Dietz, S.F. et al. (2017) International Worm Meeting "Quantitative proteomics and interactomics in Caenorhabditis elegans."

Dietz, S.F., Butter, F., Ketting, R.F., Vasconcelos Almeida, M.

[International Worm Meeting, 2017]

Quantitative proteomics by mass spectrometry is a powerful tool to investigate the interactions and expression of proteins. We applied different molecular biology and quantitative proteomics methods to define interacting proteins, the composition of complexes and differences in protein expression levels in the model organism Caenorhabditis elegans, demonstrating that the combination of these techniques can be routinely used to identify and characterize endogenous proteins in the absence of suitable antibodies. We here compare several approaches for quantitative proteomics: 1) introducing a mass label metabolically via feeding with E. coli, 2) reductive demethylation, which introduces a mass tag at the peptide level during the sample preparation and 3) label free quantitation, which compares peptide intensities across different mass spectrometry measurements. To benchmark these three techniques for their applicability and benefits, we performed DNA pull-down experiments with nuclear extracts using the the telomeric sequence of C. elegans (TTAGGCn) or a scrambled control (AGGTCAn). In all three approaches, we were able to determine proteins binding to the telomeric repeat sequence including the known binders POT-1, POT-2 and MRT-1 alongside proteins not previously implicated in telomere biology that are currently further investigated. Moreover, in a pilot experiment, we coupled native size exclusion chromatography with label-free quantitation to obtain a global overview of C. elegans protein complexes. Proteins belonging to a complex should show co-elution. This could be validated by several members of known complexes, like the CUL-4/DDB-1 ubiquitin ligase complex, the NuRD/CHD chromatin remodeling complex and the DCR-1/RDE-4 complex which co-occurred in same fractions, suggesting that protein-protein interactions can be recovered using this approach. Additionally, this extended fractionation allowed us to measure an in-depth proteome of >5000 proteins. Besides interactomics, mass-spectrometry based quantitative proteomics can be used to study differences in protein levels similar to next generation sequencing for transcriptomes. We determined significant protein abundance differences between the wildtype and the pot-2(tm1400) knockout strain by label free proteomics. Overall, we show that diverse classical molecular biology techniques can be combined with quantitative proteomics approaches to readily uncover protein functionality in C. elegans.

van Schendel, R. et al. (2017) International Worm Meeting "Polymerase theta is a key driver of genome evolution, of CRISPR/Cas9-mediated mutagenesis and of mutagen-induced deletion allele formation."

Tijsterman, M., van Schendel, R.

[International Worm Meeting, 2017]

For more than half a century, genotoxic agents have been used to induce mutations in the genome of model organisms to establish genotype-phenotype relationships. We have recently demonstrated for two of the most widely used mutagens in C. elegans, i.e. ethyl methanesulfonate (EMS) and photo-activated trimethylpsoralen (UV/TMP), that deletion mutagenesis is the result of polymerase Theta (POLQ)-mediated end joining (TMEJ) of double strand breaks (DSBs) 1, 2. This discovery allowed us to survey many thousands of available deletion alleles, which reveals a step-wise and versatile model for the in vivo mechanism of TMEJ, explaining the molecular nature of mutagen-induced deletion alleles 3. We also found that CRISPR/Cas9-induced genomic changes are exclusively generated through POLQ action, refuting a previously assumed requirement for NHEJ in their formation; Unlike somatic cells, which use NHEJ to repair DNA breaks, germ cells use an alternative route 4. Finally, through whole-genome sequencing of propagated populations, we show that only POLQ-proficient animals accumulate genomic scars that are abundantly present in genomes of wild C. elegans, pointing towards POLQ as a major driver of genome diversification. New insights into the mechanism of genome engineering, genome maintenance and evolution will be discussed. 1. Koole,W. et al. A Polymerase Theta-dependent repair pathway suppresses extensive genomic instability at endogenous G4 DNA sites. Nat. Commun. 5, 3216 (2014). 2. Roerink,S.F., van Schendel,R., & Tijsterman,M. Polymerase theta-mediated end joining of replication-associated DNA breaks in C. elegans. Genome Res. 24, 954-962 (2014). 3. van Schendel R., van Heteren J., Welten,R., & Tijsterman,M. Genomic Scars Generated by Polymerase Theta Reveal the Versatile Mechanism of Alternative End-Joining. PLoS. Genet. 12, e1006368 (2016). 4. van Schendel,R., Roerink,S.F., Portegijs,V., van den Heuvel,S., & Tijsterman,M. Polymerase Theta is a key driver of genome evolution and of CRISPR/Cas9-mediated mutagenesis. Nat. Commun. 6, 7394 (2015).

