The SEK-1 p38 MAP Kinase Pathway Modulates Gq Signaling in Caenorhabditis elegans

Gq is a heterotrimeric G protein that is widely expressed in neurons and regulates neuronal activity. To identify pathways regulating neuronal Gq signaling, we performed a forward genetic screen in Caenorhabditis elegans for suppressors of activated Gq. One of the suppressors is an allele of sek-1, which encodes a mitogen-activated protein kinase kinase (MAPKK) in the p38 MAPK pathway. Here, we show that sek-1 mutants have a slow locomotion rate and that sek-1 acts in acetylcholine neurons to modulate both locomotion rate and Gq signaling. Furthermore, we find that sek-1 acts in mature neurons to modulate locomotion. Using genetic and behavioral approaches, we demonstrate that other components of the p38 MAPK pathway also play a positive role in modulating locomotion and Gq signaling. Finally, we find that mutants in the SEK-1 p38 MAPK pathway partially suppress an activated mutant of the sodium leak channel, NCA-1/NALCN, a downstream target of Gq signaling. Our results suggest that the SEK-1 p38 pathway may modulate the output of Gq signaling through NCA-1(unc-77).

Gq is a widely expressed heterotrimeric G protein that regulates a variety of biological processes, ranging from neurotransmission to cardiovascular pathophysiology (Sánchez-Fernández et al. 2014). In the canonical Gq pathway, Gq activates phospholipase Cb (PLCb), which cleaves phosphatidylinositol 4,5-bisphosphate into the second messengers diacylglycerol (DAG) and inositol trisphosphate (Rhee 2001). In addition to PLCb, other Gq effectors have been identified including kinases, such as protein kinase Cz and Bruton's tyrosine kinase (Btk) (Bence et al. 1997;García-Hoz et al. 2010;Vaqué et al. 2013), and guanine nucleotide exchange factors (GEFs) for the small GTPase Rho, such as Trio (Williams et al. 2007;Vaqué et al. 2013). These noncanonical effectors bridge the activation of Gq to other cellular signaling cascades.
In order to study noncanonical pathways downstream of Gq, we used the nematode Caenorhabditis elegans, which has a single Gaq homolog  and conservation of the other components of the Gq signal-ing pathway (Koelle 2016). In neurons, EGL-30 signals through EGL-8 (PLCb) (Lackner et al. 1999) and UNC-73 (ortholog of Trio RhoGEF) (Williams et al. 2007). UNC-73 activates RHO-1 (ortholog of RhoA), which has been shown to enhance neurotransmitter release through both diacylglycerol kinase (DGK-1)-dependent and -independent pathways (McMullan et al. 2006).
To identify additional signaling pathways that modulate Gq signaling, we screened for suppressors of the activated Gq mutant, egl-30(tg26) (Doi and Iwasaki 2002). egl-30(tg26) mutant animals exhibit hyperactive locomotion and a "loopy" posture, in which worms have exaggerated, deep body bends and loop onto themselves (Bastiani et al. 2003;Topalidou et al. 2017). Here, we identify one of the suppressors as a deletion allele in the gene sek-1. SEK-1 is a mitogen-activated protein kinase kinase (MAPKK), the C. elegans ortholog of mammalian MKK3/ 6 in the p38 MAPK pathway (Tanaka-Hino et al. 2002). The p38 MAPK pathway has been best characterized as a pathway activated by a variety of cellular stresses and inflammatory cytokines (Kyriakis and Avruch 2012). However, the p38 MAPK pathway has also been shown to be activated downstream of a G protein-coupled receptor in rat neurons (Huang et al. 2004). Btk, a member of the Tec family of tyrosine kinases, has been shown to act downstream of Gq to activate the p38 MAPK pathway (Bence et al. 1997), but C. elegans lacks Btk and other Tec family members (Plowman et al. 1999).

