Go, Aiza et al. (2009) International Worm Meeting "The Conserved Glc7/PP1 Phosphatases GSP-3 and GSP-4 Regulate Sperm Morphogenesis."

Chu, Diana, Mirsoian, Susan, Go, Aiza, Samson, Mark, Wu, Jui-Ching, Zhou, Hongkang

[International Worm Meeting, 2009]

Sperm have evolved widely distinct modes of motility. Mammalian sperm are flagellar and rely on actin for cytoskeletal function, whereas C. elegans sperm are ameboid and utilize MSP for motility. Nevertheless, they share developmental events important for sperm function, like meiosis and the tight compaction of DNA that results in transcriptional inactivity. Thus, sperm rely heavily on post-translational regulation for morphogenesis into migration of competent cells. Interestingly, Glc7/PP1 phosphatases are required for male fertility in mouse, with roles in both sperm meiosis and sperm morphogenesis. We identified two Glc7/PP1 phosphatases, GSP-3 and GSP-4 that are 98% identical and function redundantly in hermaphrodite and male fertility. Cytological and biochemical analyses have confirmed that GSP-3 and GSP-4 are sperm specific. Though we have identified defects in chromosome segregation in gsp-3gsp-4 mutants, we also investigated the role of these proteins in sperm morphogenesis. Our studies revealed that the sterility of gsp-3 gsp-4 mutants is indeed caused by a failure of sperm morphogenesis and activation. First, in sterile gsp-3gsp-4 hermaphrodites, immotile hermaphrodite sperm block the spermatheca and prevent exogenous sperm from accessing the oocyte. However, when gsp-3gsp-4 hermaphrodites are manipulated to not produce sperm, they could be fully fertilized by wild type male sperm. Furthermore, we examined the morphology of gsp-3gsp-4 mutant male sperm and found they exhibit abnormal sizes and shapes. The gsp-3gsp-4 sperm also show severe defects in morphogenesis. Instead of growing pseudopods on one side of the cell, random protrusions form all over gsp-3gsp-4 sperm. Upon artificial activation, these protrusions can undergo such extreme extension that sperm occasionally explode. Thus GSP-3 and GSP-4 play an important role in regulating pseudopod formation and sperm motility. Further supporting this, we found that GSP-3 and GSP-4 localize as a crescent shape on the periphery of mature but inactive sperm, reminiscent of the asymmetric localization of the pseudopod. In activated sperm, GSP-3 and GSP-4 then shifts to colocalize with MSP only in pseudopods. Our current model is that GSP-3 and GSP-4 regulates sperm activation and pseudopod formation through modulating MSP dynamics. We are now conducting coimmunoprecipitation experiments to find interacting partners of GSP-3 and GSP-4 that target them to specific subcellular domains to function in either sperm meiosis or morphogenesis. Our studies will provide insight on how these evolutionarily conserved PP1 phosphatases are adapted as a core regulator for sperm formation and activity by otherwise morphologically different sperm.

Aiza Go et al. (2008) Development & Evolution Meeting "Dual Roles of the Conserved Glc7/PP1 Phosphatases, GSP-3 and GSP-4, in Chromosome Dynamics and Sperm Activation"

Diana Chu, Aiza Go, Susan Mirsoian, Bernadette Nera, Mark Samson

[Development & Evolution Meeting, 2008]

