Khan, Munzareen et al. (2021) International Worm Meeting "

Khan, Munzareen, Sengupta, Piali, Dwyer, Noelle, Hartmann, Anna, Bargmann, Cori, Piccione, Madeline

[International Worm Meeting, 2021]

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Wu S-L et al. (1996) East Coast Worm Meeting "CHARACTERIZATION OF AN ATYPICAL PROTEIN KINASE C IN C. Elegans."

Rubin CS, Wu S-L

[East Coast Worm Meeting, 1996]

Protein kinase Cz (PKCz) is an atypical member of the PKC superfamily. PKCz contains Cys-rich zinc binding and catalytic domains that are homologous with corresponding regions in other PKCs. Unlike other PKCs, the zeta isoform in not activated by diacyl glycerol, TPA or calcium. However, PKCz appears to be a target for activation by alternative lipid second messengers (3,4,5-phosphoinositol, ceramide) generated by PI-3-kinase and/or sphingomyelinase. Activated PKCz has been implicated in the transmission of regulatory signals from the cytoplasm to the nucleus, but its precise physiological roles remain to be rigorously determined. We have initiated studies on the structure, function and regulation of an atypical C. elegans PKC (designated PKC-Z) that is related to mammalian PKCz. A cDNA that encodes full-length PKC-Z was cloned and sequenced. The predicted PKC-Z protein (598 amino acid residues, molecular weight = 68,000) is only ~55% identical with mammalian PKCz. N-terminal and central portions of the two proteins are highly divergent. However, the catalytic, psuedosubstrate and Cys-rich domains are highly conserved. Comparison of the cDNA sequence with data from the C. elegans genome project indicates that the PKC-Z gene (LGII) encompasses 3.6 kbp of DNA and contains 9 exons. A C terminal fragment (120 amino acids) of PKC-Z was expressed as a His-tagged fusion protein in E. coli, purified and injected into rabbits to produce antiserum. Western immunoblot analysis revealed that PKC-Z is most abundantly expressed in the soluble fraction of embryos. The kinase is also evident in L1-L4 larvae and adult nematodes, but the bulk of the enzyme is tightly associated with the particulate fractions of post-embryonic animals. Consistent with the latter observation, immunostaining and confocal microscopy of permeabilized C. elegans revealed that PKC-Z is localized and concentrated in discrete intracellular clusters. The nature of the intracellular structure at which PKC-Z accumulates is not yet known. PKC-Z is being expressed in baculovirus/Sf9 cells and in mammalian cells for enzymic characterization. In addition, injected anti-sense oligonucleotides are being used to probe the function(s) of PKC-Z in embryogenesis.

Yu X et al. (2010) Aging, Metabolism, Stress, Pathogenesis, and Small RNAs, Madison, WI "A FYVE Domain-Containing Myotubularin Is Essential for Maintenance of Muscle Fibers and Prevention of Autophagy in Adult Caenorhabditis elegans"

Yu X, Ma J, Zhao Z

[Aging, Metabolism, Stress, Pathogenesis, and Small RNAs, Madison, WI, 2010]

The myotubularin (MTM) enzymes specifically dephosphorylate phosphotidylinositol-3-phosphate. They play a major role in membrane trafficking and have major pathological implications in muscular and neuronal disorders. We employed the Caenorhabditis elegans nematode as an animal model to study the role of these enzymes in aging associated problems. We focused our study on an enzyme designated ceMTM3. ceMTM3 contains a FYVE lipid-binding domain at its C-terminus which binds phosphotidylinositol-3-phosphate. It is expressed during the early development and adulthood of the animal and is predominantly distributed in muscles and eggs. Knockdown of the enzyme by using feeding-based RNA interference resulted in an increased level of phosphotidylinositol-3-phosphate and caused severe impairment of body movement of the worms at their post-reproductive ages and significantly shortened their lifespan. The morbidity and mortality are caused by an accelerated loss of muscle fibers which is preceded by the early onset of autophagy in the adult worm. This study thus reveals an important role of ceMTM3 in maintaining muscle functions and preventing autophagy, which may have clinical implications in prevention and treatment of sarcopenia.

