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The vesicle-associated membrane protein 7 (VAMP7) is a SNARE protein of the longin family involved in a wide range of subcellular trafficking events, including neurite sprouting and elongation. The expression of the human gene SYBL1, encoding VAMP7, is finely regulated by alternative splicing. Among the minor isoforms identified so far, VAMP7j is the one most expressed and modulated in the human brain. Therefore, we focused on gaining functional evidence on VAMP7j, which lacks a functional SNARE motif but retains both the longin and transmembrane domains. In human SH-SY5Y cells, we found VAMP7j to modulate neuritogenesis by mediating transport of L1CAM toward the plasma membrane, in a fashion regulated by phosphorylation of the longin domain. VAMP7-mediated regulation of L1CAM trafficking seems at least to differentiate humans from rats, with VAMP7j CNS expression being restricted to primates, including humans. Since L1CAM is a central player in neuritogenesis and axon guidance, these findings suggest the species-specific splicing of SYBL1 is among the fine tuners of human neurodevelopmental complexity. [ABSTRACT FROM AUTHOR]

Deficiency in the conserved oligomeric Golgi (COG) complex that orchestrates SNARE‐mediated tethering/fusion of vesicles that recycle the Golgi's glycosylation machinery results in severe glycosylation defects. Although two major Golgi v‐SNAREs, GS28/GOSR1, and GS15/BET1L, are depleted in COG‐deficient cells, the complete knockout of GS28 and GS15 only modestly affects Golgi glycosylation, indicating the existence of an adaptation mechanism in Golgi SNARE. Indeed, quantitative mass‐spectrometry analysis of STX5‐interacting proteins revealed two novel Golgi SNARE complexes—STX5/SNAP29/VAMP7 and STX5/VTI1B/STX8/YKT6. These complexes are present in wild‐type cells, but their usage is significantly increased in both GS28‐ and COG‐deficient cells. Upon GS28 deletion, SNAP29 increased its Golgi residency in a STX5‐dependent manner. While STX5 depletion and Retro2‐induced diversion from the Golgi severely affect protein glycosylation, GS28/SNAP29 and GS28/VTI1B double knockouts alter glycosylation similarly to GS28 KO, indicating that a single STX5‐based SNARE complex is sufficient to support Golgi glycosylation. Importantly, co‐depletion of three Golgi SNARE complexes in GS28/SNAP29/VTI1B TKO cells resulted in severe glycosylation defects and a reduced capacity for glycosylation enzyme retention at the Golgi. This study demonstrates the remarkable plasticity in SXT5‐mediated membrane trafficking, uncovering a novel adaptive response to the failure of canonical intra‐Golgi vesicle tethering/fusion machinery. [ABSTRACT FROM AUTHOR]

During phagocytosis, endosomes both contribute with membrane to forming phagosomes and promote phagosome maturation. However, how these vesicles are delivered to the phagocytic cup and the phagosome has been unknown. Here, we show that Protrudin-mediated endoplasmic reticulum (ER)-endosome contact sites facilitate anterograde translocation of FYCO1 and VAMP7-positive late endosomes and lysosomes (LELys) to forming phagocytic cups in a retinal pigment epithelial-derived cell line (RPE1). Protrudin-dependent phagocytic cup formation required SYT7, which promotes fusion of LELys with the plasma membrane. RPE1 cells perform phagocytosis of dead cells (efferocytosis) that expose phosphatidylserine (PS) on their surface. Exogenous addition of apoptotic bodies increased the formation of phagocytic cups, which further increased when Protrudin was overexpressed. Overexpression of Protrudin also led to elevated uptake of silica beads coated with PS. Conversely, Protrudin depletion or abrogation of ER-endosome contact sites inhibited phagocytic cup formation resulting in reduced uptake of PS-coated beads. Thus, the Protrudin pathway delivers endosomes to facilitate formation of the phagocytic cup important for PS-dependent phagocytosis. [ABSTRACT FROM AUTHOR]

Obesity is one of the most prevalent chronic metabolic diseases, and induction of apoptosis in preadipocytes and adipocytes is a potential strategy to treat obesity. Celastrol represents one of the most robust anti-obesity phytochemicals so far, yet its direct binding target remains elusive. Here, we determined that celastrol could induce apoptosis in preadipocytes via mitochondrial mediated pathway. Further study clarified that celastrol inhibited the fusion of autophagosome and lysosome to prohibit autophagy, leading to cell apoptosis. By conducting virtual screening and genetic manipulation, we verified that overexpression of VAMP7 and RAB7 could block the effects of celastrol on inhibiting autophagy and inducing apoptosis. The Surface Plasmon Resonance study confirmed the direct binding of celastrol with VAMP7 and RAB7. The functional study illustrated the inhibition of RAB7 GTPase activity after celastrol treatment. Moreover, celastrol induced comparable apoptosis in murine epididymal adipose tissue, human preadipocytes and adipocytes, but not in human hepatocytes. An inhibitory effect on differentiation of human primary visceral preadipocytes was also observed. In conclusion, celastrol exhibited inhibitory effect of autophagy via direct binding with VAMP7 and RAB7, leading to an increase in preadipocytes apoptosis. These results advance our understanding in the potential application of celastrol in treating obesity. [ABSTRACT FROM AUTHOR]

