SNAP to attention: A SNARE complex regulates neuronal progenitor polarity

SNARE vesicle targeting complex controls the polarity of neuronal progenitors. Kunii et al. (2020. J. Cell Biol. https://doi.org/10.1083/jcb.201910080) show that the SNAP23–VAMP8–Syntaxin1B complex is required for membrane targeting of N-cadherin and formation of adherence junction complexes in radial glia neuronal progenitors, the major prerequisite of cell polarity establishment.

Proliferation and maintenance of neural stem cells is crucial for proper establishment of the brain cytoarchitecture during development and correct functioning of the mature brain. In the vertebrate developing the central nervous system (CNS), neural stem cells form a monolayer that enfolds the neural tube. The rostral, brain-forming part of the neural tube develops into a chain of ventricles, the brain ventricular system, connected with the central canal of the spinal cord (1). The ventricular system and central canal are filled with cerebrospinal fluid that immerses the layer of neural stem cells in the primary proliferative zone of the developing CNS, an area called the ventricular zone (VZ). Neural stem cells divide there and produce young neurons that, after exiting mitotic cycle, migrate out of the VZ to settle in their final location in the developing brain and differentiate into mature neurons that assemble neuronal circuits.The cells of the VZ, also called neuroepithelium, are polarized like all epithelial cells. They have a basal side with which they are attached to the pial surface, while with their apical side they face the ventricular lumen. Radial glia cells (RGCs) that appear later in development are a class of polarized cells that are present in many places in the CNS but especially distinct in the forebrain. The degree of polarization of the RGC is even more pronounced than that of neuroepithelial cells, as their basal process is very long. RGC apical processes span the entire thickness of the developing neocortex and can extend as far as several millimeters in length in primates (2).The polarity of RGCs is important for both their proliferation and for the postmitotic migration of young neurons. Detachment of their basal and/or apical processes results in abnormal proliferation and apoptosis, leading to abnormal cytoarchitecture of the postnatal brain (3). If only the basal process is detached from the pia, radial glia proliferation is undisturbed but neuronal migration is disrupted, also leading to defects in the proper construction of the brain (4).It is not only the morphology of basal and apical processes that makes RGC a highly polarized cell. Presence of adherence junctions (adherence junctions complex; AJC) on the apical side of the cell membrane is another sign and determinant of asymmetry. Disruption of adherence junctions can cause disorders such as hydrocephalus and hemorrhage in humans (5).Key proteins involved in the establishment and maintenance of AJC and polarity in RGCs are transmembrane N-cadherin and its cytoplasmic partner β-catenin (6). Disruption of the N-cadherin–β-catenin complex in the brain causes complete loss of both AJC and apico-basal polarity (6). Similarly, disruption of proteins that control N-cadherin trafficking, such as LLGL1, Dlg5, Numb and Numblike, can also cause failure of adherence junction formation (7).In the current issue, Kunii et al. identified new players in this process, SNARE complex protein Snap23 and its binding partners (8). SNARE complex proteins NSF, soluble NSF attachment protein (SNAP), and its receptor SNARE were first identified as key proteins in targeted vesicular fusion events in the secretory pathway (9). Later, SNARE presynapse-specific homologues, VAMP (also known as synaptobrevin), and syntaxin and its binding partner SNAP-25 were shown to be the main controllers of the synaptic vesicles’ fusion with presynaptic neuronal membrane (10).Although it could be suspected that SNARE complex has a role in the polarity of the neuronal progenitors, knockout mice lacking components of the classical VAMP–syntaxin–SNAP25 complex did not show any abnormalities in the morphology of RGC. Kunii et al. hypothesized that other homologous SNARE complexes might control polarity of neuronal progenitors. To test this hypothesis, the authors inactivated a close homologue of Snap25, SNAP23, in the mouse CNS by conditional knockout. These mutant mice demonstrated severe hypoplasia and disorganization of the cytoarchitecture of the neocortex and hippocampus, defects in cerebellar development, hydrocephalus, and hemorrhaging. The authors suggested an explanation for why SNAP25 does not play a role in RGCs: its expression is mostly confined to neurons, while SNAP23 is expressed in both RGCs and postmitotic