Marc Bitoun
email: m.bitoun@institut-myologie.org

Keywords: Dynamin 2, Focal adhesion, endocytosis, focal adhesion disassembly

Dynamin 2 (DNM2) is involved in endocytosis and intracellular membrane trafficking through its function in vesicle formation from distinct membrane compartments. During the last decade, several studies pointed out an important role of DNM2-dependent trafficking in turnover of focal adhesions which represent a physical link between the extracellular matrix and the intracellular actin cytoskeleton, and a platform for several signaling pathways. Here, we review the involvement of DNM2 in structural and functional aspects of the focal adhesion sites. Mutations in the DNM2 gene cause two hereditary neuromuscular disorders; dominant centronuclear myopathy and Charcot-Marie-Tooth peripheral neuropathy. Potential impairment of focal adhesions as a pathophysiological hypothesis in DNM2-related human diseases is discussed.

Dynamin 2 (DNM2) is a ubiquitously expressed large GTPase, involved in various membrane trafficking events. At the plasma membrane, DNM2 is involved in the formation and release of vesicles for the clathrin-mediated [1] and clathrin-independent endocytosis [START_REF] Gold | Dynamin 2 is required for phagocytosis in macrophages[END_REF]3]. DNM2 also participates in the formation of transport vesicles from the endosomal system and the Golgi apparatus [START_REF] Van Dam | Dynamin-dependent transferrin receptor recycling by endosome-derived clathrin-coated vesicles[END_REF]5]. Moreover, several studies have demonstrated that DNM2 directly interacts with the microtubule [1,[START_REF] Tanabe | Dynamic instability of microtubules requires dynamin 2 and is impaired in a Charcot-Marie-Tooth mutant[END_REF] and actin cytoskeleton [START_REF] Mcniven | Regulated interactions between dynamin and the actin-binding protein cortactin modulate cell shape[END_REF][START_REF] Mooren | Dynamin2 GTPase and cortactin remodel actin filaments[END_REF]. DNM2 is a 98 kDa protein composed of a N-terminal catalytic GTPase domain, a middle domain involved in DNM2 self-assembly, a pleckstrin homology domain (PH) which interacts with membrane phosphatidylinositol 4,5-bisphosphate (PI4,5P2) and therefore is involved in the targeting of dynamin to membranes [START_REF] Dong | Expression and purification of dynamin II domains and initial studies on structure and function[END_REF], a GTPase effector domain regulating the GTPase activity, and a C-terminal proline rich domain (PRD) containing multiple Src homology 3 (SH3) binding motifs engaged in protein-protein interactions [START_REF] Durieux | Dynamin 2 and Human diseases[END_REF]. Heterozygous mutations in the DNM2 gene cause rare forms of the Charcot-Marie-Tooth peripheral neuropathy (CMT) [START_REF] Zuchner | Mutations in the pleckstrin homology domain of dynamin 2 cause dominant intermediate Charcot-Marie-Tooth disease[END_REF] and autosomal dominant centronuclear myopathy (CNM) [START_REF] Bitoun | Mutations in dynamin 2 cause dominant Centronuclear Myopathy[END_REF]. There is no treatment for DNM2related diseases and the pathophysiological mechanisms are still largely unknown.

The focal adhesions (FAs) represent the major site of cell attachment to the extracellular matrix (ECM) where a structural and functional link between the ECM and the intracellular actin cytoskeleton occurs. FAs are macromolecular complexes composed of transmembrane receptors including integrins, structural proteins including vinculin, talin, and -actinin, and signalling proteins such as Focal adhesion kinase (FAK) [START_REF] Kanchanawong | Nanoscale architecture of integrin-based cell adhesions[END_REF]. FAs act in cell adhesion and migration and dynamics of FA assembly-disassembly is a crucial process to achieve these functions. The molecular mechanisms leading to FA assembly have been well characterized.

In contrast, FA disassembly mechanisms are not fully understood. During the last decade, a role of DNM2 in FAs disassembly emerged. The purpose of this review is to summarize the studies showing the role of DNM2 as a regulator of FAs dynamics. In addition, we discuss the hypothesis of FA dysfunction in the DNM2-related human diseases.

DNM2-mediated endocytosis at FAs

The first link between dynamin and components of the FA was reported in 1995 in human monocyte primary cultures stimulated by the macrophage colony-stimulating factor (M-CSF) [START_REF] Kharbanda | Stimulation of human monocytes with macrophage colony-stimulating factor induces a Grb2-mediated association of the focal adhesion kinase pp125FAK and dynamin[END_REF]. Ligand-activated M-CSF receptor recruits a protein complex including dynamin, FAK and the adaptor protein Grb2 (growth factor receptor-bound protein-2). Interestingly, dynamin is phosphorylated and co-immunoprecipitated with activated FAK in M-CSF-treated monocytes suggesting a regulation of dynamin function by FAK that may contribute to receptor internalization. FAK is a non receptor tyrosine kinase involved in the signaling pathway of several agonist-activated membrane receptors including integrins and is mainly expressed at the FA. In addition, FAK is a key element of the regulation of FA disassembly [START_REF] Webb | FAK-Src signalling through paxillin, ERK and MLCK regulates adhesion disassembly[END_REF] and actin polymerization by phosphorylation of downstream targets [START_REF] Schober | Focal adhesion kinase modulates tension signaling to control actin and focal adhesion dynamics[END_REF]. This first demonstration of DNM2 association with FAK had strongly suggested a role for DNM2 at the adhesion sites.

In cultured cells, microtubule depolymerisation by nocodazole results in FA disassembly.

After washing the drug, microtubules grow, contact the FAs and lead to FA disassembly [START_REF] Kaverina | Microtubule targeting of substrate contacts promotes their relaxation and dissociation[END_REF].

