Comparative molecular physiology of mammalian formins

描述（由申请人提供）：细胞内含有肌动蛋白的结构的多样性需要以特定方式形成肌动蛋白丝的多种机制。由于纯化的肌动蛋白单体在体外聚合缓慢，细胞必须具有加速肌动蛋白聚合的因子。最近来自酵母的证据表明，肌动蛋白在体外加速肌动蛋白聚合，并且是产生特定的细胞肌动蛋白的结构，如电缆和细胞动力学裂解沟所必需的。虽然至少有12种哺乳动物formin存在，它们的细胞作用和生化活性没有很好地定义。 这项资助的总体目标是确定哺乳动物formin的生化活动如何影响其细胞功能，特别关注淋巴细胞中微绒毛的组装。循环淋巴细胞表达三种高水平的formin，一种透明的formin（DRF1）和两种高度同源的非透明的formin（FRLa和一种新的ORF，称为小鼠4）。DRF1和FRLa都能加速肌动蛋白聚合，但这些作用的细节表明了不同的机制。这些蛋白质具有几个有趣的生化特性，包括：多聚化，肌动蛋白丝结合和肌动蛋白单体结合。至少有一个DRF（DRF1）定位于淋巴细胞微绒毛。 在目标1中，淋巴细胞形成蛋白加速肌动蛋白聚合的机制将使用生物化学和生物物理技术进行研究。控制淋巴细胞形成生化特性的氨基酸序列将通过诱变作图。此外，profilin在formin介导的肌动蛋白聚合加速的作用将被检查。目的2将阐明调控机制控制formin介导的聚合加速，通过确定分子内相互作用和与其他蛋白质的相互作用的影响。在目标3中，将检查formins在淋巴细胞中的功能，包括细胞肌动蛋白定位，RNAi诱导的蛋白抑制对淋巴细胞肌动蛋白结构的影响，以及破坏肌动蛋白生化特性的突变对淋巴细胞形态的影响。使用瑞士3T3细胞的平行研究将检查formins在淋巴细胞中未发现的结构上的作用，例如板状伪足和应力纤维。目标1和2的生化结果将为目标3的细胞研究提供基础。

DESCRIPTION (provided by applicant): The diversity of cellular actin-containing structures necessitates multiple mechanisms for actin filament formation in a context-specific manner. Since purified actin monomers polymerize slowly in vitro, cells must possess factors that accelerate actin polymerization. Recent evidence from yeast shows that formin proteins accelerate actin polymerization in vitro, and is necessary for generating specific cellular actin-containing structures, such as cables and the cytokinetic cleavage furrow. Although at least 12 mammalian formins exist, their cellular roles and biochemical activities are not well defined. The overall goal of this grant is to determine how biochemical activities of mammalian formins influence their cellular function, with a particular focus on assembly of microvilli in lymphocytes. Circulating lymphocytes express three formins at high levels, one diaphanous formin (DRF1) and two highly homologous non-diaphanous formins (FRLa and a novel ORF termed mouse4). Both DRF1 and FRLa accelerate actin polymerization, but the specifics of these effects suggest different mechanisms. These proteins possess several interesting biochemical properties, including: multimerization; actin filament binding; and actin monomer binding. At least one formin (DRF1) localizes to lymphocyte microvilli. In Aim 1, mechanisms by which lymphocyte formins accelerate actin polymerization will be examined using biochemical and biophysical techniques. Amino acid sequences controlling lymphocyte forming biochemical properties will be mapped by mutagenesis. In addition, the role of profilin in formin-mediated actin polymerization acceleration will be examined. Aim 2 will elucidate regulatory mechanisms controlling formin-mediated polymerization acceleration, by determining effects of intramolecular interactions and interactions with other proteins. In Aim 3, the function of formins in lymphocytes will be examined, including cellular formin localization, effects of RNAi-induced protein suppression on lymphocyte actin structures, and effects on lymphocyte morphology of mutations disrupting formin biochemical properties. Parallel studies using Swiss 3T3 cells will examine roles of formins on structures not found in lymphocytes, such as lamellipodia and stress fibers. Biochemical results from Aims 1 and 2 will provide the basis for the cellular studies in Aim 3.