Oka T et al. (1998) European Worm Meeting "Multiple genes for vacuolar-type ATPase proteolipids in Caenorhabditis elegans"

Yamamoto R, Oka T, Futai M

[European Worm Meeting, 1998]

In the vacuolar type H+-ATPase (V-ATPase), highly hydrophobic subunits known as the proteolipids form the integral membrane VO sector. Previously, we described the identification of three different proteolipid genes in Caenorhabiditis elegans [Oka et. al. (1997) J. Biol. Chem. 272, 24387-24392]: vha-1 and vha-2 encoded 16 kDa subunits, and vha-4, a 23 kDa isoform. We report here that a third 16 kDa gene, vha-3, has been identified on chromosome IV. This is the first example in which four proteolipid genes have been found in a single organism. vha-2 and vha-3 exhibited 85 % nucleotide identity within the open reading frames which encoded the identical amino acid sequence. Northern blot analysis indicated that all four genes were expressed in a similar pattern during the worm life cycle; however, studies with transgenic worms indicated that the vha-3 gene was expressed differently from other proteolipid genes in a cell-specific manner. These results implied that the isoforms of the proteolipids may be related to functional differences of the V-ATPases in various cell types. Another new gene, vha-11, contained seven exons and was found to be located immediately downstream of vha-3. The two genes constitute a single transcriptional unit. The VHA-11 protein had 384 amino acids and shared strong sequence similarities with the C subunit, a component of the peripheral V1 sector of the V-ATPase, from yeast, bovine and human. Expression of the vha-11 cDNA complemented the null mutation of VMA5, the yeast C subunit gene, thus demonstrating that vha-11 was the functional C subunit of C. elegans.

Rand JB et al. (2000) East Coast Worm Meeting "UNC-13 in the C. elegans Nervous System"

Kohn RE, Rand JB, McManus JR, Moulder GL, Duke A, Barstead RJ, Duerr JS

[East Coast Worm Meeting, 2000]

C. elegans unc-13 and its homologues in vertebrates and Drosophila are involved in neurotransmitter release. UNC-13 has several regions homologous to PKC regulatory domains; these domains confer it with calcium, phorbol ester and phospholipid binding properties (Maruyama and Brenner, PNAS 88, 1991). A 5.9kb transcript coding for a 200kDa protein was initially identified (now designated L-R for left and right regions). We have identified two additional types of transcripts. One transcript includes a 1kb novel exon (L-M-R, M for middle region) and another transcript lacks the 5' region included in the other two transcripts (M-R). All three transcripts are identical at the 3' end (R). C. elegans with mutations in the 5' end of the gene (L) alter two types of transcripts (L-R and L-M-R) resulting in an uncoordinated coily phenotype and resistance to the anti-cholinesterease, aldicarb. A 2.7kb deletion near the 3' end (R) (identified by Bob Barstead using PCR analysis) affects all unc-13 transcripts and results in a lethal phenotype. Antibodies recognizing the N-terminal region of UNC-13 (L) label synapses, but not synaptic vesicles, of most or all neurons; many mutations in L and R remove staining with this antibody. Supported by grants from the NIH and OCAST.

Peng Huang et al. (2001) International C. elegans Meeting "CLR-1 and Fibroblast Growth Factor Receptor (FGFR) Signaling in C. elegans"

Christina Z Borland, Peng Huang, Michael J Stern

[International C. elegans Meeting, 2001]