Using this microtubule-induced FA disassembly assay, Ezratty and collaborators showed that disassembly is independent of Rho-and Rac-GTPase activities but dependent on FAKinduced recruitment of DNM2 at the FA via its PRD domain [START_REF] Ezratty | Microtubule-induced focal adhesion disassembly is mediated by dynamin and focal adhesion kinase[END_REF]. Before FA disassembly, DNM2 colocalizes with FA and co-immunoprecipitation studies suggest that DNM2 interacts with the phosphorylated, i.e. activated, form of FAK (p-FAK). By the development of a phospho-antibody against DNM2's tyrosine 231, Wang and collaborators further defined the DNM2 regulation occurring during FA turnover [START_REF] Wang | A direct interaction between the large GTPase dynamin-2 and FAK regulates focal adhesion dynamics in response to active Src[END_REF]. The authors showed that activation of Src is responsible for the phosphorylation of DNM2 at the FA where the phosphorylated DNM2 (pDNM2) interacts directly with FAK. Consequently, the formation of a pDNM2-FAK-Src complex promotes 1-integrin endocytosis and FA disassembly [START_REF] Wang | A direct interaction between the large GTPase dynamin-2 and FAK regulates focal adhesion dynamics in response to active Src[END_REF]. Such Srcmediated phosphorylation of DNM2 was already shown to induce endocytosis [START_REF] Cao | SRC-mediated phosphorylation of dynamin and cortactin regulates the "constitutive" endocytosis of transferrin[END_REF]. Inhibition of the DNM2 activity, using dominant negative DNM2-K44E mutant and DNM2-siRNA, inhibits FA disassembly [START_REF] Ezratty | Microtubule-induced focal adhesion disassembly is mediated by dynamin and focal adhesion kinase[END_REF] and impairs cell migration [START_REF] Ezratty | Microtubule-induced focal adhesion disassembly is mediated by dynamin and focal adhesion kinase[END_REF][START_REF] Ezratty | Clathrin mediates integrin endocytosis for focal adhesion disassembly in migrating cells[END_REF][START_REF] Chao | Focal adhesion disassembly requires clathrin-dependent endocytosis of integrins[END_REF][START_REF] Chao | Type I phosphatidylinositol phosphate kinase beta regulates focal adhesion disassembly by promoting beta1 integrin endocytosis[END_REF] confirming the central role of DNM2-dependent processes in FA disassembly.

Additional data strongly suggested that clathrin-mediated endocytosis is the main route involved in the DNM2-dependent endocytosis of FA components leading to FA disassembly.

A rapid accumulation of clathrin occurs in a microtubule-dependent manner as FAs disappear [START_REF] Ezratty | Clathrin mediates integrin endocytosis for focal adhesion disassembly in migrating cells[END_REF] and clathrin depletion impairs microtubule-induced FA disassembly [START_REF] Ezratty | Clathrin mediates integrin endocytosis for focal adhesion disassembly in migrating cells[END_REF][START_REF] Chao | Focal adhesion disassembly requires clathrin-dependent endocytosis of integrins[END_REF] and cell migration [START_REF] Ezratty | Microtubule-induced focal adhesion disassembly is mediated by dynamin and focal adhesion kinase[END_REF][START_REF] Ezratty | Clathrin mediates integrin endocytosis for focal adhesion disassembly in migrating cells[END_REF][START_REF] Chao | Focal adhesion disassembly requires clathrin-dependent endocytosis of integrins[END_REF][START_REF] Chao | Type I phosphatidylinositol phosphate kinase beta regulates focal adhesion disassembly by promoting beta1 integrin endocytosis[END_REF] as shown for DNM2. Clathrin-and DNM2-mediated endocytosis targets specifically 1-integrin engaged in extracellular matrix interaction towards the endosomal recycling pathway [START_REF] Ezratty | Clathrin mediates integrin endocytosis for focal adhesion disassembly in migrating cells[END_REF][START_REF] Chao | Focal adhesion disassembly requires clathrin-dependent endocytosis of integrins[END_REF][START_REF] Chao | Type I phosphatidylinositol phosphate kinase beta regulates focal adhesion disassembly by promoting beta1 integrin endocytosis[END_REF]. Among the clathrin adaptors responsible for clathrin recruitment to endocytic pits, endocytosis of 1-integrin at the FA involves either Dab2 (disabled 2) and ARH (autosomal recessive hypercholesterolemia protein) in NIH-3T3 cells [START_REF] Ezratty | Clathrin mediates integrin endocytosis for focal adhesion disassembly in migrating cells[END_REF] or AP2 (adaptor-related protein complex 2) and Dab2 in the HT1080 cell line [START_REF] Chao | Focal adhesion disassembly requires clathrin-dependent endocytosis of integrins[END_REF]. These apparent differences suggest a cell-specific combination of clathrin adaptors for integrin endocytosis and FA disassembly. Even if clathrin-mediated endocytosis seems to be the major route for integrin endocytosis leading to FA disassembly, involvement of DNM2-and caveolin-dependent endocytosis was reported [START_REF] Vassilieva | Lipid rafts mediate internalization of beta1-integrin in migrating intestinal epithelial cells[END_REF][START_REF] Bass | A syndecan-4 hair trigger initiates wound healing through caveolinand RhoG-regulated integrin endocytosis[END_REF] suggesting that caveolin-mediated endocytosis may also participate in the FA disassembly. Further studies will be necessary to better define if clathrin-or caveolae-dependent endocytosis pathways could be exploited in a cell-specific manner or if they both coexist in a single cell.

