Understanding complete transport cycles mediated by importin beta-type nuclear transport receptors in situ and in living cells

了解原位和活细胞中输入β型核转运受体介导的完整转运周期

• 批准号: 5366574
• 负责人: Professor Dr. Dirk Görlich
• 金额: --
• 依托单位: Abteilung Zelluläre Logistik
• 依托单位国家: 德国
• 项目类别: Priority Programmes
• 财政年份: 2002
• 资助国家: 德国
• 起止时间: 2001-12-31 至 2006-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/5366574?language=en
• 关键词: Understanding; complete; transport; cycles; mediated

Many molecular components of the nucleocytoplasmic transport machinery have been identified over the last ten years and we have a good mechanistic understanding of this process which even extends to the structural level at atomic resolution (Conti and Izaurralde, 2001). However, formal proof of the central hypothesis of the driving force of nucleocytoplasmic transport, the existence of a RanGTP gradient across the nuclear envelope (NE) is still lacking. Moreover, we have currently no quantitative understanding of the kinetic parameters of the transport process in the context of the intact cell and are just beginning to understand its capacity and the mechanism of translocation through the nuclear pore complex itself (Ribbeck and Görlich, 2001, Rabut and Ellenberg, 2001). This project is aimed to address theses central open questions of nucleocytoplasmic transport. It will use a combination of biochemical, cell biological, light microscopy and mathematical modeling expertise uniquely combined in the laboratories of the two applicants. By purifying and fluorescently labeling all the components of the importin beta (Impb) mediated transport cycle, we will measure all the kinetic parameters of this reaction separately both in vitro and in situ (using permeabilized and live cells, and state of the art confocal microscopy) and finally combine them in a comprehensive mathematical model of nucleocytoplasmic transport. This model should ulitmately explain all the experimentally observed kinetics and can be used to predict the behaviour of the transport machineery and so far unknown limiting parameters. In addition, this project will use fluorescence energy transfer probes to directly visualize the RanGTP gradient as well as the spatiotemporal distribution of key protein complexes if the Ran system.

在过去的十年中，已经鉴定了核质转运机制的许多分子组分，我们对这一过程有了很好的机制理解，甚至延伸到原子分辨率的结构水平（Conti和Izaurralde，2001）。然而，核质运输的驱动力的中心假设，跨核膜（NE）的RanGTP梯度的存在的正式证明仍然缺乏。此外，我们目前还没有定量了解完整细胞中转运过程的动力学参数，并且刚刚开始了解其通过核孔复合物本身转运的能力和机制（Ribbeck和Görlich，2001，Rabut和Ellenberg，2001）。本项目旨在解决核质转运的核心问题。它将使用生物化学，细胞生物学，光学显微镜和数学建模专业知识的组合，独特地结合在两个申请人的实验室。通过纯化和荧光标记的importin β（Impb）介导的运输周期的所有组件，我们将分别在体外和原位（使用透化和活细胞，和最先进的共聚焦显微镜）测量该反应的所有动力学参数，并最终将它们结合在一个全面的数学模型中的核质运输联合收割机。该模型应ulitelessly解释所有的实验观察到的动力学，并可用于预测的运输机械的行为和迄今未知的限制参数。此外，该项目将使用荧光能量转移探针直接可视化RanGTP梯度以及Ran系统中关键蛋白质复合物的时空分布。