Gilleard JS et al. (1995) International C. elegans Meeting "REGULATION OF THE CUTICULAR COLLAGEN GENE dpy-7"

Barry JD, Gilleard JS, Johnstone IL

[International C. elegans Meeting, 1995]

We are interested in identifying trans acting factors which control cuticular collagen gene expression. Sequences sufficient for regulated expression of dpy-7 appear to reside relatively close to the initiator ATG since the wild type gene only requires 310bp of 5' flanking sequence for transformation repair of phenotype. We have analysed sequences necessary and sufficient for dpy-7 expression using lac-Z fusions and we have identified a 95bp fragment, encompassing the transcritional start sites, which is sufficient to produce lac-Z expression predominantly, or possibly exclusively, in hypodermal cells. This fragment includes a region in which 65 out of 72bp are conserved in the 5' flank of the C.briggsae dpy-7 homolog and interspecies transformation rescue and lac-Z fusion experiments have shown that the dpy-7 cis regulatory elements are functionally conserved. A 15bp deletion at the 5' end of this homology which removes a AGATAA motif, also present in C.briggsae, completely ablates lac-Z expression . We are attempting to identify transcription factors which bind to this motif by South Western expression library screening. We are also searching for new C.elegans GATA factor homologs with a particular interest in any which are expressed in hypodermal cells postembryonically.

Dolinski CM et al. (1997) International C. elegans Meeting "PHYLOGENETIC IMPLICATION OF BUCCAL CAPSULE DEVELOPMENT IN SOME BACTERIAL FEEDING NEMATODES"

Baldwin JG, Dolinski CM, Thomas WK

[International C. elegans Meeting, 1997]

Bacterial feeding nematodes including Zeldia punctata (Rhabditida: Nematoda), and Caenorhabditis elegans differ profoundly in feeding adaptations and specifically in the rhabdions (cuticular thickenings), and associated cells lining in the buccal capsule. We are carrying out a range of tests to determine what rhabdions are evolutionarly homologous between the two species. Reconstruction of the buccal capsule and procorpus (pharynx) with transmission electron microscopy indicates that the lining of the buccal capsule of Z. punctata includes four sets of muscular radial cells ma, mb, mc, md in contrast the same region in C. elegans which has two sets of epithelial radial cells (e1, e3) and two sets of radial muscle cells (m1, m2). In Z. punctata ma is a set of three radial muscle cells each with 2 nuclei. In C. elegans m1 has the identical arrangement. Similarly, in Z. punctata mb is a set of 3 muscle cells each with one nucleus, similar to m1 in C. elegans. These findings could contradict all previous hypotheses of homology, and suggest instead that ma and mb in Z. punctata are homologous respectively with m1 and m2 in C. elegans. Ongoing additional tests of homology include comparative cell lineages and screening mutants to recognize genes involved in buccal capsule expression.

Mi-Mi, Lei et al. (2013) International Worm Meeting ""Ultrastructure analysis of the sarcomeres in worms that lack Z-line formins"."

Pruyne, David, Mi-Mi, Lei

[International Worm Meeting, 2013]

Even with extensive studies that revolve around actin cytoskeleton, the detailed mechanism as to how actin filaments are recruited into sarcomeres, the smallest units of striated muscle contractile lattice, is yet to be elucidated. The barbed ends of actin filaments anchor at the Z-line structures (dense bodies in C. elegans) that define the sarcomere ends. Net actin polymerization favorably occurs at the barbed end that is known to have unique association with formins, one of the highly conserved and most widespread families of actin nucleating factors. We have shown in our previous study that two nematode formins, CYK-1 and FHOD-1 are Z-line-associated proteins that work together to organize and maintain sarcomeric organization in C. elegans striated muscle - the first evidence in an intact organism. In this study, we have carried out the ultrastructure analysis on sarcomeric structures of worms lacking CYK-1 and/or FHOD-1. Not only do the current electron microscopy results complement our previous conclusions, they also suggest that CYK-1 and/or FHOD-1 may contribute in achieving normal Z-line structures in C. elegans sarcomeric organization.

Bi, Y. et al. (2017) International Worm Meeting "Genome wide screening of hybrid incompatibility identifies multiple C. briggsae loci that rescue male sterility in F1 hybrid between C. briggsae and C. nigoni."

Li, R., Zhao, Z., Ding, Q., Ren, X., Bi, Y.

[International Worm Meeting, 2017]

