The Gα12-RGS RhoGEF-RhoA signalling pathway regulates neurotransmitter release in C. elegans
2006; Springer Nature; Volume: 25; Issue: 24 Linguagem: Inglês
10.1038/sj.emboj.7601458
ISSN1460-2075
AutoresEmma Hiley, Rachel McMullan, Stephen Nurrish,
Tópico(s)Fungal and yeast genetics research
ResumoArticle30 November 2006free access The Gα12-RGS RhoGEF-RhoA signalling pathway regulates neurotransmitter release in C. elegans Emma Hiley Emma Hiley MRC Cell Biology Unit, MRC Laboratory for Molecular Cell Biology University College, London, UK Department of Pharmacology, University College, London, UK Search for more papers by this author Rachel McMullan Rachel McMullan MRC Cell Biology Unit, MRC Laboratory for Molecular Cell Biology University College, London, UK Department of Pharmacology, University College, London, UK Search for more papers by this author Stephen J Nurrish Corresponding Author Stephen J Nurrish MRC Cell Biology Unit, MRC Laboratory for Molecular Cell Biology University College, London, UK Department of Pharmacology, University College, London, UK Search for more papers by this author Emma Hiley Emma Hiley MRC Cell Biology Unit, MRC Laboratory for Molecular Cell Biology University College, London, UK Department of Pharmacology, University College, London, UK Search for more papers by this author Rachel McMullan Rachel McMullan MRC Cell Biology Unit, MRC Laboratory for Molecular Cell Biology University College, London, UK Department of Pharmacology, University College, London, UK Search for more papers by this author Stephen J Nurrish Corresponding Author Stephen J Nurrish MRC Cell Biology Unit, MRC Laboratory for Molecular Cell Biology University College, London, UK Department of Pharmacology, University College, London, UK Search for more papers by this author Author Information Emma Hiley1,2, Rachel McMullan1,2 and Stephen J Nurrish 1,2 1MRC Cell Biology Unit, MRC Laboratory for Molecular Cell Biology University College, London, UK 2Department of Pharmacology, University College, London, UK *Corresponding author. MRC Cell Biology Unit, MRC Laboratory for Molecular Cell Biology, University College, Gower Street, London WC1E 6BT, UK. Tel.: +44 207 679 7267; Fax: +44 207 679 7805; E-mail: [email protected] The EMBO Journal (2006)25:5884-5895https://doi.org/10.1038/sj.emboj.7601458 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info In Caenorhabditis elegans adults, the single Rho GTPase orthologue, RHO-1, stimulates neurotransmitter release at synapses. We show that one of the pathways acting upstream of RHO-1 in acetylcholine (ACh)-releasing motor neurons depends on Gα12 (GPA-12), which acts via the single C. elegans RGS RhoGEF (RHGF-1). Activated GPA-12 has the same effect as activated RHO-1, inducing the accumulation of diacylglycerol and the neuromodulator UNC-13 at release sites, and increased ACh release. We showed previously that RHO-1 stimulates ACh release by two separate pathways—one that requires UNC-13 and a second that does not. We show here that a non-DAG-binding-UNC-13 mutant that partially blocks increased ACh release by activated RHO-1 completely blocks increased ACh release by activated GPA-12. Thus, the upstream GPA-12/RHGF-1 pathway stimulates only a subset of RHO-1 downstream effectors, suggesting that either the RHO-1 effectors require different levels of activated RHO-1 for activation or there are two distinct pools of RHO-1 within C. elegans neurons. Introduction Rho family GTPases regulate numerous processes in eukaryotic cells, including organisation of the cytoskeleton, cell morphology and motility, gene expression, and neurotransmitter release (Jaffe and Hall, 2005; McMullan et al, 2006). They function as molecular switches and are