Hannes Lans et al. (2002) European Worm Meeting "Specificity of G-protein mediated signaling in odorant detection by C. elegans"

Gert Jansen, Hannes Lans

[European Worm Meeting, 2002]

The olfactory system of C. elegans is ideally suited for studying specificity of G-protein coupled signal transduction. C. elegans can detect more than 60 odorants, using only two pairs of sensory neurons, AWA and AWC. This is possible due to the expression of several G-protein coupled odorant receptors per neuron. Furthermore, six Ga-subunits are expressed in AWA and AWC, which probably contribute to the ability to detect different odorants and discriminate between many of them. These Ga-subunits are GPA-2, -3, -5, -6, -13 and ODR-3.

Hara, Yuki et al. (2009) International Worm Meeting "Cell-size-dependent spindle elongation in the Caenorhabditis elegans early embryo."

Hara, Yuki, Kimura, Akatsuki

[International Worm Meeting, 2009]

Cell division occurs in cells of various sizes. For appropriate chromosome segregation during anaphase, the mitotic spindle must elongate in a cell-size-dependent manner. Here, we quantified the relationship between spindle elongation and cell size in the Caenorhabditis elegans early embryo and propose possible models for cell-size-dependent spindle elongation. First, we measured the dynamics of spindle elongation quantitatively in cells with various sizes. We found that the extent and speed of spindle elongation are proportional to the cell size. To explore the mechanism underlying the cell-size-dependent spindle elongation, we investigated the role of Ga activity-sensitive force pulling astral microtubules toward the cortex by using RNAi knockdown of gpr-1/2 (G protein regulator). In the gpr-1/2 (RNAi) embryos, the spindles failed to fully elongate in cells with various sizes and the speed of spindle elongation was almost constant regardless of the cell size. The results indicated that the Ga activity-sensitive cortical pulling force is involved in the cell-size-dependent spindle elongation. In addition, this quantification demonstrated that Ga activity-insensitive force contributes to the spindle elongation as well. Next, to obtain insights into the mechanisms of Ga activity-sensitive and -insensitive force, we evaluated possible models through numerical modeling and experiments. A force generator-limited model, which is based on a previous estimation of the number of Ga activity-sensitive force generators [1], reproduced characteristics of the Ga activity-sensitive mechanism. A constant pulling model, which assumes saturated number of weak force generators at the cortex, reproduced the characteristics of the Ga activity-insensitive mechanism. When we performed a simulation using these two models together, the in vivo cell-size-dependent spindle elongation behavior could be explained. Thus, we propose that these two models act in concert to elongate the mitotic spindle in a cell-size-dependent manner. At this meeting, we will present our recent analyses on the molecular bases and theoretical features of the models. [1] Grill, S.W., Howard, J., Schaffer, E., Stelzer, E.H., and Hyman, A.A. (2003) Science, 301, 518-521.

Patikoglou GA et al. (1998) East Coast Worm Meeting "Two regulators of G protein signaling with similar expression patterns have opposite effects on egg laying"

Koelle MR, Patikoglou GA, Sigler PB

[East Coast Worm Meeting, 1998]

