Bunte, Myrna et al. (2021) International Worm Meeting "Unravelling the identity of phosphorylcholine-transferring enzymes in C. elegans"

Riksen, Joost, Patrian, Marta, Garcia Perez, Elena, Wilbers, Ruud, Kammenga, Jan, Bunte, Myrna, Schots, Arjen

[International Worm Meeting, 2021]

The attachment of phosphorylcholine on carbohydrates (PC-glycans) is a common modification for nematodes including C. elegans. The presence of PC appears to be important for the development of nematodes and for immunomodulation by parasitic nematodes. PC-glycoprotein ES-62 of Acanthoceilonema vitae directs immune cells towards an anti-inflammatory phenotype and this immunomodulatory property appears to be dependent on the presence of PC-glycans. This makes glycoproteins with PC-glycans interesting therapeutic agents to combat immune disorders such as rheumatoid arthritis, systemic lupus erythematosus and asthma. However, the biosynthetic pathway of PC-glycans is not completely understood, where especially the identity of the PC-transferring enzyme, or PC-transferase, is not fully elucidated. There are strong indications suggesting that fukutin-related genes potentially encode for this PC-transferase. In this study, we examined whether four selected fukutin-related genes of C. elegans (W02B3.4, T07A5.1, T07D3.4 and Y22D7AL.11) are involved in the biosynthetic pathway of PC glycans. With CRISPR/Cas9 technology we created C. elegans knock-out lines for each fukutin-related gene, but no significant reduction of PC was observed. Interestingly, one mutant line of W02B3.4 showed an increase of PC due to a potentially introduced signal peptide, indicating that the W02B3.4 gene could encode for a PC-transferase. Currently, we are combining mutant lines into double/triple/quadruple knock-out lines, which may provide more insight whether these fukutin-related genes encode for PC-transferases. These findings will contribute to our understanding of the pathway for PC-glycan biosynthesis, offering potential opportunities for design and synthesis of PC-glycan therapeutics.

Marta Artal-Sanz et al. (2006) European Worm Meeting "Lysosomal Biogenesis and Function is Critical for Necrotic Cell Death in C. elegans"

Chrysanthi Samara, Nektarios Tavernarakis, Marta Artal-Sanz, Popi Syntichaki

[European Worm Meeting, 2006]

Marta Artal-Sanz, Chrysanthi Samara, Popi Syntichaki and Nektarios Tavernarakis. Necrotic cell death is defined by distinctive morphological characteristics, displayed by dying cells. The cellular events that transpire during necrosis to generate these necrotic traits are poorly understood. Recent studies in our lab show that cytoplasmic acidification develops during necrosis and is required for cell death in C. elegans. However the origin of cytoplasmic acidification remains elusive. We are investigating the role of lysosomes in necrotic cell death. We observed that alkalization of endosomal and lysosomal compartments ameliorates necrotic cell death triggered by diverse stimuli. In addition, mutations in genes that result in altered lysosomal biogenesis and function markedly affect neuronal necrosis. We used a genetically encoded fluorescent marker to follow lysosome fate during neurodegeneration in vivo. Strikingly, we find that lysosomes fuse and localize exclusively around a swollen nucleus. In advanced stages of cell death, the nucleus condenses and migrates towards the periphery of the cell, while GFP-labeled lysosomal membranes fade, indicating lysosomal rupture. Our findings demonstrate a prominent role for lysosomes in cellular destruction during necrotic cell death that is likely conserved in metazoans.

Eva Brozova et al. (2006) European Worm Meeting "NHR-40, a Caenorhabditis elegans Supplementary Nuclear Receptor Regulates the Development of Embryos and L1 Larvae"

Katerina Simeckova, Zdenek Kostrouch, Eva Brozova, Marta Kostrouchova

[European Worm Meeting, 2006]

Eva Brozova1, Katerina Simeckova1, Zdenek Kostrouch2, Marta Kostrouchova1 In this study, we functionally characterized NHR-40, a member of an extended group of supplementary nuclear receptors (Robisnon-Rechavi et al, J. Mol. Evol. 2005, 60: 577-586). We show that nhr-40 is expressed in 3 isoforms that are regulated by two promoters organized in tandem. The function of NHR-40 was studied by RNA interference, by overexpression of nhr-40 and using a mutant strain, RB840, which carries a deletion in the first intron. We show that NHR-40 regulates embryonic and early larval development. This phenotype is connected with defective movement and muscle cell development. This adds NHR-40 to the growing list of nematode NHRs involved in the regulation of development.. Acknowledgement: We thank Drs. A. Fire for vectors and host used in RNAi, M.W. Krause for support and advice, The C.elegans Gene Knockout Consortium for preparation of the deletion strain RB840. The work was supported by grants 303/03/1115 and 301/ 05/0859 from the Czech Science Foundation and by the grant 0021620806 from the Ministry of Education, Youth and Sports of the Czech Republic.

