Michelle Po et al. (2007) International Worm Meeting "An unc-7 suppressor screen to identify novel active zone regulators."

Taizo Kawano, Michelle Po, Mei ZHEN, Magali BOUHOURS

[International Worm Meeting, 2007]

Synapses are asymmetric structures that facilitate the transmission of signals between neurons and their targets. The mature synapse consists of a presynaptic terminal in direct apposition to a postsynaptic terminal. Neurotransmission occurs when neurotransmitter-filled presynaptic vesicles dock and fuse with the plasma membrane. The neurotransmitter contents are released into the synaptic cleft and subsequently bind to receptors at the postsynapse. The active zone is an electron-dense region of the synapse that is required for the docking and fusion events of synaptic transmission. Our lab is interested in identifying genes that regulate the formation of active zones. In our study we are using the GABAergic nervous system of C. elegans as our model system. The active zones in this nervous system are visualized using a unique active zone specific marker SYD-2::GFP that was developed in our laboratory (Yeh et al., 2006). A forward genetic screen isolated an unc-7 loss-of-function mutant that shows a significant decrease in number of SYD-2::GFP puncta. We have determined that the innexin UNC-7 localizes to perisynaptic regions in addition to gap junctions. Subsequently, we showed that loss-of-function unc-9 mutants, which encodes an innexin protein that is 56% identical to UNC-7, shows the same synaptic defects as unc-7 mutants (Yeh and Ng, unpublished), suggesting a novel regulatory role for innexins at chemical synapses. We further investigated the role of innexins at the synapse by performing an unc-7 suppressor screen. We identified 10 dominant alleles of unc-1, a gene that encodes a stomatin homologue, that suppress the kinker behavior as well as the synaptic phenotype of unc-7 loss-of-function mutants. We found that UNC-7 and UNC-1 co-localize at peri-synaptic regions, indicating that the two proteins may functionally interact. We are investigating the physical properties of the potential UNC-7, UNC-9, and UNC-1 channel complexes. Current progress will be presented at the meeting. Yeh E, Kawano T, Weimer RM, Bessereau JL, Zhen M. 2005. Identification of genes involved in synaptogenesis using a fluorescent active zone marker in Caenorhabditis elegans. J Neurosci. 25(15):3833-41.

Barbier, Louis et al. (2011) International Worm Meeting "The Stomatins UNC-1 and STO-6 Function in the C. elegans Motor Circuit."

Barbier, Louis, Kawano, Taizo, Po, Michelle, Zhen, Mei

[International Worm Meeting, 2011]

The knowledge of the wiring diagram and functional tests (i.e. ablation studies) of the C. elegans nervous system has led to the identification of the core components of the motor circuit, which contains five pairs of command interneurons signaling to at least six classes of motoneurons to generate patterns of locomotion. The control of C. elegans locomotion, however, is not fully understood at the molecular and cellular level. At the molecular level, mutations in many neural genes lead to specific, quantifiable movement phenotypes, but the underlying circuit defects remain poorly understood. We have used a combined molecular genetic and calcium imaging approach to dissect the function of innexin genes in regulating different layers of this motor circuit. To this end, we have shown that UNC-7 and UNC-9 innexins form gap junctions that control the output balance between the forward and backward components of the motor circuit (Kawano et al. submitted; see also Po et al., 2010 C. elegans Neuro meeting). How these gap junctions are regulated at the molecular level is unknown. In a screen for suppressors of the unc-7(e5) innexin kinker mutant, we isolated gain-of-function alleles of two stomatin genes, unc-1 and sto-6, that allow unc-7(e5) animals to move forwards. Stomatins are a conserved family of membrane-associated proteins that regulate ion channels1. Stomatins contain a stomatin domain, oligomerization domain, and occasionally other functional motifs. Like innexin unc-7 and unc-9 loss of function mutants, unc-1(lf) results in a kinker phenotype, whereas a sto-6 frameshift deletion mutant does not display an overt locomotion phenotype. unc-1(gf) and sto-6(gf) mutants, however, display unique phenotypes. We will present studies that indicate a differential role of these two classes of stomatins in regulating C. elegans motor circuit function. 1) Salzer U, Mairhofer M and Prohaska R. Dynamic Cell Biology, 2007.

Xu, Tianqi et al. (2017) International Worm Meeting "Gap junction networks within the motor circuit of C. elegans facilitate undulatory wave propagation."

