Molecular Mechanisms of Cellular Motility

描述（由申请人提供）：我们的目标是了解在细胞运动和内吞作用过程中控制肌动蛋白丝组装和拆卸的分子机制，以特定反应的速率。我们将在裂变酵母中使用生化，遗传和细胞实验的组合来测试细胞如何维持肌动蛋白亚基库，启动新的肌动蛋白丝和分解老化的肌动蛋白丝。我们将使用光谱分析来绘制在profilin存在和不存在的情况下，Arp2/3复合物形成肌动蛋白丝分支的途径。我们将描述裂变酵母cofilin的温度敏感突变体的生化特性，用于活酵母细胞的实验。我们将从纯化的蛋白质中重建一个循环肌动蛋白运动系统，并通过荧光显微镜分析单个细丝的动力学。我们将使用表达功能性融合蛋白的裂变酵母的荧光显微镜来确定内吞过程中肌动蛋白丝组装和周转的分子途径，并与数学模型进行比较。我们将通过改变蛋白质浓度和/或诱变来测试细胞中的机制。我们将使用生长中的肌动蛋白细丝的直接观察来确定FH2结构域中连接肽的长度、formin FH1FH2结构体n端张力以及FH1结构域和FH2结构域中profilin结合位点之间的距离是如何影响延伸的。我们将使用单分子荧光显微镜研究肌动蛋白丝末端FH2结构域的构象变化，并研究生长中的肌动蛋白丝末端FH2结构域的“旋转悖论”。

DESCRIPTION (provided by applicant): Our goal is to understand the molecular mechanisms controlling assembly and disassembly of actin filaments during cellular locomotion and endocytosis in terms of the rates of specific reactions. We will use a combination of biochemical, genetic and cellular experiments in fission yeast to test how cells maintain a pool of actin subunits, initiate new actin filaments and disassemble aged actin filaments. We will use spectroscopic assays to map the pathway of actin filament branch formation by Arp2/3 complex in the presence and absence of profilin. We will characterize the biochemical properties of a temperature sensitive mutant of fission yeast cofilin for use in experiments with live yeast cells. We will reconstitute a recycling actin motility system from purified proteins and analyze the dynamics of individual filaments by fluorescence microscopy. We will use fluorescence microscopy of fission yeast expressing functional fusion proteins to determine the molecular pathway of actin filament assembly and turnover during endocytosis for comparisons with mathematical models. We will test mechanisms in cells by varying protein concentrations and/or mutagenesis. We will use direct observations of growing actin filaments to determine how elongation is influenced by the length of linker peptides in the FH2 domain, tension on the N-terminus of formin FH1FH2 constructs and the distance between profilin binding sites in the FH1 domain and the FH2 domain. We will use single molecule fluorescence microscopy to study conformational changes in the FH2 domain on the end of actin filaments and to investigate the "rotation paradox" of FH2 domains on the ends of growing actin filaments. PUBLIC HEALTH REVELATION: We study the molecular basis of cellular motility and cytokinesis, particularly the roles of actin filaments and myosin motors. Actin-based movements are essential for cell division, shaping organs during embryonic development, defense against microorganisms and wiring the nervous system. Movement of cells out of primary tumors is the chief cause of mortality in cancer.