DNM2 targeting to FAs

In addition to its interaction with FAK, DNM2 may be targeted to the FA by interacting with syndecan 4 mediated by the PH domain of DNM2 [START_REF] Yoo | Dynamin II interacts with syndecan-4, a regulator of focal adhesion and stress-fiber formation[END_REF]. Syndecan 4 is a heparan sulphate proteoglycan involved in the formation of FAs and actin stress fibres [START_REF] Echtermeyer | Syndecan-4 core protein is sufficient for the assembly of focal adhesions and actin stress fibers[END_REF][START_REF] Woods | Syndecan-4 binding to the high affinity heparin-binding domain of fibronectin drives focal adhesion formation in fibroblasts[END_REF]. It regulates FAK activity by increasing FAK phosphorylation in a Rho-dependent manner [START_REF] Wilcox-Adelman | Syndecan-4 modulates focal adhesion kinase phosphorylation[END_REF], and participates in integrin internalization [START_REF] Bass | A syndecan-4 hair trigger initiates wound healing through caveolinand RhoG-regulated integrin endocytosis[END_REF]. In NIH-3T3 cells treated with lisophosphatidic acid to induce FA formation, DNM2 and syndecan 4 are both recruited and interact at FAs [START_REF] Yoo | Dynamin II interacts with syndecan-4, a regulator of focal adhesion and stress-fiber formation[END_REF]. This study shows that DNM2 is recruited at the FA as soon as FAs are formed and engaged via ECM interaction. Even if syndecan 4 do target DNM2 to FAs, syndecan 4 endocytosis itself occurs via a clathrin-and DNM2-independent process [START_REF] Tkachenko | Fibroblast growth factor 2 endocytosis in endothelial cells proceed via syndecan-4-dependent activation of Rac1 and a Cdc42-dependent macropinocytic pathway[END_REF] showing a tightly regulated sorting of FA components targeted to the clathrin-and DNM2-associated endocytic vesicles. DNM2 may also be targeted at FAs by interaction via its PH domain with the PI4,5P2 enriched at the FA. In fact, local production of this phosphoinositide which also interacts with syndecan 4 [START_REF] Oh | Syndecan-4 proteoglycan cytoplasmic domain and phosphatidylinositol 4,5-bisphosphate coordinately regulate protein kinase C activity[END_REF], may increase recruitment and activation of DNM2 and the endocytic machinery leading to vesicle formation [START_REF] Paolo | Phosphoinositides in cell regulation and membrane dynamics[END_REF]. Interestingly, it was shown that FA disassembly is dependent on the type 1 phosphatidylinositol phosphate kinase beta (PIPK) located at the FA before disassembly which locally produces the PI4,5P2 allowing recruitment of clathrin, clathrin adaptors (Dab2 and AP2) and DNM2 and formation of the FAK-DNM2 complex [START_REF] Chao | Type I phosphatidylinositol phosphate kinase beta regulates focal adhesion disassembly by promoting beta1 integrin endocytosis[END_REF]. Concerning clathrin and adaptors, the cytoplasmic domain of -integrins contains NPXY motif [START_REF] De Deyne | The vitronectin receptor associates with clathrin-coated membrane domains via the cytoplasmic domain of its beta5 subunit[END_REF] necessary for their recruitment. However, an interesting possibility is that clathrin could be targeted to the FA by interacting with other components of FAs. Indeed, -actinin and vinculin, two components of the integrin-based adhesion sites [START_REF] Kanchanawong | Nanoscale architecture of integrin-based cell adhesions[END_REF], have been shown to interact directly with clathrin heavy chain [START_REF] Merisko | Alpha-actinin and calmodulin interact with distinct sites on the arms of the clathrin trimer[END_REF][START_REF] Fausser | Interaction of vinculin with the clathrin heavy chain[END_REF].

DNM2-dependent mechanisms in FA regulation

Processes involved in the turnover of FA components include the microtubule-mediated regulation of FA dynamics [START_REF] Kaverina | Microtubule targeting of substrate contacts promotes their relaxation and dissociation[END_REF][START_REF] Stehbens | Targeting and transport: how microtubules control focal adhesion dynamics[END_REF] leading to DNM2-dependent integrins endocytosis as discussed earlier. FA turnover also includes proteolysis by calpains [START_REF] Franco | Calpain-mediated proteolysis of talin regulates adhesion dynamics[END_REF][START_REF] Chan | Regulation of adhesion dynamics by calpain-mediated proteolysis of focal adhesion kinase (FAK)[END_REF] and protein dephosphorylation by phosphatases [START_REF] Burridge | Regulation of cell adhesion by proteintyrosine phosphatases. I. Cell-matrix adhesion[END_REF]. The importance of calpain 2, a calcium-dependant protease, was shown for the proteolysis of talin and the subsequent disassembly of the macromolecular complexes also including paxilin and vinculin [START_REF] Franco | Calpain-mediated proteolysis of talin regulates adhesion dynamics[END_REF]. More recently, calpain 2mediated proteolysis of FAK was also shown to contribute to the regulation of FA turnover [START_REF] Chan | Regulation of adhesion dynamics by calpain-mediated proteolysis of focal adhesion kinase (FAK)[END_REF]. FAK is composed of a N-terminal FERM domain (F for 4.1 protein, E for ezrin, R for radixin and M for moesin), which interacts with DNM2 [START_REF] Wang | A direct interaction between the large GTPase dynamin-2 and FAK regulates focal adhesion dynamics in response to active Src[END_REF], the catalytic kinase domain containing the binding site for Src and a FAT (focal adhesion targeting) domain involved in the recruitment of FAK to the FA [START_REF] Mitra | Focal adhesion kinase: in command and control of cell motility[END_REF]. The cleavage site of FAK by calpain 2 was mapped at the serine residue in position 745 located between the kinase and FAT domains [START_REF] Chan | Regulation of adhesion dynamics by calpain-mediated proteolysis of focal adhesion kinase (FAK)[END_REF]. The consequences of calpain-mediated proteolysis on the FAK-Src-DNM2 complex and on the DNM2-dependent FA disassembly were not investigated until now. It is tempting to speculate that the proteolysis contributes to the activation of the clathrin and DNM2-mediated endocytosis maybe by allowing an increased polymer formation and GTPase activity of DNM2.

Another mechanism of DNM2-dependent regulation of FA emerged from studies performed on podosomes, a transient adhesion site specific to motile cells [START_REF] Spinardi | Podosomes as smart regulators of cellular adhesion[END_REF]. In osteoclasts, DNM2 is recruited at podosomes by the activated phosphorylated form of Pyk2; i.e. a focal adhesion kinase highly homologous to FAK, which in turn induces a negative feedback loop leading to the dephosphorylation and inactivation of Pyk2 [START_REF] Bruzzaniti | Dynamin reduces Pyk2 Y402 phosphorylation and SRC binding in osteoclasts[END_REF]. The DNM2-dependent dephosphorylation of FAK was also reported [START_REF] Bruzzaniti | Dynamin reduces Pyk2 Y402 phosphorylation and SRC binding in osteoclasts[END_REF]. Recently, the mechanism of the DNM2dependent Pyk2 dephosphorylation was demonstrated to be mediated by the tyrosine phosphatase PTP-PEST leading to the arrest of Pyk2 downstream signaling [START_REF] Eleniste | Dynamin and PTP-PEST cooperatively regulate Pyk2 dephosphorylation in osteoclasts[END_REF]. PTP-PEST is known to dephosphorylate several components of FAs [START_REF] Shen | The noncatalytic domain of protein-tyrosine phosphatase-PEST targets paxillin for dephosphorylation in vivo[END_REF][START_REF] Angers-Loustau | Protein tyrosine phosphatase-PEST regulates focal adhesion disassembly, migration, and cytokinesis in fibroblasts[END_REF]. Eleniste and collaborators showed that dynamin's GTPase activity is required for the formation of the DNM-Pyk2-PTP complex [START_REF] Eleniste | Dynamin and PTP-PEST cooperatively regulate Pyk2 dephosphorylation in osteoclasts[END_REF]. The recruitment of the PTP-PEST phosphatase by DNM2 when its GTPase activity increases may be a more general mechanism at the FAs resulting to the dephosphorylation of FA components, inactivation of signaling cascades, disengagement of integrins and finally FA disassembly. Further studies will be necessary to better define the temporal regulation and coordination of these different processes. For example, it was already demonstrated that calpain is required for microtubule-mediated FA disassembly [START_REF] Bhatt | Regulation of focal complex composition and disassembly by the calcium-dependent protease calpain[END_REF] showing that FA disassembly is an intricately regulated process.

