βPIX-activated Rac1 stimulates the activation of phospholipase D, which is associated with exocytosis in neuroendocrine cells

Rho proteins constitute a subgroup of the small GTPase Ras superfamilly and comprise 20 members that are often involved in membrane trafficking through the remodelling of the actin cytoskeleton (Ridley, 2006). Exocytosis is a fundamental membrane trafficking event that requires coordinated interactions between actin and the vesicles undergoing recruitment, docking and fusion with the plasma membrane. Yet, Rho GTPases have been implicated in many processes involving exocytotic reactions, including hormone and transmitter release, cell migration, cell repair and proliferation. In neuroendocrine cells, Ca²⁺-mediated exocytosis of hormones is under the control of two members of the Rho family: RhoA bound to secretory granules and Cdc42 associated with the plasma membrane. Functional experiments based on the expression of interference RNA or constitutively active GTP-loaded proteins indicate that Cdc42 and RhoA influence the exocytotic machinery by spatially and temporally coordinating actin dynamics to the traffic of secretory granules under the plasma membrane (Bader et al., 2004; Gasman et al., 2004; Malacombe et al., 2006). Earlier experiments based on the use of clostridial toxins, which differentially impair the function of Rho GTPases, suggest that Rac might also play a role in secretion, although in an actin-independent manner (Gasman et al., 1999). Hence, the participation of Rac1 in Ca²⁺-regulated exocytosis has subsequently been demonstrated in neurons (Humeau et al., 2007; Humeau et al., 2002) and in various endocrine cells, including chromaffin cells (Li et al., 2003) and pancreatic β-cells and acini (Amin et al., 2003; Bi and Williams, 2005). The upstream events leading to the activation of Rac1 and the downstream signalling pathway by which Rac1 controls the exocytotic response, however, remains unexplored.

The present study was undertaken to investigate the mechanism by which Rac1 controls exocytosis. Using PC12 cells as a secretory cell model, we demonstrate that activation of Rac1 is an essential step in the exocytotic process. We also provide evidence that βPix is the GEF-promoting nucleotide exchange and evidence for activation of Rac1 at the plasma membrane in secretagogue-stimulated cells. We show that Scrib, the mammalian orthologue of the Drosophila tumour suppressor Scribble, controls Rac1 activation most probably by recruiting βPix to the plasma membrane (Audebert et al., 2004). Moreover, our results reveal that the downstream pathway by which Rac1 controls Ca²⁺-regulated exocytosis involves the lipid-modifying enzyme phospholipase D1, which is known to provide fusogenic lipids at the sites of exocytosis (Vitale et al., 2001; Vitale et al., 2002; Zeniou-Meyer et al., 2007). Altogether, our findings define a novel molecular pathway, for the first time linking Rac1 to the final stages of Ca²⁺-regulated exocytosis in secretory neuroendocrine cells.

To determine the activation status of Rac1 during Ca²⁺-regulated exocytosis, PC12 cells were stimulated for various periods of time with a depolarizing concentration of K⁺ and Rac activation was measured by a pull-down assay using the PAK1-binding domain as a bait to trap the GTPase in its GTP-bound form. As shown in Fig. 1A, stimulation with 59 mM K⁺ increased the amount of GTP-loaded Rac1, with a maximal level of activation reproducibly found after 10 minutes of stimulation (n=3; see semi-quantitative analysis in Fig. 1B). Comparable levels of Rac1 activation in response to K⁺-stimulation, i.e. three- to fivefold increase of GTP-loaded Rac1, were measured using an ELISA (Fig. 1C). Under similar experimental conditions, maximal release of growth hormone (GH) occurred in response to 5-10 minutes of K⁺-stimulation (Fig. 1D). Moreover, Rac1-GTP rapidly decreased to basal levels as cells returned to the resting conditions. Together, these findings indicate that Rac1 undergoes a complete activation/desactivation cycle in stimulated cells, which temporally parallels the hormone exocytotic process.

