Martel, Pier-Olivier et al. (2021) International Worm Meeting "MIG-6 PLAC domain affects Notch signaling and the extracellular matrix composition."

Martel, Pier-Olivier, Degremont, Julia, Beaulieu, Lucie, Narbonne, Patrick, Dologuele, Eden

[International Worm Meeting, 2021]

In a forward genetic screen aimed at isolating mutations that disrupt germline stem cell (GSC) regulation, we found a new allele of mig-6, which we called qz2. MIG-6 is homologous to mammalian Papilin, a component of the extracellular matrix (ECM), and exists as two isoforms. The mig-6(qz2) mutation disrupts the long isoform, which is known to cell-autonomously promote DTC migration (Kawano et al. 2009). More specifically, it affects its C-terminal well-conserved protease and lacunin (PLAC) domain, thought to be important in matrix metalloproteases and proprotein convertases. The mig-6(qz2) mutants lay 26% less eggs than wild-type and have a slight increase in embryonic (+5.9%) and larval lethality (+1,8%). In addition, the distal half of their gonad arms are 30% shorter and 43% wider than in wild type. By comparing gonad morphology in mig-6(qz2) with mig-6l(∅)/+, mig-6(qz2)/+ and wild type animals, we found the qz2 mutation to have semidominant features regarding this morphological defect. Consistent with its altered distal gonad morphology, the distal tip cell (DTC) cytonemes are shorter and spread wider in mig-6(qz2) mutants than in wild-type. Interestingly, the rate of GSC proliferation is normal in mig-6(qz2) mutants, indicating that the morphology of the distal gonad, including niche morphology, can be dramatically altered without affecting the rate of GSC proliferation. On the other hand, we noticed that mig-6(qz2) greatly aggravates the glp-1(gf) thermosensitive defects at a permissive temperature. Namely, glp-1(gf); mig-6(qz2) double mutants develop 3.8 times more tumors than glp-1(gf) single mutants, while they lay 57% less eggs that show drastic increases in embryonic (+38.7%) and larval lethality (+9.1%). Despite the changes in DTC and gonadal morphology in mig-6(qz2) animals, we failed to detect a difference in both lag-2 expression and GLP-1 levels. However, we found that the fraction of intracellular GLP-1 (cleaved NICD) was higher. Indeed, using Imaris 3D reconstructions of confocal z-stacks acquisitions of GLP-1::GFP animals (kind gift from David Greenstein), we found that there was significantly more GLP-1::GFP in the rachis of mig-6(qz2) mutants. In parallel, we noticed that the ECM in the distal gonad of mig-6(qz2) mutants was enriched in one of its main components, Collagen IV (EMB-9). Our results are thus compatible with the notion that MIG-6's PLAC domain may modify the ECM composition in a way that dampens Notch activation.

(2019) Development "The people behind the papers - Julia Brandt, Mary Rossillo and Niels Ringstad."

[Development, 2019]

A fundamental aim in developmental biology is to understand how the various cell types of the body are specified by differential gene regulation. <i>Caenorhabditis</i><i>elegans</i> nervous system development provides a powerful system for studying this, as exemplified by a new Development paper reporting on how the BAG neurons that help the worm sense oxygen and carbon dioxide are specified. We caught up with first authors Julia Brandt and Mary Rossillo and their supervisor Niels Ringstad (Associate Professor at the Skirball Institute of Biomolecular Medicine and Department of Cell Biology at New York University) to find out more about the story.

Ben Lehner et al. (2006) European Worm Meeting "Systematic Mapping of Genetic Interactions in C. elegans Suggests a New Paradigm for Human Genetic Disease"

Catriona Crombie, Andrew G. Fraser, Ben Lehner, Angelo Fortunato, Julia Tischler

[European Worm Meeting, 2006]

