![[Feedback]](/icons/banner/feedbackACT.gif){kind=icon}
![[For Subscribers]](/icons/banner/subscriberACT.gif){kind=icon}
![[Archive]](/icons/banner/archiveACT.gif){kind=icon}
![[Search]](/icons/banner/searchACT.gif){kind=icon}
![[Contents]](/icons/banner/tocACT.gif){kind=icon}
|
|
|
|
|
||
Vol. 15, Issue 2, 520-531, February 2004
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
* Centre National de la Recherche Scientifique Unité Propre de Recherche 2356, Institut Fédératif de Recherche 37, 67084 Strasbourg, France;
Cancer Research UK, Lincoln's Inn Fields Laboratories, London WC2A 3PX, England
Submitted June 16, 2003;
Revised September 16, 2003;
Accepted September 30, 2003
Monitoring Editor: Anne Ridley
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
|---|
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
|---|
). Over the past years, actin has emerged as a key player in membrane trafficking, especially in exo- and endocytotic events (Doussau and Augustine, 2000; Jeng and Welch, 2001). Neuroendocrine cells, like most secretory cells, display a dense network of filamentous actin beneath their plasma membrane. This actin web is classically viewed as a physical barrier to exocytosis by restricting the access of secretory granules to their release sites at the plasma membrane (Aunis and Bader, 1988; Vitale et al., 1995). However, evidence is emerging that actin is not simply a physical barrier but could be responsible for an active step in exocytosis. For example, agents that completely depolymerize actin filaments have been shown to inhibit secretagogue-induced exocytosis in various secretory cells, including PC12 cells (Matter et al., 1989), HIT insulin-secreting cells (Li et al., 1994), and mast cells (Pendleton and Koffer, 2001). Moreover, a recent report in PC12 cells, illustrated that actin can both hinder and mediate movements of green fluorescent protein (GFP)-labeled dense-core granules in the subplasmalemmal region (Lang et al., 2000). Thus, the actin cytoskeleton seems to be necessary for regulated exocytosis to occur. However, to what extent secretion in neuroendocrine cell requires de novo actin polymerization and the regulatory mechanisms underlying this event remains to be elucidated.
Dynamic regulation of the actin cytoskeleton has been shown to involve the Rho GTPase family (Hall, 1998). Belonging to the small GTP-binding proteins of Ras superfamily, mammalian Rho GTPases consist of at least 20 distinct members, of which RhoA, Rac1, and Cdc42 are the best characterized (Etienne-Manneville and Hall, 2002). Several recent studies have demonstrated the involvement of Rho proteins in a wide range of membrane trafficking aspects (Ridley, 2001). Indeed, RhoB and RhoD regulate endosomal trafficking (Gampel et al., 1999; Gasman et al., 2003), whereas RhoA and Rac are involved in receptor internalization (Lamaze et al., 1996), phagocytosis (Caron and Hall, 1998), and neurotransmitter release (Doussau et al., 2000). In chromaffin cells, we previously proposed that reorganization of the actin cytoskeleton underlying membrane trafficking at the site of exocytosis is under the combined control of two members of the Rho family. RhoA bound to secretory granules was found to maintain actin filaments in the vicinity of secretory granules by modulating the activity of a granule-associated phosphatidylinositol-4 kinase (Gasman et al., 1997; Gasman et al., 1998). However, inactivation of RhoA did not modify secretagogue-evoked secretion, excluding the active participation of RhoA in mediating actin structures required for the exocytotic machinery (Gasman et al., 1999). Conversely, in mast cells, RhoA has been involved in secretion but through a pathway that does not involve actin (Sullivan et al., 1999). Further investigations led us to the idea that Cdc42 might play an actin-dependent function in chromaffin cell secretion (Gasman et al., 1999). The active participation of Cdc42 in the exocytotic reaction in mast cells and pancreatic 
cells has also been described (Kowluru et al., 1997; Brown et al., 1998). In view of these observations, Cdc42 seems to be a good candidate to coordinate the actin architecture to the molecular machinery underlying exocytosis. To date, several functionally distinct Cdc42 effectors have been identified (Cotteret and Chernoff, 2002). Among them, the neural Wiskott-Aldrich syndrome protein (N-WASP) links Cdc42 to actin polymerization through the actin-related protein-2/3 (Arp2/3) complex, which promotes actin nucleation and polymerization (Carlier et al., 1999; Rohatgi et al., 1999; Higgs and Pollard, 2001).
The aim of the present work was to investigate the role of Cdc42 in the exocytotic process in PC12 cells. We report that in secretagogue-stimulated cells, subplasmalemmal Cdc42 switches to its active GTP-bound form and facilitates secretion by promoting actin polymerization in the cell periphery. Our results reveal that N-WASP and Arp2/3 are the molecules that bridge Cdc42 signaling to the actin cytoskeleton and the exocytotic machinery.
|
MATERIALS AND METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
|---|
The N-terminally GFP-tagged human Cdc42N17, Cdc42L61 (G25 isoform), and all the WASP constructs were described previously (Moreau et al., 2000). GFP-Cdc42L61A124 was generated by site-directed mutagenesis with a QuikChange mutagenesis kit from Stratagene (La Jolla, CA). Cdc42L61C40 was a gift from V. Moreau (INSERM U-441, Pessac, France). Myc-tagged PAK1 constructs (wild-type PAK1, PAK1T423E and PAK1K299R mutants; Sells et al., 1999) were given by N. Vitale (CNRS UPR-2356, Strasbourg, France). The GFP-tagged TC10L75 and TCLL79 were gifts from A. Blangy (CNRS UPR-1086, Montpellier, France).
Culture, Transfection, and Assay of Growth Hormone (GH) Release from PC12 Cells
PC12 cells were grown in DMEM supplemented with glucose (4500 mg/l) and containing 30 mM NaHCO3, 5% fetal bovine serum, 10% horse serum, and 100 U/ml penicillin/streptomycin. Mammalian expression vectors were introduced into PC12 cells together with the GH plasmid pXGH5 (six-well dishes, 80% confluence, 4 µg/well of each plasmid) by using GenePorter (Gene Therapy Systems) according to the manufacturer's instruction.
GH release experiments were performed 48 h after transfection. PC12 cells were washed twice with Locke's solution (140 mM NaCl, 4.7 mM KCl, 2.5 mM CaCl2, 1.2 mM KH2PO4, 1.2 mM MgSO4, 11 mM glucose, 0.56 mM ascorbic acid, and 15 mM HEPES, pH 7.2) and then incubated for 10 min with calcium-free Locke's solution (basal release) or stimulated with an elevated K+ solution (Locke's containing 59 mM KCl and 85 mM NaCl). The supernatant was collected and the cells harvested by scraping in 10 mM phosphate-buffered saline. The amounts of GH secreted into the medium and retained in the cells were measured using a radioimmunoassay kit (Nichols Institute, San Juan, Capistrano, CA). The amount of GH secretion is expressed as a percentage of total GH present in the cells before stimulation.
