Sandhya Koushika et al. (2007) International Worm Meeting "Understanding the function of the UNC-104 PH-domain by characterizing intragenic suppressors of unc-104."

Jitendra Kumar, Sandhya Koushika, Sowdhamini R, Raghuprasad M, Michael Nonet

[International Worm Meeting, 2007]

UNC-104 is a kinesin-like motor that transports synaptic vesicle proteins to the synapse. In unc-104 mutants, presynaptic vesicles accumulate in the cell body, and the number of synaptic vesicles at the nerve ending is reduced. unc-104 mutants also have large decrease in numbers of muscle arms. UNC-104 has a PH domain that has been shown to be involved in binding PI(4,5)P₂ in vitro and is essential for function in vivio It is suggested that the PH domain is involved in cargo binding, but the molecular players that regulate cargo binding through the PH domain of UNC-104, are poorly understood. Numerous alleles of unc-104 were sequenced among which unc-104(e1265) harbours a lesion in the cargo binding PH domain. This allele has reduced protein levels of UNC-104 which may be a consequence of inability to bind cargo. We have isolated twelve behavioral suppressors of unc-104(e1265). Of these, three are intragenic and carry a second site mutation within the PH domain. Structural modeling based on the structure of the PH domain of 1FB8 using Modeler v7.0 and GRAMM supports the idea that some of the mutated residues are involved in recognizing PI(4,5)P₂. Although the intragenic suppressors move similar to wild type, they show only partial recovery of transport and muscle arms. We are trying to understand whether the increase in transport is due to increased protein stability or improved cargo binding.

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.

Kumar J et al. (2010) Neuronal Development, Synaptic Function and Behavior, Madison, WI "The C. elegans kinesin motor UNC-104 is degraded upon loss of specific binding to cargo"

Zheng Q, Metpally R, Kumar J, Klopfenstein D R, Nonet M L, Sowdhamini R, Koushika S P, Chowdhary B

[Neuronal Development, Synaptic Function and Behavior, Madison, WI, 2010]

UNC-104 is a Kinesin-3 motor that transports synaptic vesicles from the cell body towards the synapse by binding to PI(4,5)P2 through its PH-domain. The fate of the motor upon reaching the synapse is not known. We found that wild type UNC-104 is not retrogradely transported and is degraded at synaptic regions through the ubiquitin pathway. As a possible means to regulate the motor, we tested the effect of cargo binding on UNC-104 levels. The unc-104(e1265) allele carries a point mutation (D1497N) in the PI(4,5)P2 binding pocket of the PH-domain, which greatly reduces preferential binding to PI(4,5)P2 in vitro. unc-104(e1265) animals have poor locomotion irrespective of in vivo PI(4,5)P2 levels due to reduced anterograde transport of pre-synaptic vesicles. Moreover, they show highly reduced levels of UNC-104 in vivo. To confirm that loss of cargo binding specificity reduces motor levels, we isolated two intragenic suppressors, unc-104(e1265tb107) and unc-104(e1265tb120), with additional changes in R1501Q and M1540I respectively. Both suppressors map within the PH domain and show partial restoration of in vitro PI(4,5)P2 binding specificity. These animals show improved locomotion dependent on in vivo PI(4,5)P2 levels and increased anterograde vesicle transport. They also show partial restoration of UNC-104 protein levels in vivo. For further proof we mutated the conserved W1549 residue in a D1497N M1540I background. The PH domain in this triple mutant lacked in vitro PI(4,5)P2 binding specificity and the animals again showed locomotory defects and reduced motor levels. All allelic variants show increased UNC-104 levels upon blocking the ubiquitin pathway. These data show that inability to bind cargo can target motors for degradation. In view of the observed degradation of wild type UNC-104 in synaptic regions, this further suggests that wild type UNC-104 motors gets degraded at synapses upon release of cargo.

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.

Martyna W Pastok et al. (2007) International Worm Meeting "Studies on the C. elegans Protein Kinase A (PK-A) Catalytic and Regulatory Subunit Genes."

Huw H Rees, Martyna W Pastok, Michael J Fisher, Caroline Dart, Patrica A Murray

[International Worm Meeting, 2007]

The cyclic AMP-dependent protein kinase (protein kinase A; PK-A) holoenzyme consists of two catalytic (C) subunits bound to two regulatory (R) subunits, forming an inactive heterotetramer (R2C2). Binding of cyclic AMP to the R subunits results in the dissociation of catalytically active monomeric C subunits. These C subunits catalyse the protein phosphorylation events underlying the diverse range of cellular processes triggered by alterations in cyclic AMP concentration. In C. elegans, C and R subunits are encoded by the kin-1 and kin-2 genes respectively. We have undertaken both bioinformatic and molecular studies on the structure and expression of these genes, in C. elegans. We have also undertaken comparative bioinformatic studies on orthologus genes, and gene products, in C. briggsae and C. remanei. These studies have highlighted the potential significance of alternative splicing processes, as a means of generating multiple isoforms of both the C and R subunits in C. elegans. The possible significance of such multiple isoforms is discussed in the context of the subcellular distribution of PK-A and in relation to the targeting of specific phosphorylation events, in responses to changes in intracellular cyclic AMP concentration.