active when GTP is bound and are inactive when GDP is bound. They are activated by specific guanine-nucleotide-exchange factors (GEFs), which stimulate the exchange of GTP for GDP (Schmidt and Hall, 2002). More than 85 RhoGEFs have been identified (Jaffe and Hall, 2005), all of which contain a Dbl homology (DH) domain that is responsible for the GDP–GTP exchange activity; most also contain a Pleckstrin homology (PH) domain and a variety of interaction domains that are implicated in signal transduction. We have recently shown that the single Caenorhabditis elegans Rho orthologue (RHO-1) acts in adult animals to regulate neurotransmitter release (McMullan et al, 2006). Expression of a constitutively active RHO-1 (G14V) in C. elegans cholinergic motor neurons stimulates acetylcholine (ACh) release; conversely, inhibition of endogenous RHO-1 via the Rho inhibitor C3 transferase reduces ACh release. RHO-1 regulates ACh release by at least two separate pathways. In one, presynaptic RHO-1 increases ACh release by stimulating the accumulation of diacylglycerol (DAG) and the DAG-binding protein UNC-13 at sites of neurotransmitter release; this pathway is blocked by UNC-13 mutants unable to bind DAG. A second UNC-13-independent pathway is revealed by a RHO-1 mutant unable to increase DAG levels; this RHO-1 mutant protein retains the ability to stimulate ACh release by a mechanism that is not blocked by the non-DAG-binding-UNC-13 mutant. The heterotrimeric G proteins Gq, G12, and G13 have been shown to mediate signals from G protein-coupled receptors (GPCRs) to Rho GTPases. These G proteins are believed to activate directly RhoGEFs that contain a Regulator of G protein Signalling (RGS) domain: p115 RhoGEF, for example, binds activated Gα12 or Gα13 through its RGS domain, and Gα13 binding stimulates p115 RhoGEF activity (Hart et al, 1998; Kozasa et al, 1998). Mutations in either Drosophila Gα12 or p115 RhoGEF homologues (concertina and DRhoGEF2) disrupt gastrulation, suggesting that these proteins may act in the same signalling pathway (Parks and Wieschaus, 1991; Barrett et al, 1997; Hacker and Perrimon, 1998). In C. elegans, the single Gα12 protein (GPA-12) has been implicated in the control of pharyngeal pumping (van der Linden et al, 2003). Both GPA-12 and the single RGS RhoGEF, RHGF-1, are coexpressed in the C. elegans nervous system, although GPA-12 is more strongly expressed in cells where RHGF-1 is absent, including the hypodermis, muscle, intestinal cells, and pharynx (van der Linden et al, 2003; Yau et al, 2003). To understand how RHO-1 is regulated in the adult C.elegans nervous system, we have examined the role of the GPA-12/RHGF-1 signalling pathway in neurotransmitter release in motor neurons. We show that GPA-12 stimulates ACh release by a pathway that depends on RHGF-1, RHO-1, and UNC-13. Results GPA-12 stimulates ACh release in a RHO-1-dependent manner To test for a role of GPA-12 in the control of ACh release, we used a heat shock-inducible, constitutively active Gα12 transgene (hs∷GPA-12 (Q205L)) constructed by van der Linden et al (2003) (Figure 1A). ACh release was quantified by measuring the time course of paralysis induced by the ACh esterase inhibitor aldicarb (Nonet et al, 1993; Nguyen et al, 1995; Miller et al, 1996; Nurrish et al, 1999). Aldicarb blocks removal of endogenously released ACh, resulting in hypercontraction of the muscles and paralysis. Increases in ACh release cause animals to become paralysed faster with aldicarb treatment; conversely, animals with a decrease in ACh release become paralysed more slowly and continue to move at time points when wild-type animals are completely paralysed. Heat shock-induced expression of constitutively active GPA-12 (Q205L) in adults increased aldicarb sensitivity both immediately and 24 h post-heat shock compared to controls, suggesting that GPA-12 increased ACh release (Figure 1B). Even without heat shock, the hs∷GPA-12 (Q205L) animals were slightly hypersensitive to aldicarb, probably due to a leakiness of the heat shock promoter (Figure 1B). Expression of either constitutively active GPA-12 (Q205L) or constitutively active RHO-1 (G14V) (McMullan et al, 2006) mutants resulted in aldicarb hypersensitivity, consistent with a role for GPA-12 in activating RHO-1. Figure 1.A constitutively active GPA-12 (Q205L) acts within motor neurons to increase ACh release upstream of RHO-1. (A) In C. elegans GPA-12 is the single Gα12 orthologue, and RHGF-1 is the single RGS-containing RhoGEF, see text for abbreviations. To investigate the role of GPA-12 and RHGF-1 on ACh release, we tested pk322 and ok880 deletion mutants, respectively (deletions indicated by dotted lines): pk322 removes almost the entire coding region of gpa-12 and is likely a null mutant, ok880 is an in-frame deletion that removes the N-terminal part of the DH RhoGEF domain (residues 599–804). Another mutation rhgf-1(gk217) is an in-frame deletion that removes the RHGF-1 C1 domain. The pkIs1330 integrated transgene (van der Linden et al, 2003) expresses the constitutively active GPA-12 (Q205L) mutant from a heat shock promoter. (B–D) Levels of ACh release were assessed by testing for rates of paralysis on the ACh esterase inhibitor aldicarb, animals with increased rates of ACh release paralyse faster on aldicarb. Dashed lines indicate heat shocked animals. Mean % of paralysed animals are shown. Error bars indicate s.e.m., all assays were performed at least five times. (B) The hs∷ GPA-12 (Q205L) transgene (hs∷GPA-12*) paralysed faster on aldicarb than wild-type animals (wt) even in the absence of heat shock (−hs). Rates of paralysis were increased both immediately (+hs) and 24 h (+hs 24 h) post-heat shock. (C) gpa-12(pk322) and rhgf-1(ok880) mutant animals were slightly aldicarb hypersensitive immediately after heat shock. Neither altered aldicarb hypersensitivity caused by heat shock expression of constitutively active RHO-1 (G14V). (D) Aldicarb hypersensitivity caused by heat shock expression of GPA-12 (Q205L) (hs∷GPA-12*+hs) was blocked by inhibition of endogenous RHO-1 by the specific Rho inhibitor C3 transferase expressed from a heat shock promoter (hs∷GPA-12*; hs∷C3T+hs). (E) Expression of the constitutively active GPA-12 (Q205L) in cholinergic neurons using the unc-17 promoter (N∷GPA-12*) caused aldicarb hypersensitivity that was suppressed by the rhgf-1(ok880) mutation (N∷GPA-12*;rhgf-1(ok880)). Mutations in both gpa-12(pk322) and rhgf-1(ok880) did not change the response to aldicarb. Download figure Download PowerPoint Two results suggested that GPA-12 was acting upstream of RHO-1. First, aldicarb hypersensitivity of animals expressing constitutively active RHO-1 (G14V) was not altered by the presence of the null gpa-12(pk322) mutation (Figure 1A and C). Second, inhibition of endogenous RHO-1 by C3 transferase almost completely suppressed the increase in aldicarb hypersensitivity caused by expression of the constitutively active GPA-12 (Q205L) (Figure 1D). The gpa-12 (pk322) mutation alone had no effect on the sensitivity to aldicarb in the absence of heat shock (Figure 1E) and was very slightly hypersensitive to aldicarb in the presence of heat shock (Figure 1C), suggesting that GPA-12 signalling is not required for standard rates of ACh release under laboratory conditions. To test whether GPA-12 had a role in motor neuron development, we labelled cholinergic motor neurons using GFP expressed from the acr-2 promoter. We observed no