cells. Moreover, the authors found that SNAP23 localization is higher in the apical process than in the basal process. Consistent with this, apico-basal polarity of RGCs is defective in SNAP23 knockout animals, resulting in loss of these long processes—the main morphological feature of the radial glia.Expectedly, disruption of radial glia polarity was accompanied by premature differentiation of RGC due to accelerated cell cycle exit as well as abnormal migration and increased apoptosis of their offspring neurons. This premature differentiation in turn results in exhaustion of the progenitor pool and ultimately leads to brain hypoplasia. At the cell architecture level, inactivation of SNAP23 in the RGC disrupts formation of the adherens junctions. This is accompanied by failure of targeted localization of N-cadherin and β-catenin to the apical end, as well as mislocalization of several other components of the AJC, such as Par3, ZO-1, Grb3, and Pals1.This finding was substantiated by in vitro experiments wherein RGC with low levels of SNAP23 failed to attach to each other and had reduced levels of N-cadherin. The authors also reported protein-specific transport defects. Surface proteins like Ephrin-B1 and β-integrin did not make it to the plasma membrane, while membrane localization of other proteins such as low-density lipoprotein receptor (LDLR) and Na⁺/K⁺-ATPase was not affected.Using another elegant approach, the authors showed that N-cadherin depletion from the plasma membrane is the key molecular event in SNAP23 deficiency that causes loss of polarity in RGC (Fig. 1). In this set of experiments, they deleted SNAP23 in the developing cortex using a combination of in utero DNA electroporation with CRISPR-Cas9. In genetic rescue experiments, the authors expressed either wild-type N-cadherin or a chimeric protein consisting of the extracellular domain of N-cadherin fused to the transmembrane and cytoplasmic domains of the LDLR protein. The authors found that transport of the wild-type protein was dependent on SNAP23, while the chimeric protein appeared to use an alternative pathway. Accordingly, only the chimeric protein could restore the RGC polarity and other phenotypes in the SNAP23 mutant phenotype.Open in a separate windowFigure 1.Polarized organization of radial glia cells. (A) N-cadherin membrane targeting is a key event in formation of AJC. SNARE complex targets N-cadherin to the plasma membrane, while LLGL1 removes it from the basolateral side. (B) If any of the three components of the SNARE complex is destroyed, AJC is not formed due to failure to deliver N-cadherin to the plasma membrane, preventing apico-basal polarization. This causes abnormal brain development. aPKC, atypical PKC.To identify other members of SNARE complex that act with SNAP23 in the control of RGC polarity, the authors performed pulldown assays and precipitated several interacting partners, including homologues of VAMP and syntaxin. They then inactivated the identified candidates in the developing neocortex using siRNA. Inactivation of only two of them, VAMP8 and Stx1B, caused disruption of N-cadherin plasma membrane localization and AJC formation in radial glia (Fig. 1 B). In experiments using COS7 cells, the authors also found that VAMP8 preferentially colocalizes with vesicles transporting N-cadherin.The study also raises a number of questions. For example, how is protein cargo specificity achieved? Which member of the SNARE complex can be replaced by a homologue without affecting specificity? Can SNAP25 fully replace SNAP23 in N-cadherin transport, or are other syntaxin homologues able to recapitulate Stx1B function? Another question to be addressed is whether localization of the SNAP23-containing complex is tightly regulated and, if so, how? It has been suggested that nonphosphorylated N-cadherin is removed from the basolateral side by Llgl1 via internalization (11). Conversely, atypical PKC phosphorylates N-cadherin on the apical side, preventing its interaction with Llgl1 and allowing N-cadherin retention on the apical membrane (Fig. 1 A; 11). Whether this is a mechanism that competes with SNAP23-mediated targeting remains to be clarified. It is also not clear whether the SNAP23-containing complex is required for targeting proteins located basolaterally such as Scripple-Lgl-Dgl.In summary, this study uses a beautiful combination of mouse genetics and in vitro trafficking visualization and biochemistry to demonstrate an important role for the VAMP8–Stx1B–SNAP23 SNARE complex in the regulation of N-cadherin recruitment and neural progenitor polarity setting. Future work will undoubtedly provide further insight into the mechanisms that regulate the function of this SNARE complex as well as the role of other SNARE proteins in this process.     

