrins and extracellular matrix (ECM) [START_REF] Shattil | The final steps of integrin activation: The end game[END_REF].

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b.

Diversity of adhesion structures

In addition to their priming and activation, ECM molecules induce the clustering of integrins into units called adhesion structures [START_REF] Hynes | Integrins: bidirectional, allosteric signaling machines[END_REF]. The adhesion structure size and distribution reflect the cell contractility state, which depends on the matrix properties and the cell contractile nature.

There are three distinct types of adhesions connecting the cell to the extracellular matrix: focal complexes, focal adhesions, and fibrillar adhesions. (Figure 17). These structures are distinct by their cellular localization, composition, the orientation of the actin-associated with them, and their function [START_REF] Orré | The inner life of integrin adhesion sites: From single molecules to functional macromolecular complexes[END_REF]. Focal adhesions are adhesive structures from 1 to 5 µm 2 , located at the end of the stress fibers, and attached to the actomyosin stress fibers. They are also composed of integrins α5β1 and αvβ3 but at higher densities than in early adhesions [START_REF] Ballestrem | Marching at the front and dragging behind: Differential αVβ3-integrin turnover regulates focal adhesion behavior[END_REF]. The recruitment of zyxin accompanies the formation of focal adhesions. These structures are stable with a lifespan between 30 up to 90 minutes but are nevertheless dynamic. They can develop into a third type of adhesion, fibrillar adhesions (Figure 17.b).

Focal complexes and focal adhesions are required to probe the cellular environment mediated by the β3 integrin, which are found preferentially in these adhesion structures. Focal adhesions can disassemble during the migration or slide centripetally under the cell, displacing α5β1 integrin to develop into fibrillar adhesion. [START_REF] Pankov | Integrin dynamics and matrix assembly: Tensin-dependent translocation of α5β1 integrins promotes early fibronectin fibrillogenesis[END_REF][START_REF] Roca-Cusachs | Clustering of ␣ 5 ␤ 1 integrins determines adhesion strength whereas ␣ v ␤ 3 and talin enable mechanotransduction[END_REF] Fibrillar adhesions are elongated in appearance, thinner than focal adhesions, and located in a more central cell position. They are composed of α5β1 integrin linked to fibronectin, and whose activation

or "priming" level is higher than that observed in focal adhesions [START_REF] Clark | A specific α5β1-integrin conformation promotes directional integrin translocation and fibronectin matrix formation[END_REF]. Tensin, the structural protein replacing talin in fibrillar adhesions, plays a significant role in the translocation of integrins at the level of fibrillar adhesions [START_REF] Pankov | Integrin dynamics and matrix assembly: Tensin-dependent translocation of α5β1 integrins promotes early fibronectin fibrillogenesis[END_REF] (Figure 17.c).

The contractility generated by RhoA also plays an essential role in the formation of fibrillar adhesions and the assembly of fibronectin. Cells that do not express the Src protein cannot form fibrillar adhesions. A long lifespan characterizes these structures c.

Adhesive structure mechanotransduction

As described previously, integrins are linked to the actin cytoskeleton within adhesion sites [START_REF] Albiges-Rizo | Actin machinery and mechanosensitivity in invadopodia, podosomes and focal adhesions[END_REF]. The actin cytoskeleton is connected to nuclear structural molecules, lamins, chromatin, and DNA within the nucleus (Figure 18). The mechanical forces applied to the cell surface at the integrin level activate signaling pathways from the membrane, but they also cause structural rearrangements in the cytoplasm and the nucleus. They can act at a distance and be converted into a mechanical-chemical signal directly in the nucleus in addition to the cytoplasmic mechanical transduction initiated at the plasma membrane by the mechanical sensors linked to integrins. The structural connection between the integrins and the nucleus allows the propagation of forces over long distances and the mechanical propagation of a signal faster than the propagation of a biochemical signal.

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The transduction of an extracellular mechanical signal into an intracellular biochemical signal requires different actors: mechano-receptors, actors allowing the transmission of forces, and mechano-sensors capable of adapting a biochemical response to a mechanical signal.

Figure 18 Molecular connectivity from the ECM to the nucleus . Mechanotransduction: from integrin to nucleus. The actin cytoskeleton is connected physically to the ECM. Adapted from [START_REF] Monteiro | Engineered systems to study the synergistic signaling between integrin-mediated mechanotransduction and growth factors (Review)[END_REF].

Integrin can activate several signaling pathways independently [START_REF] Assoian | Coordinate signaling by integrins and receptor tyrosine kinases in the regulation of G1 phase cell-cycle progression[END_REF], but also they act in synergy with other growth factor receptors [START_REF] Alam | The integrin -Growth factor receptor duet[END_REF]. It may not always require spatial coexistence, but integrin and growth factor co-localization facilitate cross-talk between them.

Several studies have demonstrated this cooperation, particularly the integrin receptor's nature and the main effects on cellular processes. It appears that numerous experiments in coimmunoprecipitation have demonstrated the existence of physical and sometimes specific interaction between growth factors and the integrin receptors. Other studies have revealed that this interaction can also occur in the absence of growth factors and requires the extracellular domain of Integrin [START_REF] Borges | Platelet-derived growth factor receptor beta and vascular endothelial growth factor receptor 2 bind to the beta 3 integrin through its extracellular domain[END_REF].

The integration of mechanobiology in growth factor signaling.

Several reviews have already been dedicated to growth factor signaling and its relation to the biomechanical environment. It is already known that mechanical loading is essential for bone growth and regeneration [START_REF] Boerckel | Effects of in vivo mechanical loading on large bone defect regeneration[END_REF]. BMP signaling and mechano-transduction pathways are tightly interconnected and represent an elaborate signaling network that is active during development [START_REF] Kopf | BMP growth factor signaling in a biomechanical context[END_REF]. At the cellular scale, cells interpret the biophysical properties of their microenvironment via cell surface mechanoreceptors that are coupled to the contractile cytoskeleton. Signaling is achieved via signal direct and indirect pathways that control cell fate via nuclear organization and gene expression [START_REF] Discher | Matrix Mechanosensing: From Scaling Concepts in 'Omics Data to Mechanisms in the Nucleus, Regeneration, and Cancer[END_REF][START_REF] Swift | Nuclear lamin-A scales with tissue stiffness and enhances matrix-directed differentiation[END_REF]) (Figure 19). Kopf et al. [START_REF] Kopf | BMP growth factor signaling in a biomechanical context[END_REF] and Ashe [START_REF] Ashe | Modulation of BMP signalling by integrins[END_REF] have reviewed, more specifically, the synergy between the integrin adhesion receptors and BMPs-mediated signaling. The proximity between FN 12-14 and FN 7-11 domains of FN is crucial to favor the cross-talk between BMP receptors and integrins, suggests proximity and possible physical interaction between the two receptors ((Laure Fourel, Valat, Faurobert, Guillot, Reynard, et al., 2016)). This arrangement yields a specialized biological response by stimulating integrins and growth factor receptors signaling in a confined space.

Cooperation between BMPR and integrins have multiple effects, either positively or negatively, affecting Integrin and BMP-signaling, highlighting the context-dependent nature of integrin-BMP cross-talk. The signaling cross-talk between BMP-dependent and integrin-mediated pathways has been explored mainly towards the modulation of both osteogenic differentiation and adhesion to the ECM (S. H. [START_REF] Kwon | Modulation of BMP-2-Induced chondrogenic versus osteogenic differentiation of human mesenchymal stem cells by cell-specific extracellular matrices[END_REF].

b. BMP2 on cytoskeleton and cell migration

To date, only a few studies have exposed the impact of BMP signaling on integrins and integrinmediated structures for the regulation of cell adhesion. BMP2 signaling is involved in wound healing and cancer invasiveness, likely by acting on actin cytoskeleton dynamics [START_REF] Ehata | Bi-directional roles of bone morphogenetic proteins in cancer: Another molecular Jekyll and Hyde?[END_REF][START_REF] Moustakas | The regulation of TGF signal transduction[END_REF]Padua & Massagué, 2009) and cell migration (T [START_REF] Crouzier | Presentation of BMP-2 from a soft biopolymeric film unveils its activity on cell adhesion and migration[END_REF][START_REF] Fourel | β3 integrin-mediated spreading induced by matrix-bound BMP-2 controls Smad signaling in a stiffness-independent manner[END_REF][START_REF] Sieber | Recent advances in BMP receptor signaling[END_REF] (Figure 20). The specificity of small GTPases or kinases involved in actin dynamics is microenvironment context-dependent.

It was shown that, upon BMP-binding to the BMPR complex, LIMK1 dissociates from BMPRII and phosphorylates cofilin [START_REF] Foletta | LIM kinase 1, a key regulator of actin dynamics, is widely expressed in embryonic and adult tissues[END_REF]. The activation of LIMK1 by BMP2 initiates signaling to the cytoskeleton in a PI3K-dependent manner, but a concomitant activity of Cdc42 has shown that a regulatory subunit of PI3K directed BMP2-induced cell migration [START_REF] Gamell | BMP2 induction of actin cytoskeleton reorganization and cell migration requires PI3-kinase and Cdc42 activity[END_REF][START_REF] Hiepen | BMP2induced chemotaxis requires PI3K recruitment at the cytocortex[END_REF]. Lai et al. [START_REF] Lai | αvβ integrins play an essential role in BMP-2 induction of osteoblast differentiation[END_REF] have reported that during four days of osteoblast stimulation with soluble BMP2, the expression of αv integrin is increased, BMPRs co-localize with αv and β1 integrin in focal adhesions (Figure 21. A) and co-precipitate with these receptors. However, recent studies using high-resolution microscopy could not confirm the co-localization pattern using vinculin as a focal adhesion marker (Figure 21.B) [START_REF] Medda | Challenges in imaging cell surface receptor clusters[END_REF]. In osteoblasts, BMP2 enhances the I-45 formation of focal adhesions and stress fibers by increasing α5 and β1 integrin expression and triggers migration by enhancing the association of β1 integrin into lipid rafts [START_REF] Shah | Mechanism of BMP-2 stimulated adhesion of osteoblastic cells to titanium alloy[END_REF][START_REF] Sotobori | Bone morphogenetic protein-2 promotes the haptotactic migration of murine osteoblastic and osteosarcoma cells by enhancing incorporation of integrin β 1 into lipid rafts[END_REF].

These studies indicate that BMP2 participates in cell protrusion formation and migration, acting on diverse parallel pathways related to actin reorganization. However, the dynamics and the spatialtemporal aspect of BMPR/integrin cooperation at the plasma membrane level need to be investigated. The type of integrin cooperating with BMPR will undoubtedly drive cell decision making. It should be considered for the design of biomaterials/implants for the delivery of BMP2, adhesion being the first step at the interface between cells and synthetic material. In turn, many answers to these open questions might come shortly with the aid of material science approaches that allow control over the presentation of BMP2 to cells c.

Osteogenic and adhesion signaling cross-talk

Regarding the participation of integrin signaling in the transcription of genes for osteogenic differentiation, the collagen-binding integrins α1β1 and α2β1 regulate BMP induced differentiation by acting downstream of BMPRI [START_REF] Jikko | Collagen Integrin Receptors Regulate Early Osteoblast Differentiation Induced by BMP-2[END_REF][START_REF] Reyes | α2β1 integrin-specific collagen-mimetic surfaces supporting osteoblastic differentiation[END_REF]. Additionally, FAK phosphorylation is necessary for SMAD6 activity but not for the translocation of SMAD1 [START_REF] Tamura | Focal Adhesion Kinase Activity Is Required for Bone Morphogenetic Protein -Smad1 Signaling and Osteoblastic Differentiation in Murine MC3T3-E1 Cells[END_REF] αv integrin also regulates BMP-dependent osteogenic differentiation [START_REF] Lai | αvβ integrins play an essential role in BMP-2 induction of osteoblast differentiation[END_REF], and in particular osteoblastic response to CYR61. This bone activator increases the level of BMP2 and activates the αvβ3 integrin/ILK/ERK signaling pathway [START_REF] Su | CYR61 regulates BMP-2-dependent osteoblast differentiation through the αvβ3 integrin/integrin-linked kinase/ERK pathway[END_REF].

It has been shown that the SMAD phosphorylation in the presence of sBMP2 on (PLL/HA) stiff films was similar to sBMP2 on glass, but higher phosphorylation was observed for bBMP2 on soft films in comparison to sBMP2. An unexpected finding was that, besides BMP2-mediated signaling, bBMP2 could induce cell adhesion and spreading on soft films and potentialize cell migration on soft and stiff films, the effects being more potent for soft films. This BMP2 induced cell spreading was mostly mediated by β3 integrin (L. Fourel et al., 2016)and depended on the FN already present in the cellular coat of C2C12 skeletal myoblasts. Thus, in FN/BMP2 binding, cellular spreading requires the binding sites for fibronectin and BMP2, suggesting a requirement for a juxtaposition of the two receptors (v integrin and BMPR). These receptors work together to control SMAD signaling and cell adhesion. Indeed, matrix-bound BMP2 was sufficient to induce β3 integrin-dependent cell spreading by overriding the soft biomaterial and impacting actin organization and adhesion site dynamics. In turn, v3 integrin was required to mediate BMP2-induced SMAD signaling through the LIM kinase pathway involving Cdc42-Src-FAK-ILK, independently of ROCK. β3 integrin was found to regulate a multistep process to control first the BMPR activity and second the repressive role of GSK3 on SMAD signaling. This result differed from the effect of cell spreading induced by sBMP2 on mesenchymal stem cells cultured on stiff substrates, which was found to be regulated solely by RhoA/ROCK (Y.-K.

K. [START_REF] Wang | Bone Morphogenetic Protein-2-Induced Signaling and Osteogenesis Is Regulated by Cell Shape, RhoA/ROCK, and Cytoskeletal Tension[END_REF]. Fibronectin secretion and the associated juxtaposition of integrin αvβ3 and BMPR is essential for this process as impairing fibronectin synthesis inhibits cell spreading. β3 integrin-BMPR cross-talk might be driven by the receptors ' spatial proximity rather than interactions between their respective pathways (Laure Fourel, Valat, Faurobert, Guillot, Reynard, et al., 2016) (Figure 22.A). However, it is still not proved whether such cross-talk involves membrane-proximal interactions between integrins and BMP receptors. Moreover, we lack information on whether and when these mechanosensitive growth factor receptors and integrins converge intracellularly. We can expect that the ECM's chemical composition and mechanical properties, the dose, and the I-47 gradient of BMPs, their spatial and temporal availability will impact not only receptor dynamics but also cell and tissue responses.

Cell spreading itself potentiated BMP2-dependent phosphorylation of SMAD1/5/8. This SMAD signaling was found to depend on LIM kinase-2 and ROCK, rather than myosin II activation on fibronectin pattern [START_REF] Fitzpatrick | Signal mingle: Micropatterns of BMP-2 and fibronectin on soft biopolymeric films regulate myoblast shape and SMAD signaling[END_REF]) (Figure 22.B). This result differed from the effect of cell spreading induced by sBMP2 on mesenchymal stem cells cultured on stiff substrates, which was found to be regulated solely by RhoA/ROCK (Y.-K. K. [START_REF] Wang | Bone Morphogenetic Protein-2-Induced Signaling and Osteogenesis Is Regulated by Cell Shape, RhoA/ROCK, and Cytoskeletal Tension[END_REF]. Fourel, Valat, Faurobert, Guillot, Reynard, et al., 2016) , (B) Phosphorylation and translocation of SMAD1/5/8 to the nucleus depends on cell spreading: immunofluorescence images of C2C12 myoblasts spread on small (500 μm2) and large (1500 μm2 ) micropatterns of FN/BMP2 and FN without or with BMP2 in solution [START_REF] Fitzpatrick | Micromotifs de BMP-2 et de fibronectine sur niche de cellules souches in vitro[END_REF]. Figure from [START_REF] Migliorini | Learning from BMPs and their biophysical extracellular matrix microenvironment for biomaterial design[END_REF] The same type of collaboration is illustrated in Drosophila collagen IV (Figure 23), which binds Dpp (a BMP homolog). Collagen IV enhances its interactions with BMP receptors to regulate the dorsoventral axis and the numbers of germinal stem cells through integrin activation [START_REF] Sawala | Peak BMP Responses in the Drosophila Embryo Are Dependent on the Activation of Integrin Signaling[END_REF]. However, integrin can also act as an inhibitor of BMP signaling. Indeed, in a specific cellular context and experimental conditions, integrins can act as inhibitors. It has been shown that upon interaction with collagen type-II, β1 integrins compete with BMP receptors to bind with SMAD1 and then inhibit SMAD1 activation and nuclear import [START_REF] Lian | Collagen type II suppresses articular chondrocyte hypertrophy and osteoarthritis progression by promoting integrin β1-SMAD1 interaction[END_REF]. This SMAD1 inhibition helps in suppressing articular chondrocytes hypertrophy and osteoarthritis progression. The diversity of BMP signaling may also result from the combination of BMP ligands like BMP2/BMP-6/activin A chimera, which is increasing the binding affinity to BMP receptors, notably ALK2 involved in bone formation [START_REF] Seeherman | A BMP/activin A chimera is superior to native BMPs and induces bone repair in nonhuman primates when delivered in a composite matrix[END_REF]. These examples illustrate the capacity of conserved elements of ECM proteins to regulate, either positively or negatively, the functions of various BMPs. Morphogens, like BMPs, by concept, are intimately associated with position and dose such that the signaling of BMP varies across a tissue depending on the amount of ligand, antagonist, and receptors.

The function and efficiency of cell signaling pathways are highly dependent on their collaborative organization both in space and time (Figure 24). BMP signaling also depends on the dose, the time scale of the spatial presentation, and pattern (duration, presence of gradients…). The contribution of biophysicists is and will be useful to design simplified microenvironments to decorticate the complex biological phenomena that occur in vivo during development and tissue regeneration

Spatial regulation of BMP signaling

Signaling components in cells are organized to transmit information from one region to another. This spatial organization of signaling pathways depends on the nanoscale molecular interactions between ligands and transmembrane receptors, which is a critical step in signal transduction and receptor trafficking. [START_REF] Bethani | Spatial organization of transmembrane receptor signalling[END_REF] In particular, the interactions of BMP with ECM components facilitate their localization and spatiallyregulated signaling, thanks to the high affinity and promiscuous ECM protein-binding. BMPs are recognized to have numerous interactions with GAGs and fibrillar ECM proteins. Thus, the ECM components control the availability of BMPs since they act as a localized reservoir [START_REF] Fitzpatrick | Signal mingle: Micropatterns of BMP-2 and fibronectin on soft biopolymeric films regulate myoblast shape and SMAD signaling[END_REF]. ECM proteins regulate cell signaling via their interactions with the adhesive cellular receptors

The adhesive receptors engaged by the ECM act near BMP receptors and could have an essential role as BMP co-receptors ECM proteins can thus have the role of enhancers or BMP signaling inhibitors, depending on the context. For instance, the presence of metalloproteases (BMP-1/TLD) can establish an antagonist sink regulating the bioavailability of BMPs, which is considered as the primary mechanism to drive BMP formation gradients [START_REF] Metzger | Cell-Mediated Proteolytic Release of Growth Factors from Poly(Ethylene Glycol) Matrices[END_REF]. However, the complexity of biological forms is vast, and although diffusion may appear to be the simplest way to disperse signals, the informational content of diffusion-generated distributions is limited by temporal and quantitative imprecision [START_REF] Kornberg | Distributing signaling proteins in space and time: the province of cytonemes[END_REF][START_REF] Wolpert | Positional Information and Pattern Formation[END_REF].

