Dutta A et al. (2014) Front Biosci (Landmark Ed) "Identification of C. elegans & C. briggsae miRNAs by modified miRsearch."

Chatterjee R, Chaudhuri K, Dutta A

[Front Biosci (Landmark Ed), 2014]

In this study, a modified miRsearch program was developed in C++ for the detection of miRNAs. All the mature miRNA sequences of Caenorhabditis elegans, Caenorhabditis briggsae, Caenorhabditis remanei, Drosophila melanogaster, Homo sapiens and Rattus norvegicus available in miRbase was searched by this program for homologous sequences with a maximum of 3-mismatches in the chromosomes of C. elegans excluding the miRNAs of C. elegans. The same strategy was repeated for C. briggsae excluding C. briggsae miRNAs. The probable pre-miRNA sequences with stem loop secondary structures were assessed by implementation of longest-common-subsequence (LCS) algorithm with appropriate scoring system. As miRNA genes could be on either strand, each sequence was searched in both forward and reverse strands. The putative miRNAs were viewed through Mapviewer to identify their intronic or intergenic location in C. elegans and C. briggsae genomes. Further, the quality of stem-loop formation of the remaining pre-miRNA sequences was assessed through RNAFOLD. This algorithm will be helpful in detection of potential miRNAs in future sequencing data, making this an invaluable tool for miRNA prediction.

Hyojin Lee et al. (2008) East Asia Worm Meeting "A C. elegans AMP-activated Protein Kinase Is Phosphorylated by an LKB1 Homologue and Influences the Sensitivity to Oxidative Stress and Motility of Worms."

Jeong Soo Cho, Hyeon-Sook Koo, Anton Gartner, Nils Lambacher, Hyojin Lee, Jieun Lee

[East Asia Worm Meeting, 2008]

AAK-2 is one of two alpha isoforms of the AMP-activated protein kinase in C. elegans, and is involved in life span maintenance and stress responses. In this study, we found that AAK-2 was phosphorylated in response to paraquat treatment and the phosphorylation was attenuated by mutants of PAR-4, an LKB1 homologue. Both aak-2 and par-4 mutations increased the sensitivity of C. elegans worms to paraquat, and the double deficiency did not further increase sensitivity, indicating that they affect the same pathway. Both mutations also slowed body bending during locomotion and resulted in abnormalities of head oscillation. Consistent with this abnormal motility, expression of AAK-2::GFP fusion protein was observed in the ventral cord and body wall muscle, as well as in other neurons, pharynx, vulva, and excretory cells. Our study suggests that AMPK can influence the behavior of C. elegans worms in addition to its well-known function in metabolic control.

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.

Golden JW (1980) Worm Breeder's Gazette "C elegans dauer larvae, and cyclic Amp."

Golden JW

[Worm Breeder's Gazette, 1980]

Cyclic AMP levels have been assayed in N2 synchronous cultures and starvation-induced dauer larvae as a first step in the analysis of hormonal involvement in dauer larva formation, maintenance, and recovery. N2 worms are synchronized by egg purification (after the method of Ralph Hecht), hatching into M9 buffer, and resuspension in liquid worm medium containing an adenyl cylase deletion mutant of E. coli. The worms are harvested at various time intervals by immediately microwaving to boiling and washing in glass distilled water. This is followed by lyophillization after which the dry weight is determined. The assay uses a cAMP binding protein (Calbiochem) in a competitive binding assay with [3H]cAMP. After equilibrium is reached, the protein is filtered through a nitrocellulose filter and counted in a liquid scintillation counter. Initial results indicate that changes in cAMP concentration are correlated with the life cycle. Assays performed on well fed worms at various times in larval development show a linear decrease in cAMP levels from 25 pmol/mg dry weight in L2's to 15 pmol/mg dry weight in L3's, down to 1 pmol/mg dry weight in L4's. Starvation-induced dauer larvae have a cAMP level of 5 pmol/mg dry weight, or about one-third the level present at the L2 molt. Recovering dauers isolated 6 hours after the addition of food to starvation-induced dauers, show a dramatic drop in cAMP levels to less than 0.1 pmol/mg dry weight. The data collected to this point indicate that dauer larvae have a reduced level of cAMP compared to the L2 an' L3 larval stages and that recovery from the dauer stage results in cAMP levels below that found in L4's. These preliminary studies are to be followed by a more detailed study of cAMP and cGMP levels in N2 worms at all stages of the life cycle and in selected dauer-constitutive mutants. Cyclic GMP assays and pharmacological studies using theophylline as a phosphodiesterase inhibitor are currently being developed.

Dhondt I et al. (2017) Mol Cell Proteomics "Changes of protein turnover in aging Caenorhabditis elegans."

