Temporally resolving protein and lipid colocalization at exocytic sites

FARE Award Winner

Authors

• AJ Trexler
• KA Sochacki
• JW Taraska

Abstract

Regulated exocytosis, the controlled release of vesicle-enclosed molecules from cells, is essential for eukaryotic life and human health. Release of vesicle cargo requires that the vesicle membrane fuse with the plasma membrane of the cell, and the SNARE proteins driving this process form the core of the exocytic machinery. However, there are over a dozen other molecular factors that are absolutely critical to exocytosis. It is largely unknown how all these components come together in living cells to achieve exocytosis. To address this outstanding question, we used two-color total internal reflection fluorescence microscopy to visualize individual exocytic events in live insulinoma cells which enabled us to observe proteins present at exocytic sites before, during, and after vesicle fusion. We examined over 20 proteins involved in exocytosis, and one of our most surprising findings was that tomosyn, a protein believed to negatively regulate exocytosis, was present at exocytic sites until the moment of fusion after which it diffused away rapidly. We hypothesize that tomosyn may be responsible for fine-tuning the number of properly assembled SNARE complexes at the exocytic site. We also observe the recruitment of a protein complex involving the classic endocytic protein dynamin to exocytic sites near the moment of membrane fusion. Dynamin appears to be recruited via interactions with the lipid PIP2 and the protein amphiphysin. Collectively our data begin to build a better cellular context into which the vast biochemical knowledge of exocytosis can be better understood at a functional level.

Scientific Focus Area: Cell Biology

This page was last updated on Friday, March 26, 2021