In that way, ECM, including the fibrillar matrix, helps to form a gradient, reduced diffusion rates and could lead to local accumulation of BMPs around cells that secrete BMPs. Moreover, heparan sulfate chains might sculpt BMP distribution, increasing pericellular levels of BMPs which might, in turn, activate autocrine BMP signaling if the cells also express the appropriate BMP receptors. Thus, ECM microenvironments that reduce BMP diffusion would be predicted to sensitize cells to autocrine BMP signaling. In the other hand, the presence of cytonemes, that move signaling molecules in the space between cells, offers another way to diffuse paracrine BMP signal. [START_REF] Kornberg | Distributing signaling proteins in space and time: the province of cytonemes[END_REF].

a.

Gradients and dose of BMP2

In vivo, BMP signaling's spatial organization has fundamental roles in both embryonic development and postnatal bone homeostasis [START_REF] Chau | Gene Expression Profiling Reveals Similarities between the Spatial Architectures of Postnatal Articular and Growth Plate Cartilage[END_REF]. The growth factor gradient appears to be crucial for cell fate decisions. Much of our understanding of the spatially-distinct signaling comes from studies on murine and avian limb buds where BMP2 and BMP7 were localized to the limb bud [START_REF] Badugu | Digit patterning during limb development as a result of the BMP-receptor interaction[END_REF]. A gradient in genes related to BMP2 expression has been observed across the cartilage growth plate, likely playing a zonal differentiation [START_REF] Cho | Differential temporal expression of members of the transforming growth factor β superfamily during murine fracture healing[END_REF]. The gradients for BMP2 and BMP6 signaling were mainly present within the hypertrophic zones. These spatial gradients favor the differentiation of chondrocytes in the endochondral skeleton. Furthermore, BMP signaling is one of the critical pathways regulating craniofacial development and mineralized structures. It is involved in the head's early patterning, development of cranial neural crest cells, and facial patterning [START_REF] Graf | Common mechanisms in development and disease: BMP signaling in craniofacial development[END_REF].

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The presence of BMP gradients demonstrates that BMP can be confined to a specific volume due to Several steps of embryonic development are also found during bone healing [START_REF] Tsuji | BMP2 activity, although dispensable for bone formation, is required for the initiation of fracture healing[END_REF], indicating a spatio-temporal pattern of BMP expression through the bone fracture. Indeed, the spatially and temporally restricted BMP expression patterns during bone regeneration appear to be necessary for determining the sites of coupled angiogenesis and osteogenesis that underlie bone regeneration. In Xenopus, zebrafish, and sea urchins, a gradient of NODAL signaling, counteracted by a gradient of BMP signaling, is sufficient to organize a complete embryonic axis [START_REF] De Robertis | Spemann's organizer and the self-regulation of embryonic fields[END_REF][START_REF] Lapraz | A deuterostome origin of the Spemann organiser suggested by Nodal and ADMPs functions in Echinoderms[END_REF][START_REF] Xu | Construction of a vertebrate embryo from two opposing morphogen gradients[END_REF].

The activity of the pathway as BMP active pathway, through immunofluorescence for the visualization of phosphorylated SMAD5 (p-SMAD5), has shown a uniform low level of signaling throughout the early blastula embryo (4 hpf), which progressively clears dorsally and intensifies in the ventral-most regions [START_REF] Tucker | The BMP Signaling Gradient Patterns Dorsoventral Tissues in a Temporally Progressive Manner along the Anteroposterior Axis[END_REF]. This produces a steep gradient that persists through gastrulation. Ramel and Hill further demonstrated that this same pattern of regular expression followed by dorsal clearance is seen for the transcription of the BMP ligands. [START_REF] Ramel | The ventral to dorsal BMP activity gradient in the early zebrafish embryo is determined by graded expression of BMP ligands[END_REF]. It is suggested that diffusion does not play a significant role in the establishment of the morphogen gradient, but rather that it is dependent on the spatial regulation of transcription.

Therefore, these studies suggest that the BMP gradient in zebrafish Dorso-Ventral (DV) patterning evolves through time in a manner highly dependent on the spatial pattern of transcription of the ligand, but also through interaction with other DV-localized proteins in the early embryo. While in the wing disc, the model of BMP gradient formation may show elements of a classical model of a morphogen diffusing from a local source to set up a gradient. This does not capture the variety of ways morphogen gradients are set up in biological systems. These studies on BMP signaling in early zebrafish embryos have focused mainly on which of a set of distinct models can best describe the formation of a BMP signaling gradient (Figure 25).

No single model can likely capture the entire dynamics. [START_REF] Economou | Temporal dynamics in the formation and interpretation of Nodal and BMP morphogen gradients[END_REF]. In any case, observations indicate that BMP proteins might distribute across fields, forming concentration gradients and eliciting concentration-dependent responses. However, available evidence does not support the idea that dispersal is by secretion into extracellular space, followed by some type of passive diffusion. BMP proteins would be rather either constrained in ECM or move along cytonemes and exchange between producing and receiving cells at direct contact sites. b.

The threshold of BMP2 signaling

Wolpert's fundamental proposition, contained in [START_REF] Durston | A tribute to Lewis Wolpert and his ideas on the 50th anniversary of the publication of his paper 'positional information and the spatial pattern of differentiation'. Evidence for a timing mechanism for setting up the vertebrate anterior-posterior (A-P) axis[END_REF][START_REF] Wolpert | Positional Information and Pattern Formation[END_REF], is that concentration gradients of morphogens acting on concentration thresholds can establish a developmental map in the embryo. A graded morphogen could act as a source of positional information across a tissue, whereby the concentration would encode a positional value. By interpreting the concentration of a morphogen relative to a threshold, cells in different parts of the embryo could differentiate into distinct fates [START_REF] Wolpert | Positional information and patterning revisited[END_REF]. Cells can also monitor the absolute level of SMAD proteins despite their variability, suggesting that the mechanism that produces robust cellular outcomes is downstream from SMADs [START_REF] Frick | Sensing relative signal in the Tgf-β / Smad pathway[END_REF].

I-53 c.

Dynamics of BMP receptor.

The signaling dynamics are driven by differences in the localization and internalization of receptors for each ligand, which determines cells' capability to monitor levels of an extracellular ligand. By using mathematical modeling, Miller demonstrated that ligand-receptor affinity differences might drive the distinct receptor behaviors and signaling dynamics observed. [START_REF] Miller | TGF-β family ligands exhibit distinct signalling dynamics that are driven by receptor localisation[END_REF]. In non-polarized cells, the receptors are more homogenous distributed, with high levels at sites of cell contacts and at the tips of filopodia and cytonemes or at the leading edge of migrating cells [START_REF] Heldin | Signaling Receptors for TGF-β Family Members[END_REF]Kornberg, 2017a) Besides ligand diffusion-based models [START_REF] Rogers | Morphogen Gradients: From Generation to Interpretation[END_REF], substantial evidence has emerged that ligands are presented to receptors distances that span many cell diameters through thin, filipodia-like membrane protrusions, named cytonemes [START_REF] Kornberg | Distributing signaling proteins in space and time: the province of cytonemes[END_REF]. These structures have been seen in Drosophila and vertebrate tissues, including in tumors. These observations are in line with the ability of BMP2 to induce filopodia, as observed by electron microscopy in cells spread onto the BMP2 bound matrix (L. Fourel et al., 2016). Cytoneme-mediated ligand presentation could explain the highly localized ligand-receptor activities, as well as gradients of morphogen signaling.

Considering the narrow diameter of the cytonemes and the extended surface of cells, receptor activation by cytonemes is expected to initiate in synapse-like structures at the spot of contact that as highly localized signaling cores [START_REF] Heldin | Signaling Receptors for TGF-β Family Members[END_REF] , 2005). In fish, BMP signaling sets aside tail progenitors during early gastrulation [START_REF] Agathon | The molecular nature of the zebrafish tail organizer[END_REF] then patterns these progenitors into ventral tail tissues during early somitogenesis [START_REF] Connors | Temporal and spatial action of Tolloid (Mini fin) and Chordin to pattern tail tissues[END_REF][START_REF] Pyati | Transgenic zebrafish reveal stage-specific roles for Bmp signaling in ventral and posterior mesoderm development[END_REF]. Thus, current models of the temporal requirements for BMP signaling imply only a coarse temporal program wherein a BMP signaling gradient patterns the entire embryo's DV axis, aside from the tail, at a single interval before or during gastrulation. A temporal cue might regulate a cell's competence to respond to BMP signaling, allowing the acquisition of a cell's DV and AP identity simultaneously [START_REF] Tucker | The BMP Signaling Gradient Patterns Dorsoventral Tissues in a Temporally Progressive Manner along the Anteroposterior Axis[END_REF].

In terms of signaling, interestingly, ERK activation is rapid and transient, peaking after a few minutes and disappearing in the hour; conversely, the canonical pathway is slow and more stable, with a maximal SMAD nuclear accumulation peaking after 20 minutes [START_REF] Batut | Kinesin-Mediated Transport of Smad2 Is Required for Signaling in Response to TGF-β Ligands[END_REF].

a.

Oscillations of BMP2 signaling

By quantifying the spatial distribution of the central signal transducer of the BMP2/4 pathway in response to different levels of stimuli in C2C12 cells, it has been reported that the SMAD1 localization is independent of ligand concentration [START_REF] Schul | Bursted BMP Triggered Receptor Kinase Activity Drives Smad1 Mediated Long-Term Target Gene Oscillation in c2c12 Cells[END_REF]. In contrast, SMAD1 phosphorylation levels relate proportionally to the BMP2 concentration.

Interestingly, BMP2 stimulates the expression of target genes in non-linear, wave-like forms.

Amplitudes showed a clear BMP concentration-dependency, both for sustained and transient BMP stimulation. Even burst-stimulation triggers gene-expression wave-like modulations that are detectable for at least 30 h. Furthermore, oscillations in target gene expression depend on receptor kinase activity. Interestingly, an essential role of the core clock factor BMAL1 was demonstrated for normal function and structural integrity of articular cartilage [START_REF] Dudek | The chondrocyte clock gene Bmal1 controls cartilage homeostasis and integrity[END_REF]. It might correlate with BMP regulators like gremlin [START_REF] Yeung | Gremlin-2 is a BMP antagonist that is regulated by the circadian clock[END_REF]. Gremlin-2 is a BMP antagonist that is regulated I-55 by the circadian clock. In wild-type mice, the core circadian clock transcription factor Bmal1 activates the expression of TGF-β pathway components in adipocytes, which suppresses the differentiation of brown adipocytes. In Bmal1-knockout cells, TGF-β signaling is attenuated, leading to the upregulation of BMP signaling and enhanced brown adipose formation. Therefore, in brown adipocytes, a cell's decision to differentiate appears to result from a balance between TGF-β and BMP signals, which is subjected to circadian clock modulation [START_REF] Nam | The adipocyte clock controls brown adipogenesis through the TGF-b and BMP signaling pathways[END_REF]. However, TGF and BMP signaling seems to have any input on the clock molecular pathway [START_REF] Kon | Activation of TGFβ/activin signalling resets the circadian clock through rapid induction of Dec1 transcripts[END_REF].

Spatiotemporal control of BMP signaling

Optogenetics

Optogenetics is a strategic field that combines light (opto-) and genetics (genetic) into applications able to control-influence cell behavior using light. These changes are achieved, making the cell express one or more proteins, resulting in a conformational change induced by light [START_REF] Kushibiki | Optogenetics: Novel tools for controlling mammalian cell functions with light[END_REF]. It was first used to study and develop new neurobiology regenerative therapies [START_REF] Dugué | Optimizing bone wound healing using BMP2 with absorbable collagen sponge and Talymed nanofiber scaffold 11 Medical and Health Sciences[END_REF]. Currently, it is used in different biomedical research fields, including ophthalmology, heart failure recovery, post-stroke recovery, tissue engineering, and regenerative medicine, as review recently by Spagnuolo [START_REF] Spagnuolo | The Impact of Optogenetics on Regenerative Medicine[END_REF] Transduction of signals is a highly dynamic process in which the precise outcome is the result of the exact spatial-temporal resolution of single sub-events. Whereas conventional techniques as classic genetic manipulation or drug inducer systems have been previously applied to study an extensive variety of biological processes and led an understanding of the architecture of signal transduction pathways, they are limited to resolve the spatiotemporal aspects and lack of reversibility (Figure 26). Most of these studies have been focused on the same family of receptors heterodimerization, but any crosstalk with other co-receptor has been explored. In the interest of heterodimerization, an interesting tool developed in 2015 by Guntas et al. [START_REF] Guntas | Engineering an improved light-induced dimer (iLID) for controlling the localization and activity of signaling proteins[END_REF] used computational protein design to create an efficient improved Light Inducer Dimer (iLID). The system contains a LOV2 domain to optically control the bacterial SsrA peptide binding to its natural partner SspB. In the dark state, the LOV2 domain interacts with its C-terminal helix termed the Jα helix. The blue light illumination exposure causes the Jα helix to unfold, an unmasking of a protein fused to the C-terminus, allowing it to interact with its binding partner. This optogenetic system assembles and disassembles in a few seconds (Figure 28).

I-59

The faster an optogenetic system can be reversed. Indeed, intracellular signals can be manipulated in space and time. Optogenetic tools provide a means to quantify the dose of activating light and the strength of the input, that is, the number of optogenetic proteins actively signaling.

In the future, the optogenetic toolbox will increase further. The generality and modularity of existing optogenetic systems can be used in the development of new optogenetic systems for better application , in other organisms (e.g., zebrafish) or in mammalian cells [START_REF] Buckley | Reversible Optogenetic Control of Subcellular Protein Localization in a Live Vertebrate Embryo[END_REF][START_REF] Gao | Optogenetic manipulation of cGMP in cells and animals by the tightly light-regulated guanylylcyclase opsin CyclOp[END_REF][START_REF] Gasser | Engineering of a red-light-activated human cAMP/cGMP-specific phosphodiesterase[END_REF].

PH.D. OBJECTIVES

Understanding how cells integrate or adapt multiple signaling pathways to achieve specific cell differentiation is a challenging question in cell biology.

Previous results showed that BMP receptors and β3 integrin work together to control SMAD signaling and tensional homeostasis (Laure Fourel, Valat, Faurobert, Guillot, Reynard, et al., 2016), thereby coupling cell adhesion and fate commitment -two fundamental aspects of developmental biology and regenerative medicine. From a broader perspective, this coupling between integrins and BMP signaling pathways is of high relevance in developmental processes and regenerative medicine, where cell recruitment is a prerequisite to cell differentiation to form a specific organ or repair damaged tissue. However, very little is known about whether the spatial arrangement between BMPR and integrin affects the cellular response to receptor signaling. Whether dynamics between integrin and BMP receptors are controlled in space and time to guide pivotal intracellular processes remains to be elucidated. Spatio-temporal control allows dissociating physical and biochemical cues to understand how cells integrate multiple signaling pathways to couple cell migration and cell differentiation.

The Ph.D. project aims to investigate the spatial organization of BMPRs at the cell membrane concerning 3 integrin-mediated adhesion sites and the impact of the physical proximity between integrin and BMP receptor for driving cell adhesion dynamics and cell differentiation.

For this purpose, an optogenetic approach and live-cell imaging have been developed to understand whether spatial patterns between integrins and BMP receptors impact adhesion site dynamics and nuclear transcription factor translocation.

The work's first objective consisted of designing optogenetic tools for BMP2 receptor and β3 integrin and establishing the opto-cell lines expressing the appropriate levels of fluorescent BMPRs suitable for microscopy analysis.

The second objective consists of investigating the distribution and dynamics of the BMP2 receptors at the cell surface and the impact of the physical proximity between BMPR and β3 integrin on the control of cellular processes.

II-63

CHAPTER II. MATERIALS AND METHODS.

This chapter gathers the material and methods used during the thesis work.

The first part (

Cell culture and Receptor transduction

Cell culture procedures were performed in sterile conditions. All cell culture material (dishes, media, pipette, tips) were sterile, and all procedures are done in a laminar flow hood that provides an aseptic work area. Cells are maintained in a tissue culture dish in a cell incubator at an adequate medium at 37°C and 5% CO2.

Cryo-conservation cell lines were carried out by gently detaching the adherent cells with Trypsin/EDTA, centrifuging at 1100 RPM for 3 min at RT, resuspended the cells in freeze medium (50% DMEM, 40% serum, 10% DMSO), and immediate freezing of 1 mL aliquots at -80°C in an isopropanol cooling chamber. For long-term conservation, cells were preserved in liquid nitrogen.

To re-start a cell culture, a cryovial containing frozen cells was putting at RT for 5 minutes to thaw them before diluting in DMEM (10%FBS) at high density to optimize their recovery. The medium is replaced after 4h.

Cell Lines

The diversity of BMP signaling is well known to be highly cell context-dependent. Multiple cell lines were selected to study their response to BMP2 as candidates to be used in the optogenetic system, including mesenchymal stem cells and epithelial cell lines.

MEF (CRL-2907, American Type Culture Collection ATCC) and REF52 (CVCL_6848) were cultured in DMEM medium, C2C12 (CRL-1772, American Type Culture Collection (ATCC) were cultured in DMEM: Ham's F12 medium (11320, Gibco). These mediums were supplemented with 10% FBS (FBS, PAA Laboratories) and penicillin-streptomycin mixture. EpH4 was cultured in DMEM supplemented with 10% newborn calf serum (Thermo Fischer Scientific, 26010074) and penicillin/streptomycin.

Before the transcription factor signaling experiments, cells were starved using their corresponding medium supplemented with 0.5% serum.