Dhondt I, Petyuk VA, Smith RD, Bauer S, Brewer HM, Depuydt G, Braeckman BP

[Mol Cell Proteomics, 2017]

Protein turnover rates severely decline in aging organisms, including C. elegans However, limited information is available on turnover dynamics at the individual protein level during aging. We followed changes in protein turnover at one-day resolution using a multiple-pulse (15)N-labeling and accurate mass spectrometry approach. Forty percent of the proteome shows gradual slowdown in turnover with age, while only few proteins show increased turnover. Decrease in protein turnover was consistent for only a minority of functionally related protein subsets, including tubulins and vitellogenins, while randomly diverging turnover patterns with age were the norm. Our data suggests increased heterogeneity of protein turnover of the translation machinery, whereas protein turnover of ubiquitin-proteasome and antioxidant systems are well-preserved over time. Hence, we presume that maintenance of quality control mechanisms is a protective strategy in aging worms, although the ultimate proteome collapse is inescapable.

D Polet et al. (2004) European Worm Meeting "THREE PROFILINS THAT BIND ACTIN, PI-4,5-P2, AND PROLINE-RICH SEQUENCES ARE EXPRESSED IN A TISSUE SPECIFIC MANNER IN CAENORHABDITIS ELEGANS."

K Ono, A Lambrechts, C Ampe, S Ono, J Vandekerckhove, D Polet

[European Worm Meeting, 2004]

Profilins are actin binding proteins that also interact with polyphosphoinositides and proline-rich ligands. Mining the C. elegans genome using profilin sequences yields three hypothetical profilin (PFN) isoforms, which show intermediate to low similarity to each other. Potentially, C. elegans would be the first multicellular invertebrate that expresses three highly diverse profilins. These homologues are functionally expressed. Biochemical characterization showed that PFN-1, PFN-2 and PFN-3 behave as classical profilins with respect to actin sequestering, influence on actin dynamics, poly-L-proline binding and PI-4,5-P2 binding. For PFN-2 and PFN-3 however, the latter interaction does not result in competition with actin as for other profilins [1] and for PFN-1. This suggests that for PFN-2 and PFN-3 binding of actin and PI-4,5-P2 is not mutually exclusive. PFN-1 is essential, RNAi indicated a strong cytokinesis defect [2]. By contrast, PFN-2 is not essential since knock-out and RNAi of PFN-2 did not result in visible phenotypes. Expression of PFN-3 could not be reduced by RNAi and its proof of importance awaits the availability of the PFN-3 knock-out. Immunostainings revealed different expression patterns for the isoforms suggesting different biological functions. In embryos, PFN-1 localizes in the cytoplasm and to the cell-cell contacts at the early stages, and in the nerve ring during later stages. During late embryogenesis, expression of PFN-3 was specifically detected in body wall muscle cells. In adult worms, PFN-1 is expressed in most of the non-muscle tissues, PFN-2 in the intestinal wall, the spermatheca and the pharyngeal region and PFN-3 stains in a striking dot-like fashion in body wall muscle. Searches for C. elegans proline-rich partners are ongoing, characterization of their interaction and colocalization with the profilin isoforms will shed light on their cellular functions . References [1]Lambrechts et al., BMC Biochem.(2002);3(1):12. [2]Severson et al., CurrBiol.(2002);12(24).

K Ferguson et al. (2001) International C. elegans Meeting "IDENTIFICATION OF AMP KINASE AS A MODIFIER OF INSULIN RECEPTOR SIGNALING IN VIVO IN C. elegans and Drosophila"

C Seidel-Dugan, K Shaw, L L'Archeveque, K Ferguson, T Kidd, R Chatterjee

[International C. elegans Meeting, 2001]

We are using model organism genetics to identify new genes in the Insulin Receptor (InR) signaling pathway. Such genes might encode targets for the development of novel therapeutics for the treatment of Type 2 Diabetes. Both C. elegans and Drosophila have homologs of the human insulin receptor, and genetic analyses in these organisms have demonstrated that the regulation of downstream signaling components also is conserved. We have taken several genetic approaches to identify new components of the InR signaling pathway, using InR mutants in C. elegans and Drosophila as entry points. In C. elegans , we performed large-scale RNAi of selected gene classes, including kinases, phosphatases, and proteases, and screened for suppressors of the dauer constitutive phenotype of daf-2 (InR) mutants. To facilitate detection of weak modifiers of daf-2 signaling, we screened for suppression in daf-2 backgrounds that display more moderate mutant phenotypes than the canonical e1370 allele used in the majority of previous screens. In parallel, a reverse genetics screen was done in Drosophila , using transposon insertion lines to look for modifiers of InR mutant phenotypes. Strikingly, subunits of the metabolic sensor AMP kinase were identified as one of the modifiers of InR signaling in both screens. AMP kinase is a key modulator of metabolic flux in mammalian cells, regulating pathways involved in fatty acid and carbohydrate metabolism. Furthermore, AMP kinase activity stimulates glucose transport in an insulin-independent manner. The identification of AMP kinase as a modifier of insulin signaling in C. elegans and Drosophila indicates that this interaction is conserved, and will allow genetic dissection of the mechanism of this interaction.

