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，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，并通过功能上的FH1-FH2单位的协同作用来调节肌动蛋白和MT的动力学。我们鉴定FHDC1是一个独特的MT结合形式家族成员。我们已经证明，FHDC1蛋白作用于不同的MT网络，控制间期高尔基带的组装和静止细胞中初级纤毛的组装。这些作用都是肌动蛋白和MT依赖的，这表明FHDC1在这些不同细胞器的组装过程中协调这两个细胞骨架网络的组织。作为我们工作的一部分，我们发现FHDC1的缺失或过度表达对纤毛分解和母中心粒的成熟有深远的影响。这些效应部分是通过FHDC1与Cep170的相互作用而介导的，Cep170是一种蛋白质，发现于母中心粒的亚远端附属物，将FHDC1的活性与中心体动力学的中央调节因子PLK1联系起来。该计划的短期目标是继续研究FHDC1在母中心粒组装和初级纤毛形成过程中在亚远端附属物中的作用。这些研究将包括调查FHDC1是如何被招募到基底体的，并进一步确定其在纤毛组装中作用的性质。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。目的1.测定FHDC1活性对纤毛超微结构的影响。超分辨和电子显微镜将被用来研究FHDC1耗尽和过度表达对原代纤毛超微结构的影响，并确定FHDC1在中心粒的纳米定位。目的2.确定FHDC1在中心粒成熟调控中的作用。在缺乏和过度表达FHDC1的细胞中，将使用活的和固定的细胞成像来跟踪中心体的定位和成熟。目的3.描述针对FHDC1到不同MT网络的信号通路。多管齐下的方法将被用来确定管理FHDC1招募到不同的细胞质MT网络的机制。

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 directly coordinate the regulation of both actin and MT networks. Formins are identified by the presence of two domains of homology, Formin Homology (FH) 1 and FH2, and regulate actin and MT dynamics through the concerted efforts of the functional FH1-FH2 unit. We identified FHDC1 as a unique MT-binding formin family member. We have shown that FHDC1 protein acts at separate MT networks to govern both assembly of the Golgi ribbon during interphase and assembly of the primary cilia in quiescent cells. These effects are both actin and MT-dependent, suggesting that FHDC1 coordinates the organization of these two cytoskeletal networks during the assembly of these distinct organelles. As part of our work we found that depletion or over-expression of FHDC1 has profound effects on cilia disassembly and maturation of the mother centriole. These effects are mediated in part by the interaction of FHDC1 with Cep170, a protein found at the subdistal appendages of the mother centriole that links FHDC1 activity to the kinase PLK1, a central regulator of centrosome dynamics. The short-term objective of this program is to continue to investigate the role of FHDC1 at the subdistal appendages during assembly of the mother centriole and formation of the primary cilia. These studies will include investigating how FHDC1 is recruited to the basal body and further define the nature of its role in cilia 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. Aim 1. Determine the effects of FHDC1 activity on cilia ultrastructure. Super-resolution and electron microscopy will be used to investigate the effects of FHDC1 depletion and over-expression on the ultrastructure of the primary cilia and determine the nanoscale localization of FHDC1 at the centriole. Aim 2. Determine the role of FHDC1 in the regulation of centriole maturation. Live and fixed cell imaging will be used to follow centrosome positioning and maturation in cells lacking and over-expressing FHDC1. Aim 3. Characterize the signaling pathways that target FHDC1 to distinct MT networks. A multi-pronged approach will be used to identify the mechanisms that govern recruitment of FHDC1 to distinct cytoplasmic MT networks.