Imaging the Disassembly of Syntxin Cluster during the Exocytosis of Single Secretory Granules

Abstract

During the exocytosis of synaptic vesicles or of secretory granules, three so-called SNARE proteins mediate membrane fusion. One of the SNARE proteins, Syntaxin (Syx) is known to reside in the plasma membrane. Our previous work has shown that Syx makes a submicron size cluster where a granule docks at the plasma membrane. By labeling Syx clusters and granules in different colors, we now show that the Syx cluster disassembles when the adjacent granule undergoes exocytosis.As a model system, we used Ins-1 cells, a cell line derived from pancreatic beta cells. Cells expressed tissue plasminogen activator-EGFP (tPA-EGFP) to mark granules in green, and Syx-mCherry to mark Syx in red. Cells were voltage clamped in the whole-cell mode and exocytisis was evoked by depolarizing pulse. Exocytosis of single granules was observed with TIRF. Some granules (“responders”) brightened suddenly, as their exocytosis caused the release of protons and the de-quenching of tPA-EGFP. Other granules remained quiescent and served as controls (non-responders). The fluorescence of Syx-mCherry was measured at the same times and locations as that of the granules. Before exocytosis, the Syx-mCherry signal was significantly brighter beneath responders than beneath non-responders. This is consistent with the idea that syntaxin nanodomains facilitate exocytosis. After exocytosis, the Syx-mCherry signal beneath responders diminished, while no large change occurred for non-responders. Some “responders” were not visible before fusion, suggesting that they were located apart from the plasma membrane. Even for such granules, Syx-mCherry signal was observed before exocytosis and diminished after exocytosis. These results indicate that Syx clusters beneath granules are an advantage for exocytosis but then disassembled at the time of granule fusion.