Secretion of VGF relies on the interplay between LRRK2 and post-Golgi v-SNAREs.

The neuropeptide VGF was recently proposed as a neurodegeneration biomarker. The Parkinson's disease-related protein leucine-rich repeat kinase 2 (LRRK2) regulates endolysosomal dynamics, a process that involves SNARE-mediated membrane fusion and could regulate secretion. Here we investigate potential biochemical and functional links between LRRK2 and v-SNAREs. We find that LRRK2 directly interacts with the v-SNAREs VAMP4 and VAMP7. Secretomics reveals VGF secretory defects in VAMP4 and VAMP7 knockout (KO) neuronal cells. In contrast, VAMP2 KO "regulated secretion-null" and ATG5 KO "autophagy-null" cells release more VGF. VGF is partially associated with extracellular vesicles and LAMP1+ endolysosomes. LRRK2 expression increases VGF perinuclear localization and impairs its secretion. Retention using selective hooks (RUSH) assays show that a pool of VGF traffics through VAMP4+ and VAMP7+ compartments, and LRRK2 expression delays its transport to the cell periphery. Overexpression of LRRK2 or VAMP7-longin domain impairs VGF peripheral localization in primary cultured neurons. Altogether, our results suggest that LRRK2 might regulate VGF secretion via interaction with VAMP4 and VAMP7.

Protocol to study starvation-induced autophagy in developing rat neurons.

Autophagy is being involved in an increasing number of cellular pathways. It now appears that autophagy stimulation and inhibition have complex effects in neurons. Here, we present a simple yet powerful protocol to induce autophagy in primary neurons in culture by partial nutrient deprivation, in neurons with or without transfection of plasmids encoding the Longin domain of VAMP7 or a nanobody directed against VAMP7. Although limited to cells in culture, this protocol can facilitate the study of autophagy in neurons. For complete details on the use and execution of this protocol, please refer to Wojnacki et al. (2020).

Role of VAMP7-Dependent Secretion of Reticulon 3 in Neurite Growth.

VAMP7 is involved in autophagy and in exocytosis-mediated neurite growth, two yet unconnected cellular pathways. Here, we find that nutrient restriction and activation of autophagy stimulate axonal growth, while autophagy inhibition leads to loss of neuronal polarity. VAMP7 knockout (KO) neuronal cells show impaired neurite growth, whereas this process is increased in autophagy-null ATG5 KO cells. We find that endoplasmic reticulum (ER)-phagy-related LC3-interacting-region-containing proteins Atlastin 3 and Reticulon 3 (RTN3) are more abundant in autophagy-related protein ATG5 KO and less abundant in VAMP7 KO secretomes. Treatment of neuronal cells with ATG5 or VAMP7 KO conditioned medium does not recapitulate the effect of these KOs on neurite growth. A nanobody directed against VAMP7 inhibits axonal overgrowth induced by nutrient restriction. Furthermore, expression of the inhibitory Longin domain of VAMP7 impairs the subcellular localization of RTN3 in neurons. We propose that VAMP7-dependent secretion of RTN3 regulates neurite growth.

VOPP1 promotes breast tumorigenesis by interacting with the tumor suppressor WWOX.

BACKGROUND: The WW domain-containing oxidoreductase (WWOX) gene, frequently altered in breast cancer, encodes a tumor suppressor whose function is mediated through its interactions with cancer-related proteins, such as the pro-apoptotic protein p73α. RESULTS: To better understand the involvement of WWOX in breast tumorigenesis, we performed a yeast two-hybrid screen and co-immunoprecipitation assays to identify novel partners of this protein. We characterized the vesicular overexpressed in cancer pro-survival protein 1 (VOPP1) as a new regulator of WWOX. In breast cancer cells, VOPP1 sequestrates WWOX in lysosomes, impairs its ability to associate with p73α, and inhibits WWOX-dependent apoptosis. Overexpressed VOPP1 potentiates cellular transformation and enhances the growth of transplanted tumors in vivo. VOPP1 is overexpressed in breast tumors, especially in tumors that retain WWOX. Moreover, increased expression of VOPP1 is associated with reduced survival of patients with WWOX-positive, but not with WWOX-negative, tumors. CONCLUSIONS: These findings emphasize the importance of the sequestration of WWOX by VOPP1 in addition to WWOX loss in breast tumors and define VOPP1 as a novel oncogene promoting breast carcinogenesis by inhibiting the anti-tumoral effect of WWOX.

