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The Regulation of Macropinocytosis

巨胞饮作用的调节

基本信息

批准号：
9330196
负责人：
JOEL A SWANSON
金额：
$ 31.39万
依托单位：
UNIVERSITY OF MICHIGAN AT ANN ARBOR
依托单位国家：
美国
项目类别：
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/9330196
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 Medical Methods Microscopic Modeling Molecular Morphology Movement Mus Mutate Mutation Nature Oncogenic Pharmacology Phosphatidylinositols Process Proteins Regulation Research Role Route Serum Signal Transduction Signaling Protein Testing Time Vesicle Virus Work cancer cell cell transformation drinking enzyme activity genetic regulatory protein macrophage novel novel therapeutics pathogenic bacteria public health relevance ras Proteins response

项目摘要

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（由申请人提供）：本研究的长期目标是确定巨胞饮体形成和成熟的机制。巨胞饮作用是一个多阶段的内吞过程，其中大的囊泡从富含肌动蛋白的细胞表面皱褶形成。它是生长因子刺激的细胞和致癌突变转化的细胞中的常见活性，所述致癌突变增加I型磷脂酰肌醇3-激酶（PI 3 K）或GTP酶Ras的活性。它是树突状细胞内化抗原的过程，是许多病原性细菌和病毒进入细胞的途径，也是Ras转化的癌细胞获得生长所必需的氨基酸的机制。尽管巨胞饮在许多与人类健康相关的细胞活动中具有重要意义，但调节巨胞饮体形成的机制尚不清楚。巨胞饮作用发生在杯状皱褶的局部组装，这些皱褶在其远端边缘闭合或折叠成胞内小泡。PI 3 K和GTP酶Ras、Rac和Rab 5通过调节彼此的活性和多种效应酶的活性来促进巨胞饮的组分运动。Swanson实验室最近的显微镜研究发现，伴随巨胞饮体形成的PI 3 K、Rac、Ras和Rab 5的生长因子依赖性激活是由形态学而不是由生长因子加入细胞的时机组织的。与巨胞饮体形成的每个阶段相关的酶活性取决于完整的环形皱褶的形成，其本身可以在生长因子刺激后的不同时间形成。的增订条文这一发现提供了新的机会，分析生长因子信号转导级联在稳态条件下。本研究的目的是明确PI 3 K和Ras在巨胞饮体形成中的作用和调控。中心假设是，生长因子信号放大在稳态下限于巨胞饮杯，并通过PI 3 K，Ras，Rac和Rab 5的相互作用组织成两个主要的信号传导节点。这一假设将通过解决三个具体目标进行检验。目的1将确定在巨胞饮体形成过程中响应于生长因子的运动和信号的序列，检验由不同生长因子刺激的巨胞饮运动表现出Ras、Rac、Rab 5和PI 3 K活动的共同特征，并具有来自其他细胞骨架调节剂的不同贡献的假设。在巨噬细胞、鼠胚胎成纤维细胞和人上皮细胞中巨胞饮体形成期间，在连续存在其同源生长因子的情况下，将分析细胞骨架和相关信号的动力学。目的2将确定Ras在巨胞饮体形成中的作用，测试Ras的激活促进皱皱、巨胞饮体闭合和巨胞饮体成熟的假设。Ras蛋白和Ras效应子对巨胞饮体相关信号动力学活动的贡献将通过药理学、遗传学和定量荧光显微镜方法进行分析。目的3将确定PI 3 K在巨胞饮体形成中的作用。将使用药理学、遗传学和显微镜方法来确定PI 3 K蛋白p85 β和p110 β在3'磷酸肌醇合成中的作用以及在皱褶、巨胞饮杯和巨胞饮体中Ras、Rac和Rab 5的调节。总体而言，预计单个巨胞饮体的定量分析将定义巨胞饮体连续形成期间调控信号的时间和位置，并确定巨胞饮体形成的每个阶段所必需的调控相互作用。这项研究对人类健康的影响是，它将把医学上重要的信号蛋白PI 3 K和Ras的调节置于一个基本的和医学上相关的细胞过程的背景下。