Katerina Simeckova et al. (2006) European Worm Meeting "NHR-60, a Caenorhabditis elegans Supplementary Nuclear Receptor residing at the Nuclear Periphery, Regulates Embryonic Development in Connection with Acyl-Coenzyme a Binding Protein"

Zdenek Kostrouch, Eva Brozova, Marta Kostrouchova, Jaroslav Vohanka, Katerina Simeckova, Michal Pohludka

[European Worm Meeting, 2006]

Katerina Simeckova1, Eva Brozova1, Jaroslav Vohanka1, Michal Pohludka2, Zdenek Kostrouch2, Marta Kostrouchova1 . NHR-60 is a Caenorhabditis elegans supplementary nuclear receptor that belongs to a subset of 18 of the 284 nuclear hormone receptors characterized by the P-box sequence CNGCKT. We show that NHR-60 is expressed ubiquitously and is predominantly localized at the nuclear periphery. The expression of NHR-60 is accented in seam cells, gland cells and in the germline. In seam cells, the expression of NHR-60 is dependent on CHR3 (NHR-23). Our research also focused on the possible interaction of Acyl-CoA-binding protein, which is known to interact with several nuclear hormone receptors, and NHR-60. The interaction in vivo between these two proteins was confirmed by co-immunoprecipitation and co-localization using confocal microscopy. acbp-1 inhibition by RNAi induced developmental defects similar to nhr-60 RNAi. These results indicate that regulation of development by nuclear hormone receptor NHR-60 is functionally linked to Acyl-CoA-binding protein and may constitute a connection with fat metabolism. Acknowledgement: We thank Drs. A. Fire for vectors and host used in RNAi and M.W. Krause for support and advice. The work was supported by grants 303/03/1115 and 301/05/0859 from the Czech Science Foundation and by the grant 0021620806 from the Ministry of Education, Youth and Sports of the Czech Republic.

Petr Liby et al. (2006) European Worm Meeting "Inhibition of bir-1, the Homologue of Human Survivin, Induces Changes of Expression of Developmentally Active Collagen Genes in L1 Larval Stage"

Marta Kostrouchova, Eva Brozova, Marketa Kostrouchova, Michal Pohludka, Jaroslav Vohanka, Petr Liby, Zdenek Kostrouch

[European Worm Meeting, 2006]

Petr Liby1, Marketa Kostrouchova1, Michal Pohludka1, Jaroslav Vohanka2, Eva Brozova2, Marta Kostrouchova2 and Zdenek Kostrouch1. BIR-1 is a member of the Inhibitors of Apoptosis protein family that is involved in the regulation of microtubule organization, condensation of chromosomes and cell division in C. elegans as well as in vertebrates. In C. elegans, bir-1 is organized in an operon together with SKIP, a transcription and splicing cofactor. bir-1 inhibition induces developmental defects including dpy and egl phenotypes. Here we studied the effect of bir-1 inhibition by RNAi in L1 larval stage using whole genome microarrays (Affymetrix). Microarrays detected changes in expression of several developmentally active collagen genes in bir-1 inhibited larvae. We further characterized the effect of bir-1 inhibition on selected collagen gene expression and induction of dpy phenotype. Acknowledgement: We thank Drs. A. Fire for GFP construct, vectors and host used in RNAi and M.W. Krause for support and advice. We thank the NIDDK Microarrays facility for performing the microarrays analysis. The work was supported by the grant 303/03/0333 from the Czech Science Foundation, by the grant NC-7554 from the Ministry of Health of the Czech Republic and by the grant 0021620806 from the Ministry of Education, Youth and Sports of the Czech Republic.