[3H]Noradrenaline Release from Chromaffin Cells
Chromaffin cells were isolated from fresh bovine adrenal glands by retrograde perfusion with collagenase, purified on self-generating Percoll gradients, and cultured as monolayers on 24 multiple 16-mm Costar plates at a density of 2.5 x 105 cells/well. For release experiments, cells were labeled with [3H]noradrenaline (14,68 Ci/mmol; PerkinElmer Life Sciences, Boston, MA) for 60 min, washed four times with Locke's solution, and subsequently stimulated for 10 min with Locke's solution containing 10 µM nicotine. [3H]Noradrenaline release after stimulation was determined by measuring the radioactivity present in the incubation medium and in cells after precipitation with 10% (wt/vol) trichloroacetic acid. Release of [3H]noradrenaline is expressed as a percentage of total radioactivity present in the cells before Ca2+-induced stimulation.
Pull Down Assay for Cdc42-GTP
Activated Cdc42 was precipitated using a Rac/Cdc42 activation assay kit (Up-state Biotechnology). After stimulation, PC12 cells were immediately lysed in ice-cold lysis buffer (25 mM HEPES, pH 7.5, 150 mM NaCl, 5 mM MgCl2, 5 mM EDTA, 1% NP-40, 10% glycerol, 0.1 mM Na3VO4, 4 mM NaF and mammalian protease inhibitor cocktail [1:100 dilution]; Sigma, Saint Quentin Fallavier, France). GTP-bound Cdc42 was pulled down by incubating lysates containing equal amounts of proteins (700 µg) with the Cdc42-interacting domain (CRIB) domain of PAK1 (p21 activated kinase) for 2 h at 4°C. Lysates loaded with guanosine 5'-O-(3-thio)triphosphate and GDP served, respectively, as positive and negative controls. The precipitated GTP-bound Cdc42 was resolved on 12% polyacrylamide-SDS gels and immunoblotted with antibodies specific for Cdc42 (1:400). Blots were processed using the Western-Light Plus chemiluminescent detection system (Tropix, Bedford, MA).
Immunoblotting, Immunofluorescence, Confocal Microscopy, and Image Analysis
One dimensional SDS gel electrophoresis was performed on 12% acrylamide gels in Tris-glycine buffer. The proteins were transferred to nitrocellulose sheets at a constant current of 100 mA for 2 h. Blots were processed using the Western-Light Plus chemiluminescent detection system. Immunoreactive bands from Western blot experiments were quantified using ImageJ 1.29x software (Wayne Rasband, National Institutes of Health, Bethesda, MD).
For immunocytochemistry, PC12 cells grown on poly D-lysine-coated glass coverslips were maintained in Locke's solution or stimulated with elevated K+. The cells were then fixed for 20 min in 4% paraformaldehyde in 0.12 M sodium/phosphate buffer, pH 7.0, and for a further 10 min in fixative containing 0.1% Triton X-100. Actin filaments were stained by incubation with rhodamine-conjugated phalloidin (Sigma) at concentration of 0.2 µg/ml in phosphate-buffered saline for 15 min. Immunostaining was performed as described previously (Vitale et al., 2001), and stained cells were visualized using a confocal microscope LSM 510 (Carl Zeiss, Jena, Germany). Using the Zeiss CLSM instrument software 2.8, the proportion of Cdc42/p34-Arc colocalized with SNAP-25 or p34-Arc colocalized with chromogranin B was estimated from the double-labeled pixels, expressed as an average fluorescence intensity normalized to the corresponding surface area, and calculated as a percentage of the total Cdc42/p34-Arc fluorescence detected in each cell. The amount of F-actin present in PC12 cells was measured and expressed as an average fluorescence intensity normalized to the corresponding surface area and divided by the total surface of each cell. This allows a comparison of the F-actin content in different cells.
Subcellular Fractionation
Subcellular fractionation of PC12 cells was performed as described previously (Vitale et al., 2002). Briefly, cells were washed twice with Locke's solution and then incubated for 10 min with Locke's solution (resting condition) or stimulated with an elevated K+ solution. Medium was removed and cells immediately scrapped in 1 ml of sucrose 0.32 M (20 mM Tris-HCl, pH 8.0). Cells were broken in a Dounce homogenizer and centrifuged at 800 x g for 15 min. The supernatant was centrifuged at 20,000 x g for 20 min. The resulting supernatant was further centrifuged for 60 min at 100,000 x g to obtain the cytosol (supernatant) and microsomes (pellet enriched in endosomes). The 20,000 x g pellet containing the crude membrane fraction was resuspended in sucrose 0.32 M (20 mM Tris-HCl, pH 8.0), layered on a cushion sucrose density gradient (sucrose 1-1.6 M, 20 mM Tris-HCl, pH 8.0) and then centrifuged for 90 min at 100,000 x g. The upper fractions containing the plasma membrane and the pellet containing secretory granules were collected.
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
|---|
|
Next, we examined the distribution of Cdc42 in resting and stimulated PC12 cells. As illustrated in Figure 1B, Cdc42 immunoreactivity was essentially detected in the cell periphery. Double labeling with antibodies against the plasma membrane marker SNAP25 revealed a partial association with the plasma membrane that markedly increased upon stimulation with 59 mM K+ (Figure 1B, mask). Quantification of the relative proportion of Cdc42 colocalizing with SNAP25 indicated that 23% of the detected Cdc42 immunoreactivity was found associated to the plasma membrane marker in stimulated cells compared with 5% in resting cells (Figure 1B, histogram). When the distribution of Cdc42 was examined in subcellular fractions obtained from resting and stimulated PC12 cells, most of the protein was found in the soluble fraction (Figure 1C). However, stimulation with K+ induced also a reproducible threefold increase in the amount of Ccd42 detected in the fraction containing the plasma membrane (Figure 1C, histogram).
To directly establish whether Cdc42 plays a role in exocytosis, we examined the effect of expressing a GFP-tagged Cdc42 mutant defective in GTP hydrolysis (Cdc42L61) and its corresponding dominant inactive mutant preferentially binding GDP (Cdc42N17) on exocytosis by using GH as a secretory reporter (Vitale et al., 2001). We found that Cdc42 mutants modified GH secretion in response to high potassium (Figure 1D). Expression of the constitutively active Cdc42L61 increased GH release by 
60%, whereas the dominant negative Cdc42N17 slightly but significantly decreased it (25% inhibition). Thus, Cdc42 is able to play a positive role in the exocytotic pathway of large dense-core granules, raising the question about the underlying mechanism(s).
