Park EC et al. (1985) International C. elegans Meeting "dominant behavioral and morphological mutations of C elegans."

Horvitz HR, Park EC

[International C. elegans Meeting, 1985]

Park EC et al. (1985) International C. elegans Meeting "molecular genetics of a region of C elegans linkage group II."

Park EC, Horvitz HR, Edwards MK

[International C. elegans Meeting, 1985]

Vincent P. Mancuso et al. (2007) International Worm Meeting "Ras and Notch signaling intersect in the development of the C. elegans excretory system."

Meera Sundaram, Vincent P. Mancuso

[International Worm Meeting, 2007]

The Ras and Notch signaling pathways interact to specify cell fates in various metazoan developmental pathways, in some cases cooperatively, and others antagonistically. We study the interaction of these two conserved pathways in the C. elegans excretory system. This is a useful system because it consists of a few specialized cells with well characterized development. Two important components of the excretory system are the excretory cell (EC), which extends long canals throughout the length of the worm to collect fluid, and the duct cell, which receives the fluid from the EC, before expelling the fluid from the worm. Genetic experiments have implicated Ras and Notch signaling in excretory system development. Direct cooperation of Ras and Notch signaling in the specification of a cell fate has not yet been observed in C. elegans. However, this may occur in the duct cell, providing an opportunity to identify common targets of these widely conserved pathways. Ras acts cell autonomously to promote the duct cell fate (Yochem, et al 1997). Notch pathway mutants lack the EC and duct cell. (Lambie and Kimble, 1991) Previous work in our lab has identified the EC as a source of the Ras-activating ligand LIN-3, which may be responsible for Ras activation in the duct. One model to explain the lack of the EC and duct in Notch mutants is Notch acts on the EC alone, and then the EC activates Ras in the duct. Another model is that Notch acts directly on both the EC and the duct, in which case Notch and Ras are acting cooperatively on the duct. Our preliminary data indicate that in Notch pathway mutants, the EC is always absent while the duct is variably absent, suggesting that the EC is not absolutely necessary for duct formation. We are using laser ablation to remove the EC in wild type backgrounds to determine if the partial absence of the duct in Notch mutants is caused simply by the absence of the EC, or by other effects of Notch mutation. We are also addressing whether Notch is acting directly in the duct with expression analyses of Notch targets using GFP reporters.

Vincent Mancuso et al. (2008) Development & Evolution Meeting "Ras and Notch signaling intersect in the development of the C. elegans excretory system"

Meera Sundaram, Vincent Mancuso

[Development & Evolution Meeting, 2008]

The Ras/MAP kinase and Notch signaling pathways interact to specify cell fates in various metazoan developmental pathways, in some cases cooperatively, and others antagonistically. I am investigating the interaction of these two pathways in the fate specification of the C. elegans duct cell. Duct cell fate specification may be an example of direct cooperation between Ras and Notch signaling to specify a cell fate. LET-60/Ras activity is required for the duct cell fate (Yochem, et al, 1997). Notch pathway mutants also lack a duct cell (Lambie and Kimble, 1991), indicating a common role for both pathways. However, Notch mutants also lack an Excretory Cell (EC). Previous work in our lab has identified the EC as a source of LIN-3/EGF, which may be responsible for Ras activation in the duct. One model to explain the lack of the EC and duct in Notch mutants is Notch acts on the EC alone, and then the EC activates Ras in the duct. Another model is that Notch acts directly on both the EC and the duct, in which case Notch and Ras are acting cooperatively on the duct. We have used laser ablation to remove the EC and EC parent cell in wild type backgrounds, and found that the EC is not required for duct cell formation, suggesting another role for Notch in duct fate specification. Loss-of-function mutants and RNAi experiments indicate that the canonical Notch signaling pathway is involved in duct fate specification. To determine if Notch is working directly in the duct, we are utilizing immunohistochemistry and transcriptional reporters to look for Notch receptor expression, and Notch target activation, in the duct.

Horvitz HR et al. (1983) International C. elegans Meeting "dominant mutations of C elegans."

