Dynamic Analysis of Rho GTPase-Rho GDI Cycling

Rho GTPase-Rho GDI 循环的动态分析

• 批准号: 7932880
• 负责人: Celine DerMardirossian
• 金额: $ 52.34万
• 依托单位: SCRIPPS RESEARCH INSTITUTE, THE
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-01 至 2012-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7932880
• 关键词: Activity Cycles; Address; Angiotensin II; Angiotensin II Receptor; Architecture; Binding; Biochemical; Biochemical Genetics; Biological; Biology; Biosensor; Cardiovascular system; Cell physiology; Cells; Cessation of life; Communities; Complex; Computer Simulation; Computer software; Cytoskeleton; Cytosol; Data; Data Analyses; Defect; Disease; Dissociation; Dyes; Fluorescence; Fluorescence Resonance Energy Transfer; GTP Binding; Guanine Nucleotide Dissociation Inhibitors; Guanosine Triphosphate Phosphohydrolases; Health; Image; Image Analysis; Imaging Techniques; In Vitro; Integrins; Kidney; Kidney Failure; Kinetics; Knockout Mice; Life; Mediating; Membrane; Microscopy; Modeling; Molecular; NADPH Oxidase; Outcome; Pathway interactions; Phosphorylation; Phosphotransferases; Physiology; Play; Proteins; Reactive Oxygen Species; Regulation; Renal function; Resolution; Role; Signal Transduction; Signaling Molecule; Solutions; Structure; Techniques; Testing; Time; base; cardiovascular endothelium; cellular imaging; computerized tools; design; genetic manipulation; genetic regulatory protein; image registration; improved; in vivo; models and simulation; nephrin; novel; podocyte; predictive modeling; protein protein interaction; public health relevance; receptor; research study; response; rho GTP-Binding Proteins; rhoB p20 GDI; slit diaphragm; src-Family Kinases; submicron; tool; virtual

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.

描述（由申请人提供）：Rho GTPase GDP/GTP 活性循环和细胞质/膜定位循环的关键调节剂是 RhoGDI。基于体外生化观察，我们目前对 RhoGDI 调节及其在体内细胞信号传导过程中与 Rho GTPases 相互作用的动态的理解在很大程度上仍然是假设的。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，以阐明整合素、去氧肾上腺素和血管紧张素 II 信号传导背景下 Rho GTP 酶循环的细胞内动力学和调节。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。将开发 RhoGDI 与 Cdc42 结合和 Src 激活的荧光生物传感器。这些生物传感器将开创新的、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，这些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 与 Cdc42 激活或 Src 激活结合）。数据将使用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背景下对成像实验进行建模。解决这个“正向问题”将使我们能够推导出体内浓度和速率。我们将在体内研究所提出的 GTPase 调节周期中的关键步骤，以定量评估 RhoGDI 对 Rho GTPase 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 GTP酶激活以及由此产生的生物学（即细胞骨架）结果相关。将评估通过 Src 介导的磷酸化对 Rho GTPase-RhoGDI 复合物的调节，利用体内新型 Src 激酶和 GDI 络合生物传感器，结合同步成像技术，将 RhoGDI 的激酶激活和磷酸化与 Rho GTPase 的动力学联系起来骑自行车。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，这些参数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来评估路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 GTP 酶及其关键调节蛋白 Rho GDI 在正常生理和疾病中发挥着重要作用。了解 RhoGDI 如何调节活细胞中 Rho GTP 酶活性和细胞质到膜循环的基本动力学对于了解 Rho GTP 酶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 GTP 酶在健康和疾病中的功能。