Identification of a new regulation pathway of EGFR and E-cadherin dynamics.

E-cadherin and EGFR are known to be closely associated hence regulating differentiation and proliferation notably in epithelia. We have previously shown that galectin-7 binds to E-cadherin and favors its retention at the plasma membrane. In this study, we shed in light that galectin-7 establishes a physical link between E-cadherin and EGFR. Indeed, our results demonstrate that galectin-7 also binds to EGFR, but unlike the binding to E-cadherin this binding is sugar dependent. The establishment of E-cadherin/EGFR complex and the binding of galectin-7 to EGFR thus lead to a regulation of its signaling and intracellular trafficking allowing cell proliferation and migration control. In vivo observations further support these results since an epidermal thickening is observed in galectin-7 deficient mice. This study therefore reveals that galectin-7 controls epidermal homeostasis through the regulation of E-cadherin/EGFR balance.

Mechanical Tension Drives Elongational Growth of the Embryonic Gut.

During embryonic development, most organs are in a state of mechanical compression because they grow in a confined and limited amount of space within the embryo's body; the early gut is an exception because it physiologically herniates out of the coelom. We demonstrate here that physiological hernia is caused by a tensile force transmitted by the vitelline duct on the early gut loop at its attachment point at the umbilicus. We quantify this tensile force and show that applying tension for 48 h induces stress-dependent elongational growth of the embryonic gut in culture, with an average 90% length increase (max: 200%), 65% volume increase (max: 160%), 50% dry mass increase (max: 100%), and 165% cell number increase (max: 300%); this mechanical cue is required for organ growth as guts not subject to tension do not grow. We demonstrate that growth results from increased cell proliferation when tension is applied. These results outline the essential role played by mechanical forces in shaping and driving the proliferation of embryonic organs.

Downregulation of Membrane Trafficking Proteins and Lactate Conditioning Determine Loss of Dendritic Cell Function in Lung Cancer.

Restoring antigen presentation for efficient and durable activation of tumor-specific CD8+ T-cell responses is pivotal to immunotherapy, yet the mechanisms that cause subversion of dendritic cell (DC) functions are not entirely understood, limiting the development of targeted approaches. In this study, we show that bona fide DCs resident in lung tumor tissues or DCs exposed to factors derived from whole lung tumors become refractory to endosomal and cytosolic sensor stimulation and fail to secrete IL12 and IFNI. Tumor-conditioned DC exhibited downregulation of the SNARE VAMP3, a regulator of endosomes trafficking critical for cross-presentation of tumor antigens and DC-mediated tumor rejection. Dissection of cell-extrinsic suppressive pathways identified lactic acid in the tumor microenvironment as sufficient to inhibit type-I IFN downstream of TLR3 and STING. DC conditioning by lactate also impacted adaptive function, accelerating antigen degradation and impairing cross-presentation. Importantly, DCs conditioned by lactate failed to prime antitumor responses in vivo These findings provide a new mechanistic viewpoint to the concept of DC suppression and hold potential for future therapeutic approaches.Significance: These findings provide insight into the cell-intrinsic and cell-extrinsic mechanisms that cause loss of presentation of tumor-specific antigens in lung cancer tissues. Cancer Res; 78(7); 1685-99. ©2018 AACR.

E-cadherin dynamics is regulated by galectin-7 at epithelial cell surface.

Re-epithelialisation of wounded epidermis is ensured by collective cell migration of keratinocytes. Efficient collective migration requires the maintenance of intercellular adhesion, notably through adherens junctions, to favour cell communication, support tension forces and coordinated movement . Galectin-7, a soluble lectin expressed in stratified epithelia, has been previously implicated in cell migration and intercellular adhesion. Here, we revealed a new function of galectin-7 in the control of directionality and collective behaviour in migrating keratinocytes. Consistently, we identified galectin-7 as a direct partner of E-cadherin, a key component of adherens junctions. Unexpectedly, this interaction does not require glycosylation motifs. Focusing on the underlying mechanisms, we showed that galectin-7 stabilizes E-cadherin at the plasma membrane, restraining its endocytosis. Interestingly, galectin-7 silencing decreases E-cadherin-mediated intercellular adhesion. Consequently, this study not only identifies a new stabilizer of adherens junctions but also emphasises the importance of the interplay between E-cadherin turnover and intercellular adhesion strength.

The Q-soluble N-Ethylmaleimide-sensitive Factor Attachment Protein Receptor (Q-SNARE) SNAP-47 Regulates Trafficking of Selected Vesicle-associated Membrane Proteins (VAMPs).

