Myosin II Activation and Actin Reorganization Regulate the Mode of Quantal Exocytosis in Mouse Adrenal Chromaffin Cells

Abstract

Chromaffin cells of the adrenal medulla are innervated by the sympathetic nervous system. Stimulation causes chromaffin cells to fire action potentials, leading to the exocytosis of various classes of transmitters into the circulation. Low-frequency electrical stimulation (action potentials delivered at 0.5 Hz) causes adrenal chromaffin cells to selectively release catecholamines through a kiss-and-run fusion event. Elevated electrical stimulation (action potentials at 15 Hz) evokes fusion pore dilation, full granule collapse, and additional release of the neuropeptide-containing proteinaceous granule core. Here we apply single-cell electrophysiological, electrochemical, and fluorescence measurements to investigate the cellular mechanism for this shift in exocytic behavior. We show that at low-frequency stimulation, a filamentous-actin cell cortex plays a key role in stabilizing the kiss-and-run fusion event. Increased stimulation disrupts the actin cortex, driving full granule collapse. We show that pharmacological perturbation of the actin cortex supersedes stimulus frequency in controlling exocytic mode. Finally, we show that nonmuscle myosin II activation contributes to the cytoskeleton-dependent control of the fusion event. Inhibition of myosin II or myosin light chain kinase under elevated stimulation frequencies inhibits fusion pore dilation and maintains the granule in a kiss-and-run mode of exocytosis. These results demonstrate an essential role for activity-evoked cytoskeletal rearrangement and the action of myosin II in the regulation of catecholamine and neuropeptide exocytosis and represent an essential element of the sympathetic stress response.

Decoding the Regulation of Mast Cell Exocytosis by Networks of Rab GTPases

“…Indeed, careful analysis of the dynamics of the secretory process led to the conclusion that the frequency of kiss-and-run exocytosis is 2-fold higher than that of full exocytosis in Ag-triggered RBL cells (48). Moreover, in the presence of cytochalasin D, which was previously shown to shift kiss-and-run to full exocytosis (49), the Ion/TPA-inhibitory Rabs turned inhibitory toward Ag.…”

Dynamin I plays dual roles in the activity-dependent shift in exocytic mode in mouse adrenal chromaffin cells

“…Rather, based on amperometric analysis, the fused granule remains stable as an Ω-figure for at least the duration of catecholamine release. This behavior may simply be a result of the dense proteinacious core within the granule lumen that blocks granule collapse, or it may also include other extrinsic factors such as stabilization by the cytoskeleton [33,34]. Further experimentation will be required to reveal the factors responsible for Ω-figure stabilization under light electrical stimulation.…”

“…This effect is not reflected in the more sensitive variance analysis provided in figure 3, indicating that dilation must occur to some degree, but does not proceed to completion. Indeed, recent work has suggested that mechanisms other than dynamin function contribute to fusion pore dilation [33,34].…”

Molecular Machinery Regulating Exocytosis

“…Inhibition of phosphorylation by the myosin light chain kinase (MLCK) at sites Tyr18 and Ser19 of Myosin2, slowed down the opening of the fusion pore, during the release of catecholamines and peptide transmitters in chromaffin cells (Doreian, et al, 2008;Doreian, et al, 2009;Neco, et al, 2008). Similarly, phosphorylation at Ser 19 of Myosin2 by MLCK was shown to be necessary for maintaining the opening of the fusion pore in pancreatic cells (Bhat & Thorn, 2009).…”