gross morphological differences in neuronal development between heat shocked wild-type and hs∷GPA-12 (Q205L) animals both immediately and 24 h after heat shock (Figure 2A–C). Figure 2.Expression of GPA-12 (Q205L) from the acr-2 promoter changes gross neuronal morphology. (A–G) Soluble GFP was expressed in the cholinergic motor neurons from the acr-2 promoter. In most cases, the anterior side of the animal is towards the upper left of the picture, in (B) anterior is top, and in (F) anterior is the leftmost part of the animal. Arrowheads indicate the ventral nerve cord. Motor neuron morphology was the same immediately after heat shock in wild-type (A) and in hs∷GPA-12 (Q205L) animals immediately (B), and 24 h after heat shock (C). GPA-12 (Q205L) expressed in cholinergic cells from the unc-17 promoter (unc-17∷GPA-12*) did not alter gross morphology of the motor neurons (D). Expression of GPA-12 (Q205L) from the p.acr-2 promoter (acr-2∷GPA-12*) did cause a pathfinding defect (indicated by an arrow) (E) and this was not suppressed by the rhgf-1(ok880) mutation (F). Expression of rhgf-1 cDNA from the acr-2 promoter injected at 10 ng/μl (G) or 100 ng/μl (H) in hs∷GPA-12 (Q205L); rhgf-1(ok880) animals did not alter gross neuronal morphology. Download figure Download PowerPoint Gα12 acts presynaptically to stimulate ACh release Although the hs∷GPA-12 (Q205L) transgene is expressed in the cholinergic neurons, it is also expressed in many other cells, including muscle cells. We tested whether the transgene was acting presynaptically in two ways. First, we expressed the constitutively active GPA-12 (Q205L) specifically in cholinergic cells using the promoter for the unc-17 vesicular ACh transporter (p.unc-17) and showed it caused aldicarb hypersensitivity, albeit with a weaker effect than the hs∷GPA-12 (Q205L) transgene (Figure 1E). Secondly, we tested the sensitivity of the muscles to ACh release in heat shock-induced transgenic animals expressing GPA-12 (Q205L). The drug levamisole activates the nicotinic ACh receptors in the muscle causing animals to become paralysed at a rate dependent on muscle sensitivity to ACh (Nurrish et al, 1999). The heat shocked animals showed no significant increase in levamisole sensitivity compared to controls, suggesting that the heat shock expression of GPA-12 (Q205L) did not alter muscle response to ACh (Supplementary Figure 1A and B). Thus, GPA-12, like RHO-1, can act presynaptically to increase ACh release. We also expressed constitutively active GPA-12 (Q205L) specifically in the cholinergic motor neurons using the acr-2 promoter. This transgene, however, caused neuronal pathfinding defects (Figure 2E) and, therefore, could not be used to assay for aldicarb sensitivity. By contrast, expression of GPA-12 (Q205L) from the unc-17 promoter did not cause a detectable neurite pathfinding defect (Figure 2D). RHGF-1 is required for GPA-12-mediated ACh release RHGF-1 is the single RhoGEF containing a G protein-regulated RGS domain in C. elegans. RHGF-1 binds to GPA-12 (Yau et al, 2003), and is thus a strong candidate for mediating GPA-12's ability to stimulate RHO-1. We obtained a full-length SL1 spliced cDNA for rhgf-1, which encodes a protein that contains five conserved domains-PDZ (PSD-95/Dlg/ZO-1), RGS, C1 (PKC homology domain 1), DH, and PH (Figure 1A). We received a mutant strain with a deletion in rhgf-1(ok880), from which we isolated cDNA and confirmed the presence of an in-frame deletion that removes amino acids 599–804 (Figure 1A). The predicted truncated protein lacks the first 58 residues of the DH domain, which is required for RhoGEF activity in mammalian RhoGEFs (Kristelly et al, 2004). A second allele rhgf-1(gk217) has an in-frame deletion that