REVIEW ARTICLE
Vesicular trafficincell navigation
Kathleen Zylbersztejn
1,2
and Thierry Galli
1,2
1 ‘Membrane Trafficin 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 incell 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 incell signal-dependent
migration
The importance of adhesion and of both filamentous
actin and microtubules incell 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 incellnavigation 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 incell 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 incell 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 incell 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 incellnavigation 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 incell 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 incell 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.
Acknowledgements
We are grateful to Beverly Osborne, Karl Pfenninger,
Antonia Kropfinger, Marie-Christine Simmler and
Ve
´
ronique Proux-Gillardeaux for their critical reading
of the manuscript and help with the artwork. Work in
our group was funded in part by grants from
INSERM; the Association Franc¸ aise contre les Myop-
athies (AFM); the Association pour la Recherche sur
le Cancer (ARC); the Mairie de Paris Medical
Research and Health Program; the Fondation pour la
Recherche Me
´
dicale (FRM); and the Ecole des Neuro-
sciences de Paris (ENP) (to T.G.). K.Z. was supported
by FRM.
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. 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. 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