Reproduction depends on sperm accomplishing two goals: traveling to the oocyte and delivering intact paternal DNA. During sperm formation, DNA first undergoes meiosis, then tight compaction that excludes transcription. Thus sperm rely heavily on post-translational regulation to become motile. Glc7/PP1 phosphatases are required for male fertility in mouse, with roles in sperm meiosis and sperm activation required for motility. We identified two Glc7/PP1 phosphatases in C. elegans called GSP-3 and GSP-4. Preliminary analysis has shown GSP-3 and GSP-4 are important for sperm meiosis; however, their role in sperm activation and motility is unknown. GSP-3 and GSP-4 are 98% identical and function redundantly. Single gsp-3delta or gsp-4delta mutants display only slight decreases in fertility compared to wild-type. However, gsp-3delta gsp-4delta double mutants are completely sterile. To determine GSP-3 and GSP-4 function in sperm for fertility, gsp-3delta gsp-4delta males were mated with fog-2 mutant hermaphrodites that produce only oocytes. Though fog-2 mated with wild-type produced progeny, none were generated when fog-2 was mated with gsp-3delta gsp-4delta males. Thus GSP-3 and GSP-4 are required for sperm function. To determine GSP-3 and GSP-4 function in oocytes, wild-type males were mated with gsp-3delta gsp-4delta hermaphrodites. Mating could be achieved; however, fertility was not fully rescued suggesting a factor blocks wild-type sperm from fertilizing oocytes. This factor may be immotile sperm from the mutant hermaphrodite. In fact, we have observed that gsp-3delta gsp-4delta sperm exhibit abnormal size, shape, and activation compared to wild-type sperm. Further supporting dual roles for GSP-3 and GSP-4, immunostaining shows that GSP-3 and GSP-4 localize around sperm chromatin during later stages of spermatogenesis as well as a crescent shape pattern on the cell periphery, reminiscent of the asymmetric localization of the pseudopod. Because PP1 phosphatases work in concert with regulatory subunits, we are conducting both immunolocalization and coimmunoprecipitation experiments to find interacting partners of GSP-3 and GSP-4 that target them to specific sites for function. The finding that these phosphatases have conserved function between C. elegans and mammals is remarkable, considering that mammalian sperm are flagellar and rely on actin for cytoskeletal function, whereas C. elegans sperm are ameboid and utilize MSP for motility.

Pin-An Chen et al. (2006) Neuronal Development, Synaptic Function, and Behavior Meeting "Identification of an activated allele of Go alpha"

Pin-An Chen, Erik Jorgensen

[Neuronal Development, Synaptic Function, and Behavior Meeting, 2006]

Neurotransmission can be regulated by G protein pathways. In C. elegans, Gq signaling is the main pathway that stimulates neurotransmitter release. Go plays an inhibitory role in neurotransmission, possibly by negative regulating Gq . However, the downstream effectors of Go and the linkage between Go and Gq are still unclear. We cloned the gene mutated in a dominant paralyzed mutant, unc-109(n499). We found that unc-109(n499) has a R179C mutation in Go alpha;, encoded by goa-1. This arginine residue has been shown to be important for GTPase activity of G alpha; suggesting that unc-109(n499) is an activated allele of goa-1. The phenotype of unc-109(n499) is what we would predict for an activated Go alpha;. First, the phenotype of n499 worms is remarkably similar to null alleles of Gq alpha;: worms are paralyzed and have a straight body posture. Second, activated alleles of Gq alpha;, encoded by egl-30 (tg26), can suppress the lethality of n499 homozygotes and the uncoordinated movement of the n499 heterozygote. Third, mutations in a potential downstream target of Go, DAG kinase, also suppresses n499. Thus, unc-109(n499) is a great tool to dissect the Go pathway by looking for suppressors. We have screened 24,000 haploid genomes and obtained 18 candidates. We are now characterizing and mapping these suppressor candidates.

Mendel JE et al. (1995) International C. elegans Meeting "The Heterotrimeric G protein, Go, regulates several C. elegans behaviors"

Mendel JE, Plasterk RHA, Korswagen HC, Liu KK, Simon MI, Sternberg PW, Hajdu-Cronin YM

[International C. elegans Meeting, 1995]

The C. elegans gene encoding the alpha subunit of the heterotrimeric G protein Go, goa-1, is expressed in most neurons, the pharynx, and in muscles involved in defecation, egg-laying, and male mating. The reduction of function mutations, goa-1(pk62) and goa-1(sy192), cause a variety of behavioral defects including hyperactive movement, premature egg-laying, and male impotence. Expression of activated Go alpha subunit in transgenic nematodes results in lethargic movement, slow pharyngeal pumping in the presence of food, irregular defecation, delayed egg-laying, and reduced mating efficiency. Induction of activated Go alpha subunit by heat-shock at any developmental stage results in rapid arrest of movement, pumping, and in adults, egg production and egg-laying. Go is thus involved in neuron and muscle function throughout C. elegans life. We are investigating the role of Go alpha subunit in regulating behavior by analysis of presumed dominant negative mutations, epistatic interactions between goa-1 and other G protein subunit genes, and the isolation and analysis of extragenic suppressor mutations.