Jaroslav Vohanka et al. (2006) European Worm Meeting "An Expression Study of Seven Putative Nuclear Receptors Organized in a Cluster on Chromosome V of Caenorhabditis elegans"

Marta Kostrouchova, Zdenek Kostrouch, Jaroslav Vohanka

[European Worm Meeting, 2006]

Jaroslav Vohanka1, Zdenek Kostrouch2 and Marta Kostrouchova1. Chromosome V contains a group of seven sequences recognized by the computer program GeneFinder as potential nuclear hormone receptors (NHRs). In order to identify whether genes from this cluster may be functional NHRs, we studied their expression during C. elegans development. RT-PCR identifies transcripts of all seven genes. The expression of all members of this cluster starts in embryos. Six of these genes are expressed throughout development while one member of this cluster is only expressed in embryos and early larval stages. We prepared constructs of GFP fusion genes containing various regions of genomic sequences. At least 2 different fragments containing putative promoters were prepared for each gene of the cluster; one consisting of approximately 500 bp and a second of approximately 2000 bp. The longer constructs contain parts of the coding sequences of the preceding genes. Our results show that members of this gene cluster have a diverse expression pattern and are likely to be functional NHRs. Acknowledgement: We thank Drs. A. Fire for GFP constructs, vectors and host used in RNAi, M.W. Krause for support and advice. The work was supported by grants 303/03/1115 and 301/ 05/0859 from the Czech Science Foundation and by the grant 0021620806 from the Ministry of Education, Youth and Sports of the Czech Republic.

Marta Maria Gaglia et al. (2006) European Worm Meeting "Transcriptional Changes Underlying Chemosensory Regulation of Lifespan"

Cynthia Kenyon, Marta Maria Gaglia

[European Worm Meeting, 2006]

Marta Maria Gaglia and Cynthia Kenyon. Mutations in the insulin/IGF-1-like receptor daf-2 alone can double the lifespan of C.elegans, suggesting that aging is not just a default state, but instead a genetically regulated process. However, it is not clear what physiological processes regulate the daf-2 pathway. Interestingly, mutants with altered chemosensation also have an extended lifespan (Apfeld and Kenyon, 1999). Both daf-2 and chemosensory mutant longevity are dependent on the activity of the FOXO transcription factor DAF-16. This suggests that external inputs through the chemosensory system may regulate the insulin pathway and in turn affect lifespan. Further studies have identified some of the cellular components of the sensory pathway of lifespan control (Alcedo and Kenyon, 2004). However, the molecular mechanism by which the chemosensory system modulates lifespan remains still largely unknown. To begin to elucidate the mechanisms of chemosensory regulation of lifespan, we have performed genome-wide expression analysis with microarrays on the long-lived chemosensory mutant daf-10. We are in the process of characterizing the genes whose expression was altered with mutants and RNA interference in sensitized backgrounds. In particular, we are interested in those genes that affect lifespan without causing a chemosensory defect, as they may act downstream of the sensory neurons. In addition we are comparing the expression changes to those occurring in daf-2 mutants, to determine how the two pathways interact to modulate lifespan.

Michal Pohludka et al. (2006) European Worm Meeting "Analysis of CHR-3 (nhr-23) Loss of Function in L1 Larval Stage Using Whole Genome Microarrays and Comparative Two Dimensional Protein Chromatography"

Zdenek Kostrouch, Marta Kostrouchova, Michal Pohludka, Jaroslav Vohanka

[European Worm Meeting, 2006]

Analysis of CHR-3 (nhr-23) Loss of Function in L1 Larval Stage Using Whole Genome Microarrays and Comparative Two Dimensional Protein Chromatography. Michal Pohludka1, Jaroslav Vohanka2, Marta Kostrouchova2 and Zdenek Kostrouch1. CHR3 (nhr-23, NR1F4) is an evolutionarily conserved nuclear receptor involved in the regulation of molting and body morphology in larval development. We studied the role of CHR-3 (nhr-23) by RNAi in L1 stage and analysed the phenotype on mRNA and protein levels using whole genome microarrays (Affymetrix) and a comparative two dimensional protein chromatography by ProteomeLab PF 2D Protein Fractionation System. Protein profiling was achieved by Isoelectric chromatofocusing used as the first dimension separation and reversed phase chromatography separating proteins by hydrophobicity as the second dimension. The differentially expressed proteins were detected by ProteoVue and DeltaVue Software. Microarrays data subjected to computer analysis using GeneSpring program detected clusters of similarly regulated genes. Two dimensional chromatography revealed multiple fractions containing differentially expressed proteins in CHR3 (nhr-23) inhibited fractions suitable for analysis by mass spectroscopy. Genome and proteome wide analysis may help to further characterize phenotypes of gene inhibition by RNAi. Acknowledgement: We thank Drs. A. Fire for GFP construct, vectors and host used in RNAi and M.W. Krause for support and advice. We thank the NIDDK Microarrays Facility for performing the microarrays analysis. The work was supported by the grants 301/05/0859 and 303/03/0333 from the Czech Science Foundation, and by the grant 0021620806 from the Ministry of Education of the Czech Republic.