Xu, Tianqi, Zhen, Mei, Wu, Min, Wen, Quan, Po, Michelle, Shao, Shuai, Huo, Jing

[International Worm Meeting, 2017]

Gap junction communication between neurons is prevalent in the motor circuit of invertebrates and vertebrates, yet how electrical couplings might control and coordinate locomotion during normal animal behavior remains largely elusive. The anatomical wiring diagram of C. elegans nervous system suggests that (1) the command interneuron AVB makes en passant electrical synapses with most B-type motor neurons distributed along the worm body; (2) nearby B-type motor neurons are also electrically coupled. What are the functions of gap junction highway and byway in the worm motor circuit? By combining genetic analysis, optogenetic manipulation and computational modeling, we first showed that AVB-B electrical inputs induced a bifurcation in B motor neuron dynamics, and they work synergistically with proprioceptive couplings to enhance sequential activation of motor activities and to equalize the bending amplitude along the worm body. Second, weak electrical couplings between B motor neurons could retrogradely mediate the dynamics of head bending activities. Taken together, gap junction networks within the worm motor circuit facilitate a well-formed and flexible body undulation to propagate from head to tail during C. elegans forward locomotion.

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.

Michelle S. Teng et al. (2006) European Worm Meeting "Expression of Mammalian GPCRs in C. elegans Generates Novel Behavioural Responses to Human Ligands"

John McCafferty, Martijn P.J. Dekkers, Andrew G. Fraser, Michelle S. Teng, Bee Ling Ng, Gert Jansen, Suzanne Rademakers

[European Worm Meeting, 2006]

Michelle S. Teng1, Martijn P.J. Dekkers2, Bee Ling Ng1, Suzanne Rademakers2, Gert Jansen2, Andrew G. Fraser1 & John McCafferty1. G protein coupled receptors (GPCRs) play a crucial role in many biological processes and represent a major class of drug targets. However purification of GPCRs for biochemical study is difficult and most methods of screening receptor-ligand interactions require cultured cells and endotoxin free compounds. In contrast, Caenorhabditis elegans is a soil dwelling nematode that feeds on bacteria and uses GPCRs expressed in chemosensory neurons to detect bacteria and environmental compounds. Here we report that expression of the mammalian somatostatin receptor (Sstr2) and chemokine receptor 5 (CCR5) in gustatory neurons allow C. elegans to specifically detect and respond to human somatostatin and MIP-1? respectively in a simple avoidance assay. The endogenous signalling components involved in this remarkable promiscuity of interaction, spanning 800 million years of evolution, are investigated. This system has practical utility in ligand screening. Using structure:function studies, we identified key amino acid residues involved in the interaction of somatostatin with its receptor. This in vivo system, which imparts novel avoidance behaviour on C. elegans, can therefore be used in screening impure GPCR ligands, including the identification of bacterial clones expressing agonists within recombinant libraries.

DeGenova, Sarah E. et al. (2009) International Worm Meeting "Regulation of GLD-1 turnover in the C. elegans germ line."

Nayak, Sudhir, Jarevela, Tim, DeGenova, Sarah E.

[International Worm Meeting, 2009]

The KH domain containing RNA binding protein GLD-1 (defective in germ line development) has multiple functions in germ line development. The tightly regulated accumulation of GLD-1 at the distal region and loss of GLD-1 at the loop are critical for its functions and is essential for maintaining germ line polarity and oocyte development. Unlike the requirements for GLD-1 accumulation in the distal region, the mechanisms by which GLD-1 levels fall abruptly and are sustained at low concentration in oocytes are unknown. To identify components involved in regulation of GLD-1 levels, we performed a Po RNAi screen. Since reduction of function in GLD-1 by RNAi results in Po sterility and F1 embryonic lethality, we focused on genes that resulted in the same phenotypes in high throughput screens or were expressed in the germ line based on microarray data contained in ORFeome-RNAi v1.1 (OpenBiosystems, WS180, 1498 genes). To increase the sensitivity of the screen, we utilized a transgenic line carrying a functional GLD-1::GFP that offered a real-time readout of germ line polarity and health. Animals with altered GLD-1::GFP expression were DAPI (4'',6-diamidino-2-phenylindole) stained to asses both GLD-1 accumulation and nuclear morphology. Our screen identified genes involved in two major cellular processes: targeted degradation (pbs, cul, skr) and translation (ife, eft). Importantly, in both cases, we observed dramatic changes in GLD-1 accumulation prior to changes in germ line morphology. The focus of this presentation will center on the rapid turnover of GLD-1 in the germ line. Since the blocking of translation via RNAi (ife and eft genes) resulted in the rapid down regulation of GLD-1, we suspected a rapid turnover rate. To confirm these findings, we tested chemical translational inhibitors for their ability to phenocopy the RNAi results. Our preliminary data indicates that when strains were exposed to chemical translational inhibitors, GLD-1 accumulation decreased rapidly within 2 hours and was essentially absent after 6 hours with minimal effect on germ line morphology. In total, our data suggests that down regulation of GLD-1 prior to oocyte development is mediated by inherent instability and the action of the 26S proteasome. We are confirming these results on a subset of genes isolated from the screen with complementary approaches.