Horvitz HR, Park EC

[International C. elegans Meeting, 1983]

Ramirez-Fajardo, Emilio et al. (2021) International Worm Meeting "Innate immune responses of Caenorhabditis elegans to the emerging pathogen Elizabethkingia anophelis"

Schmidt, Kristopher, Albayati, Wesam, List, Skylar, Ramirez-Fajardo, Emilio, Grossen, Sarah, Siderhurst, Matthew

[International Worm Meeting, 2021]

Elizabethkingia anophelis (Ea) is an emerging pathogen that causes sepsis, meningitis, and death in people with comorbidities. While usually innocuous, a recent community outbreak resulted in at least 20 deaths and 66 laboratory confirmed cases. The outbreak stemmed from the rapid formation of a novel and more virulent Ea sublineage created by a conjugative element that disrupted the muty DNA repair gene. The virulence associated factors, pathogenic mechanisms, and ecology of Ea are not known. C. elegans is a well-established model for the characterization of pathogen-host interactions. When we exposed mixed stage wild-type C. elegans to Ea (Ag1 strain), the animals exhibited decreased movement, slowed development, early larval death, and an overall decreased fecundity compared to animals exposed to E. coli OP50 (Ec). We also made the striking observation that worms exposed to Ea actively avoided Ea lawns. To understand the aversive response, we conducted two-choice assays between Ea and Ec in experiments using L1 and L4 animals. L1s not previously exposed to a food source initially chose Ea over Ec (-0.75 CI, Choice Index [CI] of -1.0 represents Ea selection) but actively selected Ec over time (+0.41 CI at 96hrs). L4 animals first reared on Ec chose Ec over Ea in two-choice assays (+0.49 CI). Importantly, we found that staged adults placed on Ea bacteria without another bacterial food source adopted a lawn avoidance phenotype, suggesting that they are repulsed from Ea even in the absence of Ec. These avoidance responses depend on an intact odorant detection system because lim-4(ky403) and tax-2(p671); tax-4(p678) mutant animals failed to avoid Ea lawns. We used solid-phase microextraction (SPME) gas chromatography mass spectrometry (GC-MS) to identify volatile odorant differences between Ea and Ec and observed a marked increase in the amount of indole in Ec compared to Ea. Indole is an attractive compound recently shown to modulate predator-prey interactions between C. elegans and bacteria. To examine the pathogenic mechanisms of Ea in C. elegans, we exposed N2, sek-1 (km4), tol-1(nr2033), dbl-1(nk3) and daf-2(e1370) animals to Ea and Ec. Wild-type animals displayed several pathological changes when grown on Ea and died faster than their Ec exposed counterparts. Decreased lifespan and increased pathologies were found in sek-1 while daf-2 animals appeared immune to Ea. The tol-1 and dbl-1 alleles did not influence Ea pathogenicity. To complement these immunity assays we also examined the antimicrobial activity of dod-24, irg-1 and T24B8.5 in C. elegans exposed to Ea using transcriptional reporter GFP strains. We observed decreases in the expression of T24B8.5 and increased but more variable changes in irg-1. Taken together these experiments suggest that Ea is pathogenic to C. elegans, opening new opportunities to investigate pathogen and host factors influencing Ea related disease.

Shaye, Daniel et al. (2017) International Worm Meeting "Discovery of angiogenic regulators by studying excretory canal cell tubulogenesis genes."

Greenwald, Iva, Kitajewski, Jan, Du, Jing, Kitajewski, Alison, Shaye, Daniel

[International Worm Meeting, 2017]

