Singh, Kapil Dev et al. (2013) International Worm Meeting "Studying the effect of natural variation on protein abundance in C. elegans."

Elvin, Mark, Hengartner, Michael, Poulin, Gino, Roschitzki, Bernd, Schrimpf, Sabine, Kammenga, Jan, Kamkina, Polina, Snoek, L. Basten, Rodriguez, Miriam, Singh, Kapil Dev

[International Worm Meeting, 2013]

Cancer, neurodegenerative diseases and autoimmune diseases are complex, polygenic pathologies caused by a combination of genetic and environmental factors, including lifestyle. Many of the signaling pathways (i.e. Apoptosis, Notch, MAPK and Wnt) involved in these diseases are conserved and also present in C. elegans. We are studying the influence of natural genetic variation on the abundance of proteins participating in these four signaling pathways in C. elegans. From the two genetically divergent wild-type strains Bristol N2 and Hawaii CB4856, 200 recombinant inbred lines were previously generated. Transcriptome analysis of these RILs showed significant heritable variation in gene expression at the mRNA levels, but very little is known about heritable variation at the protein level. To determine the effect of natural variation on protein abundance, we developed SRM assays for 156 proteins from the above mentioned signaling pathways. SRM measurements were acquired on a TSQ Vantage for two biological replicates of N2 and CB4856 and for one sample of four RILs (WN 31, WN 71, WN 105 and WN 186) at developmental stage L4. So far, by an automated data analysis using mProphet, 45 proteins (mostly represented by 1 or 2 peptides) could be quantified in all samples. Proteins with significant differences in abundance will be further confirmed in biological replicates of the RILs and QTL analysis will be done to determine the genetic locus (loci) responsible for the difference in protein abundance. Finally, these modifier loci will be crossed into sensitized genetic backgrounds to determine their potential to influence developmental signaling. This work is supported by the EU-FP7 HEALTH project PANACEA, contract nr. 222936.

Vaglio P et al. (2000) East Coast Worm Meeting "Protein interaction mapping in C. elegans"

Boulton SJ, Vidal MI, Walhout AJM, Thierry-Mieg N, Reboul J, Matthews LR, Vaglio P

[East Coast Worm Meeting, 2000]

The emerging field of functional genomics aims at characterizing the function of large numbers of unannotated ORFs predicted from nearly complete genome sequences by developing standardized functional assays that can be applied in high-throughput settings. Mostly gene-based functional genomics approaches have been developed so far. These typically include large-scale gene knock-outs and microarray or chip analysis. However, it is also important to develop protein-based approaches such as protein interaction mapping, protein localization mapping, and biochemical and structural genomics. Most proteins require physical interactions with other molecules to function appropriately. Therefore, it should be informative to identify potential interaction partners for large numbers of predicted gene-products. We have started such a protein interaction mapping project in C. elegans, the first multicellular organism for which a nearly complete genome sequence has been available. As a standardized functional assay, we are using a semi-automated version of the yeast two-hybrid system that we developed. Using proteins kown to be involved in vulval development, we have shown that ~50% of previously reported interactions can be detected in our two-hybrid assay, indicating that our current version of the system should allow a reasonable first coverage of protein-protein interactions. In addition, we have identified ~150 new potential protein-protein interactions which together provide a functional annotation for approximately 100 uncharacterized gene products. Several interactions seem to cluster in closed loops (X/Y/Z/Wn/X), indicating that they might belong to either macromolecular complexes. Finally ~10% of interactions corresponded to "interologs", which we define as protein-protein interactions conserved between model organisms. We have made our data available through a website on the Internet and in a version of ACeDB. We will present the data obtained with vulval proteins and data we recently obtained on other systems.

Buttner S et al. (2013) Cell Death Differ "The Ca2+/Mn2+ ion-pump PMR1 links elevation of cytosolic Ca(2+) levels to -synuclein toxicity in Parkinson's disease models."