Cdc42 Facilitates Exocytosis through a Pathway Implicating the Actin Cytoskeleton
Many downstream targets for Cdc42 have been described, including phospholipase D (PLD) (Walker et al., 2000), a protein recently proposed as a key factor for the exocytotic machinery in PC12 cells (Vitale et al., 2001). To test whether PLD might be the effector by which Cdc42 activates the exocytotic process, we superimposed the S124A mutation onto the constitutively active Cdc42L61, leading to a mutant of Cdc42 defective in PLD activation but still able to activate other effectors (Walker and Brown, 2002). Expression of Cdc42L61A124 in PC12 cells stimulated GH release to a similar extent as that of Cdc42L61, suggesting that PLD is not required for Cdc42-mediated exocytosis (Figure 2A). Given the well established role of actin in exocytosis, we next tested the double mutant Cdc42L61C40, which is unable to interact with N-WASP and PAK (Owen et al., 2000), two major Cdc42 effectors involved in actin organization (Cotteret and Chernoff, 2002). Interestingly, superimposing the Y40C mutation onto the constitutively active Cdc42L61 prevented its ability to stimulate exocytosis and instead, Cdc42L61C40 behaved as a dominant negative, inhibiting secretion to a similar extent as the inactive GDP-bound Cdc42N17 (Figure 2A). This observation suggests that N-WASP and/or PAK play a major role in the pathway by which Cdc42 regulates exocytosis.
|
To further probe the idea that Cdc42 regulates the secretory response by controlling actin dynamics, we overexpressed GFP-tagged mutants of Cdc42 and examined their effect on the cortical actin network in stimulated PC12 cells. As shown in Figure 2B, Cdc42N17 displayed a diffuse staining pattern, indicating a predominant localization to the cytosol. In contrast, the active Cdc42L61 mutant exhibited a major staining in the cell periphery consistent with the plasma membrane localization of endogenous Cdc42 (Figure 2B). In chromaffin and PC12 cells, the majority of actin filaments are concentrated in the subplasmalemmal region, forming a continuous cortical actin network that is partially disassembled upon activation of exocytosis (Gasman et al., 1997; Vitale et al., 2001). Hence, rhodamine-phalloidin staining in PC12 cells expressing Cdc42N17 revealed a classical disrupted cortical actin network commonly observed in stimulated cells (Figure 2B). Surprisingly, expression of Cdc42L61 enhanced the rhodamine-phalloidin fluorescence in the cell periphery, indicating that the GTP-bound form of Cdc42 triggers actin polymerization in stimulated cells (Figure 2B). These observations suggest that activated Cdc42 facilitates exocytosis through the de novo formation of actin filaments in the subplasmalemmal region. This implies that cortical actin, classically viewed as a barrier that hinders the recruitment of secretory granules to the plasma membrane, may also play a positive role in the exocytotic process. Accordingly, treatment of cultured chromaffin cells with latrunculin B, an actin-filament-disrupting molecule, produced a dual effect on secretion: it increased catecholamine release at a low concentration but progressively inhibited it at higher doses (Figure 2C). Similar results were observed in PC12 cells (Matter et al., 1989), in agreement with the idea that a minimal actin structure is necessary for exocytosis to occur.
N-WASP Stimulates Secretion and Promotes Actin Polymerization in PC12 Cells
To discriminate between N-WASP and PAK as the molecular effectors that link Cdc42 to the actin cytoskeleton and exocytosis, we expressed in PC12 cells wild-type and mutated N-WASP or PAK proteins and examined their effect on K+-evoked GH secretion. As shown in Figure 3A, N-WASP expression resulted in a stimulation of GH secretion as efficient as that effected by Cdc42L61. In contrast, expression of the Myc-tagged wild-type PAK1 (our unpublished data), the constitutively active PAK1E423 or the kinase-dead PAK1R299 mutants produced no effect on GH secretion (Figure 3A), electing N-WASP as the best partner for Cdc42 in the exocytotic process. Note that expression of an N-WASP mutant containing only the CRIB and lacking the other functional motifs inhibited exocytosis by 35% (Figure 3A). This effect could be due to the partial sequestration of endogenous Cdc42 because expression of CRIBD208 that is unable to interact with Cdc42 (Miki et al., 1998), had no effect on GH release (Figure 3A). On the other hand, the CRIB domain-induced inhibition could be assigned to the sequestration of other Cdc42-related GTPases, which are known to interact with N-WASP like TC10 and/or TCL (Vignal et al., 2000). To probe this idea, we examined the effect of expressing the constitutively active TC10L75 and TCLL79 mutants on GH release in PC12 cells. Both TC10L75 and TCLL79 localized in cell periphery as seen by fluorescent confocal microscopy (our unpublished data). TC10L75 inhibited GH secretion by 30% (Figure 3B), an effect that might be related to a possible competition with endogenous Cdc42 for N-WASP interaction leading to an inhibition of secretion. TCLL79 did not modify K+-evoked secretion but it increased the basal release (Figure 3B). We have currently no explanation for this effect; however, endogenous TCL has not been detected in PC12 cells (Abe et al., 2003). Together, these results indicate that, in contrast to Cdc42, neither TC10 nor TCL are able to act as a positive regulator of exocytosis in PC12 cells.
|
Next, we examined the exocytotic activity and actin filament organization in PC12 cells transfected with cDNAs encoding a series of truncated GFP-tagged N-WASP mutants differentially lacking important structural determinants, namely, 
WH1-N-WASP, WA-N-WASP, or the WA domain alone (Figure 4). The WH1 domain has an important function in N-WASP targeting (Moreau et al., 2000). The confocal images presented in Figure 5 show that overexpressed N-WASP and WH1-N-WASP displayed a predominant cytosolic staining in resting cells but partially translocated to the cell periphery in stimulated cells. This observation suggests that activation of exocytosis triggers the recruitment of N-WASP to the cell periphery and that the WH1 domain is not involved in this event. Similarly to Cdc42, these two constructs enhanced GH release by 60 and 75%, respectively (Figure 5C) and induced the formation of actin filaments in the subplasmalemmal region in high K+-stimulated cells (Figure 5, A and B). Compared with the full-length N-WASP, the WH1 protein is more efficiently recruited to the cell periphery, induces more cortical actin filaments, and triggers a stronger secretory response (Figure 5, A and B). N-WASP is maintained in an autoinhibited conformation by intramolecular bounds that are relieved upon binding of Cdc42 or phosphoinositides (Kim et al., 2000; Prehoda et al., 2000; Rohatgi et al., 2000). Deleting the WH1 domain could weaken these intramolecular interactions and result in a protein that is unregulated or less regulated than the autoinhibited full length as already suggested (Moreau et al., 2000).
|
|
To determine whether the stimulatory effect of N-WASP on secretion is dependent on its ability to promote actin polymerization in the cell periphery, we used a mutant lacking the functional WA domain interacting with G-actin and Arp2/3 (WA-N-WASP). As previously described in adipocytes (Jiang et al., 2002), WA-N-WASP displayed a predominant nuclear localization in PC12 cells. Some WAN-WASP was nevertheless recruited to the cell periphery during cell stimulation, but this mutant was clearly less efficient in promoting the formation of actin filaments (Figure 5, A and B), and it did also not modify the secretory response (Figure 5C). Conversely, expression of the WA domain (WA) alone highly stimulated actin polymerization throughout the whole cell (Figure 5A) but failed to stimulate exocytosis (Figure 5C), most likely because WA was not recruited to the sites of exocytosis in stimulated cells. Together, these data indicate that the stimulatory effect of N-WASP on secretion is closely related to its ability to be recruited to the subplasmalemmal area and its capacity to induce actin filaments at the plasma membrane. These results reinforce also the idea that exocytosis requires to some extent the formation of actin filaments at the site of granule docking and fusion with the plasma membrane.
