FORMIN HOMOLOGY 2 DOMAIN BOUND TO THE BARBED END OF AN ACTIN FILAMENT

这个子项目是许多利用 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 翻译后摘要：形成蛋白的肌动蛋白动力学是有力的监管机构。它们是大的多结构域蛋白，涉及广泛的细胞过程，如细胞极化，粘附和胞质分裂。这些蛋白质的特征在于介导与肌动蛋白相互作用的保守的FH 2结构域。FH 2结构域使未分支的肌动蛋白丝成核，并紧密结合到丝的倒刺末端。肌动蛋白聚合的动力学研究，以及FH 2结合的细丝生长的实时成像，表明FH 2结构域保持稳定地结合到肌动蛋白细丝上，因为肌动蛋白单体添加到倒刺末端上或从倒刺末端脱附，导致它们被描述为泄漏或进行性帽。与肌动蛋白单体结合的FH 2结构域的晶体结构（在罗森实验室中测定）导致了一个模型，其中FH 2结构域存在于细丝倒刺末端两种不同构象的快速平衡混合物中。 在这种“成核棘轮”模型中，肌动蛋白单体加入到这些构象之一（可接近），并与另一种构象分离（封闭）（见下文相关出版物中的图5）。 为了理解FH 2结构域的进行性加帽机制，我最近创建了FH 2突变体，其稳定地结合细丝的倒刺末端，但完全阻断倒刺末端的伸长和解聚。我们假设这些突变体中的一类被锁定在可接近构象中，第二类被锁定在封闭构象中。我的研究的目标是使用冷冻电子显微镜（cryo-EM）和图像分析来确定与肌动蛋白丝结合的FH 2突变体的三维图像。最后，我想确定一个三维图像的野生型FH 2域绑定到肌动蛋白丝，并了解它是否可以被描述为一个分布的构象观察到的突变体。 肌动蛋白细胞骨架是一种高度动态的结构，参与了大量的细胞过程，从维持细胞形状和极性，到细胞运动和细胞分裂。肌动蛋白动力学异常与多种人类疾病相关，如心血管疾病、神经退行性疾病和癌症（侵袭和转移）。肌动蛋白动力学是通过许多相互作用伴侣的作用来调节的，其中许多是从酵母到人类保守的。更好地了解这些调节蛋白的功能将扩大我们对肌动蛋白动力学基本机制的了解，并可能为人类疾病的诊断和治疗提供益处。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Abstract: Formin proteins are potent regulators of actin dynamics. They are large, multidomain proteins that are implicated in a wide range of cellular processes such as cell polarization, adhesion and cytokinesis. These proteins are characterized by a conserved formin homology 2 (FH2) domain that mediates interactions with actin. The FH2 domain nucleates unbranched actin filaments and binds tightly to the filament barbed end. Kinetic studies of actin polymerization, as well as real-time imaging of FH2-bound filament growth, suggest that the FH2 domains remain stably bound to the actin filament as the actin monomers add onto or depolymerize from the barbed ends, leading to their description as leaky or processive caps. The crystal structure of an FH2 domain bound to actin monomers (determined in the Rosen lab) led to a model in which the FH2 domain exists in a rapidly equilibrating mixture of two different conformations at the barbed end of a filament. In this "nucleating ratchet" model, actin monomers add to one of these conformations (accessible) and dissociate from the other (blocked) (see fig5 in the relevant publications below). To understand the mechanism of processive capping by FH2 domain, I recently created FH2 mutants that stably bind the barbed end of filaments, but completely block barbed end elongation and depolymerization. We hypothesize that one class of these mutants is locked in the accessible conformation and a second class is locked in blocked conformation. The goals of my research are to determine three-dimensional images of the FH2 mutants bound to actin filaments using cryo-electron microscopy (cryo-EM) and image analysis. Ultimately, I would like to determine a three-dimensional image of the wild type FH2 domain bound to actin filaments and learn whether it can be described as a distribution of the conformations observed for the mutants. The actin cytoskeleton is a highly dynamic structure that is involved in a large number of cellular processes, ranging from maintenance of cell shape and polarity, to cell motility and cell division. Abnormalities in actin dynamics are associated with a variety of human diseases, such as cardiovascular diseases, neurodegeneration, and cancer (invasion and metastasis). Actin dynamics are regulated through the action of a number of interaction partners, many of which are conserved from yeast to humans. A better understanding of the function of these regulatory proteins will expand our knowledge of the fundamental mechanisms of actin dynamics, and potentially, will provide benefits for diagnosis and therapy of human diseases.