The subcellular localization of Rac1 was examined by immunofluorescence and confocal microscopy. As illustrated in Fig. 1E, Rac1 was essentially found in the cell periphery and to a lesser extent as a punctuated and diffuse staining pattern in the cytoplasm. Double labelling experiments indicated that most of the peripheral Rac1 colocalized with the plasma membrane marker SNAP25 (Fig. 1E). Quantification of the relative proportion of Rac1 colocalizing with SNAP25 indicated that around 60% of Rac1 was found at the plasma membrane both in resting and K⁺-stimulated PC12 cells (Fig. 1F). Thus, in line with our previous observations (Gasman et al., 1999), Rac1 is predominantly present at the plasma membrane in neuroendocrine chromaffin and PC12 cells.

Previous studies have shown that expression of constitutively active or dominant-negative mutants of Rac1 affected Ca²⁺-mediated exocytosis in chromaffin and PC12 cells (Frantz et al., 2002; Li et al., 2003). To assess directly the functional importance of Rac1 in the basic exocytotic machinery, we employed a short interference RNA (siRNA) strategy (Malacombe et al., 2006; Vitale et al., 2005), using a plasmid (pGHsuper) that expresses full-length human growth hormone (GH) and a siRNA targeted against the sequence of Rac1. GH specifically stored into secretory granules is used as a reporter for exocytosis in the subpopulation of cells that transiently express the siRNA (Malacombe et al., 2006). As revealed by western blot analysis (Fig. 2A), cells expressing Rac1 siRNA exhibited a significant reduction in the level of endogenous Rac1 compared with control cells or with cells expressing an unrelated siRNA (UnR). The expression of Cdc42 or RhoA, two related members of the Rho family, was not affected (Fig. 2A). Densitometry scans from three independent experiments revealed that the level of endogenous Rac1 was reduced by ∼64% (supplementary material Fig. S1A,B). Accordingly, we found by immunocytochemistry that cells expressing Rac1 siRNA, identified by the expression of GH, presented a reduced Rac1 staining, but the expression level of GH or the distribution of GH in secretory granules was not modified (supplementary material Fig. S1C). We then examined the effect of Rac1 silencing on secretory activity. Reduction of endogenous Rac1 significantly inhibited the secretion of GH stimulated by a depolarizing concentration of K⁺ (Fig. 2B). Co-expression of Rac1 siRNAs with human Ha-tagged Rac1, which differs from the rat cDNA by six nucleotides in the sequence recognized by the siRNA (and is thereby insensitive to the siRNAs as confirmed by western blot; data not show), largely restored secretion of cells depleted of endogenous Rac1 (Fig. 2C). Altogether, these results indicate that Rac1 plays an essential function in the exocytotic pathway of large dense-core secretory granules and prompted us to investigate the underlying molecular event(s).

We have previously demonstrated that the lipid-modifying enzyme phospholipase D1 (PLD1) is essential for the late steps of exocytosis in PC12 cells. By contrast, the PLD2 isoform is not required for exocytosis (Vitale et al., 2001; Zeniou-Meyer et al., 2007). Interestingly, several members of the Rho GTPase family such as RhoA, Cdc42 and Rac1 are established activators of PLD1 in vitro (Hammond et al., 1997; Walker and Brown, 2002). However, so far there is no functional evidence for a role of a Rho GTPase in controlling membrane trafficking processes through the regulation of PLD enzymes. To probe the possible implication of Rho GTPases in PLD1 activation during exocytosis, we measured PLD activity in resting and K⁺-stimulated PC12 cells transiently expressing siRNAs against Cdc42, RhoA or Rac1. Cdc42 siRNA was characterized previously (Malacombe et al., 2006) and western blot analysis of total cell extracts indicated that expression of the RhoA silencer decreased the level of endogenous RhoA by ∼58% (n=3) without affecting the expression of Cdc42 or Rac1 (Fig. 3A; supplementary material Fig. S1B,D). As previously observed for Rac1 activation and GH secretion (Fig. 1), high K⁺ triggered an increase in PLD activity that was maximal after 5 to 10 minutes of stimulation (supplementary material Fig. S2). Neither RhoA nor Cdc42 silencing affected the secretagogue-evoked stimulation of PLD. By contrast, reduction of Rac1 expression resulted in strong inhibition of PLD activation (Fig. 3B), which was comparable with that observed in PC12 cells expressing the PLD1 siRNA that is known to specifically reduce endogenous PLD1 level (Zeniou-Meyer et al., 2007). These results elect Rac1 as the best candidate among the Rho GTPases to mediate PLD1 activation in cells stimulated for secretion.

