Anti-VPS54 monoclonal antibody Made to order

The sperm acrosome is a specialized vacuole, a member of the family of cell-specific lysosome-related organelles. Its exocytosis, the acrosome reaction, is a crucial event during fertilization. The released acrosomal contents promote sperm penetration through the investments of the oocyte, whereas the membranous components of the acrosome are involved in sperm-oocyte interaction/fusion and oocyte activation. The way that these functionally distinct acrosomal costituents reach the vacuole during its biogenesis remains poorly understood. The biosynthetic pathway and a consistent supply from the endosomal system have recently been documented. We use immunogold electron microscopy to determine the contribution of endosome cargo-sorting during step-by-step mouse acrosomogenesis. The chosen proteins of this study were UBPy (ESCRT-DUB), together with endosome compartment markers EEA1 and pallidin. The latter is described here for the first time in male germ cells. This new insight expands our knowledge of acrosomogenesis, confirming the plasticity of the endosomal system in supporting cell-type-specific functions. We also study wobbler mice, whose Vps54 mutation causes motor neuron degeneration and male infertility. Use of electron/immunoelectron microscopy and immunofluorescence enabled us to establish that the lack of an acrosome in wobbler spermatozoa is attributable to an early block in acrosome biogenesis and that the mislocalization of acrosome-destined proteins, potentially involved in the signaling events leading to oocyte activation, is possibly responsible for wobbler infertility, even after intracytoplasmic sperm injection.

The conserved oligomeric complex (COG) is a multi-subunit vesicle tethering complex that functions in retrograde trafficking at the Golgi. We have previously demonstrated that the formation of enlarged endo-lysosomal structures (EELSs) is one of the major glycosylation-independent phenotypes of cells depleted for individual COG complex subunits. Here, we characterize the EELSs in HEK293T cells using microscopy and biochemical approaches. Our analysis revealed that the EELSs are highly acidic and that vATPase-dependent acidification is essential for the maintenance of this enlarged compartment. The EELSs are accessible to both trans-Golgi enzymes and endocytic cargo. Moreover, the EELSs specifically accumulate endolysosomal proteins Lamp2, CD63, Rab7, Rab9, Rab39, Vamp7, and STX8 on their surface. The EELSs are distinct from lysosomes and do not accumulate active Cathepsin B. Retention using selective hooks (RUSH) experiments revealed that biosynthetic cargo mCherry-Lampl reaches the EELSs much faster as compared to both receptor-mediated and soluble endocytic cargo, indicating TGN origin of the EELSs. In support to this hypothesis, EELSs are enriched with TGN specific lipid PI4P. Additionally, analysis of COG4/VPS54 double KO cells revealed that the activity of the GARP tethering complex is necessary for EELSs' accumulation, indicating that protein mistargeting and the imbalance of Golgi-endosome membrane flow leads to the formation of EELSs in COG-deficient cells. The EELSs are likely to serve as a degradative storage hybrid organelle for mistargeted Golgi enzymes and underglycosylated glycoconjugates. To our knowledge this is the first report of the formation of an enlarged hybrid endosomal compartment in a response to malfunction of the intra-Golgi trafficking machinery.