Cdc42 Recruits N-WASP at the Plasma Membrane
To strengthen the idea that activated Cdc42 is involved in the recruitment of N-WASP at the plasma membrane upon cell stimulation, we coexpressed the constitutively active Cdc42L61 or the inactive Cdc42N17 with the GFP-tagged N-WASP and examined their distribution by confocal microscopy (Figure 6A). In resting PC12 cells, N-WASP was predominantly cytosolic (Figure 5A). Hence, when individually coexpressed with the GDP-bound Cdc42N17, N-WASP was also distributed into the cytosol (Figure 6A). In contrast, in cells expressing the active Cdc42L61, N-WASP was found to colocalize with Cdc42 at the plasma membrane (Figure 6A), indicating that it was efficiently recruited by the GTP-bound form of Cdc42. To identify which domain is required for the recruitment of N-WASP, we coexpressed the active Cdc42L61 with the various N-WASP truncated mutants. As shown in Figure 6B, Cdc42 recruited similarly WH1- and WA-N-WASP but not the WA domain, suggesting that the recruitment of N-WASP was mediated through its CRIB domain. Accordingly, the CRIB domain was recruited to the plasma membrane by Cdc42L61, whereas CRIBD208, unable to interact with Cdc42, was not (Figure 6B). Together, these results are in line with the idea that GTP-loaded Cdc42 in secretagogue-activated cells is able to directly interact with N-WASP and thereby to recruit it at the plasma membrane.
|
To further demonstrate that the function of N-WASP lies downstream of Cdc42, we examined the effect of the dominant negative WA-N-WASP mutant on the stimulation of exocytosis induced by the active Cdc42L61 (Figure 6C). Coexpression of WA-N-WASP completely abolished the stimulatory effect of Cdc42L61 on GH release, confirming that N-WASP acts as a downstream effector of Cdc42 in the exocytotic pathway.
Presence of Arp2/3 on Large Dense-Core Secretory Granules
The carboxy terminus of WASP proteins requires the Arp2/3 complex to stimulate actin polymerization (Prehoda et al., 2000). We examined the intracellular distribution of Arp2/3 in PC12 cells, by using antibodies against p34-Arc, a subunit of the Arp2/3 complex. Double-labeling experiments with SNAP-25 revealed that p34-Arc was not found on the plasma membrane in resting cells but it displayed a punctuate pattern of distribution, suggesting an association with some intracellular membrane-bound compartment (Figure 7A). To probe an eventual association of Arp2/3 with secretory granules, double-labeling experiments were performed with antibodies against p34-Arc and against chromogranin B (CGB), a specific marker for secretory granule in PC12 cells. As shown in Figure 7A, p34-Arc and CGB fluorescence partially overlapped, indicating that Arp2/3 complexes were associated to large dense-core granules. Arp2/3 complex or its subcomponents have not been previously localized to secretory granules, although they have been found on phagosomes (May et al., 2000) and intracellular vacuoles in yeast (Eitzen et al., 2002). However, it is interesting to remember that isolated secretory granules do contain actin and actin-binding sites (Burridge and Phillips, 1975; Meyer and Burger, 1979; Bader and Aunis, 1983) and are able to induce the assembly of actin filaments (Wilkins and Lin, 1981). Stimulation of PC12 cells with 59 mM K+ triggered a partial movement of secretory granules to the cell periphery and the docking of a portion of the granules at the plasma membrane (Vitale et al., 2002). p34-Arc and CGB remained colocalized (Figure 7A, mask p34-Arc/CGB), indicating that Arp2/3 complexes accompany the secretory granules to the periphery. Note the increased colocalization of p34-Arc with SNAP-25 in stimulated cells (Figure 7, A and B), consistent with the presence of Arp2/3 at the granule docking sites on the plasma membrane.
|
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
|---|
). Thus, the transit of granules to fuse with the plasma membrane requires a subtle remodeling of the subplasmalemmal actin filaments. Ideal candidates to orchestrate such regulation are the small GTPases Rho family members that have emerged as important players in coupling actin dynamics to membrane trafficking events in eukaryotic cells (Ridley, 2001). Investigating the function of Rho GTPases in chromaffin cell secretion, we previously proposed that, although the granule-associated RhoA maintains actin filaments at the vicinity of the secretory granules (Gasman et al., 1998), Cdc42 might play an active role in exocytosis by mediating some actin rearrangements preceding secretion (Gasman et al., 1999). The aim of the present work was to further probe the function of Cdc42 in neuroendocrine cell secretion and to dissect the downstream effector pathway integrating Cdc42 to the actin architecture required for exocytosis. Using PC12 cells, a well established model for neuroendocrine secretion studies (Vitale et al., 2001), we have demonstrated a causal relationship between activation of Cdc42, actin, and elicitation of exocytosis and proposed a molecular mechanism involving N-WASP and the Arp2/3 complex.
Intracellular Distribution of Cdc42
We previously studied the distribution of endogenous Cdc42 in cultured chromaffin cells and found that Cdc42 localizes in the subplasmalemmal region (Gasman et al., 1999). Our current observations confirm that Cdc42 is peripherally localized and partially associated to the plasma membrane in PC12 cells. Yet, upon subcellular fractionation most of the protein is recovered in a soluble fraction. A possible explanation is that cytosolic Cdc42 could not be detected by immunofluorescence because the protein is complexed to RhoGDI. Alternatively, Cdc42 might be loosely bound to the plasma membrane and detached during cell fractionation. It is interesting to note that, in stimulated cells, Cdc42 is activated and that a larger amount associates to the plasma membrane. This suggests that stimulation with a secretagogue might induce a modification in the binding of Cdc42 with some plasma membrane-bound protein, i.e., a nucleotide exchange factor and/or an effector. Thus, it is conceivable that in this set up, activation of Cdc42 does not results in a classical cytosol-membrane translocation but rather in subtle changes in the interaction of Cdc42 with its molecular partners present at the plasma membrane. Using GFP-tagged proteins, we examined the localization of the constitutively active Cdc42L61 and the dominant negative Cdc42N17 mutants. The localization of Cdc42L61 paralleled that of the endogenous protein, validating the use of this mutant for functional assays. In contrast, the dominant negative Cdc42N17 was largely present in the cytosol. This distribution was unexpected because in resting cells, endogenous Cdc42, which seems to be mostly in its GDP-bound form (as shown here), was localized in the cell periphery. Moreover, it has been recently reported that N17/19 dominant negative mutants of the Rho family fail to bind RhoGDI, most likely because the mutants are locked in a nucleotide-free state (Strassheim et al., 2000; Michaelson et al., 2001). This results in localization to membrane compartments in epithelial cell lines (Michaelson et al., 2001), a distribution that has been proposed to play a significant role in their dominant negative activity, by permitting the N17/19 mutants to sequester membrane-associated GEFs. We have currently no explanation for the basis of the cytosolic distribution of Cdc42N17 in PC12 cells. Nevertheless, the modest inhibitory effect on secretion observed in cells expressing Cdc42N17 might be related to the mislocalization of this mutant unable to efficiently behave as a dominant negative at the sites of exocytosis.