We recently demonstrated that PLD1 is responsible for the dynamic production of phosphatidic acid (PA) at the plasma membrane that is required for the late stages of exocytosis (Zeniou-Meyer et al., 2007). To further assess the role of Rac1 as an upstream activator of PLD1 in the exocytotic pathway, we examined the level of PA found at the plasma membrane in PC12 cells expressing Rac1 siRNA, using the PA-binding domain of Spo20p fused to EGFP as PA-sensor (Nakanishi et al., 2004; Zeniou-Meyer et al., 2007). As observed previously (Zeniou-Meyer et al., 2007), the PA-binding probe (PABD) accumulated in the nucleus in resting cells but a fraction of it was recruited to the cell periphery where it colocalized with SNAP25 in K⁺-stimulated cells (Fig. 3C). Interestingly, this K⁺-evoked translocation of PABD to the plasma membrane was inhibited in cells expressing Rac1 siRNAs (Fig. 3C). Quantification of the relative proportion PABD colocalizing with SNAP25 indicated that expression of Rac1 siRNAs inhibited the recruitment of PABD to the plasma membrane by 66% (n=25 cells) (Fig. 3D), whereas expression of an unrelated siRNAs had no effect (not shown). Thus, Rac 1 is an upstream component of the signalling pathway leading to the activation of PLD1 and the production of PA required for exocytosis.

As the Rac guanine nucleotide exchange factor βPix is able to stimulate secretion from PC12 cells (Audebert et al., 2004), we decided to probe the possible relationship between βPix, Rac1 and PLD1 in the exocytotic machinery. Expression of βPix siRNA in PC12 cells reduced βPix level by 67.5 % (Fig. 4A; supplementary material Fig. S3A) and resulted in a significant inhibition of K⁺-evoked GH secretion (51.8±2.6%; n=3) (Fig. 4B). Similar results were obtained with two different βPix siRNAs, which excludes possible off-target effects (not shown). These results are in line with the idea that βPix is an element of the exocytotic machinery. Reduction of βPix level by siRNA expression also significantly inhibited PLD activation in response to elevated K⁺ (Fig. 4C) and decreased the amount of GTP-loaded Rac1 detected in K⁺-stimulated cells (Fig. 4D). Knockdown of βPix increased also the basal level of Rac1-GTP estimated in resting cells (Fig. 4D), an observation for which we have currently no clear explanation. Note, however, that the net K⁺-evoked activation of Rac1 (calculated by subtracting the amount of Rac1-GTP detected in resting cells from the amount measured in K⁺-stimulated cells) is largely decreased in cells expressing βPix siRNAs (Fig. 4D) (net), indicating that βPix is indeed responsible for the K⁺-evoked Rac1 activation required for exocytosis. Moreover, the apparent activation of Rac1 triggered by the transfection of βPix siRNA neither affected the basal release of GH nor the basal activity of PLD1 (Fig. 4B,C), suggesting that the fraction of Rac1 activated in response to βPix siRNA expression was apparently not directly linked to exocytotic activity.

Previous studies combining immunofluorescence and subcellular fractionation experiments suggest that βPix is mainly cytosolic in resting PC12 cells but is recruited to the plasma membrane in cells stimulated with 59 mM K⁺ (Audebert et al., 2004). To substantiate this finding, we analyzed the relative proportion of cytosolic versus membrane-bound βPix by evaluating the release of soluble βPix after cell permeabilization with a low concentration of saponin (Botrugno et al., 2006). As illustrated in Fig. 5A,B, permeabilization of PC12 cells with 0.05% saponin in resting condition induced the release of a fraction of the endogenous βPix, indicating that βPix is present in the cytosol in resting cells. In stimulated cells, the amount of βPix released upon permeabilization significantly decreased, whereas the amount retained within permeabilized cells was clearly enhanced, in line with the idea that cytosolic βPix is recruited to a membrane-bound compartment upon cell stimulation. Accordingly, the proportion of βPix co-localizing with Rac1 at the plasma membrane increased in K⁺-stimulated PC12 cells (Fig. 5C), supporting the idea that secretagogues trigger the translocation of βPix to the plasma membrane, thereby favouring an interaction between Rac1 and βPix, and the degree of Rac1 activation.

