Kuang, Xiangyu et al. (2021) International Worm Meeting "Computable early C. elegans embryo with a data-driven phase field model"

Tang, Chao, Zhao, Zhongying, Wong, Ming-Kin, Kuang, Xiangyu, Guan, Guoye, Zhang, Lei, Chan, Lu-Yan

[International Worm Meeting, 2021]

Morphogenesis is a precise and robust dynamic process during metazoan embryogenesis, consisting of both cell proliferation and cell migration. Despite the fact that much is known about specific regulations at the molecular level, how cell proliferation and migration together drive the morphogenesis at the cellular and organismic levels is not well understood. Here, using Caenorhabditis elegans as the model animal, we present a data-driven phase field model to compute the early embryonic morphogenesis within a confined eggshell. By using three-dimensional time-lapse cellular morphological information generated by imaging experiments to set the model parameters, we can not only reproduce the precise evolution of cell location, cell shape and cell-cell contact relationship in vivo, but also reveal the critical roles of cell division and cell-cell attraction in governing the early development of C. elegans embryo. In brief, we provide a generic approach to compute the embryonic morphogenesis and decipher the underlying mechanisms (Kuang†, Guan†, et al. bioRxiv, 2020, 422560).

Attila Stetak et al. (2008) Development & Evolution Meeting "The MAGI-1 protein functions as an organizer of LET-23 localization the adherens junctions"

ALEX HAJNAL, Attila Stetak, Sander Van Den Heuvel

[Development & Evolution Meeting, 2008]

The C. elegans hermaphrodite vulva is formed by the descendents of three out of six equipotent vulval precursor cells (the VPCs P3.p through P8.p). The gonadal anchor cell produces the LIN3 EGF signal that activates the conserved EGFR/RAS/MAPK signaling pathway in the adjacent VPCs and specifies the primary cell fate in P6.p. The basolateral localization of LET-23 EGFR in the VPCs is essential for the efficient receptor activation and consequently for proper vulval induction. A ternary complex consisting of the PDZ-domain proteins LIN-7, LIN-2 and LIN-10 is required for the localization of the EGFR to the basolateral compartment. Interestingly, PDZ-domains are often found in adaptor proteins that are control the subcellular localization of other proteins such as receptors. Using a forward genetic approach, we have identified the magi-1 gene (K01A6.2) as a suppressor of the let-60(gf) Multivulva phenotype. MAGI-1 is a multi-PDZ domain protein consisting of an N-terminal guanylate kinase domain with two overlapping WW repeats followed by five PDZ domains. Mammalian MAGI is expressed in neurons and epidermal cells and is localized at the cell junctions where it interacts with several proteins including PTEN and beta-Catenin. The C. elegans magi-1 gene encodes two isoforms transcribed from alternative promoters. Using translational MAGI-1::GFP reporters, we found that the two MAGI-1 isoforms are widely expressed, however only the isoform encoded by the shorter transcript is expressed in the VPCs. Furthermore, the MAGI-1::GFP fusion protein is localized at the adherens junctions of the epidermal and gut cells. MAGI-1 interacts in GST-pull-down experiments with HMP-2 beta-Catenin via its fifth PDZ domain and with both LET-23 EGFR and LIN-7 via its guanylate kinase or WW domains. magi-1 deletion mutants display no obvious developmental phenotypes, but magi-1(lf) animals exhibit reduced basolateral LET-23 EGFR staining in the VPCs and enhanced embryonic lethality in combination with apr-1 RNAi. Thus, MAGI-1 might serve as a junctional organizer protein during embryogenesis and vulval development.

Chang-Shi Chen et al. (2010) East Asia Worm Meeting "DAF-2 Insulin-like signaling network is involved in Pore-Forming Toxin Cellular Defenses in C. elegans"

Huan-Da Chen, Audrey Bellier, Ferdinand C. O. Los, Chang-Shi Chen, Raffi V. Aroian, Ya-Luen Yang, Cheng-Yuan Kao

[East Asia Worm Meeting, 2010]

