Anne-Cécile Durieux

Bernard Prudhon

Pascale Guicheney

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

Dynamin 2 and human disease

Keywords: 2. Number of Tables: 1 dynamin 2, centronuclear myopathy, Charcot-Marie-Tooth neuropathy, endocytosis, cytoskeleton

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Dynamin 2 (DNM2) belongs to a superfamily of large GTPases, including three classical dynamins and several dynamin-like proteins, which are involved in a wide range of cell functions [START_REF] Heymann | Dynamins at a glance[END_REF]. The importance of DNM2 was emphasized in 2005 with the demonstration of DNM2 gene mutations causing two distinct human diseases [START_REF] Zuchner | Mutations in the pleckstrin homology domain of dynamin 2 cause dominant intermediate Charcot-Marie-Tooth disease[END_REF][START_REF] Bitoun | Mutations in dynamin 2 cause dominant Centronuclear Myopathy[END_REF]. Our purpose is to review the molecular and functional data on DNM2 to highlight the possible pathophysiological hypotheses in DNM2-related diseases. Knowledge of the dynamins, especially of their molecular and biochemical properties, mainly comes from numerous studies of dynamin 1 (DNM1) [START_REF] Shpetner | Identification of dynamin, a novel mechanochemical enzyme that mediates interactions between microtubules[END_REF]. However, we have focused this review on DNM2 since several studies have clearly demonstrated notable differences between DNM1 and DNM2 [START_REF] Sontag | Differential expression and regulation of multiple dynamins[END_REF][START_REF] Lin | Phosphatidylinositol (4,5)bisphosphate-dependent activation of dynamins I and II lacking the proline/arginine-rich domains[END_REF][START_REF] Rappoport | Real-time analysis of clathrin-mediated endocytosis during cell migration[END_REF][START_REF] Fourgeaud | The metabotropic glutamate receptor mGluR5 is endocytosed by a clathrin-independent pathway[END_REF][START_REF] Kabbani | Dynamin-2 associates with the dopamine receptor signalplex and regulates internalization of activated D2 receptors[END_REF]. Moreover, DNM1 and DNM2 seem to be involved in different membrane trafficking processes in cells expressing the two proteins [START_REF] Artalejo | Sustained stimulation shifts the mechanism of endocytosis from dynamin-1-dependent rapid endocytosis to clathrin-and dynamin-2-mediated slow endocytosis in chromaffin cells[END_REF][START_REF] Elhamdani | Two mechanistically distinct forms of endocytosis in adrenal chromaffin cells: Differential effects of SH3 domains and amphiphysin antagonism[END_REF].

DNM2 gene organization and isoforms

DNM2, one of three classical dynamins, was initially identified in rat liver and brain cDNA libraries [START_REF] Sontag | Differential expression and regulation of multiple dynamins[END_REF][START_REF] Cook | Identification of dynamin 2, an isoform ubiquitously expressed in rat tissues[END_REF]. A human homologue was thereafter identified by screening of a fibroblast library [START_REF] Diatloff-Zito | Isolation of an ubiquitously expressed cDNA encoding human dynamin II, a member of the large GTP-binding protein family[END_REF]. The human transcript (3.6 kilobases) is ubiquitously expressed, with higher abundance in heart and skeletal muscle [START_REF] Diatloff-Zito | Isolation of an ubiquitously expressed cDNA encoding human dynamin II, a member of the large GTP-binding protein family[END_REF]. Human DNM2 is encoded by the DNM2 gene located on the short arm of chromosome 19 (19p13.2). The gene is composed of 22 exons in a 114-kilobase region. Four isoforms are expressed by the DNM2 gene using a combination of two alternative splice sites (Figure 1A). Isoforms 1, 2, 3 and 4 are also known as isoforms aa, ba, ab and bb, respectively [START_REF] Cao | Differential distribution of dynamin isoforms in mammalian cells[END_REF]. Exons 10 and 10bis have the same length (139 base pairs encoding the amino-acids 399-445 in the middle domain) and are alternatively spliced. In addition, the exon 13bis (12-base pair length) can be spliced leading to the translation of proteins of 866 or 870 amino-acids (Figure 1) without or with the GEIL sequence at position 516-519 in the C-terminal part of the middle domain. The expression of the four isoforms have been assessed in a panel of rat tissues including brain, heart, kidney, liver, lung, pancreas and testis [START_REF] Cao | Differential distribution of dynamin isoforms in mammalian cells[END_REF]. The human tissue expression pattern is unknown, but we have shown expression of the four isoforms in skeletal muscle and peripheral nerve [START_REF] Bitoun | Dynamin 2 mutations associated with human diseases impair clathrin-mediated receptor endocytosis[END_REF]. Specific functions of these isoforms will be discussed below.