Escherichia coli Exposure Inhibits Exocytic SNARE‐Mediated Membrane Fusion in Mast Cells

Mast cells orchestrate the allergic response through the release of proinflammatory mediators, which is driven by the fusion of cytoplasmic secretory granules with the plasma membrane. During this process, SNARE proteins including Syntaxin4, SNAP23 and VAMP8 play a key role. Following stimulation, the kinase IKKβ interacts with and phosphorylates the t‐SNARE SNAP23. Phosphorylated SNAP23 then associates with Syntaxin4 and the v‐SNARE VAMP8 to form a ternary SNARE complex, which drives membrane fusion and mediator release. Interestingly, mast cell degranulation is impaired following exposure to bacteria such as Escherichia coli. However, the molecular mechanism(s) by which this occurs is unknown. Here, we show that E. coli exposure rapidly and additively inhibits degranulation in the RBL‐2H3 rat mast cell line. Following co‐culture with E. coli, the interaction between IKKβ and SNAP23 is disrupted, resulting in the hypophosphorylation of SNAP23. Subsequent formation of the ternary SNARE complex between SNAP23, Syntaxin4 and VAMP8 is strongly reduced. Collectively, these results demonstrate that E. coli exposure inhibits the formation of VAMP8‐containing exocytic SNARE complexes and thus the release of VAMP8‐dependent granules by interfering with SNAP23 phosphorylation.      

A Novel Tetanus Neurotoxin-insensitive Vesicle-associated Membrane Protein in SNARE Complexes of the Apical Plasma Membrane of Epithelial Cells

The importance of soluble N-ethyl maleimide (NEM)-sensitive fusion protein (NSF) attachment protein (SNAP) receptors (SNAREs) in synaptic vesicle exocytosis is well established because it has been demonstrated that clostridial neurotoxins (NTs) proteolyze the vesicle SNAREs (v-SNAREs) vesicle-associated membrane protein (VAMP)/brevins and their partners, the target SNAREs (t-SNAREs) syntaxin 1 and SNAP25. Yet, several exocytotic events, including apical exocytosis in epithelial cells, are insensitive to numerous clostridial NTs, suggesting the presence of SNARE-independent mechanisms of exocytosis. In this study we found that syntaxin 3, SNAP23, and a newly identified VAMP/brevin, tetanus neurotoxin (TeNT)-insensitive VAMP (TI-VAMP), are insensitive to clostridial NTs. In epithelial cells, TI-VAMP–containing vesicles were concentrated in the apical domain, and the protein was detected at the apical plasma membrane by immunogold labeling on ultrathin cryosections. Syntaxin 3 and SNAP23 were codistributed at the apical plasma membrane where they formed NEM-dependent SNARE complexes with TI-VAMP and cellubrevin. We suggest that TI-VAMP, SNAP23, and syntaxin 3 can participate in exocytotic processes at the apical plasma membrane of epithelial cells and, more generally, domain-specific exocytosis in clostridial NT-resistant pathways.     

A Highlights from MBoC Selection: Characterization of VAMP isoforms in 3T3-L1 adipocytes: implications for GLUT4 trafficking

The fusion of GLUT4-containing vesicles with the plasma membrane of adipocytes is a key facet of insulin action. This process is mediated by the formation of functional soluble N-ethylmaleimide–sensitive factor attachment protein receptor (SNARE) complexes between the plasma membrane t-SNARE complex and the vesicle v-SNARE or VAMP. The t-SNARE complex consists of Syntaxin4 and SNAP23, and whereas many studies identify VAMP2 as the v-SNARE, others suggest that either VAMP3 or VAMP8 may also fulfil this role. Here we characterized the levels of expression, distribution, and association of all the VAMPs expressed in 3T3-L1 adipocytes to provide the first systematic analysis of all members of this protein family for any cell type. Despite our finding that all VAMP isoforms form SDS-resistant SNARE complexes with Syntaxin4/SNAP23 in vitro, a combination of levels of expression (which vary by >30-fold), subcellular distribution, and coimmunoprecipitation analyses lead us to propose that VAMP2 is the major v-SNARE involved in GLUT4 trafficking to the surface of 3T3-L1 adipocytes.     

SNAP23, Syntaxin4, and vesicle-associated membrane protein 7 (VAMP7) mediate trafficking of membrane type 1–matrix metalloproteinase (MT1-MMP) during invadopodium formation and tumor cell invasion

Movement through the extracellular matrix (ECM) requires cells to degrade ECM components, primarily through the action of matrix metalloproteinases (MMPs). Membrane type 1–matrix metalloproteinase (MT1-MMP) has an essential role in matrix degradation and cell invasion and localizes to subcellular degradative structures termed invadopodia. Trafficking of MT1-MMP to invadopodia is required for the function of these structures, and here we examine the role of N-ethylmaleimide–sensitive factor–activating protein receptor (SNARE)–mediated membrane traffic in the transport of MT1-MMP to invadopodia. During invadopodium formation in MDA-MB-231 human breast cancer cells, increased association of SNAP23, Syntaxin4, and vesicle-associated membrane protein 7 (VAMP7) is detected by coimmunoprecipitation. Blocking the function of these SNAREs perturbs invadopodium-based ECM degradation and cell invasion. Increased level of SNAP23-Syntaxin4-VAMP7 interaction correlates with decreased Syntaxin4 phosphorylation. These results reveal an important role for SNARE-regulated trafficking of MT1-MMP to invadopodia during cellular invasion of ECM.     