Cytolytic pore-forming toxins (PFTs) are a class of potent virulence factor of many disease-causing bacteria that convert from a soluble form to a membrane-integrated pore to intoxicate host cells. Although PFTs play important roles in the development of bacterial disease, we have enormous gaps in our knowledge and are only beginning to understand what these toxins do to the target cells and especially to the whole organism. The DAF-2 insulin/insulin-like growth factor-1 signaling pathway, which regulates lifespan and stress resistance in C. elegans, is known to mutate to resistance to pathogenic bacteria. Here we reveal that reduction of the DAF-2 insulin-like pathway signal cascade also confers the resistance of C. elegans to Crystal toxins, which are PFTs produced by Bacillus thuringiensis. Moreover, this resistance signal diverges at 3-phosphoinositide-dependent kinase 1 (PDK-1) and feeds into a novel signal arm defined by the WW domain Protein 1 (WWP-1). Taken together, our data suggest that WWP-1 and DAF-16 function in parallel within the fundamental DAF-2 signaling network to regulate fundamental cellular responses in C. elegans.

Reed, Kailee et al. (2021) International Worm Meeting "Identifying the role of Pashas WW domain in primary microRNA recognition and processing"

Reed, Kailee, Cafferty, Erin, Montgomery, Taiowa

[International Worm Meeting, 2021]

MicroRNAs (miRNAs) are short, non-coding RNAs that together with their protein cofactors, bind to target messenger RNAs to promote mRNA decay and translational repression. miRNAs are highly conserved and are implicated in essentially all biological processes in plants and animals. In the absence of miRNAs, C. elegans arrest during embryogenesis. Even partial loss of miRNAs can lead to severe development defects. The biogenesis of miRNAs is similar from C. elegans to humans, although there are differences in how primary miRNA transcripts are distinguished from other RNAs in different species and even between different miRNA transcripts within the same species. To better understand how miRNAs are processed in C. elegans, we developed an mCherry-based sensor that is desilenced when primary miRNA processing is impaired. We then performed a forward genetic screen to identify mutations that desilence the sensor and which may therefore affect primary miRNA recognition or processing. We are now characterizing a line we identified in our screen that has a mutation in the WW domain of Pasha as we more broadly explore the role of Pasha in primary miRNA recognition and processing.

Attila Stetak et al. (2006) European Worm Meeting "The MAGI-1 Protein Functions as an Organizer Protein at the Adherens Junctions"

Erika Frohli-Hoier, ALEX HAJNAL, Attila Stetak

[European Worm Meeting, 2006]

Attila Stetak, Erika Frhli-Hoier and Alex Hajnal. The C. elegans hermaphrodite vulva is formed by the descendents of three out of six equipotent vulval precursor cells (the VPCs P3.p through P8.p). The gonadal anchor cell produces the LIN3 EGF signal that activates the conserved EGFR/RAS/MAPK signaling pathway in the adjacent VPCs and specifies the primary cell fate in P6.p. The basolateral localization of LET-23 EGFR in the VPCs is essential for the efficient receptor activation and consequently for proper vulval induction. A ternary complex consisting of the PDZ-domain proteins LIN-7, LIN-2 and LIN-10 is required for the localization of the EGFR to the basolateral compartment. Interestingly, PDZ-domains are often found in adaptor proteins that are control the subcellular localization of other proteins such as receptors.. Using a forward genetic approach, we have identified the magi-1 gene (K01A6.2) as a suppressor of the let-60(gf) Multivulva phenotype. MAGI-1 is a multi-PDZ domain protein consisting of an N-terminal guanylate kinase domain with two overlapping WW repeats followed by five PDZ domains. Mammalian MAGI is expressed in neurons and epidermal cells and is localized at the cell junctions where it interacts with several proteins including PTEN and ?-Catenin. The C. elegans magi-1 gene encodes two isoforms transcribed from alternative promoters. Using translational MAGI-1::GFP reporters, we found that the two MAGI-1 isoforms are expressed during embryogenesis, larval development and adulthood in epidermal cells, neurons, muscle cells, the gut and the somatic gonad. However only the isoform encoded by the shorter transcript is expressed in the VPCs. Furthermore, the MAGI-1::GFP fusion protein is localized at the adherens junctions of the epidermal and gut cells. MAGI-1 interacts in GST-pull-down experiments with HMP-2 -Catenin via its fifth PDZ domain and with both LET-23 EGFR and LIN-7 via its guanylate kinase or WW domains. Surprisingly, neither a deletion of the fifth PDZ domain in MAGI-1 nor RNAi against components of the adherens (HMR-1, HMP-2, APR-1) or septate junctions (AJM-1, DLG-1) causes a loss of junctional MAGI-1 localization. Moreover, magi-1 deletion mutants display no obvious developmental phenotypes, but magi-1(lf) animals exhibit reduced basolateral LET-23 EGFR staining in the VPCs and enhanced embryonic lethality in combination with apr-1 RNAi. Thus, MAGI-1 might serve as a junctional organizer protein during embryogenesis and vulval development.

