Phospholipase Cη2 Activation Redirects Vesicle Trafficking by Regulating F-actin.

PI(4,5)P2 localizes to sites of dense core vesicle exocytosis in neuroendocrine cells and is required for Ca(2+)-triggered vesicle exocytosis, but the impact of local PI(4,5)P2 hydrolysis on exocytosis is poorly understood. Previously, we reported that Ca(2+)-dependent activation of phospholipase Cη2 (PLCη2) catalyzes PI(4,5)P2 hydrolysis, which affected vesicle exocytosis by regulating the activities of the lipid-dependent priming factors CAPS (also known as CADPS) and ubiquitous Munc13-2 in PC12 cells. Here we describe an additional role for PLCη2 in vesicle exocytosis as a Ca(2+)-dependent regulator of the actin cytoskeleton. Depolarization of neuroendocrine PC12 cells with 56 or 95 mm KCl buffers increased peak Ca(2+) levels to ~400 or ~800 nm, respectively, but elicited similar numbers of vesicle exocytic events. However, 56 mm K(+) preferentially elicited the exocytosis of plasma membrane-resident vesicles, whereas 95 mm K(+) preferentially elicited the exocytosis of cytoplasmic vesicles arriving during stimulation. Depolarization with 95 mm K(+) but not with 56 mm K(+) activated PLCη2 to catalyze PI(4,5)P2 hydrolysis. The decrease in PI(4,5)P2 promoted F-actin disassembly, which increased exocytosis of newly arriving vesicles. Consistent with its role as a Ca(2+)-dependent regulator of the cortical actin cytoskeleton, PLCη2 localized with F-actin filaments. The results highlight the importance of PI(4,5)P2 for coordinating cytoskeletal dynamics with vesicle exocytosis and reveal a new role for PLCη2 as a Ca(2+)-dependent regulator of F-actin dynamics and vesicle trafficking.

Resident CAPS on dense-core vesicles docks and primes vesicles for fusion.

The Ca(2+)-dependent exocytosis of dense-core vesicles in neuroendocrine cells requires a priming step during which SNARE protein complexes assemble. CAPS (aka CADPS) is one of several factors required for vesicle priming; however, the localization and dynamics of CAPS at sites of exocytosis in live neuroendocrine cells has not been determined. We imaged CAPS before, during, and after single-vesicle fusion events in PC12 cells by TIRF micro-scopy. In addition to being a resident on cytoplasmic dense-core vesicles, CAPS was present in clusters of approximately nine molecules near the plasma membrane that corresponded to docked/tethered vesicles. CAPS accompanied vesicles to the plasma membrane and was present at all vesicle exocytic events. The knockdown of CAPS by shRNA eliminated the VAMP-2-dependent docking and evoked exocytosis of fusion-competent vesicles. A CAPS(ΔC135) protein that does not localize to vesicles failed to rescue vesicle docking and evoked exocytosis in CAPS-depleted cells, showing that CAPS residence on vesicles is essential. Our results indicate that dense-core vesicles carry CAPS to sites of exocytosis, where CAPS promotes vesicle docking and fusion competence, probably by initiating SNARE complex assembly.

CAPS and Munc13 utilize distinct PIP2-linked mechanisms to promote vesicle exocytosis.

Phosphoinositides provide compartment-specific signals for membrane trafficking. Plasma membrane phosphatidylinositol 4,5-bisphosphate (PIP2) is required for Ca(2+)-triggered vesicle exocytosis, but whether vesicles fuse into PIP2-rich membrane domains in live cells and whether PIP2 is metabolized during Ca(2+)-triggered fusion were unknown. Ca(2+)-dependent activator protein in secretion 1 (CAPS-1; CADPS/UNC31) and ubMunc13-2 (UNC13B) are PIP2-binding proteins required for Ca(2+)-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 Ca(2+)-dependent translocation to PIP2-rich plasma membrane domains is required for its activity. 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 Ca(2+) influx conditions sufficient to activate phospholipase Cη2 (PLCη2). Such conditions reduce CAPS activity and enhance Munc13 activity, establishing PLCη2 as a Ca(2+)-dependent modulator of exocytosis. These studies provide a direct view of the spatial distribution of PIP2 linked to vesicle exocytosis via regulation of lipid-dependent protein effectors CAPS and Munc13.