Directing membrane function with inositol lipids in health and disease

在健康和疾病中用肌醇脂质指导膜功能

• 批准号: 9135008
• 负责人: Gerald R Hammond
• 金额: $ 35.28万
• 依托单位: UNIVERSITY OF PITTSBURGH AT PITTSBURGH
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-08-01 至 2021-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9135008
• 关键词: Automobile Driving; Biological Process; Cell Culture Techniques; Cell membrane; Cell physiology; Cells; Cellular biology; Communicable Diseases; Disease; Ensure; Enzymes; Feedback; Foundations; Goals; Health; Hepatitis C virus; Hereditary Disease; Image; Individual; Inositol; Knowledge; Life; Lipids; Membrane; Mission; Modeling; Molecular; Mutation; Organism; Pathogenesis; Phenotype; Phosphatidylinositol 4,5-Diphosphate; Phosphatidylinositols; Phospholipids; Phosphoric Monoester Hydrolases; Physiological; Physiology; Prevention; Process; Proteins; Public Health; Regulation; Research; Resolution; Role; Route; Signal Transduction; Site; System; Virus Diseases; chemical genetics; innovation; insight; nanometer; novel strategies; operation; optical imaging; protein activation; trafficking

PROJECT SUMMARY/ABSTRACT Tightly regulated flux of materials and signals across the exquisitely organized plasma membrane (PM) is essential for healthy cellular function. Disruption of this careful choreography is a common mechanism underlying the pathogenesis of many genetic and infectious diseases. Therefore, a central problem in cell biology is to understand the key molecular components that direct this intricate organization of signaling, transport and structural machinery at the PM. A phospholipid located in the cytosolic leaflet, phosphatidylinositol 4,5-bisphosphate (PIP2), is a key regulator of PM function, controlling recruitment and/or activation of this protein machinery. Yet how PIP2 levels are regulated to ensure each PM function has access to enough lipid to ensure correct operation, and how PIP2 is able to regulate each function discretely, is poorly understood. The goal of our research is therefore to develop a detailed mechanistic understanding of how cells regulate PIP2 levels in the PM, and how this facilitates regulation of individual PIP2-dependent functions. The goal of this application is to identify fundamental mechanisms in cell culture models, and to apply the new insights and approaches to physiological and disease-relevant systems through our established network of collaborators. Firstly, we will determine the nanoscopic organization of PIP2 molecules in the PM and determine their enrichment at sites of specific PM function. To accomplish this goal, we will probe and manipulate lipid enrichment at sites of cytoskeletal, signaling or trafficking functions with nanometer resolution, using super-resolution optical imaging approaches and chemical genetics. Secondly, we will delineate the mechanisms that regulate global PM PIP2 levels, by identifying the molecular components driving negative feedback of PIP2 synthesis. Thirdly, we will identify the biological functions of PIP2 5-phosphatase enzymes, as well as the mechanism of pathogenesis for disease-associated mutations in these enzymes. To accomplish this goal, we will identify where endogenous 5-phosphatase enzymes act in the cell, where PIP2 accumulates after loss of these enzymes, and what cellular phenotypes are triggered by the resulting accumulation of PIP2. We will employ innovative approaches throughout, combining super-resolution imaging of PIP2 and its myriad effector proteins with chemical genetics to acutely manipulate PIP2 with exquisite spatial and temporal precision. The proposed research is significant because it will uncover fundamental mechanisms that choreograph the interplay of PM functions, and consequently provide a crucial first step in developing new approaches to experimentally or therapeutically manipulate these functions in isolation.

项目总结/摘要 通过精细组织的质膜（PM）的物质和信号的严格调节的通量是 对健康的细胞功能至关重要。破坏这种精心编排是一种常见的机制 是许多遗传性和传染性疾病的发病机制的基础。因此，细胞中的一个中心问题是， 生物学就是要理解指导这种复杂的信号组织的关键分子成分， 运输和结构机械一种位于胞质小叶中的磷脂， 磷脂酰肌醇4，5-二磷酸（PIP 2）是PM功能的关键调节剂，控制募集和/或 激活这种蛋白质机制。然而，如何监管PIP 2级别以确保每个PM功能都可以访问 足够的脂质来确保正确的操作，以及PIP 2如何能够独立地调节每个功能， 明白因此，我们研究的目标是对细胞如何 调节PM中的PIP 2水平，以及这如何促进个体PIP 2依赖性功能的调节。的 本申请的目的是确定细胞培养模型中的基本机制，并应用新的 通过我们建立的网络， 合作者首先，我们将确定PM中PIP 2分子的纳米级组织， 确定它们在特定PM功能部位的富集程度。为了实现这一目标，我们将探索和 以纳米分辨率操纵细胞骨架、信号传导或运输功能位点处的脂质富集， 使用超分辨率光学成像方法和化学遗传学。其次，我们将描述 调节全球PM PIP 2水平的机制，通过识别驱动负性的分子成分， PIP 2合成的反馈。第三，我们将鉴定PIP 2 5-磷酸酶的生物学功能， 以及这些酶中疾病相关突变的发病机制。完成 为了达到这个目标，我们将确定内源性5-磷酸酶在细胞中的作用部位，PIP 2在细胞中的积累部位。 这些酶的损失后，什么样的细胞表型是由PIP 2的积累所引发的。 我们将采用创新的方法，结合PIP 2的超分辨率成像及其无数的 效应蛋白与化学遗传学，急性操纵PIP 2与精致的空间和时间 精度这项拟议中的研究意义重大，因为它将揭示 编排PM功能的相互作用，从而为开发新的 实验性或治疗性地单独操纵这些功能的方法。