Ben Lehner, Catriona Crombie, Julia Tischler, Angelo Fortunato and Andrew G. Fraser Most heritable traits, including disease susceptibility, are affected by the interactions between multiple genes. However, we still understand very little about how genes interact since only a minute fraction of possible genetic interactions have been explored experimentally. To begin to address this, we are using RNA interference to identify genetic interactions in C. elegans, focussing on genes in signalling pathways that are mutated in human diseases. We tested ~65,000 pairs of genes for possible interactions and identify ~350 genetic interactions. This is the first systematically constructed genetic interaction map for any animal. We successfully rediscover most components of previously known signalling pathways; furthermore, we verify 9 novel modulators of EGF signalling. Crucially, our dataset also provides the first insight into the global structure of animal genetic interaction maps. Most strikingly, we identify a class of highly connected ''hub'' genes: inactivation of these genes greatly enhances phenotypes resulting from mutations in many different pathways. These hub genes all encode chromatin regulators, and their activity as genetic hubs appears conserved across metazoans. We propose that these genes function as general buffers of genetic variation and that these hub genes will act as modifier genes in multiple, mechanistically unrelated genetic diseases in humans.

Bo Wang et al. (2006) European Worm Meeting "Multi-Parameter Axial Profiling of Transgenic C. elegans Expressing Fluorescent Proteins from Various Cell-Specific, Tissue Specific and Developmentally Regulated Promoters"

Bruce Holcombe, Julia Thompson, Yanping Zhang, Rock Pulak, Bo Wang, Michael Herman, Mariya Lomakina

[European Worm Meeting, 2006]

Bo Wang1, Julia Thompson1, Yanping Zhang2, Michael Herman2, Mariya Lomakina1, Bruce Holcombe1, Rock Pulak1 . The COPAS Biosort instrument automates the analysis, sorting, and dispensing of all stages of C. elegans, measuring the animals size and the intensity of expressed fluorescent markers. Once analyzed, animals can be selected according to user defined criteria, and then dispensed into multi-well plates for high throughput screening or collected in bulk for further analysis. With this technology, time required for large scale screening for certain changes in the optical properties of the animals, such as changes in the levels of expression of a fluorescent protein, can be dramatically reduced and human error minimized. Recent enhancements to an add-on module, called the Profiler II, have been tested for its ability to collect positional information of fluorescent expression. The instrument can simultaneously collect fluorescence information in three separate regions of the spectrum. Here we show that the instrument can analyze multi-colored transgenic animals and can be used to compare the amounts and relative positions of expression of two or three different colors of fluorescence. Furthermore, this technology can be used to screen for independent changes in the intensity or position of each reporter protein. We have tested various transgenic animals expressing green, yellow and/or red fluorescing proteins from a collection of promoters that include myo-2, str-1, egl-17, mab-5, and various others, separately and in certain combinations. We present some proof of principle examples of how these could be used in genetic screens.

Julia Tischler et al. (2006) European Worm Meeting "Analysing the Redundancy of Duplicated Genes and the Evolutionary Conservation of Genetic Interactions Using Combinatorial RNAi in C. elegans"

Ben Lehner, Julia Tischler, Andrew G Fraser

[European Worm Meeting, 2006]

Julia Tischler, Ben Lehner and Andrew G Fraser Systematic analyses of loss-of-function phenotypes have been carried out for almost all genes in S. cerevisiae, C. elegans, and D. melanogaster, and there are major efforts to make a comprehensive collection of mouse knockouts. While such studies greatly expand our knowledge of single gene function, they do not address redundancy in genetic networks, nor do they attempt to identify genetic interactions. Developing tools for the systematic mapping of genetic interactions is thus a key step for exploring the relationship between genotype and phenotype. We thus sought to establish protocols for targeting multiple genes simultaneously by RNA interference (RNAi) in C. elegans to provide a platform for the systematic identification of genetic interactions in this key animal model system.. We set up conditions for RNAi that allow us to target multiple genes in the same animal (combinatorial RNAi) in a high throughput setting and to detect the great majority of previously known synthetic genetic interactions. We then used this assay to test the redundant functions of genes that have been duplicated in the genome of C. elegans since divergence from either S. cerevisiae or D. melanogaster, and identified 16 pairs of duplicated genes that are at least partially functionally redundant. Intriguingly, 14 of these redundant gene pairs were duplicated before the split of C. elegans and C. briggsae 80-110 million years ago. Our data provide the first systematic investigation into the redundancy of duplicated genes in any organism and strongly support population genetics models, which suggest that redundancy can be maintained over substantial periods of evolutionary time.. Furthermore, we set out to test whether systematically compiled yeast genetic interaction data can predict genetic interactions in the worm. We will present these data.