removes the C1 domain (Figure 1A), leaving the other conserved domains. Strains carrying this mutation appear to have at least one other closely linked mutation that decreases the rate of paralysis induced by aldicarb (see Materials and methods), and so we were unable to test this allele. The responses of rhgf-1(ok880) mutants in the aldicarb and levamisole assays were indistinguishable from those of wild type in the absence of heat shock, but were very slightly aldicarb hypersensitive in the presence of heat shock (Figure 1C and E and Supplementary Figure 1A and B). However, rhgf-1(ok880) significantly reduced aldicarb hypersensitivity caused by the hs∷GPA-12 (Q205L) transgene (Figure 3A and B), and it completely suppressed the aldicarb hypersensitivity of animals expressing GPA-12 (Q205L) in cholinergic neurons (from the unc-17 promoter) (Figure 1E), suggesting that GPA-12 (Q205L)-mediated increases in ACh release require RHGF-1. Figure 3.GPA-12 (Q205L) stimulation of ACh release requires RHGF-1, DAG, and UNC-13. (A, B) The RGS RhoGEF mutation (rhgf-1(ok880)) completely suppressed aldicarb hypersensitivity caused by the hs∷GPA-12 (Q205L) transgene (hs∷GPA-12*;rhgf-1(ok880)) in the absence of heat shock (A) and strongly suppresses in the presence of heat shock (B). In hs∷GPA-12 (Q205L); rhgf-1(ok880) animals, expression of RHGF-1 from the cholinergic motor neuron-specific acr-2 promoter (N∷RHGF-1) at low copy number (injected at 10 ng/μl, hs∷GPA-12*;rhgf-1(ok880);N∷RHGF-1) had no effect on the response of animals to aldicarb in the absence of heat shock (A), but restored aldicarb hypersensitivity caused by heat-shock-induced expression of GPA-12 (Q205L) (B). (C) A transgene with a 10-fold increase in p.acr-2∷RHGF-1 (injected at 100 ng/μl N∷RHGF-1 × 10) caused aldicarb hypersensitivity in hs∷GPA-12*;rhgf-1(ok880) animals without heat shock (−hs). Aldicarb hypersensitivity was decreased slightly upon heat-shock-induced expression of GPA-12 (Q205L) (+hs) and much more strongly by removal of all GPA-12 using the gpa-12(pk322) mutation. (D) Inhibition of endogenous RHO-1 by heat shock expression of C3 transferase (hs∷C3T +hs) blocked aldicarb hypersensitivity caused by N∷RHGF-1 × 10 (compare rhgf-1(ok880);N∷RHGF-1 × 10;hsC3T in the absence (−hs) or presence (+hs) of C3 transferase expression). (E, F) Replacement of endogenous UNC-13 by a mutant UNC-13S unable to bind diacylglycerol (DAG) (unc-13(s69); UNC-13S (H173K)) caused aldicarb resistance (E) that is not altered by heat shock expression of constitutively active GPA-12 (Q205L) (F). Download figure Download PowerPoint RHGF-1 acts within the cholinergic motor neurons upstream of RHO-1 To determine the site of action of RHGF-1, we rescued the rhgf-1(ok880) mutants using full-length rhgf-1 cDNA expressed from the cholinergic-motor-neuron-specific acr-2 promoter (N∷RHGF-1). This transgene restored aldicarb hypersensitivity induced by heat shock of hs∷GPA-12 (Q205L) in rhgf-1(ok880) animals (Figure 3B), but not in the absence of heat shock (Figure 3A). Transgenic animals are created by injecting DNA into the gonads of C. elegans and looking for stable transmission of the DNA in the progeny (Mello and Fire, 1995). N∷RHGF-1 transgenics were created by injecting either 10 ng/μl (N∷RHGF-1) or 100 ng/μl (N∷RHGF-1 × 10). The N∷RHGF-1 × 10 transgene caused aldicarb hypersensitivity in hs∷GPA-12 (Q205L); rhgf-1(ok880) mutants in the absence of heat shock (Figure 3C). Surprisingly, aldicarb hypersensitivity was slightly decreased upon heat shock-induced expression of the constitutively active GPA-12 (Q205L) (Figure 3C). Removal of all GPA-12 activity using null gpa-12(pk322) animals also reduced the aldicarb hypersensitivity associated with the