Gao Shangbang et al. (2010) Biology of the C. elegans Male, Madison, WI "Action Potentials Drive Body Wall Muscle Contractions in C. elegans"

Zhen Mei, Gao Shangbang

[Biology of the C. elegans Male, Madison, WI, 2010]

The sinusoidal locomotion exhibited by C. elegans predicts a tight regulation of contractions and relaxations of its body wall muscles. Vertebrate skeletal muscle contractions are driven by voltage-gated Na+channel-dependent, regenerative, all-or-none action potentials. How coordinated motor outputs are regulated in C. elegans, which does not encode voltage-gated Na+ channels and was reported to exhibit only graded potentials in the locomotory system, has been a long-standing puzzle. Here, we show that C.elegans body wall muscles fire all-or-none, regenerative, Ca2+-dependent action potentials that are driven by the L-type voltage-gated Ca2+and Kv1 voltage-dependent K+ channels. We further demonstrate that the excitatory and inhibitory motoneuron activities regulate the frequency of action potentials to coordinate the contraction and relaxation of body wall muscles, respectively. This study provides the direct evidence for the dual modulatory model of the C. elegans locomotory circuit; moreover, it reveals an elegant mechanism of controlling locomotion, where body wall muscle cells fire quantal electrical signals upon receiving graded inputs of the nervous system.

Olson, Andrew et al. (2017) International Worm Meeting "The Role of Post-Translational Modifications in the Regulation of Serotonin Signaling."

Olson, Andrew, Koelle, Michael, Kumar, Santosh

[International Worm Meeting, 2017]

C. elegans uses serotonin as a neurotransmitter to slow locomotion, and we have used this model system to discover that post-translational modifications appear to regulate serotonin signaling. Through large-scale genetic screens for mutants that fail to paralyze in response to exogenous serotonin, we found that C. elegans mutants for either of two subunits of the ELPC Elongator Protein Complex are defective for serotonin signaling. Conversely, transgenic animals overexpressing ELPC are hypersensitive to the effects of exogenous serotonin. ELPC is conserved from C. elegans to humans and functions as a cytoplasmic lysine acetylase to reversibly modify other proteins. This is the first time that ELPC or lysine acetylation has been implicated in regulating serotonin signaling. We used two-dimensional gel electrophoresis to show that in C. elegans lysates, Gao, a neural G protein encoded by the goa-1 gene through which serotonin signals to slow locomotion, exists as a complex series of species of differing charge. Acetylation eliminates the positive charge on a lysine side chain, and differential acetylation on several Lys could produce the complex pattern of Gao species we see. Our preliminary results suggest that the series of differentially charged Gao species in wild-type lysates shifts to a less complex and more positively charged set of Gao species in lysates of ELPC mutants, as would be predicted if Gao is acetylated by ELPC. We isolated Gao from both mouse brain and C. elegans lysates and analyzed the purified proteins for post-translational modifications using mass spectrometry. In both species, Gao is acetylated on several conserved Lys residues near the N-terminus. We note that the signaling defects in ELPC mutants are much more restricted than those of Gao null mutants. Both ELPC and Gao mutants are defective for response to exogenous serotonin, but Gao null mutants have additional defects not seen in ELPC mutants, including defects in response to exogenous dopamine as well as in many behavioral assays. Thus we hypothesize that ELPC may reversibly acetylate Gao to specifically regulate its ability to be activated by serotonin receptors, while not strongly affecting its ability mediate signaling by other receptors. We are using CRISPR-Cas9 technology to mutate the acetylated Lys residues of Gao to the similar but non-acetylatable amino acid arginine to determine if acetylation at specific positions is responsible for regulating serotonin signaling.

Hofler, Catherine et al. (2011) International Worm Meeting "AGS-3 alters C. elegans behavior after food deprivation via RIC-8 activation of the neural G protein Gao."