Brown, Andre et al. (2017) International Worm Meeting "A common format for worm tracking data."

Kerr, Rex, Brown, Andre, Lee, Chee Wai, Hokanson, Jim, Currie, Michael

[International Worm Meeting, 2017]

Many labs, including ours, have built a wide variety of worm trackers. These have a wide range of capabilities, from high-resolution imaging of single animals during calcium imaging, to very low-resolution imaging of animals as points. This diversity of capability enables the C. elegans community to address a wide range of problems at an appropriate scale. Most of these trackers also produce some data that is very similar to that of other trackers: animal position or spine, for example. Unfortunately, each tracker uses its own format to store data, so that any later analysis, despite being general in nature, cannot be performed on data from different machines. As the volume of tracking data grows, and the variety of downstream analysis methods expands, this limitation will pose an increasingly large barrier to replication of and extension of existing work across different labs. To address this issue, we have defined the Worm Common Object Notation, a set of rules for how to write tracking data in the ubiquitous JSON format, so that it can be easily shared between labs. To facilitate easy adoption of WCON, we have further written software in a variety of languages that will read or write data in WCON format. So far, we have implementations in Python, Scala, Matlab, and Julia, and wrapper libraries for Octave, R, and Java to use one of the main implementations. Additionally, the Tracker Commons project of which WCON is a part contains a small but rapidly growing set of pre-packaged analysis tools for routine manipulation of worm tracking data. We will also maintain a list of other WCON-compatible analysis tools as they become available. If you are involved in worm tracking, we invite you to adopt WCON and help make C. elegans behavioral data widely accessible. WCON is developed under the open source Tracker Commons project of the OpenWorm Foundation. We invite contributions and improvements!

Julia Grabitzki et al. (2006) European Worm Meeting "Identification of O-linked N-acetylglucosamine Modified Proteins in Caenorhabditis elegans"

Gunter Lochnit, Rudolf Geyer, Michael Ahrend, Brigitte Schmitz, Julia Grabitzki

[European Worm Meeting, 2006]

Julia Grabitzki1, Michael Ahrend2, Brigitte Schmitz2, Rudolf Geyer1 and Gnter Lochnit1. The posttranslational modification N-acetylglucosamine O-glycosidically linked (O-GlcNAc) to serine and threonine residues of proteins has been shown to be ubiquitous amongst eukaryotic proteins of the nucleus, cytoskeleton, cytoplasm, and has also been detected on cytosolic tails of membrane proteins [1]. O-GlcNAcylated proteins can form reversible multimeric complexes with other polypeptides or structures. The modification is often accompanied by phosphorylation/ dephosphorylation. O-GlcNAc can act either simultaneously or in a reciprocal fashion with phosphorylation. According to the Yin-Yang hypothesis, the phosphorylation/ dephosphorylation regulates O-GlcNAc-modified protein function (z.B. signal transduction and protein-protein interaction) in concert with phosphorylation [2-4]. The addition of O-GlcNAc to and the removal from the protein backbone is dynamic with rapid cycling in response to cellular signals or cellular stages.. Despite the fact, that Caenorhabiditis elegans is the best studied model organism, there have been no studies on O-GlcNAcylation in this organism so far. Therefore, to elucidate the role of O-GlcNAcylation, we investigated the proteome of a C. elegans mixed-stage population by two-dimensional gelelectrophoresis and subsequent western-blotting with the O-GlcNAc-specific antibody CTD 110.6 for the occurrence of this modification and identified the modified proteins by mass-spectrometry. We detected and identify several O-GlcNAc-modified proteins in C. elegans. Most of the identified proteins are involved in metabolic pathways. The prediction of the cellular localisation of the identified proteins revealed a predominant cytosolic occurrence of the O-GlcNAc modification.. References:. [1]. Rex-Mathes, M., J. Koch, Werner, S., Griffith, L. S and B. Schmitz. 2002. Methods Mol Biol 194: 73-87.. [2] Zachara, N.E. and G.W. Hart, Chem Rev, 2002. 102(2): p.431-8.. [3]. Griffith, L. S. and B. Schmitz. 1999. Eur J Biochem 262(3): 824-31.. [4] Wells, L. and G. W. Hart. 2003. FEBS Lett 546(1): 154-8.

