REVIEW ARTICLE Vesicular traffic in cell navigation Kathleen Zylbersztejn 1,2 and Thierry Galli 1,2 1 ‘Membrane Traffic in Neuronal & Epithelial Morphogenesis’, INSERM ERL U950, Paris, France 2 Program in Development & Neurobiology, Institut Jacques Monod, CNRS UMR 7592, Paris Diderot University, Paris, France Cell navigation Cell navigation is an important process not only dur- ing both development and adulthood in metazoans, but also during chemotaxis in protozoans. When they are developing, cells proliferate in defined areas and often migrate towards others, where they fully differen- tiate. In adults, numerous cell types, such as epithelial cells during wound closure, neutrophils, phagocytes, fibroblasts, fast-moving fish keratocytes [1], spreading tumor cells or olfactory neurons, among many others, are able to migrate in response to environmental cues. Unicellular eukaryotes such as amoebas also migrate to avoid toxin compounds and move towards a supply of nutrients. Certain cells are also able to send out cytoplasmic extensions towards distant destinations. This applies particularly to neurons, which send out axons and dendrites to generate contacts with other neurons or non-neuronal cells. This process is repeated in adults during peripheral nerve regeneration and the differentiation of olfactory neurons. Both cell migra- tion and neuronal growth cone navigation rely on the same basic steps defined by Sheetz et al. [2]: (a) exten- sion of the leading edge; (b) adhesion to matrix con- tacts; (c) contraction of the cytoplasm; (d) release from contact sites; and (e) recycling of membrane receptors from the rear to the front of the migrating cell or the navigating growth cone. It has been known for some time that both extracellular and intracellular molecular mechanisms operate during cell navigation. This is par- ticularly important for the ability of a cell to sense the environment. In this context, extracellular soluble and Keywords axon guidance; axon outgrowth; epithelial migration; SNAREs; vesicular traffic Correspondence T. Galli, Institut Jacques Monod, Bat. Buffon, 15 rue He ´ le ` ne Brion, 75205 Paris, Cedex 13, France Fax: +33 157 278 036 Tel: +33 157 278 039 E-mail: thierry.galli@inserm.fr Website: http://sites.google.com/site/ insermu950/ (Received 17 February 2011, revised 22 April 2011, accepted 6 May 2011) doi:10.1111/j.1742-4658.2011.08168.x Cell navigation is the process whereby cells or cytoplasmic extensions are guided from one point to another in multicellular organisms or, in the case of unicellular eukaryotic organisms, in the environment. Recent work has demonstrated that membrane trafficking plays an important role in this process. Here, we review the role of soluble N-ethylmaleimide-sensitive fusion attachment protein (SNAP) receptors (SNAREs), which constitute the core machinery for membrane fusion and are essential for intracellular vesicular trafficking. We discuss the important functions of several vesicu- lar- and target-SNAREs, in particular vesicular-associated membrane pro- teins 1, 2, 3, 4 and 7; vti1a ⁄ b; SNAP23 and SNAP25; and syntaxins 1, 3, 6 and 13. We conclude that endosomal SNAREs are important for cell navi- gation, a concept that opens avenues for fundamental research. There are also possible therapeutic applications because some of these SNAREs are the targets of clostridial neurotoxins. Abbreviations EGFR, epidermal growth factor receptor; SNAP, soluble N-ethylmaleimide-sensitive fusion attachment protein; SNARE, soluble N-ethylmaleimide-sensitive fusion attachment protein receptor; t-SNARE, target SNARE; VAMP, vesicular-associated membrane protein; v-SNARE, vesicular SNARE. FEBS Journal 278 (2011) 4497–4505 ª 2011 FEBS. No claim to original French government works 4497 cell-attached molecules can be chemo-attractive or -repulsive. They signal through receptors that trans- duce intracellular signals. The latter then translate into several regulatory pathways involving the cytoskeleton and gene expression [3]. Cell migration, similar to axo- nal guidance, depends on