Rho GTPases and exocytosis: What are the molecular links?

Seminars in Cell & Developmental Biology 22 (2011) 27–32

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Review

Rho GTPases and exocytosis: What are the molecular links? Stéphane Ory ∗ , Stéphane Gasman ∗ CNRS UPR 3212, Institut des Neurosciences Cellulaires et Intégratives, Université de Strasbourg, 5 rue Blaise Pascal, 67084 Strasbourg, France

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Article history: Available online 8 December 2010 Keywords: Exocyst IQGAP Exocytosis Rho Rac Cdc42 TC10 Actin

a b s t r a c t 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. © 2010 Elsevier Ltd. All rights reserved.

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Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Exocyst and IQGAP: the missing link between Rho and polarized exocytosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. Exocyst as a docking partner for RhoGTPases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2. IQGAP as an intermediate between Rho proteins and the exocyst complex? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Rho and fusion: from yeast to neuroendocrine cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1. Insights from budding yeast . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2. A lesson from calcium-regulated exocytosis in neuroendocrine cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1. Introduction Exocytosis is one of the major ways a cell delivers proteins and lipids to the plasma membrane and releases components into the extracellular space. Exocytic processes can be classified into three main types. “Constitutive exocytosis” of vesicles occurs constantly at the plasma membrane to maintain its composition. “Polarized

Abbreviations: Arp, actin related protein; GAP, GTPase activating protein; GDI, guanosine nucleotide dissociation inhibitor; GEF, guanosine nucleotide exchange factor; GTPase, guanosine triphosphatase; MMPs, metalloproteinases; PA, phosphatidic acid; PI4K, phosphatidyl inositol 4-kinase; PIP2, phosphatidyl inositol 45 bisphosphate; PLD1, phospholipase D1; SNARE, soluble N-ethylmaleimide sensitive factor attachment protein receptor; N-WASP, neural Wiscott–Aldrich syndrome protein. ∗ Corresponding authors. Tel.: +33 388 45 67 12 (S. Gasman); +33 388 45 66 80 (S. Ory). E-mail addresses: [email protected] (S. Ory), [email protected], [email protected] (S. Gasman). 1084-9521/$ – see front matter © 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.semcdb.2010.12.002

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exocytosis” provides massive amounts of membrane or proteins to specific spatial landmarks, and thus is particularly critical for processes such as yeast bud formation, pollen tube growth in plants, neurites outgrowth, cell motility or phagocytosis, to name a few. Finally, hormones and neurotransmitters stored in synaptic vesicles or secretory granules are released by “regulated exocytosis” following a burst of intracellular calcium triggered by an extrinsic stimulus. Regulated exocytosis occurs in an extremely short time frame following stimulation and, in most secretory cells, a subpopulation of vesicles is already primed and competent for fusion to ensure a prompt response. The general mechanism for vesicles to deliver or release secretory products is highly conserved. Vesicles are tethered to the plasma membrane, docked and primed before finally fusing with the plasma membrane following interaction between vesicle and plasma membrane integral proteins, the v-SNARE and t-SNARE proteins respectively [1–3]. Thus an efficient way to control and regulate exocytosis is to control the docking and/or the fusion