Anindya Ghosh Roy et al. (2008) C.elegans Neuronal Development Meeting "Cyclic AMP Promotes Axonal Regeneration in C. elegans"

Anindya Ghosh Roy, Alexandr Goncharov, Zilu Wu, Andrew CHISHOLM, Yishi Jin

[C.elegans Neuronal Development Meeting, 2008]

Most axonal processes can regenerate following injury. Adult mammalian CNS axons are unable to regrow efficiently, in part due to the inhibitory microenvironment produced by CNS myelin. Studies in several organisms show that elevation of neuronal cyclic AMP promotes axon growth and can overcome the effects of CNS myelin. We have previously shown that C. elegans axons can regenerate after laser axotomy (Yanik et al., 2004). Here we report that the cAMP pathway promotes several aspects of axonal regrowth after laser injury, suggesting regenerating C. elegans neurons use mechanisms similar to regenerating neurons in other organisms. When the PLM process is severed between the cell body and the synaptic branch, the proximal stump reforms a growth cone within 2-6 h post axotomy and extends over the next 24-48 h (Wu et al., 2007). Under our axotomy conditions most regrowing axons do not make contacts with their distal fragments, and do not regrow branches to their targets in the ventral nerve cord. To elevate cAMP we inhibited the neuronal phosphodiesterase PDE-4, which degrades cAMP to 5''AMP (Charlie et al., 2006). When PDE-4 function is reduced we find that severed PLM proximal axons regrow more rapidly and efficiently than those in the wild type, suggesting that chronically elevating cAMP enhances regrowth. Injured neurons more frequently reform a growth cone in pde-4 compared to wild type, and the growth cone begins to extend more rapidly than in WT. These regrowing processes also more frequently fuse with the severed distal fragment, and are more likely to extend a branch to the ventral cord. Ultrastructural analysis shows that these ventral branches form presynaptic structures. Current models suggest that axonal injury leads to calcium influx, which then triggers cAMP synthesis, leading to cAMP-dependent effects on the cytoskeleton and on transcription. We are using genetically encoded FRET-based sensors to visualize cAMP and PKA activity. Our preliminary observations show that cAMP levels are elevated for several hours following axotomy in the wild type. We are currently examining candidate effectors and transcriptional targets of cAMP signaling in neurons. In summary, our studies demonstrate a conserved role for cAMP in adult axon regeneration.

Simske J (2010) Aging, Metabolism, Stress, Pathogenesis, and Small RNAs, Madison, WI "Live and Let Die: The role of Glycogen Debranching Enzyme AGL-1 and AMP Kinase During C. elegans Embryogenesis and Aging"

Simske J

[Aging, Metabolism, Stress, Pathogenesis, and Small RNAs, Madison, WI, 2010]

jc11 is a partially penetrant, maternal-effect cold-sensitive allele of the glycogen debranching enzyme gene agl-1 (R06A4.8). agl-1(jc11) embryos arrest at various developmental stages including pre-morphogenesis, enclosure, and elongation. Successfully hatching animals occasionally have morphogenetic defects including tail and snout abnormalities. agl-1(jc11) is rescued by expression of R06A4.8::GFP and R06A4.8 RNAi phenocopies agl-1(jc11). In agl-1(jc11) animals, a four base pair deletion located in R06A4.8 is predicted to result in a frameshift and truncation of the debranching enzyme prior to the glycogen-binding domain. agl-1(jc11) is phenocopied by treatment with MOR-14 (N-Methyl-1-Deoxynojirimycin), an inhibitor that targets the ---1,6-glucosidase activity of glycogen-debranching enzyme. Overexpression of AGL-1::GFP results inincreased debranching activity based on reduced glycogen staining with carminic acid in both embryos and adults expressing AGL-1::GFP. Recently, the beta;1 subunit of the energy sensor AMP-activated kinase was shown to directly interact with glycogen debranching enzyme and strong binding of AMPK beta;1 to glycogen was shown to depend on the alpha;-1,6-branch; binding in turn functions to inhibit AMPK activity[1, 2]. Loss of agl-1 function in C. elegans is predicted to result in the formation of a 'limit dextrin' glycogen molecule in which outer glucose chains are digested by phosphorylase to within four glucose residues of the alpha;-1-6 branch, resulting in a glycogen molecule well-suited to bind to and inhibit AMPK activity. Consistent with this model, over-expression of the glycogen-binding domain of AAKB-2 (an AMPK beta; subunit) suppresses agl-1(jc11) phenotypes. Also, in agl-1(jc11) mutants, AMPK activity is reduced; in turn, overexpression of aak-2, which encodes the catalytic subunit of AMPK, suppresses agl-1(jc11) phenotypes. agl-1(jc11) animals that survive to adulthood have a shorter life span than wild type, similar to animals lacking AMPK activity, and preliminary results indicate that over-expression of agl-1 increases lifespan. Finally, AGL-1 and AAKB-2 co-immunoprecipitate from worm extracts, suggesting that AGL-1 and AAKB-2 physically interact to regulate energy metabolism and lifespan via glycogen. 1.Sakoda, H., et al.,. Am J Physiol Endocrinol Metab, 2005. 289(3): p. E474-81. 2.McBride, A., et al., Cell Metab, 2009. 9(1): p. 23-34.

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.