MATERIALS AND METHODS
C. elegans strains and maintenance All strains were cultured using standard methods and maintained at 20° ( Brenner 1974). The sek-1(yak42) mutant was isolated from an ENU mutagenesis suppressor screen of the activated Gq mutant, egl-30(tg26) (Ailion et al. 2014). sek-1(yak42) was outcrossed away from egl-30(tg26) before further analysis. Double mutant strains were constructed using standard methods (Fay 2006), often with linked fluorescence markers (Frokjaer-Jensen et al. 2014) to balance mutations with subtle visible phenotypes. Supplemental Material, Table S1 in File S1 contains all of the strains used in this study.
Mapping yak42 was mapped using its slow locomotion phenotype and its egl-30(tg26) suppression phenotype. yak42 was initially mapped to the X chromosome using strains EG1000 and EG1020, which carry visible marker mutations. These experiments showed that yak42 was linked to lon-2, but it was at least several map units (cM) away. yak42 was further mapped to $1 cM away from the red fluorescence insertion marker oxTi668, which is located at +0.19 cM on the X chromosome.
Whole-genome sequencing Strain XZ1233 egl-30(tg26); yak42 was used for whole-genome sequencing to identify candidate yak42 mutations. XZ1233 was constructed by crossing a yak42 strain outcrossed two times, back to egl-30(tg26). Thus, in XZ1233, yak42 has been outcrossed three times from its original isolate. DNA was isolated from XZ1233 and purified according to the Hobert Laboratory protocol (http://hobertlab. org/whole-genome-sequencing/). Ion torrent sequencing was performed at the University of Utah DNA Sequencing Core Facility. The resulting data contained 10,063,209 reads of a mean read length of 144 bases, resulting in $14· average coverage of the C. elegans genome. The sequencing data were uploaded to the Galaxy web platform, and we used the public server at usegalaxy.org to analyze the data (Afgan et al. 2016). We identified and annotated variants with the Unified Genotyper and SnpEff tools, respectively (DePristo et al. 2011;Cingolani et al. 2012). We filtered out variants found in other strains we sequenced, leaving us with 605 homozygous mutations. The X chromosome contained 94 mutations: 55 SNPs and 39 indels. Of these, four SNPs were nonsynonymous mutations in protein-coding genes, but only two were within 5 cM of oxTi668. However, we were unable to identify yak42 from the candidate polymorphisms located near oxTi668. Transgenic expression of the most promising candidate pcyt-1 did not rescue yak42. Instead, to identify possible deletions, we scrolled through 2 MB of aligned reads on the UCSC Genome Browser, starting at 24.38 cM and moving toward the middle of the chromosome (0 cM), looking for regions that lacked sequence coverage. We found a 3713-bp deletion that was subsequently confirmed to be the yak42 causal mutation, affecting the gene sek-1 located at 21.14 cM.

Locomotion assays
Locomotion assay plates were made by seeding 10 cm nematode growth medium plates with 150 ml of an Escherichia coli OP50 stock culture, spread with sterile glass beads to cover the entire plate. Bacterial lawns were grown at room temperature (22.5-24.5°) for 24 hr, and then stored at 4°until needed. All locomotion assays were performed on first-day adults at room temperature (22.5-24.5°). L4 stage larvae were picked the day before the assay and the experimenter was blind to the genotypes of the strains assayed. For experiments on strains carrying extrachromosomal arrays, the sek-1(km4) control worms were animals from the same plate that had lost the array.
Body bend assays were performed as described (Miller et al. 1999). A single animal was picked to the assay plate, the plate lid was returned, and the animal was allowed to recover for 30 sec. Body bends were then counted for 1 min, counting each time the worm's tail reached the minimum or maximum amplitude of the sine wave. All strains in an experiment were assayed on the same assay plate. For experiments with egl-8, unc-73, and rund-1 mutants, worms were allowed a minimal recovery period (until the worms started moving forward; 5 sec maximum) prior to counting body bends.
