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.

Socovich, Alexandra et al. (2021) International Worm Meeting "The kinase pig-1/MELK is a conserved cytoskeletal regulator in C. elegans tubulogenesis and in human endothelial cells."

Shaye, Daniel, Socovich, Alexandra

[International Worm Meeting, 2021]

Tubulogenesis, the process by which organisms form biological tubes, plays an integral role in blood vessel formation and function. Defects in tubulogenesis can lead to congenital vascular disease, and dysregulated tubulogenesis associated with tumor-induced angiogenesis promotes cancer progression. Kinases are critical regulators of conserved cellular functions, signal transduction pathways, and are attractive targets for therapeutic development. Therefore, finding kinases involved in vascular tubulogenesis is an important translational research goal. The C. elegans excretory canal (ExCa), a large single-celled tube, provides a tractable model to study tubulogenesis. Moreover, human orthologs of several genes that regulate ExCa tubulogenesis have been implicated in vascular development and disease. We performed an RNAi screen to find conserved kinase regulators of ExCa tubulogenesis and identified four that were not previously implicated in this process. Notably, we found that orthologs of these four kinases are expressed in human umbilical vein endothelial cells (HUVEC), a canonical model for studying angiogenesis in vitro, consistent with their possible role in vascular development (Socovich and Shaye, in preparation). We are focusing on the kinase pig-1 and its human ortholog MELK, because despite the fact that little is known about its physiological function, MELK has been the focus of great interest due to it being highly upregulated in various aggressive cancers. Our work defines a novel mode of PIG-1/MELK regulation that is independent of canonical upstream activators like PAR-4/LKB and STRD-1/STRAD. We also discovered a new role for pig-1 in regulating the conserved formin EXC-6/INF2 and cytoskeletal components (F-actin and microtubules) in the ExCa. Both MELK and INF2 are expressed in HUVEC, and shRNA-mediated knockdown of these proteins caused cell migration phenotypes consistent with cytoskeletal defects. Based on these results we propose a novel conserved PIG-1/MELK and EXC-6/INF2 pathway that regulates the cytoskeleton in tubulogenesis.

Escudero, J. et al. (2019) International Worm Meeting "A toolkit for analyzing the role of heterotrimeric G-protein-Rho/Rac signaling, and its regulation by EXC-4/CLIC, in the excretory canal cell."

Shaye, D., Arena, A., Escudero, J.

[International Worm Meeting, 2019]

We study the genetic regulation and cell biological processes that underlie biological tube formation (tubulogenesis) in C. elegans and during angiogenesis; a process of blood vessel formation. To this end, we study the excretory canal (ExCa) a unicellular tube that elongates throughout the worms' entire body. Some genes that regulate ExCa tubulogenesis are also involved in angiogenesis (see also Socovich abstract). One example is the gene exc-4, which encodes a chloride intracellular channel (CLIC). The first mutants in this gene were retrieved in a pioneering screen conducted by Buechner et al., looking for animals with cystic excretory canals (the exc screen), and exc-4 was later cloned by the Hobert Lab. Subsequently, work from our collaborator, Dr. Jan Kitajewski, and his lab implicated two vertebrate orthologs, CLIC1 and CLIC4, in angiogenesis in vitro (using cultured human umbilical vascular endothelial cells, or HUVEC) and in vivo (in mice). In collaboration with the Kitajewski Lab we have now shown that EXC-4 in the worm, and CLIC1 and CLIC4 in endothelial cells, modulate signaling pathways that act via heterotrimeric G proteins (G?/ beta /?) and the small GTPases Rho and Rac. This suggests that EXC-4/CLIC proteins are conserved regulators of Rho/Rac signaling (see also Arena abstract). We want to use genetic and expression analyses to define how EXC-4 and G?/ beta /?-Rho/Rac signaling components interact to achieve this regulation. Genetic analysis of these players is confounded by lethality caused by null mutations in some of them (e.g. gsa-1/ G?s, gpb-1/G beta , gpc-2/G?, rho-1/Rho and ced-10/Rac) or potential redundancy among others (e.g. between different G?/ beta /?-encoding genes, or between the two Rac homologs ced-10 and mig-2). To circumvent these issues, we are generating a toolkit to activate or deplete G?/ beta /?, Rho and Rac homologs specifically in the ExCa, as well as to visualize protein localization of these components in this cell. Our goal is to combine these tools with exc-4/CLIC mutants to define the conserved mechanisms by which EXC-4/CLIC regulates G?/ beta /?-Rho/Rac signaling.

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.

Quartey MO et al. (2021) Sci Rep "The A(1-38) peptide is a negative regulator of the A(1-42) peptide implicated in Alzheimer disease progression."

