Increased activity of the vesicular soluble N-ethylmaleimide-sensitive factor attachment protein receptor TI-VAMP/VAMP7 by tyrosine phosphorylation in the Longin domain.

Vesicular (v)- and target (t)-SNAREs play essential roles in intracellular membrane fusion through the formation of cytoplasmic α-helical bundles. Several v-SNAREs have a Longin N-terminal extension that, by promoting a closed conformation, plays an autoinhibitory function and decreases SNARE complex formation and membrane fusion efficiency. The molecular mechanism leading to Longin v-SNARE activation is largely unknown. Here we find that exocytosis mediated by the Longin v-SNARE TI-VAMP/VAMP7 is activated by tonic treatment with insulin and insulin-like growth factor-1 but not by depolarization and intracellular calcium rise. In search of a potential downstream mechanism, we found that TI-VAMP is phosphorylated in vitro by c-Src kinase on tyrosine 45 of the Longin domain. Accordingly, a mutation of tyrosine 45 into glutamate, but not phenylalanine, activates both t-SNARE binding and exocytosis. Activation of TI-VAMP-mediated exocytosis thus relies on tyrosine phosphorylation.

The ER tether VAPA is required for proper cell motility and anchors ER-PM contact sites to focal adhesions.

Cell motility processes highly depend on the membrane distribution of Phosphoinositides, giving rise to cytoskeleton reshaping and membrane trafficking events. Membrane contact sites serve as platforms for direct lipid exchange and calcium fluxes between two organelles. Here, we show that VAPA, an ER transmembrane contact site tether, plays a crucial role during cell motility. CaCo2 adenocarcinoma epithelial cells depleted for VAPA exhibit several collective and individual motility defects, disorganized actin cytoskeleton and altered protrusive activity. During migration, VAPA is required for the maintenance of PI(4)P and PI(4,5)P2 levels at the plasma membrane, but not for PI(4)P homeostasis in the Golgi and endosomal compartments. Importantly, we show that VAPA regulates the dynamics of focal adhesions (FA) through its MSP domain, is essential to stabilize and anchor ventral ER-PM contact sites to FA, and mediates microtubule-dependent FA disassembly. To conclude, our results reveal unknown functions for VAPA-mediated membrane contact sites during cell motility and provide a dynamic picture of ER-PM contact sites connection with FA mediated by VAPA.

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.

The induction of HAD-like phosphatases by multiple signaling pathways confers resistance to the metabolic inhibitor 2-deoxyglucose.

Anti-cancer strategies that target the glycolytic metabolism of tumors have been proposed. The glucose analog 2-deoxyglucose (2DG) is imported into cells and, after phosphorylation, becomes 2DG-6-phosphate, a toxic by-product that inhibits glycolysis. Using yeast as a model, we performed an unbiased mass spectrometry-based approach to probe the cellular effects of 2DG on the proteome and study resistance mechanisms to 2DG. We found that two phosphatases that target 2DG-6-phosphate were induced upon exposure to 2DG and participated in 2DG detoxification. Dog1 and Dog2 are HAD (haloacid dehalogenase)-like phosphatases, which are evolutionarily conserved. 2DG induced Dog2 by activating several signaling pathways, such as the stress response pathway mediated by the p38 MAPK ortholog Hog1, the unfolded protein response (UPR) triggered by 2DG-induced ER stress, and the cell wall integrity (CWI) pathway mediated by the MAPK Slt2. Loss of the UPR or CWI pathways led to 2DG hypersensitivity. In contrast, mutants impaired in the glucose-mediated repression of genes were 2DG resistant because glucose availability transcriptionally repressed DOG2 by inhibiting signaling mediated by the AMPK ortholog Snf1. The characterization and genome resequencing of spontaneous 2DG-resistant mutants revealed that DOG2 overexpression was a common strategy underlying 2DG resistance. The human Dog2 homolog HDHD1 displayed phosphatase activity toward 2DG-6-phosphate in vitro and its overexpression conferred 2DG resistance in HeLa cells, suggesting that this 2DG phosphatase could interfere with 2DG-based chemotherapies. These results show that HAD-like phosphatases are evolutionarily conserved regulators of 2DG resistance.

Comparative study of commercially available and homemade anti-VAMP7 antibodies using CRISPR/Cas9-depleted HeLa cells and VAMP7 knockout mice.

VAMP7 (vesicle-associated membrane protein) belongs to the intracellular membrane fusion SNARE (Soluble N-ethylmaleimide-sensitive factor attachment protein receptors) protein family. In this study, we used CRISPR/Cas9 genome editing technology to generate VAMP7 knockout (KO) human HeLa cells and mouse KO brain extracts in order to test the specificity and the background of a set of commercially available and homemade anti-VAMP7 antibodies. We propose a simple profiling method to analyze western blotting and immunocytochemistry staining profiles and determine the extent of the antibodies' specificity. Using this method, we were able to rank the performance of a set of available antibodies and further showed an optimized procedure for VAMP7 immunoprecipitation, which we validated using wild-type and KO mouse brain extracts.

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.

Membrane lipids tune synaptic transmission by direct modulation of presynaptic potassium channels.

Voltage-gated potassium (Kv) channels are involved in action potential (AP) repolarization in excitable cells. Exogenous application of membrane-derived lipids, such as arachidonic acid (AA), regulates the gating of Kv channels. Whether membrane-derived lipids released under physiological conditions have an impact on neuronal coding through this mechanism is unknown. We show that AA released in an activity-dependent manner from postsynaptic hippocampal CA3 pyramidal cells acts as retrograde messenger, inducing a robust facilitation of mossy fiber (Mf) synaptic transmission over several minutes. AA acts by broadening presynaptic APs through the direct modulation of Kv channels. This form of short-term plasticity can be triggered when postsynaptic cell fires with physiologically relevant patterns and sets the threshold for the induction of the presynaptic form of long-term potentiation (LTP) at hippocampal Mf synapses. Hence, direct modulation of presynaptic Kv channels by activity-dependent release of lipids serves as a physiological mechanism for tuning synaptic transmission.

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.