To directly link the GEF activity of βPix with Rac1 and PLD1 activation, we transfected PC12 cells to express the βPix_L238R-L239S mutant that lacks guanine nucleotide exchange activity (Manser et al., 1998). Expression of the βPix_L238R-L239S mutant, which was recruited to the cell periphery upon K⁺ stimulation (not shown), largely abolished secretagogue-induced activation of Rac1 (Fig. 5D). By contrast, overexpression of wild-type βPix had no significant effect (Fig. 5D). Note that the GEF-dead βPix did not change the level of GTP-loaded Rac1 in resting cells (Fig. 5D). Similarly, PLD activity was not modified in cells overexpressing wild-type βPix but expression of the GEF-dead βPix mutant significantly prevented PLD activation in response to K⁺ stimulation (Fig. 5E). Together, these results strongly support the idea that βPix is the nucleotide exchange factor that catalyses the activation of Rac1 at the plasma membrane, which in turn activates the PLD1 required for the late steps of exocytosis.

Scrib, the mammalian homologue of the Drosophila neoplastic tumour suppressor Scribble, has been described as a membrane anchor for βPix at the plasma membrane in PC12 cells (Audebert et al., 2004). This finding led us to investigate whether Scrib might play a role in exocytosis by recruiting βPix and subsequently activating Rac1-dependent PLD1 at appropriate sites. Knockdown of endogenous Scrib by siRNA expression in PC12 cells (Fig. 6A; supplementary material Fig. S3B) significantly lowered K⁺-stimulated GH secretion (Fig. 6B), confirming that Scrib is a player of the exocytotic machinery. As a control, expression of human Scrib siRNA, which differs from the rat sequence by three nucleotides, did not affect the secretory activity (supplementary material Fig. S4). Conversely, reduction of endogenous Scrib expression inhibited also the net K⁺-evoked Rac1 activation (Fig. 6C) and K⁺-dependent PLD simulation (Fig. 6D), whereas overexpression of wild-type Scrib had no effect. We examined whether βPix recruitment was required for Scrib-mediated PLD activation. Therefore, cells were transfected to express Scrib-ΔPDZ, a Scrib mutant that cannot interact with βPix because it lacks the PDZ domain (Audebert et al., 2004). Expression of Scrib-ΔPDZ inhibited K⁺-induced recruitment of βPix to the plasma membrane, as shown by immunofluorescence (compare Fig. 6E with Fig. 5C). Accordingly, permeabilization of resting or stimulated Scrib-ΔPDZ-expressing cells triggered the leakage of cytosolic βPix to a similar extent (Fig. 6F), indicating that the presence of Scrib-ΔPDZ interfered with the recruitment of βPix to the plasma membrane upon cell stimulation. Finally, both Rac and PLD activation were inhibited by the expression of Scrib-ΔPDZ in cells stimulated high K⁺ (Fig. 6C,D). Together, these data are consistent with the idea that Scrib recruits βPix to the plasma membrane to further activate Rac1 and PLD1 at the sites of exocytosis.

Over the past years, small GTPases of the Rho family have emerged as important players in membrane trafficking (Ridley, 2001; Ridley, 2006). Among them, Rac isoforms have been involved in various processes of exocytosis, including mast cell and neutrophil degranulation (Abdel-Latif et al., 2004; Hong-Geller and Cerione, 2000), endocrine and neuroendocrine secretion (Amin et al., 2003; Bi and Williams, 2005; Frantz et al., 2002; Li et al., 2003), and neurotransmitter release (Doussau et al., 2000; Humeau et al., 2007; Humeau et al., 2002). However, most of these studies rely on the use of dominant-negative and constitutively active versions of Rac proteins and do not directly address the participation of endogenous Rac1 in the basic exocytotic machinery. The aim of the present work was to probe the actual function of Rac1 in neuroendocrine cell secretion and to dissect the upstream and downstream signalling pathways integrating Rac1 to the exocytotic machinery. We show here that Rac1 is strongly activated during secretion and that reducing the levels of endogenous Rac1 significantly inhibited exocytosis. Furthermore, we found that recruitment of the guanine exchange factor βPix by Scrib at the plasma membrane is required for Rac1 activation. Finally, we demonstrate that activated Rac1 is involved in the stimulation of PLD1 underlying hormone release.

