Coupling actin and membrane dynamics during calcium-regulated exocytosis: a role for Rho and ARF GTPases

Release of neurotransmitters and hormones occurs by calcium-regulated exocytosis, a process that shares many similarities in neurons and neuroendocrine cells. Exocytosis is confined to specific regions in the plasma membrane, where actin remodelling, lipid modifications and protein-protein interactions take place to mediate vesicle/granule docking, priming and fusion. The spatial and temporal coordination of the various players to form a "fast and furious" machinery for secretion remain poorly understood. ARF and Rho GTPases play a central role in coupling actin dynamics to membrane trafficking events in eukaryotic cells. Here, we review the role of Rho and ARF GTPases in supplying actin and lipid structures required for synaptic vesicle and secretory granule exocytosis. Their possible functional interplay may provide the molecular cues for efficient and localized exocytotic fusion.     

Regulated exocytosis in neuroendocrine cells: a role for subplasmalemmal Cdc42/N-WASP-induced actin filaments

In neuroendocrine cells, actin reorganization is a prerequisite for regulated exocytosis. Small GTPases, Rho proteins, represent potential candidates coupling actin dynamics to membrane trafficking events. We previously reported that Cdc42 plays an active role in regulated exocytosis in chromaffin cells. The aim of the present work was to dissect the molecular effector pathway integrating Cdc42 to the actin architecture required for the secretory reaction in neuroendocrine cells. Using PC12 cells as a secretory model, we show that Cdc42 is activated at the plasma membrane during exocytosis. Expression of the constitutively active Cdc42(L61) mutant increases the secretory response, recruits neural Wiskott-Aldrich syndrome protein (N-WASP), and enhances actin polymerization in the subplasmalemmal region. Moreover, expression of N-WASP stimulates secretion by a mechanism dependent on its ability to induce actin polymerization at the cell periphery. Finally, we observed that actin-related protein-2/3 (Arp2/3) is associated with secretory granules and that it accompanies granules to the docking sites at the plasma membrane upon cell activation. Our results demonstrate for the first time that secretagogue-evoked stimulation induces the sequential ordering of Cdc42, N-WASP, and Arp2/3 at the interface between granules and the plasma membrane, thereby providing an actin structure that makes the exocytotic machinery more efficient.     

Exocytosis in neuroendocrine cells: new tasks for actin

Most secretory cells undergoing calcium-regulated exocytosis in response to cell surface receptor stimulation display a dense subplasmalemmal actin network, which is remodeled during the exocytotic process. This review summarizes new insights into the role of the cortical actin cytoskeleton in exocytosis. Many earlier findings support the actin-physical-barrier model whereby transient depolymerization of cortical actin filaments permits vesicles to gain access to their appropriate docking and fusion sites at the plasma membrane. On the other hand, data from our laboratory and others now indicate that actin polymerization also plays a positive role in the exocytotic process. Here, we discuss the potential functions attributed to the actin cytoskeleton at each major step of the exocytotic process, including recruitment, docking and fusion of secretory granules with the plasma membrane. Moreover, we present actin-binding proteins, which are likely to link actin organization to calcium signals along the exocytotic pathway. The results cited in this review are derived primarily from investigations of the adrenal medullary chromaffin cell, a cell model that is since many years a source of information concerning the molecular machinery underlying exocytosis.     

Intersectin-1L nucleotide exchange factor regulates secretory granule exocytosis by activating Cdc42

Rho GTPases are key regulators of the actin cytoskeleton in membrane trafficking events. We previously reported that Cdc42 facilitates exocytosis in neuroendocrine cells by stimulating actin assembly at docking sites for secretory granules. These findings raise the question of the mechanism activating Cdc42 in exocytosis. The neuronal guanine nucleotide exchange factor, intersectin-1L, which specifically activates Cdc42 and is at an interface between membrane trafficking and actin dynamics, appears as an ideal candidate to fulfill this function. Using PC12 and chromaffin cells, we now show the presence of intersectin-1 at exocytotic sites. Moreover, through an RNA interference strategy coupled with expression of various constructs encoding the guanine nucleotide exchange domain, we demonstrate that intersectin-1L is an essential component of the exocytotic machinery. Silencing of intersectin-1 prevents secretagogue-induced activation of Cdc42 revealing intersectin-1L as the factor integrating Cdc42 activation to the exocytotic pathway. Our results extend the current role of intersectin-1L in endocytosis to a function in exocytosis and support the idea that intersectin-1L is an adaptor that coordinates exo-endocytotic membrane trafficking in secretory cells.     

