Shoman LM et al. (1994) Worm Breeder's Gazette "Worm Community System Users Envision Future Application."

Grossman E, Shoman LM, Jamison DC, Schatz BR

[Worm Breeder's Gazette, 1994]

Worm Community System Users Envision Future Application. Laura Shoman, Curt Jamison, Ed Grossman, Bruce Schatz, Community Systems Laboratory, National Center for Supercomputing Applications, University of Illinois, Urbana- Champaign, 405 North Mathews, Urbana, IL 61801 (wcs@csl.ncsa.uiuc.edu)

Hao, Hongyan et al. (2021) International Worm Meeting "The KASH-independent role of ANC-1 in positioning organelles in Caenorhabditis elegans"

Hao, Hongyan, Starr, Daniel, Cain, Natalie, Guerrero, Leslie, Kalra, Shilpi, Bolivar, Jessica, Jameson, Laura

[International Worm Meeting, 2021]

The LINC complexes formed by outer nuclear KASH proteins and inner nuclear SUN proteins play important roles in connecting nuclei to the cytoskeleton. ANC-1, an ortholog of Nesprin-1/2, is proposed to physically tether nuclei to the actin cytoskeleton through its KASH domain at the C-terminus and the calponin homology (CH) actin-binding domains at the N-terminus. anc-1 null mutants show severe nuclear positioning defects in the hypodermal syncytial cells in C. elegans. However, we found that the CH domains were dispensable for hyp7 nuclear positioning and disruption of SUN/KASH interaction only caused mild nuclear positioning defects. In addition, in anc-1 null mutants, the ER, mitochondria, and lipid-droplets were unanchored and sometimes moved freely in the cytoplasm. ANC-1 contains six tandem repeats predicted to form spectrin-like structures. Deletion of the spectrin-like region caused strong nuclear and ER anchorage defects. KASH proteins also contain a transmembrane domain adjacent to the luminal KASH domain, we found that the deletion of the transmembrane domain enhanced the nuclear or the ER anchorage defects of the KASH deletion mutant. In the hyp7, ANC-1 had a similar distribution pattern as the ER, and deletion of the tandem repeats strongly affected ANC-1's localization. Here, we propose a cytoplasmic integrity model where ANC-1 functions in positioning nuclei, ER, mitochondria, and likely other organelles in place, probably through the spectrin-like region.

Clucas C et al. (1998) European Worm Meeting "The use of Green Fluorescent Protein to identify genes involved in aspects of hypodermal function in Caenorhabditis elegans."

Johnstone IL, Stewart EJ, Clucas C

[European Worm Meeting, 1998]

We have recently performed forward genetic screens for embryonic lethal mutants using strains that carry either a col-12/GFP fusion transgene or a dpy-7/GFP transgene. We have used the green fluorescent protein expression form these hypodermal specific markers to assist in the preliminary characterisation of phenotype in isolated mutants. We find a variety of phenotypes including failure to express particular col/GFP gene fusions, greatly restricted expression but relatively normal hypodermis as judged be elongation, normal hypodermal expression but failure to elongate, and a class that expresses and elongates relatively normally (at least as judged in preliminary characterisations), however seem to fail to synthesise a normal cuticle as judged by herniations of cellular material through the newly formed cuticle. These mutants are being further characterised. Several have been mapped and one with a possible role in elongation has been repaired by cosmid rescue (details of this will be presented in a separate abstract by Laura McMahon). Recently we have successfully generated monoclonal antibodies against the dpy-7 collagen which is being used to further characterise the expression/localisation of the dpy-7 protein in wild type and various mutant embryos.

Xavier Michelet et al. (2006) European Worm Meeting "VPS-32 is an Endosomal Protein Essential to Maintain the Integrity of Epithelial Cells during C. elegans Development"

Laura BENKEMOUN, Xavier MICHELET, Christophe LEFEBVRE, Renaud LEGOUIS, Nathalie ROUDIER

[European Worm Meeting, 2006]

Xavier Michelet, Laura Benkemoun, Nathalie Roudier, Christophe Lefebvre and Renaud Legouis. Class E VPS (Vacuolar Protein Sorting) proteins are involved in the formation of MultiVesicular Body in yeast. They are components of the Endosomal Sorting Complexes Required for Transport (ESCRTI, II and III). We have previously shown that the inactivation of the C. elegans class E vps genes resulted in various phenotypes ranging from lethality to an absence of obvious phenotype (1). In particular VPS-27 is involved in endosomal and autophagic pathways and its inactivation affects cholesterol traffic and larval molting (1). We present here the characterization of VPS-32, a component of ESCRT III. Phylogenetic and functional analyses of the two vps-32 homologues present in the C. elegans genome indicated that only one encodes a functional protein, meanwhile the other is a pseudogene. Using antibodies and VPS-32::GFP fusion proteins, we observed that VPS-32 is expressed in vesicular structures and enriched in epithelial cells during the morphogenesis of the embryo.. vps-32(RNAi) and vps-32 null mutant animals arrest their development at mid-embryogenesis and early L1 stage respectively, with a disorganisation and degradation of the epidermis. Moreover, vps-32 RNAi feeding of larvae result in L4 arrest with the same epidermal defect associated with abnormal molting. Optical and electron microscopy analyses of vps-32 animals, revealed the presence of enlarged VPS-27-positive endosomes and an excess of autophagy. Theses results indicate that, similarly to several VPSE, VPS-32 protein is required for endosomal and autophagic pathways. However despite these similarities, inactivation of vpsE genes results in different developmental phenotypes. A model to explain the heterogeneity between vpsE phenotypes will be presented.

