Genetic and functional analysis of intermediate filament function to cell-cell adhesion and biomechanics in C. elegans intestine

点击翻译按钮获取中文摘要

Intermediate filaments (IFs) make up one of the three major fibrous cytoskeletal systems in metazoans. Numerous IF polypeptides are synthesized in cell type-specific combinations suggesting specialized functions. C. elegans carries great promise to elucidate the still unresolved mechanisms of IF assembly into complex networks and to determine IF function in a living organism. In contrast to Drosophila, which seems to lack cytoplasmic IFs altogether, the C. elegans genome contains 11 genes coding for cytoplasmic IFs and only a single gene for a nuclear lamin. The intestinal IFs are abundant in the mechanically resilient endotube, a prominent feature of the C. elegans terminal web region. This IF-rich structure brings together all three cytoskeletal filaments that are integrated into a coherent entity by the C. elegans apical junction (CeAJ) thereby completely surrounding and stabilizing the intestinal lumen with its characteristic brush border.In the previous grant period we have identified and started to characterize the novel intestinal filament organizer IFO-1 (=TTM-4), which ensure the correct localization of IFB-2 and IFC-2 within the terminal web. ifo-1 encodes a novel histidine-rich, polyproline tract-containing nematode protein. In addition, IFO-1 contributes to epithelial integrity in concert with ERM-1 (Ezrin-Radixin-Moesin) and DLG-1 (Discs large). In the following grant period, we intend to anaylze how IFO-1 regulates the formation of homo- and/or heteropolymer of IF proteins and to investigate the impact on their transport to the apical membrane domain in the C. elegans intestine (Leube). To better understand IFO-1s molecular mode of action, we performed different mutagenesis and RNAi screens and succeeded to identify regulators, suppressors and enhancers of IFO-1, which now will be analyzed in detail (Bossinger). So far, the expression of IFO-1 and ACT-5 (F-actin) is mutually dependent on each other and controls the apical localization of intestinal IF proteins. Furthermore, IFO-1, IFB-2, IFC-2, ERM-1, ACT-5 and DLG-1 are part of a complex genetic network that finally modulates cell-cell adhesion of the C. elegans intestine. So far, the function of E-cadherin (HMR-1) and the L1CAM SAX-7 are crucial, but the role of claudins and nectins has still to be investigated. To better understand the biomechanical properties of this network within an organ in vivo, we have worked out the first mechanical stress assays of the C. elegans intestine (Merkel). These assays should allow to measure and to quantify how different filament systems (IFs / F-actin) and cell-cell adhesion complexes (cadherin-catenin, SAX-7-DLG-1, nectin-afadin and claudin-ZOO-1) contribute to epithelial integrity. Our results will be of general interest concerning a better understanding of the subapical cytoskeleton and cell adhesion complexes in epithelial differentiation processes during development and their manifold malfunctions in certain diseases.