Mutations in clr-1 result in a dramatic Clr ( Cl ea r ) phenotype characterized by extremely short, immobile, and sterile animals that accumulate clear fluid within their pseudocoelomic cavities. clr-1 encodes a receptor tyrosine phosphatase (RTP) that negatively regulates an EGL-15 FGFR signaling pathway in C. elegans ; the Clr phenotype of clr-1 mutants is the manifestation of hyperactivation of this pathway. Conversely, animals that completely lose egl-15 function arrest early in larval development, and less severe reduction of egl-15 function results in a Scr ( Sc rawn y ) body morphology. To understand the role of FGFR signaling in C. elegans better, we are investigating the cellular basis of these phenotypic defects. The results of several different lines of experimentation suggest that CLR-1 acts in the excretory cell to regulate EGL-15 signaling. To identify the cells that express CLR-1, we generated a GFP reporter driven by the clr-1 promoter. This clr-1::gfp construct is expressed in a wide variety of cells, including the excretory cell, ventral cord neurons, body wall muscle cells, some neurons in the head, adult vulval cells, and some rectal cells. This expression pattern is also observed using a clr-1::lacZ reporter construct and immunofluorescence studies of CLR-1 expressed from a rescuing transgene. To determine the cellular site of action of clr-1 , a mosaic analysis was performed. This analysis suggests that clr-1 activity is required in the AB.plp sublineage, which gives rise to a group of cells including the excretory cell. Laser ablation of the excretory cell has been shown to result in a similar Clr phenotype 1 . Based on these results, we hypothesize that the excretory cell is the site in which CLR-1 acts to regulate EGL-15 signaling. To support this hypothesis further, we have tested a number of promoters that are capable of driving GFP expression in the excretory cell for their ability to drive CLR-1 expression to rescue clr-1 mutants. Both the vha-1 promoter ( V acuolar-type H + A TPase) and itr-1 promoter ( I nositol 1,4,5- T riphosphate R eceptor) are able to drive GFP expression in the excretory cell 2,3 . We have shown that both promoters can rescue clr-1(e1745ts) when used to drive expression of clr-1 . Furthermore, consistent with EGL-15 also acting in the excretory cell, we have also shown that the vha-1 promoter can be used to drive egl-15 expression to rescue the larval lethal phenotype conferred by egl-15(null) . These data are consistent with EGL-15 regulating the function of the excretory system. It will be interesting to determine what this pathway responds to in order to regulate fluid flux in C. elegans . 1. Nelson, F. K., and Riddle, D. L. (1984) J. Exp. Zool. 231 , 45-56 2. Oka, T., Yamamoto, R., and Futai, M. (1997) J. Biol. Chem. 272 , 24387-24392 3. Baylis, H. A., Furuichi, T., Yoshikawa, F., Mikoshiba , K., and Sattelle, D. B. (1999) J. Mol. Biol. 294 , 467-476

Abergel, Z. et al. (2017) International Worm Meeting "Adaptation of the nematode C. elegans to hypoxia and reoxygenation stress reveals an unexpected function of the neuroglobin GLB-5 in innate immunity."

Zuckerman, B., Zelmanovich, V., Abergel, Z., Abergel, R., Gross, E., Smith, Y., Romero, L., Livshits, L.

[International Worm Meeting, 2017]

Deprivation of oxygen (hypoxia) followed by reoxygenation (H/R stress) is a major component in several pathological conditions such as vascular inflammation, myocardial ischemia, and stroke. However how animals adapt and recover from H/R stress remains an open question. Previous studies showed that the neuroglobin GLB-5(Haw) is essential for the fast recovery of the nematode Caenorhabditis elegans (C. elegans) from H/R stress. Here, we characterize the changes in neuronal gene expression during the adaptation of worms to hypoxia and recovery from H/R stress. Our analysis shows that innate immunity genes are differentially expressed during both adaptation to hypoxia and recovery from reoxygenation stress. Moreover, we reveal that the prolyl hydroxylase EGL-9, a known regulator of both adaptation to hypoxia and the innate immune response, inhibits the fast recovery from H/R stress through its activity in the O2-sensing neurons AQR, PQR, and URX. Finally, we show that GLB-5(Haw) acts in AQR, PQR, and URX to increase the tolerance of worms to bacterial pathogenesis. Together, our studies suggest that innate immunity and recovery from H/R stress are regulated by overlapping signaling pathways.

A V Bicknell et al. (2002) European Worm Meeting "The C. elegans F47F2.1 gene encodes a cyclic AMP-dependent protein kinase (PK-A) catalytic subunit"

A V Bicknell, M J Fisher, M Tabish, R A Clegg, H H Rees

[European Worm Meeting, 2002]

PK-A mediates all known effects of cyclic AMP on cellular activity in eukaryotes. The holoenzyme is an inactive tetramer of two regulatory (R) subunits and two catalytic (C) subunits. Following binding of cyclic AMP to the R subunits, dissociation of active C-subunits occurs. In mammals, , and isoforms of C-subunit, encoded by different genes, have been identified.

