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[
International Worm Meeting,
2015]
Proteins destined for the secretory pathway and cell surface are folded in the endoplasmic reticulum (ER). Classes of biologically important proteins that transit the ER, known as ER client proteins, include receptors, antibodies, and extra-cellular matrix components. ER client proteins enter in an unfolded state and leave only when correctly folded and assembled. This process is dependent on a set of ER-resident proteins that catalyse specific folding steps and prevent aggregation. A particular feature of proteins folded in the ER is the presence of disulphide bonds which are formed by covalent linkage of cysteine residues. The stability and function of many secreted and cell surface proteins depends on native disulphide formation which in turn relies on fine control of the balance between oxidation (favouring disulphide formation) and reduction. The protein folding capacity of the ER is therefore highly sensitive to the redox environment of this organelle.To identify novel factors regulating redox balance and protein folding in the ER we have expressed an ER-localised redox-sensitive version of GFP in C. elegans that enables changes to ER redox to be analysed in vivo. We have identified a version of this sensor that remains monomeric, confirmed its localisation to the ER, and shown that the presence of the sensor does not induce ER stress. The sensor responds to changes in ER redox induced by compounds and by RNAi, as determined by Western blotting and with live worm populations using a plate reader. We aim to use this sensor in an RNAi or genetic screen to identify novel genes required for ER redox homeostasis. In addition, we have carried out genetic screens for mutants that are resistant to highly reducing conditions using dithiothreitol (DTT) selection. This approach has identified 13 strains that are capable of withstanding extreme reducing conditions and these are currently being characterising by whole-genome sequencing. Lastly, we are examining the C. elegans quiescin sulfhydryl oxidase family to reveal the role and substrates for these poorly understood secretory pathway enzymes. Findings from our genetic analysis of C. elegans will be extended by testing in mammalian cell systems.
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[
International Worm Meeting,
2015]
The nematode cuticle is a collagenous extracellular matrix that is synthesised repeatedly during development via the moulting process. The enzymology of cuticle collagen biosynthesis and more importantly the enzymology of cuticle ecdysis and moulting represent potential novel targets for parasitic nematode control. Using C. elegans we have identified and characterized key moulting enzymes and have focused on the nematode astacin (NAS) metalloproteases. As an example, mutation of the enzyme-encoding genes NAS-36 and NAS-37 reveals an essential developmental role for these enzymes in C. elegans moulting. We have identified the orthologs in the diverse parasitic nematodes Brugia malayi and Haemonchus contortus that are able to rescue the moulting phenotypes associated with
nas-36/ -37.Using in silico-modelling we have screened available chemical libraries and have tested several hundred potential inhibitors using in vivo and in vitro assays in C. elegans, Brugia malayi and Haemonchus contortus.The screening process will be described and the main finding of this study will be presented.
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van den Ecker, Daniela, Nijtmans, Leo G J, Distelmaier, Felix, Bossinger, Olaf, Mayatepek, Ertan, van den Brand, Mariel A M
[
C. elegans: Development and Gene Expression, EMBL, Heidelberg, Germany,
2010]
Isolation of mitochondrial proteins and subsequent analysis with blue native / SDS gel electrophoresis (BN-PAGE) is an essential tool to investigate defects of the mitochondrial respiratory chain. During the last years, Caenor habditis elegans ( C. elegans ) has become an important model system to study human disease associated with mitochondrial dysfunction. However, with current BN-PAGE protocols for C. elegans , high quantities of mitochondrial protein are required to yield clear results. To obtain these protein amounts, liquid culture was used so far. However, growth in axenic medium alters metabolism and might have adverse effects on oxidative phosphorylation, which is potentially disadvantageous in view of studies about mitochondrial function . On the other hand, mitochondrial dysfunction in C. elegans is often associated with slow growth and larval arrest. Therefore, it might be difficult to culture sufficient worm quantities on agar plates. Here, we present an optimized approach to isolate mitochondria and respiratory chain complex I from C. elegans grown on solid NGM culture plates. We demonstrate that considerably lower amounts of mitochondrial protein are sufficient to isolate complex I and to display clear in-gel activity results. Moreover, we present the first complex I assembly profiles for C. elegans , obtained by two dimensional BN-PAGE.