Lysosomal exocytosis is involved in many key cellular processes but its spatiotemporal regulation is poorly known. Using total internal reflection fluorescence microscopy (TIRFM) and spatial statistics, we observed that lysosomal exocytosis is not random at the adhesive part of the plasma membrane of RPE1 cells but clustered at different scales. Although the rate of exocytosis is regulated by the actin cytoskeleton, neither interfering with actin or microtubule dynamics by drug treatments alters its spatial organization. Exocytosis events partially co-appear at focal adhesions (FAs) and their clustering is reduced upon removal of FAs. Changes in membrane tension following a hypo-osmotic shock or treatment with methyl-β-cyclodextrin were found to increase clustering. To investigate the link between FAs and membrane tension, cells were cultured on adhesive ring-shaped micropatterns, which allow to control the spatial organization of FAs. By using a combination of TIRFM and fluorescence lifetime imaging microscopy (FLIM), we revealed the existence of a radial gradient in membrane tension. By changing the diameter of micropatterned substrates, we further showed that this gradient as well as the extent of exocytosis clustering can be controlled. Together, our data indicate that the spatial clustering of lysosomal exocytosis relies on membrane tension patterning controlled by the spatial organization of FAs. [ABSTRACT FROM AUTHOR]

VAMP7 is a vesicular SNARE of the longin family that localizes to axons and dendrites during development, where it is important in neurite growth. In the adult brain, VAMP7 is enriched in a subset of nerve terminals, particularly in hippocampal mossy fibres (Mfs) originating from the dentate gyrus. We analysed the VAMP7 function in neurotransmitter release by detailed functional characterization of Mf synapses onto CA3 pyramidal cells in knockout mutant mice for VAMP7. We have evaluated the role of VAMP7 in different forms of short-term synaptic plasticity and the potential contribution of the co-release of glutamate and zinc. This analysis has not revealed any significant impact of the loss of VAMP7 for basal properties of synaptic transmission, for short-term plasticity, for asynchronous release and for the ability of Mf vesicles to release ionic zinc. Based on these findings, the potential role of VAMP7 in the regulation of presynaptic mechanisms is discussed., (© 2025 The Author(s). European Journal of Neuroscience published by Federation of European Neuroscience Societies and John Wiley & Sons Ltd.)

Electron tomography can provide additional morphological information not easily obtained by conventional transmission electron microscopy of thin sections. It uses a goniometer stage in the electron microscope to tilt the specimen and collect a series of 2D images from different orientations, which are combined to provide a 3D volume tomogram and a colored reconstruction of the morphological feature(s) of interest. Here we describe the protocols for its use in visualizing changes in organelle morphology after depletion of the SNARE proteins VAMP7 and VAMP8 and to study VAMP7 localization on endolysosomes/lysosomes., (© 2025. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Intracellular membrane protein trafficking is crucial for both normal cellular physiology and cell-cell communication. The conventional secretory route follows transport from the Endoplasmic reticulum (ER) to the plasma membrane via the Golgi apparatus. Alternative modes of secretion which can bypass the need for passage through the Golgi apparatus have been collectively termed as Unconventional protein secretion (UPS). UPS can comprise of cargo without a signal peptide or proteins which escape the Golgi in spite of entering the ER. UPS has been classified further depending on the mode of transport. Type I and Type II unconventional secretion are non-vesicular and non-SNARE protein dependent whereas Type III and Type IV dependent on vesicles and on SNARE proteins. In this review, we focus on the Type III UPS which involves the import of cytoplasmic proteins in membrane carriers of autophagosomal/endosomal origin and release in the extracellular space following SNARE-dependent intracellular membrane fusion. We discuss the role of vesicular SNAREs with a strong focus on VAMP7, a vesicular SNARE involved in exosome, lysosome and autophagy mediated secretion. We further extend our discussion to the role of unconventional secretion in health and disease with emphasis on cancer and neurodegeneration.

HPS1, a BLOC-3 subunit that acts as a guanine nucleotide exchange factor of Rab32/38, may play a role in the removal of VAMP7 during the maturation of large dense core vesicles of Paneth cells. Loss of HPS1 impairs lysozyme secretion and alters the composition of intestinal microbiota, which may explain the susceptibility of HPS-associated inflammatory bowel disease. Hermansky-Pudlak syndrome (HPS) is characterized by oculocutaneous albinism, bleeding tendency, and other chronic organ lesions due to defects in tissue-specific lysosome-related organelles (LROs). For some HPS subtypes, such as HPS-1, it is common to have symptoms of HPS-associated inflammatory bowel disease (IBD). However, its underlying mechanism is largely unknown. HPS1 is a subunit of the BLOC-3 complex which functions in the biogenesis of LROs. Large dense core vesicles (LDCVs) in Paneth cells of the intestine are a type of LROs. We here first report the abnormal LDCV morphology (increased number and enlarged size) in HPS1-deficient pale ear (ep) mice. Similar to its role in melanosome maturation, HPS1 plays an important function in the removal of VAMP7 from LDCVs to promote the maturation of LDCVs. The immature LDCVs in ep mice are defective in regulated secretion of lysozyme, a key anti-microbial peptide in the intestine. We observed changes in the composition of intestinal microbiota in both HPS-1 patients and ep mice. These findings provide insights into the underlying mechanism of HPS-associated IBD development, which may be implicated in possible therapeutic intervention of this devastating condition.

The Soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) family mediates membrane fusion during membrane trafficking and autophagy in all eukaryotic cells, with a number of SNAREs having cell type-specific functions. The endosome-trans-Golgi network (TGN) localized SNARE, Vesicle transport through interaction with t-SNAREs 1A (Vti1a), is unique among SNAREs in that it has numerous neuron-specific functions. These include neurite outgrowth, nervous system development, spontaneous neurotransmission, synaptic vesicle and dense core vesicle secretion, as well as a process of unconventional surface transport of the Kv4 potassium channel. Furthermore, the human VT11A gene is known to form fusion products with neighboring genes in cancer tissues, and VT11A variants are associated with risk in cancers, including glioma. In this review, I highlight VTI1A's known physio-pathological roles in brain neurons, as well as unanswered questions in these regards.

HPS1, a BLOC-3 subunit that acts as a guanine nucleotide exchange factor of Rab32/38, may play a role in the removal of VAMP7 during the maturation of large dense core vesicles of Paneth cells. Loss of HPS1 impairs lysozyme secretion and alters the composition of intestinal microbiota, which may explain the susceptibility of HPS-associated inflammatory bowel disease. Hermansky-Pudlak syndrome (HPS) is characterized by oculocutaneous albinism, bleeding tendency, and other chronic organ lesions due to defects in tissue-specific lysosome-related organelles (LROs). For some HPS subtypes, such as HPS-1, it is common to have symptoms of HPS-associated inflammatory bowel disease (IBD). However, its underlying mechanism is largely unknown. HPS1 is a subunit of the BLOC-3 complex which functions in the biogenesis of LROs. Large dense core vesicles (LDCVs) in Paneth cells of the intestine are a type of LROs. We here first report the abnormal LDCV morphology (increased number and enlarged size) in HPS1-deficient pale ear (ep) mice. Similar to its role in melanosome maturation, HPS1 plays an important function in the removal of VAMP7 from LDCVs to promote the maturation of LDCVs. The immature LDCVs in ep mice are defective in regulated secretion of lysozyme, a key anti-microbial peptide in the intestine. We observed changes in the composition of intestinal microbiota in both HPS-1 patients and ep mice. These findings provide insights into the underlying mechanism of HPS-associated IBD development, which may be implicated in possible therapeutic intervention of this devastating condition. [ABSTRACT FROM AUTHOR]

Macrophage migration into injured or infected tissue is a key aspect in the pathophysiology of many diseases where inflammation is a driving factor. Membrane‐type‐1 matrix metalloproteinase (MT1‐MMP) cleaves extracellular matrix components to facilitate invasion. Here we show that, unlike the constitutive MT1‐MMP surface recycling seen in cancer cells, unactivated macrophages express low levels of MT1‐MMP. Upon lipopolysaccharide (LPS) activation, MT1‐MMP synthesis dramatically increases 10‐fold at the surface by 15 hours. MT1‐MMP is trafficked from the Golgi complex to the surface via late endosomes/lysosomes in a pathway regulated by the late endosome/lysosome R‐SNAREs VAMP7 and VAMP8. These form two separate complexes with the surface Q‐SNARE complex Stx4/SNAP23 to regulate MT1‐MMP delivery to the plasma membrane. Loss of either one of these SNAREs leads to a reduction in surface MT1‐MMP, gelatinase activity and reduced invasion. Thus, inhibiting MT1‐MMP transport through this pathway could reduce macrophage migration and the resulting inflammation. [ABSTRACT FROM AUTHOR]

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 use visual scoring for immunocytochemistry staining to determine the extent of the antibodies’ specificity. Thus, 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.

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.