In addition to its role in endocytosis, DNM2 is a well known regulator of the actin network dynamics [START_REF] Schafer | Dynamin2 and Cortactin Regulate Actin Assembly and Filament Organization[END_REF][START_REF] Itoh | Dynamin and the actin cytoskeleton cooperatively regulate plasma membrane invagination by BAR and F-BAR proteins[END_REF][START_REF] Lee | Dynamin at actin tails[END_REF]. One can hypothesize a wider function of DNM2 at the FA besides its role in endocytosis of FA components. In particular, interactions of DNM2 with several other proteins need to be further characterized at the FA. DNM2 interacts with Cbl (Casitas Blineage Lymphoma) in podosomes of osteoclasts [START_REF] Bruzzaniti | Dynamin forms a Src kinase-sensitive complex with Cbl and regulates podosomes and osteoclast activity[END_REF] and with CAP (Cbl-associated protein) [START_REF] Tosoni | CAP (Cbl associated protein) regulates receptor-mediated endocytosis[END_REF] involved in remodeling of the actin cytoskeleton at adhesion sites [START_REF] Sanjay | Cbl associates with Pyk2 and Src to regulate Src kinase activity, alpha(v)beta(3) integrin-mediated signaling, cell adhesion, and osteoclast motility[END_REF][START_REF] Ribon | A role for CAP, a novel, multifunctional Src homology 3 domain-containing protein in formation of actin stress fibers and focal adhesions[END_REF] suggesting that DNM2 may also participate in actin network dynamics at the FA before disassembly. It was also shown that the CAP-DNM2 complex negatively regulates receptor-mediated endocytosis [START_REF] Tosoni | CAP (Cbl associated protein) regulates receptor-mediated endocytosis[END_REF]. A similar complex may occur at FAs and inhibit DNM2 activity before disassembly signaling occurs.

Figure 1 summarizes the known interactions of DNM2 with FA components including FAK, PTP-PEST phosphatase and syndecan 4. The available data demonstrate that DNM2 is per se a component of FA and that the endocytic machinery including clathrin adaptors, clathrin, and DNM2 probably start being recruited at the FA during their formation and maturation by interaction with specific FA components. The subsequent clathrin-and DNM2mediated endocytosis of FA components, which recycle back to the plasma membrane [START_REF] Caswell | Integrin trafficking and the control of cell migration[END_REF], allows turnover of FA.

Relevance in DNM2-related human diseases

Mutations in the DNM2 gene cause rare forms of Charcot-Marie-Tooth peripheral neuropathy (CMT) [START_REF] Zuchner | Mutations in the pleckstrin homology domain of dynamin 2 cause dominant intermediate Charcot-Marie-Tooth disease[END_REF]. Among the hypotheses proposed to explain the CMT pathogenesis, impaired intracellular transport is of particular interest due to the number of CMT genes involved in intracellular trafficking [START_REF] Bucci | Charcot-Marie-Tooth disease and intracellular traffic[END_REF]. Given that CMT-related DNM2 mutations impaired clathrin-mediated or clathrin-independent endocytosis [START_REF] Tanabe | Dynamic instability of microtubules requires dynamin 2 and is impaired in a Charcot-Marie-Tooth mutant[END_REF][START_REF] Zuchner | Mutations in the pleckstrin homology domain of dynamin 2 cause dominant intermediate Charcot-Marie-Tooth disease[END_REF][START_REF] Koutsopoulos | Mild functional differences of dynamin 2 mutations associated to centronuclear myopathy and charcot-marie-tooth peripheral neuropathy[END_REF][START_REF] Liu | Common membrane trafficking defects of disease-associated dynamin 2 mutations[END_REF], a defect in integrin internalization and FA turnover may participate in the pathomechanisms of the disease. In agreement with this hypothesis, CMT-related DNM2 mutants inhibit clathrin-mediated endocytosis in motor neuronal cells and Schwann cells and strongly impaired myelination [START_REF] Sidiropoulos | Dynamin 2 mutations in Charcot-Marie-Tooth neuropathy highlight the importance of clathrin-mediated endocytosis in myelination[END_REF]. In this study, over-expression of the K562E CMT-mutant in rat primary Schwann cells increases the plasma membrane content of 1-integrin probably by endocytosis impairment.

On the other hand, integrin-based adhesion sites play important roles in the regulation of cell surface receptor, neurotransmitter receptors, and calcium signaling in neurons [START_REF] Wu | Integrins as receptor targets for neurological disorders[END_REF]; several processes potentially impaired by DNM2 mutation.