DNM2 structure and regulation

The 98 kDa DNM2 is a large GTPase composed of a N-terminal GTPase domain, a middle domain, a pleckstrin homology domain (PH), a GTPase effector domain (GED) and a C-terminal proline rich domain (PRD) (Figure 1B). The catalytic GTPase domain is responsible for GTP binding and hydrolysis, whereas the middle domain is involved in DNM2 self-assembly [START_REF] Smirnova | A model for dynamin self-assembly based on binding between three different protein domains[END_REF] and in GTP hydrolysis-induced conformational change of the protein [START_REF] Chen | The stalk region of dynamin drives the constriction of dynamin tubes[END_REF]. The PH domain interacts with membrane phosphoinositides and therefore involved in the targeting of dynamin to plasma or Golgi membranes [START_REF] Dong | Expression and purification of dynamin II domains and initial studies on structure and function[END_REF]. Klein and collaborators reported that the DNM2-PH domain displays phosphoinositide binding affinity following the order; PI4,5P2 ≈ PI3,4,5P3 ≈ PI3,4P2 > PI4P ≈ PI3P, and DNM2 oligomerization appears crucial for high affinity [START_REF] Klein | The pleckstrin homology domains of dynamin isoforms require oligomerization for high affinity phosphoinositide binding[END_REF]. The GED probably participates in the self-assembly of DNM2 and acts as a GTPase-activating protein (GAP) [START_REF] Sever | Impairment of dynamin's GAP domain stimulates receptor-mediated endocytosis[END_REF]. The PRD contains multiple Src homology 3 (SH3) binding motifs and mediates multiple protein-protein interactions (Table 1).

In vitro at high ionic strength, DNM2 is in monomer-tetramer equilibrium. At low ionic strength, DNM2 self-assembles into higher order aggregates leading to a drastic increase in GTPase activity [START_REF] Warnock | Ubiquitously expressed dynamin-II has a higher intrinsic GTPase activity and a greater propensity for self-assembly than neuronal dynamin-I[END_REF][START_REF] Eccleston | Oligomerization and kinetic mechanism of the dynamin GTPase[END_REF]. Microtubules or phospholipid vesicles, especially those containing PI4,5P2, also induce self-assembly and increase DNM2 GTPase activity [START_REF] Lin | Phosphatidylinositol (4,5)bisphosphate-dependent activation of dynamins I and II lacking the proline/arginine-rich domains[END_REF][START_REF] Warnock | Ubiquitously expressed dynamin-II has a higher intrinsic GTPase activity and a greater propensity for self-assembly than neuronal dynamin-I[END_REF]5. Durieux 23]. Purified from baculovirus, GTP-bound and GDP-bound monomer DNM2 has Kd values of 13.2 and 7.1 µM, respectively, with GTPase activity of 37 nmol/mg/min. When in an oligomeric state, the GTPase activity of DNM2 markedly increased and Kd values decreased [START_REF] Solomaha | Conformational changes in dynamin on GTP binding and oligomerization reported by intrinsic and extrinsic fluorescence[END_REF]. DNM2 basal activity appears 10-fold higher than for DNM1 probably due to the greater propensity of DNM2 to self-assemble and a higher affinity for GTP (Km=12 µM) [START_REF] Warnock | Ubiquitously expressed dynamin-II has a higher intrinsic GTPase activity and a greater propensity for self-assembly than neuronal dynamin-I[END_REF]. When compared to small GTPases, DNM2 exhibits a relatively low affinity for GTP but high intrinsic rates of GTP hydrolysis. DNM2 activity is regulated by post-translational modifications. DNM2 becomes phosphorylated on Tyr231 (middle domain) and Tyr597 (PH domain) through Src-mediated phosphorylation, leading to its association with caveolin and thus albumin endocytosis [START_REF] Shajahan | Role of Src-induced dynamin-2 phosphorylation in caveolae-mediated endocytosis in endothelial cells[END_REF].

Similarly, IL5-induced DNM2 phosphorylation leads to redistribution of DNM2 within endocytic vesicles and is required for IL5 receptor internalization [START_REF] Gorska | Differential regulation of interleukin 5-stimulated signaling pathways by dynamin[END_REF]. In contrast, dopamine leads to the dephosphorylation of DNM2 by increasing protein phosphatase 2A activity, necessary for dopamine-induced Na + K + -ATPase endocytosis [START_REF] Efendiev | Relevance of dopamine signals anchoring dynamin-2 to the plasma membrane during Na+,K+-ATPase endocytosis[END_REF]. S-nitrosylation of Cys86 (GTPase domain) and Cys607 (PH domain) by nitric oxide (NO) increases DNM2 GTPase activity and endocytosis [START_REF] Kang-Decker | Nitric oxide promotes endothelial cell survival signaling through Snitrosylation and activation of dynamin-2[END_REF]. Finally, regulation of DNM2 by proteolysis under pathological condition has also been reported [START_REF] Sever | Proteolytic processing of dynamin by cytoplasmic cathepsin L is a mechanism for proteinuric kidney disease[END_REF]. Sever et al. identified a cathepsin L cleavage site at positions 355-360 in the middle domain (Figure 1B). In a mouse model of kidney disease, cathepsin L induction leads to the cleavage of the cytoplasmic DNM2 and then actin reorganization in renal podocytes, filtration impairment and proteinuria [START_REF] Sever | Proteolytic processing of dynamin by cytoplasmic cathepsin L is a mechanism for proteinuric kidney disease[END_REF]. It remains to be determined whether such proteolytic regulation occurs in other tissues.

Phosphorylation of DNM1 by protein kinase C (PKC) increases Ca 2+ binding to the protein, which in turn inhibits DNM1 stimulated GTPase activity [START_REF] Liu | Calcium binds dynamin I and inhibits its GTPase activity[END_REF]. Although DNM2 is not a substrate of PKC and does not bind Ca 2+ [START_REF] Sontag | Differential expression and regulation of multiple dynamins[END_REF] it was demonstrated that Ca 2+ also inhibits 6. Durieux DNM2 GTPase activity (IC 50 =150µM) and receptor mediated endocytosis in Hela cells [START_REF] Cousin | Ca(2+) influx inhibits dynamin and arrests synaptic vesicle endocytosis at the active zone[END_REF]. This may have physiological importance in excitable cells like neurons and muscle fibers.

