Spatial control of actin assembly by phosphoinositides

 说明(申请人提供)：磷脂酰肌醇是一种膜磷脂，它控制许多细胞事件，并与数十种细胞内蛋白具有不同程度的特异性结合。这些阴离子磷脂的生产或代谢缺陷与癌症、神经元缺陷和其他疾病有关。在大多数细胞类型中含量最丰富的磷脂酰肌醇，PI4，5P2，对细胞过程中细胞骨架组装的调节特别重要 运动、分化和增殖在很大程度上是通过影响许多肌动蛋白结合蛋白，包括明胶蛋白、N-WASP和福尔马林，这些都是本项目的重点。PI4，5P2的其他异构体，如PI3，5P2，在几种不同的遗传疾病中与神经退行性变有关。与产生异常磷酸肌醇产物的突变相比，人们对特定的肌醇磷脂如何影响其配体的了解要少得多。确定这些脂类如何在膜-细胞骨架界面发挥生物控制作用，可能会导致限制或逆转这些脂类在疾病中的异常功能的新方法。前人的工作和初步数据表明，在生理相关水平的钙离子存在下，膜中低摩尔分数的PI4，5P2形成纳米级的团簇，当膜由于胆固醇含量或温度的变化而发生流体相变时，PI4，5P2分配到液体无序相中。PI4，5P2重新分布到这些纳米结构域中，改变了其从脑提取液中核化肌动蛋白组装的能力，并抑制了肌动蛋白细丝切断蛋白明胶蛋白的作用。该项目的目标是结合高分辨率成像、光谱和分子动力学计算，定量定义PI4，5P2重组为纳米级膜结构域的条件，并将PI4，5P2膜分布的变化与其抑制肌动蛋白组装的能力联系起来。生化分析将测试当PI4，5P2在膜中重新分布时，肌动蛋白调节系统的哪些成分受到影响。该项目涉及三个小组之间的合作，这些小组在膜生物物理、明胶生物化学和肌动蛋白聚合机制方面具有互补的经验；膜结构和力学的计算研究；以及电子显微镜，重点是高分辨率的膜上肌动蛋白组装研究。这项多学科的研究将导致对肌醇磷脂-蛋白质相互作用的原子水平的了解，这将有助于指导旨在改变肌醇磷脂的产生、分布和信号的策略，在许多情况下，它们改变的表达或分布与疾病有关。

 DESCRIPTION (provided by applicant): Phosphoinositides are membrane phospholipids that control many cellular events and bind with variable levels of specificity to dozens of intracellula proteins. Defects in production or metabolism of these anionic phospholipids are associated with cancer, neuronal defects, and other diseases. The most abundant phosphoinositide in most cells types, PI4,5P2, is particularly important for regulation of cytoskeletal assembly during cell motility, differentiation and proliferation largely through its effects on numerous actin binding proteins including gelsolin, N-WASP, and formins, which are the focus of this project. Other isomers of PI4,5P2, such as PI3,5P2, are associated with neurodegeneration in several genetically distinct diseases. How specific phosphoinositides affect their ligands is much less understood than are the mutations that produce abnormal phosphoinositide production. Defining how these lipids exert their biological control at the membrane-cytoskeletal interface could lead to new approaches to limiting or reversing the abnormal function of these lipids in disease. Previous work and preliminary data show that PI4,5P2 at low mole fraction in membranes forms nanoscale clusters in the presence of physiologically relevant levels of Ca2+ and partitions into the liquid disordered phase when membranes undergo fluid phase transitions due to changes in cholesterol content or temperature. Redistribution of PI4,5P2 into these nanodomains alters its ability to nucleate actin assembly from brain extracts and to inhibit gelsolin, the actin filament severing protein. The goal of this project is to quantitatively define the conditions under which PI4,5P2 reorganizes into nanoscale membrane domains using a combination of high resolution imaging, spectroscopy, and molecular dynamics computations and to relate changes in PI4,5P2 membrane distribution to its ability to inhibit actin assembly. Biochemical analyses will test which elements of the actin regulatory system are affected when PI4,5P2 redistributes in membranes. This project involves collaboration among three groups with complementary experience in membrane biophysics, gelsolin biochemistry and the mechanics of actin polymerization; computational studies of membrane structure and mechanics; and electron microscopy with emphasis on high resolution studies of actin assembly at membranes. The multi-disciplinary study will lead to an atomic level understanding of phosphoinositide-protein interactions that will help direct strategies designed to alter phosphoinositide production, distribution, and signaling in the numerous contexts where their altered expression or distribution is linked to disease.