John Kalb et al. (2001) International C. elegans Meeting "Analysis of PHA-4 activity on the myo-2 promoter and identification of a new PHA-4 DNA binding site"

Pete Okkema, Jim McGhee, John Kalb

[International C. elegans Meeting, 2001]

PHA-4 is a forkhead/winged helix transcription factor that is essential for pharyngeal organogenesis. PHA-4 is expressed in all five classes of pharyngeal cells. Ectopic expression of PHA-4 leads to ectopic expression of pharyngeal markers. We wish to identify other factors that cooperate with PHA-4 to confer cell-type specificity. In pharyngeal muscles, PHA-4 binds to a site in the myo-2 promoter to activate myo-2 transcription. A potential transcriptional cofactor of PHA-4 is PEB-1, a novel DNA binding protein that is expressed in the pharynx and binds to the myo-2 promoter at a site that overlaps with PHA-4 ( 1 ). To test for PHA-4 and PEB-1 interactions we have expressed both factors in yeast to investigate their ability to activate transcription of a reporter gene regulated by the binding sites found in the myo-2 promoter. Both PHA-4 and PEB-1 (weakly) activate transcription when expressed alone. However, we detect neither synergy nor interference between PHA-4 and PEB-1 when they are coexpressed. Other experiments also suggest no evidence for cooperative binding but rather that PEB-1 may interfere with PHA-4 binding ( 1 ). To identify PHA-4 target genes, we are determining the preferred PHA-4 binding site. The pha-4 gene produces three PHA-4 protein isoforms called PHA-4A, B and C that differ only at their N-termini. After six rounds of DNA site selection from a pool of random oligonucleotides 20 bp long, baculovirus-produced PHA-4B selects the sequence TGTGG(C/T); this differs somewhat from the known TGTTTG PHA-4 binding site in the myo-2 promoter. We are currently putting this selected sequence in the promoter of a GFP reporter gene to determine if it functions as an enhancer in vivo and continuing site selection with PHA-4A and C. ( 1 ) See the abstracts by Laura Beaster-Jones and Anthony Fernandez.

Y Zhang et al. (1999) International C. elegans Meeting "Phenotypic analysis of two C. elegans endocytosis mutants rme-1 and rme-8"

D Hirsh, L Pedraza, B Grant, Y Zhang

[International C. elegans Meeting, 1999]

Yinhua Zhang, Barth Grant, Laura Pedraza and David Hirsh, Dept. of Biochemistry and Molecular Biophysics, Columbia University, New York, NY10032 We wish to complement the classical cell biology studies on receptor-mediated endocytosis in animal cells with a genetic analysis in C. elegans . We isolated mutants defective in yolk-protein uptake using our YP170-GFP assay (see abstract by Grant et al). Here we describe two endocytosis mutants: rme-1 which appears to function in the endosome, and rme-8 which appears to function early in endocytosis. rme-1 mutants have a mild block in YP170-GFP uptake, but have normal brood size and viability. rme-1 worms also form large vacuoles in their gut cells and to a lesser degree in hypodermal cells. In wildtype gut cells, RME-1 is present on small vesicles with a punctate pattern. These vesicles take up a low level of GFP from the body cavity when it is secreted from muscle. In one missense mutant, RME-1 is also present on the surface of large vacuoles. The large vacuoles accumulate GFP to a higher level than do wild type vesicles. One model would be that the large vacuoles are derived from the small vesicles and reflect a defect in endosome sorting which results in a mild reduction of YP170-GFP uptake by rme-1 mutants. Currently we are testing this model. RME-1 contains a conserved region of 555 amino acids which has 75% identity to its mammalian homologs, indicating they function similarly in mammalian cells. rme-8 is a ts lethal with a strong block in YP170-GFP uptake at all temperatures. rme-8 is required at all developmental stages; shifting mutant larvae to 25C causes arrested development. Arrested larvae fail to shed their old cuticles. Shifting adult hermaphrodites to 25C causes endoreplication in oocytes and degeneration of the gonad and intestine. The yolk receptor (RME-2) and alpha-adaptin (a component in clathrin coated pit) co-localize with yolk in discrete dots at 15C and in large patches at 25C. This colocalization suggests that the rme-8 mutation blocks the internalization step of endocytosis at the plasma membrane. rme-8 maps to a 300kb genomic interval between gld-1 and lin-10 ; we are further mapping it in order to clone it and identify its function in endocytosis.