The Regulation of Macropinocytosis

描述（由申请人提供）：本研究的长期目标是确定大肌醇体形成和成熟的机制。巨饮作用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的囊泡。在受生长因子刺激的细胞和因致癌突变而增加I型磷脂酰肌醇3-激酶（PI3K）或GTPase Ras活性的细胞中，它是一种常见的活性。它是树突状细胞内化抗原的过程，是许多致病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。尽管巨量红细胞增多症在许多与人体健康有关的细胞活动中发挥着重要作用，但调控巨量红细胞增多体形成的机制尚不清楚。巨量红细胞增多症的发生是由于杯状褶皱的局部聚集，这些褶皱在远端边缘闭合或折叠成细胞内小泡。PI3K和GTPases Ras、Rac和Rab5通过相互调节和多种效应酶的活性参与巨噬细胞增多症的组分运动。Swanson实验室最近的显微镜研究发现，伴随大脂小体形成的PI3K、Rac、Ras和Rab5的生长因子依赖性激活是由形态学组织的，而不是由生长因子加入细胞的时间组织的。与大脂小体形成的每个阶段相关的酶活性取决于完整的圆形皱褶的形成，其本身可能在生长因子作用后的不同时间形成

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.