Dynamic Analysis of Rho GTPase-Rho GDI Cycling

描述（由申请人提供）：Rho GTPase GDP/GTP活性周期和细胞质溶胶/膜定位周期的关键调节因子是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 gtpase相互作用的动力学的理解在很大程度上仍然是假设的，基于体外生化观察。我们最近发现磷酸化是Rho GTPase-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来阐明在整合素、肾素和血管紧张素II信号传导的背景下Rho GTPase周期的细胞内动力学和调控。因此，这些研究将提供与心血管和/或肾功能相关的Rho GTPase调节的基本信息。RhoGDI结合Cdc42和Src活化的荧光生物传感器将被开发。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生产互补的生物传感器对，可以在同一细胞中以亚秒和亚微米分辨率成像（Cdc42- gdi结合Cdc42激活或Src激活）。数据将用计算工具虚拟生物传感器进行分析，该工具将模拟细胞生理学背景下的成像实验。这个“向前问题”的解决将使我们能够得出体内浓度和速率。我们将在体内研究GTPase调控周期的关键步骤，以定量评估RhoGDI对Rho GTPase调控的蛋白-蛋白相互作用、动力学和协调性。络合物的形成/解离将与整个循环动力学、调节磷酸化、Rho GTPase激活以及由此产生的生物（即酶活性）有关。细胞骨架)的结果。通过Src介导的磷酸化对Rho GTPase- RhoGDI复合物的调节将被评估，利用新的Src激酶和GDI络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 GTPase循环动力学联系起来。新的“虚拟显微镜”工具的定量分析将应用于成像数据，以获得动力学模型的参数，这些模型可以通过生化和遗传操作进一步测试。计算建模和模拟将允许假设检验，以评估该途径中的关键步骤是否已被识别和正确表征。公共卫生相关性：有大量证据表明Rho gtpase及其关键调控蛋白Rho GDI在正常生理和疾病中都起着重要作用。了解RhoGDI如何调节Rho GTPase活性和活细胞中细胞溶胶-膜循环的基本动力学对于理解Rho GTPase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 GTPase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.