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

中间丝是后生动物三大纤维细胞骨架系统之一。许多IF多肽是以细胞类型特定的组合合成的，暗示着特殊的功能。线虫很有希望阐明IF组装成复杂网络的机制，并确定IF在活体中的功能。与果蝇不同的是，果蝇似乎完全缺乏细胞质IF，线虫基因组包含11个编码细胞质IF的基因，只有一个基因编码核层粘连蛋白。肠道IF在机械弹性管内丰富，这是线虫末端网状区的一个显著特征。这种富含IF的结构将所有三种细胞骨架细丝聚集在一起，这些细胞骨架细丝通过线虫的顶端连接(CeAJ)整合成一个连贯的实体，从而用其特有的刷子边界完全包围和稳定肠腔。在上一个授权期，我们已经确定并开始鉴定新的肠丝组织者IFO-1(=TTM-4)，它确保IFB-2和IFC-2在末端网中的正确定位。Ifo-1编码一种新的富含组氨酸、多聚脯氨酸束的线虫蛋白。此外，IFO-1与ERM-1(Ezrin-Radisin-Moesin)和DLG-1(Discs Large)共同促进上皮完整性。在接下来的授权期，我们打算分析IFO-1如何调节IF蛋白的同聚和/或异聚的形成，并调查它们对线虫肠道(Leube)顶膜结构域运输的影响。为了更好地了解IFO-1s的分子作用模式，我们进行了不同的突变和RNAi筛选，并成功地确定了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-肌动蛋白)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是复杂遗传网络的一部分，最终调节线虫肠道的细胞-细胞黏附。到目前为止，E-钙粘蛋白(HMR-1)和L1CAM SAX-7的功能是至关重要的，但Claudins和Nectins的作用仍有待研究。为了更好地了解活体器官内这个网络的生物力学特性，我们设计了第一个线虫肠道(Merkel)的机械应力分析。这些检测应该能够测量和量化不同的细丝系统(iS/F-肌动蛋白)和细胞-细胞黏附复合体(钙粘连蛋白-连环蛋白、SAX-7-DLG-1、粘附素-afadin和claudin-zoo-1)对上皮完整性的贡献。我们的结果将对更好地理解发育过程中上皮分化过程中的细胞骨架和细胞黏附复合体以及它们在某些疾病中的多种功能故障具有普遍意义。

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.