Julia Grabitzki et al. (2006) European Worm Meeting "Phosphorylcholine-Modified Proteins of Caenorhabditis elegans are Specific to Developmental Stages"

Michael Ahrend, Gunter Lochnit, Julia Grabitzki, Rudolf Geyer

[European Worm Meeting, 2006]

Julia Grabitzki, Michael Ahrend, Rudolf Geyer and Gunter Lochnit. The free-living nematode Caenorhabditis elegans has been found to be an excellent model system for developmental studies [1] investigating parasitic nematodes [2] and drug screening [3]. Structural analyses of glycoconjugates derived from this organism revealed the presence of nematode specific glycosphingolipids of the arthro-series, carrying, in part, phosphorylcholine (PC) substituents [2]. PC, a small haptenic molecule, is found in a wide variety of prokaryotic organisms, i. e. bacteria, and in eukaryotic parasites such as nematodes. There is evidence that PC-substituted proteins glycolipids are assumed to be responsible for a variety of immunological effects including invasion mechanisms and long-term persistence of parasites within the host [4]. In contrast to PC-modified glycosphingolipids [5], only a limited number of PC-carrying (glyco)proteins were identified so far [6-9]. We have analysed the expression of PC-modified proteins of C. elegans during developmental stages using two dimensional SDS-Page separation, 2D-Western-blot and MALDI-TOF mass spectrometry. The pattern of PC-modified proteins was found to be stage specific. The PC-modification on proteins was most abundant in the egg and dauer larvae-stages followed by the adult-stage and L4. Only small amounts of the PC-substitution were found in L3 and L2. In L1 we couldnt detect any PC-Modification. The prediction of the cellular localisation of the identified proteins revealed a predominant cytosolic and mitochondrial occurrence of the PC- modification. Most of the identified proteins are involved in metabolism or in protein synthesis.. 1.. Brenner, S., Genetics, 1974. 77(1): p. 71-94.. 2.. Lochnit, G., R.D. Dennis, and R. Geyer, Biol Chem, 2000. 381(9-10): p. 839-47.. 3.. Lochnit, G., R. Bongaarts, and R. Geyer, Int J Parasitol, 2005. 35(8): p. 911-23.. 4.. Harnett, W. and M.M. Harnett, Mod. Asp. Immunobiol., 2000. 1(2): p. 40-42.. 5.. Friedl, C.H., G. Lochnit, R. Geyer, M. Karas, and U. Bahr, Anal Biochem, 2000. 284(2): p. 279-87.. 6.. Haslam, S.M., H.R. Morris, and A. Dell, Trends Parasitol, 2001. 17(5): p. 231-5.. 7.. Cipollo, J.F., C.E. Costello, and C.B. Hirschberg, J Biol Chem, 2002. 277(51): p. 49143-57.. 8.. Cipollo, J.F., A.M. Awad, C.E. Costello, and C.B. Hirschberg, J Biol Chem, 2005. 280(28): p. 26063-72.

Julia Samann et al. (2006) European Worm Meeting "Using C. elegans to Model the Systems Biology of Stress Response and Age-Related Disorders"

Julia Samann, Enrico Schmidt, Ralf Baumeister, Mark Seifert

[European Worm Meeting, 2006]

