The Regulation of Macropinocytosis

描述(申请人提供)：本研究的长期目标是确定大针体的形成和成熟的机制。巨噬细胞吞噬是一个多阶段的内吞过程，在这个过程中，富含肌动蛋白的细胞表面褶皱形成大泡。它在生长因子刺激的细胞和通过癌基因突变转化的细胞中是一种常见的活性，这些突变增加了I型磷脂酰肌醇3-激酶(PI3K)或GTP酶RAS的活性。它是树突状细胞内化抗原的过程，是许多致病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。尽管巨噬细胞吞噬作用在许多与人类健康相关的细胞活动中具有重要作用，但调节巨噬细胞小体形成的机制尚不清楚。巨噬细胞增多症是通过杯状褶皱的局部组装发生的，这些褶皱在它们的远端边缘关闭或折叠成细胞内的小泡。PI3K和GTP酶Ras、Rac和Rab5通过调节彼此的活性和多种效应酶的活性来参与巨饮细胞吞噬的成分运动。最近在Swanson实验室进行的显微镜研究发现，伴随着大针体形成的PI3K、RAC、RAS和Rab5依赖生长因子的激活是由形态而不是生长因子添加到细胞的时间来组织的。与大胞体形成的每一阶段相关的酶活性取决于完整的圆形褶皱的形成，该褶皱本身可能在生长因子作用后的不同时间形成。 加法。这一发现为分析稳态条件下生长因子信号转导通路提供了新的机会。本工作的目的是明确PI3K和RAS在大胞体形成中的作用和调控。中心假说是，稳定状态下生长因子信号的放大仅限于巨噬细胞杯，并通过PI3K、Ras、Rac和Rab5的相互作用组织成两个主要的信号节点。这一假设将通过解决三个具体目标来检验。目的1将确定生长因子对大胞体形成过程中的运动和信号的响应序列，验证不同生长因子刺激的大胞体吞噬运动呈现Ras、Rac、Rab5和PI3K活性的共同特征的假设，以及其他细胞骨架调节因子的不同贡献。在巨噬细胞、小鼠胚胎成纤维细胞和人上皮细胞中，在相关生长因子的持续存在下，将分析巨噬细胞、小鼠胚胎成纤维细胞和人上皮细胞中巨噬细胞形成过程中细胞骨架和相关信号的动态变化。目的2确定RAS在大针体形成中的作用，验证RAS激活促进大针体皱折、闭合和大针体成熟的假设。通过药理学、遗传学和定量荧光显微镜方法分析RAS蛋白和RAS效应器对大松体相关信号动力学活性的贡献。目的3将确定PI3K在大针体形成中的作用。将利用药理学、遗传学和显微技术来确定PI3K蛋白P85�和P110�在3‘磷脂酰肌醇合成中的作用，以及Ras、Rac和Rab5在褶皱、巨噬细胞杯和大胞体中的调节。总体而言，预计对单个大管胞小体的定量分析将确定大管胞体内连续形成过程中调控信号的时间和位置，并确定对大管胞体内形成的每个阶段至关重要的调控相互作用。这项研究对人类健康的影响是，它将把对医学上重要的信号蛋白PI3K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。

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.