Gupta, Pratyush et al. (2013) International Worm Meeting "MRG-1 and RFP-1 regulate proliferation in the germline."

Leahul, Lindsay, Gupta, Pratyush, Hansen, David, Jasper, Katie

[International Worm Meeting, 2013]

We previously demonstrated that the proteasome inhibits the proliferative fate in the C. elegans germ line. Reduced proteasomal function enhances over-proliferation in a sensitized genetic background (Macdonald, Knox et al. 2008). Presumably, the proteasome is involved in degrading proteins that either promote the proliferative fate, or inhibit meiotic entry. We refer to these as proliferation promoting proteins (PPPs). To identify these PPPs, we first sought to identify the substrate recognition subunits (SRS) of E3 ubiquitin ligases that may target the PPPs for degradation by the proteasome. We screened 826 SRSs by RNAi in four sensitized genetic backgrounds and found five that enhance over-proliferation. One of these, RFP-1, specifically enhances glp-1(ar202gf) to form a germline tumor, albeit incomplete. Large-scale 2-hybrid screens identified potential binding partners for RFP-1 (Crowe and Candido 2004; Zhong and Sternberg 2006), which could be PPPs that are targeted for proteasomal degradation by RFP-1. One of these, mrg-1, partially suppresses the over-proliferation phenotype of glp-1(ar202gf); rfp-1(ok572), suggesting that it may function as a PPP. We found that MRG-1 levels do increase in an rfp-1 mutant, as well as when proteasomal function is decreased through genetic mutation or chemical inhibition. Therefore, MRG-1 protein levels are likely regulated through proteasomal-mediated degradation. We are currently determining how mrg-1 may participate in controlling the proliferative fate in the germline.

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].

GUPTA, P. et al. (2015) International Worm Meeting "MRG-1 and RFP-1 regulate proliferation in the germline."

Hansen, D., Wang, X., GUPTA, P., LEAHUL, L., WANG, C.

[International Worm Meeting, 2015]

The germ line in C. elegans is organized in an assembly line fashion, with each region containing cells at a specific point in development. As cells move proximally, they progress further through the developmental stages of gametogenesis. Presumably, different factors are expressed in each region to execute each developmental stage. As cells move proximally, some factors must be turned on, while others are turned off. We previously demonstrated that proteasomal degradation of proteins is key for cells to properly exit the proliferative fate and enter into meiotic prophase. Reduced proteasomal function enhances over-proliferation in a sensitized genetic background (Macdonald et al. 2008). We hypothesized that the proteasome is involved in degrading proteins that either promote the proliferative fate, or inhibit meiotic entry. We refer to these as proliferation promoting proteins (PPPs). To understand how proteasomal degradation contributes to maintaining the balance between proliferation and differentiation, we sought to identify these PPPs. To do this, we first focused on identifying the substrate recognition subunits (SRSs) of the E3 ubiquitin ligases to find those that may target the PPPs for degradation. Since the SRSs directly bind their target proteins, we reasoned that if we genetically identify the SRSs involved, we could then biochemically identify the PPPs as interacting proteins. We screened 826 SRSs by RNAi in four sensitized genetic backgrounds and found five proteins that enhance over-proliferation when their activities are reduced. One of these, RFP-1 (putative E3 ligase), enhances glp-1(ar202gf) to form a germline tumor, albeit incomplete. To identify the target proteins of RFP-1, and potential PPPs, we screened the interacting proteins of RFP-1 and looked for suppression of the germline tumor when their activity was reduced. Through this, we identified the chromo-domain containing protein MRG-1. mrg-1(0) partially suppresses over-proliferation in glp-1(ar202gf); rfp-1(ok572) animals, suggesting that it may function as a PPP. We found that MRG-1 levels increase in an rfp-1 mutant, as well as when proteasomal function is decreased through genetic mutation or chemical inhibition, suggesting that its activity is regulated, at least in part, through its levels of accumulation that is likely controlled through proteasomal-mediated degradation (Gupta et al. 2015). Analysis in tissue culture cells further supports that RFP-1 directly targets MRG-1 for degradation by the proteasome. Therefore, we have identified MRG-1 as a key player in regulating the proliferation vs. differentiation decision and determined that its activity is controlled, at least in part, through proteasomal degradation.

Neri C et al. (1998) European Worm Meeting "A C. elegans model of neuron dysfunction in polyglutamine expansion diseases: suggestive evidence for polyglutamine-dependent intracellular aggregation and touch receptor neuron dysfunction"

Chalfie M, Moriniere S, Neri C, Du H-P

[European Worm Meeting, 1998]

Polyglutamine expansion (PGE) underlies several neurodegenerative diseases including the Huntington!s disease (HD). An inverse correlation between the age-of-onset and the length of the polyglutamine (polyQ) tract is observed in PGE disease families. The mechanisms which cause neurodegeneration in PGE diseases is unknown. Data from human patients, transgenic mice studies, and mammalian cell-based models have suggested that a gain of toxic properties is likely to be induced by PGE in target neurons, resulting in the formation of intracellular aggregates and cell death. Studies of HD in mammalian cell-based models have suggested that both the length of the polyQ tract and the length of the disease protein fragment carrying the expanded polyQ tract influence the localization of intracellular aggregates and the progression of neurodegeneration. The aim of our study is to develop models of PGE-induced neurodegeneration in C. elegans since the genetics, well-characterized development, and the known genomic sequence make this a useful organism in which to study these problems. We used the mec-3 promoter (mec-3p) to express GFP fusions which contain a short N-terminal fragment of huntingtin with a normal (17 units) or expanded (84 units) polyQ tract. The mec-3 gene is expressed in 10 neurons including the six touch receptor neurons AVM, ALML, ALMR, PVM, PLML and PLMR. Transgenic worms were analyzed for intracellular pattern of GFP expression, touch sensitivity, and occurrence of cell death in touch receptor neurons. Mec-3p driven GFP expression typically shows a diffuse expression pattern in the cell body and process. In transgenic animals expressing the long polyQ construct, obvious protein aggregation was observed, particularly as two large and bright perinuclear GFP aggregates were detected. A diffuse GFP expression was never observed in the processes. In transgenic animals expressing the short polyQ construct, a less severe modification of GFP localization was observed, resulting in a pattern of expression closer to that of wild type animals with GFP expression driven by mec-3p. Touch tests were performed over a period of seven days starting from the young adult stage. In worms expressing the long polyQ construct, less than 30% were Mec at the tail at Day 1, and more than 50% were Mec at the tail at Day 7. In worms expressing a short polyQ construct or in animals with GFP expression driven by mec-3p, less than 7% and 30% were Mec at the tail at Day 1 and Day 7, respectively. Cell death was not observed in these experiments. Other studies have suggested that huntingtin fragments with an expanded polyQ tract (150 Glns) induce a neuronal phenotype in transgenic animals (P. Faber, J. Alter, M. MacDonald, and A. Hart, personal communication). Our preliminary data suggest that GFP fusions comprising a short N-terminal huntingtin fragment with 84 consecutive Glns induce perinuclear aggregation and touch insensitivity when expressed in touch receptor neurons. Our data also suggest that the transgenic animals obtained might be useful for studying the biochemical mechanisms involved in polyQ-induced neuron dysfunction.