For the heat-shock experiment, plates of first-day adults were parafilmed and heat-shocked in a 34°water bath for 1 hr. Plates were then unparafilmed and incubated at 20°for 5 hr before performing body bend assays.
Radial locomotion assays were performed by picking animals to the middle of an assay plate. Assay plates were incubated at 20°for 20 hr and the distances of the worms from the starting point were measured.
Quantitative analysis of the waveform of worm tracks was performed as described (Topalidou et al. 2017). Briefly, worm tracks were photographed and ImageJ was used to measure the period and amplitude. The value for each animal was the average of five period-to-amplitude ratios.

C. elegans pictures
Pictures of worms were taken at 60· magnification on a Nikon SMZ18 microscope with the DS-L3 camera control system. The worms were age-matched as first-day adults and each experiment set was photographed on the same locomotion assay plate prepared as described above. The images were processed using ImageJ and were rotated, cropped, and converted to grayscale.

Molecular biology
Plasmids were constructed using the Gateway cloning system (Invitrogen). Plasmids and primers used are found in Table S2 in File S1. The sek-1 cDNA was amplified by RT-PCR from worm RNA and cloned into a Gateway entry vector. To ensure proper expression of sek-1, an operon GFP was included in expression constructs with the following template: (promoter)p::sek-1(cDNA)::tbb-2utr::gpd-2 operon::GFP::H2B:cye-1utr (Frøkjaer-Jensen et al. 2012). This resulted in untagged SEK-1, but expression could be monitored by GFP expression.

Injections
C. elegans strains with extrachromosomal arrays were generated by standard methods (Mello et al. 1991). Injection mixes were made with a final total concentration of 100 ng/ml DNA. Constructs were injected at 5 ng/ml, injection markers at 5 ng/ml, and the carrier DNA Litmus 38i at 90 ng/ml. Multiple lines of animals carrying extrachromosomal arrays were isolated and had similar behaviors, as observed by eye. The line with the highest transmittance of the array was assayed.

Statistical analyses
At the beginning of the project, a power study was conducted on pilot body bend assays using wild-type and sek-1(yak42) worms. To achieve a power of 0.95, it was calculated that 17 animals should be assayed per experiment. Data were analyzed to check for a normal distribution (using the D'Agostino-Pearson and Shapiro-Wilk normality tests), and then subjected to the appropriate analysis using GraphPad Prism 5. For data sets with three or more groups, if the data were normal, they were analyzed with a one-way ANOVA; if they were not, they were analyzed with a Kruskal-Wallis test. Post hoc tests were used to compare data sets within an experiment. Reported P-values are corrected. Table S3 in File S1 contains the statistical tests for each experiment. Ã P , 0.05, ÃÃ P , 0.01, ÃÃÃ P , 0.001.

Data availability
Strains and plasmids are shown in Tables S1 and S2 in File S1, and are available from the Caenorhabditis Genetics Center or upon request. The authors state that all data necessary for confirming the conclusions presented in the article are represented fully within the article and Supplemental Material.

sek-1 suppresses activated Gq
To identify genes acting downstream of Gaq, we performed a forward genetic screen for suppressors of the activated Gq mutant, egl-30(tg26) (Doi and Iwasaki 2002). egl-30(tg26) worms are hyperactive and have a loopy posture, characterized by an exaggerated waveform (Figure 1, B-E). Thus, we screened for worms that were less hyperactive and less loopy. We isolated a recessive suppressor, yak42, and mapped it to the middle of the X chromosome (see Materials and Methods). Wholegenome sequencing revealed that yak42 carries a large deletion of the sek-1 gene from upstream of the start codon into exon 4 ( Figure 1A). yak42 also failed to complement sek-1(km4), a previously published sek-1 deletion allele, for the Gq suppression phenotype ( Figure 1A) (Tanaka-Hino et al. 2002).