Marida Bimonte et al. (2001) International C. elegans Meeting "Identification and characterization of the C elegans orthologue of the mammalian molecular adaptor Fe65, a cytosolic ligand of Alzheimer's beta-amyloid precursor protein"

Paolo Bazzicalupo, Davide Gianni, Salvatore Arbucci, Tommaso Russo, Marida Bimonte, Nicola Zambrano

[International C. elegans Meeting, 2001]

Mammalian fe65 genes encode three highly related proteins, Fe65, Fe65-L1 and Fe65-L2. The common modular structure of the Fe65s, composed of a WW domain and two independent phosphotyrosine binding domains, PTB1 and PTB2, suggests a potential role of molecular adaptors for these proteins. The PTB2 domains of the three proteins interact, at the level of the corresponding cytodomains, with the products of another gene family, which has the Alzheimer's beta-amyloid precursor protein gene (APP) as the most studied member. The Fe65s may interact with other cellular proteins; in the case of Fe65, its WW domain binds to several proteins, including Mena and the non-receptor tyrosine kinase Abl. The PTB1 domain may interact, at the membrane level, with the low-density lipoprotein receptor-related protein LRP, and in the nucleus with the transcription factor CP2/LSF/LBP1. APP is cleaved by alternative pathways mediated by alpha- or beta-secretase; in this latter case, the subsequent action of gamma-secretase determines the release, under pathological conditions, of the beta-amyloid peptide, the major component of the senile plaques typical to Alzheimer's disease. Gamma-secretase activity requires, or is coincident with presenilins. Most of the proteins taking part in these complex molecular machineries have been identified and, in some instances, characterized in the nematode. In C elegans , apl-1 is the nematode orthologue of APP; the sequence conservation at the C-terminal, cytosolic domain, between APL-1 and the mammalian proteins is strikingly elevated and the structural properties of the encoded protein suggest that APL-1 adopts the same topology of APP and of related proteins. Based on these evidences, we attempted to identify, in the C elegans genome, putative Fe65-like genes. We demonstrated that a single gene encodes a protein structurally and functionally related to mammalian Fe65s. In fact, the nematode protein has the same modular organization of the Fe65s, and is capable of binding both APL-1 and APP. Studies with reporters and with specific antibodies indicate that the corresponding gene is expressed in the nervous system and in the pharynx, starting in the embryo, through larval stages and in adults. By chemical mutagenesis and PCR screening we generated mutant worms bearing a null mutation of the C elegans fe65 orthologue. The mutant is homozygous lethal and its phenotype suggests that the encoded protein provides functions essential for embryogenesis and larval development. We are currently characterizing in more details the phenotype of these mutant worms. The nematode constitutes a simple, non redundant system in which to study the basic biology of the complex network of interacting proteins centred to APP and Fe65 proteins, and will hopefully give insights into the mechanisms leading to the alterations typical to Alzheimer's disease pathogenesis.

Junsu Kang et al. (2004) East Asia Worm Meeting "Which roles does the tumor suppressor gene - LATS kinase - play in C. elegans?"

Junsu Kang, Junho Lee

[East Asia Worm Meeting, 2004]