Mutations in the DNM2 gene also cause autosomal dominant centronuclear myopathy (CNM) [START_REF] Bitoun | Mutations in dynamin 2 cause dominant Centronuclear Myopathy[END_REF], a slowly progressive congenital myopathy. The mature skeletal muscle fibres have two distinct FA types: the integrin-and the -dystroglycan (-DG)-mediated adhesions [START_REF] Ervasti | Costameres: the Achilles' heel of Herculean muscle[END_REF]. The -DG is an integral membrane protein included in the dystrophin-associated glycoprotein complex (DGC) which links extracellular laminin and ECM proteins to the intracellular actin cytoskeleton. The two types of FA are concentrated at two specialized adhesives structures of muscle fibres; i.e. the muscle-tendon attachment at myotendinous junctions and the muscle-ECM attachment at costameres along the fibres. Due to muscle intrinsic functional properties, these two adhesives structures have a crucial protective role against contractile damages during contraction and relaxation cycles and are essential for maintaining integrity of sarcomeres; i.e. the contractile unit of the muscle fibres. Several muscular dystrophies are linked to mutations in components of the two FA types [START_REF] Dalkilic | Muscular dystrophies: genes to pathogenesis[END_REF]. In muscle, integrin-mediated adhesion sites are dynamic structures, crucial for structural integrity [START_REF] Perkins | Integrin-mediated adhesion maintains sarcomeric integrity[END_REF], which require clathrin-mediated endocytosis for turnover [START_REF] Yuan | Analysis of integrin turnover in fly myotendinous junctions[END_REF]. The first link between CNM and adhesion sites was recently demonstrated in drosophila [START_REF] Ribeiro | Phosphoinositide regulation of integrin trafficking required for muscle attachment and maintenance[END_REF]. MTM1, a phosphoinositide phosphatase mutated in the X-linked recessive form of CNM [START_REF] Laporte | A gene mutated in X-linked myotubular myopathy defines a new putative tyrosine phosphatase family conserved in yeast[END_REF] participates in maintenance of muscle attachment sites through a role in integrin trafficking at myotendinous junctions and FAs [START_REF] Ribeiro | Phosphoinositide regulation of integrin trafficking required for muscle attachment and maintenance[END_REF]. Given that CNM-related DNM2 mutations may impair clathrin-mediated and clathrin-independent endocytosis [START_REF] Koutsopoulos | Mild functional differences of dynamin 2 mutations associated to centronuclear myopathy and charcot-marie-tooth peripheral neuropathy[END_REF][START_REF] Liu | Common membrane trafficking defects of disease-associated dynamin 2 mutations[END_REF][START_REF] Bitoun | Dynamin 2 mutations associated with human diseases impair clathrin-mediated receptor endocytosis[END_REF], future studies will be necessary in order to determine if similar impact may be due to DNM2 mutations.

One important question is to understand the tissue-specific impact of DNM2 mutations on skeletal muscle or peripheral nervous system. In cultured neurons, DNM2 interacts with ArgBP2 (Arg and Abl Binding Protein 2) and its brain-specific isoform nArgBP2; two proteins involved in the regulation of actin cytoskeleton at the cell adhesion sites [START_REF] Cestra | The Abl/Arg substrate ArgBP2/nArgBP2 coordinates the function of multiple regulatory mechanisms converging on the actin cytoskeleton[END_REF].

Interaction of DNM2 with cell type-specific FA components may participate to the tissuespecific impact of DNM2 mutations. Alternatively, one may hypothesize a cell-type specific effect of distinct DNM2 mutants. Indeed, CMT-mutants but not CNM-mutants impair endocytosis in peripheral nerve model [START_REF] Sidiropoulos | Dynamin 2 mutations in Charcot-Marie-Tooth neuropathy highlight the importance of clathrin-mediated endocytosis in myelination[END_REF]. Similar comparative studies on FA disassembly using skeletal muscle and peripheral nerve models will be necessary for deciphering the tissue-specific impact of DNM2 mutations.

Conclusions

Consistent data now exist to show the importance of DNM2 at the cell-matrix adhesion sites. By interacting with syndecan 4 and FAK, DNM2 is per se a component of the focal adhesion sites. DNM2 is probably recruited early during the FA formation process and may regulate actin dynamics. In addition, DNM2 plays a central role in FA disassembly through the link with four disassembly signaling: i) DNM2 is a regulator of the microtubule cytoskeleton, ii) Disassembly is dependent on the activity of the PIPK which locally produces PI4,5P2 which binds DNM2, iii) Calpain-dependent proteolysis acts on FAK which interacts with DNM2, and iv) DNM2 interacts with PTP-PEST, a phosphatase involved in FA disassembly. These data highlight a new promising pathophysiological hypothesis that should be further studied in DNM2-related human diseases.

Legends Figure 1. Summary of the DNM2 interactions with FA components.

A. Several processes may participate to the recruitment of DNM2 to FA: i) interaction with FAK in a DNM2-FAK-Src complex, ii) interaction with Syndecan 4, iii) interaction with the PTP-PEST phosphatase, and iv) interaction with PI4,5P2 enriched at the FA. Clathrin adaptors and clathrin heavy chain may be also targeted to the mature FA by interacting with integrin heterodimers, vinculin and -actinin. Under disassembly signaling, the calciumdependent proteolysis by calpain cleaves FAK and other FA components. In addition, the production of PI4,5P2 by PIPK may increase DNM2 membrane recruitment. On the other hand, the dephosphorylation of FA components by phosphatases involves the PTP-PEST phosphatase which interacts with DNM2. B. The combination of the four disassembly stimuli (pink boxes in A) activates the clathrin-and DNM2-mediated endocytosis and recycling of FA components. The figure is focused on DNM2-related processes and consequently, additional interactions between FA components have been omitted.

Acknowledgements

This work was supported by the Institut National de la Santé et de la Recherche Médicale (INSERM), the Association Institut de Myologie (AIM), the Université Pierre et Marie Curie-Paris6 and the Centre National de la Recherche Scientifique (CNRS). LB is recipient of an AIM fellowship.

Disclosure of potential conflict of interest

There is no conflict of interest to disclose.


Matteo Italia

Fabrizio Croccolo

Frank Scheffold

Alberto Vailati

Inclined layer convection in a colloidal suspension with negative Soret coefficient at large solutal Rayleigh numbers

Convection in an inclined layer of fluid is affected by the presence of a component of the acceleration of gravity perpendicular to the density gradient that drives the convective motion. In this work we investigate the solutal convection of a colloidal suspension characterized by a negative Soret coefficient. Convection is induced by heating the suspension from above, and at large solutal Rayleigh numbers (of the order of 10 7 -10 8 ) convective spoke patterns form. We show that in the presence of a marginal inclination of the cell as small as 19 mrad the isotropy of the spoke pattern is broken and the convective patterns tend to align in the direction of the inclination. At intermediate inclinations of the order of 33 mrad ordered square patterns are obtained, while at inclination of the order of 67 mrad the strong shear flow determined by the inclination gives rise to ascending and descending sheets of fluid aligned parallel to the direction of inclination.

