Funktionelle Bedeutung von Intermediärfilamenten im Darm von Caenorhabditis elegans
- Functional relevance of intermediate filaments in the C. elegans intestine
Coch, Richard Alexander; Panstruga, Ralph (Thesis advisor); Leube, Rudolf (Thesis advisor); Zimmer-Bensch, Geraldine Marion (Thesis advisor)
Dissertation / PhD Thesis
Dissertation, RWTH Aachen University, 2020
The intestinal intermediate filament network in Caenorhabditis elegans shows remarkable parallels tothat in vertebrates. Both its anchorage at cell-cell contacts and its localization in the adluminalcytoplasm beneath the brush border with its actin-rich microvilli are highly conserved. Even though impairments of this system rarely elicit dramatic effects under laboratory conditions, hostileenvironmental influences may be of great disadvantage for the affected population. The comparatively simple intestine of C. elegans represents an excellent model to investigate the importance of intermediate filaments for enterocyte function and organismal survival in amenable and hostileenvironments. The C. elegans intestine contains six cytoplasmic intermediate filament polypeptides,which are believed to be integral constituents of the endotube, an electron-dense sheath surrounding the intestinal lumen circumferentially. Recently, the intestinal filament organizer IFO-1 has been identified as an upstream regulator, whose loss results in junctional accumulation of the intermediatefilament network. The goal of the current project was to investigate the distribution and function ofindividual intermediate filament polypeptides and their regulation by IFO-1.Using improved confocal laser scanning microscopy, this work showed that the intermediate filamentpolypeptides IFB-2 and IFC-2 precisely co-localize in the endotube. In contrast to IFB-2, IFC-2, however,is highly enriched along the C. elegans apical junction suggesting an isotype-specific role for junctional anchorage of the endotube. By sequentially extracting cytoskeletal components from dissected intestines, the well-known actin-binding protein ERM-1 was identified as a potential linker molecule between the actin-containing microvillar rootlets and the adjacent intermediate filament network. Knockout of either the ifb-2 or the ifc-2 gene uncovered common and isotype-specific loss of functions. In both situations, a reduced uptake of peptides and cholesterol from the intestinal lumen was observed. But only the loss of IFB-2 perturbed intracellular cholesterol transport. This transport defectwas even more prominent in the ifo-1 knockout background indicating profound impairment of lipid metabolism and transport. This notion was further supported by transcriptome analyses. They revealed a remarkable and unexpected parallel to transcriptional changes in a loss-of-function mutantof nhr-8, which is a transcriptional regulator of lipid metabolism. In agreement with the hypothesis that the intestinal intermediate filament-rich layer serves as aprotective barrier, significant endocytic activity was not observed in the C. elegans intestine. Only imminent cell damage by pore-forming toxins leads to the internalization of affected membranes. The impairment of this protective mechanism in the ifo-1 knockout background could be explained by an altered lipid composition of the apical membrane of enterocytes. Taken together, this work highlights the profound alterations of peptide transport, lipid metabolismand barrier formation in the ifo-1 knockout and most notably, identifies a novel and hitherto unknown function of the intermediate filament proteins IFB-2 and IFC-2 in these cellular processes.
- DOI: 10.18154/RWTH-2020-11385
- RWTH PUBLICATIONS: RWTH-2020-11385