Cdc42 Regulates Exocytosis by Recruiting N-WASP
We show here that overexpression of the constitutively active Cdc42L61 mutant significantly enhanced GH secretion from PC12 cells, revealing the participation of Cdc42 in large dense-core granule exocytosis. Accordingly, mast cells, pancreatic cells, and yeast require the active participation of Cdc42 for secretion (Kowluru et al., 1997; Brown et al., 1998; Adamo et al., 2001). How might plasma membrane-associated GTP-loaded Cdc42 facilitate exocytosis in PC12 cells? In yeast, Cdc42 has been shown to promote docking and/or fusion of secretory vesicles with the plasma membrane through an interaction with the exocyst, a conserved eight-subunit complex implicated in tethering vesicles to specific sites on the plasma membrane (Zhang et al., 2001). To date, there are no published reports linking Cdc42 to the mammalian exocyst. However, a connection between Cdc42 and syntaxin has been described previously (Daniel et al., 2002), suggesting that Cdc42 may have functions related to the SNARE complex formation. PLD is also an attractive candidate to be a downstream partner of Cdc42 in the exocytotic pathway. Cdc42 is an activator of PLD1 (Walker et al., 2000), and PLD1 located at the plasma membrane has been recently described as a key factor for exocytosis in neuroendocrine cells (Vitale et al., 2001). On the other hand, it is possible that the action of Cdc42 on exocytosis is related to its ability to promote the formation of actin filaments in the cell periphery as shown here in cells expressing the GTP-bound form of Cdc42. In a first approach to decipher the cascade of events initiated by Cdc42, transfection experiments with various cDNA encoding Cdc42 mutated in the effector loop ruled out the participation of PLD1 but revealed that effectors of the WASP/PAK branch linked to the actin cytoskeleton might be involved. We have demonstrated a causal role for Cdc42-regulated N-WASP in PC12 cell exocytosis by establishing that 1) upon activation of exocytosis, overexpressed N-WASP is recruited to the cell periphery where it enhances F-actin polymerization; 2) overexpression of N-WASP stimulates exocytosis in PC12 cells as efficiently as Cdc42, whereas a truncated mutant unable to polymerize actin has no effect; 3) N-WASP is actively recruited to the plasma membrane by Cdc42; and 4) coexpression of Cdc42 with a dominant negative N-WASP mutant completely abolishes the effect of active Cdc42 on exocytosis. Moreover, overexpressing PAK1 had no effect on GH secretion, electing N-WASP as the downstream effector by which Cdc42 modulates exocytosis. So far, the participation of N-WASP in the neuroendocrine exocytotic pathway has been suggested once in PC12 cells but in a manner that was independent of Cdc42 (Frantz et al., 2002). A possible explanation for this discrepancy is that the Cdc42V12 mutant used by Frantz et al. (2002) was not localized on plasma membrane but rather on some intracellular punctated structures.
The Actin Cytoskeleton at the Sites of Exocytosis
Actin plays a central role in regulated exocytosis but its exact mode of action remains unknown. Based on studies in a variety of secretory model systems, including neurons and endocrine cells, many potential roles have been proposed. Using evanescent wave microscopy in PC12 cells, Lang et al. (2000) were able to illustrate a dual function for actin by demonstrating that it can both hinder and mediate movements of GFP-labeled secretory granules in the subplasmalemmal region. In neurons, synaptic activity induces de novo actin polymerization (Colicos et al., 2001; Sankaranarayanan et al., 2003). In pancreatic acinar cells, actin polymerizes around secretory granules during exocytosis, and it has been proposed that this actin coating facilitates the movement of granules across the actin network toward their fusion site (Valentijn et al., 2000). Hence, actinomyosin-based motility has also been shown to play an important role in vesicular trafficking, because myosin II and/or myosin V are able to drive synaptic vesicles in neurons and secretory granules in neuroendocrine cells (Langford, 2002; Neco et al., 2002; Rose et al., 2003; Rudolf et al., 2003). On the other hand, recent data suggest that actin filaments may act as a scaffold in synaptic terminals by concentrating regulatory molecules near the releasable pool of synaptic vesicles (Sankaranarayanan et al., 2003). In yeast, Cdc42p triggers actin remodeling through a molecular pathway involving Las17p (the yeast WASP homolog) and the Arp2/3 complex. Of particular interest to the present study is that Cdc42p-induced actin filaments accumulate on docked vacuoles and are required for the terminal step leading to homotypic membrane fusion (Eitzen et al., 2002). Finally, actin filaments could also be important in secretory membrane recycling by providing the force for the membrane invagination or the pinching of the endocytic vesicles (Jeng and Welch, 2001).
How do the present data fit with these current views of the actin machinery underlying regulated exocytosis? N-WASP initiates the growth of actin filaments by bringing together actin monomers and the Arp2/3 complex (Prehoda et al., 2000). Although we did not directly demonstrate the functional importance of the Arp2/3 complex in secretion, we show here that Arp2/3 is present on secretory granules but not on the plasma membrane, in resting PC12 cells. An exciting speculation relates to the differential localization of N-WASP and Arp2/3 in resting versus stimulated cells (see hypothetical model in Figure 8). In resting cells, N-WASP is in the cytosol and Arp2/3 associated to secretory granules. Secretagogue-induced activation stimulates Cdc42, which in turn recruits N-WASP to the plasma membrane, and in parallel mobilizes secretory granules to the docking sites at the plasma membrane. Thus, the interaction between N-WASP, Arp2/3, and the actin monomers would take place only at the granule docking sites, providing a way to specifically target local actin filament polymerization at the interface between the granule and the plasma membrane. Recent work has established that for dense-core granules, the fusion pore opening-closing time can be modulated by various signaling pathways, allowing a control of the amount of hormones released per granule (Elhamdani et al., 2001; Taraska et al., 2003). The possibility that actin filaments formed at the granule docking site regulate expansion and/or closure of the fusion pore, thereby providing a molecular basis for control of quantal release, will be an interesting future issue.
|
To conclude, activation of secretion in neuroendocrine cells does not simply trigger the disassembly of the cortical actin barrier but rather induces a fine remodeling of the peripheral actin network into structures required for exocytosis. The present results provide a molecular support for the de novo formation of actin filaments in the course of exocytosis by sequentially ordering Cdc42, N-WASP, and Arp2/3 in a signaling pathway dedicated for the first time to the secretion of hormones. Further studies are now required to investigate whether the local production of actin filaments during exocytosis would serve for docking, scaffolding, fusion, and/or membrane retrieval.
|
ACKNOWLEDGMENTS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
|---|
|
Footnotes |
|---|
Corresponding author. E-mail address: gasman{at}neurochem.ustrasbg.fr.