The Arf4--rhodopsin complex (mediated by the VxPx motif in rhodopsin) initiates expansion of vertebrate rod photoreceptor cilia-derived light-sensing organelles through stepwise assembly of a conserved trafficking network. Here, we examine its role in the sorting of VAMP7 (also known as TI-VAMP) -- an R-SNARE possessing a regulatory longin domain (LD) -- into rhodopsin transport carriers (RTCs). During RTC formation and trafficking, VAMP7 colocalizes with the ciliary cargo rhodopsin and interacts with the Rab11--Rabin8--Rab8 trafficking module. Rab11 and Rab8 bind the VAMP7 LD, whereas Rabin8 (also known as RAB3IP) interacts with the SNARE domain. The Arf/Rab11 effector FIP3 (also known as RAB11FIP3) regulates VAMP7 access to Rab11. At the ciliary base, VAMP7 forms a complex with the cognate SNAREs syntaxin 3 and SNAP-25. When expressed in transgenic animals, a GFP--VAMP7ΔLD fusion protein and a Y45E phosphomimetic mutant colocalize with endogenous VAMP7. The GFP--VAMP7-R150E mutant displays considerable localization defects that imply an important role of the R-SNARE motif in intracellular trafficking, rather than cognate SNARE pairing. Our study defines the link between VAMP7 and the ciliary targeting nexus that is conserved across diverse cell types, and contributes to general understanding of how functional Arf and Rab networks assemble SNAREs in membrane trafficking. [ABSTRACT FROM AUTHOR]

Alteration of axonal transport has emerged as a common precipitating factor in several neurodegenerative disorders including Human Spastic Paraplegia (HSP). Mutations of the SPAST ( SPG4 ) gene coding for the spastin protein account for 40% of all autosomal dominant uncomplicated HSP. By cleaving microtubules, spastin regulates several cellular processes depending on microtubule dynamics including intracellular membrane trafficking. Axonal transport is fundamental for the viability of motor neurons which often have very long axons and thus require efficient communication between the cell body and its periphery. Here we found that the anterograde velocity of VAMP7 vesicles, but not that of VAMP2, two vesicular-SNARE proteins implicated in neuronal development, is enhanced in SPG4-KO neurons. We showed that this effect is associated with a slight increase of the level of acetylated tubulin in SPG4-KO neurons and correlates with an enhanced activity of kinesin-1 motors. Interestingly, we demonstrated that an artificial increase of acetylated tubulin by drugs reproduces the effect of Spastin KO on VAMP7 axonal dynamics but also increased its retrograde velocity. Finally, we investigated the effect of microtubule targeting agents which rescue axonal swellings, on VAMP7 and microtubule dynamics. Our results suggest that microtubule stabilizing agents, such as taxol, may prevent the morphological defects observed in SPG4-KO neurons not simply by restoring the altered anterograde transport to basal levels but rather by increasing the retrograde velocity of axonal cargoes. [ABSTRACT FROM AUTHOR]

Graded distribution of intracellular second messengers, such as Ca2+ and cyclic nucleotides, mediates directional cell migration, including axon navigational responses to extracellular guidance cues, in the developing nervous system. Elevated concentrations of cAMP or cGMP on one side of the neuronal growth cone induce its attractive or repulsive turning, respectively. Although effector processes downstream of Ca2+ have been extensively studied, very little is known about the mechanisms that enable cyclic nucleotides to steer migrating cells. Here, we show that asymmetric cyclic nucleotide signaling across the growth cone mediates axon guidance via modulating microtubule dynamics and membrane organelle transport. In embryonic chick dorsal root ganglion neurons in culture, contact of an extending microtubule with the growth cone leading edge induces localized membrane protrusion at the site of microtubule contact. Such a contact-induced protrusion requires exocytosis of vesicle-associated membrane protein 7 (VAMP7)-positive vesicles that have been transported centrifugally along the microtubule. We found that the two cyclic nucleotides counteractively regulate the frequency of microtubule contacts and targeted delivery of VAMP7 vesicles: cAMP stimulates and cGMP inhibits these events, thereby steering the growth cone in the opposite directions. By contrast, Ca2+ signals elicit no detectable change in either microtubule contacts or VAMP7 vesicle delivery during Ca2+-induced growth cone turning. Our findings clearly demonstrate growth cone steering machinery downstream of cyclic nucleotide signaling and highlight a crucial role of dynamic microtubules in leading-edge protrusion for cell chemotaxis. [ABSTRACT FROM AUTHOR]

Objectives: Five proteins from the soluble N-ethylmaleimide-sensitive factor activating protein receptor (SNARE) complex family were studied in normal hematopoietic cells in bone marrow; normal lymphocytes at different stages of maturation and differentiation in bone marrow, thymus, tonsil, and lymph node; malignant lymphomas; and leukemias.Methods: Sixty-eight reactive and 380 hematopoietic and lymphoid neoplasms were immunohistochemically stained for syntaxin 7 (STX7), vesicle-associated membrane proteins (VAMP2, VAMP7, VAMP8), and synaptosomal-associated protein 23 (SNAP23).Results: STX7 has potential for being a useful marker for distinguishing between normal B precursors (hematogones) vs B lymphoblasts, as well as between the "popcorn" cells of nodular lymphocyte-predominant Hodgkin lymphoma vs the Reed-Sternberg cells of classic Hodgkin lymphoma or the B cells of T-cell, histiocyte-rich B-cell lymphoma. VAMP2 is uniquely expressed by both reactive and malignant plasma cells, in contrast to B-cell non-Hodgkin lymphoma. There is differential expression of SNARE proteins in normal and neoplastic lymphoid tissue depending on lymphocyte maturation stage.Conclusions: Differential SNARE protein expression in the lymphoid system may have potential use in diagnosis and may offer clues to lymphoma biology. VAMP2 is a promising new plasma cell marker. [ABSTRACT FROM AUTHOR]

