508 | G. Kabachinski et al. Molecular Biology of the Cell MBoC | ARTICLE CAPS and Munc13 utilize distinct PIP2-linked mechanisms to promote vesicle exocytosis

ABSTRACT Phosphoinositides provide compartment-specific signals for membrane traffick-ing. Plasma membrane phosphatidylinositol 4,5-bisphosphate (PIP2) is required for Ca2+-trig-gered vesicle exocytosis, but whether vesicles fuse into PIP2-rich membrane domains in live cells and whether PIP2 is metabolized during Ca2+-triggered fusion were unknown. Ca2+-de-pendent activator protein in secretion 1 (CAPS-1; CADPS/UNC31) and ubMunc13-2 (UNC13B) are PIP2-binding proteins required for Ca2+-triggered vesicle exocytosis in neuroendocrine PC12 cells. These proteins are likely effectors for PIP2, but their localization during exocytosis had not been determined. Using total internal reflection fluorescence microscopy in live cells, we identify PIP2-rich membrane domains at sites of vesicle fusion. CAPS is found to reside on vesicles but depends on plasma membrane PIP2 for its activity. Munc13 is cytoplasmic, but Ca2+-dependent translocation to PIP2-rich plasma membrane domains is required for its activ-ity. The results reveal that vesicle fusion into PIP2-rich membrane domains is facilitated by sequential PIP2-dependent activation of CAPS and PIP2-dependent recruitment of Munc13. PIP2 hydrolysis only occurs under strong Ca2+ influx conditions sufficient to activate phospho-lipase Cη2 (PLCη2). Such conditions reduce CAPS activity and enhance Munc13 activity, es-tablishing PLCη2 as a Ca2+-dependent modulator of exocytosis. These studies provide a di-rect view of the spatial distribution of PIP2 linked to vesicle exocytosis via regulation of lipid-dependent protein effectors CAPS and Munc13