Angelo RG et al. (1997) International C. elegans Meeting "DISCOVERY OF A POTENTIAL ANCHOR/SCAFFOLD PROTEIN FOR C. ELEGANS PROTEIN KINASE A (PKA)"

Angelo RG, Rubin CS

[International C. elegans Meeting, 1997]

C. elegans PKA is composed exclusively of catalytic and regulatory (R) subunits encoded by the kin-1 and kin-2 genes. Since C. elegans lacks other PKA isoforms, this enzyme must disseminate signals carried by cAMP to all cell compartments. Approximately 60% of total R (PKA) is in the particulate fraction of disrupted C. elegans, indicating that protein/protein interactions may diversify PKA signaling by anchoring the kinase at specific intracellular locations. Co-localization of PKA with upstream activators and/or downstream effectors can create target sites for cAMP action. To understand the mechanism of PKA localization in C. elegans, a cDNA expression library was screened with recombinant radiolabeled-R (0.5 nM) to obtain high-affinity binding proteins. A cDNA encoding a novel, 143 kDa A kinase anchor protein (AKAP1) was retrieved and sequenced. The corresponding gene (rap-1) is located in LGII and contains 17 exons. AKAP1 is an acidic protein (pI=4.4) that is unrelated to previously characterized proteins. C. elegans R is avidly bound by both soluble and immobilized fragments of AKAP1. Competition binding studies indicate that C. elegans R is a preferred ligand, whereas mammalian RII and RI isoforms are only weakly sequestered. Scatchard analysis yielded a Kd value of ~10 nM for the R/AKAP1 complex. Deletion mutagenesis, coupled with protein expression in E. coli and in vitro assays, demonstrated that residues 235 to 255 govern high-affinity binding of R by AKAP1. A hydrophobic surface generated by amino acids with branched aliphatic side chains may be a key determinant of R binding activity. Site directed mutagenesis will pinpoint essential residues involved in PKA binding. Studies aimed at identifying the AKAP1 binding site on R are also in progress. High-affinity anti-AKAP1 IgGs are being used to determine (a) whether AKAP1 expression is developmentally-regulated and cell- specific and (b) the relationship between R (PKA) and AKAP1 in vivo.

Hicks, Tara et al. (2021) International Worm Meeting "R-loop-induced irreparable DNA damage in C. elegans meiosis"

Hicks, Tara, Koury, Emily, Williams, Cameron, Smolikove, Sarit

[International Worm Meeting, 2021]

Most DNA-RNA hybrids are formed naturally during transcription and are composed of a nascent RNA strand hybridized to DNA as part of R-loops. The accumulation of these structures in S-phase can result in replication-transcription conflict, an outcome which can lead to the formation of double strand breaks (DSBs). While R-loops' role in mitotically dividing cells has been characterized, there are only a handful of studies describing the effect of R-loops in meiosis and these studies present a complex picture of the outcome of R-loop formation on germ cells. Here we show that DSBs formed by R-loops trigger an altered cellular response to DNA damage. RNase H is an enzyme responsible for degradation of the RNA strand in DNA-RNA hybrids and plays an essential role in preventing this outcome and its deleterious consequences. Using null mutants for the two Caenorhabditis elegans genes encoding for RNase H1 and RNase H2 (hereby rnh mutants), our studies explore the effects of replication stress-induced DNA-RNA hybrid accumulation on meiosis. As expected, rnh mutants exhibit an increase in R-loop formation. Consequently, an elevation of DSBs in germline nuclei is evidenced by the accumulation of RAD-51 foci. Despite no repair mechanism abrogation, rnh mutants fail to repair all DSBs generated, leading to a fragmentation of chromosomes in diakinesis oocytes. By combining our double mutant with a spo-11 null mutation, we show that although replicative defects are the main contributor to the phenotype, R-loops formed in meiosis are likely contributors as well. We present evidence that while rnh mutants accumulate DNA-RNA hybrids and subsequent DSBs may signal a degree of checkpoint activation in mitosis, some damaged nuclei prevail past the checkpoint, enter into meiosis, and remain unrepaired throughout. Moreover, we find no evidence of an increase in apoptosis, which indicates that DNA damage generated by R-loops remain undetected by an apoptotic checkpoint. This data altogether points to DSBs initiated by R-loops representing an irreparable type of DNA damage that evades cellular machineries designed for damage recognition.