attractive and repulsive cues [4], and the same molecules that guide axons and dendrites, such as semaphorins, also regulate cell migration [5]. These two processes thus appear to share many common molecular mechanisms. Recent work has highlighted the important role of intracellu- lar membrane trafficking in cell navigation. In the present review, we focus only on the role of soluble N-ethylmaleimide-sensitive fusion attachment protein (SNAP) receptors (SNAREs) because they are major players in intracellular membrane trafficking. SNAREs Membrane trafficking operates in three main steps: (a) the formation of a vesicular or tubular intermediate generated from a donor membrane; (b) the translo- cation of this intermediate by transport along actin microfilaments or microtubules; and (c) the docking and fusion with an acceptor membrane. The last step largely depends on SNAREs for membrane fusion in eukaryotes. SNAREs are classically classified into two categories: vesicular SNAREs (v-SNAREs; vesicular- associated membrane proteins, VAMPs), localized in the donor membrane, and target SNAREs (t-SNAREs), localized in the acceptor compartment. The pairing of v- and t-SNAREs between two opposing membranes leads to the formation of a parallel a-heli- cal bundle composed of four chains, called the SNARE complex, and subsequently to membrane fusion. The central role played by SNARE proteins in membrane trafficking is best exemplified by the role of clostridial neurotoxin targets (i.e. synaptobrevin2 ⁄ VAMP2, SNAP25 and syntaxin1) in the fusion of synaptic vesi- cles with the plasma membrane at the neuronal synapse [6]. Several v- and t-SNAREs are found in different intracellular membrane compartments and SNAREs play a conserved function in all membrane fusion events in eukaryotes [7] (Fig. 1). Biophysical experi- ments reconstituting membrane fusion in vitro show that the pairing of v- and t-SNAREs provides the energy for membrane fusion and operates when the membranes are < 10 nm apart [8]. The large number of studies on SNAREs indicate that these proteins are key players in intracellular membrane fusion, and that they act at a late stage of the process (i.e. once acceptor and donor membrane are in close proximity). Further evidence indicates that other proteins involved in their regulation or other steps of vesicular trafficking (i.e. budding, transport of membrane intermediates, dock- ing, priming) perform complementary functions. Role of SNAREs in cell signal-dependent migration The importance of adhesion and of both filamentous actin and microtubules in cell migration is clearly established [9]. Recent work has revealed that intracel- lular membrane trafficking plays an important function and may even regulate the localization of molecules that control cytoskeletal dynamics [10]. This picture has emerged from studies on endosomal SNAREs in migrating and invading cells both in vitro and in vivo during development [11]. Indeed, VAMP3 morpholinos lead to a defect in blastopore closure in Xenopus [11]. The main conclusion from several studies is that endosomal SNAREs regulate the recycling of integrins and ⁄ or the secretion of matrix proteases and that this, in turn, modifies the capacity of a cell capacity to adhere, spread, migrate and invade. More specifically, two v-SNAREs (i.e. VAMP3 and VAMP7) have been implicated (Fig. 2). Using both small interfering RNA and tetanus neu- rotoxin, which is a specific protease targeting VAMPs 1 to 3, VAMP3 was shown to mediate integrin recy- cling and also be required for cells to attach, spread and migrate [11–14]. Integrin recycling is also crucial for invasion [15]. Decreased VAMP3 expression was also found in samples presenting 1p deletion, suggest- ing a possible involvement in neuroblastoma tumori- genesis [16]. Interestingly, we and others found that VAMP3-deleted cells show impaired spreading on fibronectin [11] but more rapid attachment to collagen and other b1 integrin substrates, suggesting higher affinity for the b1 integrin substrate [17]. Initially, this may appear paradoxical, although