Pennington PR, Heistad RM, Nyarko JNK, Barnes JR, Bolanos MAC, Parsons MP, Knudsen KJ, De Carvalho CE, Leary SC, Mousseau DD, Buttigieg J, Maley JM, Quartey MO

[Sci Rep, 2021]

The pool of -Amyloid (A) length variants detected in preclinical and clinical Alzheimer disease (AD) samples suggests a diversity of roles for A peptides. We examined how a naturally occurring variant, e.g. A(1-38), interacts with the AD-related variant, A(1-42), and the predominant physiological variant, A(1-40). Atomic force microscopy, Thioflavin T fluorescence, circular dichroism, dynamic light scattering, and surface plasmon resonance reveal that A(1-38) interacts differently with A(1-40) and A(1-42) and, in general, A(1-38) interferes with the conversion of A(1-42) to a -sheet-rich aggregate. Functionally, A(1-38) reverses the negative impact of A(1-42) on long-term potentiation in acute hippocampal slices and on membrane conductance in primary neurons, and mitigates an A(1-42) phenotype in Caenorhabditis elegans. A(1-38) also reverses any loss of MTT conversion induced by A(1-40) and A(1-42) in HT-22 hippocampal neurons and APOE 4-positive human fibroblasts, although the combination of A(1-38) and A(1-42) inhibits MTT conversion in APOE 4-negative fibroblasts. A greater ratio of soluble A(1-42)/A(1-38) [and A(1-42)/A(1-40)] in autopsied brain extracts correlates with an earlier age-at-death in males (but not females) with a diagnosis of AD. These results suggest that A(1-38) is capable of physically counteracting, potentially in a sex-dependent manner, the neuropathological effects of the AD-relevant A(1-42).

Danielle Thierry-Mieg et al. (2003) Worm Breeder's Gazette "www.wormgenes.org , a new gene centered database"

Vahan Simonyan, Michel Potdevin, Mark Sienkiewicz, Yuji KOHARA, Jean Thierry-Mieg, Danielle Thierry-Mieg, Tadasu SHIN-I

[Worm Breeder's Gazette, 2003]

Wormgenes is a new resource for C.elegans offering a detailed summary about each gene and a powerful query system.

Tangrodchanapong T et al. (2020) Front Pharmacol "Frondoside A Attenuates Amyloid- Proteotoxicity in Transgenic Caenorhabditis elegans by Suppressing Its Formation."

Tangrodchanapong T, Sobhon P, Meemon K

[Front Pharmacol, 2020]

Oligomeric assembly of Amyloid- (A) is the main toxic species that contribute to early cognitive impairment in Alzheimer's patients. Therefore, drugs that reduce the formation of A oligomers could halt the disease progression. In this study, by using transgenic <i>Caenorhabditis elegans</i> model of Alzheimer's disease, we investigated the effects of frondoside A, a well-known sea cucumber <i>Cucumaria frondosa</i> saponin with anti-cancer activity, on A aggregation and proteotoxicity. The results showed that frondoside A at a low concentration of 1 M significantly delayed the worm paralysis caused by A aggregation as compared with control group. In addition, the number of A plaque deposits in transgenic worm tissues was significantly decreased. Frondoside A was more effective in these activities than ginsenoside-Rg3, a comparable ginseng saponin. Immunoblot analysis revealed that the level of small oligomers as well as various high molecular weights of A species in the transgenic <i>C. elegans</i> were significantly reduced upon treatment with frondoside A, whereas the level of A monomers was not altered. This suggested that frondoside A may primarily reduce the level of small oligomeric forms, the most toxic species of A. Frondoside A also protected the worms from oxidative stress and rescued chemotaxis dysfunction in a transgenic strain whose neurons express A. Taken together, these data suggested that low dose of frondoside A could protect against A-induced toxicity by primarily suppressing the formation of A oligomers. Thus, the molecular mechanism of how frondoside A exerts its anti-A aggregation should be studied and elucidated in the future.

Hodgkin JA (1998) International Journal of Developmental Biology "Seven types of pleiotropy."

Hodgkin JA

[International Journal of Developmental Biology, 1998]

Pleiotropy , a situation in which a single gene influences multiple phenotypic tra its, can arise in a variety of ways. This paper discusses possible underlying mechanisms and proposes a classification of the various phenomena involved.

Tuck S (2011) Curr Biol "Extracellular vesicles: budding regulated by a phosphatidylethanolamine translocase."

Tuck S

[Curr Biol, 2011]

Recent work on a Caenorhabditis elegans transmembrane ATPase reveals a central role for the aminophospholipid phosphatidylethanolamine in the production of a class of extracellular vesicles.

Viney ME et al. (2004) Naturwissenschaften "Is dauer pheromone of Caenorhabditis elegans really a pheromone?"

Viney ME, Franks NR

[Naturwissenschaften, 2004]

Animals respond to signals and cues in their environment. The difference between a signal (e.g. a pheromone) and a cue (e.g. a waste product) is that the information content of a signal is subject to natural selection, whereas that of a cue is not. The model free-living nematode Caenorhabditis elegans forms an alternative developmental morph (the dauer larva) in response to a so-called 'dauer pheromone', produced by all worms. We suggest that the production of 'dauer pheromone' has no fitness advantage for an individual worm and therefore we propose that 'dauer pheromone' is not a signal, but a cue. Thus, it should not be called a pheromone.