It is still largely unknown how the expression of DMN2 is regulated. In rat, DNM2 is up-regulated during normal pancreatic development after birth [START_REF] Cook | Upregulation of dynamin II expression during the acquisition of a mature pancreatic acinar cell phenotype[END_REF] but not in the liver [START_REF] Cook | Identification of dynamin 2, an isoform ubiquitously expressed in rat tissues[END_REF] or the brain [START_REF] Cook | Three dynamin-encoding genes are differentially expressed in developing rat brain[END_REF]. In mouse, treatment with opioid agonist results in increased DNM2 protein content in the spinal cord [START_REF] Zhang | Continuous opioid agonist treatment dosedependently regulates mu-opioid receptors and dynamin-2 in mouse spinal cord[END_REF][START_REF] Yoburn | Opioid agonist and antagonist treatment differentially regulates immunoreactive mu-opioid receptors and dynamin-2 in vivo[END_REF] whereas opioid antagonist decreases DNM2 abundance [START_REF] Yoburn | Opioid agonist and antagonist treatment differentially regulates immunoreactive mu-opioid receptors and dynamin-2 in vivo[END_REF][START_REF] Rajashekara | Chronic opioid antagonist treatment dose-dependently regulates mu-opioid receptors and trafficking proteins in vivo[END_REF]. These changes in the level of DNM2 expression are inversely correlated with opioid receptor density at the plasma membrane, suggestive of feed-back regulation. A similar type of regulation has also been described in brain [START_REF] Schroeder | Regulation of dynamin 2 and G protein-coupled receptor kinase 2 in rat nucleus accumbens during acute and repeated cocaine administration[END_REF].

DNM2 function

1. Endocytosis. DNM2 has been implicated in the formation of clathrin-coated pits [START_REF] Warnock | Ubiquitously expressed dynamin-II has a higher intrinsic GTPase activity and a greater propensity for self-assembly than neuronal dynamin-I[END_REF]. In the cytosol, DNM2 forms a complex with sorting nexin 9 (SNX9) and fructose-1,6bisphosphate aldolase [START_REF] Lundmark | Sorting Nexin 9 Participates in Clathrin-mediated Endocytosis through Interactions with the Core Components[END_REF][START_REF] Lundmark | Regulated membrane recruitment of dynamin-2 mediated by sorting nexin 9[END_REF]. Phosphorylation of SNX9 releases aldolase from the SNX9-DNM2 complex which is now competent for membrane targeting [START_REF] Lundmark | Regulated membrane recruitment of dynamin-2 mediated by sorting nexin 9[END_REF][START_REF] Lundmark | Expression and properties of sorting nexin 9 in dynamin-mediated endocytosis[END_REF]. DNM2 anchorage to the membrane occurs via interaction with PI4,5P2 membrane phosphoinositide [START_REF] Zoncu | Loss of endocytic clathrin-coated pits upon acute depletion of phosphatidylinositol 4,5-bisphosphate[END_REF] and BAR (Bin1/Amphiphysin/RVS167) domain proteins; amphiphysin 1, amphiphysin 2, and SNX9 (Table 1) in curved sites of the membranes. Subsequently, DNM2 co-localizes with clathrin before and during the internalization of the coated vesicle [START_REF] Rappoport | Real-time analysis of clathrin-mediated endocytosis during cell migration[END_REF]. This suggests that DNM2 may play a role not only in the release, but also in the first steps of vesicle formation, as recently shown in the turnover of intermediates during the maturation of clathrin-coated pits [START_REF] Loerke | Cargo and dynamin regulate clathrin-coated pit maturation[END_REF]. During this process, DNM2 forms an oligomer helical structure around the neck of the nascent vesicles [START_REF] Warnock | Ubiquitously expressed dynamin-II has a higher intrinsic GTPase activity and a greater propensity for self-assembly than neuronal dynamin-I[END_REF] and GTP hydrolysis is associated with the release of the vesicles.

DNM2 is also involved in clathrin-independent endocytosis by its role in the formation of the phagosomes in macrophages and Sertoli cells [START_REF] Gold | Dynamin 2 is required for phagocytosis in macrophages[END_REF][START_REF] Otsuka | Dynamin 2 is required for actin assembly in phagocytosis in Sertoli cells[END_REF] and caveolae in hepatocytes and endothelial cells [START_REF] Henley | Dynamin-mediated internalization of caveolae[END_REF][START_REF] Oh | Dynamin at the neck of caveolae mediates their budding to form transport vesicles by GTP-driven fission from the plasma membrane of endothelium[END_REF]. Predescu et al. described a protein complex, including DNM2, intersectin and SNAP-23, that was important for the fission and internalization of caveolae [START_REF] Predescu | Intersectin regulates fission and internalization of caveolae in endothelial cells[END_REF]. In caveolae, DNM2 also interacts with endothelial nitric-oxide synthase (eNOS) in bovine aortic endothelial cells [START_REF] Cao | Direct interaction between endothelial nitric-oxide synthase and dynamin-2. Implications for nitricoxide synthase function[END_REF] where DNM2 may regulate eNOS activation and the NO signalling cascade [START_REF] Cao | Direct interaction between endothelial nitric-oxide synthase and dynamin-2. Implications for nitricoxide synthase function[END_REF][START_REF] Chatterjee | Inhibition of GTPdependent vesicle trafficking impairs internalization of plasmalemmal eNOS and cellular nitric oxide production[END_REF][START_REF] Maniatis | Novel mechanism of endothelial nitric oxide synthase activation mediated by caveolae internalization in endothelial cells[END_REF][START_REF] Sanchez | Internalization of eNOS and NO delivery to subcellular targets determine agonist-induced hyperpermeability[END_REF]. DNM2 also participates in coat-independent endocytosis processes, i.e. micropinocytosis and macropinocytosis, by which fluid droplets and specific membrane components are internalized [START_REF] Cao | Dynamin 2 mediates fluid-phase micropinocytosis in epithelial cells[END_REF][START_REF] Liu | Isoform and splice-variant specific functions of dynamin-2 revealed by analysis of conditional knock-out cells[END_REF].

