Roles of Diaphanous-Related Formins in Neurite Outgrowth

描述（由申请人提供）：透明蛋白相关Formins （DRFs）是Rho GTPases的重要效应分子，控制细胞骨架的形式和功能的多个方面，对肌动蛋白细胞骨架施加特别严格的控制。这使得DRFs处于影响神经元形态分化的关键位置。然而，对于这些信号蛋白在神经元细胞中的表达、定位或功能，我们基本上一无所知。这些研究的总体目标是确定和定义drf在神经元形态发生中的作用。特异性抗体将用于蛋白质印迹，以定量DRF蛋白在发育中的大脑和神经元分化的细胞培养模型中的表达。免疫荧光技术将用于定位神经元生长过程中细胞间室中的DRF蛋白，重点关注细胞骨架动力学区域，如生长锥。突变DRF蛋白的外源表达将用于操纵DRF活性，从而检验DRF功能积极影响神经突生长的假设。共同免疫沉淀实验将检测DRFs与主要结合伙伴延伸因子eEFla（一种丰富的肌动蛋白结合蛋白）在神经细胞生长前和生长过程中的关联，并将检测Rho调节的Rho激酶在这种相互作用形成中的作用。这些结果将用于构建神经元中DRF功能的模型，并将阐明一个主要的DRF结合伙伴eEFla的作用。这些发现将有助于揭示神经元生长过程的细胞机制，这是神经再生过程中的一种发育功能。了解这些机制对于寻找治疗退行性疾病或神经系统创伤的治疗工具至关重要。

DESCRIPTION (provided by applicant): The Diaphanous-related Formins (DRFs) are important effector molecules of Rho GTPases, and control multiple aspects of the form and function of the cytoskeleton, exerting particularly tight control over the actin cytoskeleton. This situates the DRFs in a key position to influence neuronal morphological differentiation. Essentially nothing is known, however, about expression, localization, or function of these signaling proteins in neuronal cells. The overall goal of these studies is to identify and define the roles of the DRFs in neuronal morphogenesis. Specific antibodies will be used on protein blots to quantify DRF protein expression in the developing brain and in cell culture models of neuronal differentiation. Immunofluorescence techniques will be employed to localize DRF proteins in cellular compartments during neuronal process outgrowth, focusing on areas of cytoskeletal dynamics such as the growth cone. Exogenous expression of mutant DRF proteins will be used to manipulate DRF activity and thereby to test the hypothesis that DRF function positively influences neurite outgrowth. Co-immunoprecipitation experiments will examine the association of DRFs with a major binding partner, the elongation factor eEFla, which is an abundant actin-binding protein, in neuronal cells before and during process outgrowth, and will examine the role of the Rho-regulated Rho kinase in the formation of this interaction. The results will be used to construct a model of DRF function in neurons and will shed light on the role of one major DRF binding partner, eEFla. The findings will help uncover the cellular mechanisms that underlie neuronal process outgrowth, a developmental function that is recapitulated in neural regeneration. Understanding these mechanisms is critical in the search for therapeutic tools to use in cases of degenerative disease or trauma in nervous system.