A metazoan-specific C-terminal motif in EXC-4/CLIC and Ga-Rho/Rac signaling regulate cell outgrowth during tubulogenesis in C. elegans.

Chloride intracellular channels (CLICs) are conserved proteins whose cellular and molecular functions remain mysterious. An important insight into CLIC function came from the discovery that C. elegans EXC-4/CLIC regulates morphogenesis of the excretory canal (ExCa) cell, a single-cell tube. Subsequent work showed that mammalian CLICs regulate vascular development and angiogenesis, and human CLIC1 can rescue exc-4 mutants, suggesting conserved function in biological tube formation (tubulogenesis) and maintenance. However, the cell behaviors and signaling pathways regulated by EXC-4/CLICs during tubulogenesis in vivo remain largely unknown. We report a new exc-4 mutation, affecting a C-terminal residue conserved in virtually all metazoan CLICs, that revealed a specific role for EXC-4/CLIC in ExCa outgrowth. Cell culture studies suggest a function for CLICs in heterotrimeric G-protein (Ga/b/g)-Rho/Rac signaling, and Rho-family GTPases are common regulators of cell outgrowth. Using our new exc-4 mutant we describe a previously unknown function for Ga-encoding genes (gpa-12/Ga12/13, gpa-7/Gai, egl-30/Gaq, and gsa-1/Gas), ced-10/Rac, and mig-2/RhoG in EXC-4-mediated ExCa outgrowth. Our results demonstrate that EXC-4/CLICs are primordial players in Ga-Rho/Rac-signaling-a pathway critical for tubulogenesis in C. elegans and in vascular development.