Abstract
Background: The control of exocytosis is physiologically essential. In vitro SNARE proteins are sufficient to drive membrane fusion, but in cells there are additional proteins and lipids that work together to drive efficient, fast, and timely release of secretory vesicle cargo. Growing evidence suggests that regulatory lipids act as important lipid signals and regulate various biological processes including exocytosis. Though functional roles of many of these regulatory lipids has been linked to exocytosis, the dynamic behavior of these lipids during membrane fusion at sites of exocytosis in cell culture remains unknown. Methods: We used total internal reflection fluorescence microscopy (TIRF) to observe the spatial organization and temporal dynamics of several lipids, and accessory proteins, like lipid kinases and protein kinases, in the form of protein kinase C (PRKC) relative to single sites of exocytosis of MMP-9 in living MCF-7 cancer cells. Results: After stimulating exocytosis with PMA, we observed a transient accumulation of the regulatory lipids (e.g. PIP, PIP2, and DAG), lipid kinases (e.g. PI4K2B, PI4K3A, and PIP5KA), and protein kinases (e.g. PRKCA and PRKCE) at exocytic sites centered on the time of membrane fusion, before rapidly diffusing away from the fusion sites. Additionally, the synthesis of these regulatory lipids, degradation of these lipids, and the downstream effectors activated by these lipids, are also achieved by the recruitment and accumulation of key enzymes at exocytic sites (during the moment of cargo release), including lipid kinases, protein kinases, and phospholipases that facilitate membrane fusion and exocytosis of MMP-9. Conclusions: This work suggests that these regulatory lipids and associated effector proteins are locally synthesized and/or recruited to exocytic sites, during the time of membrane fusion and cargo release, and their enrichment at fusion sites serves as an important spatial and temporal organizing “element” defining individual exocytic sites.
Probing the biogenesis pathway and dynamics of thylakoid membranes