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REGULATION OF ACTIN FILAMENT ASSEMBLIES BY COFILIN/ADF

COFILIN/ADF 对肌动蛋白丝组件的调节

基本信息

批准号：
6637246
负责人：
AMY M MCGOUGH
金额：
$ 20.12万
依托单位：
PURDUE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
1999
资助国家：
美国
起止时间：
1999-08-01 至 2005-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/6637246
关键词：
Caenorhabditis elegans SDS polyacrylamide gel electrophoresis actin binding protein actins cryoelectron microscopy microfilaments physical model polymerization protein binding protein biosynthesis protein structure function transmission electron microscopy

项目摘要

Actin plays a central role in cell motility. As cells move in response to external signals, the cytoskeleton is constantly remodeled. For this process to continue, actin subunits must be continuously recycled to the leading edge. Although the assembly rates of actin in vitro are extremely rapid, the pointed end disassembly rate constant measured in vitro is far too slow to recycle the monomers necessary for this process to continue. Recent studies have identified a family of actin binding proteins that play a central role in actin filament dynamics in cells by both severing filaments and increasing the rate of disassembly from ends. Cofilin and ADF (Actin Depolymerizing Factor) are members of a large family of proteins that is conserved across the complete spectrum of eukaryotic organisms. They are key players in actin filament turnover in yeast and are essential in all organisms tested. Failure to regulate cofilin properly leads to Williams syndrome in man, while point mutations in cofilin produce paralysis in Caenorhabiditis elegans. Thus, elucidating how cofilin regulates actin assembly has important implications for understanding both normal and diseased states in cells. In electron cryomicroscopy studies published last year, I showed that cofilin dramatically alters F-actin structure when it binds. The purpose of the proposed research is to test the hypothesis that this effect on F-actin structure represents a novel mechanism for regulating actin dynamics and assembly in cells. The first goal of this study is to model cofilin/F-actin interactions in greater detail. Cofilin will be labeled with gold on specific residues which will be used as markers to accurately position the x-ray model in our reconstruction. In addition, we will extend the resolution of the current reconstruction beyond its present limit (27 Angstrom units) using improved experimental and computational methods. The second goal of this proposal is to use both genetically-engineered and naturally-occurring variants to identify the specific residues that endow cofilin with its unique actin regulatory properties. The third goal is to explore the molecular basis of a cofilin-dependent muscle disease in C. elegans by determining how cofilin mutants responsible for this disease interact with F-actin. This project will test the hypothesis that cofilin's effects on actin filament structure are central to its function in multi-cellular organisms. Our fourth goal is to explore the hypothesis that cofilin promotes the formation of alternative actin assemblies in cells through its mode of binding to the filament. This will involve structural studies of non-helical cofilin/actin assemblies produced in vitro as well as of cofilin/actin rods found in the cytoplasm and nuclei of diseased cells.

肌动蛋白在细胞运动中起核心作用。随着细胞响应外部信号而移动，细胞骨架不断重塑。为了使这一过程继续下去，肌动蛋白亚基必须不断地循环到前沿。虽然肌动蛋白在体外的组装速率非常快，但在体外测量的尖端拆卸速率常数太慢，以至于无法回收该过程继续所需的单体。最近的研究已经确定了一个家庭的肌动蛋白结合蛋白，发挥核心作用，在肌动蛋白丝动力学在细胞中的两个切断丝和增加解体的速度从结束。 Cofilin和ADF（肌动蛋白解聚因子）是在真核生物的整个谱中保守的蛋白质大家族的成员。它们是酵母中肌动蛋白丝周转的关键参与者，在所有测试的生物体中都是必不可少的。不能正确调节cofilin导致人类威廉姆斯综合征，而cofilin的点突变导致秀丽隐杆线虫瘫痪。因此，阐明cofilin如何调节肌动蛋白组装对于理解细胞中的正常和疾病状态具有重要意义。在去年发表的电子冷冻显微镜研究中，我发现当cofilin结合时，它会显著改变F-肌动蛋白的结构。拟议的研究的目的是测试的假设，这种影响F-肌动蛋白结构代表了一种新的机制，调节肌动蛋白的动力学和组装在细胞中。本研究的第一个目标是更详细地模拟cofilin/F-actin相互作用。Cofilin将在特定残基上用金标记，这些残基将用作标记物，以在我们的重建中准确定位X射线模型。此外，我们将使用改进的实验和计算方法，将当前重建的分辨率扩展到目前的极限（27埃单位）之外。该提案的第二个目标是使用基因工程和天然存在的变体来鉴定赋予cofilin独特的肌动蛋白调节特性的特定残基。第三个目标是探索C.通过确定如何cofilin突变体负责这种疾病与F-肌动蛋白相互作用。这个项目将测试的假设，cofilin的肌动蛋白丝结构的影响是其在多细胞生物体中的功能的核心。我们的第四个目标是探索cofilin通过其与细丝结合的模式促进细胞中替代肌动蛋白组装体的形成的假设。这将涉及在体外产生的非螺旋cofilin/肌动蛋白组件的结构研究，以及在患病细胞的细胞质和细胞核中发现的cofilin/肌动蛋白棒。