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. [Display omitted] • LRRK2 interacts with the endosomal v-SNAREs VAMP4 and VAMP7 • VAMP4 and VAMP7 mediate the secretion of the Parkinson's disease biomarker VGF • LRRK2 regulates the accumulation vs. secretion of VGF Filippini et al. report the direct interaction of leucine-rich repeat kinase 2 (LRRK2) with the endosomal vesicular SNAREs VAMP4 and VAMP7. LRRK2, VAMP4, and VAMP7 regulate the secretion of the Parkinson's disease biomarker VGF. They provide evidence for the role of LRRK2 in regulating unconventional secretion in the disease. [ABSTRACT FROM AUTHOR]

Melanosomes are a class of lysosome-related organelles produced by melanocytes. Biogenesis of melanosomes requires the transport of melanin-synthesizing enzymes from tubular recycling endosomes to maturing melanosomes. The SNARE proteins involved in these transport or fusion steps have been poorly studied. We found that depletion of syntaxin 13 (STX13, also known as STX12), a recycling endosomal Qa-SNARE, inhibits pigment granule maturation in melanocytes by rerouting the melanosomal proteins such as TYR and TYRP1 to lysosomes. Furthermore, live-cell imaging and electron microscopy studies showed that STX13 co-distributed with melanosomal cargo in the tubular-vesicular endosomes that are closely associated with the maturing melanosomes. STX family proteins contain an N-terminal regulatory domain, and deletion of this domain in STX13 increases both the SNARE activity in vivo and melanosome cargo transport and pigmentation, suggesting that STX13 acts as a fusion SNARE in melanosomal trafficking pathways. In addition, STX13-dependent cargo transport requires the melanosomal R-SNARE VAMP7, and its silencing blocks the melanosome maturation, reflecting a defect in endosome--melanosome fusion. Moreover, we show mutual dependency between STX13 and VAMP7 in regulating their localization for efficient cargo delivery to melanosomes. [ABSTRACT FROM AUTHOR]

The homotypic fusion and protein sorting ( HOPS) complex is a multisubunit tethering complex that in yeast regulates membrane fusion events with the vacuole, the yeast lysosome. Mammalian homologs of all HOPS components have been found, but little is known about their function. Here, we studied the role of hVps41 and hVps39, two components of the putative human HOPS complex, in the endo-lysosomal pathway of human cells. By expressing hemagglutinin (HA)-tagged constructs, we show by immunoelectron microscopy ( immunoEM) that both hVps41 and hVps39 associate with the limiting membrane of late endosomes as well as lysosomes. Small interference RNA (siRNA)-mediated knockdown of hVps41 or hVps39 resulted in an accumulation of late endosomes, a depletion in the number of lysosomes and a block in the degradation of endocytosed cargo. Lysosomal pH and cathepsin B activity remained unaltered in these conditions. By immunoEM we found that hVps41 or hVps39 knockdown impairs homotypic fusion between late endosomes as well as heterotypic fusion between late endosomes and lysosomes. Thus, our data show that both hVps41 and hVps39 are required for late endosomal-lysosomal fusion events and the delivery of endocytic cargo to lysosomes in human cells. [ABSTRACT FROM AUTHOR]

The growing pollen tube apex is dedicated to balancing exo- and endocytic processes to form a rapidly extending tube. As perturbation of either tends to cause a morphological phenotype, this system provides tractable model for studying these processes. Vesicle-associated membrane protein 7s (VAMP7s) are members of the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) family that mediate cognate membrane fusion but their role in pollen tube growth has not been investigated. This manuscript identifies PiVAMP726 of Petunia inflata as a pollen-specific VAMP7 that localizes to the inverted cone of transport vesicles at the pollen tube tip. The endocytic marker FM4-64 was found to colocalize with yellow fluorescent protein (YFP)-PiVAMP726, which is consistent with PiVAMP726 containing an amino-acid motif implicated in endosomal localization, At high overexpression levels, YFP- PiVAMP726 inhibited growth and caused the formation of novel membrane compartments within the pollen tube tip. Functional dissection of PiVAMP726 implicated the N-terminal longin domain in negative regulation of the SNARE activity, but not localization of PiVAMP726. Expression of the constitutively active C-terminal SNARE domain alone, in pollen tubes, generated similar phenotypes to the full-length protein, but the truncated domain was more potent than the wild-type protein at both inhibiting growth and forming the novel membrane compartments. Both endo- and exocytic markers localized to these compartments in addition to YFP-PiVAMP726, leading to the speculation that PiVAMP726 might be involved in the recycling of endocytic vesicles in tip growth. [ABSTRACT FROM PUBLISHER]