Altogether, these data demonstrate a role for DNM2 in clathrin-and caveolaedependent endocytosis as well as coat-independent endocytosis, and in regulating several important cellular processes including signal transduction, cholesterol homeostasis, plasma membrane composition and turnover, cell migration and entry of pathogens.

Intracellular membrane trafficking. DNM2 targets to the Golgi apparatus where it is

predominantly localized in the trans-Golgi network (TGN) [START_REF] Maier | Dynamin II binds to the trans-Golgi network[END_REF]. Anti-DNM2 antibody injection and over-expression of DNM2 mutants impair vesicle formation from the TGN [START_REF] Jones | Role of dynamin in the formation of transport vesicles from the trans-Golgi network[END_REF][START_REF] Kreitzer | Kinesin and dynamin are required for post-Golgi transport of a plasma-membrane protein[END_REF][START_REF] Yang | Dynamin II regulates hormone secretion in neuroendocrine cells[END_REF]. Association of DNM2 with cortactin, by an arf-1 and actin-dependent mechanism, and with syndapin 2, is required for trafficking of nascent vesicles from the TGN [START_REF] Cao | Actin and Arf1-dependent recruitment of a cortactin-dynamin complex to the Golgi regulates post-Golgi transport[END_REF][START_REF] Kessels | Complexes of syndapin II with dynamin II promote vesicle formation at the trans-Golgi network[END_REF]. DNM2 is also found at the clathrin-coated buds of early endosomes [START_REF] Van Dam | Dynamin-dependent transferrin receptor recycling by endosome-derived clathrin-coated vesicles[END_REF] and in late endosomes in Hela cells, located to the tubulo-vesicular appendices [START_REF] Nicoziani | Role for dynamin in late endosome dynamics and trafficking of the cation-independent mannose 6-phosphate receptor[END_REF]. In these two cases, interfering DNM2 mutant impairs the recycling of components from the endosomal system towards the plasma membrane or TGN [START_REF] Van Dam | Dynamin-dependent transferrin receptor recycling by endosome-derived clathrin-coated vesicles[END_REF][START_REF] Nicoziani | Role for dynamin in late endosome dynamics and trafficking of the cation-independent mannose 6-phosphate receptor[END_REF]. These data highlight the role of DNM2 in the secretory pathway and in the sorting of cell components from the Golgi apparatus and endosomal compartment.

Exocytosis.

In neuroendocrine cells, monomers of DNM2 are associated with the membrane of secretory chromaffin granules in a complex including syntaxin, a member of the exocytosis machinery [START_REF] Galas | Presence of dynamin--syntaxin complexes associated with secretory granules in adrenal chromaffin cells[END_REF]. DNM2 also interacts and co-localizes with complexin I, a SNARE regulatory protein [START_REF] Zhao | Dynamin 2 associates with complexins and is found in the acrosomal region of mammalian sperm[END_REF]. Therefore, DNM2 may participate in endocytosis-exocytosis coupling as suggested in mouse pancreatic β-cells [START_REF] Min | Dynamin is functionally coupled to insulin granule exocytosis[END_REF]. However, a role for DNM2 in exocytosis alone has been reported. During cell-mediated killing by natural killer (NK) cells, DNM2 colocalizes with lytic granules after NK cell activation, and is required for fusion of the granules with the plasma membrane [START_REF] Arneson | Dynamin 2 regulates granule exocytosis during NK cell-mediated cytotoxicity[END_REF]. Similarly in macrophages, focal exocytosis is blocked after anti-DNM2 antibody microinjection [START_REF] Di | Dynamin regulates focal exocytosis in phagocytosing macrophages[END_REF] and DNM2 GTPase activity regulates the fusion of secretory vesicles at the plasma membrane [START_REF] Jaiswal | Exocytosis of post-Golgi vesicles is regulated by components of the endocytic machinery[END_REF]. Further studies will be necessary to precisely identify the molecular role played by DNM2 in the exocytosis machinery.

Actin network.

