Coordination of 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是如何被一组相互作用的哺乳动物蛋白质协调的：APC、DIA1和EB1。这项工作将定义这些蛋白质的功能和机制，并提供更深入的了解活动和相互作用，这些过程的基础，如细胞迁移和形态发生。该项目使用大量的生化实验，结合一种新的多波长单分子生物物理方法，在体外阐明复杂的、多组分的调控系统的机制。此外，从体外实验中推导出的机制将在体内进行测试，以验证它们对这些蛋白质在活细胞中的相关生物学功能(例如，定向细胞迁移)是重要的。具体目的是：(1)测试关于Dia1和APC协同促进肌动蛋白组装的两个关键假说，包括在组件之间形成拟议的物理络合物；(2)测试额外的假说，即Dia1和APC通过“火箭发射器”机制协同刺激肌动蛋白组装；以及(3)确定EB1单独和EB1+微管调节和/或组织APC/Dia诱导的肌动蛋白组装的机制。 公共卫生相关性：这项资助是一项关于肌动蛋白和微管细胞骨架的组织和调控的研究，通过三种蛋白质的联合作用：APC、DIA1和EB1。APC(结肠腺瘤性息肉病)是一种肿瘤抑制因子。人类APC基因突变被认为是超过80%的遗传性和散发性结直肠癌进展的早期步骤。此外，EB1和MLL基因的融合可导致急性淋巴细胞性白血病。我们预计，这项研究将为肿瘤发生的潜在机制提供新的见解。

DESCRIPTION (provided by applicant): The goal of this research is to determine how the dynamics of the actin and microtubule cytoskeletons are coordinated by a group of three interacting mammalian proteins: APC, Dia1, and EB1. This work will define the functions and mechanisms of these proteins, and provide a deeper understanding of the activities and interactions that underlie such processes as cell migration and morphogenesis. This project uses bulk biochemical experiments combined with a novel multi-wavelength single molecule biophysics method tailored to elucidate the mechanisms of complex, multi-component regulatory systems in vitro. In addition, the mechanisms deduced from the experiments in vitro will be tested in vivo to verify that they are important for relevant biological functions of these proteins in living cells (e.g., directed cell migration). The Specific Aims are: (1) Test two key hypotheses about the mechanism by which Dia1 and APC synergize in promoting actin assembly, involving formation of proposed physical complexes among components; (2) Test the additional hypothesis that Dia1 and APC synergize to stimulate actin assembly by a "rocket launcher" mechanism; and (3) Define the mechanisms by which EB1 alone and EB1 plus microtubules regulate and/or organize APC/Dia-induced actin assembly. PUBLIC HEALTH RELEVANCE: This grant is an investigation of the organization and regulation of actin and microtubule cytoskeletons by the combined effects of three proteins: APC, Dia1, and EB1. APC (Adenomatous polyposis coli) is a tumor suppressor. Mutation of the human Apc gene is considered to be an early step in the progression of over 80% of colorectal cancers, inherited and sporadic. In addition, fusion of the EB1 and MLL genes can lead to acute lymphoblastic leukemia. We anticipate that this research will provide new insights into the underlying mechanisms of tumorigenesis.