Julia Smann, Enrico Schmidt, Mark Seifert and Ralf Baumeister. Our lab is interested in understanding the connection of stress response and disease. According to a prevailing theory, aging is characterized by the declining activity of genome maintenance and repair mechanisms that deal with the detrimental consequences of external and internal stress. This eventually results in the accumulation of DNA mutations and increases the risk of stroke, cancer, coronary heart diseases, and degenerative disorders (the four major causes of death in our society). One focus of our research is the generation of C. elegans model to study the biological role of the affected genes. We focus here on genes whose mutations segregate with hereditary cases of Parkinsons Disease to result in an early age-of-onset and rapid progression. C. elegans homologues of four respective genes were identified and mutants are available. These are the ubiquitin ligase parkin/pdr-1, the mitochondrial kinase PINK1/pink-1, two genes with strong similarities to human DJ-1 (pdr-2 and pdr-3: Parkinsons Disease-related gene 2 and 3), and the cGMP binding protein Dardarin/LRRK2/lrk-1. All of them have been linked to intracellular mechanisms of (oxidative and unfolded protein) stress response, suggesting that they can be functionally linked to one another. We have initiated genome-wide screens to characterize the Parkinome, the interaction network of factors related to Parkinsons Disease. One purpose of this endeavour is the identifation of common targets and regulators of PD-related gene products, as well as the modeling of their roles in the onset of disease. Using the split-ubiquitin yeast two-hybrid interaction screen, we identified over 100 proteins as potential interactors for PD-related proteins. Using a medium throughput biochemical assay we are currently working on the confirmation of these interactions, and apply genetic and pharmacological assays to understand the physiological processes that are perturbed in mutants. In previous experiments, we generated screening models for parkin and human ?-synuclein mutants in C. elegans. The expression of ?-synuclein A53T in a pdr-1 mutant background that behaves similar as the Parkin mutants found in PD resulted in temperature-sensitive toxicity that enabled us to screen for suppressors/enhancers of this phenotype. Until now, we identified more than 50 modulators of this toxicity. Results of the various screens support the model of a functional connection between PD-related factors.

Wissmann AK et al. (1996) West Coast Worm Meeting "LET-502 AND HYPODERMAL CELL SHAPE IN C. ELEGANS"

Wissmann AK, Mains PE, McGhee JD

[West Coast Worm Meeting, 1996]

Cell shape in animals is determined by a complex network of interactions between extracellular components such as the extracellular matrix and the internal cytoskeleton. Linkage occurs via integral membrane proteins. Both external cues, and the cellular differentiation state, determine the specific response of a given cell in regulating its shape. The hypodermal cells in C.elegans undergo a dramatic change in cell shape during morphogenesis of the embryo. Priess & Hirsh proposed that this shape change results from hypodermal actin fiber-dependent contraction that increases internal pressure within the embryo, squeezing an elliptical ball of cells (the embryo) into a long tube (the worm)[1]. We identified a zygotic lethal mutation (ca201) defective in elongation. This mutation leads to arrest during early morphogenesis but does not affect later stage differentiation markers such as pharyngeal and gut development and cuticle formation. ca201 was shown to be an allele of the previously identified gene let-502 isolated in a saturation screen for lethals within LGI left by Howell & Rose[2] (thank you, Ann, for kindly supplying the other five let-502 alleles to us). The let-502 cDNA was cloned and the let-502 mutations were identified (see abstract by Julia Ingles et al.). Sequence analysis reveals that LET-502 is a member of a new family of serine/threonine kinases that include p160ROCK, ROK , Rho-kinase and human myotonic dystrophy kinase. p160ROCK, ROK and Rho-kinase have been shown to bind the Ras-related GTP-binding protein Rho and a sequence comparison suggests that this is also the case for LET-502. Furthermore, a PH-domain and a Cysteine rich region similar to that found in Protein Kinase C have been identified. A let-502/lacZ fusion expresses in hypodermal cells and especially in the lateral seam cells, where the most dramatic cell shape changes take place during morphogenesis. A limited screen for suppressors of ca201 embryonic lethality identified two alleles within one gene (tentatively named kis-1 for kinase suppressor). Both alleles suppress ca201 to sterile adults. When separated from ca201, one allele (sb55) is fertile, whereas the other (sb56) gives sterile adults. sb56 has now been mapped and rescued and encodes part of a phosphatase complex. A model showing how this set of genes might be related to the observed cell shape changes will be presented. 1 Priess, J.R. & Hirsh, D.L. Developmental Biology 117, 156-173 (1986) 2 Howell, A.M. & Rose, A.M. Genetics 126, 583-592 (1990)