Hofler, Catherine, Koelle, Michael

[International Worm Meeting, 2011]

Like all animals, C. elegans modifies a number of behaviors upon food deprivation in order to seek food. These behavioral changes depend on neurotransmitters and peptides that typically signal through the major neural G protein Gao, suggesting that signaling through this G protein ultimately mediates responses to food deprivation. Proteins containing the G Protein Regulator (GPR) domain bind to Gao in vitro, but the biological functions of GPR proteins in neurons and the mechanism by which they might affect G protein signaling has remained unclear. We found that the conserved GPR domain protein AGS-3 activates Gao signaling in vivo to allow C. elegans to alter several behaviors after food deprivation. These behaviors include octanol avoidance, area-restricted search, and egg laying. AGS-3 protein in whole worm lysates undergoes a progressive change in its biochemical fractionation pattern upon food deprivation, demonstrating that food deprivation changes the physical state of the protein. Cell-specific rescue and cell-specific inactivation experiments show that AGS-3 and Gao act together in the ASH chemosensory neurons to allow food deprivation to modify response to octanol. Genetic epistasis experiments show that AGS-3 activation of Gao in the ASHs requires the guanine nucleotide exchange factor RIC-8. Conversely, RIC-8 function in the ASHs also requires AGS-3. Using purified recombinant proteins, we characterized interactions of the proteins consistent with the genetic epistasis results. AGS-3 forms a complex with the the inactive, GDP-bound form of Gao, and RIC-8 can act on this complex to promote nucleotide exchange and dissociation of all the proteins, generating active Gao-GTP. These results identify a biological role for AGS-3 in response to food deprivation and indicate the mechanism for its activation of Gao signaling in vivo.

Heather A Hess et al. (2004) East Coast Worm Meeting "RGS-7 Completes a Receptor-independent Heterotrimeric G Protein Signaling Cycle to Regulate Mitotic Spindle Positioning in C. elegans"

Michael R Koelle, Heather A Hess

[East Coast Worm Meeting, 2004]

Heterotrimeric G proteins promote astral microtubule forces on centrosomes to position mitotic spindles properly during asymmetric cell division in C. elegans embryos. While all previously studied G protein functions require activation by seven-transmembrane receptors, this function appears to be receptor-independent, and the active form of the G proteins remains unclear. We obtained mutants for all 13 C. elegans regulators of G protein signaling and found that one, RGS-7, decreases the speed and magnitude of centrosome movements. The effects of RGS-7 require two redundant Gao-related G proteins and their non-receptor activators. Using recombinant proteins, we found that the non-receptor activator RIC-8 stimulates GTP binding by Gao, and the RGS domain of RGS-7 stimulates GTP hydrolysis by Gao. These results demonstrate that the active species in the receptor-independent G protein cycle is GaoGTP, and that RGS-7 completes the cycle by driving Gao to its inactive, GDP-bound form.

Olson, Andrew C et al. (2013) International Worm Meeting "The Role of Post-Translational Modifications in the Regulation of Serotonin Signalling."

Olson, Andrew C, Koelle, Michael R

[International Worm Meeting, 2013]

C. elegans uses serotonin as a neurotransmitter to slow locomotion, and we have used this model system to discover that post-translational modifications seem to regulate serotonin signaling. First, through large-scale genetic screens for mutants that fail to respond properly to serotonin (Gurel et al., 2012), we found that C. elegans strains carrying mutations in either of two subunits of the ELPC ELongator" Protein Complex are defective for response to serotonin. ELPC is highly conserved from C. elegans to humans and functions as a lysine acetylase to reversibly modify other proteins. This is first time that ELPC or lysine acetylation has been implicated in regulating serotonin signaling. Second, our lab has also shown that Gao, a G protein through which serotonin signals to slow locomotion, is post-translationally modified in a manner that alters its charge, which is a hallmark of lysine acetylation. We hypothesize that ELPC may reversibly acetylate Gao (and/or one of the other proteins that mediate serotonin signaling) to regulate serotonin response. Serotonin regulates worm movement in C. elegans by redundantly signaling through the MOD-1 serotonin-gated ion channel and the SER-4 G Protein Coupled serotonin Receptor (GPCR), the GPCR through which Gao signals (Gurel et al. 2012). It is likely that ELPC modifies signaling through one or both of these receptors. To test this genetically, we have developed an assay that optogenetically stimulates the release of serotonin from the NSM and ADF serotonergic neurons. This strongly decreases the speed of worms that have intact MOD-1 and SER-4 signaling pathways, but this serotonin response is defective in animals missing one or both of the receptors. With WormLab worm-tracking software we are able to monitor the speed of worms throughout the course of the experiment. Using this assay we will be able to genetically determine if ELPC affects signaling through the MOD-1 or SER-4 receptor. We are also working to identify the post-translational modifications of Gao using mass spectrometry. Analysis of Gao immunoprecipitated from both C. elegans lysates as well as mouse brain lysates will allow us to determine if worm and mammalian Gao are equivalently modified.

