David Weinkove et al. (2005) International Worm Meeting "Activation of AGE-1 PI 3-kinase after prolonged starvation may increase larval responsiveness to nutrition at the expense of longevity in adults"

David Weinkove, Nullin Divecha, David Gems, Jonathan R Halstead

[International Worm Meeting, 2005]

To survive long enough to find new food, starved C. elegans enter one of two diapause stages: the dauer larva, a morphologically distinct alternative to the L3 or the starved L1, in which cell division is arrested until food is encountered. The FOXO transcription factor DAF-16 is required to form dauers but not to enter L1 diapause. However, we have shown that daf-16 is needed to protect starved L1s from oxidative stress in the form of H2O2. In L1s starved for one day, DAF-16 is localised to the cell nuclei where it presumably activates expression of stress-response genes. However, after two days of starvation, DAF-16 translocates to the cytoplasm and remains there. Despite this translocation, starved L1s retain their daf-16-dependent resistance to H2O2, suggesting that transcription on the first day is sufficient to provide long-lasting stress resistance.A candidate regulator of the translocation of DAF-16 after prolonged starvation is the AGE-1 PI 3-kinase, which activates a signalling cascade resulting in the phosphorylation of DAF-16 and its translocation to the cytoplasm. We found that DAF-16 remained in cell nuclei of age-1(hx546) L1s after prolonged starvation, implicating AGE-1 in mediating the DAF-16 translocation to the cytoplasm. DAF-16 translocation could be induced in L1s starved for only one day by H2O2 treatment, which also increased levels of the AGE-1 product, PIP3. This effect was also absent in age-1(hx546) mutants. hx546 is not a null mutation and while it increases lifespan in a daf-16-dependent manner, it has little effect on DAF-16 localisation under normal culture conditions (1). Our results suggest that hx546 disrupts a starvation-specific activation of AGE-1.We hypothesised that because pharyngeal pumping diminishes greatly with age, old worms also undergo prolonged starvation. Consistent with this, we found that DAF-16 was chiefly in the cytoplasm in the cells of old worms while DAF-16 was localised to both the nuclei and cytoplasm in young adults. However, in old age-1(hx546) adults, DAF-16 appeared mainly in the cell nuclei and did not translocate to the cytoplasm with age. These results might explain why the stress resistance of age-1(hx546) adults increases with age, and why age-1(hx546) mutants can compete with N2 in the presence of food but not during cycles of starvation. We are now investigating whether we can inhibit DAF-16 translocation in old wild type adults with PI 3-kinase inhibitors and whether this intervention increases lifespan.

David Weinkove et al. (2003) International Worm Meeting "The effects of H2O2 on starved L1s provide clues to the normal functions of AGE-1 and DAF-16"

Francesca Milano, Nullin Divecha, Jonathan R Halstead, Ronald H A Plasterk, David Weinkove

[International Worm Meeting, 2003]

The age-1(hx546) mutant is long-lived and stress resistant in a daf-16 dependent manner. However, the molecular nature of the hx546 mutation is unknown and loss of daf-16 function has only a small effect of adult lifespan and stress resistance. We have found that acute H2O2 treatment of L1s causes a subsequent developmental arrest and reduction of movement. Resistance to H2O2 is completely dependent on both starvation and daf-16. We have also characterised the effects of paraquat, juglone and UV on starved L1s. The developmental arrest caused by these agents is qualitatively different to that caused by H2O2 and daf-16 mutants are not particularly sensitive to these stresses. AGE-1 is thought activate AKT-1 and AKT-2 by its product PIP3. AKT-1 and AKT-2 inhibit DAF-16 by phosphorylation that causes translocation from the nucleus. As reported by others, we find that starvation enhances the nuclear localisation of DAF-16:GFP, but we also find that, in contrast, H2O2 causes DAF-16:GFP to exit the nucleus. In mammalian cells, H2O2 is known to cause the production of PIP3, thus providing a mechanism for DAF-16 translocation to the cytoplasm. We have labelled starved L1s with 32P orthophosphate and shown that H2O2 does indeed induce PIP3 production, and in an age-1 dependent manner. We propose that AGE-1 and DAF-16 play specific roles in the starved L1 diapause state, and that these functions may influence adult lifespan.

