Abstract
Many regulatory steps precede final membrane fusion in neuroendocrine cells. Some parts of this preparatory cascade, including fusion and priming, are dependent on the intracellular Ca2+ concentration ([Ca2+]i). However, the functional implications of [Ca2+]i in the regulation of docking remain elusive and controversial due to an inability to determine the modulatory effect of [Ca2+]i. Using a combination of TIRF-microscopy and electrophysiology we followed the movement of large dense core vesicles (LDCVs) close to the plasma membrane, simultaneously measuring membrane capacitance and [Ca2+]i. We found that a free [Ca2+]i of 700 nM maximized the immediately releasable pool and minimized the lateral mobility of vesicles, which is consistent with a maximal increase of the pool size of primed LDCVs. The parameters that reflect docking, i.e. axial mobility and the fraction of LDCVs residing at the plasma membrane for less than 5 seconds, were strongly decreased at a free [Ca2+]i of 500 nM. These results provide the first evidence that docking and priming occur at different free intracellular Ca2+ concentrations, with docking efficiency being the most robust at 500 nM.
Highlights
Before fusion with the plasma membrane (PM), secretory vesicles have to complete several steps, namely docking and priming
Because total internal reflection fluorescence (TIRF) microscopy allows the observation of large dense core vesicles (LDCVs) that reside at the PM, it is ideally suited for observing both docking and priming [4,5,6]
Increased Concentration of Free Intracellular Ca2+ Led to Increased Releasable Pool Size LDCVs in bovine chromaffin cells were stained by overexpressing neuropeptide Y (NPY) fused to the red fluorescent protein mCherry, and their mobility near the PM was visualized by TIRF-microscopy
Summary
Before fusion with the plasma membrane (PM), secretory vesicles have to complete several steps, namely docking and priming. Until now, docked vesicles were typically identified in electron micrographs by their proximity to the PM. The criteria for such ‘‘morphologically’’ docked vesicles vary, with distances as far as 30 nm from the PM and as close as visibly touching the PM [1,2]. Electron micrographs do not allow the differentiation of docked and primed vesicles [3]. Because total internal reflection fluorescence (TIRF) microscopy allows the observation of large dense core vesicles (LDCVs) that reside at the PM, it is ideally suited for observing both docking and priming [4,5,6]
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