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machinery. Although much has been learned concerning the mechanisms per se of vesicle tethering/docking and fusion, how they are regulated remains a key question. Rho family members are small GTP binding proteins belonging to the Ras superfamily of GTPases. Since the identification of RhoA in 1985 [4], Rho proteins have been implicated in the control of many cellular processes including cell migration, polarity, proliferation, survival and membrane trafficking. Many comprehensive and excellent reviews are available on the function of Rho GTPases in different aspects of the biology of the cell [5–11]. Rho proteins are found in all eukaryotic organisms, forming a family of 20 members [12]. Most of our knowledge about their function and regulation are based on studies of the three “classical” members, RhoA, Rac1 and Cdc42. Their activation and inactivation cycle is similar to that of Ras. They switch from GDP-inactive to GTP-active bound state upon signaling cues. In their GTP-bound conformation, they interact with and activate downstream effectors. Unlike Ras, which is constitutively associated to membranes [13], “classical” Rho GTPases are mainly found in the cytosol when inactive and at the plasma membrane when active. Guanine nucleotide exchange factors (GEF) are responsible for their recruitment and activation at the plasma membrane [14], whereas GTPase activating proteins (GAP) inactivate them [15]. Rho guanine nucleotide dissociation inhibitors (Rho GDI) are thought to sequester Rho GTPases in the cytosol by masking the prenyl groups (farnesyl or geranyl) which are part of post-translational modifications that localize Rho proteins to membrane compartments [16,17]. Evidence for a function of Rho GTPases in membrane trafficking comes from studies addressing their localization. Most Rho GTPases localize transiently or constitutively at the plasma membrane. In addition to their plasma membrane localization, RhoD, RhoB, TCL, TC10 and RhoG have been found on endosomes [18–22], Cdc42 and Rnd3 at the Golgi apparatus [23,24], RhoU and RhoV on endomembranes, partially colocalizing with endosome markers [25–27], and RhoA on secretory granules [28,29]. Work in the last decade has provided evidence regarding the functional importance of Rho GTPases in many secretory pathways. For example, polarized growth of pollen tubes is regulated by Rac-Rop GTPases [30], and in mammals, the delivery of vesicles to the basolateral membrane of epithelial cells is controlled by Cdc42 [31]. In neurons, RhoA, Rac1 and Cdc42 activities need to be finely tuned to promote neurite outgrowth [32]. In regulated exocytosis, Rac1 and Cdc42 regulate multiple steps of mast cell degranulation, [33] and insulin secretion in pancreatic-␤-cells [34,35]. Rac1 has also been shown to regulate calcium-dependent exocytosis in neurons [36,37] and pancreatic acini [38], while Rac2 controls primary granule release in neutrophils [39]. Finally, secretion from AtT-20 corticotropes is controlled by Rac1 and RhoG [40,41]. However, the underlying mechanisms of Rho GTPasesdependent exocytosis are not well understood. In this review, we will summarize the current evidence indicating how Rho GTPases could regulate docking and fusion processes. We will mainly focus on the results obtained from studies on the role of the exocyst complex in polarized exocytosis. Additionally, we will discuss the results obtained from neuroendocrine cell models concerning the release of hormones and neuropeptides from large dense core secretory granules.

2. Exocyst and IQGAP: the missing link between Rho and polarized exocytosis 2.1. Exocyst as a docking partner for RhoGTPases The exocyst is an octameric complex initially identified through the isolation of temperature-sensitive mutants of the yeast Saccha-

romyces cerevisiae defective in secretion [42,43]. Of the 8 subunits, 6 are associated to vesicles (Sec5, Sec6, Sec8, Sec10, Sec15 and Exo84) and 2 (Exo70 and Sec3) to the plasma membrane. Exo70 and Sec3 may serve as spatial landmarks for polarized secretion since they display a characteristic cell cycle-dependent localization pattern that begins with the appearance of a small cap at sites of bud formation where active vesicle fusion occurs (see model in figure 1) [44,45]. The exocyst complex is thought to assemble as vesicles, carrying the other six subunits, arrive at fusion sites. It thus functions as a tethering complex for vesicles in the vicinity of the plasma membrane and then facilitates exocytosis by keeping the vesicle bound to the plasma membrane until the SNARE-dependent fusion machinery acts [43]. Various small GTPases, including the Rhos, have been shown to interact with the exocyst. In a search for yeast mutants in which the exocyst complex was mislocalized, Rho1 and Cdc42 were identified as critical for Sec3 localization at the bud tip. Rho1 and Cdc42 proteins were found to interact in a GTP-dependent manner with Sec3 and appeared to compete for Sec3 binding [46,47]. In addition, Rho3 and Cdc42 were found to also interact with Exo70 [48,49], providing further evidence that regulators of vesicle tethering such as the exocyst are effectors of Rho GTPases. In mammals, an interaction between recombinant Cdc42 and exocyst subunits could not be detected in vitro suggesting that Cdc42 binding to the exocyst requires other factors [50]. Alternatively, post-translational modifications may be important for Cdc42 binding to exocyst subunits. Indeed, despite clear genetic evidence of an interaction between Exo70 and Cdc42 in yeast, binding between these proteins has recently been demonstrated in vitro only when pull down assays were performed using prenylated recombinant Cdc42 [51]. The exocyst subunits may also have evolved to interact with other members of the Rho GTPases closely related to these known ones [52]. For example, in neurons and adipocytes, Exo70 interacts with TC10 a close relative of Cdc42 [53,54]. The functional interplay between Rho GTPases and the exocyst complex has been studied in various cellular processes requiring polarized exocytosis. So far, the conclusions from those data remain elusive. For example, in cdc42-6 and rho3-V51 yeast mutants, exocyst subunits are polarized indicating that defect in exocytosis is not a problem of localization [47,55]. Moreover, no obvious growth or secretory defects were observed either in cells expressing the Exo70 subunit with a deleted Rho3 binding domain [55–57] or in cells expressing a Sec3 mutant unable to interact with Rho1 and Cdc42 [47]. The absence of phenotypes may be explained by the unusual regulation of Rho proteins binding to Exo70 described recently [51]. Indeed, Rho3 and Cdc42 bind to Exo70 with a stronger affinity when they are prenylated. In addition, binding occurs in a manner that is structurally distinct from that of unmodified recombinant Rhos but still depends on their active state. This study thus confirmed that Exo70 is an effector of Rho3 and Cdc42 but unraveled additional levels of regulation of Exo70 by Rho GTPases that will require further investigations. Focal exocytosis required for membrane extension during neurite and axonal growth is regulated by Rho GTPases [58] and by the exocyst complex [54,59,60]. The functional association of Rho GTPases and exocyst complex in neuronal growth is supported by studies on TC10, a member of the RhoGTPases family, highly homologous to Cdc42. For example, insulin-like growth factor-1 (IGF1), which stimulates exocytosis of polarized vesicles at the growth cone of hippocampal neurons [61], triggered the recruitment of Exo70 at the growth cone in a TC10-dependent manner. Moreover, silencing of Exo70 or TC10 prevented IGF1 receptor externalization establishing a link between TC10, Exo70 and polarized secretion [62]. Accordingly, TC10 recruits the Exo70 subunit at the plasma membrane and controls Glut4 exocytosis in response to insulin