Opto-plasmids design

All constructs were cloned into pSICOR lentiviral backbone designed initially for a CMV promoterdriven expression (adgene ref 11579) by insertion between AGE and EcoRI. In the case of the BMPRs, a substitution of the CMV by the PGK promotor was done to get lower and more stable expression levels. ALK3, BMPRII, β1, and β3 integrin wild types were fused to both sides of the venus_ iLID-RFPsspB opto_system.

ALK3 constitutive inactive(ALK3Ci) (K261R) or ALK3 constitutively active (ALK3Ca) (Q233D) were obtained by a point mutation and fused to RFPsspB.Furthermore, a series of β3 integrin mutations on the distal NPXY site [START_REF] Milloud | αvβ3 integrins negatively regulate cellular forces by phosphorylation of its distal NPXY site[END_REF] was fused to VenusiLID by substitution of the original eGFP.

BMP receptors and their selected mutations were fused by their C-terminal using a protein linker (integrin tcaccggtcatggatcccagtgtggtggtacgtaggaattct, BMPR: ggatcccagtgtggtggtacgcaggaattc) to the opto-fluorescent protein.

The design of engineered primer pairs for each construction (

Transient transfection

Cells were transfected using Jet prime® (polyplus transfection) or lipofectamine 2000 ® (Gibco, Invitrogen) following provider's protocol. Briefly. Cells were seeded at 70% confluency and cultured overnight. DNA is diluted in Jet prime buffer (1:100 according to the well plate size) and vortexed. Jet prime reagent was added to the mix (ratio 1:2) and vortexed again to be incubated for 10 min at RT. The incubated mix is added drop by drop to the cell in serum-containing medium and incubated 24 to 48h.

a.

SiRNA against an endogenous receptor Cells were transfected with small interfering RNA (siRNA) against ALK3, BMPRII, ACTVR1a, ACTVR2a, or β1-, β3 integrin (ON-TARGET plus SMARTpool) individually or mix according to the experiment. Briefly, cells were seeded in a six-well plate at 50% confluency and culture overnight.

The transfection mix was prepared as follows: For one well, 7 µl of Lipofectamine RNAiMAX Reagent (Invitrogen) was added to 305 µl of Opti-MEM medium (Gibco), and 25 µl at 10 µM siRNA was added to another 305 µl Opti-MEM medium. Lipofectamine-containing mix was added to siRNA-containing mix and incubated for 20 min at RT. Before transfection, the DMEM of the wells was replaced by the DMEM without antibiotics. Then, 610 µl of the final mix was added to each well. After 24h of incubation at 37°C, the cells were transfected for the second time and incubated for another 24 h.

Optimized-transduction of Opto BMPR

A flowchart protocol presents in Figure 29 shows the overview steps of the establishment of the Opto_cell line. The first stage (Steps 1-4) involved the lentivirus-producer cell line transfection. The lentiviral particles encoding the transgenes are collected and concentrated. An alternative to this step, a platform producing a virus, could also be used.

In the second stage (Steps 5-8), targeted cells are infected, stably transduced. In the third stage (step 9-10), cells are enriched by FACS sorting and expanded. Finally, optional but desirable, second FACS enrichment and intensity normalization are performed to expand the cell lines to keep for maintenance. a.

Virus production

Lentiviral particles are a potential biological hazard and require biosafety level 2 (BSL2) practices. All work was performed in an approved laminar flow cabinet using sterile techniques and comply with the appropriate institutional biosafety guidelines.

The procedure to transduce expression cells starts with a lentivirus producer cell line (HEK293T), which was transiently co-transfected with the vector (encoding the transgene of interest), envelope, and packaging plasmids to generate lentiviral particles. The lentivirus-containing supernatant was used to transduce the target cell lines using their corresponding multiplicity of infection (MOI).

II--71 -Detail optimized protocol.

Step 1: seed ~1 x 10 6 HEK293FT cells in a 10 cm plate in complete medium, bringing it to ~50% confluency. Incubate the plate for 24h in a humidified incubator operated with 5% CO2. 37°C. One plate per transfection.

Step 2: The next day, prepared the following plasmid DNA transfection mix per target gene (10 μg total DNA; 2:1:1 (wt:wt:wt) vector:packaging: envelope plasmid ratio).

• 5 μg Lentiviral plasmid containing the gene of interest Replace the growth medium of the HEK293Ft culture plate (from step 1) with 5 mL. Add drop by drop of the transfection mix. Incubate at 37°C in a humidified incubator operated with 5% CO2. Change the medium after 5h and incubated in the L2 laboratory.

Step 3. Three days after transfection, it is possible to verify the level of transfection by fluorescent microscopy. Collect the 5 mL medium from the HEK293FT into a sterile 50 mL tube. LentiX concentrator solution is incubated for 30 min on the glass before centrifugation at 1500 RPM for 45 min at 4°C. After removing the supernatant, the virus is resuspended into 100 µl of PBS.

The concentration of the produced lentivirus allows higher infection efficiency. For some transgene of interest, as the case of β3 integrin, previous concentration is not required.

Furthermore, as an alternative to the first four steps, a platform produced virus can be used.

b. Infection

Step 5 A day before the infection, seed ~20 x 10 3 target cells (C2C12, MEFsv40, REF52, MSCD1, or EpH4) in 24 well plate in their corresponding growth medium supplemented with 10% FBS. Seed it to ~50% confluency. Incubate overnight at 37°C in a humidified incubator operated with 5% CO2.

Step 6 Infection of the target cell line (Day 5) • TIMING 1 d

The 24 well plate with the target cell line is now >75% confluent; remove and discard the complete growth medium; wash one time with PBS and replace each well with growth medium (low serum, without antibiotics). 50 μl for C2C12 and 100 μl for the rest of the cell lines. (Table 3). Add the lentivirus-containing solution of BMP receptor conditions, 100 μl for C2C12, and 50 μl for the rest of the cell lines Incubate the plate at 37°C, 5% CO2 for 15 min. The viral particles will now infect the cells and stably integrate their genetic material into the host cell genome.

Step 7, add the second virus, usually β3 integrin, adding 10 μl of the lentivirus containing solution.

Incubate the plate at 37°C, a humidified incubator operated with 5% CO2 for 30 min. Then add 100 (for C2C12) or 100 μl (for the rest of cell lines) of the complete growth medium, and place back to the incubator. 4h later, adds 500 μl of complete medium.

Expansion cell line (Day 8) •TIMING ~3 d

Step 8. Two days after the infection, wash the 24-well plate thoroughly, at least three times, with PBS. Pass the cells to a 6 cm plate, incubate at 37°C in a humidified incubator operated with 5% CO2. after 24h (75% of confluence), wash the plate thoroughly with PBS and pass the cell to a 10 cm plate II--73 -c.

Cell population enrichment

Step 9. Three days after infection. After reaching 80% confluency of infected cells, the cells are suspended in low volume PBS without Ca2+/Mg2+ (˜200 μl for a 10 cm plate).

Compensation control was performed with no infected cells. Cells are sorted by their BMP receptor expression level using 561nm Laser to select tagRFP fluorescence intensity and β3 integrin using a 488 nm laser using the FACS cell sorter (FACS Aria IIu, BD Biosciences).

Step 10. Split the cells to continue her expansion. A second round of FACS enrichment is strongly recommended.

Microscopy techniques Epifluorescence

In epifluorescence microscopy (Figure 30), the light is first filtered through an excitation

Optogenetic activation

The 488 nm laser excitation was used for the observation of the receptor fused to Venus_iLID simultaneously for the stimulation of the optogenetic system and therefore inducing the interaction of the complement optogenetic protein fused to sspB. The 561 nm laser excitation was used for the observation of protein fused to RFP without causing any activation in the optosystem.

A classic protocol used for optogenetic stimulation of BMP receptor recruited to focal adhesion includes three steps: i) few images using 561 nm light to get the basal state and position of the BMP Receptors. ii) Follow during three minutes with 488 nm (green channel)

and 561 nm (red channel) light to activate the opto system, iii) finally, a dissociation step with images acquisition using just the 561 nm light during 3 minutes. Frequencies and time of stimulation were optimized to observe the recruitment (until reached a maximum plateau) and total dissociation of the opto_system. All images were processed with ImageJ software to create complete videos with the images taken. BMP receptor recruitment quantity was followed on the -RFP channel of each TIRF image at an ROI limited by the focal adhesion using time series analyzer plugin on Fiji software. Each intensity was normalized using the intensity at the membrane over time Cell population photostimulation was performed using two different customize blue LED plates according to the need. The first, designed by M. Balland (Lihpy, Grenoble-Alpes University). The LED illumination was programmed by Arduino software at 10 sec, 30 sec, or 2 minutes frequency at 50% LED power for 30 min, 4h, or 12h.

d. FRAP

The second device consists of adaptation from Janovjak Laboratory [START_REF] Grusch | Spatiotemporally precise activation of engineered receptor tyrosine kinases by light[END_REF]). An incubator (PT2499; ExoTerra/ HAGEN, Holm, Germany) was equipped with 300 lightemitting diodes (JS-FS5050RGB-W30 with JS-CON-004 controller; Komerci, Ebern, Germany). Light intensity was controlled with an analog dimmer and measured with a digital power meter (Sanwa). For stimulation over extended periods (> 8 h), a box cover on aluminum was equipped with the same diodes and placed in a cell culture incubator.

Evaluation of cellular response

This work's primary goal was to study the effects of the physical proximity between BMPR and β3 integrin on different cellular processes, including adhesion, migration, proliferation, and differentiation.

Detection of transcription factors a. Immunofluorescence staining

Cells were seeded on glass coverslips and stimulated with 0, 6, 60, or 600 ng/mL BMP2 or by constant pulses of blue light illumination for a determined period. After stimulation, the cells were first rinsed in PBS and fixed in 4% formaldehyde for 10 min at Room Temperature (RT) for fixation. Then cells were once again rinsed with PBS before being permeabilized in (PBS 0.5% tween, 0.3% triton 100X) for 10 min at RT. After rinsing with PBS, samples were blocked for 30 min with PBS 0.5% tween, 1% BSA, 5% goat serum).

The sample was then incubated with primary antibodies at the right dilution described in Table 4. After the incubations with the primary antibodies for 2h at RT, cells were washed three times in PBS and incubated for 1h at RT with corresponding secondary antibodies.

When necessary, actin was labeled with phalloidin (1:1000, Sigma) simultaneously as the secondary antibody. Coverslips were mounted with mowiol (containing DAPI when required). Quantification of the nuclear transcription factor was performed by measuring the nucleus's mean intensity, delimited through DAPI staining. For the intensity of BMP receptor recruitment, or pTyrosine limited at focal adhesion, either 3 integrin or pPaxillin were used as a mask. A macro was written to semi-automatize the intensity measurement in a defined ROI.

b. Immunoblotting

Cells were seeded on 6 or 12 well plates a culture overnight. According to the experimental conditions, the cells were starved by 3h and stimulated with BMP2 or blue light pulses during the determined time. Cells were lysed in RIPA buffer (Tris-Buffer Saline (TBS), 50mM NAF, 40mM, Nappi, 1% Triton, 0.1% SDS) supplemented with protease and phosphatase inhibitors. Tampon blue Laemmli 2X was added and heated up to 95°C for 5 minutes. The lysate samples were run in 8-10% polyacrylamide gel, at 30mA/gel, and transferred in nitrocellulose or PVDF (polyvinylidene difluoride) membranes in transfer buffer containing 20% isopropanol and 0.04% SDS. After electrotransferred proteins (110v, 1h20), the membrane was incubated at room temperature for 1h in a blocking buffer containing TBS-Tween-20 (0.1% v/v) and low-fat milk (5%). Then, membranes were blotted against the protein of interest (Table 5) diluted at their corresponding final dilution in TBS-T, containing 5% BSA (bovine serum albumin) overnight at 4°C. After three rinses (TBS-T), the membrane was incubated with a secondary antibody coupled to HRP diluted in TBS-T-BSA for 1h at room temperature. After three rinses, the peroxidase activity was visualized by chemiluminescence. The membrane was incubated with the substrate from the kit ECL II--79 -(enhance chemiluminescence, Thermo scientific). The luminescence is detected using the camera fusion fx (Vilber, smart imaging). As a loading control, GAPDH was used. The intensity analysis was done using Image J.

Gene expression quantification qPCR

For real-time quantitative PCR, total RNA was prepared from the cell samples after cell lysis using an RNA extraction kit (Nucleospin RNA, Macherey-Nagel) following the providers protocol. After reverse transcription of 1 µg total RNA, PCR was performed using a set of gene-specific primers for the target gene. The sequences of primers used for real-time PCR are listed in Table 6. cDNA (equivalent to 12.5 ng) was used for real-time quantitative PCR, performed with a thermocycler (CFX96 Biorad. The 15-µl reaction mix contained 1-µl

Master SYBR Green I mix, including Taq DNA polymerase, buffer, deoxynucleoside trisphosphate mix, SYBR Green I dye, 3 mM MgCl2, and 0.5 µM of each primer. An amount of 5 µl of 25-fold-diluted cDNA was added to the mixture. Primer efficiency was established by a standard curve using sequential dilutions of gene-specific PCR fragments. Data were normalized from the quantitative RT-PCR housekeeping gene ATP50, TBP, HPRT, GAPDH, as an index of cDNA content after reverse transcription

Cell spreading and migration

Adhesion is the first and essential step of cell-substrate interactions, especially for anchorage-dependent cells. Different parameters, such as the number of adherent cells, spreading area, morphological parameters, and cytoskeletal organization, were evaluated.

Cells were seeded on soft (low-crosslinked) and stiff (high cross-linked) PLL/HA film, with bound BMP2 or soluble BMP2, with or without blue pulses of illumination (10s) during 4h.

The cells were washed with PBS and fixed with PFA 4% for 10 min. After washing with PBS, actin was labeled (Alexa Fluor™ 647 Phalloidin). Image acquisition was performed using an iMIC inverted microscope in epifluorescence mode. The data collected was analyzed by ImageJ software (v 1.44p, NIH, Bethesda) to calculates the cell area. Another macro was written for the measurement of the cell number and mean spreading area. Images of the nuclei were binarized using an intensity threshold, touching nuclei separated using the watershed function.

a. Individual migration

C2C12 cells were seeded at 15 000 cells/cm 2 in 96-well plates stiff film, allowing a spreading time of 4 h before monitoring by time-lapse microscopy using Zeiss LSM 700 microscope equipped with an incubator. Images were taken every 10 min using a Zeiss LSM 700 microscope equipped with an incubator. Images were taken every 10 min for 10h. For the II--81 -analysis, at least 20 cells were tracked using ImageJ (v1.45d, NIH, Bethesda). After cell tracking, the paths of the cells were plotted using Chemotaxis, and Migration Tool (ibidi) allows quantifying cell velocity and directionality b.

Polyelectrolyte multilayer films

Poly-L-Lysine (PLL, 2 × 10 4 g/mol, Sigma-Aldrich). (0.5 mg/ml) and Sodium hyaluronan Sodium hyaluronate (HA; 2 × 10 5 g/mol, Lifecore Biomedical) (1 mg/ml) was dissolved in a HEPES-NaCl buffer (20 mM HEPES, pH 7.4, and 0.15 M NaCl). For all experiments, (PLL/HA) 12 were prepared as previously described [START_REF] Machillot | Automated Buildup of Biomimetic Films in Cell Culture Microplates for High-Throughput Screening of Cellular Behaviors[END_REF] with an automated film buildup using a liquid handling robot, in 24 well plates, and 96-well plates glass bottom.

The films were cross-linked following the protocol previously described (Thomas Crouzier, Ren, Nicolas, Roy, & Picart, 2009) using 1-ethyl-3-(3-dimethylamino-propyl) carbodiimide at 30 mg/ml (soft films) or 70 mg/ml (stiff films) and N-hydro-sulfosuccinimide at 11 mg/ml (both purchased from Sigma-Aldrich).BMP2 (clinical grade; Medtronic) was incorporated into films preequilibrated for 30 min in the medium in which BMP2 was suspended (1 mM HCl). It was deposited onto the films and left to adsorb 1ht at 30°C. The films were washed for 1h in HEPES-NaCl [START_REF] Crouzier | Layer-by-layer films as a biomimetic reservoir for rhBMP-2 delivery: Controlled differentiation of myoblasts to osteoblasts[END_REF].

Receptor Colocalization analysis on protein matrices

Opto_cells were cultured on Lab-Tek chambers (Thermo Fisher Scientific), coated with poly-(L-lysine) (PLL, 60 kDa, P2636, Sigma, France), vitronectin (Sigma Aldrich), fibronectin, or collagen at 5µg /mL Then cells were kept in the dark or illuminated for 4 hours. Soluble BMP2 (600 ng/mL) was added to half of the conditions. The sample was fixed, and the TIRFM mode imaging was used to acquire the three-channel.

ImageJ software was used for image processing and analysis. Merging channel and Plot profiles at a focal adhesion limited region are presented. The total image intensity of both channels was quantified to provide a measure of colocalization, specifically by using the Manders coefficient calculated using an ImageJ plugin, named JACoP, that integrates current global statistic methods and a novel object-based approach. [START_REF] Bolte | A guided tour into subcellular colocalization analysis in light microscopy[END_REF].

Immunoprecipitation

REF52 was cultured in DMEM+10%FBS until they reached 80% confluency. Then cells were starved for 1h (DMEM+0.05%FBS) before ligand treatment or photostimulation.

Stimulation with 600 ng/mL BMP2 and/or constant blue light (every10sec) during 1h was performed.

Cells were washed with PBS and lysed with RIPA lysis buffer (150mM NaCl, 20mM Tris pH 7,4,1mM sodium orthovanadate, 10mM NaF, 10mM β-GCP, 1% NP40, 0,5% NaDOC, 0,1% SDS supplemented with protease inhibitor EDTA-free). The lysates were incubated for 1h using anti-RFP antibodies (MA515257, ThermoFisher scientific) or IgG at 4°C for 1h. Samples

Statistical analysis.

Graphic data visualization and statistical analysis were performed using Prism 7 (GraphPad).

The comparison of the two samples was performed using the unpaired t-test if variances were equal. Statistically significant values are reported on the figures, according to the Pvalue (Table .7).

CHAPTER III. RESULTS

This chapter is divided into three sections.

In the first section (III.A), we challenge different cell lines as in vitro study models and compared their ability to express BMPR and integrin as their ability to respond to BMP2.

We established an optimized protocol to obtain stable fluorescent BMPR cell lines to use in an optogenetic assay.

In the second section (III.B), we focus on the discrete spatial organization of BMP2 receptors concerning adhesion sites. The results presented are under preparation for journal submission.