PLD1 generates phosphatidic acid, a multifunctional lipid that has been involved in many vesicular trafficking events by altering membrane curvature and favouring fusion (Wang et al., 2006). We and others have demonstrated that PLD1 constitutes an essential regulator for exocytosis in secretory cells (Choi et al., 2002; Humeau et al., 2001; Vitale et al., 2001; Waselle et al., 2005). In vitro, Rac1 directly interacts with PLD1 to enhance its activity. Using a loss-of-function approach based on siRNA expression, we show here that a reduction of Rac1 level prevents PLD-dependent production of PA in stimulated PC12. These results led us to conclude that Rac1 is a crucial regulator of PLD1 in the course of Ca²⁺-regulated exocytosis. Interestingly, in neurons, independent experiments have led to the conclusion that both Rac1 and PLD1 control transmitter release by regulating the number of functional release sites at the synapse (Doussau et al., 2000; Humeau et al., 2007; Humeau et al., 2002; Humeau et al., 2001). Thus, by analogy with the function proposed here in neuroendocrine secretion, Rac1 may well be an upstream activator of the plasma membrane-bound PLD1 in the cascade leading to neurotransmitter release in neurons. We cannot completely rule out the possibility that Rac1 might be able to control hormone/transmitter release by additional pathways. We did not observe actin rearrangements in PC12 cells depleted of Rac1 (data not shown). However, overexpression of Rac1 mutants in chromaffin cells and isolated pancreatic acini has been reported to modify actin cytoskeletal organization (Bi and Williams, 2005; Li et al., 2003). In RBL-2H3 cells, Rac1 seems to control antigen-induced degranulation by several mechanisms, including Ca²⁺ signalling through inositol triphosphate production, protein kinase C activation and possibly PLD stimulation (Hong-Geller and Cerione, 2000; Powner et al., 2002). Rac1 has also been shown to bind and stimulate phosphatidylinositol-5 kinase (PIP5-kinase), which generates PtdIns(4,5)P₂ (PIP2), a phosphoinositide largely implicated in various exocytotic processes (Gong et al., 2005; Weernink et al., 2004). However, the present observation that Rac1 is able to stimulate secretion by activating the PLD1-dependent production of PA at the sites of exocytosis implies that a Rac1-dependent signalling pathway might be directly linked to the late stages of exocytosis and might thereby be able to modulate the formation or extension of the fusion pore.

The specific activation of Rac1 in cells stimulated by a depolarizing concentration of K⁺ raised the issue of the upstream signalling pathway linking K⁺-induced cytosolic Ca²⁺ elevation to Rac1 activation. So far, the identity of the RhoGEFs implicated in regulated secretion has not been extensively studied. Kailirin and Trio, two activators of Rac and RhoG, play a role in pituitary cell secretion (Ferraro et al., 2007), whereas intersectin 1L, a GEF for Cdc42 activates the exocytotic machinery in PC12 cells (Malacombe et al., 2006). Additionally, βPix, an exchange factor for Rac1 has been described to be recruited by Scrib at the plasma membrane during exocytosis (Audebert et al., 2004). We demonstrate here the functional relationship between the nucleotide exchange activity of βPix, the activation of Rac1 and PLD1 at the plasma membrane, and exocytosis. Moreover, we show that Scrib is essential in the pathway leading to Rac1 activation, PLD stimulation and secretion, suggesting that Scrib could define spatial landmarks in the plasma membrane where Rac1 activation is required for exocytosis to occur. This idea fits well with the observation that Rac1 stimulates the local production of PA at the plasma membrane and may therefore play a role in stages of exocytosis that occur after recruitment and docking of vesicles to the plasma membrane.