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[
International Worm Meeting,
2017]
C. elegans develops through four consecutive larval stages (L1-L4) separated by molts. During each molt, worms synthesize and secrete a new exoskeleton called cuticle underneath the existing one, followed by their separation (apolysis) and the shed of the old exoskeleton (ecdysis). Lethargus, an inactive sleep-like state characterized by a gradual decrease in general activity and feeding, occurs at the end of each larval stage coinciding with separation of the old exoskeleton from the hypodermis.1, 2 We found that lysosomes, which are labeled by either the lysosomal enzyme NUC-1 or the lysosomal membrane protein LAAT-1, appear as small puncta or thin tubules during larval development. Interestingly, worms at the lethargus stage contain extensive tubular lysosomes in the hypodermis, whereas globular lysosomes are predominant when animals exit lethargus to enter the ecdysis stage. Moreover, EGF overexpression, which induces behavioral quiescence, leads to abundant tubular lysosome formation, whereas physical disturbance of the locomotive quiescence partially disrupts tubular lysosomes formed during lethargus. This indicates a close correlation between tubular lysosome formation and lethargus quiescence. We found that morphology of other intracellular organelles including ER, Golgi, mitochondria and endosomes remain unaltered during lethargus, suggesting that lysosomes are specifically altered at this stage. In addition to morphological changes, we found that lysosome biogenesis and dynamics appear to be up-regulated during lethargus. We are currently investigating how lysosome dynamics and functions are regulated at this specific developmental stage and how they may contribute to the molting process. 1. The cuticle. Antony P. Page and Iain L. Johnstone, 2007, Wormbook. 2. Lethargus is a Caenorhabditis elegans sleep-like state. David M. Raizen et al, 2008, Nature.
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[
International Worm Meeting,
2005]
Over the past few years, development of the WormBase user interface has focused on building a consistent interface comprised of informationally rich displays and user friendly tools for data visualization. This development has been driven by a flood of data culled from literature and large scale analyses by the relentless team of curators at WormBase. User interface development has now shifted to a fundamental rearchitecture of the software that drives WormBase. What should users expect from this redesign? At first glance, nothing new. The rearchitecture is designed to replicate the current WormBase look-and-feel. Under the surface, however, will be a highly configurable and responsive user interface. A My WormBase section of the website will allow users to build a page that contains a browsing history and often-accessed information. Perhaps you would rather have WormBase look more like Ensembl, NCBI or Flybase? Maybe you would like to hide the display of references on the gene page? Such options will be configurable across the site enabling users to build displays that best suit their needs. Pages will also be more flexible. They will respond to range-based queries and display results in a contextually-sensitive manner consistent with the type of query and number of items retrieved. The new site will be directly accessible to data mining. Each human readable section label on individual pages can become a target. Such targets can be accessed by a simple programming interface, enabling users to write efffective data mining scripts quickly and easily. For example, a script could be written to fetch all of the brief identifications listed on the Gene Summary page. Many elements of the rearchitecture will be invisible to end users. Major under-the-hood features include a consistent programming interface for WormBase developers; the ability to generically support additional genomes and species; the ability to be driven from multiple data sources; and maintenance of a persistent browsing state for users. As always, WormBase welcomes your feedback. Please send questions, comments, or suggestions to help@wormbase.org.
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[
International C. elegans Meeting,
1991]
RNA trans-splicing in nematodes involves the transfer of an evolutionarily conserved 22-nucleotide spliced-leader (SL) and a trimethylguanosine (TMG) cap to the 5'-end of the recipient mRNA. The physiological role of trans-splicing, of the SL and the biological effect of TMG cap at the 5' ends of mRNAs are not yet understood. The possibility has been raised that SL itself has a catalytic role in splicing. The TMG cap has recently been shown to be an essential nuclear targeting signal. We hope to learn the functions of SL or transsplicing by identifying proteins specifically bound to SL and elucidating their functions. Electrophoretic mobility shift assays combined with competition analysis showed two proteins SLBP1 and SLPB2, bind specifically to SL1. The binding can be stimulated by a cap structure at the 5' end of SL. Although SLBP2 can bind SL1 RNA, SLBP1 seems unable to bind SL1 RNA. This may be due to the highly structured SL1 RNA interfering SLBP1 binding. We are presently testing the binding of SLBP1 and SLBP2 to SL2. UV cross-linking and SDS-PAGE revealed that SLBP1 has a molecular mass of 57kD and SLBP2 of 30kD. Nitrocellulose filter retention assay showed that the binding between SLBP1 and SL1, SLBP2 and SL1 conforms to a simple biomolecular reaction. SLBP1 has been purified to homogeneity as judged by silver staining on SDS-PAGE. Gel filtration analysis indicates that SLBP1 exists as a monomer and a dimer in solution. However we do not know whether dimerization is required for RNA binding. We intend to isolate their genes and generate antibodies against SLBP1 and SLBP2 to further characterize their biochemical properties and biological functions.
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[
International Worm Meeting,
2013]
We continue to see growth in volume and diversity of nematode genomic variation data, in large part due to increasing research effort in whole-genome sequencing (WGS) of numerous C.elegans mutant and wild-isolate strains. WormBase have responded to the challenges presented by this growth by making changes to the way in which we curate, store and display variation data. One significant change has been to more-clearly distinguish between naturally-occurring polymorphisms and laboratory-induced mutations at the display level. These are now show in two separate tables on Gene Summary pages, with laboratory-induced alleles identified by the allele designation of the laboratory of origin, and naturally-occurring polymorphisms identified by WormBase variation accessions. We have also begun to consolidate redundant data from independent wild-isolate sequencing projects. Previously, if a specific molecular variation had been identified by multiple independent projects, and/or in multiple strains, a separate variation object would have been created for each. Now, a single reference variation is created which cross-references all studies that have characterised that variation and all strains that carry it. A new version of the WormBase website was launched in early 2012, and we continue to refine and improve the display of variation data. Coloured fields in the Strain widget on the Variation Summary Page clearly show which strains carry a variation and whether the strain is available from the CGC. The Gene Summary Page now allows customisation in the the way Variations are viewed; both variation tables can be sorted by various properties, including type of molecular change, effect on the protein, and the number of associated phenotypes. We have also increased the complement of variation tracks on the genome browser, clearly separating classical alleles from those generated by large-scale sequencing projects, and creating additional tracks for single-nucleotide variants that confer a putative change-of-function on a protein. We we continue to refine the presentation of this data, and welcome feedback from the C.elegans research community.
