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

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 摘要：福尔曼蛋白是肌动蛋白动力学的有力调节者。它们是大的、多结构域的蛋白质，与广泛的细胞过程有关，如细胞极化、黏附和胞质分裂。这些蛋白质的特征是具有一个保守的FH2结构域，该结构域介导与肌动蛋白的相互作用。FH2结构域形成未分支的肌动蛋白细丝，并与细丝的带刺末端紧密结合。肌动蛋白聚合的动力学研究以及FH2结合的细丝生长的实时成像表明，当肌动蛋白单体添加到带刺末端或从带刺末端解聚时，FH2结构域仍然稳定地结合在肌动蛋白细丝上，导致它们被描述为泄漏或过程帽。FH2结构域与肌动蛋白单体结合的晶体结构(在Rosen实验室中确定)导致了一种模型，在该模型中，FH2结构域存在于细丝带刺末端两种不同构象的快速平衡混合物中。在这个“成核棘轮”模型中，肌动蛋白单体添加到这些构象中的一个(可访问的)，并与另一个解离(被阻止)(参见下面相关出版物中的图5)。为了了解FH2结构域的过程封顶机制，我最近创造了FH2突变体，它稳定地结合了细丝的带刺末端，但完全阻止了带刺末端的延长和解聚。我们假设这些突变体中的一类被锁定在可接近的构象中，第二类被锁定在被阻止的构象中。我的研究目标是使用冷冻电子显微镜(Cryo-EM)和图像分析来确定FH2突变体与肌动蛋白细丝结合的三维图像。最终，我想确定野生型FH2结构域与肌动蛋白细丝结合的三维图像，并了解它是否可以描述为观察到的突变体的构象分布。 肌动蛋白细胞骨架是一种高度动态的结构，参与了从维持细胞形状和极性到细胞运动和细胞分裂的大量细胞过程。肌动蛋白动力学异常与多种人类疾病有关，如心血管疾病、神经变性和癌症(侵袭和转移)。肌动蛋白的动态是通过许多相互作用伙伴的作用来调节的，其中许多相互作用伙伴从酵母到人类都是保守的。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有益的帮助。

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.