Escudero, Julianna et al. (2021) International Worm Meeting "Defining the molecular determinants by which EXC-4/CLICs regulate Rho-family GTPase signaling"

Escudero, Julianna, Mao, De Yu, Shaye, Daniel, Arena, Anthony, Kitajewski, Jan

[International Worm Meeting, 2021]

The excretory canal (ExCa) in C. elegans, a unicellular tube that collects excess fluid for homeostasis and osmoregulation, is a powerful model to study the genetic regulation and cell biological processes underlying biological tube formation (tubulogenesis). Several conserved genes involved in ExCa tubulogenesis have been implicated in vascular development and disease. One example is exc-4, which encodes an ortholog of the chloride intracellular channel (CLIC) family of proteins, and two vertebrate CLICs, CLIC1 and CLIC4, which regulate human umbilical vein endothelial cell (HUVEC) angiogenesis in vitro and mouse vascular development in vivo. We have recently shown that EXC-4 in the worm, and CLIC1 and CLIC4 in HUVEC, are involved in G protein-coupled receptor (GPCR), heterotrimeric G protein, and Rho-family GTPase signaling (Arena and Shaye, in preparation. Mao et al., in press). To further understand the role of CLICs in this conserved pathway we are investigating shared and unique functions of EXC-4, CLIC1, and CLIC4. In C. elegans EXC-4 constitutively localizes to the ExCa apical membrane via an N-terminal putative transmembrane domain (PTMD). In contrast, CLIC1 and CLIC4 are cytoplasmic in HUVEC despite also having N-terminal PTMDs, and they only transiently localize to the plasma membrane, in a PTMD-dependent manner, upon GPCR activation. Importantly, membrane localization is necessary for EXC-4 function in the ExCa, and CLIC1 or CLIC4 function in HUVEC. Moreover, previous work showed that ExCa-specific expression of an apical membrane-targeted CLIC1 C-terminus could rescue exc-4 null (0). These results led us to hypothesize that while EXC-4/CLIC membrane localization is achieved via the PTMD, conserved function in Rho-family signaling is achieved via shared features in the C-terminus. However, we have also found that CLIC1 and CLIC4 are not interchangeable in HUVEC: CLIC1 promotes RhoA and Rac1 activity, while CLIC4 only regulates Rac1, and overexpression of one CLIC cannot compensate for loss of the other. Therefore, we further hypothesize that CLIC-specific functions are encoded by differences in their C-termini. We are undertaking structure/function studies of EXC-4, CLIC1, and CLIC4 in the ExCa and in HUVEC to test these hypotheses in order to define molecular determinants of EXC-4/CLIC function in Rho-family GTPase signaling.

Socovich, A. et al. (2019) International Worm Meeting "The excretory canal as a platform to discover kinase regulators of tubulogenesis and angiogenesis."

Shukla, P., Shaye, D., Socovich, A., Greenwald, I., Violi, F.

[International Worm Meeting, 2019]

Tubulogenesis, the process by which organisms form biological tubes, plays an integral role in development, including in blood vessel formation (angiogenesis) and function. Moreover, defects in tubulogenesis can lead to congenital disease (e.g., capillary malformations) and dysregulated tubulogenesis due to tumor-induced angiogenesis is associated with cancer progression and metastasis. The excretory canal (ExCa) provides a tractable model to study tubulogenesis. Human orthologs of several genes that regulate ExCa tubulogenesis, for example orthologs of the CLIC channel exc-4 regulate Rho/Rac signaling in endothelial cells (see also Arena and Escudero abstracts). Moreover, the kinases gck-1, gck-3, and wnk-1, all previously implicated in ExCa tubulogenesis, regulate angiogenic behaviors in human endothelial cells, or have been implicated in vascular disease. Our hypothesis is that we can find novel regulators of angiogenesis by screening for new players in ExCa tubulogenesis. Kinases are critical regulators of key cellular functions, with approximately 30% of human proteins being phosphorylated by kinases. Using OrthoList, a compendium of C. elegans genes with human orthologs, we identified 248 C. elegans kinases that have human orthologs. An RNAi screen against these kinases led to discovery of nine whose loss leads to ExCa defects. Among these are four (gck-1, gck-3, mrck-1 and wnk-1) previously implicated in ExCa tubulogenesis, indicating a low false-negative rate. Moreover, alleles for the remaining five confirmed the RNAi results, indicating a low false-positive rate. We are characterizing the phenotypes caused by loss of these kinases, using available nulls and conditional alleles we are generating by CRISPR to understand their roles in tubulogenesis. We have found that orthologs of all the kinases identified in the ExCa screen are expressed in human umbilical vein endothelial cells (HUVEC); a canonical model for studying endothelial cell behavior and angiogenesis in vitro. We are using shRNA constructs to knockdown the expression of these kinases and performing angiogenesis assays to define conserved roles that kinases discovered in C. elegans have in angiogenesis.

Arena, A. et al. (2019) International Worm Meeting "EXC-4/CLIC proteins are ancient regulators of heterotrimeric G-protein-Rho/Rac signaling."

Mao, D., Arena, A., Kitajewski, J., Shaye, D.

[International Worm Meeting, 2019]

G-protein coupled receptors (GPCRs) regulate many aspects of physiology and are implicated in various pathological conditions. Several GPCR signaling pathways are vital for angiogenesis, the formation of new blood vessels from existing ones. We are interested in understanding this as well as other forms of biological tube formation (tubulogenesis). To this end, we use the C. elegans excretory canal (ExCa) as a model to find novel and conserved regulators of angiogenesis (see also Socovich abstract). Our collaborative studies of ExCa tubulogenesis and mammalian angiogenesis led us to discover a new class of GPCR regulators: the Chloride Intracellular Channel (CLIC) family of proteins. Although CLICs have been implicated in development and disease, their role has remained largely unknown. Here we provide evidence for a novel and conserved role for CLICs as regulators of GPCR-heterotrimeric G-protein (G?/ beta /?)-Rho/Rac signaling. A role for CLICs in tubulogenesis was first defined by studies of a C. elegans CLIC ortholog called exc-4. Subsequently, we and others showed that two mammalian CLICs, CLIC1 and CLIC4, function in endothelial (blood vessel) cells to promote migration, growth, and tube formation during angiogenesis. While EXC-4 is constitutively localized to the plasma membrane in the ExCa, and this localization is critical for its function, in mammalian cells CLIC1 and CLIC4 are cytoplasmic. However, these CLICs are recruited to the plasma membrane upon activation of various GPCRs, including the S1P family of receptors (S1PRs). These receptors are potent regulators of angiogenesis that act through G?i/o, G?12/13, RhoA and Rac1. Therefore, the regulation of CLIC localization by S1PRs led us to hypothesize that EXC-4/CLICs function in G?-Rho/Rac signaling. Our data demonstrate that CLIC1 and CLIC4 regulate RhoA and Rac1 in endothelial cells. Additionally, we find that exc-4 genetically interacts with goa-1/G?i/o, gpa-12/G?12/13 and two of the worm Rac orthologs, ced-10 and mig-2 in ExCa tubulogenesis. This is the first evidence that G? and Rac proteins function in ExCa tubulogenesis. We are currently generating more tools to further test genetic and physical interactions between exc-4 and other components of G?/ beta /?-Rho/Rac signaling in the ExCa (see Escudero abstract). Taken together, our data suggests that regulation of G?/ beta /?-Rho/Rac is a primordial function of EXC-4/CLIC proteins.