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，开发操纵聚合的方法，并确定这如何影响它们的功能。Septins是一个从酵母到人类的细胞骨架蛋白家族，在细胞迁移、胞质分裂、纤毛发生等生物学过程中发挥重要作用，通过形成细胞丝将信号分子固定在细胞需要的位点。在人类中，有13个septin基因属于4个家族。间隔蛋白作为六聚体或八聚体的复合物存在，所述复合物分别含有3个或4个间隔蛋白中的每一个的两个拷贝，其中每个家族的一个成员存在于每个八聚体复合物中。晶体学已经揭示了六聚体中septins的组织，基于这种组织原则，六聚体和八聚体应该不能共聚合，尽管我们有证据表明它们可以。尽管它们具有根本的重要性，但很少有人知道是什么调节它们的聚合，尽管一个家族的CDC42效应蛋白（CDC42EPs）已被牵连。 为了这个目标，我们已经开发了一种方法来纯化septin六聚体和八聚体在哺乳动物细胞中表达，并已使用这些建立方法来测量其聚合使用光和电子显微镜。在这个应用程序中，我建议的特性septin聚合特性，并确定小分子抑制剂，以获得前所未有的见解，这些重要的，但很少表征的蛋白质。目标1. Septin聚合影响因素的表征。通过用荧光蛋白标记单个septins，我们已经证明septins六聚体和八聚体可以在体外形成大的细丝，并且令人惊讶的是它们可以共聚合。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取代到复合物中的效果，并确定CDC42EPs对Septin聚合和稳定性的作用。目标2.寻找Septin聚合的小分子调节剂。使用我们的荧光Septin复合物和基于细胞的测定，我们将建立高通量筛选以寻找在不存在或存在CDC42EP的情况下抑制Septin聚合的小分子。在体外和体内破坏隔蛋白丝的分子将进一步表征其对隔蛋白依赖性细胞过程如细胞分裂、细胞迁移和纤毛发生的影响。本申请中提出的研究将确定所有细胞的细胞骨架的主要成分的基本性质，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.