it could suggest that integrin recycling is required for spreading and migra- tion but not necessarily for attachment. Indeed, migra- tion requires adhesion but is impaired by strong attachment [18]. Migration and spreading depend on both the clustering and activation state of integrins [19]. It is thus tempting to speculate that VAMP3 may be required for integrin clustering and ⁄ or activation. By contrast, by expressing the Longin negative regu- latory domain of VAMP7, it was shown that VAMP7 regulates exosome ⁄ lysosome and matrix metallopro- tease secretion [20], suggesting that VAMP7 may par- ticipate to matrix degradation and invasion. VAMP7 has been also implicated in chronic myeloid leukemia [21] and potentially in human cancers of the prostate [22]. Longin-expressing epithelial cells fail to repair Vesicular trafficking in cell navigation K. Zylbersztejn and T. Galli 4498 FEBS Journal 278 (2011) 4497–4505 ª 2011 FEBS. No claim to original French government works after mechanical wounding, further suggesting a role in lysosomal secretion [20]. Interestingly, VAMP7 also regulates epidermal growth factor receptor (EGFR) dynamics on the cell surface, clathrin-dependent endo- cytosis and signaling, through the exocytosis of CD82, a tetraspanin known to control EGFR localization in microdomains [23]. Recently, mutant p53 expression was shown to promote invasion, loss of directionality of migration and metastatic behavior, as well as to enhance integrin and EGFR trafficking and to result in constitutive activation of EGFR ⁄ integrin signaling [15]. Therefore, VAMP3 and VAMP7-dependent traf- ficking may interact in integrin and EGFR pathways in migrating cells. Both v-SNAREs cooperate with SNAP23, which is also involved in integrin recycling, cell spreading and migration. Similarly, the endosomal syntaxin13 is important for cell spreading, and the SNAP23–syntaxin13-VAMP3 complex is involved in extracellular matrix-induced lamellipodium formation. This complex functions in the trafficking of b1 integrin Nucleus Golgi ER Ly EE Lamellipodia protrusion Growing axon VAMP3 VAMP7 SNAP23 SNAP29 syntaxin3 syntaxin6 syntaxin12/13 VAMP2 VAMP4 VAMP7 SNAP23 SNAP25 syntaxin1 syntaxin3 syntaxin12/13 VAMP3 VAMP4 VAMP7 VAMP8 VAMP2 VAMP4 VAMP7 Fig. 1. SNAREs in a migrating cell and growing axon. Migrating cells and growing axons share similarities in their morphologies and the SNAREs involved. Several v-SNAREs were shown to be involved either in the growing protrusion (i.e. the lamellipodium in migrating cells or the axon in growing neurons). Several SNAREs also have functions in cell bodies. Listed are the v- and t-SNAREs that are involved in each process. ER, endoplasmic reticulum; Ly, lysosome; EE, early endosomes; , cleavable by clostridial neurotoxins. ER VAMP7 VAMP3 t-SNARE Metalloproteases Integrins t-SNARE VAMP7 t-SNARE CD82 ? EGF Activation, Diffusion Endocytosis of EGFR Hypothetical pathways Activated EGF Inactivated EGFR CD82 Integrin Metalloprotease Actin Nucleus Ly Golgi Direction of migration Lamellipodium RE ? RE VAMP3 VAMP7 t-SNARE Fig. 2. Roles of SNAREs in cell migration. VAMP3 is involved in the trafficking of integrins necessary for epithelial migration, whereas VAMP7 is necessary for the trafficking of metalloproteases. Both mediate trafficking at the leading edge. The potential role of SNAREs at the rear of the cell is not characterized. ER, endoplasmic reticulum; Ly, lysosome; RE, recycling endosome. K. Zylbersztejn and T. Galli Vesicular trafficking in cell navigation FEBS Journal 278 (2011) 4497–4505 ª 2011 FEBS. No claim to original French government works 4499 from a sorting endosome to a Rab11-containing recy- cling compartment. The SNAP23–VAMP4 complex is required for the formation of phorbol 12-myristate 13-acetate-induced F-actin rich membrane ruffling [24]. Finally, the endosomal and trans Golgi network syn- taxin6 is involved in vascular EGFR-induced cell pro- liferation and migration [25]. Syntaxin6 also forms SNARE complexes with VAMP3 and VAMP7 [26–28]. Thus, SNAREs regulating exocytosis and