An increasing number of studies suggest that distinct pools of synaptic vesicles drive specific forms of neurotransmission. Interspersed with these functional studies are analyses of the synaptic vesicle proteome which have consistently detected the presence of so-called "non-canonical" SNAREs that typically function in fusion and trafficking of other subcellular structures within the neuron. The recent identification of certain non-canonical vesicular SNAREs driving spontaneous (e.g., VAMP7 and vti1a) or evoked asynchronous (e.g., VAMP4) release integrates and corroborates existing data from functional and proteomic studies and implies that at least some complement of non-canonical SNAREs resident on synaptic vesicles function in neurotransmission. Here, we discuss the specific roles in neurotransmission of proteins homologous to each member of the classical neuronal SNARE complex consisting of synaptobrevin2, syntaxin-1, and SNAP-25. [ABSTRACT FROM AUTHOR]

Abstract: During reticulocyte maturation, some membrane proteins and organelles that are not required in the mature red cell are lost. Several of these proteins are released into the extracellular medium associated with the internal vesicles present in multivesicular bodies (MVBs). Likewise, organelles such as mitochondria and endoplasmic reticulum are wrapped into double membrane vacuoles (i.e., autophagosomes) and degraded via autophagy. Morphological, molecular, and biochemical studies have shown that autophagosomes fuse with MVBs forming the so-called amphisomes, a prelysosomal hybrid organelle. SNAREs are key molecules of the vesicle fusion machinery. TI-VAMP/VAMP7 and VAMP3/cellubrevin are two v-SNARE proteins involved in the endocytic and exocytic pathways. We have previously shown that in the human leukemic K562 cells, Rab11 decorates MVBs and it is necessary for fusion between autophagosomes with MVBs. In the present report, we present evidence indicating that VAMP3 is required for the fusion between MVBs with autophagosomes to generate the amphisome, allowing the maturation of the autophagosome, but it does not seem to be involved in the next step, i. e., fusion with the lysosome. On the other hand, we demonstrate that VAMP7 is necessary for this latter event, allowing the completion of the autophagic pathway. Furthermore, VAMP7 and ATPase NSF, a protein required for SNAREs disassembly, participate in the fusion between MVBs with the plasma membrane to release the internal vesicles (i.e., exosomes) into the extracellular medium. [Copyright &y& Elsevier]

Abstract: Soluble N-ethylmaleimide-sensitive-factor Attachment protein Receptors (SNAREs) participate in the specificity of membrane fusions in the cell. The mechanisms of specific SNARE sorting are still however poorly documented. We investigated the possible role of Adaptor Protein (AP) complexes in sorting of the Dictyostelium discoideum v-SNARE VAMP7. In live cells, GFP-VAMP7 is observed in the membrane of endocytic compartments. It is also observed in the plasma membrane of a small proportion of the cells. Mutation of a potential dileucine motif dramatically increases the proportion of cells with GFP-VAMP7 in their plasma membrane, strongly supporting the participation of an AP complex in VAMP7 sorting to the endocytic pathway. A partial increase occurs in knockout cells for the medium subunits of AP-2 and AP-3 complexes, indicating a role for both AP-2 and AP-3. VAMP7, as well as its t-SNAREs partners syntaxin 8 and Vti1, are co-immunoprecipitated with each of the medium subunits of the AP-1, AP-2, AP-3 and AP-4 complexes. This result supports the conclusion that VAMP7 directly interacts with both AP-2 and AP-3. It also raises the hypothesis of an interaction with AP-1 and AP-4. GFP-VAMP7 is retrieved from the endocytic pathway at and/or before the late post-lysosomal stage through an AP-independent mechanism. [Copyright &y& Elsevier]

Genitourinary disorders are the most frequent congenital defects in newborns; however, little is known about their etiology. Several studies have been carried out to find genetic risk factors in the development of these malformations. The expression of VAMP7 is found in testes, epididymis, seminal vesicles, prostatic tissues, penis, and urethra. Alterations in gene dose of VAMP7 were recently reported in a subset of male patients initially identified clinically by the presence of congenital genitourinary disorders. In 2016, the authors developed a diagnostic algorithm for early detection of sex chromosome aneuploidies by quantifying the SHOX, VAMP7, and SRY gene dose in newborns by qPCR using dried blood spot (DBS) samples. Correlate the increased gene dose of VAMP7 , obtained by qPCR using DBS, with genitourinary congenital defects attributable to disorders in virilization and verify the increased gene dose by microarrays. Samples that only presented increased VAMP7 gene dosage were selected from a previously analyzed group of 5088 males in which the early detection of sex chromosomes aneuploidies was performed. Eight males were found with an increased gene dose of VAMP7 (relative quantitation > 1.3) and were called in for a complete clinical evaluation aimed at the identification of genitourinary anomalies, qPCR and microarrays. Eight males from 5088 samples were identified with increased VAMP7 gene dosage of which six patients were clinically evaluated, of which 50% were identified with alterations in genital development (bilateral cryptorchidism, unilateral cryptorchidism, and glandular hypospadias) and speech delay, while the rest presented different types of atopy. Tannour-Louet et al. postulated on 2014 that the duplication of the Xq28 region, specifically of VAMP7 , plays a role in the human masculinization disorders of the urogenital tract. The study was based on array comparative genomic hybridization (aCGH) results performed to 116 males with disorders of sexual differentiation. In the present study, the patients were initially selected due to an increased gene dose of VAMP7 detected by qPCR, then the clinical evaluation and the aCGH were performed, inverse to what was reported previously but with similar percentages between both studies. In this work, the authors report cases of cryptorchidism, hypospadias, language delay and atopy in male preschoolers initially identified because they have an increased gene dose of VAMP7. [ABSTRACT FROM AUTHOR]

