A Cdc42-directed/formin-driven actin remodeling machine

描述(由申请人提供)： 透明相关蛋白(DRF)和Rho家族GTP酶在细胞形状、运动和细胞分裂的调节过程中相互联系，以响应生长因子和细胞外刺激；特定的DRF/GTP酶对共定位于特定的基于肌动蛋白的亚细胞结构。这项建议集中在哺乳动物的DRF India2及其与GTPase CDc42在促进丝状足或微穗形成方面的相互作用。我们的初步数据表明，mDia2在丝状伪足的发生中起到了CDC42效应器的作用，并且定位于微刺的尖端。我们认为，CDC42指导mDia2相关蛋白复合体的形成，从而导致mDia2靶向丝状伪足。这里提出的实验旨在确定驱动依赖于Cdc42的mDia2丝状靶向的分子决定因素，确定哪些蛋白质与mDia2相关，并了解CDc42指导的蛋白质如何有助于将mdia定位到迁移细胞的前沿。荧光共振能量转移(FRET)技术将被用来测量细胞内mDia2/Cdc42相互作用的空间和时间动力学，并确定靶向mDia2结构域的要求。影响靶向的CDC42导向的mDia2合作者将通过质谱学(MS)和FRET证明的共定位和直接相互作用来鉴定。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，这一结果应该有助于深入了解CDC42和mDia2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。最终，我们计划利用DRF家族作为癌症治疗的目标。了解mDia2/Cdc42相互作用的性质是实现这一目标的根本步骤。

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.