The functional interplay between Rac1 and ARF6 GTPases has been demonstrated in various cellular processes, including cell migration, neurite outgrowth and membrane trafficking (Albertinazzi et al., 2003; Cotton et al., 2007; Palacios and D'Souza-Schorey, 2003; Powner et al., 2002; Santy and Casanova, 2001). As we have previously demonstrated that ARF6 regulates exocytosis through the activation PLD1 (Vitale et al., 2002), the coordination of Rac1 and ARF6 during the secretory process is an unresolved issue. Further complicating the scheme, βPix has been described to be tightly associated with GIT1 (Premont et al., 2004), a GTPase-activating protein (GAP) that negatively regulates ARF6 activity and exocytosis in neuroendocrine cells (Meyer et al., 2006). Thus, in principle Rac1 activation could occur in the same time frame as ARF6 inactivation, leading to contradictory signals on PLD1. Obviously, several missing parameters preclude the emergence of a clear picture. First, the time course of Rac1 and ARF6 activation/inactivation in response to secretagogues is currently unknown. Second, the activity of GIT and βPix themselves, either as individual proteins or as a macromolecular complex remain unknown. Premont and collaborators have proposed that GIT and βPix constitute subunits of a large and stable macromolecular complex (Premont et al., 2004), whereas others have suggested that these two proteins function independently and can associate and dissociate in a regulated manner (Di Cesare et al., 2000; Feng et al., 2002; Zhao et al., 2000). Additionally, Scrib, which recruits the βPix/GIT complex in various tissues (Audebert et al., 2004; Meyer et al., 2006), may well act as a scaffolding protein-interacting module that spatially and temporally organize βPix and GIT1 activities at the sites of exocytosis. Hence, Scrib has been shown to organize assembly of signalling components in diverse processes including neurotransmission and synaptic plasticity (Harris and Lim, 2001; Hata et al., 1998). Clearly, further studies are now required to understand the regulation of the Scrib/βPix/GIT1 complex in stimulated neuroendocrine cells and how this complex tunes up the secretory machinery.

Endocrine tumours are often associated with a dysfunction of hormones, neurotransmitters or metabolite secretion (Gratzl et al., 2004). Rho GTPases are generally not mutated in the various human cancers. However, numerous studies have shown that it is the deregulation of Rho GTPase signalling (e.g. expression or activation of regulators and/or effectors) that plays a crucial role in the initiation and the progression of cancer (Ellenbroek and Collard, 2007; Vega and Ridley, 2008). Thus, uncovering the molecular pathways controlling the exocytotic steps that involve Rho proteins constitute a clear biological, pharmaceutical and therapeutic challenge. We describe here, for the first time, several steps of the signalling pathway linking Rac1 to Ca²⁺-regulated hormone release. An appealing feature is that all the actors identified in this pathway have been implicated in cancer. βPix, as well as many members of the Dbl family of guanine nucleotide exchange factors trigger cell transformation (Cerione and Zheng, 1996). Elevated PLD activity has been demonstrated in various tumourigenic processes, including cell proliferation, survival signalling or cell transformation (Foster, 2007; Shi et al., 2007). Finally, Scribble has been classified as a tumour suppressor according to its ability to regulate and maintain cell polarity (Humbert et al., 2003). Altogether, these observations make the Rac1 signal transduction pathway an attractive candidate for therapeutic intervention.