Bacterial toxins: useful for studying G-proteins implicated in the mechanism of exocytosis in neuroendocrine cells

In neuroendocrine cells, regulated exocytosis is a multistep process that comprises the recruitment and priming of secretory granules, their docking to the exocytotic sites, and the subsequent fusion of granules with the plasma membrane leading to the release of secretory products into the extracellular space. Using bacterial toxins which specially inactivate subsets of G proteins, we were able to demonstrate that both trimeric and monomeric G proteins directly control the late stages of exocytosis in chromaffin cells. Indeed, in secretagogue-stimulated chromaffin cells, the subplasmalemmal actin cytoskeleton undergoes a specific reorganization that is a prerequisite for exocytosis. Our results suggest that a granule-bound trimeric Go protein controls the actin network surrounding secretory granules through a pathway involving the GTPase RhoA and a downstream phosphatidylinositol 4-kinase. Furthermore, the GTPase Cdc42 plays a active role in exocytosis, most likely by providing specific actin structures to the late docking and/or fusion steps. We propose that G proteins tightly control secretion in neuroendocrine cells by coupling the actin cytoskeleton to the sequential steps underlying membrane trafficking at the site of exocytosis. Our data highlight the use of bacterial toxins, which proved to be powerful tools to dissect the exocytotic machinery at the molecular level.     

The GTP-binding protein RhoA localizes to the cortical granules of Strongylocentrotus purpuratas sea urchin egg and is secreted during fertilization

The sea urchin egg has thousands of secretory vesicles known as cortical granules. Upon fertilization, these vesicles undergo a Ca2+-dependent exocytosis. G-protein-linked mechanisms may take place during the egg activation. In somatic cells from mammals, GTP-binding proteins of the Rho family regulate a number of cellular processes, including organization of the actin cytoskeleton. We report here that a crude membrane fraction from homogenates of Strongylocentrotus purpuratus sea urchin eggs, incubated with C3 (which ADP-ribosylates specifically Rho proteins) and [32P]NAD, displayed an [32P]ADP-ribosylated protein of 25 kDa that had the following characteristics: i) identical electrophoretic mobility in SDS-PAGE gels as the [32P]ADP-ribosylated Rho from sea urchin sperm; ii) identical mobility in isoelectro focusing gels as human RhoA; iii) positive cross-reactivity by immunoblotting with an antibody against mammalian RhoA. Thus, unfertilized S. purpuratus eggs contain a mammalian RhoA-like protein. Immunocytochemical analyses indicated that RhoA was localized preferentially to the cortical granules; this was confirmed by experiments of [32P]ADP-ribosylation with C3 in isolated cortical granules. Rho was secreted and retained in the fertilization membrane after insemination or activation with A23187. It was observed that the Rho protein present in the sea urchin sperm acrosome was also secreted during the exocytotic acrosome reaction. Thus, Rho could participate in those processes related to the cortical granules, i.e., in the Ca2+-regulated exocytosis or actin reorganization that accompany the egg activation.     