Introduction

A liquid layer under the action of a temperature gradient can be either in a stable or unstable configuration, depending on the orientation of the density gradient generated by thermal dilation relative to the acceleration of gravity. The most investigated configuration is the Rayleigh-Bénard one, where the liquid is heated from below and the density gradient and the acceleration of gravity g are anti-parallel [1]. The relevant parameter to characterize the stability of this system is the Rayleigh number Ra = gΔρ T h 3 /(ηκ), where g is the acceleration of gravity, Δρ T the density difference generated by thermal dilation of the liquid, h the thickness of the layer, η the shear viscosity and κ the thermal diffusivity. Under this configuration, when Ra exceeds the threshold value Ra c = 1708, the symmetry of the system gets broken by non-equilibrium fluctuations and convective motions develop inside the liquid. The presence of a component of g perpendicular to the density gradient determined by an inclination of the liquid layer generates a Large Scale Shear Flow that strongly affects the convective motions. For simple fluids close to the threshold of the instability, it has been shown both theoretically [2][3][4] and experimentally [5][6][7][8] that for Contribution to the Topical Issue "Thermal nonequilibrium phenomena in multi-component fluids" edited by Fabrizio Croccolo and Henri Battaler.

a e-mail: alberto.vailati@unimi.it inclination angles in the range 10 • -120 • the interplay between Rayleigh-Bénard convection and Large Scale Flow gives rise to a rich phase diagram characterized by a wide range of states, which include longitudinal rolls, cross-rolls and oscillations. At large Rayleigh numbers of the order of 10 8 , a region where convection becomes turbulent, a small tilting determines a slight change in the heat transferred [9]. In a recent work [10] we have shown that the solutal convection in a binary liquid mixture with positive Soret coefficient heated from below at Ra < Ra c is strongly affected by the presence of a marginal inclination as small as 6 mrad. Remarkably, in the presence of such a small inclination the stationary square patterns that characterize the planform in the absence of inclination [11][12][13][14] evolve into Superhighway convective patterns where columns of ascending and descending liquid self-organize into ordered lanes drifting into opposite directions [10]. However, the analysis of the early stages of the instability shows that they are not affected by the presence of an inclination.

In this work we investigate the influence of a marginal inclination on the solutal convection of a colloidal suspension with negative Soret coefficient heated from above. In this case the density variation Δρ T induced by the thermal dilation of the sample stabilizes the system. However, the temperature gradient induces a Soret mass flow j = -ρD(∇c + S T c(1c)∇T ) in the upward direction and at steady state the presence of impermeable bound- aries gives rise to an unstable solutal density difference across the liquid layer Δρ c = ρ 0 βS T c(1c)ΔT [START_REF] De Groot | Nonequilibrium thermodynamics[END_REF]. Here ρ 0 is the mass density of the suspension at reference temperature, β is the solutal expansion coefficient, c is the weight fraction concentration of the denser component, and S T is the Soret coefficient. The relative contribution of Δρ T and Δρ c to the overall density difference is quantified by the separation ratio Ψ = Δρ c /Δρ T ; when Ψ < -1 the system is unstable when heated from above [START_REF] Platten | Convection in Liquids[END_REF]. A further contribution to the destabilization of the sample comes from the fact that concentration fluctuations in colloidal suspension are characterized by decay times orders of magnitudes larger than those of temperature fluctuations. The ratio between the two time scales is expressed by the Lewis number Le = D/κ, which can be typically of the order of 10 -4 for colloidal suspensions. For this reason, even a small solutal density difference can give rise to solutal convection in the presence of a stabilizing density gradient determined by the thermal dilation of the sample. The relevant parameter in this case is the solutal Rayleigh number Ra s = gΔρ c h 3 /(ηD) = RaΨ/Le. Studies on simple fluids [START_REF] Busse | [END_REF][18][19] and binary mixtures and colloidal suspensions [20][21][22][23][24][25] performed at large (solutal) Rayleigh numbers of the order of 10 5 < Ra s < 10 9 have shown that the planform of the instability is a spoke pattern made by two staggered networks (fig. 1) with loops that are on average quadrilateral [25]. In the case of an instability induced by the Soret effect, the onset of the instability is accompanied by relaxation oscillations determined by the interplay between the growth of solutal boundary layers and the triggering of the convective flow [22,23,26], a mechanism similar to that reported for the onset of solutal convection in suspensions of thermo-sensitive particles with positive Soret coefficient [27][28][29]. The main questions that we want to address with this work are: i) is there any detectable evidence of the presence of a marginal inclination during the onset of the instability in a colloidal suspension with negative Soret coefficient? ii) what is the long-term effect of the inclination on the structure of the convective planform?

Materials and methods

The sample is a colloidal suspension of silica nanoparticles with a diameter of 32 nm dispersed into water at a weight fraction concentration of 4.0%, known commercially as LUDOX TMA. The separation ratio is Ψ = -3.4 and the Lewis number Le = 1.54 × 10 -4 . Other thermophysical properties of the suspension are detailed in [30,31]. In order to investigate the development of convective patterns we need to access the sample optically from the vertical direction. For this reason, the sample is sandwiched between two sapphire windows with a diameter of 70 mm and a thickness of 12.0 mm. The configuration of the thermal gradient cell is similar to that adopted and tested in other experiments (fig. 2) [30][31][32].

The relatively high thermal conductivity of sapphire (of the order of 35 W m -1 K -1 ) allows to use the windows at the same time as thermalizing plates and as optical windows. The sample thickness is kept at 2.90 mm by means of calibrated Delrin spacers. Each sapphire window is in contact with an annular Peltier heat pump with an inner hole of diameter 27 mm. The sample is confined laterally by an o-ring gasket with an inner diameter of 27 mm. This feature guarantees that we are able to access optically the entire sample, even close to the lateral boundary. Each Peltier element is controlled by a Proportional-Integral-Derivative servocontrol and can transfer heat from/to a water circulation bath. The stability of the average temperature and of the temperature difference applied to the sample is of the order of 10 mK/24 hours. The uniformity of the temperature gradient across the observation window is better than 3%. The cell assembly is supported by a kinematic table accurately leveled by using a machinist level with an accuracy of 0.05 mm/m. Once levelled, the cell can be tilted by means of a calibrated screw and the inclination can be read with a resolution 60 μrad by using a comparator with a resolution of 0.01 mm. The optical technique of choice is shadowgraphy [START_REF] Settles | Schlieren and Shadowgraph Techniques[END_REF], a projection technique used mostly for visualization purposes in the past, which has become a highly quantitative technique during the last 15 years [START_REF] Trainoff | [END_REF][35][36]. The layout of the setup is similar to that used in [32,37]. The light source is a superluminous diode with a wavelength of 676.5 nm, FWHM of 14.1 nm and a maximum intensity of 1 mW, coupled to a monomode optical fiber. The divergent beam coming out of the fiber is collimated by an achromatic doublet with a focal length of 50 cm. The large focal length of the doublet allows to achieve a uniform illumination of the sample. The collimated beam crosses the sample in vertical direction and an achromatic doublet of focal length 10 cm images a plane at a distance z = 50 mm from the cell onto a Charged Coupled Device (CCD) detector with a resolution of 768 × 576 pixels. The signal coming from the CCD is sampled by a frame grabber and recorded digitally.

