Defining Genetic and Physical Interactors of the C. elegans’ EXC-4 Protein that Regulate Tubulogenesis

Tubulogenesis, the process of biological tube formation and maintenance, is regulated by conserved signaling pathways. We use the C. elegans excretory canal (ExCa) cell, a unicellular tube, as a model to identify and study conserved tubulogenesis regulators. EXC-4, a C. elegans Chloride Intracellular Channel (CLIC) protein, was first defined for its role in ExCa tubulogenesis and subsequent work using human endothelial cells as well as knockout mice showed that the exc-4 mammalian orthologs CLIC1 and CLIC4 regulate angiogenesis. Work from our collaborators identified a role for CLIC1/4 in S1P-mediated GPCR-RhoA/Rac signaling. Here we define genetic interactions between exc-4 and four Gα components (egl-30/Gαq, gpa-12/Gα12/13, gsa-1/Gαs, and gpa-7/Gαi) as well as genetic interactions between exc-4 and the small GTPases ced-10/Rac1 and mig-2/RhoG. Thus, our work demonstrates that EXC-4/CLICs are conserved in vivo regulators of Gα and Rho/Rac signaling, but how EXC-4/CLICs function in this pathway remains unknown. Previous studies demonstrated that EXC-4 constitutively localizes to the ExCa apical plasma membrane, while in mammalian endothelial cells CLIC1/4 are cytoplasmic at steady state but can be transiently recruited to the plasma membrane upon activation of GPCR signaling. Moreover, it was shown that human CLIC1 can rescue exc-4(0), indicating conserved function, but only when localized to the apical membrane. Therefore, in both C. elegans and human cells, EXC-4/CLIC membrane localization is critical for functionality in Gα-Rho/Rac signaling. Taking advantage of EXC-4’s constitutive apical localization we aimed to identify direct EXC-4 interactors to better understand how EXC-4/CLICs perform this conserved function. Using EXC-4 fusion proteins as the “bait” we performed ExCa specific biotin proximity labeling and co-immunoprecipitation experiments and identifies 32 EXC-4 specific protein interactors. From these 32 proteins: 85% have human orthologs, 53% are involved in angiogenesis, 22% are cytoskeletal associated, and 18% are mitochondrial related. One of the identified proteins, profilin, has previously been characterized as a direct interactor of CLIC4 in HEK293 cells. We show here that depletion of profilin by RNAi induces robust negative effects on ExCa development and morphology. Moreover, RNAi analysis of four candidate EXC-4 interactors, including profilin, resulted in 3 of the 4 (75%) proteins demonstrating ExCa phenotypes upon their loss. Thus, our proteomics findings have the potential to identify novel regulators of EXC-4-mediated tubulogenesis. Moving forward, each candidate interactor will be examined by RNAi and/or mutant allele analysis and then assessed for direct interaction(s) with EXC-4. Ultimately, any significant findings will be extrapolated to in vitro and/or in vivo studies in mammalian systems, which will drastically enhance our understanding of how EXC-4/CLICs regulate tubulogenesis and angiogenesis.