The Rho Guanine Nucleotide Exchange Factors Intersectin 1L and β-Pix Control Calcium-Regulated Exocytosis in Neuroendocrine PC12 Cells

GTPases of the Rho family are molecular switches that play an important role in a wide range of membrane-trafficking processes including neurotransmission and hormone release. We have previously demonstrated that RhoA and Cdc42 regulate calcium-dependent exocytosis in chromaffin cells by controlling actin dynamics, whereas Rac1 regulates lipid organisation. These findings raised the question of the upstream mechanism activating these GTPases during exocytosis. The guanine nucleotide exchange factors (GEFs) that catalyse the exchange of GDP for GTP are crucial elements regulating Rho signalling. Using an RNA interference approach, we have recently demonstrated that the GEFs Intersectin-1L and β-Pix, play essential roles in neuroendocrine exocytosis by controlling the activity of Cdc42 and Rac1, respectively. This review summarizes these results and discusses the functional importance of Rho GEFs in the exocytotic machinery in neuroendocrine cells.     

Regulation of neuroendocrine exocytosis by the ARF6 GTPase-activating protein GIT1

Neuroendocrine cells release hormones and neuropeptides by exocytosis, a highly regulated process in which secretory granules fuse with the plasma membrane to release their contents in response to a calcium trigger. Using chromaffin and PC12 cells, we have recently described that the granule-associated GTPase ARF6 plays a crucial role in exocytosis by activating phospholipase D1 at the plasma membrane and, presumably, promoting the fusion reaction between the two membrane bilayers. ARF6 is activated by the nucleotide exchange factor ARNO following docking of granules to the plasma membrane. We show here that GIT1, a GTPase-activating protein stimulating GTP hydrolysis on ARF6, is the second molecular partner that turns over the GDP/GTP cycle of ARF6 during cell stimulation. Western blot and immunofluorescence experiments indicated that GIT1 is cytosolic in resting cells but is recruited to the plasma membrane in stimulated cells, where it co-localizes with ARF6 at the granule docking sites. Over-expression of wild-type GIT1 inhibits growth hormone secretion from PC12 cells; this inhibitory effect was not observed in cells expressing a GIT1 mutant impaired in its ARF-GTPase-activating protein (GAP) activity or in cells expressing other ARF6-GAPs. Conversely reduction of GIT1 by RNA interference increased the exocytotic activity. Using a real time assay for individual chromaffin cells, we found that microinjection of GIT1 strongly reduced the number of exocytotic events. These results provide the first evidence that GIT1 plays a function in calcium-regulated exocytosis in neuroendocrine cells. We propose that GIT1 represents part of the pathway that inactivates ARF6-dependent reactions and thereby negatively regulates and/or terminates exocytotic release.     

Exocytosis of catecholamine (CA)-containing and CA-free granules in chromaffin cells

Recent evidence suggests that endocytosis in neuroendocrine cells and neurons can be tightly coupled to exocytosis, allowing rapid retrieval from the plasma membrane of fused vesicles for future use. This can be a much faster mechanism for membrane recycling than classical clathrin-mediated endocytosis. During a fast exo-endocytotic cycle, the vesicle membrane does not fully collapse into the plasma membrane; nevertheless, it releases the vesicular contents through the fusion pore. Once the vesicle is depleted of transmitter, its membrane is recovered without renouncing its identity. In this report, we show that chromaffin cells contain catecholamine-free granules that retain their ability to fuse with the plasma membrane. These catecholamine-free granules represent 7% of the total population of fused vesicles, but they contributed to 47% of the fusion events when the cells were treated with reserpine for several hours. We propose that rat chromaffin granules that transiently fuse with the plasma membrane preserve their exocytotic machinery, allowing another round of exocytosis.     

ECL cell morphology.

Using immunohistochemistry at the conventional light, confocal and electron microscopic levels, we have demonstrated that rat stomach ECL cells store histamine and pancreastatin in granules and secretory vesicles, while histidine decarboxylase occurs in the cytosol. Furthermore the ECL cells display immunoreactivity for vesicular monoamine transporter type 2 (VMAT-2), synaptophysin, synaptotagmin III, vesicle-associated membrane protein-2, cysteine string protein, synaptosomal-associated protein of 25 kDa, syntaxin and Munc-18. Using electron microscopy in combination with stereological methods, we have evidence to suggest the existence of both an exocytotic and a crinophagic pathway in the ECL cells. The process of exocytosis in the ECL cells seems to involve a class of proteins that promote or participate in the fusion between the granule/vesicle membrane and the plasma membrane. The granules take up histamine by VMAT-2 from the cytosol during transport from the Golgi zone to the more peripheral parts of the cells. As a result, they turn into secretory vesicles. As a consequence of stimulation (e.g., by gastrin), the secretory vesicles fuse with the cell membrane to release their contents by exocytosis. The crinophagic pathway was studied in hypergastrinemic rats. In the ECL cells of such animals, the secretory vesicles were found to fuse not only with the cell membrane but also with each other to form vacuoles. Subsequent lysosomal degradation of the vacuoles and their contents resulted in the development of lipofuscin bodies.     