Marketa Kostrouchova et al. (2006) European Worm Meeting "Valproic Acid Affects Gene Expression and Development in Caenorhabditis elegans"

Zdenek Kostrouch, Marta Kostrouchova, Marketa Kostrouchova, Petr Liby

[European Worm Meeting, 2006]

Marketa Kostrouchova1, Petr Liby1, Marta Kostrouchova2 and Zdenek Kostrouch1. Valproic acid (VPA, 2-propylpentanoic acid), a drug widely used in the treatment of epilepsy and bipolar disorder, has been shown to posses anti-cancer activity. VPA suppresses tumor growth, induces tumor cell differentiation and reduces the formation of metastases. Multiple mechanisms were shown to be part of VPAs mode of action, including inhibition of histone deacetylase activity, inhibition of glycogen synthase 3 and interaction with the regulation of nuclear hormone receptors. We studied the effect of VPA on the development and gene expression in C. elegans. VPA delivered to worms by microinjections to the ovarial syncytium or by incubating worms in various concentrations of VPA (1 to 10 mM) caused a dose dependent delay of larval development and lethality in two developmental stages: in late embryogenesis and in L1 larval stage. Surprisingly, most animals were able to overcome the effect of VPA, suggesting a compensatory or balancing mechanism. Microarray experiments show that genes affected by VPA can be divided to at least three different categories suggesting multiple mechanisms to be involved in VPA mode of action. Acknowledgement: We thank Dr. M.W. Krause for support and advice. We thank the NIDDK Microarrays facility for performing the microarrays analysis. The work was supported by the grant 303/03/0333 from the Czech Science Foundation, by the grant NC-7554 from the Ministry of Health of the Czech Republic and by the grant 0021620806 from the Ministry of Education, Youth and Sports of the Czech Republic.

Marta Artal-Sanz et al. (2006) European Worm Meeting "The Mitochondrial Prohibitin Complex Modulates Ageing In C. elegans"

Nektarios Tavernarakis, Marta Artal-Sanz

[European Worm Meeting, 2006]

Marta Artal-Sanz and Nektarios Tavernarakis. The prohibitin complex in eukaryotes consists of two prohibitin subunits (PHB1 and PHB2) that assemble into a macromolecular structure of approximately 1 MD in the inner mitochondrial membrane. The evolutionary conservation and the ubiquitous expression of PHB genes in mammalian tissues suggest an important function for the prohibitin complex. However, the physiological role of the complex has remained elusive. A number of diverse cellular functions have been attributed to these proteins, including a role in cell cycle regulation, receptor-mediated signalling, regulation of transcription, apoptosis and a role in the assembly of mitochondrial respiratory chain complexes. We are characterizing the function of prohibitin during C. elegans development and ageing. The PHB complex is essential for C. elegans embryonic development. The postembryonic depletion of PHB proteins results in gonad differentiation defects and sterility, reduced physiological rhythms and oxygen consumption rates, and increased sensitivity to oxidative stress. Muscle mitochondria appear fragmented and disorganized in phb(RNAi) animals, suggesting that the prohibitin complex has an important role in establishing or maintaining the integrity of mitochondrial membranes. We are currently investigating the effect of phb-1 and phb-2 knock down on the lifespan of wild type animals as well as in different mutant genetic backgrounds. Prohibitin depletion has no effect on the lifespan of N2 animals at 20 degrees, although sometimes a slight reduction in lifespan can be observed. However, phb-1/2(RNAi) animals show increased lifespan at 25 degrees as well as increased intrinsic thermotolerance (itt). This indicates that in PHB-deficient animals, changes in temperature have a dramatic effect on mitochondrial function and in ageing. RNAi knockdown of PHB proteins dramatically increases daf-2(e1370) lifespan in a daf-16 dependent manner. However, the increased itt observed in phb-1/2(RNAi) animals is partially daf-16 independent. Further, phb-1/2(RNAi) considerably extends the lifespan of the long lived mitochondrial mutant isp-1(qm150) as well as the caloric restricted mutant eat-2(ad465). Our results suggest an important role for prohibitins in mitochondrial metabolism that also impinges on the ageing process.