SNAREs constitute the core machinery of intracellular membrane fusion, but vesicular SNAREs localize to specific compartments via largely unknown mechanisms. Here, we identified an interaction between VAMP7 and SNAP-47 using a proteomics approach. We found that SNAP-47 mainly localized to cytoplasm, the endoplasmic reticulum (ER), and ERGIC and could also shuttle between the cytoplasm and the nucleus. SNAP-47 preferentially interacted with the trans-Golgi network VAMP4 and post-Golgi VAMP7 and -8. SNAP-47 also interacted with ER and Golgi syntaxin 5 and with syntaxin 1 in the absence of Munc18a, when syntaxin 1 is retained in the ER. A C-terminally truncated SNAP-47 was impaired in interaction with VAMPs and affected their subcellular distribution. SNAP-47 silencing further shifted the subcellular localization of VAMP4 from the Golgi apparatus to the ER. WT and mutant SNAP-47 overexpression impaired VAMP7 exocytic activity. We conclude that SNAP-47 plays a role in the proper localization and function of a subset of VAMPs likely via regulation of their transport through the early secretory pathway.

Migration Speed of Cajal-Retzius Cells Modulated by Vesicular Trafficking Controls the Size of Higher-Order Cortical Areas.

In the neocortex, higher-order areas are essential to integrate sensory-motor information and have expanded in size during evolution. How higher-order areas are specified, however, remains largely unknown. Here, we show that the migration and distribution of early-born neurons, the Cajal-Retzius cells (CRs), controls the size of higher-order areas in the mouse somatosensory, auditory, and visual cortex. Using live imaging, genetics, and in silico modeling, we show that subtype-specific differences in the onset, speed, and directionality of CR migration determine their differential invasion of the developing cortical surface. CR migration speed is cell autonomously modulated by vesicle-associated membrane protein 3 (VAMP3), a classically non-neuronal mediator of endosomal recycling. Increasing CR migration speed alters their distribution in the developing cerebral cortex and leads to an expansion of postnatal higher-order areas and congruent rewiring of thalamo-cortical input. Our findings thus identify novel roles for neuronal migration and VAMP3-dependent vesicular trafficking in cortical wiring.

Role of tetanus neurotoxin insensitive vesicle-associated membrane protein in membrane domains transport and homeostasis.

Biological membranes in eukaryotes contain a large variety of proteins and lipids often distributed in domains in plasma membrane and endomembranes. Molecular mechanisms responsible for the transport and the organization of these membrane domains along the secretory pathway still remain elusive. Here we show that vesicular SNARE TI-VAMP/VAMP7 plays a major role in membrane domains composition and transport. We found that the transport of exogenous and endogenous GPI-anchored proteins was altered in fibroblasts isolated from VAMP7-knockout mice. Furthermore, disassembly and reformation of the Golgi apparatus induced by Brefeldin A treatment and washout were impaired in VAMP7-depleted cells, suggesting that loss of VAMP7 expression alters biochemical properties and dynamics of the Golgi apparatus. In addition, lipid profiles from these knockout cells indicated a defect in glycosphingolipids homeostasis. We conclude that VAMP7 is required for effective transport of GPI-anchored proteins to cell surface and that VAMP7-dependent transport contributes to both sphingolipids and Golgi homeostasis.

Role of VAMP3 and VAMP7 in the commitment of Yersinia pseudotuberculosis to LC3-associated pathways involving single- or double-membrane vacuoles.

Yersinia pseudotuberculosis can replicate inside macrophages by hijacking autophagy and blocking autophagosome acidification. In bone marrow-derived macrophages, the bacteria are mainly observed inside double-membrane vacuoles positive for LC3, a hallmark of autophagy. Here, we address the question of the membrane traffic during internalization of Yersinia investigating the role of vesicle- associated membrane proteins (VAMPs). First, we show that as in epithelial cells, Yersinia pseudotuberculosis replicates mainly in nonacidic LC3-positive vacuoles. Second, in these cells, we unexpectedly found that VAMP3 localizes preferentially to Yersinia-containing vacuoles (YCVs) with single membranes using correlative light-electron microscopy. Third, we reveal the precise kinetics of VAMP3 and VAMP7 association with YCVs positive for LC3. Fourth, we show that VAMP7 knockdown alters LC3's association with single-and multimembrane-YCVs. Finally, in uninfected epithelial cells stimulated for autophagy, VAMP3 overexpression and knockdown led respectively to a lower and higher number of double-membrane, LC3-positive vesicles. Hence, our results highlight the role that VAMPs play in selection of the pathways leading to generation of ultrastructurally different LC3 compartments and pave the way for determining the full set of docking and fusion proteins involved in Yersinia pseudotuberculosis' intravesicular life cycle.