Plasmids encoding Flag-βPix, Flag-βPix_L238R-L239S, GFP-Scrib, GFP-ΔPDZ-Scrib, Yeast Spo20p PA-binding domain (wtPABD), siRNA against Cdc42, βPix, Scrib or PLD and the vectors pGHsuper and pEGFP-RNAi have been described previously (Audebert et al., 2004; Malacombe et al., 2006; Osmani et al., 2006; Zeniou-Meyer et al., 2007). Human Rac1 cDNA was a gift from M. Way (Cancer Research UK, London, UK) and was cloned into a N-terminal HA-tagged vector (kindly provided by H. W. Shin; Kyoto University, Japan) to generate HA-Rac1. The sequence of the Rac1 and RhoA siRNA, which are derived from the target transcript, were obtained from Ambion. The sequence of unrelated (UnR) siRNA was as reported previously (Randhawa et al., 2004). Rat DNA fragment encoding the unrelated siRNA sequence (ATTCTATCACTAGCGTGAC) and the siRNA sequences of Rac1 (CCATTTTGAACCAATGAAC) or RhoA (GAAGTCAAGCATTTCTGTC) separated from it reverse complement by a short spacer were annealed and cloned in the BglII and HindIII sites in front of the H1 promoter of either the pGHsuper vector or the pEGFP-N2-RNAi (Malacombe et al., 2006).

PC12 cells culture conditions were as described previously (Gasman et al., 2004). For immunofluorescence experiments and GH release assay, expression vectors were introduced into PC12 cells (24-well dishes, 1×10⁵ cells, 0.5 μg/well of each plasmid) using GenePorter (Gene Therapy Systems) on adherent cells according to the manufacturer's instructions. Under these conditions, the transfection efficiency reached only 10-30%. Plasmids were electroporated (Nucleofector Amaxa; 50 to 80% of transfection efficiency) for the PLD activity assay, for the Rac activation assay, for the saponin-mediated permeabilization experiments and for the production of lysates depleted through siRNA knockdown. Transfection efficiency was analyzed by flow cytometry using a FACSCalibur instrument (Becton-Dickinson, San Jose, CA, USA), CellQuest and Paint-a-gate software.

The activation of cellular Rac1 has been measured using a pull-down assay (Bagrodia et al., 1998) and/or an ELISA assay. For pull-down assay, PC12 cells (10 cm petri dish, 10⁶ cells) were immediately lysed in 500 μl ice-cold lysis buffer [25 mM HEPES (pH 7.5), 150 mM NaCl, 5 mM MgCl₂, 5 mM EDTA, 1% NP-40, 10% glycerol and mammalian protease inhibitor cocktail (Sigma)]. GTP-bound Rac was pulled down by incubating lysate containing equal amounts of proteins (500 μg) with the Rac/Cdc42-interacting domain (CRIB) domain of PAK1 (p21 activated kinase) for 2 hours at 4°C. Lysates loaded with guanosine 5′-O-(3-thio) triphosphate and GDP served, respectively, as positive and negative controls. The precipitated endogenous GTP-bound Rac1 or the expressed HA-tagged Rac1-GTP was resolved on 12% polyacrylamide-SDS gels and immunoblotted with antibodies specific for Rac1 and HA, respectively. For the ELISA Rac activation assay, PC12 cells (six-well dishes, 5×10⁵ cells) were immediately lysed after stimulation and GTP-bound Rac1 were measured in lysate containing equal amounts of proteins (170 μg) using the G-LISA Rac Activation Assay Kit (Cytoskeleton) according to the manufacturer's instructions.

GH release experiments were performed 48 or 72 hours after transfection. PC12 cells (24-well dishes, 1×10⁵ cells) were washed four times with 750 μl of 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 and 15 mM HEPES (pH 7.2)] and then incubated for 10 minutes in 500 μl of Locke's solution (Resting) or stimulated with 500 μl of elevated K⁺ solution (Locke's containing 59 mM KCl and 85 mM NaCl). The supernatant was collected, and the cells were harvested by scraping in 1 ml of 10 mM phosphate-buffered saline. The amounts of GH secreted into the medium and retained in the cells were measured in 200 μl samples using an ELISA assay (Roche). GH secretion is expressed as a percentage of total GH present in the cells before stimulation.

The following antibodies were used: monoclonal anti-Rac and anti-actin antibodies (Sigma); rabbit polyclonal anti-human GH antibodies (Dr A.F. Parlow, NIDDKs National Hormone and Pituitary Program, Torrance, CA); monoclonal anti-Cdc42 antibodies (BD Transduction Laboratories); goat polyclonal anti-Scrib and monoclonal anti-RhoA antibodies (Santa Cruz Biotechnology); rabbit polyclonal anti-βPix and anti-SNAP25 antibodies (Chemicon); monoclonal anti-Ha and anti-SNAP25 (Convance). Alexa-labelled secondary anti-mouse and anti-rabbit antibodies were obtained from Molecular Probes. Alexa-labelled secondary anti-goat antibodies were obtained from Santa Cruz.

