[
C.elegans Aging, Stress, Pathogenesis, and Heterochrony Meeting,
2008]
G-protein coupled receptors (GPCRs) play a critical role in multiple processes. The signal from these cell-surface receptors is transduced to a variety of intracellular effectors via heterotrimeric G proteins. C. elegans contains over 1200 genes that encode GPCRs, as well as 25 genes that encode alpha, beta, or gamma heterotrimeric G protein subunits. At least one C. elegans GPCR, FSHR-1, is known to act in innate immunity (J. Powell, unpublished). However, the specific heterotrimeric G protein subunits that transduce FSHR-1''s signal are unknown. It is also possible that other as-yet-unidentified GPCRs participate in the C. elegans innate immune response. To explore the role of GPCR signaling in C. elegans innate immunity, we performed a tissue-specific RNAi screen of all G-protein subunits. The intestine is the primary site of infection of worms by pathogenic bacteria, so we specifically removed the function of each G-protein subunit in the intestine. This permitted us to assay the immunity function of these genes even if they are required for other essential processes. We have identified several interesting candidates from this screen, which we will discuss.
[
International Worm Meeting,
2021]
The WD-40 repeat protein CDC-20 is the co-activator of the Anaphase Promoting Complex/Cyclosome (APC/C), the E3 ubiquitin ligase responsible for mitotic exit, as well as a core subunit of the mitotic checkpoint complex that restrains APC/C activity when chromosomes are not yet attached to the mitotic spindle. CDC-20 rapidly fluxes through the kinetochore region of chromosomes via association with a conserved binding motif, known as the ABBA motif, in BUB-1. CDC-20 flux through kinetochores is essential for mitotic checkpoint activation, which delays anaphase onset until all chromosomes attach to microtubules, and for promoting anaphase onset following kinetochore-microtubule attachment (Kim and Lara-Gonzalez et al, 2017, Genes and Development 31:1089-1094). Here, we investigate the regulation of BUB-1-dependent recruitment of CDC-20 to kinetochores. We found that mutating a conserved Polo-like Kinase 1 (PLK-1) docking site in BUB-1 eliminated CDC-20 kinetochore recruitment to the same extent as mutating the ABBA motif; in addition, the peak kinetochore recruitment of the mutant BUB-1 was reduced to ~50% of wildtype levels, likely due to a role for BUB-1-bound PLK-1 in promoting kinetochore recruitment of BUB-1. To address if the defect in CDC-20 kinetochore recruitment was a consequence of reduced BUB-1 kinetochore localization or was due to BUB-1-docked PLK-1 regulating the ABBA motif-CDC-20 interaction, we selectively mutated the BUB-1-associated protein BUB-3 to impair its recognition of the phosphorylated kinetochore scaffold KNL-1. The BUB-3 phospho-recognition mutant reduced peak BUB-1 kinetochore recruitment to ~30% of wildtype levels; however the consequences on anaphase onset and CDC-20 kinetochore localization were significantly less severe than observed for PLK-1-docking mutant BUB-1. These observations support a model in which BUB-1-associated PLK-1 is essential for CDC-20 kinetochore recruitment, either by direct phospho-regulation of the ABBA motif or by controlling access of the ABBA motif to CDC-20. Our current experiments are focused on distinguishing between these two potential mechanisms.