Role of the Diaphanous Formin FHOD1 and its interaction with nesprin-2-giant in nuclear migration

FHOD1是透明相关双Formin （DRF）蛋白家族的非典型成员，它不参与肌动蛋白聚合，但能束成肌动蛋白丝，修饰细胞肌动蛋白结构，并与微管细胞骨架协调肌动蛋白。我们之前的工作发现FHOD1是控制迁移成纤维细胞核运动机制的组成部分，这对于肌动蛋白介导的细胞核定位以及中心体重新定位至关重要。为了促进核定位，FHOD1通过n端肌动蛋白结合位点与肌动蛋白链结合，并与外核包膜蛋白nesprin2 -giant （N2G）相互作用。在确定了FHOD1如何促进肌动蛋白依赖核定位的基本原理之后，本提案的第一个目标是在结构和功能水平上深入了解这种不寻常的机制。为此，我们将精确定义FHOD1与N2G和肌动蛋白相互作用所涉及的表面，表征其生化和功能特性，并研究其调控机制。这将包括生成两个结合伙伴的相互作用域之间的配合物的x射线结构，最终目标是解决所有三个相互作用域的三方配合物的结构。这些分析还允许我们定义分子特征，以确定其在DRF蛋白中的非典型作用模式。该项目的第二个主要目标是阐明FHOD1- n2g相互作用是如何被调节的，以及哪些额外的FHOD1结合伙伴参与了FHOD1的功能。我们的初步结果确定了激酶2蛋白KIF3C是核迁移所必需的新的FHOD1结合伙伴。机制分析现在将集中在剖析FHOD1在核迁移中协调肌动蛋白和微管细胞骨架的机制，特别关注KIF3C和其他新发现的FHOD1配体的作用。这种跨学科的方法结合了Fackler和Geyer实验室在细胞生物学和结构生物学/生物化学方面的专业知识，将对非典型DRF FHOD1用于最大细胞器动态运输的分子机制产生重要的新见解。

FHOD1 is an atypical member of the Diaphanous Related Formin (DRF) protein family that does not nucleate actin polymerization but bundles actin filaments, decorates cellular actin structures, and coordinates actin with microtubule cytoskeletons. Our previous work identified FHOD1 as component of the machinery governing nuclear movement of migrating fibroblasts that is essential for actin-mediated positioning of the nucleus as well as for centrosome reorientation. To facilitate nuclear positioning, FHOD1 associates with actin cables via an N-terminal actin binding site and interacts with the outer nuclear envelope protein nesprin-2-giant (N2G). Having established this basic principle of how FHOD1 facilitates actin dependent nuclear positioning, the first goal of this proposal is to gain insight into this unusual mechanism at the structural and functional level. To this end we will define precisely the surfaces involved in the interactions of FHOD1 with N2G and actin, characterize their biochemical and functional properties, and investigate regulatory mechanisms. This will include the generation of X-ray structures of complexes between the interacting domains of two binding partners with the final goal of solving the structure of the tripartite complex of all three interacting domains. These analyses should also allow us to define the molecular signatures that determine its atypical mode of action among DRF proteins. The second main goal of this project is to elucidate how FHOD1-N2G interactions are regulated and which additional FHOD1 binding partners are involved in FHOD1 function. Our preliminary results identified the kinesin-2 protein KIF3C as novel FHOD1 binding partner essential for nuclear migration. Mechanistic analyses will now focus on dissecting the mechanisms by which FHOD1 coordinates actin and microtubule cytoskeletons in nuclear migration with particular focus on the role of KIF3C and other newly identified FHOD1 ligands. Together, this interdisciplinary approach combining the expertise in cell biology and structural biology/biochemistry of the Fackler and Geyer laboratories will yield important new insights into the molecular mechanism used by the atypical DRF FHOD1 for the dynamic transport of the largest cellular organelle.