The egl-30(tg26) allele causes an R243Q missense mutation in the Ga switch III region that has been shown to reduce both the intrinsic GTPase activity of the G protein and render it insensitive to GTPaseactivation by a regulator of G protein signaling (RGS) protein, thus leading to increased G protein activation (Natochin and Artemyev 2003). To test whether the suppression of egl-30(tg26) by sek-1 is specific for this egl-30 allele, we built a double mutant between sek-1(km4) and the weaker activating mutation egl-30(js126). egl-30(js126) causes a V180M missense mutation in the Ga switch I region immediately adjacent to one of the key residues required for GTPase catalysis (Hawasli et al. 2004). Thus, the tg26 and js126 alleles activate EGL-30 through different mechanisms. The sek-1(km4) mutant also suppresses the hyperactivity and loopy waveform of egl-30(js126) (Figure 1, G and H), demonstrating that sek-1 suppression of activated egl-30 is not allele specific.
sek-1 acts in mature acetylcholine neurons egl-30 is widely expressed and acts in neurons to modulate locomotion (Lackner et al. 1999), so it is possible that sek-1 also acts in neurons to modulate Gq signaling. sek-1 is expressed in neurons, intestine, and several other tissues (Tanaka-Hino et al. 2002), and has been shown to function in GABA neurons to promote synaptic transmission (Vashlishan et al. 2008).
To identify the cell type responsible for the sek-1 locomotion phenotypes, we expressed the wild-type sek-1 cDNA under different cellspecific promoters and tested for transgenic rescue of a sek-1 null mutant. Expression of sek-1 in all neurons (using the unc-119 promoter) or in acetylcholine neurons (unc-17 promoter) was sufficient to rescue the sek-1 mutant slow locomotion phenotype, but expression in GABA neurons (unc-47 promoter) was not sufficient (Figure 2, A and B). These results indicate that sek-1 acts in acetylcholine neurons to modulate locomotion rate.
We next tested whether sek-1 acts in neurons to suppress egl-30 (tg26). Expression of sek-1 under pan-neuronal and acetylcholine neuron promoters reversed the sek-1 suppression of egl-30(tg26). Specifically, egl-30(tg26) sek-1 double mutants expressing wild-type sek-1 in all neurons or acetylcholine neurons resembled the egl-30(tg26) single mutant (Figure 2, C-E). However, expression of sek-1 in GABA neurons did not reverse the suppression phenotype (Figure 2, C-E). Together, these data show that sek-1 acts in acetylcholine and not GABA neurons to modulate both wild-type locomotion rate and Gq signaling.
To narrow down the site of sek-1 action, we expressed sek-1 in head (unc-17H promoter) and motorneuron (unc-17b promoter) acetylcholine neuron subclasses (Topalidou et al. 2017). Expression of sek-1 in acetylcholine motorneurons rescued the sek-1 slow locomotion phenotype ( Figure S2A in File S1), suggesting that the slow locomotion of sek-1 mutants is due to a loss of sek-1 in acetylcholine motorneurons. However, expression of sek-1 in either the head acetylcholine neurons or motorneurons partially reversed the sek-1 suppression of egl-30(tg26) hyperactivity ( Figure S2B in File S1), suggesting that the hyperactivity of activated Gq mutants may result from excessive Gq signaling in both head acetylcholine neurons and acetylcholine motorneurons; sek-1 may act in Gq signaling in both neuronal cell types. By contrast, expression of sek-1 in head acetylcholine neurons but not motorneurons reversed the sek-1 suppression of the egl-30(tg26) loopy waveform ( Figure S2C in File S1), suggesting that the loopy posture of activated  sek-1 acts in acetylcholine neurons to modulate locomotion rate. sek-1 wild-type cDNA driven by the Gq mutants may result from excessive Gq signaling in head acetylcholine neurons, and sek-1 may act in those neurons to control body posture.