The keratin-binding protein Albatross regulates polarization of epithelial cells

The keratin intermediate filament network is abundant in epithelial cells, but its function in the establishment and maintenance of cell polarity is unclear. Here, we show that Albatross complexes with Par3 to regulate formation of the apical junctional complex (AJC) and maintain lateral membrane identity. In nonpolarized epithelial cells, Albatross localizes with keratin filaments, whereas in polarized epithelial cells, Albatross is primarily localized in the vicinity of the AJC. Knockdown of Albatross in polarized cells causes a disappearance of key components of the AJC at cell–cell borders and keratin filament reorganization. Lateral proteins E-cadherin and desmoglein 2 were mislocalized even on the apical side. Although Albatross promotes localization of Par3 to the AJC, Par3 and ezrin are still retained at the apical surface in Albatross knockdown cells, which retain intact microvilli. Analysis of keratin-deficient epithelial cells revealed that keratins are required to stabilize the Albatross protein, thus promoting the formation of AJC. We propose that keratins and the keratin-binding protein Albatross are important for epithelial cell polarization.     

Syntaxin 7 complexes with mouse Vps10p tail interactor 1b, syntaxin 6, vesicle-associated membrane protein (VAMP)8, and VAMP7 in b16 melanoma cells

Syntaxin 7 is a mammalian target soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) involved in membrane transport between late endosomes and lysosomes. The aim of the present study was to use immunoaffinity techniques to identify proteins that interact with Syntaxin 7. We reasoned that this would be facilitated by the use of cells producing high levels of Syntaxin 7. Screening of a large number of tissues and cell lines revealed that Syntaxin 7 is expressed at very high levels in B16 melanoma cells. Moreover, the expression of Syntaxin 7 increased in these cells as they underwent melanogenesis. From a large scale Syntaxin 7 immunoprecipitation, we have identified six polypeptides using a combination of electrospray mass spectrometry and immunoblotting. These polypeptides corresponded to Syntaxin 7, Syntaxin 6, mouse Vps10p tail interactor 1b (mVti1b), alpha-synaptosome-associated protein (SNAP), vesicle-associated membrane protein (VAMP)8, VAMP7, and the protein phosphatase 1M regulatory subunit. We also observed partial colocalization between Syntaxin 6 and Syntaxin 7, between Syntaxin 6 and mVti1b, but not between Syntaxin 6 and the early endosomal t-SNARE Syntaxin 13. Based on these and data reported previously, we propose that Syntaxin 7/mVti1b/Syntaxin 6 may form discrete SNARE complexes with either VAMP7 or VAMP8 to regulate fusion events within the late endosomal pathway and that these events may play a critical role in melanogenesis.     

Three-dimensional analysis of post-Golgi carrier exocytosis in epithelial cells

Targeted delivery of proteins to distinct plasma membrane domains is critical to the development and maintenance of polarity in epithelial cells. We used confocal and time-lapse total internal reflection fluorescence microscopy (TIR-FM) to study changes in localization and exocytic sites of post-Golgi transport intermediates (PGTIs) carrying GFP-tagged apical or basolateral membrane proteins during epithelial polarization. In non-polarized Madin Darby Canine Kidney (MDCK) cells, apical and basolateral PGTIs were present throughout the cytoplasm and were observed to fuse with the basal domain of the plasma membrane. During polarization, apical and basolateral PGTIs were restricted to different regions of the cytoplasm and their fusion with the basal membrane was completely abrogated. Quantitative analysis suggested that basolateral, but not apical, PGTIs fused with the lateral membrane in polarized cells, correlating with the restricted localization of Syntaxins 4 and 3 to lateral and apical membrane domains, respectively. Microtubule disruption induced Syntaxin 3 depolarization and fusion of apical PGTIs with the basal membrane, but affected neither the lateral localization of Syntaxin 4 or Sec6, nor promoted fusion of basolateral PGTIs with the basal membrane.     

