Relating specific biochemical activities of ADF/cofilin family members to their physiological functions

将 ADF/cofilin 家族成员的特定生化活性与其生理功能联系起来

• 批准号: 238964129
• 负责人: Professor Dr. Jan Faix
• 金额: --
• 依托单位: Institut für Biophysikalische Chemie
• 依托单位国家: 德国
• 项目类别: Priority Programmes
• 财政年份: 2013
• 资助国家: 德国
• 起止时间: 2012-12-31 至 2016-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/238964129?language=en
• 关键词: Relating; specific; biochemical; activities; ADF

The dynamic turnover of actin filament networks is essential for a number of important cellular processes including establishment and maintenance of cell morphology, endocytosis and cell migration. Key players known to catalyze subunit turnover by severing and depolymerizing filaments belong to a family of small and ubiquitous proteins jointly called actin depolymerization factor (ADF)/cofilin. Vertebrates express three ADF/cofilin family members: ADF, cofilin1 and cofilin2. The inactivation of any of the three ADF/cofilin members in the mouse leads to discrete phenotypes in spite of continuous expression of the remaining two family members. Based on these findings, we hypothesize individual ADF/cofilin family members to exert distinct biochemical activities, i.e. concerning binding to G or F-actin, concerning severing or nucleation of actin filaments, either by themselves or in alliance with the cofilin-potentiating proteins coronin, Aip1 or CAP. The objective of the proposed work is to directly compare and quantitate the activities of the three mouse proteins and of phosphomimetic mutants, either in the absence or presence of these accessory disassembly factors, in order to uncover these differences and relate them to physiology. Additionally, we seek to examine the biochemical properties of as yet uncharacterized mutations in human cofilin2 causing nemaline and myofibrilar myopathies, with the aim to directly correlate them with the development of disease. To accomplish these ambitious goals, we will first and foremost employ advanced in vitro TIRF microscopy in conjunction with other biochemical and cell culture-based assays. Owing to rapid technological progress in the field and high demand from this and eight other SPP1464 projects, we here apply for the project-relevant acquisition of an advanced state-of-the-art TIRF microscope allowing single molecule detection combined with simultaneous multi-color imaging. The requested TIRF system will be exclusively used by the project partners of the SPP1464 to study the dynamics of actin-based processes on isolated proteins or protein complexes. These analyses will focus on the functions of various formins, WH2- domain-based nucleators and other actin-binding proteins in the assembly or disassembly of actin filaments. Single molecule multi-color TIRF imaging will allow us to address numerous, contemporary scientific questions and monitor actin assembly or disassembly reactions with supreme precision and at a high temporal resolution. We are confident therefore that these studies will have significant implications for our understanding of the multitude of mechanisms underlying actin-based motility.

肌动蛋白丝网络的动态周转对许多重要的细胞过程至关重要，包括细胞形态的建立和维持、内吞作用和细胞迁移。已知通过切断和解聚细丝来催化亚基周转的关键参与者属于一个小而普遍存在的蛋白质家族，统称为肌动蛋白解聚因子(ADF)/cofilin。脊椎动物表达三个ADF/cofilin家族成员：ADF、cofilin1和cofilin2。在小鼠中，三种ADF/cofilin成员中的任何一种失活都会导致离散表型，尽管其余两种家族成员持续表达。基于这些发现，我们假设单个ADF/cofilin家族成员发挥不同的生化活性，即与G或f -肌动蛋白结合，与肌动蛋白丝的切断或成核有关，无论是单独还是与cofilin增强蛋白coronin， Aip1或CAP联合。提出的工作的目的是直接比较和定量三种小鼠蛋白和拟磷突变体的活性。无论是在没有或存在这些附属拆卸因子的情况下，为了揭示这些差异并将它们与生理学联系起来。此外，我们试图研究人类cofilin2中引起线状和肌原纤维肌病的尚未表征的突变的生化特性，目的是直接将它们与疾病的发展联系起来。为了实现这些雄心勃勃的目标，我们将首先采用先进的体外TIRF显微镜，结合其他生化和细胞培养检测。由于该领域的快速技术进步以及该项目和其他八个SPP1464项目的高需求，我们在此申请获得一台先进的最先进的TIRF显微镜，该显微镜可以同时进行单分子检测和多色成像。所请求的TIRF系统将专门用于SPP1464的项目合作伙伴，以研究分离蛋白质或蛋白质复合物上基于肌动蛋白的过程的动力学。这些分析将集中在各种形成蛋白、基于WH2结构域的核子和其他肌动蛋白结合蛋白在肌动蛋白丝的组装或拆卸中的功能。单分子多色TIRF成像将使我们能够解决许多当代科学问题，并以极高的精度和高时间分辨率监测肌动蛋白组装或拆卸反应。因此，我们相信这些研究将对我们理解基于动作蛋白的运动的多种机制具有重要意义。