The C. elegans excretory canal (EC) cell, which is required for osmoregulation, is a simple model to study biological tube formation, or tubulogenesis1. The EC extends long projections first dorsally, then anteriorly and posteriorly, each with an intra-cellular lumen. Previous genetic analyses have revealed conserved cell biological processes required for EC tubulogenesis1: canal outgrowth requires some genes that also mediate neuronal outgrowth; lumen formation is driven by regulation of cell polarity, polarized vesicular trafficking, and fluid and ion transport; and lumen maintenance depends on the apical actin cytoskeleton. Many of these cell biological processes are also involved in vertebrate angiogenesis2. Moreover, several genes required for EC tubulogenesis, such as exc-4/Clic, ccm-3/PDCD10, gck-3/STK39 and wnk-1/WNK function in angiogenesis and are linked to vascular disease1, 3-8. Therefore, the EC is a powerful model to identify and characterize novel conserved regulators of angiogenesis. To identify new conserved regulators of EC tubulogenesis we performed a feeding RNAi screen and tested the 249 kinases conserved with humans9. We identified nine conserved kinases that caused EC phenotypes. Among these nine where those previously implicated in EC tubulogenesis (gck-1, gck-3, mrck-1 and wnk-1)1, indicating a low false-negative rate for the screen. Moreover, RNAi phenotypes of the newly identified kinases were confirmed by genetic mutants, indicating a low false-positive rate. To ask whether these kinases play a role in angiogenesis, we are performing an in vitro angiogenic sprouting assay called FIBA10, which uses human umbilical cord venous endothelial cells (HUVEC) coated onto collagen-dextran beads, embedded in fibrin, and stimulated to develop angiogenic sprouts. Our RNAseq analysis shows that orthologs of all of the kinases identified in our screen are expressed in HUVEC. Currently we are validating lentiviral shRNA clones to knock-down kinase expression in HUVEC, and positive clones will be used to assess the role of kinases in angiogenic sprouting. 1) Sundaram and Buechner, 2016. 2) Betz et al., 2016. 3) Tung et al., 2009. 4) Ulmasov et. al., 2009. 5) Tung and Kitajewski, 2010. 6) Xie et al., 2013. 7) Dbouk et al., 2014. 8) Lai, et al., 2014. 9) Shaye and Greenwald, 2011. 10) Tattersall, et al., 2016.

Khan, Liakot et al. (2015) International Worm Meeting "Peri-lumenal intermediate filaments and microtubules maintain a single lumen in extending C. elegans excretory canals."

Gobel, Verena, Khan, Liakot, Membreno, Edward, Zhang, Hongjie

[International Worm Meeting, 2015]

In an RNAi-based modifier screen we identified 3 intermediate filaments (IFs: IFA-4, IFB-1, IFC-2) that enhanced the cystic ERM-1-overexpression excretory canal (EC) phenotype. Although IFB-1's function in EC morphogenesis has been noted, it remains unclear how IFs contribute to this process. Here we compare IFs' role in EC tubulogenesis with the roles of microfilaments (MFs; previously analyzed) and microtubules (MTs). IFs were found to build an intracellular perilumenal lattice, and to be non-redundantly required for lumen extension. Severe interference with IFs causes cell-body close cysts and loss of canal extension, but does not abort lumen formation. In contrast, severe interference with MFs via ERM-1/ACT-5, that we think expand the lumenal membrane via vesicle fusion, abolishes lumen formation. Selective IF removal during larval EC extension generates a "multiple lumen varicosity" phenotype with collapsed lumens between varicosities (periodic structures along extending canals that serve as osmotically sensitive "fueling stations" for wild-type growth). In contrast, larval MF removal produces short, thin lumens with small vacuoles at canal tips. IF removal generates true ectopic "lumens" (vacuoles lined with an apical membrane and cytoskeleton) not seen with MF removal. This scenario could suggest that IFs function by laterally integrating vacuoles into a lumenal membrane that has already acquired an actin coat, as opposed to MF-dependent membrane expansion via vesicle fusion at the tip that concomitantly assembles actin. A TBB-2/tubulin::GFP fusion localizes to the EC cytoplasm, but also to strands on the perilumenal IF lattice that in turn resides on perilumenal MFs. Colocalization and loss-of-function studies during EC development suggest that ERM-1 indirectly recruits lumenal IFs, whereas perilumenal lattice formation may depend on MTs. Interference with MTs copies the IF phenotypes and, unlike interference with MFs, enhances it. Thus, MTs like IFs support single lumen maintenance and lateral vacuole fusion, suggesting that one of MTs' roles in lumen extension is mediated by IFs. Effects of the 3 cytoskeletal components on endosomal and canalicular vesicles will be presented. Our findings suggest that EC lumen extension relies on tip and lateral growth and requires a resilient perilumenal IF matrix that allows vacuoles to dock at the lateral lumen (conspicuous in varicosities), thereby shaping the lumen's cylindrical tunnel structure and transmitting fluid pressure between varicosities that maintains lumen diameter and supports its anterior-posterior extension. .