A molecular network for the transport of the TI-VAMP/VAMP7 vesicles from cell center to periphery.

The compartmental organization of eukaryotic cells is maintained dynamically by vesicular trafficking. SNARE proteins play a crucial role in intracellular membrane fusion and need to be targeted to their proper donor or acceptor membrane. The molecular mechanisms that allow for the secretory vesicles carrying the v-SNARE TI-VAMP/VAMP7 to leave the cell center, load onto microtubules, and reach the periphery to mediate exocytosis are largely unknown. Here, we show that the TI-VAMP/VAMP7 partner Varp, a Rab21 guanine nucleotide exchange factor, interacts with GolginA4 and the kinesin 1 Kif5A. Activated Rab21-GTP in turn binds to MACF1, an actin and microtubule regulator, which is itself a partner of GolginA4. These components are required for directed movement of TI-VAMP/VAMP7 vesicles from the cell center to the cell periphery. The molecular mechanisms uncovered here suggest an integrated view of the transport of vesicles carrying a specific v-SNARE toward the cell surface.

Absence of TI-VAMP/Vamp7 leads to increased anxiety in mice.

Vesicular (v)- and target (t)-SNARE proteins assemble in SNARE complex to mediate membrane fusion. Tetanus neurotoxin-insensitive vesicular-associated membrane protein (TI-VAMP/VAMP7), a vesicular SNARE expressed in several cell types including neurons, was previously shown to play a major role in exocytosis involved in neurite growth in cultured neurons. Here we generated a complete constitutive knock-out by deleting the exon 3 of Vamp7. Loss of TI-VAMP expression did not lead to any striking developmental or neurological defect. Knock-out mice displayed decreased brain weight and increased third ventricle volume. Axon growth appeared normal in cultured knock-out neurons. Behavioral characterization unraveled that TI-VAMP knock-out was associated with increased anxiety. Our results thus suggest compensatory mechanisms allowing the TI-VAMP knock-out mice to fulfill major developmental processes. The phenotypic traits unraveled here further indicate an unexpected role of TI-VAMP-mediated vesicular traffic in anxiety and suggest a role for TI-VAMP in higher brain functions.

Identification of a novel Bves function: regulation of vesicular transport.

Blood vessel/epicardial substance (Bves) is a transmembrane protein that influences cell adhesion and motility through unknown mechanisms. We have discovered that Bves directly interacts with VAMP3, a SNARE protein that facilitates vesicular transport and specifically recycles transferrin and beta-1-integrin. Two independent assays document that cells expressing a mutated form of Bves are severely impaired in the recycling of these molecules, a phenotype consistent with disruption of VAMP3 function. Using Morpholino knockdown in Xenopus laevis, we demonstrate that elimination of Bves function specifically inhibits transferrin receptor recycling, and results in gastrulation defects previously reported with impaired integrin-dependent cell movements. Kymographic analysis of Bves-depleted primary and cultured cells reveals severe impairment of cell spreading and adhesion on fibronectin, indicative of disruption of integrin-mediated adhesion. Taken together, these data demonstrate that Bves interacts with VAMP3 and facilitates receptor recycling both in vitro and during early development. Thus, this study establishes a newly identified role for Bves in vesicular transport and reveals a novel, broadly applied mechanism governing SNARE protein function.

Role of Varp, a Rab21 exchange factor and TI-VAMP/VAMP7 partner, in neurite growth.

The vesicular soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) tetanus neurotoxin-insensitive vesicle-associated membrane protein (TI-VAMP/VAMP7) was previously shown to mediate an exocytic pathway involved in neurite growth, but its regulation is still largely unknown. Here we show that TI-VAMP interacts with the Vps9 domain and ankyrin-repeat-containing protein (Varp), a guanine nucleotide exchange factor (GEF) of the small GTPase Rab21, through a specific domain herein called the interacting domain (ID). Varp, TI-VAMP and Rab21 co-localize in the perinuclear region of differentiating hippocampal neurons and transiently in transport vesicles in the shaft of neurites. Silencing the expression of Varp by RNA interference or expressing ID or a form of Varp deprived of its Vps9 domain impairs neurite growth. Furthermore, the mutant form of Rab21, defective in GTP hydrolysis, enhances neurite growth. We conclude that Varp is a positive regulator of neurite growth through both its GEF activity and its interaction with TI-VAMP.

Role of HRB in clathrin-dependent endocytosis.

