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Activity-dependent bulk endocytosis (ADBE) is the dominant SV endocytosis mode under intense neuronal activity.\n\nThe authors investigated whether directed sorting of SV cargo occurs at the plasma membrane during ADBE, since this will ultimately impact on the molecular composition of SVs generated via this endocytosis mode.\n\nThey examined the retrieval of different SV cargo molecules during intense stimulation using a series of genetically encoded pH-sensitive reporters in neuronal cultures, and they found that only one was preferentially trafficked via ADBE-VAMP4.\n\nAuthor showed two important results:\n1) VAMP4 is not simply represented as an ADBE cargo, actually authors showed by using biochemical fractionation experiments, that endogenous VAMP4 was selectively enriched on bulk endosomes.\n\n2) VAMP4 is essential for ADBE to proceed. Authors determined this by using validated shRNA oligonucleotides against VAMP4. When 40Hz/10s stimulation was applied, VAMP4 knockdown abolished TMR-dextran uptake, an inhibition fully rescued by expression of wild-type VAMP4- pHluorin (Figures 7B and C). Therefore, in addition to being the first identified ADBE cargo molecule, VAMP4 is essential for this key endocytosis mode.\n\nVAMP4 Location: Figure 7A --\u003e Representative images show nerve terminals labeled with VAMP4 antibodies after immunogold detection and silver enhancement. Almost all silver particles were localized to endosomal structures after 40 Hz stimulation (indicated by arrows).. Experimental description: Rat: Primary cultures of cerebellar neurons were prepared from the cerebella of 7-day-old Sprague Dawley rat pups of both sexes (Anggono et al., 2006).\n\nMouse: Dissociated primary hippocampal neuronal cultures were prepared from E17.5 C56BL/6J mouse embryos of both sexes.\n\nCerebellar neurons were transfected between 5 and 7 days in culture, whereas hippocampal neurons were transfected between 6 and 8 days in culture with Lipofectamine 2000 (Gordon et al., 2011).\n\n7/4/2017 PIm\n- The over-expresssion of VAMP4 used in figure 7 is used as specificity control for the shRNA-mediated KD. Therefore, the species of this VAMP4 protein is not relevant and the annotation against rat VAMP4 is approved."},{"key":"https://w3id.org/biolink/vocab/in_taxon","value":"http://purl.obolibrary.org/obo/NCBITaxon_10116"}]}