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Crocker, C., Fisher, K., Xu, M., Stephney, T., Wolkow, C.A., Altun, Z.F., Hall, D.H., Herndon, L.A.
[
International Worm Meeting,
2011]
We announce the release of WormImage 2.0. The WormImage database houses over 55,000 unpublished electron micrographs and their related metadata. This new version of WormImage still includes access to this enormous collection of C. elegans micrographs, but now features a new look and a new search interface making the data more easily accessible to users. To streamline the website, users can now search directly from the front page. As before, search criteria allow users to narrow results by sex, genotype, age, body portion and tissue type of the animal, but now this is found all on one page. Additionally, with new expandable menu options, one can now select a single tissue or multiple tissue types with just a few clicks. Search results are presented in a new format that is simpler, making it easier to screen through and navigate among all the images. The dataset offered by WormImage continues to expand and welcomes all laboratories to share their best archival TEM and SEM images so that this resource can continue to grow and serve the C. elegans community.
The WormAtlas website is also growing and changing. To adapt to the increased demand for mobile-ready content, WormAtlas is now available in a format optimized for access from mobile devices. This allows for users to quickly navigate through the handbook chapters without worrying about zooming in on small icons and menu bars. We are also pleased to announce that WormAtlas will soon feature a new handbook section on the Dauer Larva. Sections for this new handbook will be posted as they become available. In addition to adding new content, we are also continually updating material on WormAtlas to keep the information available to users current. As such, the Individual Neuron pages are being revised and feature new images and data. Slidable Worm is expanding as well, with the ongoing addition of new slices.
This work is supported by NIHRR 12596 to DHH.
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[
International Worm Meeting,
2009]
We announce the release of WormAtlas2.0. This new version of WormAtlas includes all previous information, but features a new look and many new additions. The main page adopts a tighter format by using drop down menus and more image based icons for intuitive navigation throughout the site. The Handbook is reorganized and provides a table of contents in a static frame for each section to allow for easy maneuvering to subsections and figures. The Hermaphrodite Handbook has updated content including an entirely new section covering the nervous system. Each section features new and updated figures and each figure now links to a separate page with the figure and legend and also to a high resolution version showing even greater detail. The Male Handbook also has been converted to the new layout and we are planning to introduce new content in the near future. Similarly, the Individual Neuron pages are being updated to a new format and will contain new figures, including a major expansion of the Male-specific neurons and their wiring patterns (see Emmons et al., this meeting). Slidable Worm is also gaining new pages and we have initiatives underway to streamline and accelerate this process. It is our hope that WormAtlas2.0 will be simpler to navigate and that beginners will find it more accessible. We encourage feedback from members of the C. elegans community on ways to improve the new version of WormAtlas. The WormImage website houses thousands of unpublished electron micrographs and related data, and has been expanding steadily. It now presents more data from mutant animals, particularly for genes affecting the nervous system. We continue to rely heavily on MRC datasets, but we are also adding more from the Riddle and Hall lab files, among others. We encourage more laboratories to share their best archival TEM and SEM images so that this resource can continue to grow and serve the C. elegans community. We are very grateful to many people who have already contributed ideas, advice and experimental results that are featured on these websites. This work is supported by NIHRR 12596 to DHH.
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[
International Worm Meeting,
2007]
Caenorhabditis elegans is susceptible to infection by bacteria associated with insect parasitic nematodes such as Photorhabdus luminescens and Xenorhabdus nematophila (Couillault and Ewbank, 2002). We exposed several species of Pristionchus (Pristionchus pacificus California and Washington strains, Pristionchus entomophagus and Pristionchus maupasi) to P. luminescens, X. nematophila and Moraxella osloensis. Mortality was assessed over a 20-day period and compared with C. elegans. We hypothesized that Pristionchus was not susceptible to bacterial infection as it lacks a grinder and bacteria will pass through the gut alive and intact. In a second experiment we investigated the possible role of glutathione produced by Pristionchus to detoxify and protect the nematode from bacterial pathogens. Glutathione transferase proteins were extracted from nematodes exposed to pathogenic and non-pathogenic bacteria and were analysed using SDS-PAGE, 2D Electrophoresis and identified using MALDI-TOF. Total glutathione produced by the nematodes was also examined and compared with C. elegans. Couillault, C. and Ewbank, J.J. 2002. Diverse bacteria are pathogens of Caenorhabditis elegans. Infection and Immunity, 4705-4707.