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（由申请人提供）：本研究的长期目标是确定巨胞质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，其中富含肌动蛋白的细胞表面褶皱形成大囊泡。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，这些突变会增加 I 型磷脂酰肌醇 3-激酶 (PI3K) 或 GTPase Ras 的活性。它是树突状细胞内化抗原的过程，是许多病原细菌和病毒进入细胞的途径，也是Ras转化的癌细胞获取生长必需氨基酸的机制。尽管巨胞饮作用在与人类健康相关的许多细胞活动中很重要，但调节巨胞饮体形成的机制尚不清楚。巨胞饮作用是通过杯形褶边的局部组装而发生的，这些褶边在其远端边缘闭合或折叠成细胞内囊泡。 PI3K 以及 GTPases Ras、Rac 和 Rab5 通过调节彼此以及多种效应酶的活性来促进巨胞饮作用的组成部分运动。 Swanson 实验室最近的显微研究发现，伴随巨脂质体形成的 PI3K、Rac、Ras 和 Rab5 的生长因子依赖性激活是通过形态而不是通过向细胞添加生长因子的时间来组织的。与巨脂质体形成的每个阶段相关的酶活性取决于完整的圆形褶边的形成，其本身可能在生长因子作用后的不同时间形成 添加。这一发现为分析稳态条件下生长因子信号转导级联提供了新的机会。本工作的目的是明确 PI3K 和 Ras 在巨胞质体形成中的作用和调节。中心假设是稳态下生长因子信号放大仅限于巨胞饮杯，并通过 PI3K、Ras、Rac 和 Rab5 的相互作用组织成两个主要信号节点。该假设将通过解决三个具体目标来检验。目标 1 将确定巨胞饮体形成过程中响应生长因子的运动和信号序列，检验以下假设：不同生长因子刺激的巨胞饮运动表现出 Ras、Rac、Rab5 和 PI3K 活性的共同特征，以及其他细胞骨架调节因子的不同贡献。在巨噬细胞、鼠胚胎成纤维细胞和人上皮细胞的巨胞质体形成过程中，在其同源生长因子持续存在的情况下，将分析细胞骨架和相关信号的动力学。目标 2 将确定 Ras 在大胞饮体形成中的作用，检验 Ras 激活促进皱褶、大胞饮体闭合和大胞饮体成熟的假设。 Ras 蛋白和 Ras 效应子对巨脂质体相关信号动力学活性的贡献将通过药理学、遗传和定量荧光显微镜方法进行分析。目标 3 将确定 PI3K 在巨脂质体形成中的作用。将使用药理学、遗传学和显微方法来确定 PI3K 蛋白 p85� 和 p110� 在 3' 磷酸肌醇合成中的作用以及 Ras、Rac 和 Rab5 在褶边、巨胞饮杯和巨胞饮体中的调节。总体而言，预计单个巨脂质体的定量分析将确定巨脂质体连续形成过程中调节信号的时间和位置，并确定巨脂质体形成每个阶段所必需的调节相互作用。这项研究对人类健康的影响在于，它将医学上重要的信号蛋白 PI3K 和 Ras 的调节置于重要的、医学相关的细胞过程的背景下。