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Both in the presynaptic terminals of neurons, and the membranes of neuroendocrine cells, the specific site of vesicle fusion is tightly regulated. Using an advanced microscopy technique – evanescent wave or total internal reflection fluorescence (TIRF) – to assess both the extent and localization of secretion in a neurosecretory cell line (PC12 cells), we are investigating how cellular architecture specifies the site of vesicle fusion. This method relies on the evanescent field that penetrates a short distance into the low refractive index medium upon total internal reflection. Its depth of penetration depends on the angle of incidence, and decays exponentially as a function of distance away from the interface. Excitation intensity drops by a factor of >0.5 for ~80nm, and is insignificant beyond 200-300nm. It is this very tightly restricted layer of illumination that allows TRIF microscopy to observe the processes that occur only around the membrane of the cell without optical contamination from the rest of the cell. Using this approach, we have shown that the actin cytoskeleton acts as both a facilitator of secretion (by delivering vesicles to a membrane proximal zone) and an inhibitor of fusion (by restricting access to the plasma membrane). We are investigating how the lipid composition of the membrane regulates release dynamics and site specificity.
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