A typical measurement sequence (fig. 3) involves the sudden imposition of a temperature difference to the sample by heating from above. Following a latency phase, convective columns appear and gradually evolve into a spoke pattern configuration. The strength of the convective flow can be characterized by determining the contrast of each image

C(t) = [I(x, t) -I(x, t) o ] 2
x where . . . x represents the average over the pixels of an image, and I(x, t o ) is a background image at time t = 0 before applying the temperature gradient.

Results and discussion

Relaxation oscillations at the onset of the instability

The contrast signal remains stable for a latency time of a few hundred seconds following the imposition of the temperature gradient. This is apparent from fig. 4, which shows curves of the contrast as a function of time at different solutal Rayleigh numbers at an inclination angle of 19 mrad.

The onset of the instability is characterized by the development of solutal boundary layers located at the impermeable boundaries [24]. Due to the solutal mass flow generated by the Soret effect, colloidal particles tend to accumulate near the top boundary and to be removed from a region near the bottom boundary. The thickness d of the boundary layers grows diffusively d ∝ √ Dt until it reaches a critical thickness d * when it becomes unstable [START_REF] Howard | Proceedings of the Eleventh International Congress of Applied Mechanics[END_REF]24,28]. At the critical thickness the non-equilibrium fluctuations generated by velocity fluctuations at the boundary layer get amplified by the density gradient and they give rise to convective flows in the form of ascending and descending regions of liquid [START_REF] Giavazzi | [END_REF]. The convective flow partially destroys the boundary layers that generated it, and this gives rise to the relaxation oscillations shown in fig. 4 [40,41,24]. Theoretical models [42][43][44]26] and experiments [23,24] have shown that in the absence of inclination the critical time needed for the system to become unstable exhibits a power law dependence τ * = 25.3Ra -0.52 s from the solutal Rayleigh number (circles in fig. 5). Here τ * = t * /t D is the critical time t * made dimensionless by dividing it by the diffusive time across the cell height

t D = h 2 /D.
Similar results were found for the onset of convection in suspensions of thermo-sensitive particles [27,28]. In order to assess the influence of the inclination of the sample on the time τ * , we performed a series of systematic measurements with solutal Rayleigh numbers in the range 1.37 × 10 7 < Ra s < 1.29 × 10 8 and inclination angles 0 < θ < 67 mrad. The results are shown by the squares in fig. 5. One can appreciate that the data obtained in the presence and in the absence of inclination nicely collapse onto the same curve. From the latency time we are able to determine the thickness δ = √ πτ of the boundary layers at the onset of the instability normalized by the sample thickness [24]. At the large solutal Rayleigh numbers used in this work the thickness of the boundary layers is always a small fraction of the thickness of the sample.

Although the growth dynamics of the boundary layers does not appear to be affected by the inclination, a close inspection of the convective patterns at the onset [24], while squares to results with inclination angle in the range 19 mrad < θ < 67 mrad. The solid line represents the power law behavior predicted by the theory [42][43][44]26]. of the instability reveals that they are influenced by the inclination from the beginning of the instability (fig. 6). Quite generally, the start of the growth of the contrast corresponds to the appearance of white structures associated to the downwelling columnar flow of colloid-rich liquid (fig. 3b). This temporal asymmetry between the appearance of black and white structures occurs independently of the presence of an inclination, while the inclination gives rise to an additional spatial asymmetry. In the absence of inclination, when the contrast reaches its peak the pattern is characterized by a uniform distribution of ascending and descending structures (fig. 6(a)). For intermediate inclinations of 33 mrad white downwelling structures dominate the highest side (bottom left of fig. 6(b)) of the cell and black upwelling structures the lowest side (top right of fig. 6(b)). At larger inclinations of the order of 67 mrad the central part of the cell is dominated by ascending and descending sheets of liquid, and the columnar flow is confined in regions close to the highest and lowest side of the cell (fig. 6(c)).

Long-term behavior

At large times the convective pattern arranges into a spoke pattern configuration for inclination angles smaller than 66 mrad. Spoke patterns are made by two staggered polygonal networks [START_REF] Busse | [END_REF][18][19] (fig. 1). Each node of the network represents an ascending/descending column of liquid and is surrounded on average by four descending/ascending columns [25]. Spoke patterns are generated during convection in simple fluid and binary mixtures near the transition to turbulent convection, when the Rayleigh number, or solutal Rayleigh number, is of the order of 10 5 or larger. The patterns are isotropic and any orientation imposed by the lateral boundaries becomes irrelevant at a distance from them comparable to the thickness of the cell, provided that the aspect ratio of the cell is large enough. The inclination of the cell determines the presence of a component of the gravity force perpendicular to the density gradient, which gives rise to a shear motion of the boundary layers in the direction of the inclination (fig. 7). The top boundary layer, rich in colloidal particles, slides down along the top sapphire window, while the bottom boundary layer slides up along the bottom window. As a result, the vertical columns of ascending and descending liquid characteristic of the spoke pattern configuration are dragged by the boundary layers that originated them and tend to drift into opposite directions.

