Regulation of septin polymerization in vitro

我的研究计划的长期目标是了解Septin聚合是如何控制的，开发出操纵聚合的方法，并确定这如何影响它们的功能。Septins是一个细胞骨架蛋白家族，从酵母到人类都是保守的。它们在广泛的生物过程中发挥作用，包括细胞迁移、胞质分裂、纤毛发生等，通过形成丝状结构将信号分子支架到所需的位置。在人类中，有13个Septin基因，分别属于4个家族。间隔蛋白以六聚体或八聚体的复合体形式存在，分别含有3个或4个间隔蛋白的两个拷贝，每个家族的一个成员存在于每个八聚体复合体中。结晶学揭示了六聚体中间隔素的组织，根据这一组织原则，六聚体和八聚体不应该能够共聚，尽管我们有证据表明它们可以共聚。尽管它们具有基本的重要性，但人们对它们的聚合调控机制知之甚少，尽管已有一系列CDC42效应蛋白(CDC42EP)参与其中。为了这个目标，我们开发了一种方法来纯化在哺乳动物细胞中表达的Septin六聚体和八聚体，并利用这些方法建立了使用光学和电子显微镜来测量它们的聚合的方法。在这项应用中，我建议表征Septin聚合特性并识别小分子抑制剂，以获得对这些重要但特性不佳的蛋白质的前所未有的洞察。目的1.影响Septin聚合的因素表征。通过用荧光蛋白标记单个间隔蛋白，我们已经证明了间隔蛋白六聚体和八聚体在体外可以形成大的细丝，并且令人惊讶的是，它们可以共聚。我们现在将确定影响聚合的生化因素，包括蛋白质浓度、离子条件、核苷酸状态以及膜结合对细丝形成的动力学和组织的贡献。我们还将评估将不同的家族成员替换到复合体中的效果，并确定CDC42EPs对Septin聚合和稳定性的作用。目的2.寻找Septin聚合的小分子调节剂。使用我们的荧光隔膜复合体和基于细胞的分析，我们将建立一个高通量的筛选，以寻找在没有CDC42EP或存在CDC42EPs的情况下抑制Septin聚合的小分子。在体外和体内破坏Septin细丝的分子将进一步表征它们对Septin依赖的细胞过程的影响，如细胞分裂、细胞迁移和纤毛形成。本申请中提出的研究将定义所有细胞的细胞骨架的主要组成部分的基本性质，并确定其抑制剂。从长远来看，这些研究将为研究这些蛋白质提供新的工具，并研究它们在大量细胞过程中的功能。

The long-term goal of my research program is to understand how septin polymerization is controlled, develop ways to manipulate polymerization, and determine how this affects their functions. Septins are a family of cytoskeletal proteins conserved from yeast to man. They function in a wide range of biological processes including cell migration, cytokinesis, ciliogenesis and many more, by forming filaments that scaffold signaling molecules to required sites. In humans there are 13 septin genes that fall into 4 families. Septins exist as complexes of hexamers or octamers containing two copies each of 3 or 4 septins, respectively, with one member of each family is present in each octameric complex. Crystallography has revealed the organization of septins within a hexamer, and based on this organizational principle, hexamers and octamers should not be able to co-polymerize, although we have evidence that they can. Despite their fundamental importance, very little is known about what regulates their polymerization, although a family of CDC42 effector proteins (CDC42EPs) have been implicated. Toward this goal we have developed a method to purify septin hexamers and octamers expressed in mammalian cells and have used these to establish methods to measure their polymerization using light and electron microscopy. In this application I propose to characterize septin polymerization properties and identify small molecule inhibitors to gain unprecedented insights into these important yet poorly characterized proteins. Aim 1. Characterization of factors affecting septin polymerization. By tagging individual septins with fluorescent proteins, we have demonstrated that septin hexamers and octamers can form large filaments in vitro, and surprisingly that they can co-polymerize. We will now determine biochemical factors that affect polymerization including protein concentration, ionic conditions, nucleotide status and the contribution of membrane binding to the kinetics and organization of filament formation. We will also assess the effect of substituting different family members into the complexes and determine the role of the CDC42EPs on septin polymerization and stability. Aim 2. Search for small molecule regulators of septin polymerization. Using our fluorescent septin complexes, and cell-based assays we will establish a high throughput screen to search for small molecules that inhibit septin polymerization in the absence or presence of CDC42EPs. Molecules that disrupt septin filament in vitro and in vivo will be further characterized for their effects on septin-dependent cellular processes such as cell division, cell migration and ciliogenesis. The studies proposed in this application will define the fundamental properties of a major component of the cytoskeleton of all cells and identify inhibitors for it. In the long term, these studies will provide new tools to study these proteins, and to study their function in a vast array of cellular processes.