Arena, Anthony et al. (2017) International Worm Meeting "Analysis of the roles of EXC-4 and human CLICs during tubulogenesis in C. elegans and endothelial cells."

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

[International Worm Meeting, 2017]

Biological tube formation (tubulogenesis) is a key process during vascular development and angiogenesis, and its disruption leads to disease. The chloride intracellular channel (CLIC) family was first implicated in tubulogenesis by the discovery that EXC-4, a worm CLIC, is required for development of the C. elegans excretory canal (EC) cell1; a single-celled tube that is a proven model for studying conserved regulators of tubulogenesis2. We, and others, have shown that two human CLICs, CLIC1 and CLIC4, are expressed in vascular endothelial cells and are required for lumen formation during angiogenesis3-5. Moreover, CLIC1, when localized to the apical plasma membrane, rescues the exc-4 null (0) phenotype, suggesting conservation of function between EXC-4 and CLIC16. In the EC EXC-4 is constitutively localized to the apical plasma membrane1. This localization is critical for EXC-4 function1, 6, and the ability of CLIC1 to rescue exc-4(0) was dependent on apical membrane targeting6. Therefore, membrane localization is an important determinant of EXC-4/CLIC function. In contrast to EXC-4, human CLICs accumulate in the cytoplasm and are only transiently recruited to the plasma membrane upon activation of G-protein-coupled receptors (GPCRs). Additionally, our results suggest that CLIC1 and CLIC4 influence different pathways downstream of GPCRs in endothelial cells, suggesting distinct functions for these CLICs. We are investigating whether CLIC4, like CLIC1, can rescue exc-4(0) when targeted to the apical EC membrane. Results from these experiments will address whether both CLICs have similar, or distinct, functions in tubulogenesis. Dependent on these results, we will undertake structure-function analyses to further define the functions of CLIC1 and CLIC4 in the EC and in human endothelial cells. We are also addressing whether EXC-4 can replace CLIC1 and/or CLIC4 in human endothelial cells, and whether exc-4 and G-protein signaling also intersect in CeEC tubulogenesis. 1) Berry et al., 2003. 2) Sundaram and Buechner, 2016. 3) Tung et al., 2009. 4) Ulmasov et al., 2009. 5) Tung and Kitajewski, 2010. 6) Berry and Hobert, 2006.

Victoria L Scranton et al. (2004) East Coast Worm Meeting "Toward expression and biochemical characterization of EFF-1."

William A Mohler, Victoria L Scranton

[East Coast Worm Meeting, 2004]

Expression of integral transmembrane proteins has always presented a major challenge to biologists. We have explored several different systems for expressing and purifying the integral transmembrane protein EFF-1, with varying degrees of success. We have initially focused on bacterial expression, because of advantageous inducible expressions systems and a variety of sequence tags available for affinity purification. We have successfully expressed high levels of a truncated 'solublilized' form of the protein (EFF-1-EC) using the pET bacterial expression system, and are using this pure product to raise specific antibodies to EFF-1. However, EFF-1-EC is confined to inclusion bodies (and likely not in a native conformation), and we have been unable to express the full length product - possibly due to known toxic effects of transmembrane proteins in bacterial expression systems. Another drawback is that proteins expressed in bacteria lack eukaryotic post-translational modifications. To overcome these issues and to incorporate eukaryotic post-translational modifications, we have also tried to express full length protein in the TnT Coupled Wheat Germ Extract System. The yields from this in vitro system will require further optimization to be useful.