Because sek-1 acts in the development of the AWC asymmetric neurons, we asked whether sek-1 also has a developmental role in modulating locomotion by testing whether adult-specific sek-1 expression (driven by a heat-shock promoter) is sufficient to rescue the sek-1 mutant. We found that sek-1 expression in adults rescues the sek-1 slow locomotion phenotype ( Figure 2F). This result indicates that sek-1 is not required for development of the locomotion circuit, and instead acts in mature neurons to modulate locomotion.
The JNK MAPK pathway, related to the p38 MAPK family, also modulates locomotion in C. elegans. Specifically, the JNK pathway members jkk-1 (JNK MAPKK) and jnk-1 (JNK MAPK) have been shown to act in GABA neurons to modulate locomotion (Kawasaki 1999). We found that the jkk-1 and jnk-1 single mutants had slow locomotion and that the double mutants with p38 MAPK pathway members exhibited an additive slow locomotion phenotype ( Figure  S3A in File S1). Moreover, neither jkk-1 nor jnk-1 suppressed the loopy phenotype of egl-30(tg26) ( Figure S3B in File S1). Thus, the JNK and p38 MAPK pathways modulate locomotion independently, and the JNK pathway is not involved in Gq signaling.
We also tested the involvement of possible p38 MAPK pathway effectors. One of the targets of PMK-1 is the transcription factor ATF-7 (Shivers et al. 2010). Both the atf-7(qd22 qd130) loss-of-function mutant and the atf-7(qd22) gain-of-function mutant moved slowly compared to wild-type animals ( Figure S3C in File S1). However, atf-7(qd22 qd130) did not suppress the loopy waveform of egl-30(tg26) ( Figure S3B in File S1), suggesting that atf-7 is not a target of this pathway, or else it acts redundantly with other downstream p38 MAPK targets. We also tested gap-2, the closest C. elegans homolog of ASK1-interacting protein (AIP1), which activates ASK1 (the ortholog of C. elegans NSY-1) in mammalian systems (Zhang et al. 2003). A C. elegans gap-2 mutant showed no locomotion defect ( Figure S3D in File S1). Finally, we tested VHP-1, a phosphatase for p38 and JNK MAPKs that inhibits p38 MAPK signaling (Kim et al. 2004). However, the vhp-1(sa366) mutant also showed no locomotion defect ( Figure S3D in File S1).
egl-30(tg26) animals are loopy and hyperactive, so we tested whether increased activation of the TIR-1/p38 MAPK signaling module causes similar phenotypes. The tir-1(ky648) allele leads to a gain-offunction phenotype in the AWC neuron specification (Chang et al. 2011), but does not cause loopy or hyperactive locomotion ( Figure  S3, E and F in File S1).

Genetic interactions of sek-1 with pathways acting downstream of Gq
Our forward genetic screen for suppressors of egl-30(tg26) identified mutants that fall into three different categories: mutants in the canonical Gq pathway, such as the PLC egl-8 ( (Lackner et al. 1999); mutants in the RhoGEF Trio pathway, such as unc-73 (Williams et al. 2007); and mutants that affect dense-core vesicle biogenesis and release (Ailion et al. 2014;Topalidou et al. 2016).
To test if sek-1 acts in any of these pathways, we built double mutants between sek-1 and members of each pathway. Loss-of-function alleles of egl-8(sa47), unc-73(ox317), and rund-1(tm3622) have slow locomotion (Figure 4, A-C). We found that sek-1 enhances the slow locomotion phenotype of egl-8 and rund-1 single mutants, suggesting that sek-1 does not act in the same pathway as egl-8 or rund-1 (Figure 4, A and B). By contrast, sek-1 does not enhance the slow locomotion phenotype of unc-73 mutants ( Figure 4C), suggesting that sek-1 may act in the same genetic pathway as the Trio RhoGEF unc-73.