Woldemariam, Sarah et al. (2015) International Worm Meeting "Elucidating the role of cGMP dynamics in behavioral plasticity."

Schneider, Martin, Krzyzanowski, Michelle, Bethke, Mary, Nagpal, Jatin, Gottschalk, Alexander, Woldemariam, Sarah, Ferkey, Denise, L'Etoile, Noelle

[International Worm Meeting, 2015]

cGMP is a ubiquitous second messenger implicated in many important biological processes. In neurons, cGMP dynamics can regulate the function of ion channels and kinases, resulting in physiological changes. In the context of learning and memory, these changes result in short-term and long-term behavioral changes based on the organism's experience. We attempt to understand the molecular basis for long-term plasticity by studying the behavioral responses of the nematode C. elegans. Along with our collaborator Michelle Krzyzanowski from the Denise Ferkey lab in SUNY Buffalo, we are interested in how food might modulate behaviors. One food-modulated behavior is repulsion from quinine. This repulsion is mediated by the ASH neuron and it is down regulated by food withdrawal and the cGMP-dependent protein kinase EGL-4. This poses a conundrum since no guanylyl cyclases, which produce cGMP, are expressed in ASH. Genetic evidence suggests that guanylyl cyclases in other neurons are required for the food-modulated repulsion from quinine in ASH and that gap junctions are required for the transmission of cGMP from these neurons to ASH. In order to understand how cGMP dynamics in these neurons are modulated, we need a tool to visualize cGMP. To this end, we are using a cGMP sensor that will allow us to image cGMP dynamics in ASH and other neurons in the living behaving animal in the presence and absence of food.

Bijaya Kumar Dhakal et al. (2002) East Coast Worm Meeting "Vacuolar-type H + -ATPase E subunit is required for embryogenesis and yolk transfer in C. elegans."

Jae-Ran Yu, Kyu Yeong Choi, Yon Ju Ji, Bijaya Kumar Dhakal, Joohong Ahnn

[East Coast Worm Meeting, 2002]

Vacuolar H+ ATPase (V-ATPase) is a multi-subunit proton pump composed of peripheral V1 sector and membrane-bound Vo sector. It is localized in the membrane of intracellular acidic organelles including endosomes, lysosome, golgi apparatus and clathrin coated vesicle as well as in plasma membrane. Because of its regulation in acidification, V-ATPase is required for many intracellular processes such as, receptor mediated endocytosis, zymogen activation, accumulation of neurotransmitters and protein sorting. We have characterized the E subunit of V-ATPase in C. elegans. This subunit is one of the well conserved subunit showing 57 % identity in amino acid sequences to human homologue, ATP6E. GFP expression and antibody staining showed that vha-8 is abundantly expressed in large H-shaped excretory cell, which is consistent with other subunit expression patterns. RNA mediated interference (both dsRNA injection and bacterial feeding) targeted to vha-8 resulted in embryonic lethal or larval arrest phenotypes. In addition, we also observed endomitotic oocytes and defect in receptor-mediated yolk transfer in Po animals. Interestingly, RNAi affected animals showed resistance to high osmotic environment compared to wild type. We are in the process of characterizing these phenotpes. The observed phenotypes are possibly caused by disrupted pH homeostasis due to the defective V-ATPase. Our results suggest that E subunit of V-ATPase is involved in embryogenesis and receptor mediated endocytosis.