Biomechanical Control of Lysosomal Secretion Via the VAMP7 Hub: A Tug-of-War between VARP and LRRK1.

The rigidity of the cell environment can vary tremendously between tissues and in pathological conditions. How this property may affect intracellular membrane dynamics is still largely unknown. Here, using atomic force microscopy, we show that cells deficient in the secretory lysosome v-SNARE VAMP7 are impaired in adaptation to substrate rigidity. Conversely, VAMP7-mediated secretion is stimulated by more rigid substrate and this regulation depends on the Longin domain of VAMP7. We further find that the Longin domain binds the kinase and retrograde trafficking adaptor LRRK1 and that LRRK1 negatively regulates VAMP7-mediated exocytosis. Conversely, VARP, a VAMP7- and kinesin 1-interacting protein, further controls the availability for secretion of peripheral VAMP7 vesicles and response of cells to mechanical constraints. LRRK1 and VARP interact with VAMP7 in a competitive manner. We propose a mechanism whereby biomechanical constraints regulate VAMP7-dependent lysosomal secretion via LRRK1 and VARP tug-of-war control of the peripheral pool of secretory lysosomes.

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.

Soluble N-ethylmaleimide-sensitive factor attachment protein receptors required during Trypanosoma cruzi parasitophorous vacuole development.

Trypanosoma cruzi, the etiologic agent of Chagas disease, is an obligate intracellular parasite that exploits different host vesicular pathways to invade the target cells. Vesicular and target soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) are key proteins of the intracellular membrane fusion machinery. During the early times of T. cruzi infection, several vesicles are attracted to the parasite contact sites in the plasma membrane. Fusion of these vesicles promotes the formation of the parasitic vacuole and parasite entry. In this work, we study the requirement and the nature of SNAREs involved in the fusion events that take place during T. cruzi infection. Our results show that inhibition of N-ethylmaleimide-sensitive factor protein, a protein required for SNARE complex disassembly, impairs T. cruzi infection. Both TI-VAMP/VAMP7 and cellubrevin/VAMP3, two v-SNAREs of the endocytic and exocytic pathways, are specifically recruited to the parasitophorous vacuole membrane in a synchronized manner but, although VAMP3 is acquired earlier than VAMP7, impairment of VAMP3 by tetanus neurotoxin fails to reduce T. cruzi infection. In contrast, reduction of VAMP7 activity by expression of VAMP7's longin domain, depletion by small interfering RNA or knockout, significantly decreases T. cruzi infection susceptibility as a result of a minor acquisition of lysosomal components to the parasitic vacuole. In addition, overexpression of the VAMP7 partner Vti1b increases the infection, whereas expression of a KIF5 kinesin mutant reduces VAMP7 recruitment to vacuole and, concomitantly, T. cruzi infection. Altogether, these data support a key role of TI-VAMP/VAMP7 in the fusion events that culminate in the T. cruzi parasitophorous vacuole development.

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.

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.

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.

A methodological approach to unravel organ-specific breast cancer metastasis.

Breast cancer is the most commonly diagnosed and the second highest cause of cancer-related mortality. Although major breakthroughs have emerged during the past decades concerning the characterization of major malignant tumors hallmarks, little is known about the molecular process that sustains the most deadly feature of cancer: metastasis to distant organs. In fact, this colonization of tumor cells to secondary sites is not random but rather orientated, and depends on several signalling events that are not fully elucidated yet. Understanding the precise molecular and cellular mechanisms accountable for the specific invasion of tissues by breast cancer cells is likely to be important for developing new therapeutic strategies to effectively prevent metastasis in patients diagnosed with early cancer lesions. Here, we briefly describe a multidisciplinary approach based on the molecular profiling of breast cancer metastases, the elaboration of prognostic gene signatures, the clinical validation and the experimental confirmation using cell and animal models to better address breast cancer metastasis. This methodology can be considered as a useful workflow to identify and validate the genes that trigger and support organ tropism of breast cancer cells during metastasis.

