FLUORESCENT INDICATORS OF RHO AND RAN NUCLEOTIDE STATE

该提案涉及监管GTP酶Ran和Rho， 参与控制极化细胞行为和核输入。 新的荧光蛋白类似物将被设计来报告 这些小GT3信号蛋白的核苷酸状态， 个体的活细胞 将制定替代办法， 使得能够基于多种GTP酶产生此类类似物， 这些方法将在这里应用于产生Ran的指标， Rho家族GTP酶。因为小GTP酶无处不在， 不同的基本细胞行为，新技术验证， 这些研究将具有广泛的用途和意义。 全 了解Rho族和Ran GTdR函数需要了解 完整细胞内信号的空间组织。 的 核苷酸状态指示剂将用于研究空间和 活细胞中GT3活化的时间动力学。 的机制 Rho GTPases通过其控制细胞极化和运动性， 在伤口愈合成纤维细胞中通过表征 亚细胞定位，空间和时间的动态， 激活，并通过将这些行为与 细胞骨架 激活的位置和级别都将 量化。 关于整联蛋白和生长调节的假设 通过GTP酶定位和激活变化的因子串扰 水平将被探索。 在Ran函数的研究中， 将揭示Ran在不同亚细胞中的核苷酸状态 地点，区分潜在的监管机制 细胞核输入率和细胞周期反馈控制 有丝分裂到DNA复制的过程。 的基本过程 由这些GTP酶控制，包括细胞运动，核输入， 和细胞形态的调节，对正常的体内平衡至关重要 与多种疾病和免疫功能有关。

This proposal concerns the regulatory GTPases Ran and Rho, which are involved in the control of polarized cell behavior and nuclear import. Novel fluorescent protein analogs will be designed to report the nucleotide state of these small GTPase signalling proteins within individual, living cells. Alternate approaches will be developed to enable production of such analogs based on a wide variety of GTPases, and these methods will be applied here to produce indicators of Ran and Rho family GTPases. Because small GTPases are ubiquitous and impact diverse fundamental cell behaviors, the new technology validated in these studies will be of broad utility and significance. A full understanding of Rho family and Ran GTPase function requires knowledge of the spatial organization of signalling within intact cells. The nucleotide state indicators will be used to study the spatial and temporal dynamics of GTPase activation in living cells. The mechanisms by which Rho GTPases control cell polarization and motility will be examined in wound healing fibroblasts by characterizing changes in subcellular localization, the spatial and temporal dynamics of activation, and by correlating these behaviors with the dynamics of the cytoskeleton. Both the location and levels of activation will be quantified. Hypotheses regarding modulation of integrin and growth factor crosstalk through GTPase localization and variation of activation levels will be explored. In studies of Ran function, the indicators will reveal the nucleotide state of Ran in different subcellular locations, distinguishing between potential mechanisms for regulation of nuclear import rate and cell cycle feedback controls linking progression of mitosis to DNA replication. The fundamental processes controlled by these GTPases, including cell motility, nuclear import, and regulation of cell morphology, are critical to normal homeostasis and relevant to many diseases and immune function.