Crystal structure and biophysical properties of a complex between the N-terminal SNARE region of SNAP25 and syntaxin 1a

SNARE proteins are required for intracellular membrane fusion. In the neuron, the plasma membrane SNAREs syntaxin 1a and SNAP25 bind to VAMP2 found on neurotransmitter-containing vesicles. These three proteins contain "SNARE regions" that mediate their association into stable tetrameric coiled-coil structures. Syntaxin 1a contributes one such region, designated H3, and SNAP25 contributes two SNARE regions to the fusogenic complex with VAMP2. Syntaxin 1a H3 (syn1aH3) and SNAP25 can form a stable assembly, which can then be bound by VAMP2 to form the full SNARE complex. Here we show that syn1aH3 can also form a stable but kinetically trapped complex with the N-terminal SNARE region of SNAP25 (S25N). The crystal structure of this complex reveals an extended parallel four-helix bundle similar to that of the core SNARE and the syn1aH3-SNAP25 complexes. The inherent ability of syn1aH3 and S25N to associate stably in vitro implies that the intracellular fusion machinery must prevent formation of, or remove, any non-productive complexes. Comparison with the syn1aH3-SNAP25 complex suggests that the linkage of the N- and C-terminal SNAP25 SNARE regions is kinetically advantageous in preventing formation of the non-productive syn1aH3-S25N complex. We also demonstrate that the syn1aH3-S25N complex can be disassembled by alpha-SNAP and N-ethylmaleimide-sensitive factor.     

Differential roles for actin polymerization and a myosin II motor in assembly of the epithelial apical junctional complex

Differentiation and polarization of epithelial cells depends on the formation of the apical junctional complex (AJC), which is composed of the tight junction (TJ) and the adherens junction (AJ). In this study, we investigated mechanisms of actin reorganization that drive the establishment of AJC. Using a calcium switch model, we observed that formation of the AJC in T84 intestinal epithelial cells began with the assembly of adherens-like junctions followed by the formation of TJs. Early adherens-like junctions and TJs readily incorporated exogenous G-actin and were disassembled by latrunculin B, thus indicating dependence on continuous actin polymerization. Both adherens-like junctions and TJs were enriched in actin-related protein 3 and neuronal Wiskott-Aldrich syndrome protein (N-WASP), and their assembly was prevented by the N-WASP inhibitor wiskostatin. In contrast, the formation of TJs, but not adherens-like junctions, was accompanied by recruitment of myosin II and was blocked by inhibition of myosin II with blebbistatin. In addition, blebbistatin inhibited the ability of epithelial cells to establish a columnar phenotype with proper apico-basal polarity. These findings suggest that actin polymerization directly mediates recruitment and maintenance of AJ/TJ proteins at intercellular contacts, whereas myosin II regulates cell polarization and correct positioning of the AJC within the plasma membrane.     

Specificity of botulinum protease for human VAMP family proteins

The botulinum neurotoxin light chain (BoNT-LC) is a zinc-dependent metalloprotease that cleaves neuronal SNARE proteins such as SNAP-25, VAMP2, and Syntaxin1. This cleavage interferes with the neurotransmitter release of peripheral neurons and results in flaccid paralysis. SNAP, VAMP, and Syntaxin are representative of large families of proteins that mediate most membrane fusion reactions, as well as both neuronal and non-neuronal exocytotic events in eukaryotic cells. Neuron-specific SNARE proteins, which are target substrates of BoNT, have been well studied; however, it is unclear whether other SNARE proteins are also proteolyzed by BoNT. Herein, we define the substrate specificity of BoNT-LC/B, /D, and /F towards recombinant human VAMP family proteins. We demonstrate that LC/B, /D, and /F are able to cleave VAMP1, 2, and 3, but no other VAMP family proteins. Kinetic analysis revealed that all LC have higher affinity and catalytic activity for the non-neuronal SNARE isoform VAMP3 than for the neuronal VAMP1 and 2 isoforms. LC/D in particular exhibited extremely low catalytic activity towards VAMP1 relative to other interactions, which we determined through point mutation analysis to be a result of the Ile present at residue 48 of VAMP1. We also identified the VAMP3 cleavage sites to be at the Gln 59-Phe 60 (LC/B), Lys 42-Leu 43 (LC/D), and Gln 41-Lys 42 (LC/F) peptide bonds, which correspond to those of VAMP1 or 2. Understanding the substrate specificity and kinetic characteristics of BoNT towards human SNARE proteins may aid in the development of novel therapeutic uses for BoNT.     