endocytosis may control the repertoire and density of many cell surface proteins and, in particular, integrins and EGFR, which are key players in the capacity of a cell to sense its environment. Exocytosis and endocytosis also regulate cell surface tension. The former decreases surface tension, whereas the latter increases surface tension, which may have profound effects on the capacity of a cell to remodel its shape during migration [29–31]. This view is particu- larly interesting if exocytosis and endocytosis are polar- ized along the migration axis (e.g. rear endocytosis and front exocytosis). This would generate a flux of intra- cellular membrane with profound effects on surface tension in the rear and front of the cell. It is tempting to speculate that VAMP3 and VAMP7 participate in integrin flux, a hypothesis that requires investigation. Role of SNAREs in neuronal growth cone navigation Although many analogies can be drawn between cell migration and neuronal growth cone navigation, partic- ularly the same five major steps proposed by Sheetz et al. [2] and their similar sets of guidance cues [3,32], there is a major difference. In the case of neuronal out- growth, nucleokinesis does not occur but, instead, the cell surface increases. It is still unclear what determines how and when a neuron will stop moving in toto and start growing neurites. The cell surface increase needed for neurite growth can be as high as 20% per day and total 1000-fold by the time the neuron is fully differenti- ated, which represents a truely herculean task for mem- brane biogenesis [33]. The biochemical nature of the secretory compartments contributing to growth is not fully known. It may contain Golgi-derived membranes [34] and specific microdomains, especially a glycosyl- phosphatidylinositol-anchored protein compartment, could be of particular relevance in this process [35]. Although endocytosis compensates for exocytosis in cells at equilibrium, the growth of neurites requires a net surplus of exocytosis and cytoskeletal stabilization of the growing protrusions [36]. As in the case of cell migration, the role of SNAREs in neurite growth and navigation is beginning to be explored. Neuronal growth cones principally express two v-SNAREs: VAMP2 and VAMP7. VAMP2 was ini- tially considered not to be involved in neurite growth and navigation because tetanus neurotoxin-treated neu- rons show normal growth and Syb-2⁄ VAMP2 null mice do not show any striking brain developmental defect [37,38]. Recent data suggest, however, that VAMP2 may mediate axonogenesis in neurons grown on poly-d-lysine [39]. VAMP7 was shown to mediate axonal and dendritic growth in cultured neurons [40]. Again, this mechanism was recently shown to depend on the substratum. Indeed, axonogenesis relies on the integrin-dependent activation of FAK and Src and uses coordinated activity of the arp2 ⁄ 3 complex and VAMP7-mediated exocytosis in the presence of laminin [39]. Finally, a perhaps novel form of neurite growth, induced by the activation of rac1 in PC12 cells, was shown to be sustained by another pool of exocytic organelles, the enlargosomes [41]. These organelles comprise a membrane compartment distinct from Golgi and trans Golgi network vesicles and endosomes, and exist in some cortical neurons of the embryonic and neonatal brain. The rapid neurite outgrowth observed was regulated by VAMP4-mediated exocyto- sis, most probably in the cell body in neurons [42,43]. Botulinum neurotoxin C1, which cleaves syntaxin1 and SNAP25, impairs axonal growth [44]. Using small interfering RNA silencing in cultured neurons, syn- taxin3, but not syntaxin1, was shown to be involved in axonal and dendritic growth [45]. Syntaxin1 gene knockout in the mouse, however, produced conflicting results. Although no major developmental defect was detected in one case [46], another study reported embryonic lethality [47], potentially suggesting environ- mental ⁄ epigenetic regulation of redundant pathways (such as between syntaxins 1 and 3). The endosomal syntaxin13, which interacts with both VAMP2 