Cell Adhesion and the Regulation of Rho GTPases

细胞与其他细胞和细胞外基质的黏附是所有多细胞的基本特征 有机体。粘连不仅在细胞内骨架和细胞外之间提供了结构上的联系 环境，但也提供了影响细胞行为的许多方面的信号转导部位。这笔赠款是 旨在了解来自细胞基质和细胞间黏附的信号如何调节Rho家族成员 GTP酶本身就是细胞骨架和许多细胞内过程的关键调节者。目标是 第一个目的是确定与细胞外基质蛋白纤维连接蛋白的黏附如何刺激RhoA 活动。将分别研究整合素和Syndecan-4的分子。我们将探索其贡献 整合素的特定类型、表达密度和聚集性对RhoA活性的影响。我们将检验这一假设 Syndecan-4‘S的部分作用是通过激活另一种低分子量GTP酶Rapl来实现的。 RhoA激活相关鸟嘌呤核苷酸交换因子(GEF)的鉴定和分离策略 将会被使用。我们将探索调节这些GEF的信号通路。细胞可以感知细胞的物理状态 他们附着的表面，我们将检验等长张力可以刺激RhoA的假设 活动。RhoA活性将使用单细胞表达的生物传感器和活细胞成像来测量。我们会 确定对单细胞施加张力是否局部激活RhoA。第二个目标是指导 部分有助于识别响应钙粘素参与而被激活的RACL GEF。在初步阶段 工作中，我们发现许多Rho家族的GEF与PDZ结构域结合。带有PDZ结构域的蛋白质有 通常在细胞-细胞连接中富含。我们将确定GEF与PDZ结构域的绑定是否 将它们招募到细胞连接，以及这种相互作用是否调节它们的活动。

Adhesion of cells to other cells and to the extracellularmatrix is a fundamental characteristicof all multicellular organisms. Adhesions providenot onlystructurallinksbetween the intracellularcytoskeletonand theextracellular environment, but also provide sites of signal transductionthat affect many aspects of cell behavior. This grant is aimed at understandinghow signals from cell-matrix and cell-cell adhesions regulate members of the Rho family of GTPases, which are themselves key regulators of the cytoskeleton and many intracellular processes. The goal of the first aim is to determine how adhesion to the extracellular matrix protein fibronectin stimulates RhoA activity. The respectiveroles of integrins and syndecan-4 will be investigated. We will explore the contribution of specific integrin type, density of expression and clustering on RhoA activation. Wewill test thehypothesis that some of syndecan-4's effects are mediated through activation of Rapl, another low molecular weight GTPase. Strategies for identifying and isolating guanine nucleotideexchange factors (GEFs) involved in RhoA activation will be used. We will explore signaling pathwaysthat regulate these GEFs. Cells can sense the physical state of the surface to which they adhere and we will test the hypothesis that isometric tension can stimulate RhoA activity. RhoA activity will be measuredusing biosensors expressed in singlecells and live cell imaging. We will determine whetherthe applicationof tension to singlecells locally activatesRhoA. The second aim is directedin part toward identifying the Racl GEFs that are activated in response to cadherin engagement. Inpreliminary work, we have discovered that many Rho family GEFs bind to PDZ domains. Proteins with PDZ domains are typically enriched in cell-cell junctions. We will determine whether the binding of GEFs to PDZ domains serves to recruit them to cell junctions and whether this interaction regulates their activity.