REGULATION OF ACTIN FILAMENT ASSEMBLIES BY COFILIN/ADF

肌动蛋白在细胞运动中起核心作用。当细胞响应外部信号移动时，细胞骨架不断被重塑。为了使这一过程继续进行，肌动蛋白亚基必须不断地再循环到前缘。尽管肌动蛋白在体外的组装速度非常快，但体外测量的尖端拆卸速率常数太慢，无法继续回收该过程所需的单体。最近的研究发现了一个肌动蛋白结合蛋白家族，它通过切断纤维和增加末端的分解速率，在细胞的肌动蛋白丝动力学中起核心作用。Cofilin和ADF （Actin Depolymerizing Factor，肌动蛋白解聚因子）是真核生物中保守的一大家族蛋白的成员。它们是酵母中肌动蛋白丝周转的关键参与者，在所有被测试的生物体中都是必不可少的。在人类中，不能适当地调节cofilin导致Williams综合征，而在秀丽隐杆线虫中，cofilin的点突变导致瘫痪。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病变状态具有重要意义。在去年发表的电子低温显微镜研究中，我展示了cofilin在结合时显著改变了F-actin的结构。本研究的目的是验证这种对f -肌动蛋白结构的影响代表了调节肌动蛋白动力学和细胞内组装的新机制。本研究的第一个目标是更详细地模拟cofilin/F-actin相互作用。Cofilin将用金标记在特定的残基上，这些残基将用作标记物，以便在我们的重建中准确定位x射线模型。此外，我们将利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。本提案的第二个目标是使用基因工程和自然发生的变体来识别赋予cofilin独特的肌动蛋白调节特性的特定残基。第三个目标是通过确定导致这种疾病的cofilin突变体如何与F-actin相互作用来探索线虫cofilin依赖性肌肉疾病的分子基础。这个项目将测试一个假设，即cofilin对肌动蛋白丝结构的影响是其在多细胞生物中的功能的核心。我们的第四个目标是探索cofilin通过其与丝的结合模式促进细胞中替代肌动蛋白组装形成的假设。这将涉及在体外产生的非螺旋型cofilin/actin组合的结构研究，以及在患病细胞的细胞质和细胞核中发现的cofilin/actin棒。

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.