and VAMP7, is also required for neurite growth in cultured neurons [48]. The potential role of SNAP25 is not clear because botulinum neurotoxin A, which cleaves SNAP25, blocks axonal growth [49,50], although SNAP25 knockout does not appear to impair brain development [51]. SNAP23 gene constitutive knockout induces early embryonic lethality [52,53] and, thus, does not allow for the analysis of potential specific brain developmental defects. Overall, neurite growth is likely to rely on redundant pathways involving VAMPs 2, 4 and 7; SNAP23 and SNAP 25; syntaxins 1, 3 and 13; and possibly more. A recent study further supports this notion because double knockout mice lacking the endosomal v-SNAREs, Vit1a and Vti1b, show perina- tal lethality and massive defects in brain development, which is not observed in single knockout mutants [54]. Vesicular trafficking in cell navigation K. Zylbersztejn and T. Galli 4500 FEBS Journal 278 (2011) 4497–4505 ª 2011 FEBS. No claim to original French government works The growth of neurites occurs in concert with growth cone navigation (i.e. the ability of axonal or dendritic growth cones to probe their molecular environment via specific receptors). The guidance cues and their key receptors comprise attractants, such as netrin and semaphorins, and repellents, such as net- rins, semaphorins, slits, ephrins or myelin-associated glycoprotein [3]. Whether or not (and how) membrane trafficking participate in growth cone navigation is poorly understood. We can propose at least two possi- ble roles for SNAREs in growth cone navigation: (a) a direct role through trafficking of membrane and guid- ance receptors and (b) an indirect role through traf- ficking of regulatory molecules, which would control guidance receptors or ion channels (Fig. 3). These two roles are complementary. Concerning the former hypothesis, the guidance of the growth cone was previously defined as an asym- metric balance between exocytosis (i.e. that would induce attraction) and endocytosis (i.e. that would induce repulsion) induced by Ca 2+ release [55,56]. In agreement with this hypothesis, recent data suggest that VAMP2 mediates Ca 2+ -evoked exocytosis, induc- ing a positive turning response in neurons grown on an artificial L1 substrate [57]. By contrast, semapho- rin3A-mediated repulsion requires endocytosis of its receptors [58,59]. To maintain the repulsion, a resensi- tization of the growth cone is necessary, which may occur by exocytosis of the receptors originating from recycling compartments or from the neo-synthesized pool of the secretory pathway [60]. In agreement with this hypothesis, VAMP2 has been shown to be essen- tial for fast- endocytosis in synapses [61]. Thus, it is tempting to speculate that a VAMP2-dependent endo- cytosis may also occur in the growth cone to redistrib- ute semaphorin3A receptors. However, this hypothesis has not yet been directly tested. Two independent groups have also shown that netrin1 induces cluster formation of DCC (i.e. deleted in colorectal cancer) receptors at the surface of axon shafts in an exocyto- sis-dependent manner [63] and that this DCC insertion at the cell surface is insensitive to tetanus neurotoxin [64]. Thus, we can speculate that the tetanus insensitive v-SNARE VAMP7 may mediate this exocytosis. SNARE-mediated vesicular trafficking could regulate growth cone responses to guidance molecules by con- trolling cell surface expression of receptors via the bal- ancing of endocytosis ⁄ exocytosis [65,66]. With respect to the second hypothesis noted above, considered as an additional modulatory mechanism, SNARE-depen- dent fusion could be necessary for trafficking of mole- cules regulating the growth cone homeostasis and response to guidance cues. V-ATPase interacts with VAMP2 [67] and this interaction regulates exocytosis [68]. In V-ATPase knockout flies, endocytosis is impaired [69] and guidance receptors cluster in the endosomal compartment and are directed to degrada- tion [70]. Recent data suggest that synaptic exocytosis allows for V-ATPase-mediated proton secretion