REGULATION OF ISOACTIN DYNAMICS IN LIVING CELLS

描述：本文件中描述的研究的实验结果 五年研究计划应为等肌动蛋白提供重要的新见解 异肌动蛋白的功能多样性及其调控的分子机制 对细胞运动的控制。建议书中提供的初步数据和 最近发表的结果确定了一种蛋白质复合体，b肌动蛋白，Ezrin和 一种名为bcap73的b肌动蛋白特异性结合蛋白，它在 膜突出。在具体目标1中，调查员将使用细胞 微量注射和荧光光活化技术定量测定体内异肌动蛋白 野生型和突变型细胞系中的细丝动力学/功能 肌动蛋白缺乏或有缺陷。批判性地评估非肌肉组织 研究人员称，等肌动蛋白细丝具有独特的细胞特异性功能 将向细胞中引入阻断功能的异肌动蛋白特异性抗体 可以区分b和g肌动蛋白。他预计，以下各项的综合结果 荧光肌动蛋白等蛋白的光激活与抗等肌动蛋白 野生型和突变型细胞的功能丧失研究将揭示 B肌动蛋白细丝动力学足以驱动细胞质在 领先优势。在特定目标2中，调查员将测试 Bcap73单独或与Ezrin共同调控b的特定假说 肌动蛋白成核和微丝组装。这些活动对于 细胞运动过程中的细胞质重塑。定量iso-f-actin 使用全长或截短bcap73和bcap73的组合的结合分析 Ezrin，将识别核化或覆盖b-肌动蛋白的结构域，但不识别其他 肌动蛋白亚型。来自这些体外检测的数据将与 两种新型荧光鬼臼蛋白检测方法的实验结果 揭示异肌动蛋白聚合动力学和柔韧性是否 唯一；以及bcap73或其他肌动蛋白封顶/切断蛋白 影响这些行为。与此同时，他们将把bcap73描述为 野生型和突变型细胞系。这些结果应该会提供新的见解 等肌动蛋白、它们的结合蛋白和膜之间的相互作用 在发育和疾病过程中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.