N∷RHGF-1 × 10 transgene, although not back to wild-type levels (Figure 3C). Inhibition of endogenous RHO-1 by heat shock expression of C3 transferase almost completely suppressed the aldicarb hypersensitivity of N∷RHGF-1 × 10 to levels observed with expression of C3 transferase alone, consistent with a role for RHGF-1 upstream of RHO-1 (Figure 3D). Gross neuronal morphology of transgenic animals expressing both low levels and high levels of RHGF-1 were normal (Figure 2G and H). GPA-12 increases ACh release at existing synapses GPA-12 could be stimulating an increase in ACh release through either an increase in the number of ACh-releasing synapses, an increase in the release of ACh from existing synapses, or both. To test the first possibility, we measured the number of synapses in hs∷GPA-12 (Q205L) animals using CFP-labelled synaptobrevin (SNB-1∷CFP) expressed specifically in cholinergic motor neurons using the acr-2 promoter. Synaptobrevin is an integral membrane protein enriched in synaptic vesicles and thus acts as a marker for release sites. We observed no significant difference in the number of SNB-1∷CFP puncta before heat shock or immediately and 24 h post-heat shock in either wild-type (2.48±0.11 versus 2.51±0.10 and 2.38±0.11 puncta per 10 μm, all errors are s.e.m.) or hs∷GPA-12 (Q205L) animals (2.49±0.09 versus 2.56±0.11 and 2.3±0.11 puncta per 10 μm) (Figure 4A and B). Thus, heat shock induction of GPA-12 (Q205L) in adults did not increase the number of release sites in cholinergic motor neurons, suggesting that constitutively active GPA-12 (Q205L) increases ACh release at pre-existing release sites. Mutation of rhgf-1, either in the presence or absence of heat shock-induced GPA-12 (Q205L) also failed to change the number of SNB-1∷CFP puncta (2.58±0.10 versus 2.54±0.09 puncta per 10 μm, Figure 4B), suggesting that the rhgf-1(ok880) mutation does not suppress GPA-12 (Q205L) by reducing the number of ACh release sites. We also observed no change in SNB-1∷CFP puncta numbers in gpa-12(pk322) mutants (2.5±0.1 puncta per 10 μm, Figure 4B). The rhgf-1(ok880) mutants did have a decrease in the number of release sites in the dorsal cord (2.27±0.06 puncta per 10 μm, Figure 4), but this just failed to reach a statistically significant difference from the non-heat-shocked wild type (P=0.051 in a two-tailed t-test). In all mutant and transgenic animals tested, there was no significant difference from wild type in average size (non-heat-shocked wild type puncta size 0.7 μm2±0.05) (Figure 4C) or fluorescence (Figure 4D) of the SNB-1 puncta. The only notable difference was a much increased variation in SNB-1 puncta size in hs∷GPA-12 (Q205L) animals immediately, but not 24 h, post-heat shock. Figure 4.hs∷GPA-12 (Q205L) does not increase the number, size, or fluorescence of SNB-1∷CFP puncta. (A) Dorsal cord neuromuscular junctions were labelled by expressing a CFP-tagged version of synaptobrevin (SNB-1∷CFP, which is enriched in synaptic vesicles) in cholinergic motor neurons (using the acr-2 promoter). Animals with an integrated array expressing the constitutively active GPA-12 (Q205L) from a heat shock promoter (hs∷GPA-12 (Q205L)) either without heat shock (top panel) or with heat shock (lower panel) had the same density of SNB-1∷CFP puncta. (B–D) Numbers (B), average size (C), and average fluorescence (D) of SNB-1∷CFP puncta in dorsal nerve cords were measured using Image J for animals with the indicated genotypes and heat shock conditions (see Materials and methods). Bars are means±s.e.m., numbers of animals counted are given in brackets. Statistical analysis was performed using a two-tailed t-test and no significant differences were detected (P>0.05). Download figure Download