Allyson V McCormick et al. (2003) International Worm Meeting "Analysis of neuronal synchrony in Caenorhabditis elegans"

James H Thomas, Allyson V McCormick

[International Worm Meeting, 2003]

Mutations in a few C. elegans genes can result in a convulsive phenotype marked by repeated end-to-end full body contractions caused by the simultaneous excitation of dorsal and ventral body wall muscles. unc-43 encodes the only CaM Kinase II homologue in C. elegans. unc-43(lf) worms have rare spontaneous convulsions that are intensified in frequency and severity by exposure to neurostimulants (e.g. pentylenetetrazole [PTZ] and pilocarpine). Mutants affecting GABA function have anteriorly restricted convulsions in response to PTZ exposure and full body convulsions on high concentrations of pilocarpine. To investigate the underlying molecular mechanisms of these convulsions, we are looking in detail at the specific neuronal requirement for UNC-43. Transgenic expression of a wild-type unc-43 cDNA solely in neurons can alleviate drug-induced convulsions in unc-43(lf) animals. Using neural subtype specific promoters, we have delineated the requirement of UNC-43 to the motor neurons. Aided by the complete reconstruction of the worm nervous system1, we have a likely network of neurons involved in controlling convulsions. We are following up this finding by expressing cameleon proteins2 (fluorescent calcium sensors) in these neurons and will be pursuing a comparative in vivo analysis of wild type, CaM Kinase II defective and GABA defective neuronal patterns of excitation. Additionally, we have conducted screens to discover other mutations able to confer convulsion susceptibility in worms, and screens for modifiers, which either attenuate or enhance this convulsion phenotype. These experiments establish C. elegans as a model for mechanisms underlying simultaneous excitation of neuronal networks. Similar types of neuronal synchrony may cause human epilepsies, since -CaM Kinase II3 and GAD654 mouse knockouts are susceptible to spontaneous and drug-induced seizures. We believe that our use of C. elegans, as a well-studied and tractable model organism combining sophisticated genetics, a "mapped" nervous system, and calcium imaging microscopy, may be a powerful way to characterize key molecular components controlling seizures and perhaps identify new drug targets for treatment. 1White, J.G., et al. (1986). Phil. Trans. R. Soc. Lond. 314:1-340; 2Miyawaki, A., et al. (1999). PNAS 96:2135-2140; 3Butler, L.S., et al. (1995). PNAS 92:6852-6855; 4Kash S.F., et al. (1997). PNAS 94:14060-5.

Wolkow CA et al. (1998) East Coast Worm Meeting "REGULATION OF DAUER FORMATION AND LONGEVITY BY AGE-1"

Wolkow CA, Ruvkun GB

[East Coast Worm Meeting, 1998]

In C. elegans, an insulin-like signaling pathway regulates dauer formation and life span. One component of this pathway, age-1, encodes a homolog of the p110 catalytic subunit of mammalian phosphatidylinositol 3-kinase (PI(3)K) (1). Mutations in age-1 are maternal effect and cause constitutive dauer formation. The phenotype of maternally rescued age-1 mutants is a two-fold extension in adult life span. PI(3)K is a known effector for insulin receptor signaling in mammals. In C. elegans, age-1 acts in a pathway with daf-2, which encodes an insulin receptor homolog (2). Although the physiological effects of insulin have been well-studied in mammalian systems, relatively little is known of the pathways between PI(3)K activation and the physiological outputs of insulin. To clarify this problem, we have undertaken cell biological and genetic studies to identify the function of AGE-1 PI(3)K in dauer formation and aging. To identify potential functions for age-1, the expression pattern and subcellular localization of age-1 was examined using two age-1::gfp transgenes. One transgene expresses the full-length AGE protein with GFP fused to the AGE-1 carboxyl terminus. The second transgene fuses GFP to a truncated AGE-1 protein lacking the carboxyl terminal catalytic domain. Both the full-length and truncated age-1::gfp reporters are consistently expressed in 3 pairs of head neurons. Although age-1::gfp expression is observed at all stages of development and in adults, expression is strongest in L1 larvae. age-1::gfp is also expressed weakly in body wall muscle at all stages and in the spermatheca of adults. In addition, age-1 expression appears to be autoregulated because age-1::gfp expression in neurons increases significantly in daf-2 dauers. Fluorescence from the truncated AGE-1::GFP reporter appears in a punctate pattern in the cell bodies and processes of neurons. The punctate subcellular distribution of AGE-1::GFP may suggest that AGE-1 functions in specific subcellular compartments, such as vesicles or synaptic junctions in neural processes. Work done by others suggests that, upon insulin stimulation, mammalian PI(3)K traffics away from the plasma membrane into distinct cellular compartments (3). We are currently testing the functional relevance of the AGE-1::GFP subcellular distribution. REFERENCES: (1) Morris, J. Z., Tissenbaum, H. A. and Ruvkun, G. (1996) Nature 382: 536-539. (2) Kimura, K. D., Tissenbaum, H. A., Liu, Y. and Ruvkun, G. (1997) Science 277: 942-946. (3) Clark, S.F., Martin, S., Carozzi, A.J., Hill, M.M., James, D.E. (1998) J. Cell Biol. 140: 1211-1225.

