Geisler, Florian et al. (2021) International Worm Meeting "Perturbed intermediate filament regulation causes aggregate toxicity"

Leube, Rudolf, Remmelzwaal, Sanne, Richardson, Christine, Geisler, Florian, Boxem, Mike

[International Worm Meeting, 2021]

Intermediate filaments are major components of the metazoan cytoskeleton. A long-standing debate concerns the question whether intermediate filament network organization only reflects or also determines cell and tissue function and dysfunction. This is particularly relevant for aggregate-forming diseases involving intermediate filaments. Using C. elegans as a genetic model organism, we have recently described mutants of signaling and stress response pathways with perturbed intermediate filament network organization. In a mutagenesis screen, we now identify the intermediate filament polypeptide IFB-2 as a highly efficient suppressor of these phenotypes restoring not only intestinal morphology but also rescuing compromised development, growth, reproduction and stress resilience. Ultrastructural analyses show that downregulation of IFB-2 leads to depletion of the aggregated intermediate filaments. The findings provide compelling evidence for the toxic function of deranged intermediate filaments and reveal novel insights into the cross talk between signaling and structural functions of the intermediate filament cytoskeleton.

Carberry, Katrin et al. (2010) C. elegans: Development and Gene Expression, EMBL, Heidelberg, Germany "Identification and characterization of a novel cytoskeletal organizer in C. elegans"

Leube, Rudolf, Bonn, Barbara, Wiesenfahrt, Tobias, Bossinger, Olaf, Carberry, Katrin

[C. elegans: Development and Gene Expression, EMBL, Heidelberg, Germany, 2010]

While numerous studies have focused on the characterization of microtubules and the F-actin network, comparatively little is known about the regulatory mechanisms determining the organization and function of intermediate filaments. The nematode Caenorhabditis elegans provides an excellent model system to study intermediate filaments in vivo. We performed a classical F2 EMS mutagenesis screen to identify mutants with aberrant intestinal expression patterns of the intermediate filament reporter IFB-2::CFP. Two alleles, kc2 and kc3, of the ifo-1 (intermediate filament organizer) gene were isolated, mapped, cloned and further characterized at the cellular level. IFO-1 colocalizes with the interm ediate filaments IFB-2 and IFC-2 in the terminal web of intestinal cells. In ifo-1 mutants, IFB-2 and IFC-2 become mislocalized to cytoplasmic aggregates and accumulate at the C. elegans apical junction. Structural defects of the terminal web are readily apparent by electron microscopy while microvilli are still intact. Yet, we observe a considerable decrease of terminal web F-actin. This finding suggests a more general role of IFO-1 for cytoskeletal organization in the C. elegans intestine and, considering the substantial growth retardation of mutant worms, assigns an important contribution of the ordered cytoskeleton to proper nutrition.

Katrin Hasken et al. (2006) European Worm Meeting "Identification of Intermediate Filament Organizers in Caenorhabditis elegans"

Katrin Hasken, Rudolf E. Leube

[European Worm Meeting, 2006]

Katrin Hsken and Rudolf E. Leube. Besides actin filaments and microtubules, intermediate filaments (IFs) constitute the third fibrous component of the cytoskeleton. Functionally, IFs play a role in maintaining mechanical integrity of cells. It is believed that their phosphorylation state is essential for correct assembly and network maintenance. Alterations of IF assembly are pathological features of several diseases (e.g. epidermolysis bullosa simplex, amyotrophic lateral sclerosis).. Compared to the 65 genes coding for human IFs, the cytoplasmic IF cytoskeleton in C. elegans consists of only 16 differentially expressed polypeptides, several of which fulfil essential functions. C. elegans IFs are expressed mainly in epithelial organs. They occur as three dimensional networks in the uterus, as polarized networks in the terminal web of intestinal cells or as dense bundles in the marginal cells of the pharynx. To examine molecular mechanisms that are responsible for these different assembly types we designed an optical screen. To this end we established transgenic strains with distinct and easily discernible fluorescence patterns. They are being subjected to a genome wide RNAi screen to identify gene products altering these patterns. We will further assess the consequence of altered IF organization on cell functions in the nematode.