Quantitative and robust assay to measure cell-cell contact assembly and maintenance.

Epithelial junction formation and maintenance are multistep processes that rely on the clustering of macromolecular complexes. These events are highly regulated by signalling pathways that involve Rho small GTPases. Usually, when analysing the contribution of different components of Rho-dependent pathways to cell-cell adhesion, the localisation of adhesion receptors at junctions is evaluated by immunofluorescence. However, we find that this method has limitations on the quantification (dynamic range), ability to detect partial phenotypes and to differentiate between the participation of a given regulatory protein in assembly and/or maintenance of cell-cell contacts.In this chapter, we describe a suitable method, the aggregation assay, in which we adapted a quantitative strategy to allow objective and reproducible detection of partial phenotypes. Importantly, this methodology estimates the ability of cells to form junctions and their resistance to mechanical shearing forces (stabilisation).

Ajuba is required for Rac activation and maintenance of E-cadherin adhesion.

Maintenance of stable E-cadherin-dependent adhesion is essential for epithelial function. The small GTPase Rac is activated by initial cadherin clustering, but the precise mechanisms underlying Rac-dependent junction stabilization are not well understood. Ajuba, a LIM domain protein, colocalizes with cadherins, yet Ajuba function at junctions is unknown. We show that, in Ajuba-depleted cells, Rac activation and actin accumulation at cadherin receptors was impaired, and junctions did not sustain mechanical stress. The Rac effector PAK1 was also transiently activated upon cell-cell adhesion and directly phosphorylated Ajuba (Thr172). Interestingly, similar to Ajuba depletion, blocking PAK1 activation perturbed junction maintenance and actin recruitment. Expression of phosphomimetic Ajuba rescued the effects of PAK1 inhibition. Ajuba bound directly to Rac·GDP or Rac·GTP, but phosphorylated Ajuba interacted preferentially with active Rac. Rather than facilitating Rac recruitment to junctions, Ajuba modulated Rac dynamics at contacts depending on its phosphorylation status. Thus, a Rac-PAK1-Ajuba feedback loop integrates spatiotemporal signaling with actin remodeling at cell-cell contacts and stabilizes preassembled cadherin complexes.

Newly formed E-cadherin contacts do not activate Cdc42 or induce filopodia protrusion in human keratinocytes.

BACKGROUND INFORMATION: The appropriate regulation of cell-cell adhesion is an important event in the homoeostasis of different cell types. In epithelial cells, tight adhesion mediated by E-cadherin receptors is essential for the differentiation and functionality of epithelial sheets. Upon assembly of cadherin-mediated cell-cell contacts, it is well established that the small GTPases Rho and Rac are activated and are necessary for junction stability. However, the role of the small GTPase Cdc42 in cadherin adhesion is less clear. Cdc42 can be activated by E-cadherin in a breast tumour cell line, but the requirement for Cdc42 function for new junction assembly or maintenance has been contradictory. Cdc42 participation in cell-cell contacts has been inferred from the presence of filopodia, the typical F-actin structure induced by Cdc42 activation, as cells approach each other to establish cell-cell contacts. Yet, under these conditions, the contribution of migration to filopodia protrusion cannot be excluded and the results are difficult to interpret. RESULTS: In the present study, we set out to address (a) whether Cdc42 is activated by new E-cadherin cell-cell contacts when junction assembly occurs without prior migration and (b) whether Cdc42 function is necessary for cadherin stability. We found that junction formation in confluent keratinocytes or upon E-cadherin clustering decreased Cdc42-GTP levels. In the absence of serum- and migration-induced Cdc42 activation, we demonstrated that cell-cell contacts do not induce filopodia or require Cdc42 function to assemble. CONCLUSION: We conclude that Cdc42 does not participate in the early events that initiate stable cadherin adhesion in keratinocytes. Yet, it is feasible that Cdc42 may be activated at later time points or by other receptors. Cdc42 can then participate in additional functions during polarization, such as Golgi re-positioning or basolateral trafficking.

