Imaging the rapid yet transient accumulation of regulatory lipids, lipid kinases, and protein kinases during membrane fusion, at sites of exocytosis of MMP-9 in MCF-7 cells.

BACKGROUND: The regulation of exocytosis is physiologically vital in cells and requires a variety of distinct proteins and lipids that facilitate 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: Total internal reflection fluorescence microscopy (TIRF) was used to observe the spatial organization and temporal dynamics (i.e. spatial positioning and timing patterns) of several lipids, and accessory proteins, like lipid kinases and protein kinases, in the form of protein kinase C (PRKC) associated with sites of exocytosis of matrix metalloproteinase-9 (MMP-9) in living MCF-7 cancer cells. RESULTS: Following stimulation with phorbol myristate acetate (PMA) to promote exocytosis, a transient accumulation of several distinct regulatory lipids, lipid kinases, and protein kinases at exocytic sites was observed. This transient accumulation centered at the time of membrane fusion is followed by a rapid diffusion 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). This includes key enzymes like 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 sites of exocytosis, during membrane fusion and cargo release. More importantly, their enrichment at fusion sites serves as an important spatial and temporal organizing "element" defining individual exocytic sites.

Spatiotemporal organization and protein dynamics involved in regulated exocytosis of MMP-9 in breast cancer cells.

Altered regulation of exocytosis is an important mechanism controlling many diseases, including cancer. Defects in exocytosis have been implicated in many cancer cell types and are generally attributed to mutations in cellular transport, trafficking, and assembly of machinery necessary for exocytosis of secretory vesicle cargo. In these cancers, up-regulation of trafficking and secretion of matrix metalloproteinase-9 (MMP-9), a proteolytic enzyme, is responsible for degrading the extracellular matrix, a necessary step in tumor progression. Using TIRF microscopy, we identified proteins associated with secretory vesicles containing MMP-9 and imaged the local dynamics of these proteins at fusion sites during regulated exocytosis of MMP-9 from MCF-7 breast cancer cells. We found that many regulators of exocytosis, including several Rab GTPases, Rab effector proteins, and SNARE/SNARE modulator proteins, are stably assembled on docked secretory vesicles before exocytosis. At the moment of fusion, many of these components are quickly lost from the vesicle, while several endocytic proteins and lipids are simultaneously recruited to exocytic sites at precisely that moment. Our findings provide insight into the dynamic behavior of key core exocytic proteins, accessory proteins, lipids, and some endocytic proteins at single sites of secretory vesicle fusion in breast cancer cells.