S Eimer et al. (2002) European Worm Meeting "Loss of spr-5 bypasses sel-12 defects by stage-specific derepression of hop-1."

S Eimer, Ralf Baumeister, Bernard Lakowski, R Donhauser

[European Worm Meeting, 2002]

sel-12 and hop-1 are two C. elegans genes which are structurally and functionally homologous to the human presenilins, PS1 and PS2. Mutations in the human presenilins contribute to the majority of familial Alzheimer's disease cases. Work in C. elegans revealed that presenilins are also involved in Notch signaling, as sel-12 was initially isolated as a suppressor of a lin-12 gain- of-function allele (Levitan and Greenwald, 1995). Several lines of evidence suggest that presenilins mediate the transmembrane cleavage event that releases the LIN-12/Notch intracellular domain which is crucial for nuclear Notch signaling. As a consequence, a sel-12; hop-1 double mutant causes phenotypes that resemble that of Notch (lin-12 and glp-1) null mutants (Westlund et al., 1999).

S Roehrig et al. (1999) International C. elegans Meeting "UNC-86: The POUer of Interaction"

R Donhauser, R Baumeister, I Roeckelein, S Roehrig

[International C. elegans Meeting, 1999]

The POU transcription factor UNC-86 is necessary for the determination and differentiation of 57 neurons. These neurons differ in function, position, lineage and their time of expression of unc-86 , suggesting that unc-86 regulates different downstream genes in these distinct neural cell types. In order to understand how unc-86 determines cell fates we pursued two strategies. In the first approach, mutations in partial loss of function ( u5 , u168 ) and null alleles ( n412 ) of unc-86 were identified. These mutations were assayed for their effect on the DNA and protein interaction of the UNC-86 protein in vivo using a modified one-hybrid yeast system. In the second approach this system was also used to identify novel proteins that interact with UNC-86. By the first approach we found that single amino acid substitutions in the first helix ( u168 ) and in the linker region ( u5 ) between the third and fourth helix of the POU specific domain eliminate the interaction between UNC-86 and the LIM homeodomain protein MEC-3 in the mechanosensory neurons. This and other data show that specific residues in the POU specific and homeodomain mediate protein interactions of UNC-86. A single amino acid substitution in the recognition helix of the POU homeodomain ( n412 ) leads to a failure in DNA-binding. However, it was previously shown that unc-86 regulates its own expression and that the UNC-86 protein levels in n412 are similar to wild-type. This suggests that the autoregulation of unc-86 is independent of the ability of UNC-86 to bind DNA. Using the modified one-hybrid system six candidate interactors were identified. One candidate gene, which encodes a cAMP dependent protein kinase, is especially promising, since we were able to show that UNC-86 is phosphorylated by PKA in vitro . Furthermore, UNC-86, which is mutated in a serine residue known to be phosphorylated in other POU proteins, shows altered DNA-binding and a dominant phenotype when expressed in wild-type animals. Other candidates include nuclear proteins, whose mammalian homologues have been shown to interact with POU proteins. In addition at least two of the candidates are expressed in some of the unc-86 expressing neurons. We propose that UNC-86 is regulated by phosphorylation and is able to determine cell fates by interacting with different proteins in different cells.

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.

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.

Kagoshima H et al. (1997) International C. elegans Meeting "THE HOMEOBOX GENES CEH-14 AND CEH-7"

Cassata G, Kagoshima H, Burglin TR

[International C. elegans Meeting, 1997]

ceh-14 is an orthologue of the vertebrate Lim3 homeobox genes. From the lacZ and GFP transgenic lines we observe the following expression patterns: Expression in blast cells in the anterior and posterior during embryogenesis. The expression in the anterior, i.e., the head, disappears or is strongly reduced in larval stages. Additional neurons, which are generated during postembryonic cell divisions are expressing ceh-14 in the tail. The following neurons in the body and tail were identified: BDUL/R, PVT, DVC, PHAL/R, PHBL/R, PHCL/R, PVNL/R, and tentatively PVWL/R, PVR. Apart from the neuronal expression, nuclear staining is seen in the spermatheca and in the central hypodermis of young hermaphrodites. The hypodermal expression has the appearance of a gradient, being stronger in the center and getting weaker towards the anterior and posterior. To test this hypothesis, we examined the expression in male animals. Indeed, the hypodermal gradient was now localized in the male tail, rather than in the central body, suggesting that signaling systems responsible for the sexual dimorphism also control the expression of ceh-14. We tested whether the mutations of known signaling systems would change the pattern of ceh-14 expression. However, expression in the background of a lin-3 mutation did not show any difference. Neither did we observe any differences in a lin-12 mutation. Experiments using laser ablation of the gonad precursor cells are currently underway in collaboration with Ralf Sommer. We isolated cDNAs for ceh-14, which confirmed the predicted gene structure of our genomic sequence. ceh-7 is a tiny orphan homeobox gene. A GFP construct shows specific restricted expression in only a few male tail cells, probably B decendents. This is the first case of a sex-specific homeobox gene.