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.

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.

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.

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.

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.

Ectopic expression of syntaxin 1 in the ER redirects TI-VAMP- and cellubrevin-containing vesicles.

SNARE proteins are key mediators of membrane fusion. Their function in ensuring compartmental specificity of membrane fusion has been suggested by in vitro studies but not demonstrated in vivo. We show here that ectopic expression of the plasma membrane t-SNARE heavy chain syntaxin 1 in the endoplasmic reticulum induces the redistribution of its cognate vesicular SNAREs, TI-VAMP and cellubrevin, and its light chain t-SNARE SNAP-23. These effects were prevented by co-expressing nSec1. Expression of syntaxin 1 alone impaired the cell surface expression of TI-VAMP and cellubrevin but not the recycling of transferrin receptor. TI-VAMP, cellubrevin and SNAP-23 associated in vivo with exogenous syntaxin 1. Redistribution of TI-VAMP in the ER of syntaxin-1-expressing cells was microtubule dependent and impaired the trafficking of CD63, a cargo of TI-VAMP-containing vesicles. We conclude that the destination of v-SNAREs is driven by their specific interaction with cognate t-SNAREs. Our in vivo data provide strong support for the theory that highly specific v-SNARE-t-SNARE interactions control compartmental specificity of membrane fusion.

D53 is a novel endosomal SNARE-binding protein that enhances interaction of syntaxin 1 with the synaptobrevin 2 complex in vitro.

Synaptobrevin 2 (Sb2), syntaxin1 (Stx1), and synaptosomal-associated protein of 25 kDa (SNAP-25) are the main components of the soluble N -ethylmaleimide-sensitive fusion protein attachment protein receptor (SNARE) complex involved in fusion of synaptic vesicles with the presynaptic plasma membrane. We report the characterization of D53, a novel SNARE-binding protein preferentially expressed in neural and neuro-endocrine cells. Its two-dimensional organization, established by the hydrophobic cluster analysis, is reminiscent of SNARE proteins. D53 contains two putative helical regions, one of which includes a large coiled-coil domain involved in the interaction with Sb2 in vitro. Following subcellular fractionation, endogenous D53 was specifically detected in the membrane-containing fraction of PC12 cells, where it co-immunoprecipitated with Sb2. Analysis by confocal microscopy showed that, in these cells, endogenous D53 co-localized partially with the transferrin receptor in early endosomes. In vitro assays revealed that binding properties of D53 to Stx1 and Sb2 are comparable with those of SNAP-25. Furthermore, D53 forms Sb2/Stx1/D53 complexes in vitro in a manner similar to SNAP-25. We propose that D53 could be involved in the assembly or disassembly of endosomal SNARE complexes by regulating Sb2/Stx interaction.