RGS (Regulator of G protein Signaling) proteins were identified through genetic studies as negative regulators of G protein signaling (reviewed in Koelle, 1997). Degenerate PCR and genomic sequence analyses predict the existence of many RGS proteins both in mammals and in C. elegans. In vitro studies using randomly chosen pairs of RGS and Ga proteins show that RGS proteins are potent GTPase Activating Proteins (GAPs), accelerating the slow intrinsic rate of GTP hydrolysis by Ga proteins and thus converting them to their inactive GDP-bound forms. Surprisingly, these assays show that a number of RGS proteins have promiscuous GAP activity for the same set of Ga proteins in vitro. However, little is known about the specificity of RGS proteins in vivo. One possibility is that each RGS protein is expressed in a restricted set of cells and interacts with many Ga proteins in those cells. Another possibility is that RGS proteins are widely expressed but are not promiscuous in vivo, and have specificity for only one or a few physiologic Ga targets. While all RGS proteins contain a core RGS domain that is required for GAP activity, a subset of RGS proteins also contain an additional N-terminal conserved region of unknown function. In C. elegans, this subset consists of the EGL-10 and C16C2 RGS proteins. EGL-10 was the first C. elegans RGS protein to be genetically characterized and was shown to inhibit the Ga protein GOA-1, which in turn inhibits egg laying (Koelle and Horvitz, 1996). We overexpressed all worm RGS proteins by generating 11 types of transgenic worm strains carrying multiple copies of the genomic DNA for each of the 11 RGS genes. We found that overexpression of EGL-10 increases the frequency of egg laying, while overexpression of C16C2 decreases it. These results suggest that C16C2 may inhibit signaling by the Ga protein EGL-30, which is an activator of egg laying. Furthermore, GFP reporter transgenes for EGL-10 and C16C2 indicate that they are both expressed in most or all neurons, as are the G proteins EGL-30 and GOA-1. Thus, it is possible that EGL-10 and C16C2 function in the same cells but are biochemically specific for different Ga protein subunit partners to achieve different functions. Our hypothesis is that the N-terminal conserved region may confer specificity for the correct Ga protein and thus direct GAP activity only to the appropriate protein target. To test this idea, we are conducting domain swapping experiments between C16C2 and EGL-10 to see if they result in a specificity swap in vivo. We are also analyzing the functions of the two conserved RGS protein domains by expressing them individually in C. elegans to assess their effect on egg laying. In addition, biochemical and X-ray crystallographic methods will be used to determine how the two conserved regions of the EGL-10 protein contribute to its interaction with GOA-1. Koelle, M. R. (1997) A new family of G-protein regulators - the RGS proteins. Curr Opin in Cell Biol, 9, 143-7. Koelle, M. R. and Horvitz, H. R. (1996) EGL-10 Regulates G Protein Signaling in the C. elegans Nervous System and Shares a Conserved Domain with Many Mammalian Proteins. Cell, 84, 115-125.

Patikoglou GA et al. (2000) East Coast Worm Meeting "G-protein beta-2 regulates the activity of two RGS proteins"

Chase DL, Patikoglou GA, Koelle MR

[East Coast Worm Meeting, 2000]

Heterotrimeric G proteins are signaling molecules that permit cells to adapt to changing extracellular conditions. Termination of the G protein signal is stimulated by a class of GTPase activating proteins known as regulators of G protein signaling or RGS proteins. We want to understand how RGS proteins select their proper G protein target in vivo. The Ga proteins GOA-1 and EGL-30, while normally expressed in the same cells of the C. elegans nervous system, have opposite effects on several behaviors including egg laying. Genetic analysis has identified two RGS proteins, EGL-10 and EAT-16, that negatively regulate GOA-1 and EGL-30, respectively. Such specificity of RGS proteins for Ga is not generally observed in vitro and encouraged further study of these signaling pathways. EGL-10 and EAT-16 share a region of similarity to Gg proteins known as the G-gamma-like or GGL domain. Mammalian GGL-containing RGS proteins have been shown to bind to the unique Gb protein, Gb5. To determine if the C. elegans ortholog of Gb5, GPB-2, plays a role in G protein signaling, or if GPB-2 contributes to signaling specificity, we isolated a deletion allele of gpb-2. This allele, missing the first two exons and 500 bases of promoter sequence (and thus a presumptive null allele), has little affect on egg-laying behavior in a wild-type background apparently because it has equal and opposing effects through EGL-10 and EAT-16. In mutants of either egl-10 or eat-16 in which we can isolate one RGS protein in the absence of the other, the gpb-2 deletion allele has dramatic effects. These effects are consistent with GPB-2 being required for full activity of these RGS proteins. If EGL-10 and EAT-16 physically interact with GPB-2 there may exist novel types of heterotrimeric complexes consisting of Ga, GPB-2 and RGS protein. Activation of these complexes by a receptor would then liberate GTP-bound Ga from the Gb-RGS complex. One might imagine that the proximity of the active Ga and Gb-RGS molecules could lead to a dramatically increased rate of GTP hydrolysis and hence termination of the Ga signal. We are currently using in vitro biochemical analyses of purified proteins to independently establish the interaction of GPB-2 with both EGL-10 and EAT-16. If these complexes form we will also determine the effect of GPB-2 binding on RGS activity and Ga subunit selection.

