Frondoni, Julia et al. (2021) International Worm Meeting "Identifying the key players of phosphatidylserine externalization in non-apoptotic dying cells"

Frondoni, Julia, Wehman, Ann, Jorg, Lisa, Stetter, Maurice

[International Worm Meeting, 2021]

Throughout development, cells are programmed to die for homeostatic control. Corpse clearance is key for animals to avoid inflammation and auto-immune disease. To better understand the "eat me" signal of phosphatidylserine (PS) exposure for cell clearance, we are attempting to disrupt the externalization of PS on C. elegans polar bodies. The second polar body undergoes a non-apoptotic form of cell death, externalizes PS, and is cleared by embryonic cells during early development. PS exposure is known to be caused by lipid scramblases, enzymes present in the plasma membrane that translocate phospholipids between the leaflets of the lipid bilayer. However, the scramblases that regulate PS exposure in non-apoptotic dying cells are unknown. A combination of RNAi and the ced-8(n1891) loss-of-function allele suggested that the scramblase CED-8 plays a redundant role in externalizing PS on polar bodies with the mitochondrial factor WAH-1 as well as the scramblases SCRM-1, SCRM-2, and SCRM-3. As CED-8, WAH-1, and SCRM-1 are involved in PS externalization during apoptosis, we tested whether PS exposure depends upon the apoptotic caspase CED-3. However, PS was still externalized on polar bodies in ced-3(n717) mutants, confirming that scramblase activation is independent of the apoptotic pathway. To confirm the role of the redundant scramblases, we generated a scrm-4 scrm-1 scrm-2; scrm-3; ced-8 quintuple scramblase mutant strain. These mutants still externalized PS on polar bodies, indicating that there are other ways to externalize PS on these cells. We are currently testing other scramblase proteins, including SCRM-6, SCRM-7, SCRM-8, and CED-7. Thus, we will define the molecular players in the regulation of PS exposure for non-apoptotic cell death, which will allow us to define the role of PS in phagocytic uptake as well as later steps of phagosome maturation.

Pitts, Lauren R et al. (2023) MicroPubl Biol

Wehman, Ann M, Pitts, Lauren R, Nguyen, Alexander T, Frondoni, Julia

[MicroPubl Biol, 2023]

Cells release extracellular vesicles (EVs) from their surface, but the mechanisms that govern EV release by plasma membrane budding are poorly understood. The lipid flippase TAT-5 inhibits EV release from the plasma membrane in C. elegans , but how the level of flippase activity regulates EV release was unknown. We generated point mutations in the DGET motif of TAT-5 predicted to lead to a partial or complete loss of ATPase activity. We discovered that tat-5(E246Q) mutants were sterile, while tat-5(D244T) mutants produced embryos that arrested during development. Using degron-based reporters, we found that EV release was increased in tat-5(D244T) mutant embryos and that phagocytosis was also disrupted. These data suggest that a low level of flippase activity can promote fertility, while a higher level of flippase activity is required to inhibit EV release, allow phagocytosis, and carry out embryonic development.

(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.