and nerve terminal alkalinization following depolarization [71]. If a similar mechanism occurred in growth cones, it could be important for regulating ion homeostasis and possibly resting potential. Intracellular calcium is a second messenger downstream of several guidance receptors. For example, netrin1 and brain-derived neurotrophic factor depolarize, whereas semaphorin3A and slit2 hyperpolarize the growth cone [72]. Several SNARE-channel interactions have been identified. The ATP Low repulsion A ttraction H+ V-ATP Ca(v)2.1 K(v)2.1 VAMP2 SNAP25 Syntaxin1 Ca2+ K+ K+ Ca2+ TRP3 Na+ Ca2+ Fig. 3. Potential mechanisms of the function of SNAREs in growth cone navigation. Little is known about the roles of SNAREs in growth cone navigation. Here, we hypothesized that they could have direct roles in the trafficking of membrane and ⁄ or receptors mediating attrac- tion ⁄ repulsion, and ⁄ or indirect roles through the transport of ionic channels, such as V-ATPse, Ca(v)2.1, K(v)2.1 or TRP3. K. Zylbersztejn and T. Galli Vesicular trafficking in cell navigation FEBS Journal 278 (2011) 4497–4505 ª 2011 FEBS. No claim to original French government works 4501 calcium channel Ca(v)2.1 and SNAP25 directly interact [73,74] and SNAP25 is known to be important for Ca 2+ -dependent exocytosis [75]. At the same time, semaphorin2A genetically interacts with Ca(v)2.1 and Na(v)1 and mutations in any of them lead to ectopic neuromuscular contacts in the fly [76]. Moreover, syntaxin1 directly interacts with the potassium channel K(v)2.1 [77,78]. VAMP2 interacts with TRPC3, which is a nonselective cation-permeable channel, and tetanus neurotoxin inhibits carbachol-evoked calcium influx [79]. It is thus tempting to speculate that SNAREs may regulate the transport and ⁄ or activity of several channels that, in turn, would modify the response of a growth cone to guidance cues. Moreover, VAMP2 mediates the traffic of b1 integrin to the plasma mem- brane [80] and integrin traffic regulates the repulsive response of myelin-associated glycoprotein in the growth cone [81]. We have shown that trafficking of L1 is mediated by VAMP7 [82] and that the formation of L1 homophilic contacts depends equally on exocyto- sis and diffusion of L1 molecules at the cell surface [83]. L1 is a regulator of semaphorin3A repulsion [84] and L1 mutant mice show severe developmental defects that depend on the genetic background [85–87]. Therefore, VAMP7-dependent transport of L1 could regulate the capacity of growth cones to respond to semaphorin3A and possibly other guidance molecules. Even though much has been learned in recent years, it is still unclear how SNAREs and vesicular traffic mediate neurite growth and navigation. Much work remains to be carried out with the aim of developing a more integrated picture of the functions of SNAREs and their possible redundancy. Perspectives From the data discussed above, it is clear that SNARE-mediated vesicular trafficking plays an impor- tant role in cell migration, neurite growth and possibly neuronal growth cone guidance. The case of Vti1a ⁄ b [54] may suggest that functional redundancy among SNAREs is high. The early embryonic lethality of SNAP23 knockout [52] indicates the importance of performing additional mouse genetic studies to allow a firm conclusion to be reached. Further work in differ- ent organisms carrying mutations in single and multi- ple SNAREs is clearly needed. Even if the regulation of integrin traffic appears to be a common mechanism between cell migration and growth cone navigation, the traffick of guidance receptors and channels still remains largely unknown and is expected to greatly contribute to cell navigation. Again, this requires further investigation in cultured cells, as well as in vivo. Finally, controlling cell migra- tion and growth cone navigation by impairing SNARE-dependent trafficking with neurotoxins may harbor potential benefits for inhibiting tumorigenesis and tumor spreading, and this hypothesis also requires investigation in vivo. 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