David Weinkove et al. (2002) European Worm Meeting "The roles of developmental arrest, phosphoinositides, Type II PIP kinase and DAF-16 in the larval response to oxidative stress."

David R Jones, David Weinkove, Nullin Divecha, Jonathan R Halstead, Ronald H A Plasterk

[European Worm Meeting, 2002]

Since the appearance of terrestrial oxygen, it has been essential to deal with oxidative stress. For a nematode, the important issue is to lay eggs without germline damage. Rather than study the response in adults, for which it is probably too late to do anything useful, we have investigated how L1 larvae respond (1) developmentally, (2) genetically and (3) biochemically to a burst of hydrogen peroxide.

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.

Soltesz, Z. et al. (2011) International Worm Meeting "Carbon dioxide avoidance is mediated by a diverse set of sensory neurons in C. elegans."

Soltesz, Z., de Bono, M., Bretscher, A. J.

[International Worm Meeting, 2011]

Monitoring carbon dioxide levels has a twofold importance for many living organisms: CO2 can act as a sensory cue for food or other animals, while regulating internal CO2 levels is an important part of homeostasis. C. elegans relies on diffusion for gas exchange, and avoids CO2 levels as low as 1%. We are interested in the neural and molecular mechanisms underlying the C. elegans CO2 avoidance behaviour. Mutants defective in the tax-4 or tax-2 genes, which encode the a and b subunits, respectively, of a cGMP-gated ion channel, showed reduced CO2 avoidance in behavioural assays1,2. By expressing tax-2 cDNA from neuron-specific promoters in tax-2 mutants to rescue the avoidance behaviour, and by imaging neurons using the genetically encoded calcium indicator YC3.60, we have shown that sensory neurons previously implicated in oxygen, temperature, and salt-sensing, including BAG, AFD and ASE, are CO2 sensors as well3. We have observed both persistent and transient cell-intrinsic calcium-responses in several sensory neurons, suggesting that CO2 stimuli could modulate neural activity in C. elegans in a complex manner. We are therefore investigating how CO2 stimuli can affect neural processing in downstream neurons. 1 Andrew Jonathan Bretscher, Karl Emanuel Busch, and Mario de Bono, A carbon dioxide avoidance behavior is integrated with responses to ambient oxygen and food in Caenorhabditis elegans. PNAS 105(23):8044-8049 2 Elissa A. Hallem and Paul W. Sternberg, Acute carbon dioxide avoidance in Caenorhabditis elegans. PNAS 105(23):8038-8043 3 Andrew Jonathan Bretscher, Eiji Kodama-Namba, Karl Emanuel Busch, Robin Joseph Murphy, Zoltan Soltesz, Patrick Laurent and Mario de Bono, Temperature, Oxygen, and Salt-Sensing Neurons in C. elegans Are Carbon Dioxide Sensors that Control Avoidance Behavior. Neuron 69(6):1099-1113.

Edgley ML et al. (1995) International C. elegans Meeting "CYBERWORMS"

Collins D, Bryer J, Baillie DL, Edgley ML, Rose AM

[International C. elegans Meeting, 1995]

We are engaged in two projects to improve the alignment of the genetic and physical maps for C. elegans. The Genetic Toolkit project uses PCR to tie the endpoints of balanced deficiencies to the physical map; and the CGAT project uses microinjection of cloned cosmids from the worm sequencing project to generate transgenic arrays that are used to rescue mapped, balanced lethal mutations. Both projects are generating large amounts of genetic map information, which is being integrated into the CGC map (compiled by Jonathan Hodgkin) using the ACEDB program (Richard Durbin and Jean Thierry-Mieg). We also have established a World-Wide Web hypertext server to provide access to information generated by these projects. This Genetic Toolkit web site (http: genetic balancers and transgenic strains available from our laboratories. For stable transgenic strains, listed cosmids are linked to modified GenBank entries describing the sequence. This work is funded by the NIH National Center for Research Resources (NCRR) and the Canadian Genome Analysis and Technology Program (CGAT).

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.