Plakophilin 3 and Par3 facilitate desmosomes’ association with the apical junctional complex

Desmosomes (DSMs), together with adherens junctions (AJs) and tight junctions (TJs), constitute the apical cell junctional complex (AJC). While the importance of the apical and basolateral polarity machinery in the organization of AJs and TJs is well established, how DSMs are positioned within the AJC is not understood. Here we use highly polarized DLD1 cells as a model to address how DSMs integrate into the AJC. We found that knockout (KO) of the desmosomal ARM protein Pkp3, but not other major DSM proteins, uncouples DSMs from the AJC without blocking DSM assembly. DLD1 cells also exhibit a prominent extraDSM pool of Pkp3, concentrated in tricellular (tC) contacts. Probing distinct apicobasal polarity pathways revealed that neither the DSM’s association with AJC nor the extraDSM pool of Pkp3 are abolished in cells with defects in Scrib module proteins responsible for basolateral membrane development. However, a loss of the apical polarity protein, Par3, completely eliminates the extraDSM pool of Pkp3 and disrupts AJC localization of desmosomes, dispersing these junctions along the entire length of cell–cell contacts. Our data are consistent with a model whereby Par3 facilitates DSM assembly within the AJC, controlling the availability of an assembly competent pool of Pkp3 stored in tC contacts.     

The role of VAMP7/TI-VAMP in cell polarity and lysosomal exocytosis in vivo

VAMP7 or tetanus neurotoxin-insensitive vesicle- associated membrane protein (TI-VAMP) has been proposed to regulate apical transport in polarized epithelial cells, axonal transport in neurons and lysosomal exocytosis. To investigate the function of VAMP7 in vivo, we generated VAMP7 knockout mice. Here, we show that VAMP7 knockout mice are indistinguishable from control mice and display a similar localization of apical proteins in the kidney and small intestine and a similar localization of axonal proteins in the nervous system. Neurite outgrowth of cultured mutant hippocampal neurons was reduced in mutant neurons. However, lysosomal exocytosis was not affected in mutant fibroblasts. Our results show that VAMP7 is required in neurons to extend axons to the full extent. However, VAMP7 does not seem to be required for epithelial cell polarity and lysosomal exocytosis.     

A direct interaction between Cdc42 and vesicle-associated membrane protein 2 regulates SNARE-dependent insulin exocytosis

In pancreatic beta cells, insulin granule exocytosis is regulated by SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor protein (SNAP) receptor) proteins, and this is coupled to cortical F-actin reorganization via the Rho family GTPase Cdc42 by an unknown mechanism. We investigated interactions among the target SNARE protein Syntaxin 1A and the vesicle-associated membrane SNARE protein (VAMP2) with Cdc42 and compared these structural interactions with their functional importance to glucose-stimulated insulin secretion in MIN6 beta cells. Subcellular fractionation analyses revealed a parallel redistribution of Cdc42 and VAMP2 from the granule fraction to the plasma membrane in response to glucose that temporally corresponded with the glucose-induced activation of Cdc42. Moreover, within these fractions Cdc42 and VAMP2 were found to co-immunoprecipitate under basal and glucose-stimulated conditions, suggesting that they moved as a complex. Furthermore, VAMP2 bound both GST-Cdc42-GTPgammaS and GST-Cdc42-GDP, indicating that the Cdc42-VAMP2 complex could form under both cytosolic GDP-bound Cdc42 and plasma membrane GTP-bound Cdc42 conformational conditions. In vitro binding analyses showed that VAMP2 bound directly to Cdc42 and that a heterotrimeric complex with Syntaxin 1A could also be formed. Deletion analyses of VAMP2 revealed that only the N-terminal 28 residues were required for Cdc42 binding. Expression of this 28-residue VAMP2 peptide in MIN6 beta cells resulted in the specific impairment of glucose-stimulated insulin secretion, indicating a functional importance for the Cdc42-VAMP2 interaction. Taken together, these data suggest a mechanism whereby glucose activates Cdc42 to induce the targeting of intracellular Cdc42-VAMP2-insulin granule complexes to Syntaxin 1A at the plasma membrane.     