Figure 8 shows some configurations obtained at late times larger than 6 × 10 4 s under various inclination conditions. The first panel (fig. 8(a)) corresponds to the base state in the absence of inclination. Under these conditions the pattern is a spoke pattern. From its power spectrum (fig. 8(b)) one can appreciate that the pattern is nearly isotropic. Panel 8(c) shows the pattern obtained in the presence of an inclination of 19 mrad. Near the lateral boundaries of the cell the geometry of the pattern is constrained by the boundary conditions. However, in the central part of the cell the boundary conditions become ir- relevant and one can appreciate that the spoke patterns rearrange into square cells aligned approximately parallel to the direction of inclination. The presence of a square pattern is apparent in the power spectrum of the pattern (fig. 8(d)), which shows the presence of two characteristic wave vectors aligned parallel and perpendicular to the direction of inclination. At larger inclinations of the order of 67 mrad the large-scale shear flow becomes stronger and the columnar flow disappears. Solutal convection takes the form of ascending and descending sheets aligned approximately parallel to the direction of inclination (fig. 8(e)). The pattern becomes strongly anisotropic, as witnessed by the power spectrum, which develops two wings perpendicular to the direction of inclination (fig. 8(f)). Under this condition the pattern is very similar to those obtained for simple fluid in the presence of shear induced by a movement of the horizontal boundaries into opposite directions [45]. By performing systematic measurements at various solutal Rayleigh numbers and inclinations we were able to obtain a phase diagram for the long-term behavior of the convective patterns (fig. 9). Quite generally, in the absence of inclination the pattern is a spoke pattern (stars in fig. 9). In the presence of small inclinations, the solutal columnar flow is efficient in dissipating the excess of mass at the boundary layers, and the effect of the inclination is limited to a rearrangement of the position of the columns located at the vertexes of the spoke pattern; this gives rise to two staggered square patterns aligned parallel to the inclination (squares in fig. 9). Under this condition each ascending (descending) column is caged by four descending (ascending) columns and this gives rise to a jamming of the pattern that prevents further motion in the direction of inclination. At larger inclinations the caging mechanism is not effective any more and the convective flow is dominated by the large-scale flow, which favors the rearrangement of aligned columns into ondulated rolls (waves in fig. 9).

Conclusions

Convection in a suspension of silica colloidal particles in water with negative Soret coefficient gives rise to spoke patterns when the system is heated from above at solutal Rayleigh numbers larger than 10 5 . The presence of a marginal inclination as small as 19 mrad strongly affects the pattern formation. The overall growth of solutal boundary layers preceding the onset of convection is not affected much. However, the inclination affects even the early stages of the instability and gives rise to a nonisotropic destabilization of the boundary layers. At large times, the interplay between solutal convection and largescale shear flow gives rise to an orientation of the patterns at inclinations smaller than about 50 mrad and to the development of ondulated rolls at inclinations of 67 mrad.

Fig. 1 .

 1 Fig. 1. Spoke pattern obtained during the solutal convection of a suspension of silica nanoparticles in water. The suspension has a negative Soret coefficient and convection is induced by heating from above at Ra s = 5.9 × 10 7 after 6 × 10 4 s from the onset of convection.

Fig. 2 .

 2 Fig. 2. Cross section of the thermal gradient cell. The heart of the cell is made by two sapphire windows that act at the same time as thermalizing plates and optical windows. Heat is injected from/to the windows by using annular Peltier elements.

Fig. 3 .

 3 Fig. 3. Time evolution of the convective pattern at Ras = 1.29 × 10 8 and θ = 33 mrad. The solid line represents the direction of the inclination. The cell is higher at the bottom left of each panel. Dark features represent regions of upwelling flow of water-rich liquid, while light features regions of downwelling flow of colloid-rich fluid. (a) At time t = 0, when the gradient is turned on, the pattern is featureless. (b) After t = 373 s downwelling columns of liquid appear at the bottom left. (c) At t = 508 s the pattern is divided into nearly symmetric halves: upwelling column of liquid at the top right of the panel and downwelling columns at the bottom left. (d) After 1130 s the pattern has developed two staggered networks of ascending and descending liquid. (e) At t = 20744 s the networks have started the transition to a spoke pattern. (f) At t = 61617 s the pattern has become a fully developed spoke pattern made mostly of quadrilateral cells aligned in the direction of inclination.

Fig. 4 .

 4 Fig. 4. Time evolution of contrast at various solutal Rayleigh numbers for an inclination of 19 mrad with respect to the horizontal direction.

Fig. 5 .

 5 Fig. 5. (a) Latency time as a function of solutal Rayleigh number. (b) Boundary layer thickness as a function of solutal Rayleigh number. Circles correspond to results in the absence of inclination[24], while squares to results with inclination angle in the range 19 mrad < θ < 67 mrad. The solid line represents the power law behavior predicted by the theory[42][43][44]26].

Fig. 6 .

 6 Fig. 6. Effect of the inclination of the patterns at the onset of the instability. Patterns correspond to the first peak of the contrast, when the convective flow is stronger. The solid line represents the direction of the inclination. (a) Isotropic pattern at Ra s = 5.9 × 10 7 and θ = 0 mrad. (b) At Ras = 1.3 × 10 8 and θ = 19 mrad upwelling and downwelling flows tend to develop into opposite halves of the cell. (c) Shear pattern at Ra s = 8.1 × 10 7 and θ = 67 mrad.

Fig. 7 .

 7 Fig. 7. Large-scale shear flow. The columnar solutal flow in the central part of the cell is accompanied by a shear motion of the boundary layers into opposite directions. The top boundary layer is denser than the liquid below it and tends to slide down along the top sapphire window. The bottom boundary layer is lighter than the fluid above it and tends to slide up along the bottom sapphire window.

Fig. 8 .

 8 Fig. 8. Long-term convective patterns and power spectra. The solid lines correspond to the direction of inclination. (a) Spoke pattern obtained in the absence of inclination at Ra s = 5.9 × 10 7 . (b) Isotropic power spectrum of (a). (c) Pattern obtained at θ = 19 mrad and Ra s = 1.3 × 10 8 . Cells at the center of the pattern are on average quadrilateral and aligned in the direction of the inclination. This is reflected in the power spectrum in (d). (e) Shear pattern obtained at θ = 67 mrad and Ra s = 8.1 × 10 7 . The spectrum in (f) shows the presence of ondulated rolls, mostly parallel to the direction of inclination.

Fig. 9 .

 9 Fig. 9. Phase diagram of long-term convective behavior. Stars: spoke patterns; squares: square patterns; waves: ondulated rolls.

AV acknowledges discussion with Roberto Cerbino, Fabio Giavazzi and Stefano Mazzoni. We thank Grace Davison for the supply of the LUDOX sample. Work supported by the Swiss National Science Foundation (projects 132736 and 149867).