MCC, a new interacting protein for Scrib, is required for cell migration in epithelial cells.

To further characterize the molecular events supporting the tumor suppressor activity of Scrib in mammals, we aim to identify new binding partners. We isolated MCC, a recently identified binding partner for beta-catenin, as a new interacting protein for Scrib. MCC interacts with both Scrib and the NHERF1/NHERF2/Ezrin complex in a PDZ-dependent manner. In T47D cells, MCC and Scrib proteins colocalize at the cell membrane and reduced expression of MCC results in impaired cell migration. By contrast to Scrib, MCC inhibits cell directed migration independently of Rac1, Cdc42 and PAK activation. Altogether, these results identify MCC as a potential scaffold protein regulating cell movement and able to bind Scrib, beta-catenin and NHERF1/2.

Scrib regulates PAK activity during the cell migration process.

Genetic studies have highlighted the key role of Scrib in the development of Metazoans. Deficiency in Scrib impairs many aspects of cell polarity and cell movement although the mechanisms involved remain unclear. In mammals, Scrib belongs to a protein complex containing betaPIX, an exchange factor for Rac/Cdc42, and GIT1, a GTPase activating protein for ARF6 implicated in receptor recycling and exocytosis. Here we show that the Scrib complex associates with PAK, a serine-threonine kinase family crucial for cell migration. PAK colocalizes with members of the Scrib complex at the leading edge of heregulin-treated T47D breast cancer cells. We demonstrate that the Scrib complex is required for epithelial cells and primary mouse embryonic fibroblasts to efficiently respond to chemoattractant cues. In Scrib-deficient cells, the pool of cortical PAK is decreased, thereby precluding its proper activation by Rac. Loss of Scrib also impairs the polarized distribution of active Rac at the leading edge and compromises the regulated activation of the GTPase in T47D cells and mouse embryonic fibroblasts. These data underscore the role of Scrib in cell migration and show the strong impact of Scrib in the function of PAK and Rac, two key molecules implicated in this process.

Junctional recruitment of mammalian Scribble relies on E-cadherin engagement.

Members of the LAP protein family, LET-413 in Caenorhabditis elegans, Scribble in Drosophila melanogaster, and Erbin, Lano, Densin-180 and hScrib in mammals, have conserved structural features. LET-413 and Scribble are junctional proteins involved in establishing and maintaining epithelial cell polarity. scribble also behaves as a neoplastic tumor suppressor gene. We show here that, in epithelial cells, hScrib is recruited at cell-cell junctions in an E-cadherin-dependent manner as shown by calcium switch assays in MDCK cells, re-expression of E-cadherin in MDA-231 cells treated by 5-Aza-2'-deoxycytidine (5Aza), and siRNA experiments. hScrib is restricted at the basolateral membrane of epithelial cells by its LRR domain, and is enriched in Triton X-100-insoluble fractions. In breast cancers, most lobular tumors did not express hScrib and E-cadherin while ductal tumors had a less frequent downregulation of hScrib. Our data provide additional insights on the modalities of recruitment of hScrib at the cell-cell junctions, and establish a potential link between the E-cadherin and hScrib tumor suppressors.

[LAP proteins: new gatekeepers of epithelial homeostasis]. / Protéines LAP: de nouvelles clés de voûte de l'architecture épithéliale.

Cell proliferation and cell differentiation are balanced processes required for the correct development and maintenance of tissues, including epithelial tissues. Disruption of this balance by downregulation or loss of function of gatekeepers of epithelial homeostasis may unleash tumor suppressing activities leading ultimately to tumorigenesis. Among the newcoming actors involved in epithelial cell polarity, recent data shed light on the crucial role played by the LAP (LRR And PDZ) protein family. LAP proteins assemble receptors, cytoplasmic adaptors and enzymes in multimolecular networks important for the different steps of epithelial differentiation : adhesion, building of tight junctions and trafficking of proteins along the secretory pathway. Furthermore, genetic studies in invertebrates and vertebrates have installed LAP proteins not only as crucial determinants for epithelial integrity but also as key regulators of cell proliferation and embryonic development.