Yang, Z. et al. (2011) International Worm Meeting "Tissue specific 14-3-3 activities in Caenorhabditis elegans lifespan regulation."

Yang, Z., Wang, Y., Yu, Z., Zhao, D., Yu, S.

[International Worm Meeting, 2011]

The 14-3-3 proteins are a family of ubiquitiously expressed and highly conserved proteins. They play key roles in the regulation of central physiological processes through interactions with multiple proteins. Our previous studies demonstrated that two C. elegans 14-3-3 proteins, PAR-5 and FTT-2 can interact with DAF-16 and SIR-2.1 to regulate lifespan and overexpression of PAR-5 and FTT-2 in C. elegans extended longevity significantly. To further explore the role of 14-3-3 proteins in the regulation of DAF-16 and SIR-2.1 for the longevity, we investigated in which tissue(s) PAR-5 and FTT-2 activity may be required for the lifespan regualtion of C. elegans. By using tissue specific promoters to drive PAR-5::GFP and FTT-2::GFP expression, we could specifically express PAR-5::GFP and FTT-2::GFP in different tissues of the transgenic worms. Since par-5 is widely expressed in different tissues of C. elegans, we used unc-119 promoter, ges-1 and spl-1 promoters, lin-26 promoter and unc-54 promoter to drive PAR-5::GFP expression in the nervous system, the intestine, hypodermis and the body-wall muscle, respectively. For the tissue specific expression of FTT-2::GFP, we used unc-119, ges-1 and spl-1, lin-26, unc-54 and myo-2 promoters to specifically express FTT-2::GFP in the nervous system, the intestine, hypodermis, the body-wall and pharyngeal muscles, respectively. Then we examined the lifespan of these transgenic lines. Compared with the control, the transgenic line overexpressing PAR-5::GFP in the nervous system and hypodermis, where both DAF-16 and SIR-2.1 are expressed, extended mean lifespan significantly. The transgenic lines overexpressing PAR-5::GFP in other tissues had no obvious influence on the lifespan. The transgenic lines overexpressing FTT-2::GFP in the intestine and pharygenal muscle extended the lifespan significantly. Taken together, our results suggest that PAR-5 and FTT-2 may regulate lifespan through interaction with DAF-16 and SIR-2.1 in different tissues. Key words: C. elegans, 14-3-3 protein, tissue specificity, lifespan.

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.

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.

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.

Cockrum, Chad et al. (2017) International Worm Meeting "Transmission of an epigenetic "MESsage" for germline development in C. elegans."

Strome, Susan, Kreher, Jeremy, Cockrum, Chad

[International Worm Meeting, 2017]

During C. elegans development, two primordial germ cells (PGCs) must acquire germ fate and proliferate into hundreds of germ cells to form a complete and fertile adult germline. How PGCs acquire and maintain germ cell fate is poorly understood. Two important contributors are the chromatin regulators MES-4, a histone methyltransferase, and MRG-1, a chromodomain-containing protein. Without maternal delivery of these proteins to embryos, germ cells in early larvae fail to proliferate and die, resulting in sterile adults. We hypothesize that MES-4 and MRG-1 transmit the memory of a germline gene expression program from parent germ cells to progeny germ cells to ensure that PGCs express the appropriate genes for their survival and proliferation. Our working model is that 1) during embryogenesis, MES-4 maintains a memory of which genes were previously expressed in the parental germline by maintaining H3K36me3 on nucleosomes that package those genes, and 2) MRG-1 effects the memory by binding to H3K36me3 generated by MES-4 and activating an appropriate germline transcription program in PGCs. To test our model, I am determining whether PGCs from L1s that lack MES-4 or MRG-1 fail to launch an appropriate germline gene expression program. Due to the previous limitation in gathering enough PGCs for mRNA profiling, I first profiled mRNAs from mutant adult germlines that inherited maternally provided MES-4 and MRG-1, but cannot express one of the gene products (M+Z-). mes-4 M+Z- and mrg-1 M+Z- mutant adult germlines misexpress a similar set of genes that are normally repressed in germ cells: somatic genes and genes on the X chromosome. Thus, MES-4 and MRG-1 are required for a proper germline gene expression program and they regulate similar genes in adult germlines. We predict that PGCs in L1 progeny of these mutant adults, which completely lack MES-4 or MRG-1 (M-Z-), fail to acquire germ cell fate because they fail to launch a proper germline transcription program and more severely misexpress somatic genes and genes on the X chromosome. To test this prediction, I developed a method to isolate PGC pairs from wild-type and mutant L1 larvae to profile their mRNAs. I showed that this approach can successfully profile the mRNA population from single sister pairs of wild-type PGCs. This dissection approach offers advantages over FACS sorting large populations of PGCs: I can analyze mutant PGCs instead of RNAi PGCs, PGCs are of a uniform age, and PGCs spend minimal time in buffer before RNA preparation. I am currently analyzing the transcriptome of PGCs isolated from mes-4 and mrg-1 M-Z- mutant larvae, to learn how maternal MES-4 and MRG-1 ensure PGC survival and proliferation.