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in adipocytes [53,63]. Furthermore, NGF treatment of PC12 cells, activated TC10 and promoted TC10-Exo70 interaction. Fluorescence lifetime imaging microscopy (FLIM) showed that TC10 and Exo70 interaction occurs at discrete sites where spines are formed. In addition, local activation of the TC10-Exo70 interaction led to inhibition of WASP, but did not prevent formation of thin actin protrusions. Overexpression of Exo70 alone induced some neurite outgrowth. This may be related to the ability of Exo70 to bind and activate the actin-nucleating complex Arp2/3 [64]. Such results have led to the notion that while Cdc42-WASP interaction may be required for neurite outgrowth through global actin remodeling in axon [32,54], the TC10-Exo70 complex could locally counteract the signaling between Cdc42 and N-WASP to promote spine-like extension by activating other actin nucleation factors such as Arp2/3 [54]. Whether this is coupled to a local increase in exocytosis has not yet been addressed. In contrast to what has been described for Cdc42 and Sec3 in yeast [57], GTP hydrolysis of TC10 seems to be required for exocytosis [65]. These findings suggest that a balance exists between Cdc42 and TC10 and this may serve additional roles in neuron maturation.

2.2. IQGAP as an intermediate between Rho proteins and the exocyst complex? Recent studies have shed some light on the relationship between Rho GTPases, the exocyst complex and secretion at invadopodia. Invadopodia are dot-like actin-rich cellular protrusions that are formed at contact site between tumor cells and the extracellular matrix (ECM). Focal exocytosis occurs at site of invadopodia releasing metalloproteinases (MMPs) in order to degrade the ECM and favor tumor cell dissemination [66]. Cdc42, N-WASP and Arp2/3 have been implicated in invadopodia formation, but whether this involves focal exocytosis is not known [67]. Secretion of MMPs was found to depend on exocyst subunit functions (Exo70, Sec3 and Sec8) and IQGAP1 [50,68]. IQGAP1 is a scaffold protein known to regulate cadherin-mediated cell-cell adhesion and actin dynamics. It binds to multiple proteins including actin, E-cadherin, ␤-catenin and active Rac1 and Cdc42. Accordingly, IQGAP1 has been involved in neoplasm processes [69,70]. Recently, IQGAP1 has been shown to interact with Sec3, Sec8, Exo70 and Exo84 [50]. However, direct interaction with IQGAP was shown only for Sec3 and Sec8 suggesting that other partners are required for the interaction of IQGAP with Exo70 and Exo84. Interestingly, while active RhoA and Cdc42 stimulated binding of IQGAP1 to Sec3 and Sec8 subunits, Rac1 did not [50]. Furthermore, RhoA and Cdc42 bound to the Sec3–Sec8 complex, but the binding of Sec3 and Sec8 to IQGAP1 was independent of Rho proteins. The interaction of Rho proteins with Sec3/Sec8 complex has been proposed to trigger the association of IQGAP1 with these exocyst subunits, and thus promote focal exocytosis. In addition, in ␤-pancreatic cells, IQGAP1 was found to interact with Exo70 and regulate insulin secretion in response to mastoparan, a peptide from wasp venom that stimulates Ca2+ -independent exocytosis by activating heterotrimeric G-proteins and Cdc42 [71,72]. However, while Cdc42 stimulated exocyst association with IQGAP1 and increased MMPs secretion in tumor cells [50], Cdc42 dissociated the exocyst and IQGAP1 in response to mastoparan in ␤-pancreatic cells and reduced insulin secretion [72]. Although further study will be required to understand how IQGAP1, the exocyst complex and Cdc42 regulate secretion, it is noteworthy that yeast IQGAP1 (Iqg1p) controls polarized localization of Cdc42 and binds the exocyst subunit Sec3 to regulate septin organization during yeast cytokinesis [73,74]. This suggests that coordination of focal exocytosis by IQGAP, Rho proteins and exocyst is a highly conserved process, although its exact role remains obscure (Fig. 1).

