Formin-dependent Regulation of Subcellular Organization

亚细胞组织的形式依赖性调节

• 批准号: RGPIN-2016-05921
• 负责人: Copeland, John
• 金额: $ 2.26万
• 依托单位: University of Ottawa
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2020
• 资助国家: 加拿大
• 起止时间: 2020-01-01 至 2021-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=710063
• 关键词: Formin; dependent; Regulation; Subcellular; Organization

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.

肌动蛋白和微管（MT）网络密切协调工作，以管理细胞极性，细胞运动和亚细胞组织。因此，在这两个系统之间存在着必要的交互作用，以确保它们协同工作。例如，肌动蛋白应力纤维充当将动态MT引导至细胞皮层处的受体以促进MT稳定的轨道，而动态MT反过来通过调节Rho家族GTP酶的活性来调节肌动蛋白动力学。形成蛋白同源蛋白，或形成蛋白，是一个高度保守的家族的细胞骨架重塑蛋白，能够直接协调的肌动蛋白和MT网络的调节。形成蛋白通过存在两个同源结构域，形成蛋白同源性（FH）1和FH 2来鉴定，并且通过功能性FH 1-FH 2单元的协同作用来调节肌动蛋白和MT动力学。我们鉴定了INF 1（也称为FHDC 1）作为一个独特的MT-结合的成员，该家族。在最近提交的工作（附录A）中，我们发现，一个池的INF 1蛋白聚集在高尔基体衍生的MT网络，在那里它参与肌动蛋白和MT依赖的组装的核周高尔基带。高尔基带组装依赖于中心体和高尔基体衍生的MT网络，中心体重新定向和中心体完整性也依赖于其与高尔基体的连接。 这两种结构的定位是指导亚细胞组织的关键标志。我们将使用我们的工作与INF 1作为起点，研究协调调节肌动蛋白和MT动态高尔基体组装过程中，以及如何建立细胞极性。 该计划的短期目标是继续研究INF 1在高尔基体组装中的作用。这些研究将集中在这种蛋白质是如何被招募到高尔基体衍生的MT网络，并进一步定义其在高尔基体组装中的作用的性质。我们研究计划的长期目标是确定不同的细胞骨架网络如何协调以建立和维持细胞的内部组织，以及这些系统如何整合到细胞的调节机制中。 目的1：研究高尔基体组装蛋白质网络的结构。我们将使用BioID在一个无偏见的发现为基础的方法来确定INF 1相互作用的蛋白质。 目标2：建立细胞模型研究高尔基体的组装。CRISPR技术将用于产生敲除和敲入细胞系，以促进对控制高尔基体组装的因素的研究。 目的3描述高尔基体衍生的MT组装的信号网络。一个多管齐下的方法将被用来确定的机制，管理招聘的INF 1高尔基体。