We next tested whether sek-1 interacts with rho-1, encoding the small G protein Rho that is activated by Trio. Because rho-1 is required for viability (Jantsch-Plunger et al. 2000), we used an integrated transgene overexpressing an activated rho-1 mutant allele specifically in acetylcholine neurons. Animals carrying this activated RHO-1 transgene, referred to here as rho-1(gf), have a loopy posture reminiscent of egl-30(tg26) (McMullan et al. 2006), and a decreased locomotion rate (Figure 4, D-F). rho-1(gf) sek-1(km4) double mutants had a loopy body posture like rho-1(gf) animals, and an even slower locomotion rate (Figure 4, D-F), suggesting that sek-1 and rho-1(gf) mutants have additive locomotion phenotypes. However, both sek-1(km4) and sek-1(yak42) weakly suppress the slow growth rate of the rho-1(gf) mutant (data not shown). Because sek-1 does not enhance unc-73 mutants and suppresses some aspects of the rho-1(gf) mutant, sek-1 may modulate output of the Rho pathway, although it probably is not a direct transducer of Rho signaling.
sek-1 and nsy-1 partially suppress activated NCA To clarify the relationship of the SEK-1 p38 MAPK pathway to the Rho pathway acting downstream of Gq, we examined interactions with nca-1, a downstream target of the Gq-Rho pathway (Topalidou et al. 2017). NCA-1 and its orthologs are sodium leak channels associated with rhythmic behaviors in several organisms (Nash et al. 2002;Lu et al. 2007;Shi et al. 2016). In C. elegans, NCA-1 potentiates persistent motor circuit activity and sustains locomotion (Gao et al. 2015).
We tested whether sek-1 and nsy-1 mutants suppress the activated NCA-1 mutant ox352, referred to as nca-1(gf). The nca-1(gf) animals are coiled and uncoordinated; thus, it is difficult to measure their locomotion rate by the body bend assay because they do not reliably propagate sinusoidal waves down the entire length of their body. Instead, we used a radial locomotion assay in which we measured the distance animals moved from the center of a plate. nca-1(gf) double mutants with either sek-1(km4) or nsy-1(ok593) uncoiled a bit, but still exhibited uncoordinated locomotion ( Figure 5A). In fact, although these double mutants showed more movement in the anterior half of their bodies than nca-1(gf), they propagated body waves to their posterior half even more poorly than the nca-1(gf) mutant. However, both sek-1 and nsy-1 partially suppressed the loopy waveform of the nca-1(gf) mutant ( Figure 5, A and B), and in radial locomotion assays, sek-1 and nsy-1 weakly suppressed the nca-1(gf) locomotion defect ( Figure 5C). Additionally, both sek-1 and nsy-1 partially suppressed the small body size of nca-1(gf) ( Figure S4A in File S1). Together, these data suggest that mutations in the SEK-1 p38 MAPK pathway suppress some aspects of the nca-1(gf) mutant.
Given that sek-1 acts in acetylcholine neurons to modulate wild-type and egl-30(tg26) locomotion, we tested whether sek-1 also acts in these neurons to suppress nca-1(gf). Expression of sek-1 in all neurons or in acetylcholine neurons of nca-1(gf) sek-1(km4) animals restored the nca-1(gf) loopy phenotype (Figure 5,D and E). By contrast, expression of sek-1 in GABA neurons did not affect the loopy posture of the nca-1(gf) sek-1 double mutant (Figure 5,D and E). These data suggest that sek-1 acts in acetylcholine neurons to modulate the body posture of nca-1(gf) as well. However, in radial locomotion assays, expression of sek-1 in any of these neuron classes did not significantly alter the movement of the nca-1(gf) sek-1 double mutant ( Figure S4B in File S1), although the weak suppression of nca-1(gf) by sek-1 in this assay makes it difficult to interpret these negative results. To further narrow down the site of action of sek-1 for its NCA suppression phenotypes, we expressed it in subclasses of acetylcholine neurons. Surprisingly, expression of sek-1 in acetylcholine motorneurons but not head acetylcholine neurons was sufficient to restore the loopy posture of the nca-1(gf) mutant ( Figure 5E), the opposite of what we found for sek-1 modulation of the loopy posture of the activated Gq mutant, suggesting that the loopy posture of nca-1(gf) mutants may result from excessive NCA-1 activity in acetylcholine motorneurons. Additionally, expression of sek-1 in either the head acetylcholine neurons or the motorneurons restored the nca-1(gf) small body size phenotype ( Figure S4C in File S1). We make the tentative conclusion that sek-1 acts in acetylcholine neurons to modulate nca-1(gf) body posture and size, but we are not able to conclusively narrow down its site of action further, possibly due to the uncoordinated phenotype of nca-1(gf) and the weaker suppression of nca-1(gf) by sek-1.

