Directing membrane function with inositol lipids in health and disease

项目摘要/摘要 组织严密的质膜(PM)上的材料和信号流量受到严格控制 对于健康的细胞功能是必不可少的。打乱这种仔细的编排是一种常见的机制 许多遗传性和传染性疾病的发病机制。因此，细胞中的一个中心问题 生物学就是要了解指导这种复杂的信号组织的关键分子成分， 总理办公室的运输和建筑机械位于胞质小叶中的磷脂， 磷脂酰肌醇4，5-二磷酸(PIP2)是PM功能的关键调节因子，控制募集和/或 这种蛋白质机制的激活。然而，如何监管PIP2级别以确保每个PM功能都可以访问 要有足够的脂质来确保正确的操作，以及PIP2如何能够离散地调节每个功能，是很差的 明白了。因此，我们的研究目标是从机制上详细了解细胞如何 调节PM中的PIP2水平，以及这如何促进对依赖于PIP2的单个功能的调节。这个 这项应用的目标是识别细胞培养模型中的基本机制，并应用新的 通过我们已建立的网络对生理和疾病相关系统的见解和方法 合作者。首先，我们将确定PM和PM中PIP2分子的纳米结构 确定它们在特定PM功能部位的富集度。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、信号或运输功能部位的脂质浓缩， 使用超分辨率光学成像方法和化学遗传学。其次，我们将划定 通过确定驱动负值的分子成分来调节全球PM PIP2水平的机制 PIP2合成的反馈。第三，我们将鉴定PIP25-磷酸酶的生物学功能，如 以及这些酶与疾病相关的突变的发病机制。要完成 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，即PIP2在哪里积累 在失去这些酶之后，以及由此产生的PIP2的积累触发了哪些细胞表型。 我们将在整个过程中采用创新的方法，结合PIP2及其无数的超分辨率成像 具有化学遗传学的效应蛋白以精细的空间和时间敏锐地操纵PIP2 精确度。这项拟议的研究具有重要意义，因为它将揭示 规划PM功能的相互作用，从而为开发新的 从实验上或治疗上孤立地操纵这些功能的方法。

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.