Cortical filamentous actin disassembly and scinderin redistribution during chromaffin cell stimulation precede exocytosis, a phenomenon not exhibited by gelsolin

Department of Pharmacology, Faculty of Medicine, University of Ottawa, Ontario, Canada.
The Journal of Cell Biology (Impact Factor: 9.83). 07/1991; 113(5):1057-67.
Source: PubMed


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 Ca(2+)-dependent actin filament severing proteins, in the cortical surface of the chromaffin 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-beta-hydroxylase suggest that exocytosis sites are preferentially localized to areas of F-actin disassembly.

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    • "Much of what is known about Ads was discovered through detailed examination of exocytosis in bovine chromaffin cells of the adrenal medulla. Immunocytochemistry analysis of chromaffin cells co-localize Ads with a cortical F-actin ring [24], [46], while numerous studies have demonstrated the role of Ads in depolymerizing the cortical F-actin ring and allowing for vesicular exocytosis [47]-[51]. To our knowledge, the microarray by Yang et al. [35] aimed at identifying genes differentially regulated by RANKL in mature osteoclasts was the first published report of Ads in the osteoclast biological system. "
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    ABSTRACT: Adseverin (Ads), a member of the Gelsolin superfamily of actin binding proteins, regulates the actin cytoskeleton architecture by severing and capping existing filamentous actin (F-actin) strands and nucleating the assembly of new F-actin filaments. Ads has been implicated in cellular secretion, exocytosis and has also been shown to regulate chondrogenesis and megakaryoblastic leukemia cell differentiation. Here we report for the first time that Ads is involved in regulating osteoclastogenesis (OCG). Ads is induced during OCG downstream of RANK-ligand (RANKL) stimulation and is highly expressed in mature osteoclasts. The D5 isoform of Ads is not involved in regulating OCG, as its expression is not induced in response to RANKL. Three clonal Ads knockdown RAW264.7 (RAW) macrophage cell lines with varying degrees of Ads expression and OCG deficiency were generated. The most drastic OCG defect was noted in the clonal cell line with the greatest degree of Ads knockdown as indicated by a lack of TRAcP staining and multinucleation. RNAi mediated knockdown of Ads in osteoclast precursors resulted in distinct morphological changes characterized by altered F-actin distribution and increased filopodia formation. Ads knockdown precursor cells experienced enhanced migration while fusion of knockdown precursors cells was limited. Transient reintroduction of de novo Ads back into the knockdown system was capable of rescuing TRAcP expression but not osteoclast multinucleation most likely due to the transient nature of Ads expression. This preliminary study allows us to conclude that Ads is a RANKL induced early regulator of OCG with a potential role in pre-osteoclast differentiation and fusion.
    PLoS ONE 10/2014; 9(10):e109078. DOI:10.1371/journal.pone.0109078 · 3.23 Impact Factor
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    • "The cortical actin network plays an important and well-described role during vesicle exocytosis (5, 7, 9, 10), and in recent years new functions for actin and its associated proteins have emerged (2, 9, 10, 20–24). Ca2+-dependent reorganization and remodeling of the cortical actin network help vesicles move toward the plasma membrane by partial disassembly of the cortical layer (Figure 1B) (3, 6). At the same time, this remodeling provides tracks that extend further toward the center of the cell allowing the mobilization of SGs from the reserve pool (25) to their docking and fusion sites at the plasma membrane (4, 26, 27). "
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    ABSTRACT: Dysregulation of regulated exocytosis is linked to an array of pathological conditions, including neurodegenerative disorders, asthma, and diabetes. Understanding the molecular mechanisms underpinning neuroexocytosis including the processes that allow neurosecretory vesicles to access and fuse with the plasma membrane and to recycle post-fusion, is therefore critical to the design of future therapeutic drugs that will efficiently tackle these diseases. Despite considerable efforts to determine the principles of vesicular fusion, the mechanisms controlling the approach of vesicles to the plasma membrane in order to undergo tethering, docking, priming, and fusion remain poorly understood. All these steps involve the cortical actin network, a dense mesh of actin filaments localized beneath the plasma membrane. Recent work overturned the long-held belief that the cortical actin network only plays a passive constraining role in neuroexocytosis functioning as a physical barrier that partly breaks down upon entry of Ca(2+) to allow secretory vesicles to reach the plasma membrane. A multitude of new roles for the cortical actin network in regulated exocytosis have now emerged and point to highly dynamic novel functions of key myosin molecular motors. Myosins are not only believed to help bring about dynamic changes in the actin cytoskeleton, tethering and guiding vesicles to their fusion sites, but they also regulate the size and duration of the fusion pore, thereby directly contributing to the release of neurotransmitters and hormones. Here we discuss the functions of the cortical actin network, myosins, and their effectors in controlling the processes that lead to tethering, directed transport, docking, and fusion of exocytotic vesicles in regulated exocytosis.
    Frontiers in Endocrinology 10/2013; 4:153. DOI:10.3389/fendo.2013.00153
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    • "The dynamics of actin depolymerization plays a key role in regulation of vesicle secretion (Aunis and Bader, 1988; Muallem et al., 1995; Yoneda et al., 2000; Eitzen, 2003). Depolymerization of F-actin has been shown to promote vesicle fusion and secretion in mammalian cells (Vitale et al., 1991). Here, we have tested the potential role of actin depolymerization in microneme secretion by P. falciparum merozoites. "
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    ABSTRACT: Plasmodium falciparum invades host erythrocytes by multiple invasion pathways. The invasion of erythrocytes by P. falciparum merozoites is a complex process that requires multiple interactions between host receptors and parasite ligands. A number of parasite proteins that mediate interaction with host receptors during invasion are localized to membrane-bound apical organelles referred to as micronemes and rhoptries. The timely release of these proteins to the merozoite surface is crucial for receptor engagement and invasion. It has been demonstrated previously that exposure of merozoites to a low potassium (K(+) ) ionic environment as found in blood plasma leads to a rise in cytosolic calcium (Ca(2+) ), which triggers microneme secretion. The signaling pathways that regulate microneme discharge in response to rise in cytosolic Ca(2+) are not completely understood. Here, we show that a P. falciparum Ca(2+) -dependent protein phosphatase, calcineurin (PfCN), is an essential regulator of Ca(2+) -dependent microneme exocytosis. An increase in PfCN activity was observed in merozoites following exposure to a low K(+) environment. Treatment of merozoites with calcineurin inhibitors such as FK506 and cyclosporin A prior to transfer to a low K(+) environment resulted in inhibition of secretion of microneme protein apical merozoite antigen-1 (PfAMA-1). Inhibition of PfCN was shown to result in reduced dephosphorylation and depolymerization of apical actin, which appears to be critical for microneme secretion. PfCN thus serves as an effector of Ca(2+) -dependent microneme exocytosis by regulating depolymerization of apical actin. Inhibitors that target PfCN block microneme exocytosis and limit growth of P. falciparum blood stage parasites providing a novel approach towards development of new therapeutic strategies against malaria.
    Cellular Microbiology 08/2013; 16(1). DOI:10.1111/cmi.12177 · 4.92 Impact Factor
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