Insight in the exocytotic process in chromaffin cells: regulation by trimeric and monomeric G proteins

Catecholamine secretion from chromaffin cells has been used for a long time as a general model to study exocytosis of large dense core secretory granules. Permeabilization and microinjection techniques have brought the possibility to dissect at the molecular level the multi-protein machinery involved in this complex physiological process. Regulated exocytosis comprises distinct and sequential steps including the priming of secretory granules, the formation of a docking complex between granules and the plasma membrane and the subsequent fusion of the granule with the plasma membrane. Key proteins involved in the exocytotic machinery have been identified. For instance, SNAREs which participate in the docking events in most intracellular transport steps along the secretory pathway, play a role in exocytosis in both neuronal and endocrine cells. However, in contrast to intracellular transport processes for which the highest fusion efficiency is required after correct targeting of the vesicles, the number of exocytotic events in activated secretory cells needs to be tightly controlled. We describe here the multistep control exerted by heterotrimeric and monomeric G proteins on the progression of secretory granules from docking to fusion and the molecular nature of some of their downstream effectors in neuroendocrine chromaffin cells.     

Rho GTPases and exocytosis: what are the molecular links?

Delivery of proteins or lipids to the plasma membrane or into the extracellular space occurs through exocytosis, a process that requires tethering, docking, priming and fusion of vesicles, as well as F-actin rearrangements in response to specific extracellular cues. GTPases of the Rho family have been implicated as important regulators of exocytosis, but how Rho proteins control this process is an open question. In this review, we focus on molecular connections that drive Rho-dependent exocytosis in polarized and regulated exocytosis. Specifically, we present data showing that Rho proteins interaction with the exocyst complex and IQGAP mediates polarized exocytosis, whereas interaction with actin-binding proteins like N-WASP mediates regulated exocytosis.     

A cell-free system for regulated exocytosis in PC12 cells

We have developed a cell-free system for regulated exocytosis in the PC12 neuroendocrine cell line. Secretory vesicles were preloaded with acridine orange in intact cells, and the cells were sonicated to produce flat, carrier-supported plasma membrane patches with attached vesicles. Exocytosis resulted in the release of acridine orange which was visible as a disappearance of labeled vesicles and, under optimal conditions, produced light flashes by fluorescence dequenching. Exocytosis in vitro requires cytosol and Ca(2+) at concentrations in the micromolar range, and is sensitive to Tetanus toxin. Imaging of membrane patches at diffraction- limited resolution revealed that 42% of docked granules were released in a Ca(2+)-dependent manner during 1 min of stimulation. Electron microscopy of membrane patches confirmed the presence of dense-core vesicles. Imaging of membrane patches by atomic force microscopy revealed the presence of numerous particles attached to the membrane patches which decreased in number upon stimulation. Thus, exocytotic membrane fusion of single vesicles can be monitored with high temporal and spatial resolution, while providing access to the site of exocytosis for biochemical and molecular tools.     