Actin-based dynamic processes are crucial for late stage endocytosis and vesicle formation, and DNM2 interacts with several actin-binding proteins. Direct interactions have been identified with Abp1 (actin-binding protein 1) [START_REF] Kessels | Mammalian Abp1, a signal-responsive F-actin-binding protein, links the actin cytoskeleton to endocytosis via the GTPase dynamin[END_REF] and cortactin [START_REF] Mcniven | Regulated interactions between dynamin and the actin-binding protein cortactin modulate cell shape[END_REF][START_REF] Schafer | Dynamin2 and Cortactin Regulate Actin Assembly and Filament Organization[END_REF]. Abp1 is a Src kinase which provides a physical bridge between the endocytosis machinery and the cortical actin network, and cortactin is a component of the clathrin-mediated endocytosis machinery [START_REF] Cao | Cortactin is a component of clathrin-coated pits and participates in receptor-mediated endocytosis[END_REF]. In addition, DNM2 is a component of actin-based motile vesicles (actin comets) [START_REF] Orth | The large GTPase dynamin regulates actin comet formation and movement in living cells[END_REF][START_REF] Lee | Dynamin at actin tails[END_REF] which provide transport through the cytoplasm for vesicles formed by the Golgi apparatus or plasma membrane. Expression of DNM2-K44A mutant, defective for the GTPase activity, strongly reduces the number, length and velocity of the comets [START_REF] Orth | The large GTPase dynamin regulates actin comet formation and movement in living cells[END_REF][START_REF] Lee | Dynamin at actin tails[END_REF].

Interaction between DNM2 and the actin cytoskeleton may have another cytoskeletal role such as in the formation of membrane tubules and protrusions. Furthermore, a recent study showed the crucial function played by the DNM2-cortactin complex in the global 9. Durieux organisation and remodelling of the actomyosin cytoskeleton [START_REF] Mooren | Dynamin2 GTPase and cortactin remodel actin filaments[END_REF]. In addition, DNM2 is present in cortical ruffles and lamellipodia, both important in cell migration [START_REF] Cao | Differential distribution of dynamin isoforms in mammalian cells[END_REF][START_REF] Mcniven | Regulated interactions between dynamin and the actin-binding protein cortactin modulate cell shape[END_REF]. The supramolecular complex including DNM2, cortactin and Arp2/3 mediates the reorganization of actin allowing lamellipodia formation at the leading edge of migrating cells [START_REF] Krueger | A Dynamin-Cortactin-Arp2/3 Complex Mediates Actin Reorganization in Growth Factor-stimulated Cells[END_REF].

Disruption of DNM2 function by DNM2-K44A mutant or small interfering RNA (siRNA), inhibits the formation of lamellipodia [START_REF] Schlunck | Modulation of Rac localization and function by dynamin[END_REF]. Similarly, under PDGF stimulation, DNM2 is concentrated within the leading ruffles of migrating fibroblasts where it co-localizes with cortactin [START_REF] Mcniven | Regulated interactions between dynamin and the actin-binding protein cortactin modulate cell shape[END_REF]. To allow cell migration, DNM2 participates in disassembly of focal adhesions, as well as β-integrin internalization at the rear of the cell [START_REF] Ezratty | Microtubule-induced focal adhesion disassembly is mediated by dynamin and focal adhesion kinase[END_REF][START_REF] Vassilieva | Lipid rafts mediate internalization of beta1-integrin in migrating intestinal epithelial cells[END_REF]. Additionally, DNM2 is enriched in specialized membrane protrusions such as podosomes and invadipodia.

Podosomes represent attachment sites between cells and substratum [START_REF] Ochoa | A functional link between dynamin and the actin cytoskeleton at podosomes[END_REF] and invadipodia are focalized matrix degradation sites [START_REF] Baldassarre | Dynamin participates in focal extracellular matrix degradation by invasive cells[END_REF]. Inhibition of DNM2 diminishes the amount of such structures [START_REF] Baldassarre | Dynamin participates in focal extracellular matrix degradation by invasive cells[END_REF]. It has also been shown that DNM2 regulates the formation of actin stressfibers by interaction with the cell surface heparin sulphate proteoglygan syndecan-4 [START_REF] Yoo | Dynamin II interacts with syndecan-4, a regulator of focal adhesion and stress-fiber formation[END_REF] Expression of DNM2-mutant, truncated for the PRD domain mediating interaction with cortactin, increases the number of actin-stress fibers, which is associated with abnormal cell shape [START_REF] Mcniven | Regulated interactions between dynamin and the actin-binding protein cortactin modulate cell shape[END_REF].

Microtubule network and MTOC.

It is noteworthy that the first isolated dynamin, i.e. DNM1, was initially reported as a microtubule associated protein [START_REF] Shpetner | Identification of dynamin, a novel mechanochemical enzyme that mediates interactions between microtubules[END_REF]. A similar binding property has been evidenced for DNM2 in vitro [START_REF] Warnock | Ubiquitously expressed dynamin-II has a higher intrinsic GTPase activity and a greater propensity for self-assembly than neuronal dynamin-I[END_REF] and the binding region was located to the PRD [START_REF] Lin | Phosphatidylinositol (4,5)bisphosphate-dependent activation of dynamins I and II lacking the proline/arginine-rich domains[END_REF][START_REF] Hamao | New function of the proline rich domain in dynamin-2 to negatively regulate its interaction with microtubules in mammalian cells[END_REF]. More recently, it was shown that down-regulation of DNM2 by siRNA increases the amount of acetylated tubulin, a more stable form of tubulin in microtubules, and reduces their growing capacity [START_REF] Tanabe | Dynamic instability of microtubules requires dynamin 2 and is impaired in a Charcot-Marie-Tooth mutant[END_REF], suggesting that DNM2 may regulate the polymerizationdepolymerization equilibrium of microtubules. Through its interaction with microtubules, 10. Durieux DNM2 appears involved in Golgi apparatus cohesion [START_REF] Tanabe | Dynamic instability of microtubules requires dynamin 2 and is impaired in a Charcot-Marie-Tooth mutant[END_REF]. Moreover, DNM2 has been identified as a component of the centrosome, the main microtubule organizing center (MTOC), where it binds to γ-tubulin [START_REF] Thompson | Dynamin 2 binds gamma-tubulin and participates in centrosome cohesion[END_REF]. The centrosome consists of a pair of centrioles embedded in a filamentous pericentriolar matrix, where γ-tubulin is essential for microtubule nucleation. The function played by DNM2 at the centrosome is still unknown, but DNM2 silencing by siRNA suggests a role in centrosome splitting [START_REF] Thompson | Dynamin 2 binds gamma-tubulin and participates in centrosome cohesion[END_REF]. Likewise, participation of DNM2 in all the phases of mitosis has also been reported. DNM2 is detected in the 2 MTOC during early prophase, along the mitotic spindle during metaphase and in the spindle midzone region during anaphase and early telophase [START_REF] Thompson | The large GTPase dynamin associates with the spindle midzone and is required for cytokinesis[END_REF]. Thereafter, DNM2 is accumulated at the intracellular bridge where the final separation occurs. The time required for separation of the two daughter cells is longer in DNM2 knock-out cells [START_REF] Liu | Isoform and splice-variant specific functions of dynamin-2 revealed by analysis of conditional knock-out cells[END_REF]. Taken together, these data suggest that DNM2 may regulate microtubule-dependent processes by acting on microtubule dynamics and organisation.