Precise regulation of cell proliferation and apoptosis is important to decide the organ size during development. It is not well known how these processes are tightly regulated. It seems true that many tumor suppressor genes play essential roles in these processes. Novel class of tumor suppressor genes -RASSF1A, WW domain protein, Mst kinase and LATS kinase - are known in the mammalian system to control to promote appropriate termination of cell proliferation and to stimulate apoptosis. They are highly conserved from nematodes to humans. One of them - LATS kinase - is a Ser/Thr kinase. Mammalian studies showed that overexpression of LATS1 inhibits cdc2 kinase activity and causes G2/M arrest. Also, LATS1 mutant mouse developed soft-tissue sarcomas, ovarian tumors and pituitary dysfunction; and Warts(LATS) mutant flies result in overgrowth. What would a tumor suppressor, such as LATS kinase, if existent, do in an animal such as C. elegans in which no tumors have been established? There is one homolog of LATS kinase in C. elegans . We found that Ce_LATS is expressed in muscle cells - body wall muscle, pharyngeal muscle, and sphincter muscle- in the embryo, larvae, and adult animals. It is possible that the functions of the nematode LATS indeed resembles those in other animals regardless of the absence of tumors by nature. We will pursue the cellular and molecular events in which LATS in involved in the hope that our results will facilitate the future research on the mammalian homologs.

Sebastien Holbert et al. (2001) International C. elegans Meeting "The Gln-Ala repeat transcriptional activator CA150 interacts with huntingtin: neuropathologic and genetic evidence for a role in Huntington's disease pathogenesis"

Robert J Ferrante, Isabelle Denghien, Sebastien Holbert, Jean Dausset, Tamara Kiechle, Christopher A Ross, Christian Neri, Cheryll Wellington, Michael R Hayden, Adam Rosenblatt, Russell L Margolis

[International C. elegans Meeting, 2001]

Huntington's disease (HD) is a neurodegenerative disease caused by polyglutamine (polyQ) expansion in the protein huntingtin (htt). Pathogenesis in HD appears to involve the formation of ubiquitinated neuronal intranuclear inclusions containing N-terminal mutated htt, abnormal protein interactions, and the aggregate sequestration of a variety of proteins (noticeably transcription factors). To identify novel htt-interacting proteins in a simple model system, we used a yeast two-hybrid screen with a Caenorhabditis elegans activation domain library (R. Barstead, ORMF, Oklahoma City, OK). We found a predicted WW domain protein (ZK1127.9) that interacts with N-terminal fragments of htt in two-hybrid tests. A human homologue of ZK1127.9 is CA150, a transcriptional coactivator with a N-terminal insertion that contains an unperfect (Gln-Ala)38 tract encoded by a polymorphic repeat DNA. CA150 interacted in vitro with full length htt from lymphoblastoid cells. The immunohistochemical expression of CA150 was markedly increased in human HD brain tissue, in comparison to normal age-matched human brain tissue, and showed aggregate formation with colocalization to ubiquitin-positive aggregates. In 432 HD patients, the CA150 repeat length explains a small but statistically significant amount of the variability in HD onset age. Our data suggest that abnormal expression of CA150, as mediated by interaction with polyQ-expanded htt, may alter transcription, and have a role in HD pathogenesis. Our data illustrate the value of using C. elegans for the direct identifcation of novel biochemical and genetic modifiers of human neurodegenerative disease pathogenesis [Holbert et al. (2001) Proc. Natl. Acad. Sci. USA 98 1811-1816].

Julie McCleery et al. (2005) International Worm Meeting "Regulation of GABAergic motor neuron fate specification by the sumoylation pathway and chromatin modifying factors"

Yishi Jin, Chris Li, James Meir, Julie McCleery

[International Worm Meeting, 2005]

The C. elegans ventral cord GABAergic DD and VD neurons have identical cell morphology but differ in several aspects. They are born from separate lineages at different stages of development, they form synapses to opposite body wall muscles and the juvenile DDs undergo developmental synaptic remodeling during L1 to L2 larval development (1). Thus far, only two genes have been shown to be differentially expressed in these two groups of neurons. The unc-55/coupTF nuclear hormone receptor is expressed in the VDs (2) and the flp-13 gene is expressed in the DDs (3). We have identified a consensus COUPII DNA binding site in the flp-13 promoter. In unc-55 mutants, flp-13 promoter driven transgenes are ectopically expressed in VD neurons suggesting that unc-55 specifies VD neuron fate by repressing DD neuron-specific genes including flp-13. To further understand the mechanism that distinguishes DD and VD neurons, we performed a genetic screen utilizing a Pflp-13-SNB-1::GFP marker. We isolated a group of mutants that show ectopic expression of the Pflp-13-SNB-1::GFP marker in VD neurons. These genes encode the small ubiquitin-like-modifier (SUMO) E1 and E2 enzymes, aos-1 and ubc-9, the ATRX homolog SWI/SNF chromatin remodeler xnp-1, and the MYST histone acetyltransferase mys-1. Sumoylation has been shown to play a role in transcriptional repression by modification of nuclear receptor transcription factors and chromatin modifiers. Our double mutant analysis is consistent with this possibility in that it indicates that the chromatin modifiers and sumoylation pathway components may function in the same pathway, and that this pathway may modulate the VD neuron specification program controlled by unc-55. P-xnp-1-xnp-1-GFP is altered in sumo E1 and E2 mutants indicating XNP-1 may be modified by sumoylation. We are currently performing biochemical experiments to determine if XNP-1, MYS-1 or UNC-55 may interact with the sumo E2 enzyme UBC-9.1.White J Nature 271, 764-766 (1978)2.Zhou MH, Walthall WW The Journal of Neuroscience 18(24), 10438-10444 (1998)3.Kyuhyung K, Li C The Journal of Comparative Neurology 475, 540-550 (2004)