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
|---|
Adamo, J.E., Moskow, J.J., Gladfelter, A.S., Viterbo, D., Lew, D.J., and Brennwald, P.J. (2001). Yeast Cdc42 functions at a late step in exocytosis, specifically during polarized growth of the emerging bud. J. Cell Biol. 155, 581-592.
Aunis, D., and Bader, M.F. (1988). The cytoskeleton as a barrier to exocytosis in secretory cells. J. Exp. Biol. 139, 253-266.
Bader, M.F., and Aunis, D. (1983). The 97-kD alpha-actinin-like protein in chromaffin granule membranes from adrenal medulla: evidence for localization on the cytoplasmic surface and for binding to actin filaments. Neuroscience 8, 165-181.[CrossRef][Medline]
Borisy, G.G., and Svitkina, T.M. (2000). Actin machinery: pushing the envelope. Curr. Opin. Cell Biol. 12, 104-112.[CrossRef][Medline]
Brown, A.M., O'Sullivan, A.J., and Gomperts, B.D. (1998). Induction of exocytosis from permeabilized mast cells by the guanosine triphosphatases Rac and Cdc42. Mol. Biol. Cell 9, 1053-1063.
Burridge, K., and Phillips, J.H. (1975). Association of actin and myosin with secretory granule membranes. Nature 254, 526-529.[CrossRef][Medline]
Carlier, M.F., Ducruix, A., and Pantaloni, D. (1999). Signalling to actin: the Cdc42-N-WASP-Arp2/3 connection. Chem. Biol. 6, R235-R240.[CrossRef][Medline]
Caron, E., and Hall, A. (1998). Identification of two distinct mechanisms of phagocytosis controlled by different Rho GTPases. Science 282, 1717-1721.
Colicos, M.A., Collins, B.E., Sailor, M.J., and Goda, Y. (2001). Remodeling of synaptic actin induced by photoconductive stimulation. Cell 107, 605-616.[CrossRef][Medline]
Cotteret, S., and Chernoff, J. (2002). The evolutionary history of effectors downstream of Cdc42 and Rac. Genome Biol. 3, reviews0002.0001-0008.
Daniel, S., Noda, M., Cerione, R.A., and Sharp, G.W. (2002). A link between Cdc42 and syntaxin is involved in mastoparan-stimulated insulin release. Biochemistry 41, 9663-9671.[CrossRef][Medline]
Doussau, F., and Augustine, G.J. (2000). The actin cytoskeleton and neurotransmitter release: an overview. Biochimie 82, 353-363.[Medline]
Doussau, F., Gasman, S., Humeau, Y., Vitiello, F., Popoff, M., Boquet, P., Bader, M.F., and Poulain, B. (2000). A Rho-related GTPase is involved in Ca(2+)-dependent neurotransmitter exocytosis. J. Biol. Chem. 275, 7764-7770.
Eitzen, G., Wang, L., Thorngren, N., and Wickner, W. (2002). Remodeling of organelle-bound actin is required for yeast vacuole fusion. J. Cell Biol. 158, 669-679.
Elhamdani, A., Palfrey, H.C., and Artalejo, C.R. (2001). Quantal size is dependent on stimulation frequency and calcium entry in calf chromaffin cells. Neuron 31, 819-830.[CrossRef][Medline]
Etienne-Manneville, S., and Hall, A. (2002). Rho GTPases in cell biology. Nature 420, 629-635.[CrossRef][Medline]
Frantz, C., Coppola, T., and Regazzi, R. (2002). Involvement of Rho GTPases and their effectors in the secretory process of PC12 cells. Exp. Cell Res. 273, 119-126.[CrossRef][Medline]
Gampel, A., Parker, P.J., and Mellor, H. (1999). Regulation of epidermal growth factor receptor traffic by the small GTPase rhoB. Curr. Biol. 9, 955-958.[CrossRef][Medline]
Gasman, S., Chasserot-Golaz, S., Hubert, P., Aunis, D., and Bader, M.F. (1998). Identification of a potential effector pathway for the trimeric Go protein associated with secretory granules. Go stimulates a granule-bound phosphatidylinositol 4-kinase by activating RhoA in chromaffin cells. J. Biol. Chem. 273, 16913-16920.
Gasman, S., Chasserot-Golaz, S., Popoff, M.R., Aunis, D., and Bader, M.F. (1997). Trimeric G proteins control exocytosis in chromaffin cells. Go regulates the peripheral actin network and catecholamine secretion by a mechanism involving the small GTP-binding protein Rho. J. Biol. Chem. 272, 20564-20571.
Gasman, S., Chasserot-Golaz, S., Popoff, M.R., Aunis, D., and Bader, M.F. (1999). Involvement of Rho GTPases in calcium-regulated exocytosis from adrenal chromaffin cells. J. Cell Sci. 112, 4763-4771.[Abstract]
Gasman, S., Kalaidzidis, Y., and Zerial, M. (2003). RhoD regulates endosome dynamics through Diaphanous-related Formin and Src tyrosine kinase. Nat. Cell Biol. 5, 195-204.[CrossRef][Medline]
Hall, A. (1998). Rho GTPases and the actin cytoskeleton. Science 279, 509-514.
Higgs, H.N., and Pollard, T.D. (2001). Regulation of actin filament network formation through ARP2/3 complex: activation by a diverse array of proteins. Annu. Rev. Biochem. 70, 649-676.[CrossRef][Medline]
Jeng, R.L., and Welch, M.D. (2001). Cytoskeleton: actin and endocytosis-no longer the weakest link. Curr. Biol. 11, R691-694.[CrossRef][Medline]
Jiang, Z.Y., Chawla, A., Bose, A., Way, M., and Czech, M.P. (2002). A phosphatidylinositol 3-kinase-independent insulin signaling pathway to N-WASP/Arp2/3/F-actin required for GLUT4 glucose transporter recycling. J. Biol. Chem. 277, 509-515.
