Dynamic Analysis of Rho GTPase-Rho GDI Cycling

描述（由申请人提供）：Rho GTP酶GDP/GTP活性循环和胞质溶胶/膜定位循环的关键调节因子是RhoGDI。我们目前对RhoGDI调控及其在体内细胞信号传导过程中与Rho GTP酶相互作用的动力学的理解在很大程度上仍然是基于体外生化观察的假设。我们最近发现磷酸化是Rho GTP酶-RhoGDI循环的关键调节因子。我们建议开发新的生物传感器和计算方法，以阐明整合素、肾蛋白和血管紧张素II信号传导背景下Rho GTPase循环的细胞内动力学和调节。因此，这些研究将提供有关Rho GT3调节与心血管和/或肾功能相关的基本信息。将开发RhoGDI结合Cdc 42和Src激活的荧光生物传感器。这些生物传感器将开拓新的，普遍适用的方法，调整染料的波长将使我们能够产生互补的生物传感器对，可以在同一细胞中成像亚秒和亚微米分辨率（Cdc 42-GDI结合Cdc 42激活或Src激活）。这些数据将用一种计算工具虚拟生物传感器进行分析，该工具将在细胞生理学的背景下对成像实验进行建模。这个“正问题”的解决方案将允许我们推导出体内浓度和速率。将在体内研究所提出的GT3调节周期的关键步骤，以定量评估RhoGDI对Rho GT3调节的蛋白质-蛋白质相互作用、动力学和协调。复合物的形成/解离将与整体循环动力学、调节磷酸化、Rho GT3活化以及所产生的生物学（即，细胞骨架）结果。通过Src介导的磷酸化调节Rho GTdR- RhoGDI复合物将被评估，利用新的Src激酶和GDI复合生物传感器在体内，结合同步成像技术，将RhoGDI的激酶激活和磷酸化与Rho GTdR循环的动力学联系起来。使用新的“虚拟显微镜”工具进行定量分析将应用于成像数据，以获得动力学模型的参数，这些参数可以通过生化和遗传操作进行进一步测试。计算建模和模拟将允许进行假设检验，以评估是否已确定并正确表征了途径中的关键步骤。公共卫生相关性：有大量证据表明，Rho GTP酶和它们的关键调节蛋白Rho GDI在正常生理和疾病中起重要作用。了解RhoGDI如何调节Rho GTPases活性和活细胞中胞质-膜循环的基本动力学对于了解Rho GTPases在生物学中的功能至关重要。通过我们的研究，我们希望提供一个定量详细的预测模型，可用于研究Rho GTdR在健康和疾病中的功能。

DESCRIPTION (provided by applicant): A key regulator of both the Rho GTPase GDP/GTP activity cycle and the cytosol/membrane localization cycle is RhoGDI. Our current understanding of RhoGDI regulation and the dynamics of its interactions with Rho GTPases during in vivo cell signaling remains largely hypothetical, based upon in vitro biochemical observations. We have recently shown phosphorylation to be a key regulator of the Rho GTPase-RhoGDI cycle. We propose to develop new biosensor and computational approaches to elucidate the intracellular dynamics and regulation of the Rho GTPase cycle in the context of integrin, nephrin, and Angiotensin II signaling. These studies will thus provide basic information about Rho GTPase regulation relevant to cardiovascular and/or renal function. Fluorescent biosensors of RhoGDI binding to Cdc42, and of Src activation will be developed. These biosensors will pioneer new, generally applicable approaches, Adjusting wavelengths of dyes will enable us to produce complementary biosensor pairs that can be imaged in the same cell with subsecond and submicron resolution (Cdc42-GDI binding with Cdc42 activation or Src activation). The data will be analyzed with a computational tool, Virtual Biosensor, that will model the imaging experiments in the context of the cell physiology. Solution of this "forward problem" will permit us to derive in vivo concentrations and rates. Key steps in the proposed GTPase regulatory cycle will be investigated in vivo to quantitatively evaluate the protein-protein interactions, kinetics, and coordination of Rho GTPase regulation by RhoGDI. Complex formation/dissociation will be related to overall cycle dynamics, to regulatory phosphorylations, to Rho GTPase activation, and to the resulting biological (ie. cytoskeletal) outcomes. Regulation of Rho GTPase- RhoGDI complexes through Src-mediated phosphorylation will be evaluated, making use of the novel Src kinase and GDI complexation biosensors in vivo, combined with simultaneous imaging techniques, to relate kinase activation and phosphorylation of RhoGDI to the dynamics of Rho GTPase cycling. Quantitative analysis with new "Virtual Microscopy" tools will be applied to imaging data to derive parameters for kinetic models that can be further tested through biochemical and genetic manipulations. Computational modeling and simulation will permit hypothesis testing to assess whether the key steps in the pathway have been identified and correctly characterized. PUBLIC HEALTH RELEVANCE: There is substantial evidence that Rho GTPases and their critical regulatory protein Rho GDI play important roles in both normal physiology and in disease. Understanding the basic dynamics of how RhoGDI regulates Rho GTPase activity and cytosol-to- membrance cycling in live cells is critical to understanding the function of Rho GTPases in biology. With our studies, we hope to provide a quantitatively detailed, predictive model that can be used to investigate Rho GTPase function in health and disease.