Abstract
The small GTPases Rab3A and Rab27A are endogenously expressed and specifically localized to dense-core vesicles in PC12 cells and are implicated in the late steps of dense-core vesicle exocytosis. What remains unknown is the individual and collective impact of these proteins on the size of the releasable pools and the rate at which vesicles are primed into these pools. Chromaffin cells isolated from Rab3A-/-, Rab27-/-, and double knock-out mice were used to examine the density of docked vesicles, granule mobility at the membrane, and exocytotic activity. Docked granules were visualized using electron micrographs and quantified based on their placement with respect to the plasma membrane. Mobility of granules was examined using total internal reflection fluorescence (TIRF) microscopy with virus-infected chromaffin cells expressing NPY-cherry. Changes in membrane capacitance were recorded from chromaffin cells in adrenal slices using patch clamp electrophysiology, providing data reflecting fusion kinetics and the refilling of releasable pools. The present study has identified the contributions of Rab3 and Rab27 to release readiness of dense-core vesicles in primary mouse chromaffin cells.
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