Morphological, biochemical, and membrane capacitance measurements were used to study the role of cortical filamentous actin (F-actin) in exocytosis. Fluorescence and electron microscopy of resting chromaffin cells revealed a cortical actin network that excluded secretory vesicles from the subplasmalemmal area. Phorbol ester (PMA) treatment disrupted cortical F-actin and increased both the number of vesicles within the 0-50 nm subplasmalemmal zone and the initial rate of stimulated catecholamine release. In PMA-pretreated cells, membrane capacitance studies showed an increased number of vesicles fusing with the plasmalemma during the first two depolarizations of a train. PMA did not affect voltage-dependent Ca2+ influx. The total number of vesicles fused with the plasma membrane correlated well with the number of vesicles occupying the 0-50 nm cortical zone. Therefore, cortical F-actin disassembly allows translocation of vesicles to the plasmalemma in preparation for exocytosis.

Abstract.Immunofluorescence and cytochemical studies have demonstrated that filamentous actin is mainly localized in the cortical surface of the chromaffin cell. It has been suggested that these actin filament networks act as a barrier to the secretory granules, impeding their contact with the plasma membrane. Stimulation of chromaffin cells produces a disassembly of actin filament networks, implying the removal of the barrier. The presence of gelsolin and scinderin, two Ca2+-dependent actin filament severing proteins, in the cortical surface of the chromatfin cells, suggests the possibility that cell stimulation brings about activation of one or more actin filament severing proteins with the consequent disruption of actin networks. Therefore, biochemical studies and fluorescence microscopy experiments with scinderin and gelsolin antibodies and rhodamine-phalloidin, a probe for filamentous actin, were performed in cultured chromaffin cells to study the distribution of scinderin, gelsolin, and filamentous actin during cell stimulation and to correlate the possible changes with catecholamine secretion. Here we report that during nicotinic stimulation or K+-evoked depolarization, subcortical scinderin but not gelsolin is redistributed and that this redistribution precedes catecholamine secretion. The rearrangement of scinderin in patches is mediated by nicotinic receptors. Cell stimulation produces similar patterns of distribution of scinderin and filamentous actin. However, after the removal of the stimulus, the recovery of scinderin cortical pattern of distribution is faster than F-actin reassembly, suggesting that scinderin is bound in the cortical region of the cell to a component other than F-actin. We also demonstrate that peripheral actin filament disassembly and subplasmalemmal scinderin redistribution are calcium-dependent events. Moreover, experiments with an antibody against dopamine-~-hydroxylase suggest that exocytosis sites are preferentially localized to areas of F-actin disassembly.

Chromaffin cell exocytosis is a fascinating interplay between secretory vesicles and cellular components. One of these components is the cytoskeleton and its associated regulatory proteins. Transport of chromaffin secretory granules from their site of biosynthesis towards the active site of exocytosis requires both F-actin fine remodelling as well as microtubule trails. At least two molecular motors, myosins II and V, seem to play a crucial role in the control of F-actin dynamics and vectorial vesicle displacement respectively. Vesicle movement experiences spatial restrictions as they approach the cell cortical region, where the F-actin meshwork constitutes a barrier-limiting vesicle access to the plasmalemma. During secretion, cortical F-actin is locally disrupted providing access of vesicles to release sites on the plasmalemma. Removal of the stimulus restores cortical F-actin. Two pathways (Ca2+-scinderin and PKC-MARCKS) control F-actin changes during the secretory cycle . Furthermore, GTPases such as RhoA, that controls F-actin network integrity, and Cdc42 signalling which induces the formation of local actin filaments at active sites, provide additional evidence on the importance of F-actin as a key element in vesicle transport and in the exocytotic machinery of chromaffin cells.

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The presence of myosin II and V in chromaffin cells and their subcellular distribution is described. Myosin II and V distribution in sucrose density gradients showed only a strong correlation between the distribution of myosin V and secretory vesicle markers. Confocal microscopy images demonstrated colocalization of myosin V with dopamine b-hydroxylase, a chromaffin vesicle marker, whereas myosin II was present mainly in the cell cortex. Cell depolarization induced, in a Ca 2+ and time-dependent manner, the dissociation of myosin V from chromaffin vesicles suggesting that this association was not permanent but determined by secretory cycle requirements. Myosin II was also found in the crude granule fraction, however, its distribution was not affected by cell depolarization. Myosin V head antibodies were able to inhibit secretion whereas myosin II antibodies had no inhibitory effect. The pattern of inhibition indicated that these treatments interfered with the transport of vesicles from the reserve to the release-ready compartment, suggesting the involvement of myosin V and not myosin II in this transport process. The results described here suggest that myosin V is a molecular motor involved in chromaffin vesicle secretion. However, these results do not discard an indirect role for myosin II in secretion through its interaction with F-actin networks.