Geisler, Florian et al. (2010) C. elegans: Development and Gene Expression, EMBL, Heidelberg, Germany "The intermediate filament organizer IFO-1 is a multifunctional linker of the intestinal cytoskeleton"

Geisler, Florian, Bonn, Barbara, Leube, Rudolf, Bossinger, Olaf, Felkl, Marco, Keul, Dominik

[C. elegans: Development and Gene Expression, EMBL, Heidelberg, Germany, 2010]

The lumen of the C. elegans intestine is surrounded by the resilient endotube that is mainly composed of F-actin and six different intermediate filament proteins (e.g. IFC-2 and IFB-2). The conspicuous intestine-specific expression pattern of IFB-2::CFP translational fusion makes this organ ideally suited to identify intermediate filament organizers (IFO). After chemical mutagenesis, we succeeded in the identification of several alleles of ifo-1, which are associated with a highly impaired organization of intestinal intermediate filaments and also display defects in the organization of F-actin. To elucidate the mechanism of ifo-1 function in vivo, we performed a classical suppressor screen, used yeast two hybr id analyses, accomplished RNAi by feeding against several F-actin organizers and generated anti-IFO-1 antibodies. In this way, we isolated suppressor strains, identified potential binding partners of IFO-1, determined cross-talk between the actin and intermediate filament cytoskeleton and examined IFO-1 expression. Taken together, we conclude that IFO-1 is a versatile and multifunctional cytoskeletal linker in the C. elegans intestine.

Christian Abraham et al. (2006) European Worm Meeting "Evolutionary Conservation of Synaptic Tetraspan Vesicle Membrane Proteins but Lack of Defects in Mutant C. elegans Strains"

Robby Weimer, Gabriele Spatkowski, Harald Hutter, Erik Jorgensen, Reinhard Windoffer, Christian Abraham, Lin Bai, Rudolf E. Leube, Mark Palfreyman

[European Worm Meeting, 2006]

1Christian Abraham, 1Lin Bai, 2Harald Hutter, 3Mark Palfreyman, 1Gabriele Spatkowski, 4Robby Weimer, 1Reinhard Windoffer, 3Erik Jorgensen and 1Rudolf E. Leube. Tetraspan vesicle membrane proteins (TVPs) comprise a major portion of synaptic vesicle proteins, yet their contribution to the synaptic vesicle cycle is poorly understood. TVPs are grouped in three mammalian gene families, the physins, gyrins and secretory carrier associated membrane proteins (SCAMPs). Remarkably, each family is represented by only a single polypeptide in C. elegans and all nematode TVPs co-localize to the same vesicular compartment when expressed in mammalian cells. To examine their function, C. elegans null-mutants were isolated for each gene. Triple mutants were generated by combination of all three mutant alleles. Careful analyses of these worms revealed no morphological or behavioral phenotypic defects. Nervous system architecture and synaptic contacts appear normal in the triple mutants. Mutant worms moved normally and released normal levels of neurotransmitter as assayed pharmacologically and electrophysiologically. Worms also responded normally to chemical or mechanosensory stimuli. We therefore conclude that TVPs are not needed for the basic neuronal machinery. Current transrciptome analyses identify potential regulatory components whose expression is altered in the mutant strains.

Remmelzwaal, Sanne et al. (2021) International Worm Meeting "BBLN-1 is essential for intermediate filament organization and apical membrane morphology"

Jarosinska, Olga, Leube, Rudolf, van der Horst, Suzanne, Boxem, Mike, Altelaar, Maarten, Ramalho, Joao, Pasolli, Milena, Akhmanova, Anna, Stucchi, Riccardo, Remmelzwaal, Sanne, Richardson, Christine, Geisler, Florian, Kroll, Jason

[International Worm Meeting, 2021]

Epithelial tubes are essential components of metazoan organ systems that control the flow of fluids and the exchange of materials between body compartments and the outside environment. The size and shape of the central lumen confer important characteristics to tubular organs and need to be carefully controlled. Here, we identify the small coiled-coil protein BBLN-1 as a regulator of lumen morphology in the C. elegans intestine. Loss of BBLN-1 causes the formation of bubble-shaped invaginations of the apical membrane into the cytoplasm of intestinal cells, and abnormal aggregation of the subapical intermediate filament (IF) network. BBLN-1 interacts with IF proteins and localizes to the IF network in an IF-dependent manner. The appearance of invaginations is a result of the abnormal IF aggregation, indicating a direct role for the IF network in maintaining lumen homeostasis. Finally, we identify bublin (BBLN) as the mammalian ortholog of BBLN-1. When expressed in the C. elegans intestine, bublin recapitulates the localization pattern of BBLN-1 and can compensate for the loss of BBLN-1. In mouse intestinal organoids, bublin localizes subapically, together with the IF protein keratin 8. Our results therefore may have implications for understanding the role of IFs in regulating epithelial tube morphology in mammals.

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.