MICROTUBULE/ACTIN INTERACTIONS IN CELL MOTILITY

细胞运动中的微管/肌动蛋白相互作用

• 批准号: 6181997
• 负责人: Clare Michal Waterman
• 金额: $ 29.11万
• 依托单位: SCRIPPS RESEARCH INSTITUTE, THE
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-09-01 至 2005-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6181997
• 关键词: Xenopus oocyte; actins; cell motility; digital imaging; fluorescence microscopy; fluorescent dye /probe; guanine nucleotide binding protein; guanine nucleotide exchange factors; intermolecular interaction; microtubules; tubulin

Cell locomotion is critical to development, wound healing and tissue remodeling, and a central problem during metastasis in cancer. Thus, it is important to gain an understanding of cell motility at the basic level. The cytoskeletal polymers, microtubules and filamentous actin (f-actin) are required for cell motility. Polarized f-actin assembly and movement is thought to drive cell motility, while microtubules are believed to spatially direct sites of f-actin assembly. There is evidence that microtubules may achieve this by activation of cellular signaling via small GTPases of the Rho family. However, the organization and dynamics of microtubules and how they are affected by actin is not thoroughly characterized, and the molecules involved in mediating their interactions during cell motility are unknown. I approach these problems at 3 levels: First, by examining microtubules and f-actin dynamics in living cells using multimode microscopy, by exploring with both microscopy and biochemistry microtubule/f-actin interactions in a cellular extract model systems, and finally by examining the biochemical interactions of purified components of the motile machinery in vitro. The combination of unique microscopy methods and molecular biochemistry is a novel means of carefully characterizing cellular phenomenon and then going directly to the test tube test hypotheses about the molecular mechanisms underlying the phenomena. In this proposal, I describe three specific aims to shed light on our understanding of microtubule/f- actin interactions in cell motility. 1. To develop new microscopy methods for the simultaneous visualization and analysis of f-actin and microtubule dynamics and to carefully analyze microtubule/actin interactions in living migrating cells. 2. To biochemically and microscopically probe the molecular mechanism dynamic microtubule/actin interactions in Xenopus egg extracts. 3. To biochemically analyze microtubule mediation of Rho family signaling and study the mechanism by which growing microtubules activate Rac1 during cell motility. The results of these studies will greatly enhance our understanding of the mechanisms and mechanics of cell locomotion and will provide new methods for analyzing protein dynamics in living cells.

细胞运动对于发育、伤口愈合和组织重塑至关重要，并且是癌症转移过程中的中心问题。 因此，重要的是在基础水平上了解细胞运动。 细胞骨架聚合物、微管和丝状肌动蛋白（f-肌动蛋白）是细胞运动所必需的。 极化的肌动蛋白组装和运动被认为是驱动细胞运动，而微管被认为是空间上指导肌动蛋白组装的网站。 有证据表明，微管可以通过Rho家族的小GTP酶激活细胞信号传导来实现这一点。 然而，微管的组织和动力学以及它们如何受到肌动蛋白的影响还没有完全表征，并且在细胞运动期间参与介导它们相互作用的分子也是未知的。 我接近这些问题在3个层次：首先，通过检查微管和f-肌动蛋白在活细胞中的动态使用多模显微镜，通过探索与显微镜和生物化学微管/f-肌动蛋白的相互作用在细胞提取物模型系统，最后通过检查的生化相互作用的纯化组件的能动机械在体外。 独特的显微镜方法和分子生物化学的结合是一种新的手段，仔细表征细胞现象，然后直接进入试管测试有关现象背后的分子机制的假设。 在这个建议中，我描述了三个具体的目标，阐明我们对微管/f-肌动蛋白在细胞运动中的相互作用的理解。 1.开发新的显微镜方法，用于同时观察和分析f-肌动蛋白和微管动力学，并仔细分析活迁移细胞中微管/肌动蛋白的相互作用。 2.从生物化学和显微镜两方面探讨非洲爪蟾卵提取物中微管/肌动蛋白动态相互作用的分子机制。 3.从生物化学的角度分析微管对Rho家族信号传导的调节作用，并研究细胞运动过程中微管激活Rac 1的机制。 这些研究的结果将大大提高我们对细胞运动机制和力学的理解，并将为分析活细胞中的蛋白质动力学提供新的方法。