Abstract
Upon cell stimulation, the network of cortical actin filaments is rearranged to facilitate the neurosecretory process. This actin rearrangement includes both disruption of the preexisting actin network and de novo actin polymerization. However, the mechanism by which a Ca2+ signal elicits the formation of new actin filaments remains uncertain. Cortactin, an actin-binding protein that promotes actin polymerization in synergy with the nucleation promoting factor N-WASP, could play a key role in this mechanism. We addressed this hypothesis by analyzing de novo actin polymerization and exocytosis in bovine adrenal chromaffin cells expressing different cortactin or N-WASP domains, or cortactin mutants that fail to interact with proline-rich domain (PRD)-containing proteins, including N-WASP, or to be phosphorylated by Ca2+-dependent kinases, such as ERK1/2 and Src. Our results show that the activation of nicotinic receptors in chromaffin cells promotes cortactin translocation to the cell cortex, where it colocalizes with actin filaments. We further found that, in association with PRD-containing proteins, cortactin contributes to the Ca2+-dependent formation of F-actin, and regulates fusion pore dynamics and the number of exocytotic events induced by activation of nicotinic receptors. However, whereas the actions of cortactin on the fusion pore dynamics seems to depend on the availability of monomeric actin and its phosphorylation by ERK1/2 and Src kinases, cortactin regulates the extent of exocytosis by a mechanism independent of actin polymerization. Together our findings point out a role for cortactin as a critical modulator of actin filament formation and exocytosis in neuroendocrine cells.
Highlights
The exocytotic release of neurotransmitters and neuropeptides is a highly regulated process triggered by a rise in cytosolic Ca2+, and that relies on the formation of the SNARE complex as well as the Ca2+ sensor synaptotagmin (Südhof, 2013)
Given the role of cortactin in the remodeling of the cortical actin cytoskeleton, we examined whether cortactin colocalizes with actin filaments in the DMPP-stimulated condition
Given that the SH3 domain of cortactin promotes actin polymerization, whereas the neural Wiskott-Aldrich syndrome (N-WASP) proline-rich domain (PRD) inhibits the formation of new actin filaments (Figures 2B,C), we subsequently investigated the impact of these actions on exocytosis
Summary
The exocytotic release of neurotransmitters and neuropeptides is a highly regulated process triggered by a rise in cytosolic Ca2+, and that relies on the formation of the SNARE complex as well as the Ca2+ sensor synaptotagmin (Südhof, 2013). During the fusion process a narrow channel called “fusion pore” is formed This channel remains open for a variable period of time. The cortical F-actin meshwork is dynamically remodeled following stimuli that elevate cytosolic Ca2+ concentrations (Wollman and Meyer, 2012; Olivares et al, 2014) In this regard, Ca2+ concentrations that induce exocytosis promote disruption of the preexisting cortical actin network, as well as formation of new actin filaments (Trifaró et al, 2000; Gasman et al, 2004; Olivares et al, 2014). The mechanism by which a Ca2+ signal elicits the formation of actin filaments remains poorly understood
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