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3. Rho and fusion: from yeast to neuroendocrine cells 3.1. Insights from budding yeast During cell division, the budding yeast S. cerevisiae adopts a polarized morphology that results in the emergence of a bud which will eventually separate and give rise to a daughter cell. During the early stage of bud formation, a vectorial actin-dependent traffic takes place to deliver secretory vesicles to the bud tip, and provide the proteins, lipids and membrane necessary for proper growth of the bud. Genetic approaches have shown that Cdc42 is crucial for cell polarity and bud growth. Indeed, Cdc42 has been shown to be recruited by its exchange factor to the spatial landmark of the bud where it dictates the polymerization of F-actin cables that target secretion to the selected bud sites [75]. Further studies demonstrated that the temperature-sensitive Cdc42 mutant (cdc42-6) yeast strain failed to divide because vesicle fusion was impaired and bud growth arrested. Nonetheless, vesicles accumulated in the nascent bud testifying to normal polarized vesicle transport [55]. While looking for a suppressor of the cold-sensitive Rab GTPase sec4-p48 mutants, Rho3 and the SNARE Sec9 were identified as potent suppressors. The cold-sensitive effector domain mutant of Rho3 (rho3-V51) demonstrated a severe secretory defect with an accumulation of post-Golgi vesicles, but no significant effect on actin cytoskeleton organization [57,76]. This phenotype is reminiscent of the cdc42-6 allele suggesting that a common pathway is shared by Rho3 and Cdc42. Interestingly, growth defects observed in both Rho3 and Cdc42 mutants were suppressed by a common set of genes, including the Rab GTPase Sec4 and the SNARE Sec9 indicating that the effector pathways of these two GTPases overlap [55]. These findings indicate that Rho3 and Cdc42 regulate polarized exocytosis by controlling vesicle docking or fusion during exocytosis. However, the underlying mechanism is not currently understood. Further evidence of the importance of Rho GTPases in fusion in yeast comes from the homotypic vacuole fusion assay, an elegant approach that reproduces in a controlled manner all steps (tethering, docking, priming, fusion) of exocytosis in vitro [77]. Vacuoles purified from strains with thermosensitive Cdc42 or Rho1 were thermolabile for fusion. Antibodies against Cdc42 or Rho1 or recombinant RhoGDI Rdi1p (known to extract Rho GTPases from membranes) also inhibited vacuole fusion [77,78]. Interestingly, Rho1 and Cdc42 were shown to act between the Rab GTPase Ypt7, important for tethering vesicles, and the trans-SNARE complex required for membrane fusion suggesting that Rho GTPases act between the tethering and fusion steps [78,79]. In addition to Rho1 and Cdc42, other proteins required for actin polymerization including Las17/Bee1p (WASp homolog), Vrp1 (WIP homolog), Arp3 (Actin related protein 3) and actin itself have been detected on purified vacuoles. The functional relationship between Rho proteins, actin polymerization machinery and vesicle fusion was further established by modifying actin dynamics through the use of drugs, actin mutants or inhibition of several actin regulators. Modifications in actin dynamics all led to defects in vacuole fusion, but did not affect docking [80]. Cdc42-dependent actin polymerization on vacuoles was also required to facilitate vacuole fusion [80,81] indicating that Rho GTPases (e.g. Rho1, Rho3 and Cdc42) control exocytosis by catalyzing changes in actin polymerization at specific sites of vesicle docking and fusion. 3.2. A lesson from calcium-regulated exocytosis in neuroendocrine cells Secretion in neuroendocrine cells is a good cellular model to establish the mechanistic links between Rho GTPases signaling and exocytosis. In neuroendocrine chromaffin cells and PC12 cells, Rho proteins have distinct localization. Rac1 and Cdc42 are local-

