SUBCELLULAR LOCALIZATION OF PI 3-KINASE SIGNALING

这些研究的目的是阐明分子机器的结构和动力学机制 由磷脂酰肌醇/蛋白质复合体组成，因为它们在调节膜过程中起作用。我们 胰岛素介导的GLUT4葡萄糖转运体向脂肪细胞质膜转位的研究进展 作为我们的模型系统，生物学中的一个开创性问题合并了细胞信号和膜领域 贩卖人口。在本计划项目中已经开发的独特TIRF技术的应用，以及新的 将在项目4中开发的技术，使我们能够回答该领域以前难以回答的问题。 我们建议定义包含GLUT4的囊泡在大约200 nm范围内的轨迹和动力学 质膜(TIRF区)，使用新方法，使粒子能够在分辨率为 50 nm或更小。自上次提交以来获得的新数据表明，我们现在可以识别和量化 在胰岛素介导的这些囊泡在TIRF区的胞吐过程中的对接和融合事件。 因此，我们现在可以解决中心假设，即胰岛素调节GLUT4的动力学- 通过调节与2或2相关的囊泡来包含囊泡对接/融合过程 基于磷脂酰肌醇的特定功能蛋白质复合体。我们假设其中一个子集 含GLUT4的胞外囊泡通过一种依赖于 PI(3)P/Rabenosyn-5/EHD1/EHBP1/Rab5/Rab4配合物。我们假设GLUT4的第二个子集- 包含胞外囊泡的循环和融合速度更慢，通过一种依赖于 PI(3，4，5)P3/FIP2/EHD1/EHBP1/Rab11络合物。我们现在的所有努力都是为了测试这些重点 与含GLUT4的胞外囊泡进行对接和融合的性质相关的假说 与质膜结合。我们建议定义包含GLUT4的囊泡穿越的3D路径 在回收和胞吐过程中相对于其他货物(项目2中的EGFR和转铁蛋白受体)。vbl.使用 高分辨率TIRF显微镜与表面标记的myc-GLUT4-EGFP，我们建议跟踪轨迹 对含有GLUT4的胰岛素敏感的囊泡进行检测，以测试GLLJT4是否存在于Rab5阳性的早期内吞体内 快速循环，以“短路”方式与质膜融合。独特的组合 具有生化方法的成像工具提供了一种强有力的方法来剖析 含EHD1和Rab蛋白的肌醇磷脂复合物在GLUT4动力学中的作用。我们还将 测试PI(3，4，5)P3/Akt2/底物的络合物在含GLUT4的对接/融合中的作用 并调节TIRF区这些通路中的具体步骤。在项目3中，我们还将定义 作用于含GLUT4囊泡的分子复合体的膜形貌和结构 TIRF区，并阐明其行动所涉及的机制。

The aim of these studies is to elucidate structural and dynamic mechanisms of molecular machines composed of phosphoinositide/protein complexes as they function in modulating membrane processes. We focus on insulin-mediated GLUT4 glucose transporter translocation to the plasma membrane of adipocytes as our model system, a seminal problem in biology that merges the fields of cell signaling and membrane trafficking. Application of unique TIRF technology already developed within this Program Project, and new technology to be developed in Project 4, positions us to answer previously intractable questions in this field. We propose to define trajectories and dynamics of GLUT4-containing vesicles within about 200nm of the plasma membrane (TIRF zone), using novel methods that enable localization of particles at resolutions of 50nm or less. New data obtained since the last submission shows we can now identify and quantify docking and fusion events during insulin-mediated exocytosis of these vesicles in the TIRF zone. Thus, we can now address the central hypothesis that insulin regulates the kinetics of GLUT4- containing vesicle docking/fusion processes through modulating vesicles associated with either of 2 specific functional phosphoinositide-based protein complexes. We hypothesize that one subset of GLUT4-containing exocytic vesicles rapidly recycle and fuse through a mechanism that depends upon PI(3)P/Rabenosyn-5/EHD1/EHBP1/Rab5/Rab4 complexes. We hypothesize that a second subset of GLUT4- containing exocytic vesicles more slowly recycle and fuse through a mechanism that depends upon PI(3,4,5)P3/FIP2/EHD1/EHBP1/Rab11 complexes. All our efforts are now directed to testing these focused hypotheses related to the nature of GLUT4-containing exocytic vesicles that proceed to docking and fusion with the plasma membrane. We propose to define the 3D paths that GLUT4-containing vesicles traverse during recycling and exocytosis relative to other cargo(EGFR and transferrin receptor with Project 2). Using high resolution TIRF microscopy with exofacially labeled myc-GLUT4-EGFP, we propose to track trajectories of insulin-sensitive, GLUT4-containing vesicles to test whether GLLJT4 in Rab5-positive early endosomes rapidly recycles to fuse with the plasma membrane in a "short circuit" pathway. The combination of unique imaging tools with biochemical approaches provides a powerful way to dissect out the functions of phosphoinositide-based complexes containing EHD1 and Rab proteins in GLUT4 dynamics. We shall also test whether complexes of PI(3,4,5)P3/Akt2/substrates function in docking/fusion of GLUT4-containing vesicles and regulate specific steps in these pathways in the TIRF zone. With Project 3, we will also define the membrane topographies and structures of molecular complexes that act on GLUT4-containing vesicles in the TIRF zone, and elucidate the mechanisms involved in their actions.