Hybrid incompatibility (HI) between related species plays a key role in speciation. The genetic basis of HI has been intensively studied in yeast, Drosophila, and mouse species, but remains largely unexplored in nematodes until recent isolation of a C. briggsae sister species, C. nigoni, which can mate with each other and produce a few viable progeny. We have recently generated a genome-wide HI map between the two species by phenotyping 111 hybrid strains, each carrying a defined introgression fragment derived from GFP-labeled C. briggsae chromosome. Notably, the HI phenotypes were scored essentially in the C. nigoni background produced by GFP-aided backcrossing for at least 15 generations. However, many HI phenotypes were manifested in the F1 generation, often in a crossing direction-dependent manner. Unfortunately, few HI loci have been mapped in F1 hybrid between the two species. We address this issue using the large collection of introgression strains. This is because the crossings between individual C. nigoni strains carrying a defined C. briggsae fragment and C. briggsae wild isolate AF16 will produce selective homozygosity or hemizygosity of the C. briggsae genomic fragment in the F1 hybrid, whereas the remaining genomic contents would be comparable to those of the F1 hybrids between the wild isolates of the two species. Contrasting the hybrid F1 phenotypes in the crossings of AF16 with C. nigoni wild isolate or with an introgression strain permits isolation of HI loci in the F1 generation. We perform the crossings in both directions between AF16 and the introgression strains that are prioritized based on the even distribution of introgressions over the C. briggsae genome. Altogether, the introgression fragments cover approximately 80% of the C. briggsae genome. Surprisingly, we identified numerous C. briggsae loci that produced an opposite HI phenotype in F1 hybrid compared to that in the introgression lines, including rescue of male sterility and killing of female, which is consistent with the BDM model of speciation. In addition, some autosomal HI loci demonstrate crossing direction-dependent HI phenotypes, for example, selective male killing or global killing of all progeny, while others show crossing direction-independent killing of all progeny. In summary, we are able to identify numerous C. briggsae loci that produce crossing direction-dependent or independent incompatible phenotypes in F1 hybrids, which provide a foundation for molecular characterization of HI between the two nematodes.

Britton C et al. (1997) International C. elegans Meeting "Regulation of the gut cysteine protease gene gcp-1"

Johnstone IL, Britton C, McKerrow JH

[International C. elegans Meeting, 1997]

Nematode proteolytic enzymes are often expressed in a stage- and tissue-specific manner to carry out defined functions. We have been examining the regulatory mechanisms controlling expression of a cysteine protease gene, gcp-1, of C. elegans. Northern blot analysis and in situ hybridisation have shown that gcp-1 is expressed specifically in the gut of all stages except the embryo; this is also demonstrated by gcp-1/lac Z reporter constructs. Promoter deletion analysis has indicated that 210bp upstream of the transcription start site is sufficient for gut-specific expression. Within this region are three GATA motifs, mutation of which individually results in a reduction in reporter gene expression. To examine the role of GATA motifs in gcp-1 regulation, lac Z reporter constructs were generated containing multimerised copies of this motif linked to a 100bp region upstream of the gcp-1 start site. In contrast to the native gcp-1/lac Z constructs, this concatamer-containing construct resulted in strong expression in the embryonic gut and in adult hypodermal cells, as well as larval gut cells. More constructs are currently being examined to try to determine the possible mechanisms involved in this alteration in stage and tissue specificity.

Jiang LI et al. (1997) International C. elegans Meeting "STUDY OF NEUROGENESIS DURING C. ELEGANS MALE SPICULE DEVELOPMENT"

Sternberg PW, Jiang LI

[International C. elegans Meeting, 1997]

SPD, SPV and SPC are three spicule associated neurons in C. elegans males responsible for multiple steps during male mating behaviors including spicule insertion and sperm transfer. These three neurons are generated from b and z sublineages, descendants of the male B blast cell. b and z sublineages share similarity with the Drosophila sensory organ precursor lineage, where one cell divides twice to generate neuron and non-neuronal structural cells, sheath and socket. We are interested in how these neuronal precursors are generated, and how the neuronal vs. non-neuronal fates are determined. Previous studies have shown that b and z fates are specified via multiple cell-cell interactions. We are using a spicule neuron-specific molecular marker, gpa-1, to further analyze these lineages. Cell ablation experiments have shown that gpa-1::lacZ is expressed in SPD neurons specifically, while gpa-1::GFP is expressed in all three spicule associated neurons. Using this molecular marker, we have started to examine neurogenesis during male spicule develop-ment in several types of mutants with either defective spicule morphology or defective mating behavior. We have also started an F1 clonal screen to look for mutants with misexpression of this marker. From a pilot screen of 3,200 gametes, several classes of mutants have been isolated, including those that lack gpa-1 expression and those with additional expression specifically in the spicule region.

Memar, Nadin et al. (2009) International Worm Meeting "A view on the embryogenesis of the Caenorhabditis Family."

Martin, Katharina, Schnabel, Ralf, Memar, Nadin

[International Worm Meeting, 2009]

The molecular differences of the four Caenorhabditis species C. elegans, C. briggsae, C. remanei and C. brenneri are currently of great interest, however little is known about development. Zhao et al. (2008) reported an automatic lineage of C. briggsae and came - based mostly on the cleavage pattern and cell positions - to the conclusion that the embryogenesis of the two species is very similar. We now present detailed 4D analyses of the species including the terminal differentiation patterns. All analyses including bioinformatical quantifications of cell behaviour show a huge similarity between those species. Immunochemical analyses of the tissue distributions only reveal a difference in the intestinal differentiation of C. brenneri. Interestingly hybrid embryos always appear to fail in different ways in embryogenesis.