Kim, A.S., Kakalis, L.T., Abdul-Manan, N., Liu, G.A., and Rosen, M.K. (2000). Autoinhibition and activation mechanisms of the Wiskott-Aldrich syndrome protein. Nature 404, 151-158.[CrossRef][Medline]
Kowluru, A., Li, G., Rabaglia, M.E., Segu, V.B., Hofmann, F., Aktories, K., and Metz, S.A. (1997). Evidence for differential roles of the Rho subfamily of GTP-binding proteins in glucose- and calcium-induced insulin secretion from pancreatic beta cells. Biochem. Pharmacol. 54, 1097-1108.[CrossRef][Medline]
Lamaze, C., Chuang, T.H., Terlecky, L.J., Bokoch, G.M., and Schmid, S.L. (1996). Regulation of receptor-mediated endocytosis by Rho and Rac. Nature 382, 177-179.[CrossRef][Medline]
Lang, T., Wacker, I., Wunderlich, I., Rohrbach, A., Giese, G., Soldati, T., and Almers, W. (2000). Role of actin cortex in the subplasmalemmal transport of secretory granules in PC-12 cells. Biophys. J. 78, 2863-2877.[Medline]
Langford, G.M. (2002). Myosin-v, a versatile motor for short-range vesicle transport. Traffic 3, 859-865.[CrossRef][Medline]
Li, G., Rungger-Brandle, E., Just, I., Jonas, J.C., Aktories, K., and Wollheim, C.B. (1994). Effect of disruption of actin filaments by Clostridium botulinum C2 toxin on insulin secretion in HIT-T15 cells and pancreatic islets. Mol. Biol. Cell 5, 1199-1213.[Abstract]
Matter, K., Dreyer, F., and Aktories, K. (1989). Actin involvement in exocytosis from PC12 cells: studies on the influence of botulinum C2 toxin on stimulated noradrenaline release. J. Neurochem. 52, 370-376.[CrossRef][Medline]
May, R.C., Caron, E., Hall, A., and Machesky, L.M. (2000). Involvement of the Arp2/3 complex in phagocytosis mediated by FcgammaR or CR3. Nat. Cell Biol. 2, 246-248.[CrossRef][Medline]
Meyer, D.I., and Burger, M.M. (1979). The chromaffin granule surface: the presence of actin and the nature of its interaction with the membrane. FEBS Lett. 101, 129-133.[CrossRef][Medline]
Michaelson, D., Silletti, J., Murphy, G., D'Eustachio, P., Rush, M., and Philips, M.R. (2001). Differential localization of Rho GTPases in live cells: regulation by hypervariable regions and RhoGDI binding. J. Cell Biol. 152, 111-126.
Miki, H., Sasaki, T., Takai, Y., and Takenawa, T. (1998). Induction of filopodium formation by a WASP-related actin-depolymerizing protein N-WASP. Nature 391, 93-96.[CrossRef][Medline]
Moreau, V., Frischknecht, F., Reckmann, I., Vincentelli, R., Rabut, G., Stewart, D., and Way, M. (2000). A complex of N-WASP and WIP integrates signalling cascades that lead to actin polymerization. Nat. Cell Biol. 2, 441-448.[CrossRef][Medline]
Neco, P., Gil, A., Del Mar Frances, M., Viniegra, S., and Gutierrez, L.M. (2002). The role of myosin in vesicle transport during bovine chromaffin cell secretion. Biochem. J. 368, 405-413.[CrossRef][Medline]
Owen, D., Mott, H.R., Laue, E.D., and Lowe, P.N. (2000). Residues in Cdc42 that specify binding to individual CRIB effector proteins. Biochemistry 39, 1243-1250.[CrossRef][Medline]
Pendleton, A., and Koffer, A. (2001). Effects of latrunculin reveal requirements for the actin cytoskeleton during secretion from mast cells. Cell Motil. Cytoskeleton 48, 37-51.[CrossRef][Medline]
Prehoda, K.E., Scott, J.A., Mullins, R.D., and Lim, W.A. (2000). Integration of multiple signals through cooperative regulation of the N-WASP-Arp2/3 complex. Science 290, 801-806.
Ridley, A.J. (2001). Rho proteins: linking signaling with membrane trafficking. Traffic 2, 303-310.[CrossRef][Medline]
Rohatgi, R., Ho, H.Y., and Kirschner, M.W. (2000). Mechanism of N-WASP activation by CDC42 and phosphatidylinositol 4, 5-bisphosphate. J. Cell Biol. 150, 1299-1310.
Rohatgi, R., Ma, L., Miki, H., Lopez, M., Kirchhausen, T., Takenawa, T., and Kirschner, M.W. (1999). The interaction between N-WASP and the Arp2/3 complex links Cdc42-dependent signals to actin assembly. Cell 97, 221-231.[CrossRef][Medline]
Rose, S.D., Lejen, T., Casaletti, L., Larson, R.E., Pene, T.D., and Trifaro, J.M. (2003). Myosins II and V in chromaffin cells: myosin V is a chromaffin vesicle molecular motor involved in secretion. J. Neurochem. 85, 287-298.[Medline]
Rudolf, R., Kogel, T., Kuznetsov, S.A., Salm, T., Schlicker, O., Hellwig, A., Hammer, J.A., 3rd, and Gerdes, H.H. (2003). Myosin Va facilitates the distribution of secretory granules in the F-actin rich cortex of PC12 cells. J. Cell Sci. 116, 1339-1348.
Sankaranarayanan, S., Atluri, P.P., and Ryan, T.A. (2003). Actin has a molecular scaffolding, not propulsive, role in presynaptic function. Nat. Neurosci. 6, 127-135.[CrossRef][Medline]
Sells, M.A., Boyd, J.T., and Chernoff, J. (1999). p21-activated kinase 1 (Pak1) regulates cell motility in mammalian fibroblasts. J. Cell Biol. 145, 837-849.
Strassheim, D., Porter, R.A., Phelps, S.H., and Williams, C.L. (2000). Unique in vivo associations with SmgGDS and RhoGDI and different guanine nucleotide exchange activities exhibited by RhoA, dominant negative RhoA(Asn-19), and activated RhoA(Val-14). J. Biol. Chem. 275, 6699-6702.
Sullivan, R., Price, L.S., and Koffer, A. (1999). Rho controls cortical F-actin disassembly in addition to, but independently of, secretion in mast cells. J. Biol. Chem. 274, 38140-38146.
Taraska, J.W., Perrais, D., Ohara-Imaizumi, M., Nagamatsu, S., and Almers, W. (2003). Secretory granules are recaptured largely intact after stimulated exocytosis in cultured endocrine cells. Proc. Natl. Acad. Sci. USA 100, 2070-2075.
Trifaro, J., Rose, S.D., Lejen, T., and Elzagallaai, A. (2000). Two pathways control chromaffin cell cortical F-actin dynamics during exocytosis. Biochimie 82, 339-352.[Medline]
Valentijn, J.A., Valentijn, K., Pastore, L.M., and Jamieson, J.D. (2000). Actin coating of secretory granules during regulated exocytosis correlates with the release of rab3D. Proc. Natl. Acad. Sci. USA 97, 1091-1095.
Vignal, E., De Toledo, M., Comunale, F., Ladopoulou, A., Gauthier-Rouviere, C., Blangy, A., and Fort, P. (2000). Characterization of TCL, a new GTPase of the rho family related to TC10 andCcdc42. J. Biol. Chem. 275, 36457-36464.
Vitale, M.L., Seward, E.P., and Trifaro, J.M. (1995). Chromaffin cell cortical actin network dynamics control the size of the release-ready vesicle pool and the initial rate of exocytosis. Neuron 14, 353-363.[CrossRef][Medline]
Vitale, N., Caumont, A.S., Chasserot-Golaz, S., Du, G., Wu, S., Sciorra, V.A., Morris, A.J., Frohman, M.A., and Bader, M.F. (2001). Phospholipase D 1, a key factor for the exocytotic machinery in neuroendocrine cells. EMBO J. 20, 2424-2434.[CrossRef][Medline]
Vitale, N., Chasserot-Golaz, S., Bailly, Y., Morinaga, N., Frohman, M.A., and Bader, M.F. (2002). Calcium-regulated exocytosis of dense-core vesicles requires the activation of ADP-ribosylation factor (ARF)6 by ARF nucleotide binding site opener at the plasma membrane. J. Cell Biol. 159, 79-89.
