REGULATION OF ISOACTIN DYNAMICS IN LIVING CELLS

描述：实验结果来源于本文中描述的研究。 五年研究计划将为isoactin提供重要的新见解 功能多样性和调节isoactin的分子机制 控制细胞运动。 提案中提供的初步数据， 最近发表的结果鉴定了一种蛋白质、B肌动蛋白、埃兹蛋白和 一种名为bcap 73的B肌动蛋白特异性结合蛋白，其在 膜突起 在具体目标1中，研究者将使用细胞 显微注射和荧光光活化以定量体内异肌动蛋白 在野生型和突变细胞系中的丝动力学/功能， 在B肌动蛋白上缺乏或有缺陷。 为了严格评估非肌肉 异肌动蛋白丝执行独特的细胞特异性功能， 将向细胞中引入功能阻断isoactin特异性抗体， 可以区分B和g肌动蛋白。 他预计， 荧光肌动蛋白同工蛋白光活化和抗同工蛋白 在野生型和突变型细胞中进行的“功能丧失”研究将揭示 B肌动蛋白丝动力学足以驱动细胞质扩张， 领先的优势。 在具体目标2中，研究者将测试 bcap 73单独或与ezrin一起调节B的特定假设 肌动蛋白成核和细丝组装。 这些活动对于以下方面至关重要： 细胞运动过程中的细胞质重塑。 定量异F-肌动蛋白 使用全长或截短的BCAP 73和 埃兹蛋白，将确定的结构域，核或帽B肌动蛋白，而不是其他 肌动蛋白亚型。 这些体外试验的数据将与 设计的两种新的荧光鬼笔环肽测定的实验结果 以揭示异肌动蛋白聚合动力学和柔性是否 以及bcap 73或其他肌动蛋白加帽/切断蛋白 影响这些行为。 与此同时，他们将表征bcap 73在 野生型和突变型细胞系。 这些结果应该提供新的见解 异肌动蛋白，它们的结合蛋白和膜之间的相互作用 其在发育和疾病期间引起功能性细胞运动。

DESCRIPTION: Experimental results derived from studies described in this five year research plan should provide essential new insights into isoactin functional diversity and the molecular mechanisms regulating isoactin-based control of cell motility. Preliminary data presented in the proposal and recent published results identify a complex of proteins, b actin, ezrin and a b actin-specific binding protein named bcap73, which play a role in membrane protrusion. In specific aim 1, the investigator will use cell microinjection and fluorescence photoactivation to quantify in vivo isoactin filament dynamics/function in wild-type and mutant cell lines which are deficient or defective in b actin. To assess critically whether non-muscle isoactin filaments perform unique cell-specific functions, the investigator will introduce into cellsfunction-blocking isoactin-specific antibodies that can discriminate b vs g actin. He anticipates that the combined results of fluorescent actin isoprotein photoactivation and anti-isoactin 'loss-of-function' studies in wild type and mutant cells will reveal whether b actin filament dynamics is sufficient to drive cytoplasmic expansion at the leading edge. In specific aim 2, the investigator will test the specific hypothesis that bcap73, either alone or with ezrin, regulates b actin nucleation and filament assembly. These activities are essential for cytoplasmic remodelling during cell motility. Quantitative iso-f-actin binding assays using combinations of full length or truncated bcap73 and ezrin, will identify the domains that nucleate or cap b actin but not other actin isoforms. Data from these in vitro assays will be compared with results of experiments from two novel fluorescent-phalloidin assays designed to reveal whether isoactin polymerization kinetics and flexibility are unique; and whether bcap73 or other actin capping/severing proteins influence these behaviors. Concomitantly, they will characterize bcap73 in wild type and mutant cell lines. These results should offer novel insights into interactions between isoactin, their binding proteins, and the membrane which give rise to functional cell motility during development and disease.