Coordination of 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如何通过一组三种相互作用的哺乳动物蛋白：APC，Dia 1和EB 1进行协调。这项工作将确定这些蛋白质的功能和机制，并提供更深入的了解的活动和相互作用的基础，如细胞迁移和形态发生等过程。该项目采用批量生化实验结合一种新的多波长单分子生物物理学方法，旨在阐明复杂的多组分体外调控系统的机制。此外，将在体内测试从体外实验中推导出的机制，以验证它们对于这些蛋白质在活细胞中的相关生物学功能是重要的（例如，定向细胞迁移）。具体目标是：（1）检验关于Dia 1和APC协同促进肌动蛋白组装的机制的两个关键假设，包括组分之间形成拟议的物理复合物;（2）检验Dia 1和APC协同通过“火箭发射器”机制刺激肌动蛋白组装的附加假设;（3）明确EB 1单独和EB 1+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：这项资助是对肌动蛋白和微管细胞骨架的组织和调节的研究，这些细胞骨架是由三种蛋白质：APC，Dia 1和EB 1的联合作用引起的。APC（Adenomatous polyposis coli）是一种肿瘤抑制因子。人类Apc基因的突变被认为是超过80%的遗传性和散发性结直肠癌进展的早期步骤。此外，EB 1和MLL基因的融合可导致急性淋巴细胞白血病。我们预计，这项研究将提供新的见解肿瘤发生的潜在机制。

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.