The Regulation of Macropinocytosis

巨胞饮作用的调节

• 批准号: 8921225
• 负责人: JOEL A SWANSON
• 金额: $ 33.05万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-09-05 至 2018-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8921225
• 关键词: 1-Phosphatidylinositol 3-Kinase; Actins; Acute; Address; Antigens; Biological; Cell membrane; Cell physiology; Cell surface; Cells; Communicable Diseases; Cytoskeleton; Dendritic Cells; Disease; Distal; Embryo; Enzymes; Epithelial Cells; Essential Amino Acids; Exhibits; Fibroblasts; Fluorescence; Fluorescence Microscopy; Generations; Genetic; Goals; Growth; Growth Factor; Guanosine Triphosphate Phosphohydrolases; HRAS gene; Health; Human; Immunity; Individual; Lead; Location; Malignant Neoplasms; Measures; Mediating; Methods; Microscopic; Modeling; Molecular; Morphology; Movement; Mus; Mutate; Mutation; Nature; Oncogenic; Phosphatidylinositols; Process; Proteins; Quantitative Genetics; Regulation; Research; Role; Route; Serum; Shapes; Signal Transduction; Signaling Protein; Staging; Testing; Time; Vesicle; Virus; Work; cancer cell; cell transformation; drinking; enzyme activity; genetic regulatory protein; macrophage; novel; novel therapeutics; pathogenic bacteria; ras Proteins; response; trafficking

DESCRIPTION (provided by applicant): The long-term goal of this research is to determine the mechanisms of macropinosome formation and maturation. Macropinocytosis is a multistage endocytic process in which large vesicles form from actin-rich, cell surface ruffles. It is a common activity in cells stimulated by growth factors and in cells transformed by oncogenic mutations which increase the activities of type I phosphatidylinositol 3-kinase (PI3K) or the GTPase Ras. It is the process by which dendritic cells internalize antigen, the route by which many pathogenic bacteria and viruses enter cells and the mechanism used by Ras-transformed cancer cells to acquire amino acids essential for growth. Despite the importance of macropinocytosis in many cellular activities related to human health, the mechanisms which regulate macropinosome formation are not known. Macropinocytosis occurs by the localized assembly of cup-shaped ruffles which close at their distal margins or fold into intracellular vesicles. PI3K, and the GTPases Ras, Rac and Rab5 contribute to the component movements of macropinocytosis by regulating the activities of each other and of multiple effector enzymes. Recent microscopic studies in the Swanson lab discovered that the growth factor-dependent activation of PI3K, Rac, Ras and Rab5 which accompany macropinosome formation is organized by morphology rather than by the timing of growth factor addition to cells. Enzyme activities associated with each stage of macropinosome formation are contingent on the formation of a complete circular ruffle, which itself may form at various times after growth factor addition. This discovery offers the novel opportunity to analyze growth factor signal transduction cascades under steady state conditions. The objectives of the present work are to define the roles and regulation of PI3K and Ras in macropinosome formation. The central hypothesis is that growth factor signal amplification at steady state is confined to macropinocytic cups and organized into two major signaling nodes by the mutual interactions of PI3K, Ras, Rac and Rab5. This hypothesis will be tested by addressing three specific aims. Aim 1 will determine the sequence of movements and signals during macropinosome formation in response to growth factors, testing the hypothesis that the movements of macropinocytosis stimulated by different growth factors exhibit a common profile of Ras, Rac, Rab5 and PI3K activities, with varied contributions from other cytoskeletal regulators. The dynamics of the cytoskeleton and related signals will be analyzed during macropinosome formation in macrophages, murine embryonic fibroblasts and human epithelial cells in the continuous presence of their cognate growth factors. Aim 2 will determine the role of Ras in macropinosome formation, testing the hypothesis that activation of Ras promotes ruffling, macropinosome closure and the maturation of macropinosomes. The contributions of Ras proteins and Ras effectors to the activities of macropinosome-associated signal dynamics will be analyzed by pharmacological, genetic and quantitative fluorescence microscopic methods. Aim 3 will determine the role of PI3K in macropinosome formation. Pharmacological, genetic and microscopic methods will be used to determine the roles of the PI3K proteins p85� and p110� in 3' phosphoinositide synthesis and the regulation of Ras, Rac and Rab5 in ruffles, macropinocytic cups and macropinosomes. Overall, it is anticipated that quantitative analysis of individual macropinosomes will define the timing and location of regulatory signals during the continuous formation of macropinosomes and identify regulatory interactions essential to each stage of macropinosome formation. The impact of this research for human health is that it will put the regulation of medically important signal proteins PI3K and Ras into the context of an essential and medically relevant cellular process.

描述（由应用程序提供）：这项研究的长期目标是确定大斑肌体形成和成熟的机制。大型细胞增多症是一种多阶段的内吞过程，其中大型蔬菜由富含肌动蛋白的细胞表面荷叶边形成。它是由生长因子刺激的细胞和通过致癌突变转化的细胞的常见活性，这些突变会增加I型磷脂酰肌醇3-激酶（PI3K）或GTPase RA的活性。这是树突状细胞内化抗原的过程，抗原是许多致病性细菌和病毒进入细胞的途径以及RAS转换的癌细胞使用的机制以获取生长所必需的氨基酸。尽管大型细胞增多症在与人类健康有关的许多细胞活性中的重要性，但调节大斑体形成的机制尚不清楚。大型细胞增多症是通过杯状荷叶边PI3K的局部组装而发生的，而GTPases RAS，RAC和RAB5通过反射彼此和多种效应酶的活性来促进大型细胞增多症的成分运动。 Swanson实验室中最近的微观研究发现，可容纳大分子体形成的PI3K，RAC，RAS和RAB5的生长因子依赖性激活是通过形态学而不是通过形态学的时间来组织的。与大型形成的每个阶段相关的酶活性取决于完整的圆形荷叶边的形成，生长因子后的不同时间可能形成 添加。这一发现提供了一个新的机会，可以在稳态条件下分析生长因子信号转导级联。本工作的目的是定义PI3K和RAS在大型形成中的作用和调节。中心假设是，稳态下的生长因子信号扩增仅限于大型细胞杯，并通过PI3K，RAS，RAC和RAB5的相互作用而组织为两个主要信号节点。该假设将通过解决三个具体目标来检验。 AIM 1将根据生长因子来确定大型形成过程中运动和信号的序列，以检验以下假设：巨型细胞增多症的运动刺激了不同生长因子刺激了RAS，RAC，RAC5和PI3K活性的共同特征，并从其他细胞骨架调节剂中造成了不同的贡献。在巨噬细胞，鼠类胚胎成纤维细胞和人类上皮细胞中，将分析细胞骨架和相关信号的动力学。 AIM 2将确定RAS在大型形成中的作用，检验以下假设：RAS的激活促进了荷叶边，大型刺激体闭合和大质体的成熟。 RAS蛋白和RAS效应对大型体体相关信号动力学活性的贡献将通过药理，遗传和定量荧光显微镜方法分析。 AIM 3将确定PI3K在大型体体形成中的作用。药理学，遗传和微观方法将用于确定PI3K蛋白p85和p110在3'磷酸肌醇合成中的作用，以及RAS，RAC和RAB5在荷叶边，大型杯子，大型杯和大杯中的调节。总体而言，可以预见，对大型大型体的单个大型体的定量分析将定义大型体体的连续形成过程中调节信号的时间和位置，并确定对大型体体组成的每个阶段必不可少的调节相互作用。这项研究对人类健康的影响是，它将调节医学上重要的信号蛋白PI3K和RAS进入基本和医学相关的细胞过程的背景。