Maro, Geraldine et al. (2013) International Worm Meeting "Neuroligin organizes C. elegans GABAergic NMJs."

Zhen, Mei, Liu, Michael, Shen, Kang, Maro, Geraldine, Gao, Shangbang

[International Worm Meeting, 2013]

The trans-synaptic adhesion complex neurexin-neuroligin plays key roles in synapse maturation and excitatory/inhibitory balance. Multiple neuroligins have been hypothesized to function both redundantly, and distinctly to influence synaptic connectivity or validate specific types of chemical synapses. The C. elegans genome contains a single neuroligin and neurexin encoding genes, and therefore represents a simplified genetic system to address the role of these molecules in the developing and mature nervous system. We found that at the C. elegans NMJs, NLG-1 localizes specifically at inhibitory GABAergic postsynaptic terminals. Moreover, postsynaptic NLG-1 is required for clustering of the GABAA receptor UNC-49, and nlg-1 mutants show a decrease in both the frequency and amplitude of spontaneous inhibitory postsynaptic events. Interestingly, nrx-1 null mutants do not share GABAergic NMJ morphological defects. We propose that NLG-1 is recruited to GABAergic postsynaptic terminals through a trans-synaptic interaction that is largely independent of NRX-1.

Meng, Jun et al. (2021) International Worm Meeting "A central role of AVA in regulating overall motor states activity"

Meng, Jun, Hung, Wesley, Zhen, Mei, Boulin, Thomas, Chen, Lili, El Mouridi, Sonia, Gao, Shangbang, Mercier, Marine

[International Worm Meeting, 2021]

The descending neurons transmit and process neural signals to guide the locomotor behaviour. In the C. elegans motor circuit, premotor interneurons AVA and AVB potentiate backward and forward locomotion, respectively. Previously, we and others have shown that AVA hold an unusually depolarized RMP of ~-23mV that implicates tonic activities. We show that a two-pore K+ leak channel TWK-40 plays a critical role to establish this RMP, and maintaining this RMP is crucial for C. elegans to sustain not only reversal, but also forward movement. We demonstrate that AVA's positive role in forward movement requires AVB. Specifically, the chemical synaptic release of AVA is critical for promoting the forward speed, whereas neuropeptide synthesis in AVA and AVB regulates forward bending curvature. These results are in disagreement with the mutual inhibition model of AVA and AVB, but instead, propose an alternative, new model of AVA's central role in controlling both forward and backward movement.

Kulkarni, Rashmi P et al. (2011) International Worm Meeting "Comparative functional analysis of hSTIM1 and C. elegans STIM-1."

Machaca, Khaled, Kulkarni, Rashmi P, Courjaret, Raphael

[International Worm Meeting, 2011]

STIM1, the ER Calcium sensor couples to Orai1, the Calcium channel to mediate Store-Operated Calcium Entry (SOCE). hSTIM1 is localized to the ER membrane when Ca++ stores are full. On Ca++ store depletion, hSTIM1 forms puncta that localize to the cortical ER and bind hOrai1 to allow Ca++ influx. hSTIM1 when expressed in Xenopus oocytes can gate XOrai1 to induce SOCE on store-depletion. The C. elegans STIM1 (CeSTIM-1a) when expressed in HEK293 cells shows puncta formation without store-depletion (Gao et. al. 2009). These puncta however can only gate CeORAI-1 on store-depletion. We expressed CeSTIM-1a in Xenopus oocytes and found it to be localized in a similar punctate pattern. These puncta failed to recruit XOrai1 either under store-full or store-depleted conditions similar to the reported results (Gao et. al. 2009). We are conducting comparative structure-function analysis of the hSTIM1 and CeSTIM-1a to better define the structural features within the STIM1 molecule required for its clustering into puncta and for its ability to interact with and gate Orai1. CeSITM-1a has two other isoforms (CeSTIM-1b and c) that remain uncharacterized. We have detected these isoforms by RT-PCR using total RNA from a mixed-stage hermaphrodite population and cloned them. Currently we are characterizing the localization patterns of these isoforms and studying their function in Ca++ signaling.

