Imaging Regulatory Lipids and Protein Kinase C at Sites of Exocytosis

Abstract

The control of exocytosis is physiologically essential. In vitro SNARE proteins are sufficient to drive model membrane fusion, but in cells there are over twenty additional proteins and lipids that work together to drive efficient, fast, and timely release of vesicular cargo. Many of these important accessory proteins are controlled via phosphorylation, and protein kinase C (PKC) has previously been linked to the modulation of exocytosis. We use total internal reflection fluorescence microscopy to observe the spatiotemporal dynamics of dozens of proteins and lipids relative to single sites of exocytosis in living endocrine INS-1 cells. INS-1 cells are a model system for insulin secretion. We use a vesicle cargo marker NPY-GFP to identify where exocytosis occurs and tag proteins-of-interest with a red fluorescent protein. Lipid species are visualized using lipid sensors: fluorescent proteins fused to protein domains with known specificity for single lipids. After stimulating exocytosis using ionomycin, we observe a transient accumulation of PIP2- and DAG-sensors at exocytic sites centered on the time of membrane fusion and only lasting a few seconds. Intriguingly, we observe a concomitant recruitment of conventional PKC isoforms with kinetics broadly similar to PIP2 and DAG. Novel and atypical PKCs do not appear to be recruited. PIP2, DAG, and PKC recruitment only occur at sites of exocytosis and not at control docked-vesicles nearby to sites of exocytosis. PKC is known to target several critical exocytic proteins, including munc18 and dynamin-1, and we are currently investigating whether these targets are phosphorylated in response to the recruitment of PKC that we observe. These data suggest that a regulatory lipid cascade may recruit and activate PKC to sites of exocytosis to regulate membrane fusion or cargo release.