Transbilayer asymmetry of phospholipids in the plasma membrane regulates exocytotic release in mast cells

To investigate the role of the asymmetric distribution of phospholipids of the plasma membrane in exocytosis, we examined the effects of disruption of this asymmetrical distribution of lipids on exocytotic release from mast cells (RBL-2H3). Lipid scramblase, which is activated by divalent cations and catalyzes the transbilayer movement of phospholipids, was overexpressed in mast cells. Exogenous lipid scramblase was expressed in the plasma membrane and the cytoplasm. Activation of scramblase by divalent cations disrupted the asymmetrical distribution of phospholipids in the plasma membrane. Exocytotic release induced by calcium ionophore and phorbol ester was significantly inhibited in the cells transfected with wild-type scramblase. This inhibition was observed with time lag of about 5 min. Furthermore, when the asymmetric distribution of lipids was disrupted before induction of exocytosis, the inhibition of exocytotic release was obvious from the beginning without time lag. These results suggest that the asymmetric distribution of phospholipids in the plasma membrane plays an essential role in fusion between secretory granules and the plasma membrane. This finding also demonstrates that the transbilayer asymmetry of phospholipids regulates exocytosis and gives a new insight into the significance of lipid asymmetry in the plasma membrane.     

The Microtubule-Associated Rho Activating Factor GEF-H1 Interacts with Exocyst Complex to Regulate Vesicle Traffic

The exocyst complex plays a critical role in targeting and tethering vesicles to specific sites of the plasma membrane. These events are crucial for polarized delivery of membrane components to the cell surface, which is critical for cell motility and division. Though Rho GTPases are involved in regulating actin dynamics and membrane trafficking, their role in exocyst-mediated vesicle targeting is not very clear. Herein, we present evidence that depletion of GEF-H1, a guanine nucleotide exchange factor for Rho proteins, affects vesicle trafficking. Interestingly, we found that GEF-H1 directly binds to exocyst component Sec5 in a Ral GTPase-dependent manner. This interaction promotes RhoA activation, which then regulates exocyst assembly/localization and exocytosis. Taken together, our work defines a mechanism for RhoA activation in response to RalA-Sec5 signaling and involvement of GEF-H1/RhoA pathway in the regulation of vesicle trafficking.     

Nadrin, a novel neuron-specific GTPase-activating protein involved in regulated exocytosis

It has been proposed that the cortical actin filament networks act as a cortical barrier that must be reorganized to enable docking and fusion of the synaptic vesicles with the plasma membranes. We identified a novel neuron-associated developmentally regulated protein, designated as Nadrin. Expression of Nadrin is restricted to neurons and correlates well with the differentiation of neurons. Nadrin has a unique structure; it contains a GTPase-activating protein (GAP) domain for Rho family GTPases, a potential coiled-coil domain, and a succession of 29 glutamines. In vitro the GAP domain activates RhoA, Rac1, and Cdc42 GTPases. Expression of Nadrin in NIH3T3 cells markedly reduced the number of the actin stress fibers and the formation of the ruffled membranes, suggesting that Nadrin regulates actin filament reorganization. In PC12 cells, Nadrin colocalized with synaptotagmin in the neurite termini and also with cortical actin filaments in the subplasmalemmal regions. Expression of Nadrin or its mutant composed of the coiled-coil and GAP domain enhanced Ca(2+)-dependent exocytosis of PC12 cells, but a mutant lacking the GAP domain inhibited exocytosis. These results suggest that Nadrin plays a role in regulating Ca(2+)-dependent exocytosis, most likely by catalyzing GTPase activity of Rho family proteins and by inducing the reorganization of the cortical actin filaments.     

Exocytosis in pheochromocytoma cells after exposure to a high concentration of potassium

An electron-microscopic study revealed exocytosis of electron-dense granules in rat pheochromocytoma (PC12) cells after 1-min exposure to a high concentration of potassium medium. Numerous omega profiles indicative of the exocytosis were seen in the surface membrane of the PC12 cells. Most omega profiles showed clear or did not contain the granule contents. Sometimes, the granular, flocculent or dense materials were seen within the omega profiles that were exposed directly to the extracellular medium. Moreover, noncoated or coated omega profiles were observed. Coated vesicles were also visible under the surface membrane. The present study suggests that PC12 cells retain the exocytotic release mechanism for catecholamines.     

