Comparative molecular physiology of mammalian formins

哺乳动物福明的比较分子生理学

• 批准号: 7175337
• 负责人: HENRY N HIGGS
• 金额: $ 26.53万
• 依托单位: DARTMOUTH COLLEGE
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-02-01 至 2009-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7175337
• 关键词: Ablation; Acceleration; Actins; Amino Acid Sequence; Binding; Biochemical; Cell physiology; Cells; Fibroblasts; Goals; Grant; In Vitro; Kinetics; Length; Localized; Lymphocyte; Maps; Mediating; Microfilaments; Molecular; Morphology; Mutagenesis; Mutation; Open Reading Frames; Physiology; Property; Proteins; RNA Interference; RNA Splicing; Regulation; Role; Stress Fibers; Structure; Swiss 3T3 Cells; Techniques; Testing; Variant; Yeasts; base; cellular microvillus; comparative; gastrointestinal microvillus; interest; monomer; novel; polymerization; profilin; rho GTP-Binding Proteins

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.

描述（由申请人提供）：含细胞肌动蛋白结构的多样性需要以特定于上下文特定方式来形成肌动蛋白丝形成的多种机制。由于纯化的肌动蛋白单体在体外缓慢聚合，因此细胞必须具有加速肌动蛋白聚合的因素。来自酵母菌的最新证据表明，在体外，formin蛋白在体外加速了肌动蛋白聚合，并且对于产生特定的含细胞肌动蛋白的结构，例如电缆和细胞力学裂解沟。尽管至少存在12种哺乳动物肌肉，但它们的细胞作用和生化活性尚未得到很好的定义。 该赠款的总体目标是确定哺乳动物造型的生化活性如何影响其细胞功能，特别着重于淋巴细胞中微绒毛的组装。循环淋巴细胞在高水平，一个diaphanous formin（DRF1）和两个高度同源的非脑形formins（FRLA和一种新型的ORF称为Mouse4）上表达三个formins。 DRF1和FRLA都加速了肌动蛋白聚合，但是这些作用的细节表明了不同的机制。这些蛋白质具有几种有趣的生化特性，包括：多聚化；肌动蛋白丝结合；和肌动蛋白单体结合。至少一个formin（DRF1）定位于淋巴细胞微绒毛。 在AIM 1中，将使用生化和生物物理技术检查淋巴细胞formins加速肌动蛋白聚合的机制。控制淋巴细胞形成生化特性的氨基酸序列将由诱变绘制。此外，将研究叶酸蛋白酶在formin介导的肌动蛋白聚合加速度中的作用。 AIM 2将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来阐明控制形式介导的聚合加速的调节机制。在AIM 3中，将检查造型在淋巴细胞中的功能，包括细胞甲素定位，RNAI诱导的蛋白抑制对淋巴细胞肌动蛋白结构的影响，以及对破坏formin formin formin生化特性突变的淋巴细胞形态的影响。使用瑞士3T3细胞的平行研究将检查造型在淋巴细胞中未发现的结构（例如薄片和应激纤维）中的作用。 AIM 1和2的生化结果将为AIM 3中的细胞研究提供基础。