Western blots were performed by chemiluminescence using the Super Signal West Dura Extended Duration Substrate system (Pierce). Immunoreactive bands were detected using the image acquisition system Chemi-smart 5000 and quantified using Bio-1D software (Vilber Lourmat).

For immunocytochemistry, PC12 cells grown on poly D-lysine-coated glass coverslips (24-well plates, 80% confluent) were fixed and immunostained as described previously (Gasman et al., 1998; Gasman et al., 2004). Stained cells were visualized using a confocal microscope LSM 510 (Carl Zeiss, Jena, Germany). Using the Zeiss CLSM instrument software 3.2, the proportion of PABD or Rac1 colocalized with SNAP25 was estimated from the double-labelled pixels, expressed as the average fluorescence intensity and calculated as a percentage of the total PABD or Rac1 fluorescence, respectively, detected in each cell.

PC12 cells grown on 24-well dishes (1×10⁵ cells) were washed twice with 750 μl of Ca²⁺-free Locke's solution and then incubated for 10 minutes in 500 μl of Ca²⁺-free Locke's solution or stimulated with of 500 μl elevated K⁺ solution. Medium was then replaced by 100 μl of an ice-cold Tris 50 mM (pH 8.0) solution and cells were broken by three freeze and thaw cycles. Samples were collected, mixed with an equal amount of the Amplex Red reaction buffer (Amplex Red Phospholipase D assay kit, Molecular Probes, USA) and PLD activity was estimated after 45 minutes incubation at 37°C with a Mithras (Berthold) fluorimeter using an excitation wavelength of 530 nm and a detection wavelength of 640 nm. A standard curve was performed with purified PLD from Streptomyces chromofuscus (Sigma). In order to estimate PLD activity in equal number of cells, the data are normalized according to the transfection efficiency. To do so, the PLD activity of the non-transfected cell population was subtracted and the PLD activity value from the transfected cells was multiplied by 1/transfection efficiency.

Resting and stimulated PC12 cells (six-well dishes, 5×10⁵ cells) were washed twice with 2 ml of Locke's solution and then permeabilized with 300 μl of 0.05 % saponin in Ca²⁺-free KG medium (resting: 150 mM potassium glutamate, 10 mM Pipes, pH 7.2, 5 mM nitrilotriacetic acid, 0.5 mM EGTA, 5 mM Mg²⁺-ATP, 4.5 mM magnesium acetate and mammalian protease inhibitor cocktail from Sigma) or in KG medium containing 20 μM free Ca²⁺ (stimulated: 150 mM potassium glutamate, 10 mM PIPES, pH 7.0, 5 mM nitrilotriacetic acid, 0.5 mM EGTA, 5 mM Mg²⁺-ATP, 3.75 mM magnesium acetate, 1.14 mM CaCl₂ and mammalian protease inhibitor cocktail from Sigma). After rocking on ice for 15 minutes, the supernatants were recovered and cleared by centrifugation. Membrane fractions of remaining cells were further solubilized with Triton X-100 to a final concentration of 0.5% and cleared by centrifugation. Endogenous βPix or Flag-tagged βPix were resolved on 4-12% polyacrylamide-SDS gels and detected on western blots with antibodies specific for βPix and Flag, respectively. When cells were treated without saponin, no proteins leaked out and βPix was entirely recovered in the Triton-X fraction (Total). The relative amount of βPix in the fractions was estimated by scanning densitometry and semi-quantitative analysis using Bio-1D software (Vilber Lourmat).

In all the figures, data are given as the mean values±s.d. obtained at least in three independent experiments performed on different cell cultures. n represents the number of experiments. Data were analyzed with Minitab statistical software. Statistical significance has been established using ANOVA test and data were considered significantly different when the P value was lower than 0.05. Gaussian distribution and variance equality of the data were verified.