DISCUSSION
The p38 MAPK pathway has been best characterized as a pathway activated by a variety of cellular stresses and inflammatory cytokines (Kyriakis and Avruch 2012), but it has also been implicated in neuronal function, including some forms of mammalian synaptic plasticity (Bolshakov et al. 2000;Rush et al. 2002;Huang et al. 2004). In this study, we identified a new neuronal role for the MAPKK SEK-1 and the p38 MAPK pathway as a positive modulator of locomotion rate and Gq signaling. The physiological importance of this pathway is clear under conditions of elevated Gq signaling but is less obvious during normal wild-type locomotion, consistent with the observation that sek-1 mutations have a relatively weak effect on synaptic transmission in a wildtype background (Vashlishan et al. 2008). Thus, the SEK-1 p38 MAPK pathway may be more important for modulation of Gq signaling and synaptic strength than for synaptic transmission per se.
In addition to SEK-1, we identified other p38 pathway components that modulate Gq signaling. Specifically, we found that tir-1, nsy-1, and pmk-1 pmk-2 mutants exhibit locomotion defects identical to sek-1 and suppress activated Gq, suggesting that they act in a single p38 pathway to modulate signaling downstream of Gq. These results indicate a redundant function for PMK-1 and PMK-2 in modulating locomotion rate and Gq signaling. PMK-1 and PMK-2 also act redundantly for some other neuronal roles of the p38 pathway, such as the development of the asymmetric AWC neurons and to regulate induction of serotonin biosynthesis in the ADF neurons in response to pathogenic bacteria (Shivers et al. 2009;Pagano et al. 2015). By contrast, PMK-1 acts alone in the intestine to regulate innate immunity, and in interneurons to regulate trafficking of the GLR-1 glutamate receptor (Pagano et al. 2015;Park and Rongo 2016).
What are the downstream effectors of the SEK-1 p38 MAPK pathway that modulate locomotion? There are several known downstream effectors of p38 MAPK signaling in C. elegans, including the transcription factor ATF-7 (Shivers et al. 2010). Our data indicate that ATF-7 is not required for the p38 MAPK-dependent modulation of Gq signaling. The p38 MAPK pathway may activate molecules other than transcription factors, or may activate multiple downstream effectors.
Consistent with the observation that Gq acts in acetylcholine neurons to stimulate synaptic transmission (Lackner et al. 1999), we found that sek-1 acts in acetylcholine neurons to modulate the locomotion rate in both wild-type and activated Gq mutants. sek-1 also acts in acetylcholine neurons to modulate the loopy waveform of both activated Gq and activated nca-1 mutants, and the size of activated nca-1 mutants. However, our data obtained from attempting to narrow down the site of action of sek-1 suggest that it may act in both head acetylcholine neurons and acetylcholine motorneurons, and that the waveform is probably controlled by at least partially distinct neurons from those that control locomotion rate. Further work will be required to identify the specific neurons where Gq, NCA-1, and the SEK-1 pathway act to modulate locomotion rate and waveform, and determine whether they all act together in the same cell.