Fielmich, Lars-Eric et al. (2017) International Worm Meeting "Spatiotemporal control of gene knock-out and protein-protein heterodimerization to study mitotic spindle positioning."

Akhmanova, Anna, Fielmich, Lars-Eric, Van den Heuvel, Sander, Schmidt, Ruben

[International Worm Meeting, 2017]

Tissue development and homeostasis depend on the correct axis and plane of cell division, which are determined by the position of the mitotic spindle. Studies of the C. elegans one-cell embryo have provided much insight in the regulation of spindle positioning during asymmetric cell division. Pulling forces that act from the cell cortex on astral microtubules were found to position the spindle. These pulling forces depend on a force-generating complex (FG) that anchors the dynein motor to the cortex. The FG is a conserved complex consisting of a membrane-bound Ga protein that, in the GDP-bound form, binds the GPR-1/2 linker protein, which interacts with the LIN-5 coiled-coil protein. The discovery of a non-canonical role of Ga as a membrane-anchor for the FG was surprising at the time, and still is only partly understood. The Guanine-nucleotide Exchange Factor (GEF) RIC-8 and GTPase-Activating Protein (GAP) RGS-7 regulate the GDP/GTP cycle of Ga and are also required for proper force generation. It remains unclear how this corresponds to Ga functioning as a membrane anchor. Hampering analysis, complete inactivation of ric-8 and rgs-7 appears difficult to achieve by RNAi and null mutants are not viable. We therefore developed an inducible, tissue-specific gene knock-out system based on Cre-Lox recombination. We introduced Lox sites in the endogenous ric-8 and rgs-7 loci to enable KO of the genes. To assure spatiotemporal control of the KO, we use a two-component recombinase system. Thus, gene KO can be induced in the tissue of choice, for example the germline, while maintaining a healthy strain. A complementary approach focuses on the individual functions of FG components. Hereto, we use the light-inducible heterodimerization of LOV and ePDZ to reconstitute cortical FG complexes that lack one or several components. Using a PH::LOV as membrane anchor and an ePDZ::dynein knock-in, we were able to recruit endogenous dynein directly to the membrane and bypass the FG. This was insufficient for pulling force generation and suggests that the FG is more than a dynein anchor. To test the role of Ga signaling, we generated and are currently analyzing an ePDZ::GPR-1 knock-in to reconstitute a cortical FG that lacks the Ga component. We will present our latest results at the meeting.

Park, Dae Hwi et al. (2011) International Worm Meeting "LET-99, a novel G protein regulator for asymmetric division."

Ye, Anna, Rose, Lesilee S., Park, Dae Hwi

[International Worm Meeting, 2011]