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Fig. 1. Schematic summary of molecular assembly of Rho GTPases and exocyst complex subunits during polarized exocytosis. (A) For bud growth in yeast, Rho GTPases and PIP2 are required to target Sec3 and Exo70 to the bud tip. Sec3 and Exo70 bind to common and different subsets of Rho GTPases. Cdc42 binds to both Exo70 and Sec3 whereas Rho1 binds to Sec3 and Rho3 to Exo70. Yeast IQGAP1 (Iqg1p, Cyk1p) has been reported to bind Sec3 and Cdc42. It may determine and maintain the axis polarity by recruiting vesicles loaded with the other components of the exocyst complex. (B) During metalloproteinases (MMPs) secretion at invadopodia, Sec3 and Sec8 binding to IQGAP1 is enhanced by active RhoA and Cdc42. It controls the delivery of MMPs-containing vesicles to invadopodia. Although RhoA and Cdc42 can bind to IQGAP1 and the Sec3/Sec8 complex, Sec3 and Sec8 binding is independent on RhoA/Cdc42 binding to IQGAP1. It was proposed that RhoA/Cdc42 binding to Sec3/Sec8 triggers Sec3/Sec8 association to IQGAP1. Rho GTPases could also target the complex at the site of invadopodia formation. Cdc42, N-WASP and Arp2/3 are known to control invadopodia formation by stimulating actin polymerization. By extension, since Exo70 is found in the IQGAP-Sec3/Sec8 complex and can stimulate Arp2/3 dependent polymerization, it suggests that Cdc42 could coordinate the tethering of MMPs containing vesicles at site of actin polymerization and invadopodia formation.

ized at the plasma membrane whereas RhoA is associated to the secretory granules membranes. Stimulation of PC12 cells by a secretagogue led to activation of Rac1 and Cdc42 but only Cdc42 was found to induce actin filament polymerization at the plasma membrane [29,82,83]. Interestingly, Cdc42-dependent actin rearrangement was associated with a strong increase in secretion that was suppressed by knocking down Cdc42 [83,84]. The correlation between Cdc42-dependent exocytosis and actin polymerization has been further investigated by looking at N-WASP function in regulated exocytosis. N-WASP and Arp2/3 initiate actin polymerization once N-WASP is activated upon binding to active Cdc42 and phosphatidyl PIP2 [85]. Overexpression of N-WASP in PC12 cells enhanced secretion [83,86], and the N-WASP mutant unable to stimulate actin polymerization abolished Cdc42-dependent secre-

tion [83]. In addition, since part of the Arp2/3 complex was detected on secretory granules membranes and translocated to the plasma membrane upon stimulation [83], secretagogue-evoked activation of Cdc42/N-WASP complex was proposed to trigger local Ap2/3dependent actin polymerization at the exocytic sites. This view implies that actin polymerization takes place at a late post-docking step, once secretory granules have brought the Arp2/3 complex (Fig. 2). Interestingly, a recent study in the nematode C. elegans identified WSP-1, the N-WASP ortholog, as essential for synaptic transmission at the neuromuscular junction [87] suggesting that the Cdc42/N-WASP pathway controlling regulated exocytosis is conserved between species. RhoA activation also induced polymerization of cortical actin, but unlike Cdc42, it inhibited secretion in PC12 cells [88]. Although RhoA inhibition had no incidence

