Molecular Mechanisms coordinating the actin and microtubule cytoskeletons

 描述(申请人提供)：这项研究的目标是确定肌动蛋白和微管细胞骨架的动力学和空间组织是如何由一组相互作用的哺乳动物蛋白质协调的：APC、DIA1、EB1、CLIP-170和帽蛋白。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，从而加深对细胞迁移、细胞黏附和细胞和组织形态发生等过程中的分子活性和相互作用的理解。本项目利用大量的生化实验，结合我们量身定制的新颖的多波长单分子荧光方法，在体外直接观察复杂的多组分调控系统的机制。此外，从体外实验中推导出的机制将在体内进行测试，以确认它们对这些蛋白质的特定细胞功能是重要的。具体目的是：(1)测试微管加末端结合蛋白EB1直接调控APC-Dia1成核的假说；(2)检验肌动蛋白细丝延长的速度和持续时间受Dia1、覆盖蛋白、微管和/或带刺末端EB1的动态结合作用控制的假说；以及(3)确定APC、Dia1、EB1和CLIP-170在控制微管+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.