Finally, a third section (III.C) presents two results not included in the article within particularly the impact of the β3 integrin-ALK3 proximity on ALK3 phosphorylation and the behavior of the short form of BMPRII concerning adhesive sites.

III--85 -

Optimized lentiviral transduction of BMP receptors for optogenetic assessment in Mesenchymal and Epithelial models Abstract

The host lab has previously shown that BMP receptors and β3 integrin work together to control SMAD signaling and tensional homeostasis (Laure Fourel, Valat, Faurobert, Guillot, Reynard, et al., 2016), thereby coupling cell adhesion and fate commitment -two fundamental aspects of developmental biology and regenerative medicine. However, there is insufficiently known about whether their spatial arrangement affects the cellular response to receptor signaling. Whether dynamics between integrin and BMP receptors is controlled in space and time to guide pivotal intracellular processes remains elucidated.

Analyzing the BMP receptors subunit distribution and possible interactions with other proteins is challenging to achieve with conventional microscopy approaches with labeling methods and imaging processing as limiting factors [START_REF] Medda | Challenges in imaging cell surface receptor clusters[END_REF]. The establishment of a fluorescent-BMP receptor cell line is presented. The cell lines are envisioned to be used to study BMP receptor and integrin interaction through Optogenetics.

While C2C12 is an excellent cellular model to study BMP2 signaling, C2C12 has been described as highly challenging to be transfected [START_REF] Balci | Efficient transfection of mouse-derived C2C12 myoblasts using a matrigel basement membrane matrix[END_REF], producing suboptimal transduction efficiencies. Optimization of the infection process was assessed to obtain the most stable and reliable BMPR expression suitable microscopy analysis.

Mesenchymal (C2C12, MEFsv40, REF52) and epithelial (EpH4) cells were considered as possible study models. As a first step, we test the cell's ability to respond to BMP2 in classic in vitro conditions. Our results showed that all considered cell lines respond to BMP2 through SMAD1/5/8 phosphorylation and nuclear translocation. Second, we investigated the feasibility of achieving high transgene expression. We compared the four cell lines achievability to be infected by the viral gene transfer method. Optimizing the conditions for obtaining a polyclonal BMPR optogenetic cell line with an adequate receptor expression level to be used for optogenetic assay. Fibroblast (mouse and rat) represented the best suitable model to study BMP signaling and fluorescent BMP receptors.

.

Introduction

Bone Morphogenetic Proteins (BMPs) are members of the TGF-β superfamily and have diverse functions during development and organogenesis. BMPs play a significant role in developing most of the tissue [START_REF] Wagner | BMPs: from bone to body morphogenetic proteins[END_REF]. BMPs have different physiological and pathological functions, including an essential role in the development of the musculoskeletal system [START_REF] Bandyopadhyay | Genetic Analysis of the Roles of BMP2, BMP4, and BMP7 in Limb Patterning and Skeletogenesis[END_REF], and the regulation of normal and cancer stem cells, in particular in the hematopoietic system, in brain cancer, and neurological diseases [START_REF] Wagner | BMPs: from bone to body morphogenetic proteins[END_REF]. In the cellular context in vitro, most published rhBMP2 assays use W2017 (mouse stromal), MC3T3 (preosteoblast), or C2C12 (myoblast) cell lines [START_REF] Fung | In Vitro Evaluation of Recombinant Bone Morphogenetic Protein-2 Bioactivity for Regenerative Medicine[END_REF]. Indeed, using the cell line C2C12, it was first evidenced that BMP receptors (BMPR)-β3 integrin cooperation upon BMP2 treatment in the context of bone regeneration(L. Fourel et al., 2016). While this has led to an understanding of signal transduction pathways' architecture, the spatiotemporal aspects were impossible to resolve.

Optogenetics is based on genetically encoded light-responsive proteins and it has the potential to revolutionize the manipulation of signal transduction processes. Light can be easily applied with the highest precision and minimal invasiveness. [START_REF] Mühlhäuser | Optogenetics -Bringing light into the darkness of mammalian signal transduction[END_REF]. Using the Opto couple BMPR/β3 integrin, light serves as an observational tool to follow biological events and central manipulation to modulate the components' proximity with high spatial and temporal resolution.

While fluorescent-receptors could be studied through transient transfection, they often produce low transfection efficiencies (<10%) when not optimized and up to 50% in the best conditions. [START_REF] Trivedi | Liposome-Mediated Gene Transfer into Normal and Dystrophin-Deficient Mouse Myoblasts[END_REF][START_REF] Yamano | Comparison of transfection efficiency of nonviral gene transfer reagents[END_REF] . Moreover, probing signaling pathways using biochemical assays brought forth the need to obtained stable cell lines. Lentiviral transduction is an efficient and widely used method for the delivery of transgenes to mammalian cells. It has been described previously that the genetic manipulation of skeletal muscle cells in vitro is notoriously challenging, especially when using undifferentiated muscle cell lines (like C2C12 myoblasts) [START_REF] Balci | Efficient transfection of mouse-derived C2C12 myoblasts using a matrigel basement membrane matrix[END_REF].

Considering this limitation, cell line alternatives to study BMP2 signaling and, in particular, their capacity to transduced the BMP receptor/β3 integrin construct was explored. Indeed, whether the cellular context can drive specific phenotypes upon BMP2 stimulation needs to be investigated.

Results

a.

Cell type-dependent BMP2 signaling.

β3 integrin and BMPRs cooperation upon BMP2 treatment was first evidenced in the context of bone regeneration by the mouse myoblast cell line C2C12. The mouse myoblast cell line (C2C12) was used due to its ability to differentiate towards either muscle or osteoblastic lineage. Indeed, if left at high confluency in the growth medium, C2C12 cells fused to form myotubes whereas, in the presence of BMP2, this differentiation will be biased towards bone characterized by expression of Alkaline Phosphatase (ALP) and other specific osteoblastic markers [START_REF] Bouyer | Surface delivery of tunable doses of BMP-2 from an adaptable polymeric scaffold induces volumetric bone regeneration[END_REF][START_REF] Cipitria | Mechanotransduction and Growth Factor Signalling to Engineer Cellular Microenvironments[END_REF]. As a second model, Mouse Embryonic Fibroblast (MEFsv40) has been described to possess mesenchymal stem cell-like properties and represent an excellent alternative to study differentiation to osteoblasts [START_REF] Saeed | Mouse Embryonic Fibroblasts (MEF) Exhibit a Similar but not Identical Phenotype to Bone Marrow Stromal Stem Cells (BMSC)[END_REF]. Rat Embryonic Fibroblast (REF52) cells were chosen for their ability to develop essential adhesion sites and actin cytoskeleton [START_REF] Cavalcanti-Adam | Cell spreading and focal adhesion dynamics are regulated by spacing of integrin ligands[END_REF], even though the literature is not widely used to study growth factor signaling. [START_REF] Owens | Inhibition of BMP signaling suppresses metastasis in mammary cancer[END_REF][START_REF] Sloan | Tumor-specific expression of αvβ3 integrin promotes spontaneous metastasis of breast cancer to bone[END_REF]. However, the roles of BMPs in epithelial cells are poorly understood [START_REF] Saitoh | Basolateral BMP Signaling in Polarized Epithelial Cells[END_REF].

The cell lines' ability to respond to soluble BMP2 through the canonical pathway was challenge following SMAD 1/5/9 phosphorylation by immunoblotting (Figure 33.A). After 2h starvation (low serum DMEM), followed by 4h of soluble BMP2 (600ng/mL), all cell lines presented an increase in pSMAD in response to BMP2, C2C12 being the most responsive. In the absence of BMP2, no pSMAD has been detected (Figure 33.B).

By immunofluorescence, the nuclear translocation of pSMAD was tested in two conditions at 24h, first on glass with soluble BMP2 and second in a soft matrix-bound BMP2 presentation. In both situations, all cell lines presented a translocation of pSMAD to the nucleus, which confirms that SMAD is not only phosphorylated but active as a transcription factor (Fig 33 .C andD).

III--89 - The ability of cells to respond to a specific ligand relies on the expression of the ligandspecific receptor. In the case of BMP2/BMP4 signals via two types of receptors BMPR type-I and type-II are expressed at the cell surface as homomeric or heteromeric complexes [START_REF] Nohe | The mode of bone morphogenetic protein (BMP) receptor oligomerization determines different BMP-2 signaling pathways[END_REF]. Real-time qPCR was performed, revealing a similar receptor expression pattern between the cell lines (Figure 33 E). ALK2 and ALK3 are the most expressed type-I receptors, while BMPRII is the most expressed type-II receptor. Furthermore, we examine the integrin expression, while on C2C12, both β1 integrin and β3 integrin are highly expressed in a similar way, MEFsv40 and EpH4 express around 30 times less.

a. Enrich subpopulation of transduced cells.

CMV vs. pGK promoter

It is known that promoters can influence viral transduction efficiency. We initially developed the lentiviral transfer plasmid using CMV (Immediate-early Cytomegalovirus virus)

promoter. The CMV promoter/enhancer is one of the strongest promoters with a high-level expression in a wide variety of mammalian cell lines. The use of this promoter was efficient in the transduction of CAAX-RFP in the four cell lines. However, the transduction of ALK3

and BMPRII in C2C12 and MEfsv40 were systematically low even in the optimized infection conditions (Figure 34.B).

Additionally, the induced expression is lost relatively fast (around 2 weeks-data no shown).

Like other strong viral promoters, it is prone to transcriptional silencing regulated by DNA methylation after prolonged activity [START_REF] Brooks | Transcriptional silencing is associated with extensive methylation of the CMV promoter following adenoviral gene delivery to muscle[END_REF]He, Yang, & Chang, 2005;[START_REF] Hsu | Targeted methylation of CMV and E1A viral promoters[END_REF]. [START_REF] Brooks | Transcriptional silencing is associated with extensive methylation of the CMV promoter following adenoviral gene delivery to muscle[END_REF] reported CMV methylation and silencing in skeletal muscle. Similar inactivation could be problematic with muscle cells in vitro.

To overcome this problem, we have chosen the pGK promoter (Murine Phosphoglycerate

Kinase-1). The pGK promoter is described to present a long-term persistent expression in cells susceptible to promoter silencing from methylation or histone deacetylation, such as undifferentiated embryonic stem cells. It is considered robust in most cell types, including primary cell and stem cell. This promoter can efficiently drive the expression of reporter genes (i.e., LacZ and GFP) and therapeutic genes, such as tumor-associated antigens [START_REF] Gerolami | Gene transfer to hepatocellular carcinoma: Transduction efficacy and transgene expression kinetics by using retroviral and lentiviral vectors[END_REF][START_REF] Lizée | Lentivirus vector-mediated expression of tumorassociated epitopes by human antigen presenting cells[END_REF]. Furthermore, It has been reported that pGK superiority in myoblasts [START_REF] Schul | Bursted BMP Triggered Receptor Kinase Activity Drives Smad1 Mediated Long-Term Target Gene Oscillation in c2c12 Cells[END_REF], while to date, comparative efficiencies tests have not been performed in a wide variety of cell lines. Therefore, we tested the efficacy of two commonly used promoters, CMV, and pGK, to determine which suits better our system of BMPR expression.

All our lentiviral optogenetic vectors have unique restriction sites at the promoter level, allowing their easy replacement with the choice promoter, and then follow the previous infection and enrichment protocols.

Our results (Figure 35) show that the pGK promoter was the most effective. Even though CMV and pGK appeared equally efficient in transduced simple proteins as CAAX, the promoter activity of both was comparatively weak when BMP receptors were the target protein. The pGK increases the % of positive cells expressing ALK3-RFP in C2C12, MEFsv40, III--93 -and REF52. Additionally, a sufficient level of expression was sustained for a more extended period (around 4 weeks), allowing a second round of enrichment if it is required.

In summary, Lentiviral/pGK transduction is optimal when transducing fibroblast and epithelial cells following the optimized protocol. However, not only the cell type represents a challenge to obtain high transduction levels but also the type of receptor to be expressed has an impact.

Conclusion and discussion.

Using different cell types, both mesenchymal (C2C12, MEF, and REF52) and epithelial (EpH4), we demonstrated that all selected cell lines respond to BMP2 stimulation through the canonical pathway SMAD 1/5/8.

We also evaluated the BMP receptor transduction efficiency on distinct cell lines using lentivirus along with comparing CMV and pGK promoters. Cell line C2C12 was highly challenging to infect with any of the conditions above and challenging to maintain in culture.

Like fibroblasts (mouse and rat), other cell lines represent the best option since it is possible to obtain a considerate infection rate. Furthermore, it is possible to maintain the fibroblast in culture for up to 4 weeks. It is essential to consider that the mouse fibroblast is closer in signaling to C2C12, being the best model to study BMP2 signaling. The Viral platform reported low virus production (BMPRII: 0 UI/mL), confirmed the difficulties to express this receptor in distinct cell lines (as HEK293T typically used to produce the viral particles).

Following the optimized protocol, we infected enough cells even though the virus titration was low.

The genetic manipulation of in vitro muscle cells, whether myoblast cell lines, primary myosatellites or myotubes, is notoriously tricky. Viral transduction is intermediated by interactions between viral components and host proteins, including receptors and other expressed proteins that influence infection efficiency [START_REF] Brass | Identification of host proteins required for HIV infection through a functional genomic screen[END_REF]Schwartz et al., III--95 -2007) whose presence may vary between cell types and possibly with differentiation status or the type of protein to be transduced

We evaluated the BMP receptor transduction efficiency on distinct cell lines using lentivirus and comparing CMV and pGK promoters. C2C12Cell line was highly challenging to infect with any of the conditions above and challenging to maintain in culture. Like fibroblasts (mouse and rat), other cell lines represent the best option since it is possible to obtain a considerate infection rate. Furthermore, it is possible to maintain the fibroblast in culture for up to 4 weeks. It is essential to consider that the mouse fibroblast is closer in signaling to C2C12, being the best model to study BMP2 signaling. Two strategies are traditionally used; antibiotic selection and enrichment by fluorescence-activated cell sorting (FACS). We opted for FACS because it is faster than antibiotic selection, and fluorescent marker proteins can have multiple downstream applications. The use of FACS to obtain high proteinexpressing clonal cell lines has long precedence [START_REF] Knox | A streamlined implementation of the glutamine synthetase-based protein expression system[END_REF][START_REF] Mancia | Optimization of protein production in mammalian cells with a coexpressed fluorescent marker[END_REF][START_REF] Oberbek | Generation of stable, high-producing cho cell lines by lentiviral vector-mediated gene transfer in serum-free suspension culture[END_REF] Genetic modification, including gene therapy and optogenetics, is a promising approach for therapeutics and basic science, although success has been limited due to obstacles associated with gene delivery and immunogenicity [START_REF] Cossu | New therapies for Duchenne muscular dystrophy: challenges, prospects and clinical trials[END_REF]; I. [START_REF] Kwon | Designer gene delivery vectors: Molecular engineering and evolution of adeno-associated viral vectors for enhanced gene transfer[END_REF][START_REF] Mingozzi | Therapeutic in vivo gene transfer for genetic disease using AAV: Progress and challenges[END_REF]. Better tools for investigating in vitro would help overcome these obstacles and study intracellular signaling in skeletal muscle.

However, for the analysis of BMP receptor localization in cells, tagged-receptors' successful expression has been achieved only for cells overexpressing HA-tagged BMPR II. So far, this has not been possible for BMPR type-I [START_REF] Medda | Challenges in imaging cell surface receptor clusters[END_REF]. Using standard confocal microscopy Lai et al. [START_REF] Lai | αvβ integrins play an essential role in BMP-2 induction of osteoblast differentiation[END_REF] and indirect immunostaining using myc-tagged ALK6 receptors [START_REF] Hirschhorn | Differential molecular regulation of processing and membrane expression of Type-I BMP receptors: implications for signaling[END_REF]. However, analyzing the BMPR subunit distribution and possible interactions with other proteins is challenging to achieve. In our hands.The opto-β3 integrin was efficiently expressed by lentiviral infection in all mesenchymal and epithelial cell lines used in this study. However, cells expressing the Opto_BMP receptors were far more challenging to obtain. Not only the difficulty but also the particular proteins or receptors that can be challenging as ALK3. Indeed, ALK3 regulates essential cellular response, and its overexpression leads to low infection rates and quick loss of protein expression in culture. Better molecular tools (like antibodies) for investigating BMPR in vitro would help overcome these obstacles and would help study intracellular signaling in myoblast.

III--97 -β3 integrin integrates BMP2 signaling through BMP Receptors segregation Preliminary results a.

Impact of β3 integrin/ALK3 proximity on ALK3 phosphorylation Kau et al. previously described [START_REF] Kua | C-Abl promotes osteoblast expansion by differentially regulating canonical and non-canonical BMP pathways and p16 INK4a expression[END_REF] that ALK3 tyrosine phosphorylation (pTyr) at the cytoplasmic tail is required before its heterodimerization with BMPRII. ALK3 tyrosine phosphorylation (Y-Phosphorylation) has been described as an essential step to increase the ALK3 affinity for BMPRII and favor BMPRs tetramerization. We wondered whether β3 integrin might play a role in ALK3 phosphorylation upon its proximal interaction with ALK3.

First, we evaluated the ability of the Opto-ALK3 to be phosphorylated. Using REF52 cells expressing the opto-CAAX/ β3, Opto-ALK3/ β3, and Opto-BMPRII/β3. The opto-receptors were immunoprecipitated via its tagRFP by using specific monoclonal anti-RFP antibodies.

The immunoprecipitated proteins were separated on SDS polyacrylamide gel before transferring onto nitrocellulose membrane and blotting against pTyr to assess phosphorylation levels of ALK3 with or without sBMP2 stimulation (Figure .37.A). Signal quantification was normalized based on the expression level of ALK3 detected by its RFP tag on total cell lysates. We found out that Opto-ALK3 was able to be phosphorylated by sBMP2 as previously described for endogenous receptors. (Figure .37.B).

In a second step, we analyzed the effect of β3 integrin/ ALK3 proximity induced by blue light stimulation on tyrosine phosphorylation (pTyr) of the ALK3 cytoplasmic tail. For that, ALK3 was immunoprecipitated in conditions where cells were treated with sBM2 and with blue light (Figure . 37C andD). In the dark, when the optogenetic system was inactive, ALK3 Yphosphorylation was observed upon BMP2 stimulation. However, the induction of proximity between β3 integrin and opto-ALK3 by blue light stimulation hindered BMP2induced ALK3 Y-phosphorylation.

