Regulation of septin polymerization in vitro

我的研究计划的长期目标是了解如何控制septin聚合，开发操纵聚合的方法，并确定这如何影响它们的功能。septin是一个细胞骨架蛋白家族，从酵母到人类都保存了下来。它们在广泛的生物过程中发挥作用，包括细胞迁移、细胞分裂、纤毛发生等，通过形成细丝将信号分子支撑到所需的位点。人类有13个septin基因，属于4个家族。七聚体以六聚体或八聚体的复合体形式存在，分别含有3或4个七聚体的两个拷贝，每个八聚体复合体中存在每个家族的一个成员。晶体学揭示了六聚体内的九聚体的组织，根据这一组织原理，六聚体和八聚体不应该能够共聚合，尽管我们有证据表明它们可以共聚合。尽管CDC42效应蛋白家族（CDC42EPs）与它们的聚合有关，但人们对它们的聚合调控机制知之甚少。

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.