Cell Adhesion and the Regulation of Rho GTPases

细胞与其他细胞和细胞外基质的粘附是所有多细胞肿瘤的基本特征。 有机体粘附不仅在细胞内细胞粘附和细胞外细胞粘附之间提供结构联系， 环境，而且还提供了影响细胞行为许多方面的信号转导位点。这笔赠款是 旨在了解来自细胞-基质和细胞-细胞粘附的信号如何调节Rho家族成员 GTP酶本身是细胞骨架和许多细胞内过程的关键调节剂。目标 第一个目标是确定细胞外基质蛋白纤连蛋白的粘附如何刺激RhoA 活动将研究整合素和多配体蛋白聚糖-4的各自作用。我们将探讨 特定整合素类型、表达密度和聚集对RhoA活化的影响。我们将测试这个假设， 多配体蛋白聚糖-4的某些作用是通过另一种低分子量GT3-Rap 1的活化介导的。 RhoA激活相关的鸟嘌呤核苷酸交换因子（GEFs）的鉴定和分离策略 将用于我们将探索调节这些GEF的信号通路。细胞可以感知 它们粘附的表面，我们将测试等长张力可以刺激RhoA的假设 活动RhoA活性将使用在细胞中表达的生物传感器和活细胞成像来测量。我们将 确定张力对细胞的应用是否局部激活RhoA。第二个目标是 这有助于鉴定响应钙粘蛋白接合而被激活的Racl GEF。初步的 工作中，我们发现许多Rho家族的GEF与PDZ结构域结合。具有PDZ结构域的蛋白质是 通常在细胞-细胞连接处富集。我们将确定GEFs与PDZ结构域的结合是否 以将它们募集到细胞连接处，以及这种相互作用是否调节它们的活性。

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.