Wu, You et al. (2021) International Worm Meeting "Relatives of Ras regulate function of the C. elegans exocyst complex in development"

Wu, You, Reiner, David

[International Worm Meeting, 2021]

Among three Ras effectors of roughly equivalent oncogenicity, Raf and PI3K are well studied but the downstream mechanisms of Ras-RalGEF-Ral signaling remain poorly understood. Ral (Ras-like) is a small GTPase related to Ras. Ral uses a heterooctameric protein complex named exocyst as a signaling intermediary (as exocyst components-Exo84 and Sec5 are Ral effectors) and also performs essential activities to regulate exocytosis functions of the exocyst, which precludes conventional biochemical bootstrapping to identify signal transduction components downstream of the exocyst. Rap1 (Ras proximal) is another small GTPase that sometimes signals in parallel with Ras. Delineating the mysterious functions of Ral in signaling and Ral and Rap1 in functions of the exocyst are important for therapeutic targeting of oncogenic Ras and understanding the cell biological functions of the exocyst complex. We are using genetic, biochemical and cell biological analyses in C. elegans to identify roles of Ral and Rap1 in control of exocyst functions during development. The three aims of this project are: 1. Determine the contribution of Ral to functions of the exocyst complex. 2: Investigate whether the Ral signaling function works to regulate exocytic functions of the exocyst separately from the signaling-independent functions. 3: Delineate Rap1 contributions to exocyst functions. We found that Ral is needed for the exocyst function, as deletion of Ral aggravates developmental defects conferred by maternally rescued (M+Z-) deletion of Sec5. Yet signaling-defective Ral does not alter the phenotype of M+Z- Sec5 mutant animals. Furthermore, constitutively activated Ral reduces the severity of M+Z- Sec5 mutant defects, implicating signaling-dependent and -independent contributions of Ral to exocyst-dependent development. Rap1 similarly interacts with the M+Z- Sec5 mutant and the M+Z- Ral mutant, suggesting a model similar to that of Ral. We will continue to test the contributions of Ral and Rap1 to exocyst-dependent development using the chemogenetic tool Auxin-Inducible degron (AID). We will also use the development of elaborate arborization of the axons of PVD neurons as a more precise readout of exocyst function, and also assay the impact of Sec5, Ral and Rap1 depletion on the transport of marked exocytic cargoes. Besides, we will use biochemical tools and will do confocal imaging to measure the physical interaction and colocalization of HA and fluorescent tagged Ral and exocyst components. In conclusion, this study will further investigate the role of two Ras relatives, Ral and Rap1, in the exocyst. This will help delineate the downstream effectors of the poorly understood Ras&gt;RalGEF&gt;Ral pathway, and help better understand the relationship of Rap1 with the exocyst, as well as the crosstalk of Rap1 and Ras effectors.