Src kinases regulate de novo actin polymerization during exocytosis in neuroendocrine chromaffin cells.

María José Guerra,María José Olivares,Narcisa Martínez-Quiles,Laszlo Buday,Arlek M. González-Jamett,Ana M. Cárdenas,Valentina Haro-Acuña,Ximena Baez-Matus

doi:10.1371/journal.pone.0099001

María José Guerra, María José Olivares + Show 6 more

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https://doi.org/10.1371/journal.pone.0099001

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Abstract

The cortical actin network is dynamically rearranged during secretory processes. Nevertheless, it is unclear how de novo actin polymerization and the disruption of the preexisting actin network control transmitter release. Here we show that in bovine adrenal chromaffin cells, both formation of new actin filaments and disruption of the preexisting cortical actin network are induced by Ca2+ concentrations that trigger exocytosis. These two processes appear to regulate different stages of exocytosis; whereas the inhibition of actin polymerization with the N-WASP inhibitor wiskostatin restricts fusion pore expansion, thus limiting the release of transmitters, the disruption of the cortical actin network with cytochalasin D increases the amount of transmitter released per event. Further, the Src kinase inhibitor PP2, and cSrc SH2 and SH3 domains also suppress Ca2+-dependent actin polymerization, and slow down fusion pore expansion without disturbing the cortical F-actin organization. Finally, the isolated SH3 domain of c-Src prevents both the disruption of the actin network and the increase in the quantal release induced by cytochalasin D. These findings support a model where a rise in the cytosolic Ca2+ triggers actin polymerization through a mechanism that involves Src kinases. The newly formed actin filaments would speed up the expansion of the initial fusion pore, whereas the preexisting actin network might control a different step of the exocytosis process.

Highlights

The subplasmalemmal actin mesh plays a pivotal function during the secretory process in neuroendocrine cells
The first studies conducted to understand the role of cortical actin filaments (F-actin) in exocytosis proposed that it constitutes a physical barrier that restricts the secretory vesicle access to the plasma membrane
It was observed that Factin forms trails that favor the secretory vesicle motion to the plasma membrane [4] and that the subplasmalemmal actin network regulates the expansion of the fusion pore [5,6], an intermediate structure formed during the exocytotic process [7], and the amount of catecholamine released per individual events [6], [8]

Summary

Introduction

The subplasmalemmal actin mesh plays a pivotal function during the secretory process in neuroendocrine cells. It was observed that Factin forms trails that favor the secretory vesicle motion to the plasma membrane [4] and that the subplasmalemmal actin network regulates the expansion of the fusion pore [5,6], an intermediate structure formed during the exocytotic process [7], and the amount of catecholamine released per individual events [6], [8] In these latter reports [5,6], [8], the role of F-actin was evaluated using pharmacological agents that indiscriminately disrupt the new F-actin formation and the preexisting cortical actin network, making it difficult to separate the differential roles of de novo actin polymerization from the preexistent actin mesh during exocytosis

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