Cintra, Thais Godoy et al. (2011) International Worm Meeting "GSP-3 and GSP-4 are required for sperm meiotic chromosome segregation in C. elegans."

Go, Aiza, Cintra, Thais Godoy, Wu, Jui-Ching, Chu, Diana

[International Worm Meeting, 2011]

Errors in meiotic chromosome segregation during spermatogenesis have serious consequences: male infertility, embryonic lethality, and developmental abnormalities. Meiosis is distinctly regulated in spermatocytes and oocyes to control the developmental timing, size, and number of gametes produced. However, the molecular factors that differentiate chromosome segregation events during sperm meiosis from oocyte meiosis or mitosis are largely unknown. We have found that two nearly identical PP1 phosphatases in C. elegans, GSP-3 and GSP-4 (GSP-3/4) are required for male infertility and sperm meiosis. Using live-imaging of histone-GFP tagged strains deleted for both gsp-3/4, we find multiple defects in chromosome segregation during sperm meiosis. First, in the absence of GSP-3/4 during meiosis I, the partitioning of the X chromosome is delayed, causing the X chromosome to become stretched before finally resolving to one pole. Second, in the second meiotic division, sister chromatids are unable to segregate properly, forming bridged nuclei. These bridged nuclei either end up in the same spermatids or the residual body, a repository for cellular components, resulting in either multinucleated sperm or anucleated sperm, respectively. AIR-2 (Aurora kinase) and REC-8 (meiotic cohesin) are normally localized and removed in gsp-3/4 double mutant spermatids, confirming that cohesin removal does not contribute to chromosome segregation defects. Immunolocalization experiments show GSP-3/4 localization around sperm meiotic chromosomes in a pattern similar to that of kinetochore proteins, key connectors of chromosomes and microtubules. The chromosome segregation defects and localization pattern of GSP-3/4 suggest these PP1 phosphatases may regulate chromosome-microtubule dynamics. Thus, we are investigating microtubule dynamics during sperm meiosis in the absence of GSP-3/4. In addition, we are also characterizing the role of GSP-3/4 regulation of kinetochore components in sperm meiosis. In particular, we are investigating changes in HCP-2 and HCP-4 localization, as we have previously shown they are differentially enriched in sperm chromatin compared to oocyte chromatin.

Kaplan JM et al. (2000) West Coast Worm Meeting "Is the DAG kinase DGK-1 an effector of Go alpha (GOA-1)?"

Dybbs M, Kaplan JM, Nurrish SJ

[West Coast Worm Meeting, 2000]

We (and others) have previously described two competing G-protein pathways acting in motor neurons to either facilitate or inhibit synaptic transmission at neuromuscular junctions (NMJs) [1-4]. Facilitation of release occurs via a pathway composed of a Gq alpha (EGL-30), a phospholipase C beta (EGL-8), and the diacylglycerol (DAG) binding protein UNC-13 [2,4]. The Gq alpha pathway can be activated by muscarinic agonists leading to the formation of the membrane bound second messenger DAG by EGL-8 and the subsequent recruitment of UNC-13 to sites of Acetylcholine release [2]. UNC-13 is essential for neurotransmitter release and it's enrichment at release sites correlates with an increase in neurotransmitter release. Addition of serotonin agonists inhibits acetylcholine release at NMJs. Inhibition of synaptic transmission by serotonin requires Go alpha (GOA-1) and a diacylglycerol kinase (DGK-1) which phosphorylates DAG to Phosphatididic Acid which is unable to bind UNC-13. Serotonin antagonists and goa-1 mutations result in the recruitment of UNC-13 to NMJs suggesting that the Gq and Go pathways converge to regulate UNC-13 localization, most likely by regulating levels of DAG [1,2]. As yet the Go signaling pathway is not well defined. Go may reduce DAG levels by activating DGK-1, by negatively regulating the Gq signaling pathway, or by an as yet undefined pathway. We are currently testing the first of these models. Co-expression of DGK-1 and activated Go alpha in human tissue culture cells (HEK cells) has no effect on the kinase activity of DGK-1. This suggests that there is no direct interaction between Go alpha and DGK-1. However, it is possible that Go alpha indirectly regulates DGK-1 via intermediates that are not conserved in HEK cells. Therefore we have generated a rescuing MYC tagged GFP::DGK-1 construct. The myc-DGK-1 protein can be immunoprecipitated from adult animals and possesses DAG kinase activity. We are currently testing whether the DGK-1 DAG kinase activity changes in response to levels of Go alpha signaling. We are also testing whether Go regulates the subcellular localization of DGK-1. Nurrish et al.,(1999) Neuron:24 p231-242 Lackner et al.,(1999) Neuron:24 p335-346 Hajdu-Cronin et al.,(1999) Genes Dev 13: 1780-93 Miller et al.,(1999) Neuron 24: 323-33

