Rashmi Deshpande et al. (2003) International Worm Meeting "Genetic regulation of POP-1 asymmetry."

Chris R Merritt, Russell J Hill, Rashmi Deshpande, Komudi Singh

[International Worm Meeting, 2003]

The majority of cell divisions in C. elegans produce an anterior sister and a posterior sister that have distinct developmental fates. One mechanism that distinguishes the fates of some sister cells is the regulated nuclear localization of the transcription factor POP-1. POP-1 is the C. elegans ortholog of TCF-1 which is a major transcriptional effector of the Wnt (Wingless) signal transduction pathway. During the majority of early embryonic divisions, the anterior sister has a higher nuclear level of POP-1 than its posterior sister, a phenomenon referred to as POP-1 asymmetry. While it is known that proper function of the Wnt pathway and of a MAPK-like pathway is required for sister cells to have different nuclear levels of POP-1, the mechanisms that distinguish the anterior from the posterior sisters are less well understood. To help examine these mechanisms, a reporter transgene called buEx44 that expresses a GFP::POP-1 fusion protein was produced. This reporter has a structure similar to a previously described reporter (Maduro et al., Dev. Biol. 248: 128-42 (2002)) but uses a heat shock promoter that is capable of driving expression of the fusion protein in most somatic tissues. When this promoter is induced by moderate heat shock, buEx44 animals localize GFP in a pattern that matches the localization pattern of the endogenous POP-1 protein. Time lapse (5D) videomicroscopy is being done to examine the localization of this POP-1 reporter in mutant backgrounds. Current work is examining whether the proper localization of POP-1 along the anterior/posterior axis is dependent on the function of the par genes which have important functions in polarizing the anterior/posterior axis during early embryogenesis.

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.

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.

Chris Merritt et al. (2007) International Worm Meeting "A Modular Vector Kit For Transgenesis in C. elegans."

Ekaterina Voronina, Geraldine Seydoux, Christopher Gallo, Dominique Rasoloson, Chris Merritt, Darae Ko

[International Worm Meeting, 2007]

We have constructed a new vector kit for transgenic expression in somatic and germline tissues of C. elegans. This kit was created to facilitate the cloning of promoter, ORF and 3UTR sequences in any desired combination. By allowing users to choose both promoters and 3UTR sequences, this kit can be used for genes expressed in the soma (promoter-dependent) as well as genes expressed in the germline (3 UTR-dependent, see abstract by Merritt et al.). The vector kit is based on MultiSite GATEWAY recombination cloning technology, which permits simultaneous ligation of up to three DNA fragments in one step. The kit consist of 1) a destination vector containing the unc-119 gene for selection in biolistic transformation, and 2) three sets of donor clones containing either promoter sequences (heat shock promoter for inducible expression and pie-1 promoter for germline expression), ORFs (including GFP:actin, GFP:tubulin, GFP:H2B and GFP), or 3UTR sequences (including unc-54 and several germline 3UTRs). The kit is also compatible with the Vidal/Hope collections of promoters and ORFs. The modular vector kit can be used 1) to define gene expression patterns, 2) to express ORFs ectopically and 3) to define the contributions of promoter, ORF and 3UTR sequences in specifying expression patterns. For example, we are currently using this system to systematically characterize the role of 3UTRs in specifying expression patterns in the germline. Using a generic germline promoter (pie-1) and a generic ORF (GFP:H2B), we have found that many germline expression patterns are specified by 3UTR sequences (see abstract by Merritt et al.).

S Golik et al. (1999) International C. elegans Meeting "FMRFamide-related peptides and egg-laying"

W Schafer, L Hardaker, S Golik

[International C. elegans Meeting, 1999]

The egg-laying motorneurons release multiple neurotransmitters, each of which appears to control a specific aspect of egg-laying behavior. Under favorable conditions, wild-type worms fluctuate between alternative behavioral states for egg-laying: an active state, during which eggs are laid in clusters, and an inactive state, during which eggs are retained. Switching between states, as well as egg-laying within the active state, are stochastic, Poisson-like processes. We showed previously that serotonin, released primarily from the HSNs, facilitates the switch from the inactive to the active state, while acetylcholine, released from both the HSNs and VCs, promotes egg-laying within the active state. Li and Chalfie have shown that both the HSNs and VCs contain one or more FMRFamide-related peptides, and provided pharmacological evidence that these neuropeptides may control of egg-laying behavior. To investigate the roles of these peptides in more detail, we have analyzed the egg-laying behavior of deletion mutants (kindly provided by Chris Li) that are defective in production of specific FaRP precursors. We have found that one of these mutants, defective in the gene flp-1, have dramatically lengthened inactive phases, but completely normal egg-laying within the active phase. Moreover, although these mutants are stimulated to lay eggs by serotonin with approximately normal dose responses, their rate and pattern or egg-laying on serotonin are abnormal. Thus, we hypothesize that the FLP-1 peptides and serotonin act in parallel to affect the same step in egg-laying behavior--inducing the switch between the inactive and active phases. Experiments to investigate the cellular and molecular mechanisms for the FLP-1 peptides' affects on egg-laying will be described. Thanks to Chris Li for discussions and for providing strains.

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.

Zeiser, Eva A. et al. (2009) International Worm Meeting "MosSCI and Gateway compatible toolkit for germline expression of transgenes."

Frokjaer-Jensen, Christian, Ahringer, Julie, Jorgensen, Erik, Zeiser, Eva A.

[International Worm Meeting, 2009]

Transgene silencing in the germline has hampered research in the germline and early embryo. The creation of "complex" extrachromosomal arrays using genomic DNA and then the development of technology for low copy number insertions using microparticle bombardment finally allowed germline expression of transgenes (Mello et al., 1991; Praitis et al., 2001). However, bombardment is very labor intensive and complex extrachromosomal arrays often are silenced. Recently, the Mos1 mediated Single Copy Insertion (MosSCI) method was developed to insert single copies of transgenes into defined sites in the genome of Caenorhabditis elegans (Frokjaer-Jensen et al., 2008). The technique is based on the MosTIC technique (Robert & Bessereau, 2007). Directed integration of a single copy avoids problems like overdosage, passage related loss of transgenes and locus mediated phenotypes. It also avoids extrachromosomal array related gene silencing in the germline which makes it especially attractive for studies on the gonad, and early embryos. We are using the MultiSite Gateway system to generate transgenes for germline gene expression. This method allows easy fusion of three different sequences in a defined order (5'', middle, and 3''). We have generated a set of 5'' constructs containing the germline active promoter pmex-5 (Merritt et al., 2008) on its own or fused to the sequences of GFP, EGFP, Citrine and Tag RFP-T. Similarly, we have generated a set of 3'' constructs by fusing GFP, EGFP or Citrine to the tbb-2 3''UTR (Merritt et al., 2008). Using an appropriate combination of 5'' and 3'' constructs with a middle ORF of one''s choice allows easy generation of N- or C-terminal fluorescently tagged transgenes for expression in the germline or early embryo. We also show that the hsp-16.2 and hsp-16.41 heat shock promoters can be used for inducible germline and early embryo gene expression as single copy MosSCI generated insertions.

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.