Role of SNAP23 in insulin-induced translocation of GLUT4 in 3T3-L1 adipocytes. Mediation of complex formation between syntaxin4 and VAMP2

Both syntaxin4 and VAMP2 are implicated in insulin regulation of glucose transporter-4 (GLUT4) trafficking in adipocytes as target (t) soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNARE) and vesicle (v)-SNARE proteins, respectively, which mediate fusion of GLUT4-containing vesicles with the plasma membrane. Synaptosome-associated 23-kDa protein (SNAP23) is a widely expressed isoform of SNAP25, the principal t-SNARE of neuronal cells, and colocalizes with syntaxin4 in the plasma membrane of 3T3-L1 adipocytes. In the present study, two SNAP23 mutants, SNAP23-DeltaC8 (amino acids 1 to 202) and SNAP23-DeltaC49 (amino acids 1 to 161), were generated to determine whether SNAP23 is required for insulin-induced translocation of GLUT4 to the plasma membrane in 3T3-L1 adipocytes. Wild-type SNAP23 (SNAP23-WT) promoted the interaction between syntaxin4 and VAMP2 both in vitro and in vivo. Although SNAP23-DeltaC49 bound to neither syntaxin4 nor VAMP2, the SNAP23-DeltaC8 mutant bound to syntaxin4 but not to VAMP2. In addition, although SNAP23-DeltaC8 bound to syntaxin4, it did not mediate the interaction between syntaxin4 and VAMP2. Moreover, overexpression of SNAP23-DeltaC8 in 3T3-L1 adipocytes by adenovirus-mediated gene transfer inhibited insulin-induced translocation of GLUT4 but not that of GLUT1. In contrast, overexpression of neither SNAP23-WT nor SNAP23-DeltaC49 in 3T3-L1 adipocytes affected the translocation of GLUT4 or GLUT1. Together, these results demonstrate that SNAP23 contributes to insulin-dependent trafficking of GLUT4 to the plasma membrane in 3T3-L1 adipocytes by mediating the interaction between t-SNARE (syntaxin4) and v-SNARE (VAMP2).     

Fibroblast growth factor receptor signaling in Xenopus retinal axon extension

Fibroblast growth factor receptors (FGFRs) and N-cadherin both regulate axon extension in developing Xenopus retinal ganglion cells (RGCs). Cultured cerebellar neurons have been shown to require FGFR activity for N-cadherin–stimulated neurite outgrowth, raising the possibility that N-cadherin is a FGFR ligand. To investigate this possibility in the developing visual system, retinal neurons were transfected with a dominant-negative FGFR (XFD) and plated on purified N-cadherin substrates. XFD-expressing neurons extended markedly shorter processes than control GFP-expressing neurons, implicating a role for FGFRs in N-cadherin–stimulated neurite outgrowth. To examine whether N-cadherin and FGFRs share the same pathway or use distinct second messenger pathways, specific inhibitors of implicated signaling molecules were added to neurons stimulated by N-cadherin, basic fibroblast growth factor (bFGF), or brain-derived nerve factor (BDNF) (which stimulates RGC outgrowth by a FGFR-independent mechanism). Diacylglycerol (DAG) lipase and Ca²⁺/calmodulin kinase II inhibitors both significantly reduced outgrowth stimulated by N-cadherin or bFGF but not by BDNF. Furthermore, we show that inhibiting DAG lipase activity in RGC axons extending in vivo toward the optic tectum reversibly slows axon extension without collapsing their growth cones. Thus, a common second-messenger signaling pathway mediating both N-cadherin– and bFGF-stimulated neurite extension is consistent with a model in which N-cadherin directly modulates the FGFR or a model whereby both FGFR and N-cadherin regulate the same second-messenger system. © 1998 John Wiley & Sons, Inc. J Neurobiol 37: 633–641, 1998     

Soluble NSF attachment protein receptors (SNAREs) in RBL-2H3 mast cells: functional role of syntaxin 4 in exocytosis and identification of a vesicle-associated membrane protein 8-containing secretory compartment

Mast cells upon stimulation through high affinity IgE receptors massively release inflammatory mediators by the fusion of specialized secretory granules (related to lysosomes) with the plasma membrane. Using the RBL-2H3 rat mast cell line, we investigated whether granule secretion involves components of the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) machinery. Several isoforms of each family of SNARE proteins were expressed. Among those, synaptosome-associated protein of 23 kDa (SNAP23) was central in SNARE complex formation. Within the syntaxin family, syntaxin 4 interacted with SNAP23 and all vesicle-associated membrane proteins (VAMPs) examined, except tetanus neurotoxin insensitive VAMP (TI-VAMP). Overexpression of syntaxin 4, but not of syntaxin 2 nor syntaxin 3, caused inhibition of FcepsilonRI-dependent exocytosis. Four VAMP proteins, i.e., VAMP2, cellubrevin, TI-VAMP, and VAMP8, were present on intracellular membrane structures, with VAMP8 residing mainly on mediator-containing secretory granules. We suggest that syntaxin 4, SNAP23, and VAMP8 may be involved in regulation of mast cell exocytosis. Furthermore, these results are the first demonstration that the nonneuronal VAMP8 isoform, originally localized on early endosomes, is present in a regulated secretory compartment.     

