Formin-dependent Regulation of Subcellular Organization

肌动蛋白和微管(MT)网络密切协调，控制细胞的极性、细胞的运动和亚细胞的组织。因此，这两个系统之间存在着重要的相互作用，以确保它们协调行动。例如，肌动蛋白应激纤维作为轨迹将动态的MT引导到细胞皮质的受体，以促进MT的稳定，而动态的MT反过来通过调节Rho家族GTP酶的活性来调节肌动蛋白的动力学。Forin同源蛋白，或Forins，是一个高度保守的细胞骨架重塑蛋白家族，能够直接协调肌动蛋白和MT网络的调节。Formins是通过存在两个同源结构域，Forin Homology(FH)1和FH2来识别的，并通过FH1-FH2单位的协同作用来调节肌动蛋白和MT的动力学。我们发现INF1(也称为FHDC1)是Forin家族中唯一的MT结合成员。在最近提交的工作(附录A)中，我们发现INF1蛋白池聚集在高尔基体衍生的MT网络上，在那里它参与了肌动蛋白和MT依赖的核周高尔基体带的组装。高尔基体的组装依赖于中心体和高尔基体衍生的MT网络，中心体的重新定位和中心体的完整性也依赖于它与高尔基体的连接。这两个结构的定位是指导亚细胞组织的关键里程碑。我们将以我们与INF1的工作为起点，研究高尔基体组装过程中肌动蛋白和MT动力学的协调调节，以及这是如何作用于建立细胞极性的。*该计划的短期目标是继续调查INF1在高尔基组装中的作用。这些研究将集中于该蛋白如何被招募到高尔基体衍生的MT网络，并进一步确定其在高尔基体组装中作用的性质。我们研究计划的长期目标是确定如何协调不同的细胞骨架网络来建立和维护细胞的内部组织，以及这些系统如何整合到细胞的调节机制中。*目标1：控制高尔基体组装的蛋白质网络的特征。我们将使用BioID以一种无偏见的发现为基础的方法来识别INF1相互作用的蛋白质。*目标2：发展基于细胞的模型来研究高尔基体组装。CRISPR技术将被用来产生敲除和敲入细胞系，以便于研究控制高尔基体组装的因素。*目标3控制高尔基体来源MT组装的信号网络的特征。将采用多管齐下的方法来确定管理INF1招募到高尔基人的机制。

The actin and microtubule (MT) networks work in close coordination to govern cell polarity, cell motility and subcellular organization. Thus there is an essential cross-talk between these two systems to ensure they act in tandem. For example, actin stress fibers serve as the tracks that guide dynamic MTs to receptors at the cell cortex to facilitate MT stabilization while dynamic MTs in turn regulate actin dynamics by modulating the activity of Rho family GTPases. Formin homology proteins, or formins, are a highly conserved family of cytoskeletal remodeling proteins that are able to coordinate directly the regulation of both the actin and MT networks. Formins are identified by the presence of two domains of homology, Formin Homology (FH) 1 and FH2 and regulate both actin and MT dynamics through the concerted efforts of the functional FH1-FH2 unit. We identified INF1 (also known as FHDC1) as a unique MT-binding member of the formin family. In recently submitted work (appendix A) we find that a pool of INF1 protein accumulates on the Golgi-derived MT network where it participates in the actin and MT-dependent assembly of the perinuclear Golgi ribbon. Golgi ribbon assembly is dependent upon both centrosome- and Golgi-derived MT networks and centrosome re-orientation and centrosome integrity are also dependent upon its connections with the Golgi. Positioning of both these structures are key landmarks that guide subcellular organization. We will use our work with INF1 as a starting point to investigate the coordinated regulation of actin and MT dynamics during Golgi assembly and how this works to establish cell polarity. ***The short-term objective of this program is to continue to investigate the role of INF1 in Golgi assembly. These studies will focus on how this protein is recruited to the Golgi-derived MT network and further define the nature of its role in Golgi assembly. The long-term objective of our research program is to determine how distinct cytoskeletal networks are coordinated to establish and maintain the internal organization of the cell and how these systems are integrated into the cell's regulatory machinery.***Objective 1: Characterization of protein networks governing Golgi assembly. We will use BioID in a non-biased discovery-based approach to identify INF1 interacting proteins.***Objective 2: Development of cell-based models for studying Golgi assembly. CRISPR technology will be used to generate knockout and knock-in cell-lines to facilitate the investigation of the factors governing Golgi assembly.***Objective 3 Characterization of signaling networks governing assembly of Golgi-derived MTs. A multi-pronged approach will be used to identify the mechanisms that govern recruitment of INF1 to the Golgi.