Marida Bimonte et al. (2002) European Worm Meeting "feh-1 and apl-1, the orthologues of Fe65 and Alzheimer's -amyloid precursor APP control a common pathway involved in the regulation of the pharyngeal activity in C. elegans"

Nicola Zambrano, Marida Bimonte, Davide Gianni, Tommaso Russo, Salvatore Arbucci, Paolo Bazzicalupo

[European Worm Meeting, 2002]

The main histopathological feature of Alzheimer's disease is the occurrence of the senile plaques in the brain. These are made of aggregated forms of the amyloid peptide (A ), derived from the proteolytic processing of a membrane precursor, APP. The cytosolic domain of APP contains the YENPTY sequence, which is the site of interaction for various PTB domain-containing adaptors. These proteins, interacting simultaneously with APP and other ligands, generate macromolecular complexes, which may underlie their biological functions. Emerging evidence indicates that such complexes may constitute a scaffolding site for kinases such as Abl and Jnk. Among the APP ligands, the mammalian Fe65 proteins share a common modular organisation, made of a WW domain and two PTB domains. Their PTB2 modules are engaged in the interaction with the cytodomains of APP and related proteins APLP1 and APLP2. A functional significance of Fe65/APP interaction may reside in the cytosolic retention of Fe65 by APP. Gamma-secretase cleavage of APP, one of the events which determines release of A , results in the nuclear translocation of Fe65. In the nucleus, likely through its interaction with other proteins, including the transcriptional factor CP2/LSF and the histone acetyltransferase Tip60, Fe65 is involved in transcriptional regulation. The highly redundant system of Fe65 and APP multigenic families renders the mammalian models hard to dissect by reverse genetics. In C.elegans, we have identified a gene, feh-1 (Fe65 homolog-1), which encodes a protein structurally and functionally related to mammalian Fe65s, that is capable of binding APL-1, encoded by the nematode orthologue of APP. The unicity of both feh-1 and apl-1 in the nematode genome prompted us to analyze the C. elegans system. feh-1 is expressed in the nervous system and in the pharynx. We generated a null allele of feh-1 (gb561): the homozygous mutants arrest as embryos or as L1 larvae. No pharyngeal pumping can be detected in L1s, which do not develop further, before dying, suggesting that they cannot feed. Surprisingly, the heterozygous worms (gb561/+) have an increased pharyngeal pumping. Using RNAi by injection, as well as RNAi by feeding, we have observed that strong reductions in FEH-1 levels determine pharyngeal arrest, which results in the lethal phenotypes, while milder reductions are associated to viability and increased pharyngeal activity. Interestingly, apl-1 RNAi similarly determines an increased pharyngeal contraction rate. APL-1 reduction in the gb561/+ background unexpectedly restores the normal contraction rate of the pharynx. Taken together, such data suggest that FEH-1 and APL-1 act in a common pathway, regulating feeding behaviour. The pharyngeal activity is finely tuned by the stoichiometry of the complex between the two proteins. In order to gain information about the molecular events involving FEH-1 and APL-1 in pharyngeal activities, we are exploring potential interactions between these and other genes, which are involved in these phenomena. These results will be useful for the understanding of the basic biology of Fe65-APP interaction and of the molecular phenomena regulated by this evolutionarily conserved system of interacting proteins.