PowerPoint GPA-12 increases UNC-13 levels at release sites via RHGF-1 We have previously shown that RHO-1 acts via two pathways in motor neurons to increase ACh release: one dependent on the DAG-binding neuromodulator UNC-13 and the other UNC-13 independent (McMullan et al, 2006). In the UNC-13 dependent pathway, RHO-1 increases the short form of UNC-13 (UNC-13S) at release sites via a spatially restricted increase in DAG levels. The enrichment of UNC-13 at release sites has been shown to correlate with increased neurotransmitter release (Betz et al, 1998; Lackner et al, 1999; Nurrish et al, 1999). Expression of constitutively active RHO-1 (G14V) in cholinergic motor neurons causes an UNC-13S∷YFP fusion protein to localise to punctate structures that colocalise with the SNB-1∷CFP marker for synaptic vesicles (McMullan et al, 2006). Both immediately and 24 h after heat shock expression of constitutively active GPA-12 (Q205L), the time points we observed aldicarb hypersensitivity, UNC-13S∷YFP in the dorsal nerve cord was localised into punctate structures, whereas it was not so localised in non-heat-shocked animals (1.84±0.11 pre-heat shock versus 2.29±0.12 and 2.2±0.14 versus puncta per 10 μm, using two-tailed t-test P=0.036 between before and immediately after heat shock) (Figure 5A–D and I), as we previously observed for RHO-1 (G14V) (McMullan et al, 2006). These UNC-13S∷YFP puncta colocalise with the release-site marker SNB-1∷CFP, confirming that GPA-12 (Q205L) causes UNC13S to localize to sites of neurotransmitter release (Figure 5H). In the rhgf-1(ok880) mutant animals, UNC-13S∷YFP fails to become punctate in response to heat-shock-induced GPA-12 (Q205L) expression (1.97±0.09 versus 1.86±0.11 puncta per 10 μm) (Figure 5E, F and I). Thus, rhgf-1(ok880) blocks both GPA-12 (Q205L)-mediated increases in ACh release and relocalisation of UNC-13S∷YFP, suggesting that GPA-12 (Q205L) stimulates ACh release via changes in UNC-13 localisation in a RHGF-1-dependent manner. Neither the gpa-12(pk322) nor the rhgf-1(ok880) mutant altered UNC-13∷YFP puncta density in the dorsal cord (Figure 5I). Figure 5.GPA-12 (Q205L) regulates the distribution of UNC-13S∷YFP in the dorsal nerve cord. UNC-13∷YFP and SNB-1∷CFP transgenes and image analysis were as described previously (McMullan et al, 2006). In (A–G), digital images were converted from greyscale into a 32-colour look-up table (Image J) to visualize pixel intensities. (A–F) UNC-13S∷YFP (expressed from the internal unc-13S promoter) was diffusely distributed in the dorsal nerve cord axons of untreated (A) and heat shocked (B) wild-type animals as well as non-heat-shocked animals containing the hs∷GPA-12 (Q205L) array (hs∷GPA-12*) (C). UNC-13S∷YFP became more punctate (as indicated by arrows) in the dorsal cords of hs∷GPA-12 (Q205L) (hs∷GPA-12*) animals immediately after heat shock (D). However, a mutation in the single RGS RhoGEF, rhgf-1(ok880) (E, F), blocked this increase, compare (D) with (F). (G) Unlike the wild-type protein, UNC-13S (H173K)∷GFP, which is predicted not to bind to DAG, remains diffusely distributed in heat shocked hs∷GPA-12 (Q205L) (hs∷GPA-12*) animals. (H) CFP-tagged synaptobrevin expressed from the acr-2 promoter (top), and YFP-tagged UNC-13S expressed from the unc-13s promoter (middle) were simultaneously visualized in the dorsal cord of heat shocked hs∷GPA-12 (Q205L) animal. In the merged image (bottom), the SNB-1∷CFP puncta colocalise with UNC-13S∷YFP puncta. (I) Numbers of either UNC-13S∷YFP or non-DAG-binding UNC-13S (H173K)∷GFP puncta in dorsal nerve cords were counted in the indicated genotypes and heat shock conditions (see Materials and methods). Bars are means±s.e.m., numbers of animals counted are given in