Asymmetric divisions that generate cell diversity are required for normal development and stem cell maintenance. Spindle positioning is a key aspect of asymmetric division. The mitotic spindle must be aligned with the axis of cell polarity in order for cell fate determinants to be differentially segregated to daughter cells to give them different fates. The conserved PAR polarity proteins establish cell polarity, and they also regulate spindle movements via a complex involving Ga subunits, GPR and LIN-5. The Ga/GPR/LIN-5 complex is necessary for the cortical forces the pull on astral microtubules, potentially by recruiting regulators of the microtubule motor dynein. In C. elegans one-cell embryos, GPR and LIN-5 are asymmetrically localized in a dynamic pattern that correlates with changes in the pulling forces. GPR and LIN-5 first show an overall anterior enrichment during prophase, when pulling forces center the nuclear-centrosome complex and rotate it on to the polarity axis. GPR/LIN-5 enrichment then switches to the posterior during the spindle displacement movements that produce an unequal division. We previously showed that LET-99 is a key component of this pathway and acts downstream of the PAR proteins during both nuclear centration/rotation and spindle displacement. LET-99 antagonizes G protein signaling by inhibiting the localization of GPR at the cortex. The highest levels of LET-99 are present in a posterior-lateral cortical band, which results in the lowest levels of cortical GPR in this region; nonetheless, LET-99 function is also needed for the overall anterior and posterior enrichment of GPR through the cell cycle. To determine the mechanism of LET-99 action, we tested LET-99 for interaction with other components of the pathway. In vitro pull down and yeast two-hybrid assays show that LET-99 can interact with GOA-1 and GPA-16, the two Ga subunits used in this system. Direct binding of LET-99 to Ga could directly interfere with formation of a Ga/GPR/LIN-5 complex; alternatively, LET-99 could regulate an upstream step involving other pathway components such as Gb or RIC-8. To further elucidate the molecular mechanism of LET-99 action, we are carrying out additional genetic and interaction studies with Gb and Ric-8.

Milan, Jennifer A. et al. (2013) International Worm Meeting "LET-99 regulates G protein signaling and spindle positioning during asymmetric division."

Milan, Jennifer A., Park, Dae Hwi, Rose, Lesilee S.

[International Worm Meeting, 2013]

Asymmetric divisions that generate cell diversity are required for normal development and stem cell maintenance. During asymmetric division, the mitotic spindle is aligned with the cell polarity axis so that cell fate determinants are differentially segregated to daughter cells, giving them different fates. The conserved PAR polarity proteins establish cell polarity, and regulate spindle movements via a complex involving Ga subunits, GPR, and LIN-5. The Ga/GPR/LIN-5 complex is necessary for the cortical forces that pull on astral microtubules by recruiting regulators of the microtubule motor dynein. In C. elegans one-cell embryos, GPR and LIN-5 are present at higher levels in the anterior during prophase, when pulling forces center the nuclear-centrosome complex and rotate it onto the polarity axis. During the subsequent spindle displacement movements, the cortical localization of GPR and LIN-5 becomes bipolar, with more at the posterior cortex. We previously showed that LET-99 acts downstream of the PAR proteins during both nuclear centration/rotation and spindle displacement. LET-99 antagonizes G protein signaling by inhibiting the localization of GPR at the cortex. The highest levels of LET-99 are present in a posterior-lateral cortical band, which results in the lowest levels of GPR in this region; LET-99 is also needed for the overall anterior and posterior enrichment of GPR throughout the cell cycle. Gbg sequesters Ga in an inactive state, and in gpb-1 mutants GPR is uniform on the cortex, as in let-99 mutants. Additionally, RIC-8 has been shown biochemically to be needed for Ga/GPR/LIN-5 complex formation. To gain more insight into the mechanism of LET-99 action, we compared the let-99 and gpb-1 phenotypes and performed double mutant analysis with ric-8. Consistent with previous reports we found that loss of Gb bypasses the need for RIC-8 activity in terms of force generation. In contrast, loss of LET-99 does not bypass RIC-8. This and other genetic analyses suggest that LET-99 action requires Gb, and that LET-99 acts upstream of Ga/GPR/LIN-5 complex formation. To further elucidate the molecular mechanism of LET-99 action, we are testing LET-99 for interactions with the components of the pathway.

