A Cdc42-directed/formin-driven actin remodeling machine

描述（由申请人提供）： 透明相关的formins（Drfs）和Rho家族GTP酶在调节细胞形状，运动性和细胞分裂过程中响应于生长因子和细胞外刺激相关联;特定的Drf/GTP酶对共定位于特定的亚细胞肌动蛋白为基础的结构。该建议的重点是哺乳动物的Drf india 2和它的相互作用与GTdR Cdc 42在促进丝状伪足或microspike形成。我们的初步数据表明，mDia 2作为Cdc 42效应器在产生丝状伪足，并定位于微刺的尖端。我们建议，Cdc 42指导形成的mDia 2相关的蛋白质复合物，导致mDia 2针对丝状伪足。这里提出的实验旨在定义驱动Cdc 42依赖性mDia 2丝状伪足靶向的分子决定因素，以确定哪些蛋白质与mDia 2相关，并了解Cdc 42指导的蛋白质如何有助于mDia定位到迁移细胞的前沿。荧光共振能量转移（FRET）技术将用于测量细胞内mDia 2/Cdc 42相互作用的空间和时间动力学，并确定靶向mDia 2结构域的要求。将通过质谱（MS）和通过FRET证明的共定位和直接相互作用来鉴定影响靶向的Cdc 42定向mDia 2协作者。由于肌动蛋白细胞骨架的动态重塑有助于细胞形状的变化，迁移和特定组织内的侵袭，结果应该提供关于Cdc 42和mDia 2之间的相互作用如何在正常和迁移的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点。了解mDia 2/Cdc 42相互作用的性质是实现这一目标的基本步骤。

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.