We still do not know whether the absence of ALK3 phosphorylation results from an inhibition of ALK3 phosphorylation or an increase in the dephosphorylation rate. Moreover, this result must be taken with caution as this experiment with light stimulation has been performed only once. Replicates are needed to support this data and to investigate whether the stimulation time affects ALK3 phosphorylation. III--139 - Role of the cytoplasmatic tail of BMPRII in controlling BMP2 signaling.

Although all type-II BMP receptors have similar structures, BMPRII is the only receptor from the TGF-β superfamily expressed in two splice variants, the long-form (BMPRII) and the short-form (BMPRIIsf). They differ by a cytoplasmic tail of 512 amino acids extension. Both forms are broadly expressed in diverse tissues [START_REF] Morrell | Pulmonary Hypertension Due to BMPR2 Mutation: A New Paradigm for Tissue Remodeling[END_REF]. Furthermore, while this cytoplasmic tail is proposed to modulate BMPRII induced signal actin as a docking site for several proteins, the tail has no enzymatic activity or known regulatory functions.

A recent study by Amsalem [START_REF] Amsalem | Differential regulation of translation and endocytosis of alternatively spliced forms of the type II bone morphogenetic protein (BMP) receptor[END_REF] compared these alternatively spliced forms. They fused BMPRII and BMPRIIsf to an epitope (myc-tagged) for a biochemistry based study. The long-form and short-form presented differences in their steady-state levels, the kinetics of degradation, intracellular distribution, and internalization rate. We wondered whether we observed differences in the receptor organization or interaction with β3 integrin by using these two variants fused to the optogenetic system.

We proceed by lentiviral infection of the mesenchymal (C2C12, MEFsv40, and RE52) and epithelial cell lines (EpH4). Under the same conditions, the REF52 cell transduced more consistently both types of BMPRII. In all cell lines, BMPRIIsf was always higher expressed, as observed by the population enrichments performed by Fluorescent Activated Cell sorting (FACs) (Figure .38.A). This result correlates with the fact that the C-terminal extension in BMPRII regulated its synthesis, and BMPRIIsf presented higher expression levels [START_REF] Amsalem | Differential regulation of translation and endocytosis of alternatively spliced forms of the type II bone morphogenetic protein (BMP) receptor[END_REF]. Additionally, the western blotting using the tag-RFP revealed the molecular weight difference between BMPRII and BMPRIIsf, 150kDa +30 kDa, and 60kDa + 30 kDa, respectively (Figure .38.B).

sBMP2 treatment revealed that REF52 expressing opto-BMPRsf was able to induce two times higher levels of SMAD1/5/8 phosphorylation than the overexpression of opto-CAAX, opto-ALK3, or opto-BMPRII (Figure .38.C). The previous studies also showed that enhanced expression of BMPRIIsf at the plasma membrane led to increased activation of SMAD protein-mediated signaling [START_REF] Amsalem | Differential regulation of translation and endocytosis of alternatively spliced forms of the type II bone morphogenetic protein (BMP) receptor[END_REF], suggesting that the C-terminal region may be modulating the activity of BMPRII. Additionally, when seeded at the same conditions with previous cell quantification, not only higher levels of pSMAD 1/5/8 and BMPR expression were observed, but also an increase of the housekeeping protein GAPDH A) after blue light stimulation, while BMPRIIsf was able to be recruited to focal adhesion sites, BM PRII was not able to be recruited. scale bar 15 μm.B) The intensity of BMPR at focal adhesion was followed. First, a basal state was recorded, followed by constant pulses (10s) of blue light for 4 minutes, then one pulse every 1 minute reveals the tight spatial and temporal control of the optogenetic system. The curve showed the mean of 10 cells and SD.

Finally, all these results demonstrate the spatial segregation of BMPRII from β3 integrin even after sBMP2 o blue light treatment. The cytoplasmatic tail's deletion presented a similar spatial organization mainly excluded from adhesion sites; however, the opto system reveals the ability to be highly colocalized with β3 integrin. This recruitment induced by the blue light stimulation correlates with the slight increase of colocalization between the BMPRIIsf and β3 integrin after sBMP2 treatment, suggesting that BMPR cytoplasmatic tail is responsible for the exclusion acting as a docking site for several proteins outside focal adhesion. However, it remains to investigate what domain is responsible. Additionally, While the overexpression of BMPRIIsf can increase SMAD 1/5 phosphorylation, the proximity BMPRIIsf and β3 integrin does not optimize this BMP2 signal, as it is the case for the proximity ALK3/β3 (Data not shown). Further investigation of the ability of the BMPRIIsf to interact with integrin β3 and its signaling consequences requires more investigation.

IV--145 -CHAPTER IV.

CONCLUSION, DISCUSSION AND PERSPECTIVES.

The receptors' spatial organization and trafficking is a critical step in signal transduction.

Similar to TGF-β receptors, BMPR are regulated by complex formation among numerous coreceptors [START_REF] Sánchez-Duffhues | Bone morphogenetic protein signaling in bone homeostasis[END_REF]. Receptors do not function as individual signaling units but tend to associate in multimolecular complexes that can accommodate hundreds of molecules in high order clusters to form signaling platforms [START_REF] Bethani | Spatial organization of transmembrane receptor signalling[END_REF].

Very little is known about the control of the spatial arrangement of BMPR subunits and whether this spatial arrangement affects cellular responses to receptor signaling. Many studies have reported the role of BMP2 in regulating cell cytoskeleton dynamics, and the association of BMPR with integrins through biochemical approaches [START_REF] Lai | αvβ integrins play an essential role in BMP-2 induction of osteoblast differentiation[END_REF] and biomaterial design [START_REF] Crouzier | Presentation of BMP-2 from a soft biopolymeric film unveils its activity on cell adhesion and migration[END_REF][START_REF] Fourel | β3 integrin-mediated spreading induced by matrix-bound BMP-2 controls Smad signaling in a stiffness-independent manner[END_REF]. The host lab has previously reported the cooperation between β3 integrin and BMPR to couple cell migration and BMP2

signaling (Laure Fourel, Valat, Faurobert, Guillot, Reynard, et al., 2016). Particularly, the role of β3 integrin was identified as crucial in the control of the downstream BMP2 signaling.

However, information about the events between BMPR subunits and integrins occurring at

BMP signaling relies on BMPR localization to specific adhesive microdomains

Adherent cells use their surface receptors to sense and respond to physical and chemical cues presented in the ECM, regulating different processes, like adhesion, migration, proliferation, and differentiation [START_REF] Giancotti | Integrin signaling[END_REF]. At the ventral side, multimolecular assemblies, recruited upon clustering of cell surface receptors, build up discrete adhesion structures at the interface with a surface, i.e., a 2D substrate [START_REF] Geiger | Molecular complexity and dynamics of cell-matrix adhesions[END_REF]. Receptor clustering is a purely mechanistic function that enhances cellular contacts' strength to the extracellular matrix and contributes to increased cellular sensitivity to external stimuli or can even determine signal specificity.

Integrins are transmembrane glycoproteins and the primary receptors for cell adhesion to the extracellular matrix mainly through adhesive structures named focal adhesions located at the end of the stress fibers and attached to the actomyosin stress fibers. The focal adhesion are mainly composed of α5β1 and αvβ3 integrins [START_REF] Ballestrem | Marching at the front and dragging behind: Differential αVβ3-integrin turnover regulates focal adhesion behavior[END_REF]. The spatial compartmentalization of β1 and β3 integrins in adhesion sites is associated with distinct mechanical properties due to different time of residence in response to tension through local mechanical coupling between integrin tension and actin protrusion [START_REF] De Mets | Cellular tension encodes local Src-dependent differential β1 and β3 integrin mobility[END_REF][START_REF] Milloud | αvβ3 integrins negatively regulate cellular forces by phosphorylation of its distal NPXY site[END_REF][START_REF] Roca-Cusachs | Integrin-dependent force transmission to the extracellular matrix by α-actinin triggers adhesion maturation[END_REF][START_REF] Rossier | Integrins β 1 and β 3 exhibit distinct dynamic nanoscale organizations inside focal adhesions[END_REF][START_REF] Schiller | β 1 -And α v -class integrins cooperate to regulate myosin II during rigidity sensing of fibronectin-based microenvironments[END_REF]. Integrins do not just mediate the adhesive interactions between cells and the ECM. Indeed, a plethora of transmembrane proteins, such as growth factor receptors, have been identified as co-receptors, being not just distributed homogeneously in the plasma membrane, but instead probably confined to discrete regions [START_REF] Ashe | Modulation of BMP signalling by integrins[END_REF][START_REF] Zhang | Cross-talk between TGFbeta/SMAD and integrin signaling pathways in regulating hypertrophy of mesenchymal stem cell chondrogenesis under deferral dynamic compression[END_REF]Zhou, Lee, Lee, Wei, Lim, Lin, Chien, Chiu, et al., 2013). The cooperation of integrins with their co-receptors coordinates specific cell contractility and drives specific mechanosensitive pathways to regulate cell fate, impacting the extracellular matrix (ECM) remodeling and maintaining tissue integrity. In response to different micro-environmental stimuli, cells build diverse contractile networks of actin filaments to orchestrate cell shape under the control of molecular switches or nodes connected to adhesion sites. IV--149 -

Segregation of ALK3 in focal adhesion upon BMP2 treatment depends on β3 integrin engagement.

Thanks to the design of fluorescent and optogenetics tools, this present study demonstrates a novel spatiotemporal regulation of the BMPR such that ALK3 and β3 integrin localized at sites of adhesions upon BMP2 stimulation. We have shown that this segregation requires both BMP2 and integrin engagement to the extracellular matrix.

Upon BMP2 treatment, ALK3 is no longer distributed homogeneously in the plasma membrane but instead confined to discrete regions, namely focal adhesions, whereas BMPRII subunits show no tendency to cluster. This type of segregation is not restricted to BMP receptors and reminds the differential distribution of TGFβRI and TGFβRII in focal adhesion upon TGF-β treatment [START_REF] Rys | Discrete spatial organization of TGF?? receptors couples receptor multimerization and signaling to cellular tension[END_REF]. In that case, TGFβRI colocalizes with α2β1 and αvβ6 integrin in focal adhesions while TGFβRII is excluded, forming a ring at focal adhesion's periphery. Unlike BMPRs, cellular tension is crucial for driving TGFβRs segregation.

This segregation in respect to focal adhesion was shown to be correlated with a decrease of both heterodimerizations of TGFβRs and SMAD3 phosphorylation, highlighting its role in regulating signal transduction. This suggests that the boundaries between TGFβRI and TGFβRII are crucial for the integration of mechanical and biochemical cues. As such, the physical barrier between ALK3, retained inside focal adhesions, and BMPRII, strictly excluded at the periphery, prevents their physical proximity and thus their multimerization.

Additionally, the activated form of ALK3 (ALKCa) is sufficient to promote adhesion sites' formation and its recruitment in adhesion sites independently of BMP2. This also supports the notion that type-I receptors may elicit divergent biological responses by signaling to distinct and specific downstream pathways. In conclusion, the microdomain clustering of ALK3 with β3 integrin is highly regulated by both BMP2 stimulation and β3 integrin engagement, indicating that the spatial control of ALK3 might have specific functional implications for mechano-transduction, cell adhesion processes or BMP signaling.

The control of ALK3/β3 integrin proximity through the optogenetic approach mimics BMP2 stimulation by targeting ALK3, but not BMPRII, into β3 integrin containing focal adhesions offering the opportunity to induce rapid and local signal activation. This approach allowed us to apply or withdraw the light signal to induce the proximity between ALK3 and β3 integrin. BMPR under optical control (Opto-BMPR/β3) provides a powerful approach to actuate and understand ALK3/β3 integrin clustering.

BMPRs might act as adhesive receptors to control adhesion and actin cytoskeleton.

Many studies have reported the role of BMP2 in regulating cell cytoskeleton dynamics and a close association of BMPRs with integrins through biochemical approaches [START_REF] Lai | αvβ integrins play an essential role in BMP-2 induction of osteoblast differentiation[END_REF] and biomaterial design [START_REF] Crouzier | Presentation of BMP-2 from a soft biopolymeric film unveils its activity on cell adhesion and migration[END_REF][START_REF] Fourel | β3 integrin-mediated spreading induced by matrix-bound BMP-2 controls Smad signaling in a stiffness-independent manner[END_REF].

The host lab has previously shown that BMP2, as a biochemical cue, can induce cytoskeletal reorganization that precedes the differentiation onset (osteogenic switch in the case of C2C12 muscle cells). It was previously reported the cooperation between β3 integrin and BMPR to couple cell migration and BMP2 signaling (Laure Fourel, Valat, Faurobert, Guillot, Reynard, et al., 2016). BMP2 presentation by soft biomaterials induces the generation of stress fibers and filopodia formation under the control of Cdc42 (Laure Fourel, Valat, Faurobert, Guillot, Reynard, et al., 2016). Our current results demonstrate the enrichment of ALK3 clusters in focal adhesions, which indicates a specific contribution of ALK3 in actin dynamics. In turn, ALK3 partitioning in adhesion sites is correlated with integrin-mediated responses, including adhesive and migratory processes suggesting a decoupling between ALK3 and BMPRII function in cell adhesive processes. By the optogenetic approach, the proximity between ALK3 and β3 integrin increases cell spreading behavior. We have also observed a change in cell shape upon induction of the proximity between β3 integrin and BMPRII upon light treatment. We assumed that these modifications of cell shape might be due to the formation of β3 integrin nanoclusters to form adhesive patches [START_REF] Saltel | New PI(4,5)P2-and membrane proximal integrin-binding motifs in the talin head control β3-integrin clustering[END_REF][START_REF] Fourel | β3 integrin-mediated spreading induced by matrix-bound BMP-2 controls Smad signaling in a stiffness-independent manner[END_REF]. However, these patches do not seem to be associated with cell migration as the deletion of BMPRII has no impact on cell migration as a contrast to ALK3 deletion.

Additionally, the deletion of ALK3 significantly decreases the focal adhesion area and the number of focal adhesions. Furthermore, this deletion decreases the migration on soft matrix-bound with BMP2. Only ALK3 and not BMPRII was shown to be involved in adhesion and migration. As such, the localization of ALK3 in β3 integrin containing focal adhesions might also indicate a specific contribution of ALK3 in actin dynamics [START_REF] Saeed | Mouse Embryonic Fibroblasts (MEF) Exhibit a Similar but not Identical Phenotype to Bone Marrow Stromal Stem Cells (BMSC)[END_REF].

More importantly, our study identifies ALK3 as an adhesive-like receptor, participating in fibronectin and BMP2 ligand's coincidence detection. This result adds another piece of IV--151 -evidence for the role of β3 integrin and ALK3 coupling in a defined architecture or dynamics of actin cables for SMAD signaling induced by BMP2

Different research groups also reported a regulatory interaction between LIMK1 and BMPRII, where BMPRII promotes LIMK phosphorylation (Foletta, Lim, Soosairajah, Kelly, Stanley, Shannon, He, Das, Massagué, et al., 2003;[START_REF] Lee-Hoeflich | Activation of LIMK1 by binding to the BMP receptor , BMPRII , regulates BMP-dependent[END_REF]. However, the molecular events leading to LIMK1 being phosphorylated and thereby activated in different contexts have remained unresolved. In the neuronal model, Yamauchi et al. [START_REF] Yamauchi | Type Ib BMP receptors mediate the rate of commissural axon extension through inhibition of cofilin activity[END_REF] have shown an inverse correlation between the activity of ALK6 and both the activity of cofilin and the rate of axon outgrowth. When signaling through ALK6 was increased, the activity of cofilin decreased, and the growth rate of commissural axons slowed down. Their studies have also implied that the active to inactive cofilin ratio is a crucial part of the mechanism that controls axon growth speed. In line with this, some unpublished results from our group shown that the LIMK activity is also under the control of β3 integrin as judged by p-Cofilin's decrease (LIMK substrate) after deletion of β3 integrin.

It is known that actin cable turnover depends on cofilin function in vivo [START_REF] Okada | Aip1 and Cofilin Promote Rapid Turnover of Yeast Actin Patches and Cables: A Coordinated Mechanism for Severing and Capping Filaments □ D[END_REF]. The involvement of β3 integrin and LIMK in the control of the phosphorylation of cofilin might support the need for temporal control of actin turnover, the necessity of continuous repression of actin depolymerization, or its participation in the formation of actin-cofilin rods necessary to initiate or support the osteogenic program [START_REF] Dopie | Active maintenance of nuclear actin by importin 9 supports transcription[END_REF][START_REF] Munsie | Cofilin nuclear-cytoplasmic shuttling affects cofilin-actin rod formation during stress[END_REF][START_REF] Sen | Intranuclear Actin Regulates Osteogenesis[END_REF].

For the future, it would be essential to investigate whether ALK3 might also participate in actin dynamics by modulating the activity of the LIMK1-cofilin pathway to control the rate at which the actin cytoskeleton polymerizes. It is known that cell contractility is not involved in SMAD translocation since ROCK inhibition does not affect SMAD nuclear translocation in soft conditions [START_REF] Fourel | β3 integrin-mediated spreading induced by matrix-bound BMP-2 controls Smad signaling in a stiffness-independent manner[END_REF].

Indeed, tensional homeostasis through the modulation of G-actin levels has to be precisely controlled by β3 integrin. The spreading induced by a stiff matrix-like plastic is not sufficient to maintain the phosphorylation of SMAD in conditions where β3 integrin is deleted. The control of β3 integrin/ALK3 proximity by optogenetics opens new avenues for investigating the precise involvement of β3 integrin/ALK3 coupling in cofilin-dependent actin dynamics and SMAD nuclear transport.

BMPRs segregation may balance canonical/non-canonical signaling

The Optogenetics approach allowed us to optimize cell-fate decisions generally driven by BMP2 stimulation. This result confirms the importance of BMP2 stimulation in the induction of SMAD1/5/8 signaling. The proximity by BMPR and β3 integrin at the cytoplasmic domain level was insufficient to induce the SMAD1/5/8 signaling activation, likely indicating the requirement of extracellular domains of integrins and BMP receptors in BMP2 signaling.

Indeed, it is known that fibronectin can bind BMP2/4 through the FN 12-14 domain, which is localized very close to the FN 7-11 domain known to interact with integrins, providing functional proximity between integrins and BMP receptors [START_REF] Migliorini | Learning from BMPs and their biophysical extracellular matrix microenvironment for biomaterial design[END_REF].

However, our data show that the temporal dynamics between β3 integrin and ALK3 are also essential to optimize the BMP2 downstream signaling pathway.