Wei, K. et al. (2019) International Worm Meeting "The Role of Gao-coupled Neurotransmitter GPCRs in the C. elegans Egg-Laying Circuit."

Fernandez, R.W., Wei, K., Koelle, M.R.

[International Worm Meeting, 2019]

The C. elegans egg-laying circuit is among the best-studied model neural circuits, as the function of each cell and the neurotransmitters it releases have been characterized. Our work has shown there are 20 neurotransmitter GPCRs detectably expressed in this circuit (see abstract by Fernandez et al.), yet their functions remain largely unstudied. We screened knockout mutants for every one of these neurotransmitter GPCRs to look for egg-laying defects. The single receptor knockouts showed at most mild egg-laying defects. We hypothesized that the weakness of these defects could be due to functional redundancy among the receptors. For example, knocking out GaO, a G protein known to inhibit neurotransmitter release in neurons of the egg-laying circuit, causes a very strong hyperactive egg-laying phenotype, and we found seven GaO- coupled neurotransmitter GPCRs are expressed in the egg-laying circuit that might redundantly activate this G protein. To test this idea, we generated various knockout combinations of five of the GaO-coupled neurotransmitter GPCRs. However, our results showed that even the quintuple knockout resulted in only a weak egg-laying defect. As a strategy to identify the functions of these GPCRs despite their apparent very high functional redundancy, we examined transgenic overexpressors of the GPCRs, as overexpression of this type of receptor can result in a gain-of-function effect. We found that the GABA receptor GPCR, GBB-1, when overexpressed from a high-copy transgene, showed a strong hyperactive egg laying phenotype. GBB-1 is thought to form a functional GABA receptor by forming an obligate heterodimer with a second GPCR called GBB-2. Knocking out gbb-2 suppressed most but not all of the effect of the GBB-1 overexpressor on egg laying. The residual effect of GBB-1 overexpression in the absence of GBB-2 suggests that GBB-1 may have additional dimerization partners besides GBB-2. These other partners may be other members of the family of class C GPCRs, which are known to form heterodimers with each other.

Bouhours, Magali et al. (2011) International Worm Meeting "A Co-operative Regulation of Neuronal Excitability by UNC-7 Innexin and NCA/NALCN Leak Channel."

Li, Hang, Roder, John, Georgiou, John, Po, Michelle, Zhen, Mei, Xie, Lin, Bouhours, Magali, Hung, Wesley, Gao, Shangbang

[International Worm Meeting, 2011]

Gap junctions mediate the electrical coupling and intercellular communication between neighboring cells. Some gap junction proteins, namely connexins and pannexins in vertebrates, and innexins in invertebrates, may also function as hemichannels. A conserved NCA/Dma1U/NALCN family cation leak channel regulates the excitability and activity of vertebrate and invertebrate neurons. In the present study, we describe a genetic and functional interaction between the innexin UNC-7 and the cation leak channel NCA in Caenorhabditis elegans neurons. While the loss of the neuronal NCA channel function leads to a reduced evoked postsynaptic current at neuromuscular junctions, a simultaneous loss of the UNC-7 function restores the evoked response. The expression of UNC-7 in neurons reverts the effect of the unc-7 mutation; moreover, the expression of UNC-7 mutant proteins that are predicted to be unable to form gap junctions also reverts this effect, suggesting that UNC-7 innexin regulates neuronal activity, in part, through gap junction-independent functions. We propose that, in addition to gap junction-mediated functions, UNC-7 innexin may also form hemichannels to regulate C. elegans' neuronal activity cooperatively with the NCA family leak channels.