Daniel L Chase et al. (2003) International Worm Meeting "Dopamine-resistant mutants of C. elegans identify components of Go and Gq signaling pathways"

Daniel L Chase, Judy S Pepper, Michael R Koelle

[International Worm Meeting, 2003]

Dopamine, a major neurotransmitter in the mammalian central nervous system, acts through two classes of G protein-coupled receptors known as D1-like and D2-like receptors. These two classes of receptor signal to antagonistically regulate neural activity. Despite intensive research, the signaling mechanisms responsible for the antagonistic effects of dopamine in the brain remain debated. We describe the first genetic screen designed to identify proteins required for dopamine signaling. C. elegans uses endogenous dopamine to inhibit locomotion behavior1, and treatment with excess exogenous dopamine paralyzes worms2. We isolated mutants unresponsive to exogenous dopamine and found that they are also defective for endogenous dopamine signaling. Eleven mutations identified five different genes. Four of these genes encode components of the Go and Gq signaling pathways, including the G protein Go itself and subunits of the RGS complex that inhibits Gq signaling. The Go and Gq signaling pathways act antagonistically in C. elegans to control behavior and their components are conserved in humans. Mutations in these signaling components that decrease Go signaling or increase Gq signaling cause dopamine resistance, while mutations that have opposite effects on these two signaling pathways cause dopamine hypersensitivity. Thus the physiological effects of dopamine are mediated by Go in C. elegans and are antagonized by Gq signaling. Such a dopamine signaling mechanism in mammals could explain many of the opposing effects of D1-like and D2-like receptor activation. Our screen also identified one gene that appears to encode a new Go/Gq signaling component. 1. Sawin, E.R., Ranganathan, R., and Horvitz, H.R. (2000). Neuron 26, 619-631; 2. Schafer, W.R., and Kenyon, C.J. (1995). Nature 375, 73-78.

Sarah Straud Anselmo et al. (2002) West Coast Worm Meeting "Developing a screen for HERG blocking drugs using the C. elegans pharynx"

Sarah Straud Anselmo, Leon Avery

[West Coast Worm Meeting, 2002]

The HERG (human ether-a-go-go related gene) potassium channel is expressed in the human heart and is responsible for proper repolarization of cardiac muscle at the end of the action potential. Compromising HERG function gives rise to Long QT Syndrome, a disorder in which one is predisposed to ventricular fibrillation and sudden death. Many drugs, including Seldane (an anti-histamine) and Propulsid (an acid reflux inhibitor), block HERG as an unwanted side effect and consequently have been taken off of the market.

Nurrish SJ et al. (1996) East Coast Worm Meeting "Downstream of goa-1; characterization of a serotonin signaling pathway."