Allyson V McCormick et al. (2004) West Coast Worm Meeting "Mutational analysis of genes disrupting neuronal synchrony in Caenorhabditis elegans"

James H Thomas, Gennifer E Merrihew, Allyson V McCormick

[West Coast Worm Meeting, 2004]

Convulsions in C. elegans , as we have defined them, are simultaneous repetitive contractions of body wall muscles. The largest class of mutants that present a convulsion phenotype have contractions which are restricted to anterior portions of the worm. Our initial list of anterior convulsers originated from a mutagenesis screen for mutants sensitize to neurostimulant ( e.g., pentylenetetrazole or pilocarpine). Roughly three-quarters of the mutant alleles resulted in anteriorly-restricted convulsions. (The remaining quarter display full body convulsions and represent new alleles of unc-43 1 .) Phenotypic analysis revealed that every mutant had secondary drug-independent locomotion phenotypes. This led us to expand our search for convulsers to include the set of already identified uncoordinated mutants. Roughly a quarter of these showed some convulsion-sensitivity to drug treatment. These mutants define three functional classes: defects in GABAergic neurotransmission ( e.g., unc-25 ), in general synaptic function ( e.g., unc-32 , unc-64 ) and in early axonal guidance ( e.g., unc-76 ). The head of the worm receives a multitude of synaptic inputs from environmental and internal cues. Head muscles are either innervated by motor neurons localized in the nerve ring or the ventral nerve cord 2 . Based on our analysis of the full body convulsion phenotype which implicates the involvement of ventral cord motor neurons 1 , we hypothesize that anterior convulsions are likewise the result of motor neuron dysfunction but only of those that innervate anterior muscle groups. Furthermore, we believe that disruption of GABAergic signaling may be the common thread that links the three functional groups found in our screens. This suggests that the RME inhibitory motor neurons (GABA containing neurons) may be solely or partially responsible for mediating anterior convulsions. We are in the process of combining neuron-specific promoter rescue and double mutant analysis to verify this hypothesis. These experiments broaden our worm model of mechanisms underlying simultaneous excitation of neuronal networks. Recurrent and aberrant neuronal synchrony is the cause of human and mammalian epilepsies and our model has striking parallels with the current mouse model of epilepsy. For instance, there are mouse knockouts that represent the same functional classes we found in our screens ( e.g., GAD65 3 , synapsin I 4 , uPAR 5 ). We believe that we are characterizing evolutionarily ancient strategies for coordinating and controlling nervous system synchronizations. Our use of C. elegans , as a well-studied and tractable model organism combining sophisticated genetics and a mapped nervous system 2 may be a powerful way to characterize key molecular components controlling seizures and perhaps identify new drug targets for treatment. 1 See abstract McCormick, A.V. et al. (2004). Molecular analysis of UNC-43 control of neuronal synchrony in C. elegans . West Coast Worm Meeting 2 White, J.G., et al. (1986). Phil. Trans. R. Soc. Lond. 314:1-340 3 Kash S.F., et al. (1997). PNAS 94:14060-14065 4 Terada, S. et al. (1999). J Cell Biol 145:1039-1048 5 Powell, E.M. et al. (2003). J Neurosci 23:622-631