The cystic fibrosis transmembrane conductance regulator's expanding SNARE interactome

The cystic fibrosis transmembrane conductance regulator (CFTR) interacts with multiple N-ethylmaleimide sensitive factor attachment protein (SNARE) molecules largely via its N-terminal cytoplasmic domain. The earliest known among these SNAREs are the cognate Q-SNARE pair of Syntaxin 1A (STX1A) and SNAP23 on the plasma membrane. These SNAREs affect CFTR chloride channel gating. CFTR exocytosis/recycling in intestinal epithelial cells is dependent on another SNARE located in the apical plasma membrane, STX3. Members of the STX8/STX7/vesicle transport through interaction with t-SNAREs homolog 1b/VAMP8 SNARE complex, which function in early to late endosome/lysosome traffic, are all known to interact with CFTR. Two SNAREs, STX6 and STX16 that function at the trans-Golgi network (TGN), have now been revealed as members of the CFTR SNARE interactome. We summarize here the SNAREs that interact with CFTR and discuss the roles of these SNAREs in the intracellular trafficking of CFTR and CFTR-associated pathophysiology.     

N-cadherin specifies first asymmetry in developing neurons

The precise polarization and orientation of developing neurons is essential for the correct wiring of the brain. In pyramidal excitatory neurons, polarization begins with the sprouting of opposite neurites, which later define directed migration and axo-dendritic domains. We here show that endogenous N-cadherin concentrates at one pole of the newborn neuron, from where the first neurite subsequently emerges. Ectopic N-cadherin is sufficient to favour the place of appearance of the first neurite. The Golgi and centrosome move towards this newly formed morphological pole in a second step, which is regulated by PI3K and the actin/microtubule cytoskeleton. Moreover, loss of function experiments in vivo showed that developing neurons with a non-functional N-cadherin misorient their cell axis. These results show that polarization of N-cadherin in the immediate post-mitotic stage is an early and crucial mechanism in neuronal polarity.     

Identification of signals and mechanisms of sorting of plasma membrane proteins in intestinal epithelial cells]

In epithelial cells the plasma membrane is divided into domains that are biochemically and functionally different. In intestinal cells for example the apical domain is facing the intestinal lumen and is involved in the uptake of nutriments while the basolateral domain is mediating cell-cell adhesion and signalisation. We are interested in deciphering the mechanisms underlying the creation and maintenance of such specialized domains. As an epithelial model we have used the intestinal cell line Caco-2 and we have studied the transport and sorting of the human neurotrophin receptor (p75 NTR) in these cells. Newly synthesized p75 NTR is first transported to the basolateral membrane and then is accumulated on the apical membrane after transcytosis. This final apical localization is controlled by the presence of a membrane anchor and a cluster of O-glycosylation sites located in the part of the ectodomain close to the membrane. Among the mechanisms likely to be involved in the sorting of apical components we have looked for a role of lipid-protein microdomain formation in the Golgi apparatus. These membrane microdomains are highly enriched in glycosylphosphatidyl inositol (GPI) anchored proteins, glycosphingolipids and apical proteins such as sucrase isomaltase (SI). Such a composition is also found for endocytic structures called caveolae which are made of caveolin 1. We have expressed caveolin 1 in Caco-2 cells which do not express it and also caveolin 2, a related protein of unknown function. Expression of caveolin 1 led to formation of caveolae indicating that this protein is necessary for caveolae formation while caveolin 2 is restricted to the Golgi apparatus and has no effect on caveolae formation. However Caveolin 2 increased the amount of SI incorporated in microdomains suggesting a role in recruitment into the apical pathway. The choice for a site of fusion for transport vesicles is the last step of control during exocytosis. To identify proteins involved in that step we have cloned and characterized two members of the t-SNARE family, namely syntaxin 3 and SNAP23. Syntaxin 3 is present on the apical membrane and forms a complex with SNAP23 which is also localized on the basolateral membrane where it forms a complex with syntaxin 4. Overexpression of syntaxin 3 in Caco-2 led to a decrease of SI exocytosis towards the apical membrane confirming that syntaxin 3 is involved in targeting the fusion of apical transport vesicles to the apical pole of the cells.