REGULATION OF ACTIN FILAMENT ASSEMBLIES BY COFILIN/ADF

肌动蛋白在细胞运动中起着核心作用。随着细胞对外界信号的反应而移动，细胞骨架不断重塑。为了继续这一过程，肌动蛋白亚基必须不断地循环到前沿。尽管肌动蛋白的体外组装速度非常快，但在体外测量的尖端拆解速率常数太慢，无法回收这一过程继续下去所需的单体。最近的研究发现了一类肌动蛋白结合蛋白家族，它们通过切断肌动蛋白细丝和增加末端的拆解速度，在细胞内的肌动蛋白细丝动力学中发挥核心作用。Cofilin和ADF(肌动蛋白解聚因子)是一个蛋白质大家族的成员，在真核生物的整个光谱中都是保守的。它们是酵母中肌动蛋白细丝周转的关键角色，在所有被测试的生物体中都是必不可少的。未能正确调控Cofilin会导致人类的Williams综合征，而Cofilin的点突变会导致秀丽隐杆线虫的瘫痪。因此，阐明cofilin如何调节肌动蛋白组装对于理解细胞的正常和疾病状态都具有重要的意义。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。这项研究的目的是验证这样一种假设，即这种对F-肌动蛋白结构的影响代表了一种调节细胞内肌动蛋白动态和组装的新机制。这项研究的第一个目标是更详细地模拟cofilin/F-肌动蛋白的相互作用。胶原蛋白将被金标记在特定残基上，这些残基将被用作标记，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限制(27埃单位)。这项提案的第二个目标是使用基因工程和自然产生的变体来识别赋予Cofilin独特肌动蛋白调控特性的特定残基。第三个目标是通过确定导致这种疾病的粘连蛋白突变体如何与F-肌动蛋白相互作用来探索线虫粘连蛋白依赖性肌肉疾病的分子基础。这个项目将检验这样一种假设，即粘连蛋白对肌动蛋白细丝结构的影响是其在多细胞生物体中发挥功能的中心。我们的第四个目标是探索这一假说，即Cofilin通过其与细丝结合的方式促进细胞中替代肌动蛋白组装的形成。这将包括对体外产生的非螺旋粘连蛋白/肌动蛋白组合体以及在病变细胞的细胞质和细胞核中发现的粘连蛋白/肌动蛋白棒的结构研究。

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.