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

肌动蛋白丝网络的动态更新对于许多重要的细胞过程是必不可少的，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共同称为肌动蛋白解聚因子（ADF）/cofilin的小而普遍存在的蛋白质家族。脊椎动物表达三种ADF/cofilin家族成员：ADF，cofilin 1和cofilin 2。小鼠中三个ADF/cofilin成员中的任何一个的失活导致离散的表型，尽管剩余的两个家族成员连续表达。基于这些发现，我们假设个别ADF/cofilin家族成员发挥不同的生化活动，即关于G或F-肌动蛋白的结合，关于切断或成核的肌动蛋白丝，无论是通过自己或与cofilin增强蛋白冠状蛋白，Aip 1或CAP联盟。拟议的工作的目的是直接比较和定量的三种小鼠蛋白质和磷酸化模拟突变体的活动，无论是在这些辅助拆卸因子的存在或不存在，以揭示这些差异，并将它们与生理学。此外，我们试图研究人类cofilin 2中尚未表征的突变的生化特性，这些突变导致线状体和肌原纤维肌病，目的是将它们与疾病的发展直接相关。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显微镜与其他生物化学和细胞培养为基础的测定。由于该领域的快速技术进步以及该项目和其他八个SPP 1464项目的高需求，我们在此申请与项目相关的先进的TIRF显微镜的采购，该显微镜允许单分子检测与同时多色成像相结合。所要求的TIRF系统将由SPP 1464的项目合作伙伴专门用于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formins，WH 2结构域为基础的成核剂和其他肌动蛋白结合蛋白在肌动蛋白丝的组装或拆卸的功能。单分子多色TIRF成像将使我们能够解决许多当代科学问题，并以最高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肌动蛋白运动的多种机制产生重要影响。

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.