Elkes DA, Nurrish SJ, Segalat LS, Kaplan JM

[East Coast Worm Meeting, 1996]

Addition of the neurotransmitter serotonin to C.elegans results in a reduction of locomotion and foraging behaviors; in contrast, mutations which reduce levels of endogenous serotonin cause animals to locomote and forage hyperactively. To identify genes required for serotonin signaling we have identified mutants displaying hyperactive locomotion and foraging behaviors which could not be corrected by the addition of exogenously added serotonin. These mutants are likely to define genes encoding proteins required at or downstream of the serotonin receptor; 17 such serotonin resistant mutants have been identified. Two of the genes represented by these mutants have been cloned, the first of which, the goa-1 gene encodes the alpha subunit of the G-protein Go, demonstrating that serotonin modulates behavior via a G-protein coupled receptor. Goa-1 mutants are hyperactive for locomotion, whereas transgenic animals that overexpress Go alpha mimic some of the effects of adding exogenous serotonin, i.e. display a severely reduced rate of locomotion . These results demonstrate that the levels of Go alpha are critical for the control of at least some serotonin modulated C.elegans behaviors. A second serotonin resistant mutant has identified the dgk-1 gene which encodes a diacylglycerol kinase(DGK) . In Drosophila DGK has been shown to be required for signaling downstream of light activated rhodopsin in a pathway that also requires the Gq alpha subunit and Phospholipase C (PLC). These results suggest a model in which at least some of the behavioral effects of serotonin are mediated by activation of PLC, perhaps via Go alpha. We will test this model by identifying further components of the serotonin signaling pathway. As a first step we have determined which serotonin resistant mutants are able to suppress the reduced locomotion phenotype associated with overexpression of goa-1. We are also testing the ability of the serotonin resistant mutants to suppress the reduced locomotion defect of animals expressing a constitutively active Go alpha mutant expressed from a heat shock promoter (kindly provided by P.Sternberg). Those mutants which suppress the defects resulting from overexpression of both wild type and constitutively active Go alpha are likely to define effectors downstream of Go alpha and will be further characterized.

Elkes DA et al. (1995) International C. elegans Meeting "SEROTONIN SIGNALING AND G-ALPHA O"

Kaplan JM, Segalat LS, Elkes DA

[International C. elegans Meeting, 1995]

We are interested in identifying the molecules required for serotonin (5-HT)signaling. Mutants that have low levels of 5-HT(e.g.cat-4) locomote and forage hyperactively. Therefore, to identify effectors of 5-HT signaling, we screened for hyperactive mutants whose hyperactivity is resistant to exogenous 5-HT. 28 mutants corresponding to at least 13 different genes fall into this category. In addition to the mutants we isolated, the following pre-existing mutations were also shown to disrupt 5-HT signaling: egl-21 IV, phm-2 I, and the allelic mutations n1134 and n363 I, which were isolated by Ambros and Horvitz. We have cloned the gene defined by n1134 andn363 and determined that it corresponds to goa-1, which encodes the alpha subunit of the heterotrimeric G-protein Go. Several lines of evidence suggest that Go mediates signaling by 5-HT. First, null mutations and overexpression of goa-1 cause reciprocal defects in three 5-HT controlled behaviors: locomotion, egg laying, and feeding. This indicates that the level of Go alpha activity is a critical determinant of these behaviors. Second, goa-1 mutants are resistant to the effects of 5- HT on locomotion and defecation but not feeding. Thus for feeding, 5-HT may act through other G proteins as well as Go for signaling. Third, goa-1 mutants are fully sensitive to other metabotropic agonists. Interestingly, Go appears to act differently in locomotion and egg laying. For locomotion, goa-1 animals behave like animals that lack 5-HT whereas for egg laying these mutants behave like worms in exogenous 5-HT. We propose that 5-HT inhibits locomotion and defecation via G-alpha o (hence goa-1 mutants lack signaling), whereas 5-HT stimulates egg laying through Go beta gamma (hence goa-1 mutants constitutively signal). Studies in mammalian neurons indicate that G- alpha o may play a role in axon outgrowth or guidance. We found that nearly all axons are normal in goa-1 mutants. However, a small but significant portion of HSN neurons have defects in cell and axon migration. Thus, in C. elegans, Go has a relatively modest role in axon guidance.