Human immunodeficiency virus Rev-binding protein (HRB), also called human Rev-interacting protein (hRIP) or Rev/Rex activation domain binding (RAB) is a partner of the tyrosine kinase substrate EPS15, and it has been recovered in the AP-2 interactome. EPS15 and AP-2 are involved in endocytosis, but the function of HRB in this process is still unknown. Here we identified HRB as a partner of the vesicular SNARE tetanus neurotoxin-insensitive vesicle-associated membrane protein (TI-VAMP, also called VAMP7) in yeast two-hybrid screens and using biochemical assays. In HeLa cells, HRB localized both in the nucleus and in the cytoplasm. In the cytoplasm, HRB colocalized with clathrin-, AP-2-, EPS15-, and transferrin receptor-containing vesicles. We did not see significant colocalization between HRB and TI-VAMP in HeLa cells, and we saw partial colocalization with green fluorescent protein-TI-VAMP in stably expressing Madin-Darby canine kidney cells. Nevertheless using a pHLuorin-tagged TI-VAMP construct, we found that HRB and TI-VAMP colocalize close to the plasma membrane after 5 min of anti-green fluorescent protein antibody uptake. These results suggest that TI-VAMP and HRB may interact only during the early stages of endocytosis. Furthermore uptake experiments followed by fluorescence-activated cell sorting showed that the endocytosis of fluorescent transferrin and pHLuorin-TI-VAMP is strongly reduced in HRB knockdown cells. Altogether these results suggest that HRB is involved in clathrin-dependent endocytosis and recruits TI-VAMP in this process.

Targeting the epithelial SNARE machinery by bacterial neurotoxins.

Clostridial neurotoxins are responsible for botulism and tetanus by cleaving the synaptic SNAREs (soluble N-ethylmaleimide-sensitive factor attachment protein receptors) synaptobrevin/VAMP2 (Vesicle-Associated Membrane Protein 2) and its partners SNAP-25 (synaptosome-associated protein of 25 kDa) and syntaxin 1. SNARE proteins mediate membrane fusion, a crucial step in intracellular trafficking. There are seven isotypes of botulinic neurotoxins with different target specificities and one tetanus neurotoxin (TeNT), which targets synaptobrevin. Regarding the high sequence similarities between synaptobrevin and its nonneuronal homolog cellubrevin/VAMP3, different groups developed the use of TeNT to study cellubrevin (Cb). Here, we show how we have introduced the light chain of the TeNT into nonneuronal cells and selected clones expressing this toxin by Western blotting and by immunofluorescence. We also present how we identified which cells express TeNT by searching for a soluble green fluorescent protein (GFP) pattern of expression corresponding to cleaved GFP-tagged cellubrevin in living GFP-cellubrevin and TeNT transfected cells.

Activity-dependent phosphorylation of Ser187 is required for SNAP-25-negative modulation of neuronal voltage-gated calcium channels.

Synaptosomal-associated protein of 25 kDa (SNAP-25) is a SNARE protein that regulates neurotransmission by the formation of a complex with syntaxin 1 and synaptobrevin/VAMP2. SNAP-25 also reduces neuronal calcium responses to stimuli, but neither the functional relevance nor the molecular mechanisms of this modulation have been clarified. In this study, we demonstrate that hippocampal slices from Snap25(+/-) mice display a significantly larger facilitation and that higher calcium peaks are reached after depolarization by Snap25(-/-) and Snap25(+/-) cultured neurons compared with wild type. We also show that SNAP-25b modulates calcium dynamics by inhibiting voltage-gated calcium channels (VGCCs) and that PKC phosphorylation of SNAP-25 at ser187 is essential for this process, as indicated by the use of phosphomimetic (S187E) or nonphosphorylated (S187A) mutants. Neuronal activity is the trigger that induces the transient phosphorylation of SNAP-25 at ser187. Indeed, enhancement of network activity increases the levels of phosphorylated SNAP-25, whereas network inhibition reduces the extent of protein phosphorylation. A transient peak of SNAP-25 phosphorylation also is detectable in rat hippocampus in vivo after i.p. injection with kainate to induce seizures. These findings demonstrate that differences in the expression levels of SNAP-25 impact on calcium dynamics and neuronal plasticity, and that SNAP-25 phosphorylation, by promoting inhibition of VGCCs, may mediate a negative feedback modulation of neuronal activity during intense activation.

v-SNARE cellubrevin is required for basolateral sorting of AP-1B-dependent cargo in polarized epithelial cells.