Dayadevi Jirage et al. (2004) East Coast Worm Meeting "Structural and functional studies of the C.elegans Hsp90 ortholog DAF21"

Dayadevi Jirage, Harold Smith

[East Coast Worm Meeting, 2004]

The molecular chaperone Hsp90 is required for the function of multiple eukaryotic growth regulatory signaling proteins and in the cellular response to stress. The protein sequences of Hsp90s are highly conserved across species. Hsp90 protein is the target of the antitumor drug geldanamycin (GA) - a derivative of which is currently in clinical trials for cancer treatment. C. elegans possesses a single ortholog of Hsp90 called Daf21, whose protein sequence is 74% and 76% identical to human Hsp90 alpha and beta respectively. Despite this conservation in protein sequence, DAF21 is the only Hsp90 examined to date that does not bind GA. The first goal of our research is to determine the 3-dimensional structure of DAF21 in order to identify the structural features that confer this resistance to GA. Our second goal is to test the function of human Hsp90 in C. elegans daf21 mutants (RNAi and deletion) in order to develop a C. elegans -based system for drug assays and screening. For the structural studies, we have expressed His-tagged versions of the N-terminal domain (shown to be involved in GA-binding in other species) as well as the full-length DAF21 in E.coli , and purified the proteins to homogeneity using Ni-column affinity chromatography followed by ion-exchange chromatography. The structure of these proteins will be determined using X-ray crystallography and NMR-spectroscopy. For the functional studies, we first performed RNAi of daf21 using the feeding method. We observed a range of phenotypes - slow growth, embryonic lethality, vulval defects, dauer-like appearance and sterility. We made constructs comprising cDNAs of the human Hsp90 alpha and daf-21 under the control of the daf-21 promoter. We have created transgenic worms expressing these plasmids using microparticle bombardment. We are currently analyzing these transgenic worms for complementation of the RNAi phenotypes.

Plasterk RHA et al. (2000) European Worm Meeting "Extensive modulation of G-protein mediated signaling in olfaction"

Plasterk RHA, Jansen G

[European Worm Meeting, 2000]

C. eleganscan detect at least 60 volatile attractants and discriminate among many of them using only two pairs of sensory neurons, AWA and AWC. This complex task is probably accomplished by the expression of multiple different chemosensory receptors per cell and the presence of 6 different heterotrimeric G-protein a subunits in the 2 pairs of olfactory neurons: gpa-2, 3, 5, 6, 13 and odr-3. However, loss-of-function of only odr-3 resulted in significantly altered odorant perception. What would be the function of the other 5 a subunits? Is there functional redundancy between the Ga proteins? Do they have negative regulatory functions? Or are they not involved in the perception of the odorants tested? To determine the interactions between these V Ga genes we generated animals that had lost the function of 2 to 5 Ga subunits and tested their behavior in odorant chemotaxis. Our results show that C. elegans uses ODR-3 as main stimulatory signaling route. 2 to 4 G a proteins modulate this signal. These a subunits may have stimulatory or inhibitory roles, depending on the odorant and its concentration. E.g. pyrazine detection uses both GPA-3 and ODR-3 as stimulatory signals, and GPA-2, GPA-5 and GPA-6 as redundant negative regulators. Our findings reveal extensive modulation of what seemed a simple G-protein mediated signal transduction cascade.

Kagias, Konstantinos et al. (2015) International Worm Meeting "GPA-9, a G-protein alpha subunit, regulates a food preference decision."

Liu, He, Kagias, Konstantinos, Choi, Myung-kyu, Harris, Gareth, Zhang, Yun

[International Worm Meeting, 2015]

G-protein alpha (Ga) subunits are important protein components that act in the signal transaction downstream of G protein-coupled receptors (GPCRs). C. elegans has 21 Ga subunit-encoding genes, with each gene expressed in distinct sets of neurons as well as in other tissues. We identified one of them, namely GPA-9, as a regulator of food preference in worms. In a chemotaxis assay using two different bacterial strains, a non-pathogenic E. coli strain - OP50 - and a pathogenic P. aeruginosa - PA14 - we observe a biased migration of the gpa-9 (pk348) mutant worms towards the non-pathogenic OP50. The wild type animals show no preference between these two foods. Despite this bias in preference, the gpa-9 (pk348) mutant worms do not have any obvious defects in smelling OP50 and PA14. This indicates that the observed phenotype might be due to a change in processing of the two food odors linked to generating a locomotion decision. Here, we report on our findings towards understanding the mode of action of GPA-9 during this food-preference paradigm. .