Finally, the control of ALK3/ β3 integrin proximity allows coupling between adhesive functions and differentiation processes. As ALK3, BMPRII, and β3 integrin are the three protagonists essential for SMAD1/5/8 phosphorylation and its nuclear translocation (Laure Fourel, Valat, Faurobert, Guillot, Reynard, et al., 2016), our results suggest a positive retrocontrol onto SMAD1/5/8 signaling resulting from the β3 integrin/ALK3 proximity.

A single receptor's activation is translated into different cellular responses depending on a differential organization and activation in space and time. Receptor-mediated signaling is a highly complex, evolutionary conserved mechanism that allows communication between receptors in microdomains at the cell membrane is crucial for binding to the ligand and activation of signaling cascades [START_REF] Guzman | SMAD versus Non-SMAD Signaling Is Determined by Lateral Mobility of Bone Morphogenetic Protein (BMP) Receptors[END_REF][START_REF] Zuo | Specific activation of mitogen-activated protein kinase by transforming growth factor-β receptors in lipid rafts is required for epithelial cell plasticity[END_REF]. Additionally, the different receptor oligomerization modes dictate the downstream signaling pathways and the resulting transcriptional responses [START_REF] Bengtason | PP2A regulates BMP signalling by interacting with BMP receptor complexes and by dephosphorylating both the C-terminus and the linker region of Smad1[END_REF][START_REF] Schwappacher | Novel crosstalk to BMP signalling: cGMP-dependent kinase I modulates BMP receptor and Smad activity[END_REF].

It has been reported that many of the molecular components that regulate cell-ECM adhesion are associated with cholesterol and sphingolipid-enriched detergent-resistant membrane microdomains (DRM), which are also enriched in the acidic phospholipid IV--153 -PI(4,5)P2 [START_REF] Márquez | Is DRM lipid composition relevant in cellextracellular matrix adhesion structures?[END_REF][START_REF] Pande | The role of membrane lipids in regulation of integrin functions[END_REF]. The ability of ALK3 to mainly associate with detergent-resistant membrane (DRM) fractions, likely through its ability to be S-acylated [START_REF] Wegleiter | Palmitoylation of BMPR1a regulates neural stem cell fate[END_REF], might explain the segregation between ALK3 and BMPRII. Whether the ALK3/ β3 integrin receptor clusters form through a physical process known as 'phase separation' to build molecular platforms to drive key cellular functions remains to be explored [START_REF] Banjade | Phase transitions of multivalent proteins can promote clustering of membrane receptors[END_REF][START_REF] Chong | Liquid-liquid phase separation in cellular signaling systems[END_REF][START_REF] Li | Phase transitions in the assembly of multivalent signalling proteins[END_REF].

We have shown that the confined population of ALK3 at focal adhesions has lower mobility than the freely diffusive receptor population far from adhesion sites, identifying different populations of BMP receptors with distinct behavior of lateral mobility. A significant portion of ALK3 is in more stable complexes with β3 integrin at the cell surface, dependently on BMP2 ligand stimuli or induce by blue light stimulation, as revealed by the Fluorescent Recovery After Photobleaching technique. Finally, our data reveal that BMP receptor activation requires a distinct lateral movement of type-I and type-II receptors within the plasma membrane through ALK3 trapping within focal adhesions upon BMP2 treatment.

This distinct lateral mobility has and impacts on BMP2 induced-SMAD and -non-SMAD signaling [START_REF] Guzman | SMAD versus Non-SMAD Signaling Is Determined by Lateral Mobility of Bone Morphogenetic Protein (BMP) Receptors[END_REF] In line with this, our preliminary results have shown that controlling the proximity between β3 integrin and ALK3 by optogenetics led to a decrease of ALK3 pTyr at the cytoplasmic site.

This modification has been described as essential for ALK3 heterodimerization with BMPRII [START_REF] Kua | C-Abl promotes osteoblast expansion by differentially regulating canonical and non-canonical BMP pathways and p16 INK4a expression[END_REF]. our results suggest that maintaining ALK3 close to β3 integrin would impair ALK3/BMPRII heterodimerization. In turn, this would favor signaling pathwayspecific of ALK3 by preferentially activating non-SMAD (like pERK/p16-dependent senescence) pathway rather than SMAD-dependent differentiation as already described in osteoblast model [START_REF] Kua | C-Abl promotes osteoblast expansion by differentially regulating canonical and non-canonical BMP pathways and p16 INK4a expression[END_REF]. Our results show that the proximity between BMPR and β3 integrin controlled by optogenetic tools might influence the balance between SMAD and ERK signaling pathways.

Furthermore, ALK3 palmitoylation has been described as necessary for receptor mobility and signaling regulation. ALK3 can be S-acylated on two different sites near its transmembrane domain, each modification is linked to different effects on the downstream signaling. One affects canonical signaling, while the other seems to regulate non-canonical signaling [START_REF] Wegleiter | Palmitoylation of BMPR1a regulates neural stem cell fate[END_REF]. In light of these findings, it would be interesting to analyze whether these post-translational modifications are linked to ALK3/BMPRII segregation and if the proximity with β3 integrin regulates ALK3 modifications. With that in mind, we started with the design of a vector containing opto-ALK3 mutated at these palmitoylation sites. Our results showed that OptoALK3 is able to be tyrosine phosphorylated by the presence of sBMP2 but whether ALK3/β3 proximity affects BMPR heterodimerization and whether the time of ALK3/β3 proximity participates in switching from SMAD to non-SMAD pathway warrants further investigation.

On the other hand, it was assumed that the type-II receptors have the unique task to activate the type-I kinase by phosphorylating threonine/serine residues (Rotraud [START_REF] Wieser | GS domain mutations that constitutively activate T R-I, the downstream signaling component in the TGF-,B receptor complex[END_REF]. This assumption is no longer assumed from the fact that BMPRII is the only receptor from the entire TGFβ family receptor containing a unique ~550 amino acid long cytoplasmic extension downstream of the intracellular kinase domain (C. Dupas, P. Houdy, 2007), even though no function has been assigned to this domain, and it is not required for BMP signaling through the SMAD pathway [START_REF] Nishihara | Functional heterogeneity of bone morphogenetic protein receptor-II mutants found in patients with primary pulmonary hypertension[END_REF][START_REF] Wieser | Signaling activity of transforming growth factor beta type II receptors lacking specific domains in the cytoplasmic region[END_REF] Further investigation must be carried out to understand the fine-tuned balance between non-SMAD and SMAD signaling pathways. The optogenetic approach, combined with the distinct mutation in the receptors, could decipher key physiological and morphological aspects of cell-fate decisions generally driven by BMP2 stimulation.

Impact of BMPR segregation on physio-pathological situations

Our data show that the location of ALK3 relative to other receptors, such as β3 integrin, is a key aspect of BMP2 signaling in cell adhesion and migration processes. The control of cell spreading, cell migration and cell metastasis might be considered as a new non-SMAD pathway downstream BMP2 signaling. BMPR segregation should be further studied to have a comprehensive view of ALK3 in physio-pathological situations. This is predominantly pertinent to breast cancer, which frequently metastasizes to bone, and in which studies have revealed aberrations of both BMP and BMPR type-I expression and signaling, which correlate clinically with breast cancer progression, Although integrins' primary function is

Résumé

Comprendre comment les cellules intègrent les signaux biochimiques et physiques de la matrice extracellulaire pour spécifier une différenciation cellulaire au travers de la signalisation des récepteurs adhésifs est un défi majeur en biologie cellulaire. Le BMP2 est un facteur de croissance crucial pour le développement osseux normal chez les vertébrés en induisant une différenciation ostéoblastique des cellules mésenchymateuses pluripotentes. Le laboratoire hôte a précédemment montré que les récepteurs BMP (BMPR) et l'intégrine β3 collaborent pour contrôler la signalisation SMAD et l'homéostasie tensionale (Fourel, 2016), couplant ainsi l'adhésion cellulaire et le devenir de la cellule deux aspects fondamentaux de la biologie du développement et de la médecine régénérative. Il reste à déterminer si la dynamique entre l'intégrine et les BMPR sont contrôlée dans l'espace et dans le temps pour guider les processus intracellulaires.

Mon travail pendant ce doctorat a consisté à étudier la distribution des récepteurs BMP2 (ALK3 et BMPRII) et de l'intégrine β3 à la surface cellulaire et l'impact de leur proximité physique sur le comportement cellulaire en combinant l'imagerie, la technique de FRAP (Fluorescence Recovery After Photobleaching), les substrats biomimétiques et une approche optogénétique. Dans une perspective plus large, ce couplage entre les intégrines et les voies de signalisation BMP pourrait être exploité dans les processus de développement et de médecine régénérative, où le recrutement cellulaire est une condition préalable à la différenciation cellulaire pour former un organe spécifique ou réparer un tissu endommagé. L'identification de la signalisation fournira de nouvelles stratégies thérapeutiques pour optimiser la réparation et la régénération osseuse.

Mot clés : BMP, BMP receptor, integrin, adhésion, spatio-temporal

Figure 1

 1 Figure 1 Transforming Growth Factor β superfamily phylogenetic tree. TGF-β family is composed of 4 subfamilies of a total of 30 structurally related growth factors

Figure 2 .

 2 Figure 2. Microcomputed tomography reconstructions of bone repair induced by Material load BMP2 A) sized calvarial defect healing from above (above) and in cross-section (below) using acellular collagen sponge (ACS) (Durham et al., 2018) B) an extensive bone defect repair by an alginate-based hybrid system[START_REF] Kolambkar | Spatiotemporal delivery of bone morphogenetic protein enhances functional repair of segmental bone defects[END_REF]) C) Critical size volumetric bone femoral defect repair after implantation using a polyelectrolyte film coating over an implant loaded with BMP2[START_REF] Bouyer | Surface delivery of tunable doses of BMP-2 from an adaptable polymeric scaffold induces volumetric bone regeneration[END_REF]

Figure 3 .

 3 Figure 3. BMP2 tetrameric complex receptor. BMP2 binds to two type-I receptors and two type-II receptors, while Type-II receptors have a longer carboxy-terminal, the Type-I contain a domain rich in glycine and serine (GS domain). The cytoplasmic domain of BMP receptors can interact with distinct proteins that contribute to receptor regulation and fine -tuning or receptor signaling progression. aa: amino acids.

Figure 5 .

 5 Figure 5.Live COS7 cells transfected with Myc-tagged ALK3 (R1AWT) and ALK6 (R1B-Is1 WT) BMP receptors.chicken-anti-myc and Alexa 546-labeled donkey-anti-chicken antibodies for detection of membrane-external receptors (red). Following fixation, samples were permeabiliz ed and labeled with mouse-anti-myc and Alexa 488-labeled goat-anti-mouse antibodies for the detection of internal receptors (green). Micrographs depict single confocal planes of representative cells. Bar 10 µm. Adapted from[START_REF] Hirschhorn | Differential molecular regulation of processing and membrane expression of Type-I BMP receptors: implications for signaling[END_REF]

Figure 6 .

 6 Figure 6. BMP receptors type-I and II have different lateral mobilities. A) Single-particle tracking traces and analysis of Human influenza hemagglutinin-tagged ALK3, ALK6, or BMPRII.Representative movement trajectories of individual receptor molecules demonstrate the confinement of receptor mobility to a particular area. B) after 5-min, BMP2 ALK3, ALK6, and BMPRII show confined movement. Adapted from[START_REF] Guzman | SMAD versus Non-SMAD Signaling Is Determined by Lateral Mobility of Bone Morphogenetic Protein (BMP) Receptors[END_REF].

Figure 7

 7 Figure 7 Localization of Labeled-BMP Receptor. A) Confocal microscopy (right) and STED microscopy (left) images of ALK6 (in green) and BMPRII (in red) of human umbilical vein endothelial cells (HUVECs).In the absence of BMP2, the two different receptors rarely co-localized (upper white arrowhead), and BMPRII did not cluster (lower arrowhead). When cells were exposed to BMP2, BMPRII was associated with the largest ALK6 assemblies. This different behavior could not be appreciated with confocal microscopy. Image adapted[START_REF] Medda | Challenges in imaging cell surface receptor clusters[END_REF].

Figure 8 .

 8 Figure8. SMAD BMP-induce signaling. A) Canonical SMAD signaling pathway involves the phosphorylation of SMAD1/5/8, which translocate into the nucleus in a complex with the Co-SMAD (SMAD4) and regulate the expression of BMP responsive genes Adapted from(Gámez, Rodriguezcarballo, & Ventura, 2013)

Figure 9 .

 9 Figure 9. BMP receptor complexes modulate several parallel signaling cascades (non-SMAD), as described for MAPK (i.e., p38, ERK, and JNK) and NF-κB, Jak/Stat, LIMK, PI3K, mTORC, and RhoGTPases. Adapted from[START_REF] Gámez | BMP signaling in telencephalic neural cell specification and maturation[END_REF]

  BMP and other proteins that tightly regulate signal transduction networks play essential roles in the body. Mouse knock-out models of various BMP signaling pathway components result in embryonic lethality or significant defects, highlighting the essential functions of BMPs. Also, the disruption of these signaling could lead to severe diseases. Recently widely reviewed by (Gomez-Puerto, Iyengar, García de Vinuesa, ten Dijke, & Sanchez-Duffhues, 2019; R. N.Wang et al., 2014), this section is dedicated to the prominent human pathologies associated with BMP signaling dysregulation with a particular focus on BMP2, ALK3, and BMPRII.

Figure 10 .

 10 Figure 10.Representative members of the BMP signaling pathway have been demonstrated to cause or be associated with human diseases. Adapted from (R. N.Wang et al., 2014)

Figure 11 .

 11 Figure11. BMP ligands, receptors, co-receptors, and interacting receptors. BMP signal transduction involves some ligands, type-I and type-II serine/threonine kinase receptors, and coreceptors, which regulate the activation of intracellular mediators upon interaction with extracellular stimuli. The BMP signaling interplays with cellular cascades initiated by a variety of membrane receptors, resulting in a cross-talk that provides fine-tuning. LRP, lipoprotein receptorrelated protein; Ror, receptor tyrosine kinase -like orphan receptor; VEGFR, vascular endothelial growth factor receptor; RGM. Adapted from (Sánchez-duffhues,[START_REF] Sánchez-Duffhues | Bone morphogenetic protein signaling in bone homeostasis[END_REF]

Figure 12 .

 12 Figure12. An illustration of the components of the extracellular matrix . A matrix of fiber proteins and proteoglycans with specific physical characteristics as the fiber strength and pore size. These proteins also influence the biochemical cues and the ability to act as a growth factor reservoir. Adapted from[START_REF] Migliorini | Learning from BMPs and their biophysical extracellular matrix microenvironment for biomaterial design[END_REF]

Figure 13

 13 Figure 13 Growth factor binding to ECM proteins A) measured by enzyme-linked immuneabsorbent assay, adapted from (Martino, 2014). B) The complex domain structures of ECM proteins. Representative ECM proteins are illustrating multiple, independently folded domains fibronectin (FN). Encoded by a single gene but alternatively spliced at three regions [blue circles and box and V (variable) segment]. Binding sites for other matrix proteins are marked. RGD ( *) and LDV (Leu-Asp-Val) (#). FN also binds the proangiogenic growth factors VEGF and HGF . Fibrillin-1.Fibrillins include EGF-like domains, found in many ECM proteins and TGFb-binding, denoted by T) and hybrid (H) domains, specific to fibrillins and LTBPs. LTBP-1. Four-gene family with structures related to fibrillins. Known binding sites for TGF -b/LAP latent complex (SLC, blue), fibrillin, and F N are marked. RGD (*) sequences in fibrillins and LTBPs may bind integrins. Thrombospondin-1 (TSP-1). TSPs contain TSP1 repeats (also found in other ECM proteins), EGF -like repeats, and a VWC domain, known in other proteins to bind BMPs. TSP3 repeats (purple) and C -terminal domains are unique to TSPs and bind multiple Ca++ ions. The RGD (*) sequence is known to bind to integrins. Adapted from[START_REF] Hynes | The Extracellular Matrix: Not Just Pretty Fibrils[END_REF].

  and respond to them by generating reciprocal forces proportional to those perceived, which control the assembly, maturation, and disassembly of these adhesion sites. The direct binding of BMPs to the ECM proteins suggests that ECM might spatially integrate different distinct signals in the microenvironment, facilitating integrin and BMP receptor cross-talk. These interactions are crucial in controlling various cell behaviors, including proliferation, survival, and differentiation.

Figure 15 .

 15 Figure 15. The integrin receptor Family. Integrins are αβ heterodimers. The Figure, despite the mammalian subunits and their αβ association. *: alternative spliced cytoplasmatic domain. (Hyneset al., 2002)

Figure 17 .

 17 Figure 17. Illustration of integrin structures. a)Focal complexes, b)Focal adhesion, c)fibrillar adhesion. (Emily Joo & Yamada, 2015)The focal complexes are small punctate structures (0.25 μm 2 for the focal complexes[START_REF] Nobes | Rho, Rac, and Cdc42 GTPases regulate the assembly of multimolecular focal complexes associated with actin stress fibers, lamellipodia, and filopodia[END_REF] and 0.1 μm 2 for the initial adhesions[START_REF] Choi | Actin and α-actinin orchestrate the assembly and maturation of nascent adhesions in a myosin II motor-independent manner[END_REF] composed mainly of integrins α5β1 and αvβ3[START_REF] Ballestrem | Marching at the front and dragging behind: Differential αVβ3-integrin turnover regulates focal adhesion behavior[END_REF]. Their localization is at the interface between the lamellipodia and the lamella, with a lifespan of a few minutes. Their formation requires

Figure 19 .

 19 Figure 19.illustation of signal transduction cascade. Growth factor binding to its BMP receptor, and integrin to the ECM. A possible synergy is optimized by the proximity of the ECM domain interacting with integrin and BMP ligand. Further investigations are needed. a. BMP signaling in a biomechanical context With the discovery of multiple BMP receptor-interacting proteins involved in actin organization and adhesive structures, it has been suggested that BMP signaling is coupled to mechano-sensitive proteins or cellular structures (da Silva Madaleno, Jatzlau, & Knaus, 2020). BMP receptors

  Figure 20. Cell polarization and migration in a BMP context. Cell polarity is characterized by the temporal and spatial distribution of proteins leading to migration. BMP -induced chemotaxis initiates cytoskeletal rearrangements via activation of Rho-GTPases (upper left). Proteins contributing to migration are arranged from the leading tip to the rear end (A-D).Figure from[START_REF] Sieber | Recent advances in BMP receptor signaling[END_REF] adapted from[START_REF] Ridley | Cell Migration: Integrating Signals from Front to Back[END_REF].

