Harrington, Sean et al. (2017) International Worm Meeting "The discovery of neuroactive small molecule tools using a high-throughput egg laying screening pipeline."

Yip, Chris, Harrington, Sean, Roy, Peter, Giuliani, Maximiliano, Au, Aaron

[International Worm Meeting, 2017]

The neuromuscular circuit that governs C. elegans egg-laying is well understood and the majority of regulatory elements within it are highly conserved. The egg-laying system has been exploited previously to probe the polypharmacology of established neuromodulators such as clozapine (Karmacharya et al. (2011), Brain Res., 1393(1), 91-99) and imipramine (Weinshenker et al. (1999), J of Neurosci., 19(22), 9831-9840). Findings from these studies translated to a mouse model demonstrating the conserved nature of components of the egg-laying system. Here, we aim to identify novel small-molecule modulators of neuromuscular function by screening for those that modulate egg-laying behaviour in the worm. To do so, we have developed a high-throughput screening pipeline that takes advantage of the amenability of the egg-laying phenotype to computational counting. Using this screening pipeline, we have screened through 4642 molecules and identified 83 molecules that either stimulate or suppress egg laying behaviour at a concentration of 60 M or less. Many of these molecules have no reported relevant bioactivities in the literature. Several of our hits are bioactive in a vertebrate model (Danio rerio) suggesting the possibility of a conserved mechanism of action (Burns et al. (2015), Nature Comm., 6(1), doi:10.1038/ncomms8485). We are currently characterizing the mechanism of action of these molecules using four parallel approaches: 1) testing their interaction with a panel of human neuronal targets (NIH Psychoactive Drug Screening Project - Besnard et al. (2012), Nature, 492(7428), 215-220); 2) thermal proteome profiling using quantitative mass spectrometry to identify candidate targets in worms (Franken et al. (2015), Nature Protoc., 10(10), 1567-1593); 3) forward genetics screens to identify candidate targets (Kwok et al. (2006), Nature, 441(7089), 91-95); & 4) understanding their interaction with a panel of C. elegans egg laying mutants. We hope that our hits will become useful tools for future biological investigations, and that a subset might have therapeutic potential to modulate neuromuscular activity in more complex systems.

Chang, Yu-Tai et al. (2011) International Worm Meeting "TOCA-1 and actin polymerization contribute to P-cell nuclear migration."

Starr, Daniel A., Chang, Yu-Tai

[International Worm Meeting, 2011]

Moving the nucleus to an intracellular location is essential for a wide variety of cell and developmental processes, including the formation of polarized cells, fertilization, differentiation, and cellular migration. Defects in nuclear migration block development and lead to disease. However, the mechanisms of how nuclei are moved are poorly understood. We employ larval P-cells in C. elegans as a model for nuclear migration. 12 P-cell nuclei migrate from lateral to ventral positions during the mid-L1 stage. They subsequently divide to form neurons and the vulva. Null mutations in unc-83 or unc-84 inhibit nuclear migration by disrupting interactions between nuclei and microtubule motors. Nuclear migration defects lead to the death of P-cells resulting in uncoordinated (Unc) and egg-laying defective (Egl) animals missing P-cell derived lineages. Interestingly, the unc-83 and unc-84 P-cell nuclear migration defect is temperature sensitive. P-cell nuclear migration is disrupted in unc-83 or unc-84 null animals at 25 deg C, but at 15 deg C, P-cell nuclear migration occurs normally. We therefore hypothesize that an additional pathway functions in part redundantly to the unc-83/unc-84 pathway to migrate P-cell nuclei at 15 deg C. To test our hypothesis, we carried out forward genetic screens and isolated nine emu (enhancer of the nuclear migration defect of unc-83 or unc-84) alleles at 15 deg C in unc-84 null animals in the background of an unc-84 rescuing array. To quantify the severity of the P-cell nuclear migration defect of emu alleles, we scored the number of UNC-47::GFP-positive GABA neurons in adults. Compared to unc-84 null animals, emu; unc-84 double mutants had significantly fewer GABA neurons at all temperatures. The emu alleles are recessive and have been partially mapped. Using whole-genome sequencing, we have determined that the yc20 allele is a lesion in toca-1 and are currently sequencing other alleles. toca-1(RNAi) phenocopied yc20, suggesting that TOCA-1 functions in parallel to the UNC-84/UNC-83 pathway to move P-cell nuclei. TOCA-1 is conserved in flies and mammals. It consists of an F-bar domain that binds to curved membranes and two domains that recruit cdc42 and WAVE to induce F-actin polymerization. Thus, TOCA-1 functions to regulate actin dynamics at the membrane-cytoskeleton interface; previous studies show that TOCA-1 facilitates clathrin-mediated endocytosis (Giuliani et al., 2009. PLoS Genetics). That same study also had data suggesting that TOCA-1 localizes to the nuclear envelope of oocytes. We are currently investigating the subcellular localization of TOCA-1 in P-cells. Together, our results implicate a novel mechanism for nuclear migration based on the nucleation of actin filaments.