Walker, S.J., and Brown, H.A. (2002). Specificity of Rho insert-mediated activation of phospholipase D1. J. Biol. Chem. 277, 26260-26267.
Walker, S.J., Wu, W.J., Cerione, R.A., and Brown, H.A. (2000). Activation of phospholipase D1 by Cdc42 requires the Rho insert region. J. Biol. Chem. 275, 15665-15668.
Wilkins, J.A., and Lin, S. (1981). Association of actin with chromaffin granule membranes and the effect of cytochalasin B on the polarity of actin filament elongation. Biochim. Biophys. Acta 642, 55-66.[Medline]
Zhang, X., Bi, E., Novick, P., Du, L., Kozminski, K.G., Lipschutz, J.H., and Guo, W. (2001). Cdc42 interacts with the exocyst and regulates polarized secretion. J. Biol. Chem. 276, 46745-46750.
This article has been cited by other articles:
|
|
 |
|
  Z. Wang and D. C. Thurmond Mechanisms of biphasic insulin-granule exocytosis - roles of the cytoskeleton, small GTPases and SNARE proteins J. Cell Sci., April 1, 2009; 122(7): 893 - 903. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
F. Momboisse, E. Lonchamp, V. Calco, M. Ceridono, N. Vitale, M.-F. Bader, and S. Gasman {beta}PIX-activated Rac1 stimulates the activation of phospholipase D, which is associated with exocytosis in neuroendocrine cells J. Cell Sci., March 15, 2009; 122(6): 798 - 806. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. Berberian, A. J. Torres, Q. Fang, K. Kisler, and M. Lindau F-Actin and Myosin II Accelerate Catecholamine Release from Chromaffin Granules J. Neurosci., January 21, 2009; 29(3): 863 - 870. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. Mitchell, A. Lo, M. R. Logan, P. Lacy, and G. Eitzen Primary granule exocytosis in human neutrophils is regulated by Rac-dependent actin remodeling Am J Physiol Cell Physiol, November 1, 2008; 295(5): C1354 - C1365. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Y. J. Lee, A. Szumlanski, E. Nielsen, and Z. Yang Rho-GTPase-dependent filamentous actin dynamics coordinate vesicle targeting and exocytosis during tip growth J. Cell Biol., October 22, 2008; 181(7): 1155 - 1168. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L. Grumolato, H. Ghzili, M. Montero-Hadjadje, S. Gasman, J. Lesage, Y. Tanguy, L. Galas, D. Ait-Ali, J. Leprince, N. C. Guerineau, et al. Selenoprotein T is a PACAP-regulated gene involved in intracellular Ca2+ mobilization and neuroendocrine secretion FASEB J, June 1, 2008; 22(6): 1756 - 1768. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. L. Jewell, W. Luo, E. Oh, Z. Wang, and D. C. Thurmond Filamentous Actin Regulates Insulin Exocytosis through Direct Interaction with Syntaxin 4 J. Biol. Chem., April 18, 2008; 283(16): 10716 - 10726. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. Krzewski, X. Chen, and J. L. Strominger WIP is essential for lytic granule polarization and NK cell cytotoxicity PNAS, February 19, 2008; 105(7): 2568 - 2573. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L. Cao, K. Yu, C. Banh, V. Nguyen, A. Ritz, B. J. Raphael, Y. Kawakami, T. Kawakami, and A. R. Salomon Quantitative Time-Resolved Phosphoproteomic Analysis of Mast Cell Signaling J. Immunol., November 1, 2007; 179(9): 5864 - 5876. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Z. Wang, E. Oh, and D. C. Thurmond Glucose-stimulated Cdc42 Signaling Is Essential for the Second Phase of Insulin Secretion J. Biol. Chem., March 30, 2007; 282(13): 9536 - 9546. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
I. M. Sehring, C. Reiner, J. Mansfeld, H. Plattner, and R. Kissmehl A broad spectrum of actin paralogs in Paramecium tetraurelia cells display differential localization and function J. Cell Sci., January 1, 2007; 120(1): 177 - 190. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Tomas, B. Yermen, L. Min, J. E. Pessin, and P. A. Halban Regulation of pancreatic {beta}-cell insulin secretion by actin cytoskeleton remodelling: role of gelsolin and cooperation with the MAPK signalling pathway J. Cell Sci., May 15, 2006; 119(10): 2156 - 2167. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Z. Meyer, N. Deliot, S. Chasserot-Golaz, R. T. Premont, M.-F. Bader, and N. Vitale Regulation of Neuroendocrine Exocytosis by the ARF6 GTPase-activating Protein GIT1 J. Biol. Chem., March 24, 2006; 281(12): 7919 - 7926. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 P. Alberts, R. Rudge, T. Irinopoulou, L. Danglot, C. Gauthier-Rouviere, and T. Galli Cdc42 and Actin Control Polarized Expression of TI-VAMP Vesicles to Neuronal Growth Cones and Their Fusion with the Plasma Membrane Mol. Biol. Cell, March 1, 2006; 17(3): 1194 - 1203. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
G. Baldini, A. M. Martelli, G. Tabellini, C. Horn, K. Machaca, P. Narducci, and G. Baldini Rabphilin Localizes with the Cell Actin Cytoskeleton and Stimulates Association of Granules with F-actin Cross-linked by {alpha}-Actinin J. Biol. Chem., October 14, 2005; 280(41): 34974 - 34984. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y. Gu, Y. Fu, P. Dowd, S. Li, V. Vernoud, S. Gilroy, and Z. Yang A Rho family GTPase controls actin dynamics and tip growth via two counteracting downstream pathways in pollen tubes J. Cell Biol., April 11, 2005; 169(1): 127 - 138. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Li, K. L. O'Connor, G. H. Greeley Jr., P. J. Blackshear, C. M. Townsend Jr., and B. M. Evers Myristoylated Alanine-rich C Kinase Substrate-mediated Neurotensin Release via Protein Kinase C-{delta} Downstream of the Rho/ROK Pathway J. Biol. Chem., March 4, 2005; 280(9): 8351 - 8357. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. Ehre, A. H. Rossi, L. H. Abdullah, K. De Pestel, S. Hill, J. C. Olsen, and C. W. Davis Barrier role of actin filaments in regulated mucin secretion from airway goblet cells Am J Physiol Cell Physiol, January 1, 2005; 288(1): C46 - C56. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. Kissmehl, I. M. Sehring, E. Wagner, and H. Plattner Immunolocalization of Actin in Paramecium Cells J. Histochem. Cytochem., December 1, 2004; 52(12): 1543 - 1559. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
X. Xin, F. Ferraro, N. Back, B. A. Eipper, and R. E. Mains Cdk5 and Trio modulate endocrine cell exocytosis J. Cell Sci., September 15, 2004; 117(20): 4739 - 4748. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
-HA), and analyzed by confocal microscopy. HA antibodies were visualized with Cy3-conjugated secondary antibodies. Bar, 5 µm. (C) K+-evoked GH-secretion from cells expressing 