Figure 21 A

 21 Figure 21 A) Co-localization (indicated by arrows) of ALK6( BMPRIb) and BMPRII (red) with αvβ5 integrins (green) detected by confocal microscopy. Images adapted from (Lai & Cheng, 2005). B) Two-color confocal image of vinculin and ALK6. (A) Immunolabeled vinculin in a HUVEC cell is depicted before the addition of BMP2. B) Shows vinculin (red) and ALK6 (green) at the cell periphery before the addition of BMP2, whereas in (C), vinculin and ALK6 I are shown 10 min after the addition of BMP2.[START_REF] Medda | Challenges in imaging cell surface receptor clusters[END_REF].

Figure 22 .

 22 Figure 22. BMP-mediated cell signaling is influenced by its presentation via a biomaterial and by the engagement of BMP receptors and co-receptors. (A) Schematic view of β3 integrin-GSK3β and BMP2-SMAD cooperation. BMP receptors and β3 integrin signaling converge to control both focal adhesion dynamics and SMAD signaling to couple cell migration and fate commitment. Reproduced from Fourel et al. (LaureFourel, Valat, Faurobert, Guillot, Reynard, et al., 2016) , (B) Phosphorylation and translocation of SMAD1/5/8 to the nucleus depends on cell spreading: immunofluorescence images of C2C12 myoblasts spread on small (500 μm2) and large (1500 μm2 ) micropatterns of FN/BMP2 and FN without or with BMP2 in solution[START_REF] Fitzpatrick | Micromotifs de BMP-2 et de fibronectine sur niche de cellules souches in vitro[END_REF]. Figure from[START_REF] Migliorini | Learning from BMPs and their biophysical extracellular matrix microenvironment for biomaterial design[END_REF]

Figure 23 .

 23 Figure23. BMP-responsive transcription in the Drosophila embryo is not simply a readout of the BMP concentration but also requires integrin-mediated enhancement of BMP signal transduction.[START_REF] Sawala | Peak BMP Responses in the Drosophila Embryo Are Dependent on the Activation of Integrin Signaling[END_REF]

Figure 24 .

 24 Figure24. Several standard parameters impact the biological responses such as the chemical composition and mechanical properties of the extracellular matrix,, the dose and gradients of growth factors, and the spatial distribution of the cell receptors that will impact on the temporal signal response.[START_REF] Migliorini | Learning from BMPs and their biophysical extracellular matrix microenvironment for biomaterial design[END_REF].

  a spatially restricted expression pattern. A recent study elaborated on a model to propose different mechanisms to explain how components work together to create and maintain the BMP signaling gradient in the embryo. This model relies on a restricted diffusion BMP antagonist acting as a sink that drives BMP signaling through a dorsal gradient. This data suggests that the spatio-temporal patterns of chordin and BMPs gene expression are dominant drivers of shape in 3D (L. Li, Wang, Mullins, & Umulis, 2020). Another mathematical model demonstrated the importance of BMPR localization in human embryonic stem cells in vitro and mouse embryos in vivo to shape morphogen signaling during embryogenesis. It shows that the receptor localization facilitates a robust BMP signaling gradient in the mouse embryo. The BMP receptor signaling during embryogenesis depends on the restricted, basolateral localization of BMPR since the BMP receptor localization at the apical side (mislocalization) results in ectopic BMP signaling in the mouse epiblast in vivo (Z. Zhang, Zwick, Loew, Grimley, & Ramanathan, 2019).

Figure 25 .

 25 Figure25. BMP gradients in the early zebrafish embryo gradient of BMP signaling along the DV axis of a 60% epiboly embryo seen from the animal pole, as visualized by immunofluorescence for p-SMAD1/5 (red), shown as a maximum projection. Nuclei are stained with DAP I (blue).[START_REF] Economou | Temporal dynamics in the formation and interpretation of Nodal and BMP morphogen gradients[END_REF]

  . Additionally, cytonemes are selective for ligands or receptors. They could confer differential signaling in distinct and different subcellular signaling centers. The anchorage of the growth factor to the ECM or a surface still conveys signaling by prolonged activation of receptors and differential phosphorylation[START_REF] Pohl | Surface immobilization of bone morphogenetic protein 2 via a self-assembled monolayer formation induces cell differentiation[END_REF]. Hence, ligand-receptor interaction at the cell membrane might be sufficient to obtain a sustained signaling response. It remains to elucidate if a mechanical component from the matrix causes deformation of the membrane and affects internalization signaling and If co-recruitment of other adhesion receptors such as integrins might occur in these cases, BMP2 molecules cannot be internalized.Temporal regulation of BMP signalingThe intensity, duration, and fluctuation of BMP signaling pathway stimulation are essential in determining the output from the downstream transduction network. Improving our understanding of how these differences in the input are transformed into different outputs will be essential to unravel cell fate decisions' mechanisms. However, signaling by morphogen gradients involves questions of timing.The temporal action of the BMP signaling gradient in DV patterning has been studied in several contexts. BMP activity has been visualized in Xenopus by following the spatial distribution of phosphorylated SMAD1/5, with high levels ventrally and low levels dorsally throughout gastrulation[START_REF] Faure | Endogenous patterns of TGFbeta superfamily signaling during early Xenopus development[END_REF][START_REF] Schohl | Beta-catenin, MAPK and Smad signaling during early Xenopus development[END_REF], delineating a potentially long window of time available for cells to respond to BMP signaling. Studies in Xenopus and zebrafish using inducible BMP components show that BMP inhibition initiated during midblastula stages disrupts DV axial patterning (K.[START_REF] Marom | Temporal analysis of the early BMP functions identifies distinct anti-organizer and mesoderm patterning phases[END_REF][START_REF] Pyati | Transgenic zebrafish reveal stage-specific roles for Bmp signaling in ventral and posterior mesoderm development[END_REF][START_REF] Wawersik | Somatic control of germline sexual development is mediated by the JAK/STAT pathway[END_REF]

  Diverse engineering and molecular tools have been developed to visualize and control BMP activity in vitro and in vivo allowing a better understanding of the complex ways in which BMP activity is regulated spatially. The regulation of BMP activity by the presentation of the ligand by biomimetic ECM, and at the level of BMP receptor, by modulation of co-receptors through biomaterials and optogenetics.

Furthermore

  , technological advances in microfluidics and bioactive biomaterial-based approaches can improve control of signal inputs, but they are limited by their complexity and low throughput[START_REF] Defail | Controlled release of bioactive TGF-β1 from microspheres embedded within biodegradable hydrogels[END_REF][START_REF] Tabata | Multiscale microenvironmental perturbation of pluripotent stem cell fate and self-organization[END_REF]. Optogenetics is also used better to understand cellular signaling transduction[START_REF] Mühlhäuser | Optogenetics -Bringing light into the darkness of mammalian signal transduction[END_REF].

Figure 26 .

 26 Figure 26. Manipulation of mammalian signaling at several stages of signal transduction. A)Comparing cell lines expressing genetic mutants, chemical inducers, and optogenetics in terms of spatiotemporal resolution. B) Optogenetic tools for manipulating mammalian signaling at several stages of signal transduction Overview of optogenetic systems applications at the membrane, the cytoplasm, and the nucleus. POI: protein of interest. Adapted from[START_REF] Mühlhäuser | Optogenetics -Bringing light into the darkness of mammalian signal transduction[END_REF] a.Optogenetic modulesLight is a ubiquitous, crucial environmental stimulus for organisms across all kingdoms of life. Thus, a plethora of different photoreceptor proteins has evolved. They vary in their biochemical andphysical properties (e.g., peak absorbance). However, all of them display a specific response when excited with light of specific wavelengths (UV, blue, green, and red light-responsive system) in the form of a reversible conformational change. Most display altering binding affinities for interacting proteins, depending on their current conformation. Frequently employed photoreceptors include flavoprotein blue light response sensors and red light-sensitive phytochromes[START_REF] Conrad | Photochemistry of flavoprotein light sensors[END_REF][START_REF] Levskaya | Spatiotemporal control of cell signalling using a light-switchable protein interaction[END_REF]. The light-oxygen-voltage (LOV) flavoprotein

Figure 27

 27 Figure 27 Optogenetic modules use to study transmembrane receptors A) Schematic representation of wild-type (WT) FGFR1 and optoFGFR1(N.[START_REF] Kim | Spatiotemporal control of fibroblast growth factor receptor signals by blue light[END_REF]. B) Fibroblast growth factor receptor 1 (consists of the extracellular ligand-binding domain (LBD), single-span transmembrane domain (TMD), and intracellular domain (ICD). In mFGFR1-LOV domain chimeras, only the ICD is retained to render the protein insensitive to endogenous ligand[START_REF] Grusch | Spatiotemporally precise activation of engineered receptor tyrosine kinases by light[END_REF] C) TGF-beta receptors (Y.[START_REF] Li | Spatiotemporal Control of TGF-β Signaling with Light[END_REF], D) BMP receptors[START_REF] Humphreys | Optogenetic control of the Bone Morphogenetic Protein signalling pathway through engineered blue[END_REF].

Figure 28 .

 28 Figure 28. Improved light inducer dimer system (iLID) consists of a Light Oxygen Voltage domain (LOV2) with a ssRA peptide and fused to a fluorescent protein. The second part of the system consists of the sspB peptide fused to a tagRFP. Adapted from[START_REF] Guntas | Engineering an improved light-induced dimer (iLID) for controlling the localization and activity of signaling proteins[END_REF]

  I.A) will describe cell culture and molecular biology approaches to obtain a stable Optogenetic cell line, including information about the different cell types, the optoplasmids design, cell transfection and cell infection protocols and SiRNA treatment. A second part (I.B) will describe a wide range of microscopy techniques, including the fluorescent activated cell sorting (FACs), epifluorescence imaging and live-cell imaging techniques, confocal Spinning Disk, video-microscopy and total internal reflection fluorescence microscopy (TIRFM), fluorescent recovery after photobleaching (FRAP), and the set-up of Opto-activation LED box. The third part (I.C) will compile the methods used to evaluate the cellular behavior and signaling, including immunofluorescence, immunoblotting, qPCR, cell migration and cell spreading assays, an overview of the image analysis, and the description of the statistic used. II-65

Figure 29 .

 29 Figure 29. Overview steps of the establishment of the Opto_cell line. The first stage (Steps 1-4) involved the lentivirus-producer cell line transfection, the lentiviral particles encoding the transgenes are collected and concentrated . Alternative to this step, a platform produced virus could also be used. In the second stage (Steps 5-8), targeted cells are infected, stably transduced, and FACS enriched and expanded. In the third stage, optional but desirable, second FACs enriched and intensity normalization is performed to finally expanded the cell lines to keep for maintenance.

•

  3.5 μg pC57GPBEB GagPol MLV packaging plasmid • 1.5 μg pSUSVSVG envelope plasmid Top up the transfection mix with OptiMEM to a total volume of 0.3 mL. Vortex the tube gently for 10 sec, then centrifuge briefly at low speed to collect the liquid at the bottom. Prepare 5μl lipofectamine 2000 in a sterile tube with 0.3 mL of optiMEM and gently mix the suspension. When both solutions are ready, add the Lipofectamine mix to the plasmid DNA transfection mix drop by drop and leave it incubating in the flow cabinet for 15-20 min.

  filter and passes through a dichroic mirror to be sent to the objective. The objective serves as a condenser of the excitation light. The objective is then used to collect the fluorescence light that is emitted by the sample. The emitted light passes through an emission filter before being detected by the camera (or observed by the eye using the ocular ports). In this work, we used an inverted epifluorescence microscope equipped with 10X, 20X air, and 63X immersion oil objective (iMIC inverted microscope (FEI)). The epifluorescence stabilized fiber source X-Cite 120, 100W HBO lamp with the FluoArc attenuation system. The software of acquisition is MetaMorph (Universal Imaging).

Figure 30 .

 30 Figure 30. iMIC 2.0 inverse motorized (Dynamic): Video-Fast epifluorescence microscope.

Figure 31 .

 31 Figure 31. OBFR (Optogenetique Bifocal Rapide): Fast confocal microscope (Spinning Disk) coupled to a TIRF / FRAP photoactivation module for the study and pho tomanipulation on living cells a. Confocal SD The scanning of the sample in X, Y direction by the laser allows an image of the focal plane to be taken (Figure 31.B). This scanning is obtained via motorized mirrors that are disposed on the optical path of the laser. For the scanning in the Z direction, the objective is mounted on a piezoelectric mirror. It is thus possible to obtain images of different focal planes and to reconstitute a 3D structure. Confocal images were acquired to observe internal structures in the opto cells. The image obtained in confocal has a good signal to noise ratio.

Fluorescence

  photobleaching was followed over time. The fluorescence of the total cell was also tracked over time as a control.

Figure 32 Fluorescent

 32 Figure 32 Fluorescent Recovery After Photobleaching. A) Example of a region of interest in a FRAP experiment. B) typical graph experiment. Fluorescence recovery reaches a plateau; the difference of intensity in comparison with the pre -bleach intensity of the ROI represents the immobile fraction of a molecule. Half the time of recovery (t1/2) corresponds to the time required to reach half of the plateau. FRAP analysis was performed on OA offline analysis software using the offline FRAP tool/option. A single exponential model applied to the fluorescence intensity recorded on the bleach region A*(1-exp[-t*tau_frap]) after normalization on the total cell intensity and the camera background. The characteristic recovery time (τ) of BMPR-RFP from the fit of the experimental data was the mean of at least 20 cells.

  were then incubated with Protein G sepharose beads (GE17-0618-01 SIGMA Protein G Sepharose® 4 Fast Flow 5ml) for 4h. Beads were washed three times in lysis buffer before resuspension in 50 µL Laemmli 4X supplemented with β-mercaptoethanol. The mix was centrifuged at 3000 RPM for 2 minutes at 4°C. The pellet was suspended in 50μL of 20X Laemmli before protein denaturation at 95°C for 5 min. Results were assessed by immunoblotting with an anti-P-TYR monoclonal antibody (4G10, Millipore ref sigma and anti-RFP polyclonal (AB233, Evrogen) antibodies.

Furthermore, the

 the Mouse Mammary Epithelial (EpH4) is a standard breast epithelial model to study morphogenesis in physiological and pathological situations. As both BMP signaling and αvβ3 integrin over-expression have been linked to breast cancer and metastasis, EpH4 cells might represent an excellent model to decipher the potential role of β3 integrin/ BMPRs cooperation in the destruction of epithelial organization in the tumoral context. (P

Figure 33 .

 33 Figure 33. Mesenchymal and epithelial cell lines respond to BMP2 through the SMAD pathway. A) The cell lines were starved for 2h in DMEM medium, then cultured with 0,5% serum and stimulated with no or 600ng/mL BMP2 for 4h B), and signal intensity was quantified. C) pSMAD nuclear translocation was observed by immunofluorescence after 4h stimulation upon 600ng/mL of soluble BMP2, D) quantification of nuclear pSMAD 1/5/8. E) Reportoir of BMPR and integrin expression by qPCR.

Fluorescent

  Activated Cell sorting (FACS) presents a fast and convenient method to enrich transduced cell populations. Fluorescent marker proteins allow enrichment by FACS. When performing multi-color FACS, fluorescence compensation is crucial because of the overlap of fluorochrome emission spectra. Non-transduced cells serve as a negative control sample. These control samples serve to set the photomultiplier tube (PMT) detector voltages properly and to calculate the fluorescence compensation. It is an excellent idea to cryopreserve the polyclonal stable cell line after establishment or enrichment in liquid nitrogen and the presence of a cryoprotective agent such as dimethyl sulfoxide (DMSO). Compensation color using wild type cells is done (Figure 34.A). Cell lines as REF52 can reach a higher percentage of the positive cells since round 1 (Figure 34.B). III--91 -

Figure 34 .

 34 Figure 34. A) Representative FACs enrichment of opto_MEFsv40 cell lines for single (BMPR) and double infection (BMPR+β3 integrin). Compensation color using wild type was performed. A similar window of expression was selected for all receptors. B) Example of infection round population enrichment per cell line C: C2C12, M: MEFsv40, R: REF52. E: EpH4

Figure 35 .

 35 Figure 35. Comparison of Promoter pGK and CMV in C2C12, MEFsv40, and REF52 cells, under the same infection conditions. While proteins as CAAX and β3integrin are highly expressed under both promoters, pGK resulted in a higher and more stable BMP receptor expression C: C2C12, M: MEfsv40, R: REF52 c.Opto_BMP receptor expression and localizationIn order to analyze the quality and functionality of target gene expression, immunoblotting, and membrane localization by Total Internal Reflection Microscopy (TIRFM) was performed.

Figure 36 .

 36 Figure 36. Opto BMP receptor expression and localization A) Immunoblot. of REF52 cell expressing the double construction B) TIRFM microscopy onto REF52 shows the clustering ofβ3 integrin in focal adhesion sites. In contrast, BMP receptors were localized homogenous on the cell membrane. Scale bar: 10 μm.

Figure 37 .

 37 Figure 37. Immunoprecipitation of REF52 cells A) expressing opto CAAX/3, BMPRII/3, and ALK3/3 integrin. Stimulated by sBMP2, B), the ratio of intensity between the phosphorylated signal and the Receptor expression reveals the phosphorylation after sBMP2 stimulation. C) ALK3 was stimulated with sBMP2 , and light illumination, before Immunoprecipitation (IP) followed by immunoblotting (IB)against p-Tyr or tag-RFP (upper panel).WB quantification reveals that the proximity induced between ALK3 and β3 integrin inhibits the ALK3 phosphorylation-induced by sBMP2

  III--141 -was routinely detected, suggesting a possible increase in proliferation rates after the overexpression of BMPRIIsf (Figure.38.B and C). Using Total Internal Reflection Microscopy, we can observe the expression of β3 integrin localized mainly in focal adhesion and the expression of Opto-BMPRII and Opto-BMPRIIsf at the cell membrane. Both Opto-BMPRIIs presented shadow regions where BMPRII or BMPRIIsf is absent in areas corresponding to adhesion sites. This characteristic is readily observed when significant focal adhesions are presented. The profile plot of both intensity channels at a focal adhesion site confirms the exclusion (Figure. 38.D).

Figure 38

 38 Figure 38 Transduction of BMPRII and BMPRIIsf on mesenchymal and epithelial cell lines A) Percentage of population enrichment after lentiviral infection, BMPRIIsf present higher levels of expression B) immunoblotting reveals the distinct molecular weight between BMPRII and BMPRIIsf C)SMAD 1/5/8 phosphorylation upon sBMP2 treatment (600ng/mL). 4h D) Total internal Reflection Microscopy reveals the spatial organization of Opto-BMPRII and Opto-BMPRIIsf. Using TIRFM to observe the REF52 cell expressing BMRII/β3 and BMPRIIsf/β3, it was observed with or without sBMP2 treatment (Figure.39.A), the localization of BMPRII remains excluded from focal adhesion sites as reflected by their profile plots. Manders Coefficient was used to quantify the colocalization index between BMPRII or BMPRIIsf and

Figure 39 .