The epithelial cell-specific adaptor complex AP-1B is crucial for correct delivery of many transmembrane proteins from recycling endosomes to the basolateral plasma membrane. Subsequently, membrane fusion is dependent on the formation of complexes between SNARE proteins located at the target membrane and on transport vesicles. Although the t-SNARE syntaxin 4 has been localized to the basolateral membrane, the v-SNARE operative in the AP-1B pathway remained unknown. We show that the ubiquitously expressed v-SNARE cellubrevin localizes to the basolateral membrane and to recycling endosomes, where it colocalizes with AP-1B. Furthermore, we demonstrate that cellubrevin coimmunoprecipitates preferentially with syntaxin 4, implicating this v-SNARE in basolateral fusion events. Cleavage of cellubrevin with tetanus neurotoxin (TeNT) results in scattering of AP-1B localization and missorting of AP-1B-dependent cargos, such as transferrin receptor and a truncated low-density lipoprotein receptor, LDLR-CT27. These data suggest that cellubrevin and AP-1B cooperate in basolateral membrane trafficking.

Expression of the Longin domain of TI-VAMP impairs lysosomal secretion and epithelial cell migration.

BACKGROUND INFORMATION: TI-VAMP (tetanus neurotoxin-insensitive vesicle-associated membrane protein; also called VAMP7) belongs to the Longin subfamily of v-SNAREs (vesicular soluble N-ethylmaleimide-sensitive fusion protein-attachment protein receptors). The regulatory N-terminal extension, called the Longin domain, of TI-VAMP has been shown previously to have a dual biochemical function: it inhibits the capacity of TI-VAMP to form SNARE complexes and it binds to the delta subunit of the AP-3 (adaptor protein 3) complex in early endosomes, thereby targeting TI-VAMP to late endosomes. RESULTS: We have generated MDCK (Madin-Darby canine kidney) cell lines expressing the Longin domain of TI-VAMP coupled to GFP (green fluorescent protein) in a doxycycline-dependent manner. As expected, AP-3delta (AP-3 delta subunit) is not properly localized in Longin-expressing cells. We have shown that the expression of the Longin domain impairs lysosomal secretion, as determined by the release of a pre-internalized fluorescent fluid-phase marker and by electron microscopy of the membrane-associated released particles. Membrane repair following mechanical wounding, a process requiring lysosomal secretion, is also impaired in cells expressing the Longin domain. Furthermore, cell migration, assessed by wound healing of MDCK monolayers, is also inhibited. CONCLUSIONS: The results of the present study suggest that the expression of the Longin domain of TI-VAMP regulates lysosomal secretion of epithelial cells and provide molecular evidence for a role of the late endocytic system in cell migration.

Tetanus neurotoxin-mediated cleavage of cellubrevin impairs epithelial cell migration and integrin-dependent cell adhesion.

A role for endocytosis and exocytosis in cell migration has been proposed but not yet demonstrated. Here, we show that cellubrevin (Cb), an early endosomal v-SNARE, mediates trafficking in the lamellipod of migrating epithelial cells and partially colocalizes with markers of focal contacts. Expression of tetanus neurotoxin, which selectively cleaves Cb, significantly reduced the speed of migrating epithelial cells. Furthermore, expression of tetanus neurotoxin enhanced the adhesion of epithelial cells to collagen, laminin, fibronectin, and E-cadherin; altered spreading on collagen; and impaired the recycling of beta1 integrins. These results suggest that Cb-dependent membrane trafficking participates in cell motility through the regulation of cell adhesion.

The tetanus neurotoxin-sensitive and insensitive routes to and from the plasma membrane: fast and slow pathways?

Intracellular membrane trafficking in eukaryotes involves the budding of vesicles from a donor compartment, their translocation, and subsequent fusion with a target membrane. This last step has been shown to involve SNARE proteins, classified into two categories, vesicular (v)-SNAREs and target (t)-SNAREs. It is the pairing of v- and t-SNAREs that is responsible for bringing the lipid bilayers together for membrane fusion. Key to the discovery of SNAREs is the sensitivity of their neuronal synaptic prototypes, which mediate the release of neurotransmitters, to clostridial neurotoxins. In this review, we focus on tetanus neurotoxin-sensitive and tetanus neurotoxin-insensitive v-SNAREs, in particular synaptobrevin and cellubrevin, both tetanus neurotoxin-sensitive and Tetanus neurotoxin-Insensitive Vesicle-Associated Membrane Protein (TI-VAMP, also called VAMP7). The brevins are characterized by an RD sequence in the middle of their SNARE motif whereas TI-VAMP has an RG sequence. These two categories of exocytic v-SNAREs define two important routes to and from the plasma membrane: one sensitive, the other insensitive to tetanus neurotoxin. We also discuss the central role of the endosomal system that could be considered, as already suggested for Rab proteins, as a mosaic of v-SNAREs, thus raising the question of whether or not these two routes can merge, and if so, how and where.