Biogenesis of synaptic-like microvesicles in perforated PC12 cells

We have established a system that reconstitutes the biogenesis of synaptic-like microvesicles (SLMVs) in perforated cells of the rat neuroendocrine cell line PC12. The system is based on the biotinylation of synaptophysin, a marker of synaptic vesicles and SLMVs. Biotinylation is performed at 18 degrees C, a temperature at which formation of SLMVs is blocked and biotinylated synaptophysin accumulates in the SLMV donor compartment. The biotinylated PC12 cells are then perforated by scraping and incubated at 37 degrees C in the presence of ATP and cytosolic proteins, conditions required for SLMV biogenesis. After the perforated-cell reaction, the newly formed SLMVs are isolated by differential centrifugation followed by either glycerol gradient centrifugation or a simple single-glycerol-step centrifugation. The latter allows the analysis of many perforated-cell reactions in parallel and, hence, the dissection of the molecular machinery mediating SLMV biogenesis. Using this system, we have found that clathrin, dynamin, phosphatidylinositol transfer protein, and SH3P4 are involved in SLMV biogenesis.