Apoptosis.

In order to establish a stable Hela cell line over-expressing DNM2 isoform 2, Fish et al. have reported a significant cell toxicity in dividing cells [START_REF] Fish | Evidence that dynamin-2 functions as a signaltransducing GTPase[END_REF]. The cytotoxicity occurred via induction of apoptosis by a p53-dependent mechanism. Similar results were gained in vascular smooth muscle cells [START_REF] Kashiwakura | Dynamin-2 regulates oxidized low-density lipoprotein-induced apoptosis of vascular smooth muscle cell[END_REF]. The capacity to trigger apoptosis appears DNM2-specific as DNM1 over-expression does not induce apoptosis [START_REF] Fish | Evidence that dynamin-2 functions as a signaltransducing GTPase[END_REF]. The GTPase domain of DNM2 is crucial to induce apoptosis [START_REF] Soulet | Domain requirements for an endocytosisindependent, isoform-specific function of dynamin-2[END_REF]. Besides, a point mutation (p.I684K) in the DNM2 GED enhances the apoptosis induction by the wild-type DNM2 suggesting that GED negatively regulates this DNM2 function [START_REF] Soulet | Domain requirements for an endocytosisindependent, isoform-specific function of dynamin-2[END_REF]. Mitochondria are key actors in apoptosis and, interestingly, DNM2 has been detected in isolated mitochondria from bovine lymphoblastoid BL-3 cells [START_REF] Atapattu | Dynamin-2dependent targeting of mannheimia haemolytica leukotoxin to mitochondrial cyclophilin D in bovine lymphoblastoid cells[END_REF]. However, to our knowledge, such localization has not been reported in other cell lines or tissues. DNM2 also regulates the apoptosis-inducing Fas-Fas 11. Durieux ligand pathway by facilitating the transport of Fas from the trans-Golgi network to the plasma membrane [START_REF] Ivanov | Opposite roles of FAP-1 and dynamin in the regulation of Fas (CD95) translocation to the cell surface and susceptibility to Fas ligandmediated apoptosis[END_REF].

Specific functions of DNM2 isoforms.

In a cultured rat epithelial cell line (clone 9), both DNM2 isoforms 1 and 3 show punctuate labeling of clathrin heavy chain-positive or -negative structures, but only isoform 1, with the GEIL sequence in the middle domain, appears located to the Golgi apparatus [START_REF] Cao | Differential distribution of dynamin isoforms in mammalian cells[END_REF]. These data suggest a role for the GEIL sequence in targeting to the Golgi apparatus. However, cell-type specificity probably exists, as isoforms without the GEIL sequence were also shown to be targeted to the Golgi apparatus in MDCK cells [START_REF] Kreitzer | Kinesin and dynamin are required for post-Golgi transport of a plasma-membrane protein[END_REF],

3T3L1 adipocytes [START_REF] Kao | Functional comparison of the role of dynamin 2 splice variants on GLUT-4 endocytosis in 3T3L1 adipocytes[END_REF] and fibroblastoid-like cells derived from mouse embryonic stem cells [START_REF] Liu | Isoform and splice-variant specific functions of dynamin-2 revealed by analysis of conditional knock-out cells[END_REF]. Nevertheless, this possible differential localization argues for distinct functions. Indeed, in clone 9 cells, the K44A mutants of isoforms 2 and 4 are able to inhibit fluid-phase endocytosis, whereas the mutant forms of isoforms 1 and 3 do not [START_REF] Cao | Dynamin 2 mediates fluid-phase micropinocytosis in epithelial cells[END_REF], and are more potent inhibitors of clathrin-mediated endocytosis. Similarly in a hepatocyte cell line, the K44Aisoform 1 inhibits caveolae-dependent internalization, but not the other K44A mutant isoforms [START_REF] Yao | Caveolin-1 interacts directly with dynamin-2[END_REF]. In fibroblastoid-like cells derived from mouse embryonic stem cells, isoforms 2 and 4 are the most efficient at rescuing export from the Golgi in DNM2 knock-out cells [START_REF] Liu | Isoform and splice-variant specific functions of dynamin-2 revealed by analysis of conditional knock-out cells[END_REF]. Altogether, these data suggest a preferential involvement of isoforms 1 and 3 in clathrinand caveolae-dependent endocytosis, whereas isoforms 2 and 4 participate in uncoated endocytosis and trafficking from the Golgi apparatus. However, cell-type specificity also occurs as the four isoforms exhibit a similar subcellular distribution in 3T3L1 adipocytes and dominant negative mutants of each isoform similarly affect basal and insulin-stimulated GLUT4 trafficking [START_REF] Kao | Functional comparison of the role of dynamin 2 splice variants on GLUT-4 endocytosis in 3T3L1 adipocytes[END_REF].