Together with the proteasome, macroautophagy is a main pathway for the degradation of intracellular elements. Endoplasmic reticulum (ER)-autophagy i.e . reticulophagy/ER-phagy leads to the encapsulation of pieces of the ER in forming autophagosomes. This is generally followed by fusion with lysosomes and degradation of these ER components by lysosomal hydrolases. Recent work by our group shows that ER elements could also be incorporated into late endosomes and later be released by a secretory mechanism which we will herein refer to as secretory reticulophagy/ER-phagy (SERP). In the absence of macroautophagy, such as by knocking out Atg5 , SERP is more efficient, leading to an increased secretion of MAP1LC3B-II and LC3-interacting region (LIR)-containing proteins of the ER, reticulons and atlastins. In this scenario, neurites grow longer and neuronal polarity is altered. In the absence of SERP, such as by knocking out Vamp7 , secretion of MAP1LC3B-II, ER-LIR containing proteins and neurite growth are severely inhibited. We argue that SERP might be a main secretory mechanism bypassing the Golgi apparatus, and that it is particularly active and important in neurite growth.

Background: Diet manipulation is the basis for prevention of obesity and diabetes. The molecular mechanisms that mediate the diet-based prevention of insulin resistance are not well understood. Here, as proof-of-concept, ginger-derived nanoparticles (GDNP) were used for studying molecular mechanisms underlying GDNP mediated prevention of high-fat diet induced insulin resistance. Methods: Ginger-derived nanoparticles (GDNP) were isolated from ginger roots and administered orally to C57BL/6 high-fat diet mice. Fecal exosomes released from intestinal epithelial cells (IECs) of PBS or GDNP treated high-fat diet (HFD) fed mice were isolated by differential centrifugation. A micro-RNA (miRNA) polymerase chain reaction (PCR) array was used to profile the exosomal miRs and miRs of interest were further analyzed by quantitative real time (RT) PCR. miR-375 or antisense-miR375 was packed into nanoparticles made from the lipids extracted from GDNP. Nanoparticles was fluorescent labeled for monitoring their in vivo trafficking route after oral administration. The effect of these nanoparticles on glucose and insulin response of mice was determined by glucose and insulin tolerance tests. Results: We report that HFD feeding increased the expression of AhR and inhibited the expression of miR-375 and VAMP7. Treatment with orally administered ginger-derived nanoparticles (GDNP) resulted in reversing HFD mediated inhibition of the expression of miR-375 and VAMP7. miR-375 knockout mice exhibited impaired glucose homeostasis and insulin resistance. Induction of intracellular miR-375 led to inhibition of the expression of AhR and VAMP7 mediated exporting of miR-375 into intestinal epithelial exosomes where they were taken up by gut bacteria and inhibited the production of the AhR ligand indole. Intestinal exosomes can also traffic to the liver and be taken up by hepatocytes, leading to miR-375 mediated inhibition of hepatic AhR over-expression and inducing the expression of genes associated with the hepatic insulin response. Altogether, GDNP prevents high-fat diet-induced insulin resistance by miR-375 mediated inhibition of the aryl hydrocarbon receptor mediated pathways over activated by HFD feeding. Conclusion: Collectively our findings reveal that oral administration of GDNP to HFD mice improves host glucose tolerance and insulin response via regulating AhR expression by GDNP induced miR-375 and VAMP7., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)

Axonal outgrowth is a fundamental process during the development of central (CNS) and peripheral (PNS) nervous system as well as in nerve regeneration and requires accurate axonal navigation and extension to the correct target. These events need proper coordination between membrane trafficking and cytoskeletal rearrangements and are under the control of the small GTPases of the Rho family, among other molecules. Reelin, a relevant protein for CNS development and synaptic function in the adult, is also present in the PNS. Upon sciatic nerve damage, Reelin expression increases and, on the other hand, mice deficient in Reelin exhibit an impaired nerve regeneration. However, the mechanism(s) involved the Reelin-dependent axonal growth is still poorly understood. In this work, we present evidence showing that Reelin stimulates dorsal root ganglia (DRG) regeneration after axotomy. Moreover, dissociated DRG neurons express the Reelin receptor Apolipoprotein E-receptor 2 and also require the presence of TC10 to develop their axons. TC10 is a Rho GTPase that promotes neurite outgrowth through the exocytic fusion of vesicles at the growth cone. Here, we demonstrate for the first time that Reelin controls TC10 activation in DRG neurons. Besides, we confirmed that the known CNS Reelin target Cdc42 is also activated in DRG and controls TC10 activity. Finally, in the process of membrane addition, we found that Reelin stimulates the fusion of membrane carriers containing the v-SNARE protein VAMP7 in vesicles that contain TC10. Altogether, our work shows a new role of Reelin in PNS, opening the option of therapeutic interventions to improve the regeneration process., (© 2020 Wiley Periodicals LLC.)

