Spatial control of actin assembly by phosphoinositides

 描述（由申请人提供）：磷脂酰肌醇是一种膜磷脂，可控制许多细胞事件，并以不同水平的特异性与数十种细胞内蛋白质结合。这些阴离子磷脂的产生或代谢缺陷与癌症、神经元缺陷和其他疾病有关。在大多数细胞类型中最丰富的磷酸肌醇，PI 4，5 P2，对于细胞生长过程中细胞骨架组装的调节特别重要。 运动，分化和增殖，主要是通过其对众多肌动蛋白结合蛋白的影响，包括凝溶胶蛋白，N-WASP，和formins，这是该项目的重点。PI 4，5 P2的其他异构体，如PI 3，5 P2，与几种遗传上不同的疾病中的神经变性有关。具体的磷酸肌醇是如何影响其配体的理解比突变，产生异常磷酸肌醇生产少得多。确定这些脂质如何在膜-细胞骨架界面发挥其生物控制作用，可能会导致限制或逆转这些脂质在疾病中的异常功能的新方法。先前的工作和初步数据表明，PI 4，5 P2在膜中的低摩尔分数形成纳米级簇的存在下，生理相关水平的Ca 2+和分区到液体无序相时，膜进行流体相变由于胆固醇含量或温度的变化。PI 4，5 P2重新分布到这些纳米结构域中改变了其使来自脑提取物的肌动蛋白组装成核和抑制凝溶胶蛋白（肌动蛋白丝切断蛋白）的能力。该项目的目标是定量定义PI 4，5 P2重组成纳米级膜域的条件下，使用高分辨率成像，光谱学和分子动力学计算的组合，并将PI 4，5 P2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PI 4，5 P2在膜中重新分布时，肌动蛋白调节系统的哪些元件受到影响。该项目涉及三个小组之间的合作，在膜生物物理学，凝溶胶蛋白生物化学和肌动蛋白聚合力学的互补经验;膜结构和力学的计算研究;和电子显微镜，重点是肌动蛋白组装在膜的高分辨率研究。多学科研究将导致对磷酸肌醇-蛋白质相互作用的原子水平的理解，这将有助于指导旨在改变磷酸肌醇生产，分布和信号传导的策略，在许多情况下，它们的改变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.