brackets. Significance was determined by two-tailed t-test. *Significant difference (P<0.05). Download figure Download PowerPoint The GPA-12/RHGF-1 pathway activates a subset of RHO-1 effectors Replacement of wild-type UNC-13 by the non-DAG-binding UNC-13S (H173K) mutant causes resistance to aldicarb (Lackner et al, 1999) (Figure 3E). Constitutively active RHO-1 (G14V) causes aldicarb hypersensitivity, an effect that is only partially suppressed by UNC-13S (H173K), demonstrating the existence of both UNC-13-dependent and UNC-13-independent pathways downstream of RHO-1 (McMullan et al, 2006). In contrast to constitutively active RHO-1, the aldicarb hypersensitivity of animals expressing constitutively active GPA-12 (Q205L) was completely suppressed by UNC-13S (H173K) (Figure 3E and F). Thus, GPA-12-mediated aldicarb hypersensitivity requires binding of UNC-13 to DAG, whereas its downstream effector RHO-1 can act via both UNC-13-dependent and UNC-13-independent pathways. Heat shock expression of the constitutively active GPA-12 (Q205L) did not significantly alter the localisation of the non-DAG-binding UNC-13S (H173K)∷GFP (1.33±0.19 versus 1.53±0.14 puncta per 10 μm. P=0.55, two-tailed student's t-test) (Figure 5 G and I), suggesting that GPA-12 (Q205L) alters UNC-13S localization via a spatially restricted increase in DAG levels at sites of neurotransmitter release. There appeared to be a difference in numbers of UNC-13S (H173K)∷GFP puncta between heat shocked wild-type animals and heat shocked hs∷GPA-12 (Q205L) transgenic animals (1.16±0.19 versus 1.53±0.14) (Figure 5I); this was not statistically significant (P=0.07 two-tailed t-test), however. Constitutively active GPA-12 (Q205L) causes defects in development and pharyngeal pumping independent of RHGF-1 Previous work has shown that the constitutively active GPA-12 (Q205L) causes developmental growth arrest, which is secondary to decreased pharyngeal pumping. It is possible that the aldicarb hypersensitivity and changes in UNC-13 localization caused by GPA-12 (Q205L) is also secondary to reduced pharyngeal pumping. However, both the growth arrest and decreased pharyngeal pumping associated with GPA-12 (Q205L) were suppressed by mutations in the tpa-1 gene, whereas aldicarb hypersensitivity was not suppressed by tpa-1 (Supplementary Figure 2). We have repeated the experiments of van der Linden et al (2003) and confirmed the ability of tpa-1 mutations to suppress growth and pharyngeal pumping defects caused by GPA-12 (Q205L) expression (Figure 6A and B). The rhgf-1(ok880) mutation, which suppressed GPA-12 (Q205L) aldicarb hypersensitivity, failed to suppress the pharyngeal pumping and growth defects caused by expression of GPA-12 (Q205L) (Figure 6A and B). Thus, the hs∷GPA-12 (Q205L);rhgf-1(ok880) animals have reduced pharyngeal pumping but an almost normal response to aldicarb, indicating that changes in aldicarb sensitivity associated with expression of constitutively active GPA-12 (Q205L) appear to be independent of changes in pharyngeal pumping. The rhgf-1(ok880) mutation also failed to suppress the neuronal pathfinding defect caused by expression of the constitutively active GPA-12 (Q205L) from the acr-2 promoter (Figure 2F). Figure 6.TPA-1 and RHGF-1 define at least two different GPA-12 signalling pathways regulating distinct behaviours. (A) Heat shock expression of the constitutively active GPA-12 (Q205L) severely reduced pharyngeal pumping, and this was suppressed by a PKC mutation tpa-1(pk1585) (van der Linden et al, 2003), whereas the rhgf-1(ok880) mutation has no effect. (B) Twenty L1-stage animals were placed on plates, immediately heat shocked, and photographed 4 days lat
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