A Cdc42-directed/formin-driven actin remodeling machine

Cdc42 定向/formin 驱动的肌动蛋白重塑机器

• 批准号: 6928501
• 负责人: KATHRYN M EISENMANN
• 金额: $ 5.35万
• 依托单位: VAN ANDEL RESEARCH INSTITUTE
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-08-01 至 2007-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6928501
• 关键词: RNA interference; actins; binding sites; biological signal transduction; cell migration; fluorescence resonance energy transfer; gene expression; guanine nucleotide binding protein; guanosinetriphosphatases; intracellular transport; mass spectrometry; microfilaments; molecular assembly /self assembly; postdoctoral investigator; protein binding; protein localization; protein protein interaction; protein quantitation /detection; protein structure function; protein transport

DESCRIPTION (provided by applicant): The Diaphanous-related formins (Drfs) and Rho family GTPases associate during the regulation of cell shape, motility, and cell division in response to growth factors and extracellular stimuli; Specific Drf/GTPase pairs co-localize to specific subcellular actin-based structures. This proposal focuses on the mammalian Drf india2 and its interaction with the GTPase Cdc42 in promoting filopodia or microspike formation. Our preliminary data indicate that mDia2 acts as a Cdc42 effector in the generation of filopodia and is localized to the tips of microspikes. We propose that Cdc42 directs the formation of an mDia2-associated protein complex leading to mDia2 targeting to filopodia. Experiments proposed here aim to define molecular determinants driving Cdc42-dependent mDia2 filopodial targeting, to ascertain which proteins associate with mDia2 and to understand how Cdc42-directed proteins contribute to mDia localization to the leading edge of migrating cells. Fluorescent resonance energy transfer (FRET) technology will be used to measure spatial and temporal dynamics of mDia2/Cdc42 interactions within cells and to determine mDia2 domain requirements for targeting. Cdc42-directed mDia2 collaborators affecting targeting will be identified by mass spectroscopy (MS) and co-localization and direct interactions demonstrated by FRET. As dynamic remodeling of the actin cytoskeleton facilitates changes in cell shape, migration and invasion within specific tissues, the results should provide insight as to how the interaction between Cdc42 and mDia2 can function in both normal and migrating tumor cells. Ultimately, we plan on exploiting the DRF family as targets for cancer therapy. Understanding the nature of the mDia2/Cdc42 interaction is a fundamental step towards that goal.

描述（由申请人提供）： 透明相关福明 (Drfs) 和 Rho 家族 GTP 酶在响应生长因子和细胞外刺激而调节细胞形状、运动和细胞分裂过程中联合作用；特定的 Drf/GTPase 对共定位于特定的基于肌动蛋白的亚细胞结构。该提案重点关注哺乳动物 Drf india2 及其与 GTPase Cdc42 在促进丝状伪足或微刺形成中的相互作用。我们的初步数据表明，mDia2 在丝状伪足的生成中充当 Cdc42 效应子，并且定位于微刺的尖端。我们认为 Cdc42 指导 mDia2 相关蛋白复合物的形成，导致 mDia2 靶向丝状伪足。这里提出的实验旨在定义驱动 Cdc42 依赖性 mDia2 丝状伪足靶向的分子决定因素，以确定哪些蛋白质与 mDia2 相关，并了解 Cdc42 导向的蛋白质如何促进 mDia 定位到迁移细胞的前缘。荧光共振能量转移 (FRET) 技术将用于测量细胞内 mDia2/Cdc42 相互作用的空间和时间动态，并确定 mDia2 域的靶向要求。 Cdc42 指导的影响靶向的 mDia2 合作者将通过质谱 (MS) 进行识别，并通过 FRET 证明共定位和直接相互作用。由于肌动蛋白细胞骨架的动态重塑促进了特定组织内细胞形状、迁移和侵袭的变化，因此这些结果应该提供关于 Cdc42 和 mDia2 之间的相互作用如何在正常和迁移肿瘤细胞中发挥作用的见解。最终，我们计划利用 DRF 家族作为癌症治疗的靶标。了解 mDia2/Cdc42 相互作用的本质是实现这一目标的基本一步。