Birsoy B et al. (2010) Biology of the C. elegans Male, Madison, WI "Novel Left-Right Asymmetries of Male C.elegans"

Rothman J H, Choi S, Birsoy B, Downes J C, Bloss T A

[Biology of the C. elegans Male, Madison, WI, 2010]

While the mechanism that breaks left-right (L-R) anatomical asymmetry has been described in C. elegans, we have found that at least one additional mechanism must exist to generate L-R differences in the deployment of alternative cell death pathways in the male tail, male mating behavior and patterning of the male gonad. Upon recognizing a hermaphrodite, males initiate backward locomotion and continuously scan along the hermaphrodite's body. When their tails reach either the head or tail of the hermaphrodite, males turn to maintain contact and then continue backward locomotion on the opposite side of the hermaphrodite. We found that this turning behavior shows a distinct right-handed bias: when individual males were allowed to mate with lin-2(e1309) vulvaless hermaphrodites, >70% of the turns when made over the hermaphrodites' body (away from the agar surface) and >55% of turns when made under the hermaphrodites' body (toward the agar surface) are right-handed. To determine whether this bias in turning direction correlated with L-R asymmetry in the male mating structure, which results from stochastic EGL-1-dependent loss of sensory rays, we examined the turning bias in egl-1(n1084n3082) mutants. Wild-type males lack rays more frequently on the right and egl-1 mutant males have no ray loss but still display the right-hand turning bias. To assess whether this L-R behavioral bias is dependent on anatomical asymmetry, we analyzed gpa-16(it143) mutants in which the L-R asymmetry of the internal organs is reversed as a result of the reversal in the early embryonic symmetry break. Males with reversed anatomical asymmetry showed a virtually identical right hand bias in turning, demonstrating that an asymmetry-determining system that is independent of the previously described L-R symmetry breaking event must control this behavior. During the characterization of the gonad reversals of males, we also observed a previously unreported temperature sensitive reversal of L-R asymmetry of the male gonads in N2 (Bristol) and a Hawaiian wild isolate. We will describe our recent findings on these novel L-R asymmetries of the C.elegans male anatomy and behavior.

L C Bowen et al. (2002) European Worm Meeting "The C. elegans cyclic AMP-dependent protein kinase (PK-A) catalytic subunit gene (kin-1)"

L C Bowen, M Tabish, M J Fisher, H H Rees, R A Clegg

[European Worm Meeting, 2002]

PK-A is one of the major multifunctional ser/thr protein kinases found in eukaryotic organisms. The holoenzyme is comprised of two catalytic (C) and two regulatory (R) subunits. Both R and C subunits occur in multiple isoforms encoded by distinct genes. In many organisms, the genes that encode the C subunits also show alternative splicing behaviour. This generates an even broader array of C subunit heterogeneity. The diversity of C subunit polypeptides may enable this protein kinase to selectively target substrate polypeptides for phosphorylation.

Stefan Taubert et al. (2004) West Coast Worm Meeting "Biochemical Characterization of the C. elegans Nuclear Hormone Receptor NHR-49"

Keith R Yamamoto, Stefan Taubert, Marc Van Gilst

[West Coast Worm Meeting, 2004]

Nuclear Hormone receptors (NHRs) are a family of ligand-regulated transcription factors involved in diverse metazoan functions, such as sexual differentiation, organ development, and regulation of metabolism. The C. elegans genome contains over 270 NHR genes, approximately five times as many as the human genome. For most of these no function has been described yet. We focus on characterizing NHR-49, which regulates fat metabolism, and lifespan (Marc van Gilst and Keith R. Yamamoto, unpublished). Interestingly, this mirrors the functions of human Peroxisome-proliferator-activated-receptor a (PPARalpha), while the sequence of NHR-49 suggests similarity to another human receptor, Hepatocye Nuclear Receptor 4alpha (HNF4alpha). Since HNF4alpha dimerizes via its ligand-binding-domain (LBD), we utilized yeast two-hybrid based technology to assess whether the LBDs of NHR-49 and 12 related HNF4alpha-like NHRs, can also homodimerize. While several of these LBDs readily dimerized (including NHR-49), some others did not (such as DAF-12). Since the homodimerization of NHR-49's LBD is predicted to occur via two conserved salt-bridges (E5/K55 and E50/R93), we mutated the E50 and K55 residues to alanines. We will present the effect of these mutations on LBD homodimerization and activation of a transcriptional reporter in yeast. In an attempt to identify ligands of NHR-49 we treated worms with clofibrate, a synthetic PPARa ligand used to treat hyperlipidemia. This treatment caused a strong reduction of worm body fat levels, and was accompanied by lethality at higher concentrations. However, similar effects were observed in a strain harboring an NHR-49 deletion, suggesting that the effects of clofibrate are independent of NHR-49. We are currently characterizing changes in the transcriptional program in response to clofibrate treatment.