Autophagosome and lysosome fusion is an important macroautophagy/autophagy process for cargo degradation, and SNARE proteins, including STX17, SNAP29, VAMP7 and VAMP8, are key players in this process. However, the manner in which this process is precisely regulated is poorly understood. Here, we show that VAMP7B, a SNARE domain-disrupted isoform of R-SNARE protein VAMP7, competes with SNARE domain functional isoform VAMP7A to bind to STX17 and inhibits autophagosome-lysosome fusion. Moreover, we show that DIPK2A, a late endosome- and lysosome-localized protein, binds to VAMP7B, which inhibits the interaction of VAMP7B with STX17 and enhances the binding of STX17 to VAMP7A, thus enhancing autophagosome-lysosome fusion. Furthermore, DIPK2A participates in autophagic degradation of mitochondria proteins and alleviates apoptosis. Thus, we reveal a new aspect of autophagosome-lysosome fusion in which different isoforms of VAMP7 compete with STX17 and their regulation by DIPK2A. Abbreviations : DIPK2A: divergent protein kinase domain 2A; EEA1: early endosome antigen 1; GOLGA2: golgin A2; LAMP1: lysosomal associated membrane protein 1; MAP1LC3B/LC3: microtubule associated protein 1 light chain 3 beta; MFN2: mitofusin 2; MT-CO2: mitochondrially encoded cytochrome c oxidase II; PARP1: poly(ADP-ribose) polymerase 1; PRKN: parkin RBR E3 ubiquitin protein ligase; RAB5A: RAB5A, member RAS oncogene family; RAB7A: RAB7A, member RAS oncogene family; REEP: receptor accessory protein; RTN4: reticulon 4; SNARE: SNAP receptor; SQSTM1/p62: sequestosome 1; STX17: syntaxin 17; TOMM20: translocase of outer mitochondrial membrane 20; VAMP7: vesicle associated membrane protein 7; VAMP8: vesicle associated membrane protein 8.

Syntaxin 16, a Qa-SNARE (soluble N-ethylmaleimide-sensitive factor activating protein receptor), is involved in a number of membrane-trafficking activities, particularly transport processes at the trans-Golgi network (TGN). Recent works have now implicated syntaxin 16 in the autophagy process. In fact, syntaxin 16 appears to have dual roles, firstly in facilitating the transport of ATG9a-containing vesicles to growing autophagosomes, and secondly in autolysosome formation. The former involves a putative SNARE complex between syntaxin 16, VAMP7 and SNAP-47. The latter occurs via syntaxin 16's recruitment by Atg8/LC3/GABARAP family proteins to autophagosomes and endo-lysosomes, where syntaxin 16 may act in a manner that bears functional redundancy with the canonical autophagosome Qa-SNARE syntaxin 17. Here, I discuss these recent findings and speculate on the mechanistic aspects of syntaxin 16's newly found role in autophagy. [ABSTRACT FROM AUTHOR]

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 use visual scoring for immunocytochemistry staining to determine the extent of the antibodies' specificity. Thus, 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., Competing Interests: No competing interests were disclosed.

Intestinal dietary triacylglycerol absorption is a multi-step process. Triacylglycerol exit from the endoplasmic reticulum (ER) is the rate-limiting step in the progress of the lipid from its apical absorption to its basolateral membrane export. Triacylglycerol is transported from the ER to the cis Golgi in a specialized vesicle, the pre-chylomicron transport vesicle (PCTV). The vesicle-associated membrane protein 7 (VAMP7) was found to be more concentrated on PCTVs compared with ER membranes. VAMP7 has been previously identified associated with post-Golgi sites in eukaryotes. To examine the potential role of VAMP7 in PCTV trafficking, antibodies were generated that identified a 25 kDa band consistent with VAMP7 but did not crossreact with VAMP1,2. VAMP7 was concentrated on intestinal ER by immunofluorescence microscopy. Immunoelectron microscopy showed that the ER proteins Sar1 and rBet1 were present on PCTVs and colocalized with VAMP7. Iodixanol gradient centrifugation showed VAMP7 to be isodense with ER and endosomes. Although VAMP7 localized to intestinal ER, it was not present in the ER of liver and kidney. Anti-VAMP7 antibodies reduced the transfer of triacylglycerol, but not newly synthesized proteins, from the ER to the Golgi by 85%. We conclude that VAMP7 is enriched in intestinal ER and that it plays a functional role in the delivery of triacylglycerol from the ER to the Golgi. [ABSTRACT FROM AUTHOR]