Aidyl S Gonzalez-Serricchio et al. (2005) International Worm Meeting "Paternal mitochondrial inheritance: No longer restricted to Moms"

Craig LaMunyon, Aidyl S Gonzalez-Serricchio

[International Worm Meeting, 2005]

A vast majority of eukaryotic organisms solely inherit maternal mitochondrial DNA (mtDNA) with the exception of mussels (males inherit paternal mtDNA and females inherit maternal mtDNA) (Zouros, 2000; Zouros et al., 1994). Paternal mtDNA disappear early in embryogenesis either due to selective destruction, inactivation or simple dilution by the numerous of oocyte mitochondria. Is there a deleterious effect if offspring inherits paternal mtDNA? Our aim is to further understand the mechanism of mitochondrial inheritance. We were able to isolate a strain with the capability to pass onto its offspring its paternal mtDNA. UadDf-5 is a stable heteroplasmic strain with a 3kb deletion on its mt genome (removes 11 genes; Tsang and Lemire, 2002). UaDf-5; him-8 (e1489) worms were mutagenized with ethylmethanesulfonate (EMS). F1 worms were isolated and allowed to clone in order to isolate F2 males. Next, the F2 males were crossed into fer-1 (hc13ts) hermaphrodites. Finally, F3 worms were isolated and underwent a nestedPCR assay to detect the mutant UaDf-5 band inherited from the Po: UaDf-5; him-8 (e1489) males. As we further confirm, homozygosis (if stable), and characterize the DNA lesion of the mutagenized strain(s), we will have a strong tool to use in explaining the mechanism that eliminates the sperm mitochondria from the embryo. Also, we will observe offspring that inherit some paternal mitochondria to see if it results in reduced fitness. Either initially, or after numerous generations, understand the evolution of maternal inheritance.Zouros E, 2000. The exceptional mitochondrial DNA system of the mussel family Mytilidae. Genes Genet Syst 75:313-8.Zouros E, Oberhauser Ball A, Saavedra C, Freeman KR, 1994. An unusual type of mitochondrial DNA inheritance in the blue mussel Mytilus. Proc Natl Acad Sci U S A 91:7463-7Tsang WY, Lemire BD, 2002. Stable heteroplasmy but differential inheritance of a large mitochondrial DNA deletion in nematodes. Biochem Cell Biol 80:645-54.

Telfer A et al. (1991) International C. elegans Meeting "IDENTIFICATION AND MOLECULAR ANALYSIS OF A MATERNALLY EXPRESSED GENE IN THE PAR-l REGION."

Telfer A, Stinchcomb DT

[International C. elegans Meeting, 1991]

Mutations in the par-l gene of C. elegans cause a maternal effect lethalphenotype. Embryoslacking maternallyderivedpar-lgeneproduct exhibit numerous defects, beginning in the first cell cycle. These include symmetric cleavage of PO and the failure to partition P granules into the presumptive germline (Kemphues, K.J., et al., Cell 52: 311-321 (1988)). We previously described our efforts to clone par- l by walking from linked Tcl elements and our identification of a maternally expressed gene on two cosmid clones identified in that walk (WBG 11(2): 62). Northern analysis reveals that this gene produces a 3. 3 kb transcript that is abundant in adults undergoing oogenesis. The transcript is relatively rare in larvae and in adults that lack germ cells. cDNA clones were isolated from libraries provided by Irene Schauer and Bob Barstead. Sequence analysis of the cDNAs and the corresponding genomic DNA indicates that the mRNA is transcribed from a 4 kb genomic region and is trans-spliced to the splice leader SLl. It contains a 2.8 kb open reading frame. The deduced amino acid sequence was used to search the protein databases but no significant similarities to known protein sequences were found. We have been unable to obtain direct evidence that this maternally expressed gene is par-land are seeking to ascertain its role in development by isolating mutants in the gene. We are using the polymerase chain reaction (PCR) strategy described by Rushforth, Saari and Anderson ( WBG 11(5): 65) to screen the strain TR1690, which has mutator activity conferred by mut-2(r459), for animals bearing Tcl insertions within the coding region. We screened 3.5 x 105 animals in 35 independent populations. Four of the populations contained animals with Tcl insertions in the gene. In two populations, the Tcl insertion sites were mapped to exons. These populations are being subdivided in hopes of isolating individuals bearing the insertions.