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，并刺激细胞生长，迁移和其他细胞过程的主机。ECM信号向细胞内的传递通过整联蛋白受体进行。这些受体将ECM连接到肌动蛋白细胞骨架并激活许多不同的信号传导途径。为了了解细胞如何协调细胞外信号与细胞内过程，需要深入了解ECM的组织以及有关ECM整合素相互作用激活的信号分子的信息。我们正在应用一个综合的方法来解决这个问题，使用哺乳动物细胞培养和线虫秀丽隐杆线虫。拟议的实验利用这两个系统提供的独特和互补的优势。在HT 1080人纤维肉瘤细胞中，Src家族激酶似乎在纤维连接蛋白（FN）基质组装和基质稳定性中发挥重要作用。我们将确定Src-行动的时间在组装过程中，其对基质营业额的影响，以及其在细胞表面形成基质组装位点的作用。这些实验将使用洗涤剂不溶性基质的生化分析、FN原纤维组织的显微镜分析以及添加抑制剂和突变蛋白。基质周转降低细胞粘附，因为它促进迁移。因此，我们将使用体外细胞迁移试验检查基质组装对细胞迁移率的影响。梭elegans，它只有20个整合素受体，将进行遗传分析，以确定体内ECM-整合素相互作用下游的主要信号。我们能够拯救β整联蛋白（pat-3）无效的动物与保守的β整联蛋白胞质酪氨酸残基突变的基因。获救的线虫是可行的和肥沃的，但显示缺陷的性腺形态发生，一个过程依赖于整合素介导的细胞运动。为了鉴定β尾内的其他功能序列，将分析具有其他尾突变的拯救线虫系的性腺形成缺陷和其他整联蛋白相关表型。线虫系也将用于RNA干扰和EMS诱变筛选，以分离在整合素下游途径中起作用的新基因。这些目标的结果将提供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分子和遗传信息。

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.