DNM2 and human disease

Durieux

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][START_REF] Fabrizi | Two novel mutations in dynamin-2 cause axonal Charcot-Marie-Tooth disease[END_REF][START_REF] Bitoun | A novel mutation in the dynamin 2 gene in a Charcot-Marie-Tooth type 2 patient: Clinical and pathological findings[END_REF][START_REF] Gallardo | Magnetic resonance imaging findings of leg musculature in Charcot-Marie-Tooth disease type 2 due to dynamin 2 mutation[END_REF][START_REF] Claeys | Phenotypic spectrum of dynamin 2 mutations in Charcot-Marie-Tooth neuropathy[END_REF] and autosomal dominant centronuclear myopathy (CNM) [START_REF] Bitoun | Mutations in dynamin 2 cause dominant Centronuclear Myopathy[END_REF][START_REF] Bitoun | Dynamin 2 mutations associated with human diseases impair clathrin-mediated receptor endocytosis[END_REF][START_REF] Echaniz-Laguna | Subtle central and peripheral nervous system abnormalities in a family with centronuclear myopathy and a novel dynamin 2 gene mutation[END_REF][START_REF] Bitoun | Dynamin 2 mutations cause sporadic centronuclear myopathy with neonatal onset[END_REF][START_REF] Bitoun | A new centronuclear myopathy phenotype due to a novel dynamin 2 mutation[END_REF]. The 19 reported heterozygous mutations affect only the Middle domain, the PH domain and the GED (figure 1B). DNM2-related CNM is a slowly progressive congenital myopathy characterized by frequent centrally located nuclei in muscle fibers. The most common clinical features are delayed motor milestones, facial and generalized muscle weakness, ptosis and ophthalmoplegia [100]. Nevertheless, the severity of DNM2-CNM is variable, ranging from severe neonatal to mild late-onset forms. DNM2-CMT is a peripheral neuropathy characterized by progressive muscle weakness and atrophy. DNM2 mutations can cause axonal CMT (CMT2) and dominant intermediate CMT (DI-CMT-B). In CMT2, the nerve conduction velocity is usually normal (> 38 m/s for the median nerve, which represents the cut-off value between the demyelinating CMT1 and the axonal CMT2). In the group of rare patients affected by DI-CMT-B, the nerve conduction velocity values are intermediate (between 25 and 45 m/s). In some CMT patients, neuropathy is associated with neutropenia [START_REF] Zuchner | Mutations in the pleckstrin homology domain of dynamin 2 cause dominant intermediate Charcot-Marie-Tooth disease[END_REF][START_REF] Bitoun | A novel mutation in the dynamin 2 gene in a Charcot-Marie-Tooth type 2 patient: Clinical and pathological findings[END_REF][START_REF] Claeys | Phenotypic spectrum of dynamin 2 mutations in Charcot-Marie-Tooth neuropathy[END_REF] but this association has not been described in DNM2-CNM patients. Clinical overlap could exist in some patients, but the majority of patients are affected by a tissuespecific disorder targeting either skeletal muscle or peripheral nerve [START_REF] Bitoun | A novel mutation in the dynamin 2 gene in a Charcot-Marie-Tooth type 2 patient: Clinical and pathological findings[END_REF][START_REF] Bitoun | A new centronuclear myopathy phenotype due to a novel dynamin 2 mutation[END_REF]100]. Among the 19 distinct DNM2 mutations identified to date, there are no mutations common to the two disorders and no mutations in the regions of variation due to alternative splicing. No clear genotype-phenotype relationship can be generated, except for the de novo mutations located in the C-terminal part of the PH domain, which are all associated with a severe neonatal CNM phenotype [START_REF] Bitoun | Dynamin 2 mutations cause sporadic centronuclear myopathy with neonatal onset[END_REF]. In these patients, the phenotype progressively improves, suggesting compensatory mechanisms. Of note is that the CMT-mutation G358R, is located in the 13. Durieux cathepsin L cleavage site [START_REF] Sever | Proteolytic processing of dynamin by cytoplasmic cathepsin L is a mechanism for proteinuric kidney disease[END_REF] and therefore may impair the regulation of DNM2 by proteolysis.

More recently, the DNM2 gene has been described as a susceptibility gene for lateonset Alzheimer disease [101], and DNM2 expression was subsequently found to be mutations may lead to a decrease in receptor stimulated signaling as shown for the MAPK ERK1/2 pathway [START_REF] Bitoun | Dynamin 2 mutations associated with human diseases impair clathrin-mediated receptor endocytosis[END_REF]108]. On the other hand, DNM2 mutations may lead to a prolonged 14. Durieux half-life of various proteins at the cell surface due to a defect in protein removal, similar to that as suggested for the EnaC sodium channel [109, 110], KCNQ1 potassium channel subunits [111] or the GLUT4 glucose transporter [START_REF] Kao | Functional comparison of the role of dynamin 2 splice variants on GLUT-4 endocytosis in 3T3L1 adipocytes[END_REF][112][113][114]. A deregulation of glucose transport in patients with DNM2 mutations could have a strong impact on muscle fibers given their high glucose consumption. Of note, the microtubule network plays an important role in GLUT4 trafficking [115] and a decrease in muscle weight has been reported for a transgenic mouse over-expressing GLUT4 [116].

