Anna Deplazes et al. (2003) International Worm Meeting "In vivo studies of CYB-1 in C. elegans"

Anna Deplazes, Francesco R Wagner, E Randal Hofmann, Michael O Hengartner

[International Worm Meeting, 2003]

Loss of a cell's DNA repair system or apoptotic program can contribute to tumor development. Studies in yeast and mammalian cells have shown that distinct genes called checkpoint genes are involved in DNA damage response. Activation of sensor genes by a DNA lesion followed by signal transduction to downstream effector molecules, promotes programmed cell death or cell cycle arrest depending on the amount of damage inflicted on the genome. A major DNA damage checkpoint has been established between the G2/M phases of the cell cycle where Cyclin B1 plays an important role in the mitotic entry of the cell [1]. We are using C. elegans as a tool to study how cell cycle progression is regulated in response to DNA damage [2]. We are currently studying the role of CYB-1 in cell cycle arrest following DNA damage. To follow the expression of this cell cycle regulator in the germ line of C. elegans, transgenic lines were generated containing a promoter sequence fragment followed by the complete coding sequence of cyb-1 fused to YFP. Expression of this reporter in the germ line was observed in the nuclei of the mitotic germ cells at different intensities, suggesting that its expression levels vary depending on the stage of the cell's cycle. A point mutation (S310P) within the cycling box resulted in cytoplasmic localization in the germ line, implying that this mutation interferes with nuclear import in the germ line. We are now testing the functionality of this transgene by attempting a rescue of a cyb-1 mutant strain. This would provide us with a valuable tool to better understand the interplay between the components of the cell cycle and those of the DNA damage response pathways. [1] Jin P., Hardy S. and Morgan D.O. Nuclear localization of cyclin B1 controls mitotic entry after DNA damage. J Cell Biol. 1998 May 18;141(4):875-85; [2] Gartner A., Milstein S., Ahmed S., Hodgkin J. and Hengartner M.O. A conserved checkpoint pathway mediates DNA damage-induced apoptosis and cell cycle arrest in C. elegans. Mol Cell. 2000 Mar;5(3):435-43.

Oliver Speer et al. (2005) International Worm Meeting "Analysis of cyclin B1 cyb-1 expression during germ line development and following DNA damage"

Francesco R Wagner, E Randal Hofmann, Oliver Speer, Michael O Hengartner, Anna Deplazes

[International Worm Meeting, 2005]

Loss of a cell's DNA maintenance system can contribute to genome instability and lead to tumor development. Studies of these repair mechanisms in yeast and mammalian cells have uncovered distinct checkpoint genes involved in DNA damage responses. Activation of sensor genes by DNA lesions followed by signal transduction to effector molecules promotes cell cycle arrest and programmed cell death. We are using the C. elegans germ line to study the pathways that drive these two biological responses to DNA damage [1]. A major checkpoint in response to DNA damage has been established at the G2/M border of the cell cycle. Previous work has shown that Cyclin B1 plays an important role in promoting entry into mitosis [2]. To better understand how DNA damage induces cell cycle arrest in the C. elegans mitotic germ line, we have undertaken an analysis of the B1 type cyclin cyb-1. To follow the expression of cyb-1 in the germ line of C. elegans, we generated transgenic lines containing the promoter and coding sequence of cyb-1 fused to yfp. A construct integrated on chromosome I, named opIs76[Pcyb-1::cyb-1::yfp], partially rescued the deletion mutation cyb-1(gk35). The CYB-1::YFP expression pattern was followed by fluorescence microscopy and quantified using software analysis. Expression of opIs76 in the mitotic region of the germ line was uneven, suggesting that its expression levels might vary depending on the cell cycle stage. Three to twelve hours after ionizing radiation, mitotic germ cell nuclei uniformly acquired strong CYB-1::YFP staining, as the cells possibility underwent a transient G2/M phase cell cycle arrest. In the DNA damage checkpoint mutants hus-1, mrt-2 and rad-5/clk-2, IR had no effect on the mitotic germ cells, which showed the irregular CYB-1::YFP fluorescence pattern observed in non irradiated animals. In contrast, in the C. elegans p53 homologue (cep-1) background, both cell cycle arrest and uniform nuclear CYB-1::YFP fluorescence were observed. These results confirm previous observations that cell cycle arrest following IR requires checkpoint genes but not the p53 homolog cep-1. The opIs76 transgene might be an attractive tool for the identification of novel mutations that affect germ cell proliferation and DNA damage-induced cell cycle arrest. [1] Gartner A., Milstein S., Ahmed S., Hodgkin J. and Hengartner M.O. Mol Cell. 2000 Mar;5(3):435-43 ; [2] Jin P., Hardy S. and Morgan D.O. J Cell Biol. 1998 May 18;141(4):875-85.

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

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

[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.

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

[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.

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."

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

[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.

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

Rubin CS, Angelo RG

[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.

Shane J Woods et al. (2005) International Worm Meeting "A genome-wide approach to examine genetic robustness through knock-down of duplicate genes using RNAi."

Anthony Rogers, Julie Ahringer, David M Rivers, Shane J Woods

[International Worm Meeting, 2005]

Redundant paralogs have been partially attributed to genetic robustness against null mutations (Gu et al., 2003). In an attempt to assess the genetic robustness of the C. elegans genome we have identified ~ 2000 putative duplicate gene pairs. We have optimized a double RNAi feeding method using our RNAi feeding library (Fraser et al., 2000; Kamath et al., 2003) and an RNAi-supersensitive worm strain to test candidate gene pairs for redundant functions. We will present the data from this screen, where we score embryonic lethality, sterility, and other directly observable phenotypes. This screen presents a resource for novel gene function complementing existing functional genomic screens. In addition, it will allow us to further understand how genomic redundancy is globally organised, as well as the process driving genetic backup. Previous studies of genome redundancy in C. elegans have relied on fitness data from single gene loss-of-function screens (Kamath et al., 2003; Conant & Wagner, 2004). This will be the first genome-wide screen in any higher order organism directly examining genetic robustness on a functional level.

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

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

[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"

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

[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)"

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

[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.