 39 Figure 39. Spatial segregation of BMPRII and BMPRIIsf from β3 integrin-A) By Total Internal Reflection Microscopy, the exclusion of BMPRII and BMPRIIsf by β3 integrin is revealed by the profile plot independently of sBMP2 presence. B) The colocalization index reveals a small significative difference after sBMP2 stimulation. BMPRII remains and increases its exclusion while BMPRIIsf slightly increases colocalization.

Figure 40 .

 40 Figure40.REF52 co-expressing opto-BMPRII/β3 or opto-BMPRIIsf/β3 A) after blue light stimulation, while BMPRIIsf was able to be recruited to focal adhesion sites, BM PRII was not able to be recruited. scale bar 15 μm.B) The intensity of BMPR at focal adhesion was followed. First, a basal state was recorded, followed by constant pulses (10s) of blue light for 4 minutes, then one pulse every 1 minute reveals the tight spatial and temporal control of the optogenetic system. The curve showed the mean of 10 cells and SD.

  the cell membrane such as localization and clustering is missing due to a lack of tools to monitor BMPR subunit dynamics.During this thesis work, optogenetic cell lines were established to monitor and control BMPR organization at the cell membrane. We explored whether the physical proximity between integrin and BMP receptor is essential for driving cell adhesion dynamics and cell fate. Thanks to the design of fluorescent/optogenetic BMPR tools, this present study demonstrates a novel spatial segregation of ALK3 and BMPRII (Figure41) with a discrete organization of ALK3 at the level of adhesion sites upon BMP2 treatment and β3 integrin engagement, suggesting that BMPR localization and consequently BMPR signaling is spatially regulated.

Figure 41 .

 41 Figure 41. ß3 integrin integrates BMP2 signaling through BMP receptors segregation. While Opto-ALK3 can colocalize with β3 integrin by sBMP2 or blue light stimulation, Opto BMPRII remains mainly excluded and never enriched at focal adhesion sites. The deletion of ALK3 affects spreading and migration on matrixbound soft film and reduce integrin expression and focal adhesion quantity and area in cell spread on the glass. The proximity induced by bBMP2 or blue light on soft film can induced or optimized cell spreading. The same proximity optimized the SMAD 1/5/8 phosphorylation upon sBMP2 presentation.

  Photoactivated Localization Microscopy (PALM). In this matter, we already designed BMPR labeled to mEOS2 to be used in single-particle PALM (SptPALM). PALM approach has shown integrins' ability to undergo fast free-diffusion outside focal adhesion. They evolve by cycles of slow free-diffusion and immobilization within focal adhesion[START_REF] Rossier | Integrins β 1 and β 3 exhibit distinct dynamic nanoscale organizations inside focal adhesions[END_REF] 1, and 3 Integrin exhibit distinct dynamic nanoscale organizations within focal adhesion. This technique should help determine the single-molecule localization and give information about ALK3 dynamics inside the focal adhesion nanodomains.

  cells and their environment. Efficiency, high precision and specificity are required to transmit only relevant signals to the appropriate target cells. To ensure the transmission of even weak signals, the receptor and their associated complexes can be modified by dynamic and reversible post-translational modifications, which promote amplification and diversification of signaling in the cell (Lissanu Deribe, Pawson, & Dikic, 2010). In the case of BMPR type-I, the confinement of the lateral mobility and accumulation of signaling

  our preliminary results using the optogenetic system showed that the BMPRIIsf could colocalize with β3 integrin The localization of BMPRIIsf in focal adhesion correlates with the activation of the SMAD1/5/8 pathway at elevated intensities. Altogether, these data might indicate that the alternative splicing of BMPRII may be an essential regulator of the balance of activation of canonical versus non-canonical signals by BMPs.

  Mes résultats ont montré que les BMPR sont discrètement organisés dans des domaines membranaires distincts dévoilant une ségrégation entre ALK3 et BMPRII à la surface cellulaire. J'ai montré que ALK3 est enrichi au niveau des adhérences focales contenant l'intégrine β3 après stimulation BMP2. Contrairement à ALK3, le BMPRII est souvent exclu de ces sites d'adhésion. De plus, le recrutement de ALK3 au niveau des sites d'adhésion requiert l'engagement des intégrines 3 sur leur matrice extracellulaire. Par ailleurs, la forme activée d'ALK3 est recrutée dans les sites d'adhésion indépendamment de la stimulation par le BMP2. Ces résultats suggèrent que le recrutement de ALK3 au niveau des sites d'adhésion dépend de l'activité de l'intégrine et de l'activation d'ALK3. Les expériences de FRAP supportent le modèle de ségrégation des BMPR montrant non seulement des mobilités latérales différentes entre les BMPR mais un aussi un piégeage de ALK3 dans les sites adhésifs résultant de la stimulation BMP2.Pour contrôler de manière réversible l'interaction entre le BMPR et l'intégrine β3 une approche optogénétique basée sur le système Venus iLID (improved Light Inducer Dimer) (Guntas, 2016) a été développée. Ces approches imitent la stimulation BMP2 en ciblant ALK3, mais pas BMPRII, dans les sites d'adhésion contenant l'intégrine β3. Mes résultats montrent que cette proximité entre l'intégrine β3 et ALK3 induite par la lumière est suffisante pour induire l'étalement cellulaire sur un substrat mou. De plus, le ciblage d'ALK3 dans les sites adhésifs est responsable de la migration cellulaire induite par le BMP2 présenté par la matrice.Tous ces résultats montrent que la coopération entre l'intégrine 3 et ALK3 converge déjà à la surface cellulaire de par la proximité physique lors de la stimulation BMP2. Ces données démontrent que le regroupement de ALK3 sous forme de microdomaines avec les intégrines β3 est fortement régulé à la fois par la stimulation BMP2 et l'engagement des intégrines β3 à la matrice extracellulaire. Ces résultats suggérent que le contrôle spatial d'ALK3 pourrait avoir des implications fonctionnelles spécifiques pour la mécanotransduction ou la signalisation.

Table 2

 2

	) was carried out using CLC

subsequent Gibson assembly (NEB) before transforming competent bacteria and screening by site restriction digestion. The DNA was isolated from the culture using the Nucleo bond Xtra plasmid DNA purification kit (Machery-Nagel). The constructs were verified by sequencing using the target sequences from (Table

1

) (Light run, Eurofins, Genomics) Table 1. Primer sequence for sequence verification (F): forward, (R): reverse. ECD: extracellular domain.

Table 2 .

 2 Primers for vector construction. Wild type ALK3, BMPRII, and β3 integrin were fused to the Opto_VenusiLID system. Several mutations were also designed. R: reverse primer, F: forward.

	Receptor insert

Table 3 .Titration value of platform produced-virus

 3

	virus	Titer (UI/mL)	C2C12 in 50 μl GM	Others in 100 μl GM
	740_β3 integrin	1.50E+08
	1382_ALK3_pgk	1.00E+05	100 μl	50 μl
	1288_BMPRII_pgk 00E-	100 μl	50 μl
	1289_BMPrSF_pgk 8.40E+06	100 μl	50 μl
	1372_CAAX_pgk	1.30E+07	10 μl	10 μl

Table 4 .

 4 Antibodies used for immunofluorescence. Details are listed.

	Primary Antibodies
	Target	Reference	Provider	Animal	Final Dilution
	pSMAD1/5/9	13820	Cell signaling	rabbit	1/250
	pTyrosine	4G10	Millipore	Rabbit
	β3 chain	M030-0	EMFRET	Rat	1/100
	Secondary Antibodies
	Target	Reference	Provider	Animal	Final Dilution
	647-Alexa fluor		ThermoFisherSc.	1/1000
	Others
	Product	Reference	Provider		Final Dilution
	Phalloidin	A22287	Thermo	Fischer	1:1000
	Alexa Fluor™ 647		Scientific
	Phalloidin

Table 5 Antibodies used for immunoblotting

 5

	Primary Antibodies
	Antibody	kDA	Reference	Animal	Final Dilution
	Phospho-SMAD1/5 (Ser463/465)	60	Cell signaling-9516	Rabbit mAb 1:2000
	β3 integrin (LucA.5)	90	EMFRET M030-0		1:500
	Tag-RFP	30	Evrogen AB233		1:1000
	GAPDH	30	Genetex-Gt239	mouse	1 :1000
	others
	HRP-rabbit
	HRP-Rat

Table 6 . Primer Sequence used in qPCR for the target genes

 6

	Target gene	ID	Animal	Sequence 1	Sequence 2
	3 integrin	16416	mouse	GATTGCCCTTCGACTACGGC	GTCCACGGGGTAATCCTCCA
	BMPRII	12168	mouse	GATCCTGGGCCATCAAAGCC	TGCCATCTTGTGTTGACTCAC CT
	ALK3	12166	mouse	AGGATTCACCGAAAGCCCAG	ATCACGGTTGTAACGACCCC
	BMPRII	659	human	TGGCAGCAGTATACAGATAGGT G	ATGGTTGTAGCAGTGCCTCC
	ALK3	657	human

Table 7 . Statistical significance value in GraphPad formatting

 7

	P-value	Wording	Summary
	< 0.0001	Extremely significant	****
	0.0001 to 0.001	Extremely significant	***
	0.001 to 0.01	Very significant	**
	0.01 to 0.05	Significant	*
	≥ 0.05	Not significant	ns

during mouse embryogenesis. Mechanisms ofDevelopment, 100(2),[327][328][329][330]

Acknowledgments I would like to express my gratitude to my thesis directors Corinne Albiges-Rizo and Catherine Picart, for entrusting me with this challenging but awesome project. Your constant encouragement and guidance are invaluable. I would like to thank the jury members: Caroline Hill, Ellen Van-Obberghen Schilling, Dominique Bourgeois, and Mathieu Coppey, to give me the honor to revise my work. I am sure that your recommendations and comments will enrich it. Your leadership in your fields is genuinely III--99 -III--101 -III--103 -III--105 -III--107 -III--109 -III--111 -III--113 -III--115 -III--117 -III--119 -III--121 -III--123 -III--125 -III--127 -III--129 -III--131 -III--133 -III--135 -

thought to be the coordination of cell-matrix communication to influence intracellular signaling cascade, β3 integrin in cooperation with BMPR type-I might be involved in cancer biology and chemotherapy resistance. Indeed, in breast cancer, BMP2/4 have both protumor and antitumor functions [START_REF] Balboni | Δnp63α-mediated activation of bone morphogenetic protein signaling governs stem cell activity and plasticity in normal and malignant mammary epithelial cells[END_REF][START_REF] Clement | Bone morphogenetic protein 2 (BMP-2) induces in vitro invasion and in vivo hormone independent growth of breast carcinoma cells[END_REF]. Even though BMP2 interacts with the BMPR complex, there is a contradictory role between BMPR type-I and BMPR type-II in cancer development and suppression by promoting seemingly contradictory cell processes. ALK3/BMPRII segregation might partly explain the duality between ALK3 and BMPRII. The attenuation of BMPRII signaling in mammary carcinoma cells enhances metastasis, whereas BMPR type-I deletion impairs mammary tumor formation and metastasis. Transcription studies on primary tissue of breast cancer unveiled overexpression of BMP2 and ALK6, uncovering a role for the BMP pathway in the origin of luminal breast cancer and drug resistance [START_REF] Zylbersztejn | The BMP pathway: A unique tool to decode the origin and progression of leukemia[END_REF]. Finally, even though multiple BMP signaling abnormalities have been reported in many cancers, the molecular mechanisms controlling BMP duality remain to be identified.

In this matter, we have established an OptoBMPR epithelial model of breast cells (EpH4). These cells could be interesting to explore how ALK3/ BMPRII segregation and the cooperation between ALK3 and β3 integrin might involve tumor progression and metastasis, to explain the contradictory role of BMP signaling. It would allow to explore whether this segregation can drive specific signaling depending on the cellular context including the ECM's biophysical properties. Another way to tackle the question will be to determine if the BMP2 presentation and the matrix's mechanical cues play a role in BMPR segregation and subsequent signaling. Our preliminary results have shown the ability of EpH4, to respond to BMP2 in terms of SMAD1/5/8 phosphorylation and nuclear translocation, actin reorganization and cell differentiation. Considering the importance of ECM stiffness in cancer, it will be useful to analyze their behavior in BMP2 bound matrix with controlled rigidities and their organization in both 2D and 3D environments.

On the other hand, our preliminary results using the optogenetic system showed that the opto-BMPRIIsf could colocalize with β3 integrin. The physiological importance of the Cterminal region of BMPRII has been emphasized by identifying C-terminal truncation mutations of BMPRII gene in familial primary pulmonary hypertension (PPH) patients (M. J. [START_REF] Kim | Clinical significance linked to functional defects in bone morphogenetic protein type 2 receptor, BMPR2[END_REF]. However, the exact molecular function of the C-terminal region of BMPRII remains elusive [START_REF] Deng | Familial Primary Pulmonary Hypertension (Gene PPH1) Is Caused by Mutations in the Bone Morphogenetic Protein Receptor-II Gene[END_REF]Foletta, Lim, Soosairajah, Kelly, Stanley, Shannon, He, Das, Massagué, et al., 2003;[START_REF] Lane | Heterozygous germline mutations in BMPR2, encoding a TGF-β receptor, cause familial primary pulmonary hypertension[END_REF]. This C-terminal deletion or mutation in the cytoplasmatic tail might disrupt or favor interaction with other receptor subunits or binding proteins [START_REF] Chan | A Novel Regulatory Mechanism of the Bone Morphogenetic Protein (BMP) Signaling Pathway Involving the Carboxyl-Terminal Tail Domain of BMP Type II Receptor[END_REF]. Indeed, growing evidence has shown that the BMPRII gene mutations are also implicated in other diseases, which might be due to the loss of BMPRII functions in specific combination with the ligands and their type-I binding partners.

The use of biomaterials of various rigidity and transcriptomics analyses will allow us to assess how mechanical forces impact the regulation of genes involved in the cell's early and late response to BMP2. Optogenetics needs to be combined with cell imaging to monitor cell migration and breast cell phenotypes and analyze the inter-conversion between epithelial and mesenchymal states and finally discriminate the role between ALK3 and BMPRII in coordination with 3 integrin. Furthermore, actin dynamics and the BMP pathway have been recently shown to drive apical extrusion to fulfill a developmental fate [START_REF] Clement | Bone morphogenetic protein 2 (BMP-2) induces in vitro invasion and in vivo hormone independent growth of breast carcinoma cells[END_REF]. It would be interesting to explore whether the control of ALK3/3 integrin proximity contributes to the mode of cell extrusion from a cellular monolayer or acini, recreated in 2D culture, thanks to biomaterials. Our hypothesis is that ALK3/3 integrin proximity may favor either luminal extrusion or basal extrusion. The analysis of cell populations composed of both wild type cells and opto cells needs to be investigated to mimic tumoral heterogeneity i

Annexes Curriculum Vitae

Amaris Guevara García

Born in México, April 12 th , 1990 Tel: +33 7 83399302 Email: guevara.amaris@gmail.com LinkedIn: /Amaris Guevara

Education

• Ph.D. in Chemistry and Life Sciences, Cell biology.

LABEL Industrial Research and Innovation

Abstract

How cells integrate biochemical and physical input from the extracellular matrix to achieve specific cell differentiation through adhesive receptor signaling is a big challenge in cell biology. BMP2 is a crucial molecule for normal bone development in vertebrates and induces osteoblastic differentiation of pluripotent mesenchymal cells, likely through coordination with matrix elements such as fibronectin. The host lab has previously shown that BMP receptors and β3 integrin work together to control SMAD signaling and tensional homeostasis (Fourel, 2016), thereby coupling cell adhesion and fate commitment -two fundamental aspects of developmental biology and regenerative medicine. However, still require to identify whether their spatial arrangement affects the cellular response to receptor signaling. Whether dynamics between integrin and BMP receptors is controlled in space and time to guide pivotal intracellular processes remains to be elucidated.

My work during this Ph.D. consisted of investigating the distribution of BMP2 receptors (ALK3 and BMPRII) and β3 integrin at the cell surface and the impact of their physical proximity on cell behavior by combining live imaging, fluorescent recovery after photobleaching (FRAP), biomimetic substrates, and an optogenetic approach.

My results showed that BMP receptors (BMPR) are discretely organized into segregated spatial domains at the cell surface. I have shown that ALK3 is enriched at β3-integrin focal adhesions after BMP2 stimulation. Unlike ALK3, BMPRII is often excluded from these sites and never enriched. Furthermore, the use of specific matrix protein reveals that the recruitment of ALK3 in focal adhesion is visible when β3 integrin is prone to form focal adhesions upon its engagement. The activated form of ALK3 is able to be localized in adhesion sites independently on BMP2. These results suggested that ALK3 organization at adhesion sites is dependent on integrin activity and ALK3 activation by BMP2 stimulation. For a reversible and light-controlled interaction between BMPR and β3 integrin in space and time, an optogenetic approach is based on the Venus-iLID (improved Light Inducer Dimer) system (Guntas, 2016) has been developed. These approaches mimic BMP2 stimulation by targeting ALK3, but not BMPRII, into β3 integrin containing focal adhesions. Additionally, the optogenetic approach allows us to apply or withdraw the light signal to induce the proximity between ALK3 and β3 integrin offering the opportunity to induce rapid and local signal activation. My results show that this light-induced proximity of ALK3 and β3 integrin is sufficient to induce cell spreading on a soft substrate independently on matrix-bound BMP2. Moreover, the targeting of ALK3 into β3 integrin containing focal adhesion upon BMP2 treatment is responsible for cell migration induced by matrix-bound BMP2.

These results show that the cooperation between β3 integrin and ALK3 converges already at the cell surface upon BMP2 stimulation. These data demonstrate that the microdomain clustering of ALK3 with β3 integrin is highly regulated by both BMP2 stimulation and β3 integrin engagement, indicating that the spatial control of ALK3 might have specific functional implications for mechanotransduction or BMP signaling.

From a broader perspective, this coupling between integrins and BMP signaling pathways is of great relevance in developmental processes and regenerative medicine, where cell recruitment is a prerequisite to cell differentiation to form a specific organ or repair damaged tissue. Identification of signaling will provide new therapeutic strategies for optimizing bone repair and regeneration.

Keyword: BMP, BMP receptor, integrin, adhesion, spatio-temporal