To date, the impact of disease-associated DNM2 mutants on other membrane trafficking processes in which DNM2 is involved, especially in endosomal and Golgi pathways, has not been studied. In cells over-expressing the K44A-DNM2 mutant, an impairment in the trafficking from the Golgi apparatus has been reported [117,118]. We cannot exclude a participation of these pathways in the pathomechanisms of DNM2-related disorders.

2.

Cytoskeleton. In DNM2-CNM, the majority of patients harbour a mutation in the middle domain, which is essential for the centrosomal localization of DNM2 and for its interaction with γ-tubulin [START_REF] Thompson | Dynamin 2 binds gamma-tubulin and participates in centrosome cohesion[END_REF]. Previous results in skin fibroblasts indicate that transfected GFP-DNM2mutants failed to be correctly targeted to the centrosome, suggesting that DNM2 mutations might cause CNM by interfering with centrosome function [START_REF] Bitoun | Mutations in dynamin 2 cause dominant Centronuclear Myopathy[END_REF]. In addition, CMT-related DNM2 mutants can disorganize the microtubule cytoskeleton [START_REF] Zuchner | Mutations in the pleckstrin homology domain of dynamin 2 cause dominant intermediate Charcot-Marie-Tooth disease[END_REF] and one particular CMTmutant was shown to impair microtubule-dependent membrane transport [START_REF] Tanabe | Dynamic instability of microtubules requires dynamin 2 and is impaired in a Charcot-Marie-Tooth mutant[END_REF]

Concluding remarks and open questions

Given the numerous distinct functions in which the ubiquitously expressed DNM2 is involved, the identification of pathophysiological mechanisms will be a challenge. The phenotypes encountered in CNM and CMT patients are probably due to impairment of the various functions of the protein. DNM2 is engaged in numerous protein-protein interactions (Table 1) but the relevance of these interactions in skeletal muscle and the nervous system, is largely unexplored. For example, DNM2 interacts with β-catenin in rat testis in relation to the maintenance of the blood-testis barrier integrity [123] but no data are available on this interaction in skeletal muscle where β-catenin plays an important role [124,125]. Another essential unresolved question is whether each particular mutation can similarly affect the functions of the four DNM2 isoforms. Finally, whereas some data emerge on the impact of disease-related DNM2 mutations on the microtubule network, their impact on the actin cytoskeleton is totally unknown. Future developments and characterization of animal models will certainly be useful to better determine the main functions of DNM2 in vivo, especially in skeletal muscle and peripheral nerves where membrane trafficking displays different characteristics depending to the length of the cells.

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 1 decreased in the brains of late-onset Alzheimer patients [102]. Cognitive impairments have been reported in some CNM patients harboring the p.E368Q [97], p.R465W [103; Family 1] and p.R369Q [103; Families 2 and 3] DNM2 mutations. Future studies will be necessary to determine the prevalence of central nervous system involvement in DNM2-related diseases. Pathophysiological hypotheses Membrane trafficking and signaling pathway hypothesis. In addition to the DNM2 mutations in autosomal dominant CNM, mutations in the BIN1 gene encoding amphiphysin 2, a partner of DNM2 in the endocytic process, can cause the autosomal recessive form of the disease [104]. This suggests that endocytic impairment is implicated in the pathophysiological mechanisms of autosomal CNM. Indeed, impairment of clathrin-mediated endocytosis was also reported in cultured cells expressing CNM-or CMT-DNM2 mutants [2, 15, 83]. The crucial question which remains to be explored is how a defect in clathrin-mediated endocytosis can alter the cell function. Endocytosis (via clathrin-coated vesicles, caveolae or uncoated vesicles) regulates fundamental processes including nutrient uptake, membrane composition and turnover, cell adhesion or migration, pathogen entry, and signaling of G protein-coupled receptors, tyrosine kinase receptors or channels [105-107]. Thus, DNM2 mutations may have a large spectrum of functional consequences. On one hand, DNM2

  . During skeletal muscle differentiation a profound reorganization of the microtubule network occurs, changing from a classical network centered on the juxtanuclear centrosome in myoblasts, to a longitudinal organization along the axis of differentiated myotubes [119]. One can hypothesize that mutated-DNM2 can impair functions associated with this specific 15. Durieux cytoskeleton reorganization. In addition to their roles in intracellular trafficking, the microtubule and actin networks regulate cellular architecture including nuclear positioning [120, 121]. Thus, cytoskeletal impairment may be implicated in the abnormal central location of the nuclei in the muscle fibers in CNM. In CMT, DNM2 mutations could also induce a destabilization of the microtubule network leading to abnormal axonal transport and protein trafficking, a pathophysiological mechanism described previously in various forms of CMT [122].

Figure 1 :

 1 Figure 1: DNM2 gene organization and mutations.

Figure 2 :

 2 Figure 2: DNM2 cellular functions

Acknowledgements

We thank Dr Rachel Peat and Dr Edgar Gomes for helpful advices. Anne-Cécile Durieux was a recipient of fellowship from the Association Française contre les Myopathies (AFM).

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