Molecular Mechanisms coordinating the actin and microtubule cytoskeletons

 描述（由申请人提供）：本研究的目的是确定肌动蛋白和微管细胞骨架的动力学和空间组织如何通过一组五种相互作用的哺乳动物蛋白质：APC，Dia 1，EB 1，CLIP-170和帽蛋白进行协调。这项工作将确定这些蛋白质在微管-肌动蛋白交叉调节中的功能和机制，从而对细胞迁移、细胞粘附、细胞和组织形态发生等过程的分子活性和相互作用有更深入的了解。该项目使用大量生物化学实验结合新颖的多波长单分子荧光方法，我们定制了该方法以直接观察复杂的多组分体外调控系统的机制。此外，从体外实验中推导出的机制将在体内进行测试，以确认它们对于这些蛋白质的特定细胞功能是重要的。具体目标是：（1）验证APC-Dia 1与微管和末端结合蛋白EB 1直接调控肌动蛋白丝成核的假设;（2）验证肌动蛋白丝伸长的速率和持续时间受Dia 1、帽蛋白、微管和/或EB 1在倒刺末端的动态结合相互作用控制的假设;（3）明确APC、Dia 1、EB 1和CLIP-170在调控微管+末端动力学和触发微管+末端超快肌动蛋白聚合中的作用。

 DESCRIPTION (provided by applicant): The goal of this research is to determine how both the dynamics and spatial organization of the actin and microtubule cytoskeletons are coordinated by a group of five interacting mammalian proteins: APC, Dia1, EB1, CLIP-170, and capping protein. This work will define the functions and mechanisms of these proteins in microtubule-actin cross-regulation, and will thereby provide a deeper understanding of the molecular activities and interactions that underlie such processes as cell migration, cell adhesion, and cell and tissue morphogenesis. This project uses bulk biochemical experiments combined with novel multi-wavelength single molecule fluorescence methods that we have tailored to directly observe the mechanisms of complex multi- component regulatory systems in vitro. Further, the mechanisms deduced from the experiments in vitro will be tested in vivo to confirm that they are important for specific cellular functions of these proteins. The Specific Aims are: (1) Test the hypothesis that the microtubule plus end-binding protein EB1 directly regulates nucleation of actin filaments by APC-Dia1; (2) Test the hypothesis that the rate and duration of actin filament elongation is controlled by dynamic binding interactions of Dia1, capping protein, microtubules, and/or EB1 at barbed ends; and (3) Define the roles of APC, Dia1, EB1, and CLIP-170 in controlling microtubule plus end dynamics and in triggering ultrafast actin polymerization from microtubule plus ends.