Phosphorylation of myristoylated alanine-rich C kinase substrate (MARCKS) protein is associated with bovine luteal oxytocin exocytosis

The ruminant corpus luteum, in addition to producing progesterone, synthesizes and secretes oxytocin (OT) during the estrous cycle. Secretion of oxytocin occurs by exocytosis of membrane-encapsulated granules of this hormone. Exocytosis of oxytocin involves transport of granules through a cytoskeletal matrix including an actin cortex closely associated with the plasma membrane (PM). Actin filaments crosslinked by various proteins give rise to the structural integrity of the cortex. Myristoylated alanine-rich C kinase substrate (MARCKS), a protein specifically phosphorylated by protein kinase C (PKC), crosslinks actin filaments and anchors the actin network to the inner leaflet of the PM. There is evidence that the intact actin cortex may serve as a barrier, precluding fusion of transport vesicles with the PM. In some secretory cells, phosphorylation of MARCKS has resulted in its translocation from the PM to the cytoplasm with an associated disassembly of the actin cortex. Prostaglandin F(2alpha) (PGF(2alpha)) stimulation of the bovine corpus luteum during the midluteal phase of the estrous cycle activates PKC, which is associated with an increase in OT secretion in vivo and in vitro. Data are presented demonstrating that stimulation of bovine luteal cells with PGF(2alpha) on Day 8 of the cycle promotes rapid phosphorylation of MARCKS protein and causes its translocation from the PM to the cytoplasm and concomitant, enhanced exocytosis of OT. These data are consistent with the premise that MARCKS plays a role in the exocytotic process.     

Annexin A2–dependent actin bundling promotes secretory granule docking to the plasma membrane and exocytosis

Annexin A2, a calcium-, actin-, and lipid-binding protein involved in exocytosis, mediates the formation of lipid microdomains required for the structural and spatial organization of fusion sites at the plasma membrane. To understand how annexin A2 promotes this membrane remodeling, the involvement of cortical actin filaments in lipid domain organization was investigated. 3D electron tomography showed that cortical actin bundled by annexin A2 connected docked secretory granules to the plasma membrane and contributed to the formation of GM1-enriched lipid microdomains at the exocytotic sites in chromaffin cells. When an annexin A2 mutant with impaired actin filament–bundling activity was expressed, the formation of plasma membrane lipid microdomains and the number of exocytotic events were decreased and the fusion kinetics were slower, whereas the pharmacological activation of the intrinsic actin-bundling activity of endogenous annexin A2 had the opposite effects. Thus, annexin A2–induced actin bundling is apparently essential for generating active exocytotic sites.     

Vesicular trafficking through cortical actin during exocytosis is regulated by the Rab27a effector JFC1/Slp1 and the RhoA-GTPase-activating protein Gem-interacting protein

Cytoskeleton remodeling is important for the regulation of vesicular transport associated with exocytosis, but a direct association between granular secretory proteins and actin-remodeling molecules has not been shown, and this mechanism remains obscure. Using a proteomic approach, we identified the RhoA-GTPase-activating protein Gem-interacting protein (GMIP) as a factor that associates with the Rab27a effector JFC1 and modulates vesicular transport and exocytosis. GMIP down-regulation induced RhoA activation and actin polymerization. Importantly, GMIP-down-regulated cells showed impaired vesicular transport and exocytosis, while inhibition of the RhoA-signaling pathway induced actin depolymerization and facilitated exocytosis. We show that RhoA activity polarizes around JFC1-containing secretory granules, suggesting that it may control directionality of granule movement. Using quantitative live-cell microscopy, we show that JFC1-containing secretory organelles move in areas near the plasma membrane deprived of polymerized actin and that dynamic vesicles maintain an actin-free environment in their surroundings. Supporting a role for JFC1 in RhoA inactivation and actin remodeling during exocytosis, JFC1 knockout neutrophils showed increased RhoA activity, and azurophilic granules were unable to traverse cortical actin in cells lacking JFC1. We propose that during exocytosis, actin depolymerization commences near the secretory organelle, not the plasma membrane, and that secretory granules use a JFC1- and GMIP-dependent molecular mechanism to traverse cortical actin.