Freudenberger P, Sigrist SJ, Ruli D, Habernig L, Faes L, Eisenberg T, Tavernarakis N, Reichelt WN, Kroemer G, Ghillebert R, Broeskamp F, Kourtis N, Callewaert G, Harger A, Winderickx J, Carmona-Gutierrez D, Madeo F, Franssens V, Pieber TR, D'hooge P, Buttner S, Benke S

[Cell Death Differ, 2013]

Parkinson's disease (PD) is characterized by the progressive loss of dopaminergic neurons, which arises from a yet elusive concurrence between genetic and environmental factors. The protein -synuclein (Syn), the principle toxic effector in PD, has been shown to interfere with neuronal Ca(2+) fluxes, arguing for an involvement of deregulated Ca(2+) homeostasis in this neuronal demise. Here, we identify the Golgi-resident Ca(2+)/Mn(2+) ATPase PMR1 (plasma membrane-related Ca(2+)-ATPase 1) as a phylogenetically conserved mediator of Syn-driven changes in Ca(2+) homeostasis and cytotoxicity. Expression of Syn in yeast resulted in elevated cytosolic Ca(2+) levels and increased cell death, both of which could be inhibited by deletion of PMR1. Accordingly, absence of PMR1 prevented Syn-induced loss of dopaminergic neurons in nematodes and flies. In addition, Syn failed to compromise locomotion and survival of flies when PMR1 was absent. In conclusion, the Syn-driven rise of cytosolic Ca(2+) levels is pivotal for its cytotoxicity and requires PMR1.

Shakir MA et al. (1990) Worm Breeder's Gazette "Transposon tagging of genes affecting axon growth and process placement of sensory lumbar neurons in C. elegans."

Siddiqui SS, Shakir MA

[Worm Breeder's Gazette, 1990]

A pair of lumbar ganglia are situated in the posterior-most region of the tail nervous system in C. elegans, each consisting of 12 neurons. The neurons have a simple morphology and most of them are either monopolar or bipolar cells (White et al. 1986). We have begun screening the mutator strains TR679 and RW7097 to isolate mutations affecting the morphology of lumbar neurons by transposon tagging, by light microscopic techniques. These include, FITC dye filling of the phasmid neurons PHA and PHB (Hedgecock et al. 1985), immunocytochemical staining of PHC and PVN neurons (Siddiqui and Culotti, 1984), and staining of PLM and PVR neurons (Siddiqui et al. 1989). It was shown previously by Hedgecock et al. that mutations in unc-6, unc-44, he axonal growth and guidance of PHA and PHB neurons, using FITC staining of live animals. In addition, to these genes, mutants in unc-13, unc-71, wn by immunocytochemical staining with anti-HRP antibodies, to be affected in the process placement of PHC and PVN lumbar neurons (Siddiqui and Culotti). The mechanosensory neurons PLML and PLMR, were shown to be affected in unc-53 and unc-73 mutants by staining these neurons with anti-tubulin antibody 6-11B-1. Several mutants have been isolated using the mutator strains, and we are using the well established techniques to clone these genes by identifying the Tc1 element rendering these genes mutant. SQ141 is a strain which fails to stain PHA and PHB neurons with FITC dye, but shows normal PLM and PVR neurons, as determined cytochemically. The postembryonic neurons PHC and PVN are also normal in this strain, suggesting that this mutation is specific for the dye uptake in the phasmid neurons PHA and PHB, whereas, it does not affect other lumbar neurons (PLM, PVR, PHC, and PVN). Similarly other strains show specific neuronal defects limited to one class of lumbar neurons. We are also examining the morphology of the male tail neurons, in Tc1 tagged mutants. Antibodies to mouse neural cell adhesion molecule (N-CAM) were previously reported to stain the male specific neurons CEMs and a set of ray neurons. We would like to request members of the worm community to kindly send us their isolates of unc mutants in Tc1 induced mutants, or mutants abnormal in the tail morphology.