Extracellular Matrix Modulation of Cell Phenotype

描述(由申请人提供)： 细胞外基质(ECM)在细胞决策中起着关键作用。它作为组织结构的框架，刺激细胞生长、迁移和一系列其他细胞过程。细胞外基质信号通过整合素受体传递到细胞内。这些受体将细胞外基质连接到肌动蛋白细胞骨架，并激活许多不同的信号通路。要了解细胞如何协调细胞外信号和细胞内过程，需要对ECM的组织以及由ECM-整合素相互作用激活的信号分子的信息有深入的了解。我们正在应用哺乳动物细胞培养和线虫线虫相结合的方法来解决这个问题。拟议的实验利用了这两个系统提供的截然不同和互补的优势。在HT1080人纤维肉瘤细胞中，Src家族激酶在纤维连接蛋白(FN)的基质组装和基质稳定性中起着至关重要的作用。我们将确定在组装过程中Src作用的时机，它对基质周转的影响，以及它在细胞表面形成基质组装部位中的作用。这些实验将使用洗涤剂不溶基质的生化分析、FN原纤维组织的显微分析以及添加抑制剂和突变蛋白。基质周转减少了细胞的粘附性，因为它促进了迁移。因此，我们将使用体外细胞迁移实验来检测基质组装对细胞迁移速率的影响。对于只有2个整合素受体的线虫，将进行遗传分析，以确定体内ECM-整合素相互作用的主要信号。我们能够拯救β整合素(PAT-3)缺失的动物，这些动物的基因突变在保守的β整合素细胞质酪氨酸残基中。被拯救的线虫是活的和有生育能力的，但在性腺形态发生方面存在缺陷，性腺形态发生是一个依赖于整合素介导的细胞运动的过程。为了确定Beta尾巴中的其他功能序列，将分析带有其他尾巴突变的挽救线虫品系的性腺形成缺陷和其他整合素相关表型。线虫株系还将用于RNA干扰和EMS突变筛选，以分离作用于整合素下游途径的新基因。这些目标的结果将提供关于ECM-整合素相互作用在组织形态发生过程中调节细胞迁移和基质组装的作用的分子和遗传学信息。

DESCRIPTION (provided by applicant): The extracellular matrix (ECM) plays a critical role in cellular decision-making. It serves as a framework for tissue architecture and it stimulates cell growth, migration, and a host of other cell processes. Transmission of ECM signals into cells is carried out by integrin receptors. These receptors link the ECM to the actin cytoskeleton and activate a number of different signaling pathways. To understand how cells coordinate extracellular signals with intracellular processes requires insights into the organization of the ECMas well as information about the signaling molecules that are activated by ECM-integrin interactions. We are applying a combined approach to this problem using mammalian cell culture and the nematode Caenorhabditis elegans. The proposed experiments make use of the distinct and complementary advantages provided by these two systems. In HT1080 human fibrosarcoma cells, Src family kinases appear to play an essential role in fibronectin (FN) matrix assembly and matrix stability. We will determine the timing of Src-action during assembly, its effect on matrix turnover, and its role in formation of matrix assembly sites at the cell surface. Biochemical analysis of detergent-insoluble matrix, microscopic analysis of FN fibril organization, and addition of inhibitors and mutant proteins will be used for these experiments. Matrix turnover decreases cell adhesion as it promotes migration. Therefore, we will examine the effects of matrix assembly on cell migration rate using in vitro cell migration assays. With C. elegans, which has only 2integrin receptors, genetic analyses to identify major signals downstream of ECM-integrin interactions in vivo will be performed. We are able to rescue beta integrin (pat-3) null animals with genes mutated in conserved beta integrin cytoplasmic tyrosine residues. Rescued nematodes are viable and fertile, but show defects in gonad morphogenesis, a process dependent on integrin-mediated cell movements. To identify additional functional sequences within the beta tail, rescued nematode lines with other tail mutations will be analyzed for defects in gonad formation and other integrin-related phenotypes. Nematode lines will also be used in RNA interference and EMS mutagenesis screens to isolate novel genes that act in pathways downstream of integrins. Results from these aims will provide molecular and genetic information about the role of ECM-integrin interactions in regulating cell migration and matrix assembly during tissue morphogenesis.