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

中间纤维（IF）是后生动物三大纤维细胞骨架系统之一。许多IF多肽在细胞类型特异性组合中合成，表明专门的功能。C. elegans进行了很大的承诺，阐明IF组装成复杂的网络，并确定IF功能在一个活的有机体仍然悬而未决的机制。与果蝇相反，果蝇似乎完全缺乏细胞质IFs，C。线虫基因组包含11个编码胞质IFs的基因和仅一个编码核纤层蛋白的基因。肠内IFs在具有机械弹性的内管中丰富，这是C. elegans终端web区域。这种富含IF的结构将所有三种细胞骨架丝聚集在一起，并通过C。在上一个资助期，我们已经鉴定并开始表征新型肠丝组织者IFO-1（=TTM-4），其确保IFB-2和IFC-2在终末网内的正确定位。IFO-1编码一种新的富含组氨酸的、含有聚脯氨酸片段的线虫蛋白。此外，IFO-1与ERM-1（Ezrin-Radixin-Moesin）和DLG-1（椎间盘较大）一起促进上皮完整性。在接下来的资助期内，我们打算分析IFO-1如何调节IF蛋白的同源和/或杂聚体的形成，并研究它们在C. elegans intestinal（Leube）.为了更好地理解IFO-1的分子作用模式，我们进行了不同的诱变和RNAi筛选，并成功地鉴定了IFO-1的调节子、抑制子和增强子，现在将对其进行详细分析（Bossinger）。到目前为止，IFO-1和ACT-5（F-actin）的表达是相互依赖的，并控制着肠道IF蛋白的顶端定位。此外，IFO-1、IFB-2、IFC-2、ERM-1、ACT-5和DLG-1是最终调节C.线虫肠到目前为止，E-cadherin（HMR-1）和L1 CAM SAX-7的功能是至关重要的，但claudins和nectin的作用仍有待研究。为了更好地了解在体器官中该网络的生物力学特性，我们首次进行了C。elegans intestinal（默克尔）。这些测定应允许测量和量化不同的细丝系统（IF/F-肌动蛋白）和细胞-细胞粘附复合物（钙粘蛋白-连环蛋白、SAX-7-DLG-1、连接蛋白-阿法丁和密蛋白-ZOO-1）如何促进上皮完整性。我们的研究结果将是普遍的兴趣，关于更好地了解亚顶端细胞骨架和细胞粘附复合物在上皮分化过程中的发展和他们的多种功能障碍，在某些疾病。

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.