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.

Henderson ST et al. (1994) Worm Breeder's Gazette "The lag-2 Gene Encodes a Protein With Homology to Drosophila Delta and is Expressed in the Distal Tip Cell"

Gao DL, Henderson ST, Lambie EJ, Kimble JE

[Worm Breeder's Gazette, 1994]

The lag-2 gene encodes a protein with homology to Drosophila Delta and is expressed in the distal tip cell. Sam Henderson, Dali Gao, Eric Lambie#, and Judith Kimble, University of Wisconsin, Madison WI 53706, Dartmouth College, Hanover, NH 03755

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.

Pena-Ramos, Omar et al. (2021) International Worm Meeting "Autophagy and the degradation of apoptotic cells"

Chiao, Lucia, Wang, Ying, Gao, Lidan, Liu, Xianghua, He, Henry, Yao, Tianyou, Zhou, Zheng, Pena-Ramos, Omar

[International Worm Meeting, 2021]

The autophagy pathway generates autophagosomes, double-membrane structures responsible for degrading protein aggregates, intracellular organelles, and other cellular components. This intracellular trafficking process is essential for the cell stress response, recycling the degraded material for use in different cellular processes. During C. elegans embryonic development, 113 somatic cells undergo apoptosis; these cells are then swiftly internalized by neighboring engulfing cells through phagocytosis, generating phagosomes. The newly formed phagosome then enters a maturation process during which acidic vesicles are fused to the phagosome promoting its degradation. Components of the autophagy pathway have previously been linked to phagosome maturation in mammalian cells in a process called LC3-associated phagocytosis (LAP). The autophagy machinery generates a single membrane-vesicle to conjugate LC3 directly onto the phagosome membrane promoting phagosome-lysosome fusion. Using live imaging on developing C. elegans embryos, we discovered that LC3-labeled puncta generated in engulfing cells are recruited to the surface of phagosomes and subsequently fuse to them, depositing their content into the phagosomal lumen. The observed fusion pattern suggests that the puncta are of a double-membrane nature, indicative of double-membranous autophagosomes and not single-membrane LAP vesicles. We also observed that these autophagosomes form two distinct populations, those that are labeled with LGG-1 and those marked with LGG-2, both homologs of yeast Atg8 and mammalian LC3. Lacking either group of autophagosomes significantly delay the degradation of apoptotic cells, indicating that both LGG-1 and LGG-2 function in the clearance of apoptotic cells. Finally, we identified that a signaling pathway with a previously known role in phagosome-lysosome fusion also facilitates the integration of autophagosomes to phagosomes. Our findings have collectively added autophagosomes to the list of intracellular organelles involved in phagosome maturation. Furthermore, we have demonstrated that in C. elegans, it is autophagosomes, not LAP vesicles, that facilitate phagosome maturation, and we have revealed a novel function of the autophagy pathway in the clearance of apoptotic cells.

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.

Crittenden SL et al. (1997) International C. elegans Meeting "TEMPERATURE-SENSITIVE REPRESSION OF TRANSGENES CARRYING A fem-3(gf) MUTANT 3'UTR"

Crittenden SL, Kimble JE

[International C. elegans Meeting, 1997]

We are developing a method to control gene expression in a temperature dependent manner. Our strategy is to regulate reporter expression with a well-defined promoter at the 5' end and a mutant fem-3 3'UTR at the 3' end. The fem-3(gf) mutations map to the fem-3 3'UTR and release fem-3 from post-transcriptional repression at 25 C, but not at 15 C (1,2). We have tested expression of a lag-2 promoter::GFP construct that carries either a strong or a weak fem-3(gf) 3'UTR. The lag-2 promoter drives expression in the distal tip cells of adults (3-5). We find that transgenes express GFP at much lower levels at 15 C than at 25 C when carrying a fem-3(gf) 3'UTR. We are currently trying to optimize repression at 15 C using the lag-2 promoter and GFP as a reporter. In addition, we are testing whether temperature sensitive repression can be achieved in other tissues, and we are starting to explore the biological activity of various proteins expressed in the distal tip cell. 1. Ahringer, J., and Kimble, J. (1991). Nature 349, 346-348. 2. Barton, M. K., Schedl, T. B., and Kimble, J. (1987). Genetics 115, 107-119. 3. Fitzgerald, K., and Greenwald, I. (1995). Development 121, 4275-4282. 4. Gao, D. and J. Kimble, midwest worm meeting 1996. 5. Henderson, S. T., Gao, D., Lambie, E. J., and Kimble, J. (1994). Development 120, 2913-2924.

Sternberg PW et al. (2000) West Coast Worm Meeting "Characterization and Suppression of eat-16; sag-1/dgk-1 lethality"

Sternberg PW, Chen WJ, Hajdu-Cronin YM

[West Coast Worm Meeting, 2000]

goa-1 (Gao) modulates many behaviors in C. elegans, including locomotion, egg-laying, and feeding. Reducing function in goa-1 causes hyperactive locomotion and constitutive egg laying (1, 2); in contrast, overexpressing the constitutively activated Q205L mutation causes lethargy and cessation of egg laying (1). Previously, we screened for suppressors of the paralysis of syIs17, an integrated transgene containing goa-1(Q205L) under control of a heat shock promoter. Hyperactive mutants were isolated in two genes, eat-16 and sag-1/dgk-1. We found that in addition to suppressing the phenotype of activated Gao, eat-16 and sag-1/dgk-1 mutations also partially suppressed the phenotype of several reduction of function mutations in egl-30 ( C. elegans Gaq). eat-16 encodes a regulator of G protein signaling, which we believe functions as a GAP for EGL-30, and sag-1/dgk-1 encodes a diacyl glycerol kinase (3) which likely functions to reduce the levels of diacyl glycerol (DAG), a second messenger produced upon stimulation of PLCb by activated EGL-30. eat-16(sy438) and sag-1/dgk-1(sy428) double mutants arrest during larval development. This lethal phenotype is highly penetrant (>99%). We hypothesized that the lethality of eat-16; sag-1/dgk-1 is caused by excessive levels of second messengers produced downstream of Gaq, such as DAG. In support of this hypothesis, the triple mutant egl-30(md186) eat-16(sy438); sag-1/dgk-1(sy428) is viable to adulthood and fertile. To further understand the cause of death and identify more components in the pathway, we are seeking other suppressors of eat-16; sag/dgk-1 lethality besides egl-30, both by testing mutations in genes that have already been described, and by screening for new suppressors. To date we have screened about 7000 genomes and have backcrossed 7 suppressor mutants. We are beginning to characterize the lethal phenotype by Nomarski optics and plan to determine the site of action. References: Mendel et al., 1995. Science 267: 1652-1655. Segalat et al., 1995. Science 267: 1648-1651. Nurrish et al., 1999. Neuron 24: 231-242