Fig. 2. Hypothetical model for the function of Rho GTPases in regulated exocytosis. In resting chromaffin cells, active RhoA is associated to secretory granules whereas Rac1 and Cdc42, in their inactive state, are found at the plasma membrane. RhoA stimulate PI4K and participates to the cortical actin network stabilization. Secretagogue stimulation triggers the recruitment, docking and fusion of secretory granules with the plasma membrane. RhoA is presumably inactivated to facilitate actin reorganization. In contrast, Cdc42 is activated to promote actin polymerization through N-WASP and the granule-bound Arp 2/3 complex and Rac1 activation produces fusogenic lipids (phospholipids in red) through PLD1 activation at exocytotic sites. Although not demonstrated in chromaffin cells, Cdc42 may control SNARE function during regulated exocytosis.

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on secretion, lowered RhoA activity is predicted to facilitate exocytosis [29]. It also indicates that RhoA controls the formation of actin structures distinct from those induced by Cdc42. In this respect, there is an interesting parallel between regulated exocytosis and the release of vaccinia virus. Efficient release of the virus required cortical actin depolymerization and inactivation of RhoA and this process involved inhibition of the RhoA-mDia1, but not the RhoA-ROCK pathway [89]. Whether mDia1 is involved in regulated exocytosis has not been tested. However, in chromaffin cells, RhoA has been proposed to regulate actin dynamics through a phosphatidylinositol 4-kinase (PI 4-Kinase) which is also associated with secretory granules [29]. By generating phosphatidyl-inositol4 phosphate, a precursor of PIP2 , the RhoA-PI4-kinase may trigger local production of PIP2 which is sufficient to elicit actin polymerization and stabilize cytoskeleton-membrane interaction [90]. In this way, RhoA would maintain a local increase in PIP2 and help to trap secretory granules in the cortical actin network. Once Ca2+ enters the cell, RhoA would be inactivated and secretory granules would be freed and ready to fuse with the plasma membrane. In addition, activated Rac1 has been shown to facilitate secretory granule fusion by stimulating the phospholipase D-induced formation of phosphatidic acid (PA) at the exocytotic site [82]. Accordingly, in neurons, PLD1 and Rac1 are found on synaptosomes, and both required for neurotransmitter release [36,37,91]. Although no functional links have been established between Rac1 and PLD1 in neurons, actin dynamics is not required for fast neurotransmitter release [92]. This suggests that Rac1 is unlikely to act through actin remodeling, but rather, as in chromaffin cells, the Rac1-PLD pathway may locally modify plasma membrane composition and favor membrane fusion. Finally, although the exocyst machinery appears dispensable for regulated exocytosis [59], Rho GTPases may regulate the activity of the docking/fusion machinery by interfering with SNARE functions. For example, in ␤-pancreatic cells, Cdc42 interacts with Syntaxin and VAMP2 to control insulin release in response to glucose [71,93]. In chromaffin cells and neurons, the RhoA/ROCK pathway may govern the secretory response by phosphorylating Syntaxin1A and promoting the association of Syntaxin1A with negative regulators [94,95]. These findings indicate that Rho proteins may modulate exocytosis by directly modifying the SNARE and docking complex functions. 4. Conclusion Evidence of the function of Rho GTPases in controlling exocytosis is accumulating. They appear to be involved at multiple stages, in actin remodeling, lipid biosynthesis and regulation of the docking/fusion machinery. Although progress has been made with the identification of the exocyst complex subunits as conserved direct or indirect effectors of Rho proteins in polarized exocytosis, how Rho proteins control exocyst function is yet to be determined. The role of Rho proteins in the switch between the docking step and the fusion step of exocytosis also needs to be elucidated. Finally, in regulated exocytosis, the coordination of the different Rho protein activities and their synchronization with actin dynamics, lipid synthesis and vesicle fusion remains a fertile field of exploration. Acknowledgments We wish to thank Dr. Nancy Grant for critical reading of the manuscript. The work presented in this review was supported by a Human Frontier Science Program (HFSP) grant (RGY40-2003C), an ANR grant (ANR-07-JCJC-088-01), a “Association pour la Recherche sur le Cancer” grant (ARC #1055) to S.G.

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