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 由磷酸肌醇/蛋白质复合物组成，因为它们在调节膜过程中起作用。我们 重点研究胰岛素介导的GLUT 4葡萄糖转运蛋白向脂肪细胞质膜的转位 作为我们的模型系统，这是生物学中的一个开创性问题， 贩卖人口本计划项目中已经开发的独特TIRF技术的应用，以及新的 在项目4中开发的技术，使我们能够回答这个领域以前难以解决的问题。 我们建议定义轨道和动力学的GLUT 4-含有囊泡内约200纳米的 质膜（TIRF区），使用新的方法，使粒子定位在分辨率为 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区的胞吐作用期间的对接和融合事件。 因此，我们现在可以解决胰岛素调节GLUT 4动力学的中心假设， 含有通过调节与2种或3种蛋白质相关的囊泡的囊泡对接/融合过程 特异性功能性基于磷酸肌醇的蛋白质复合物。我们假设， 含有GLUT 4的胞吐囊泡通过依赖于以下的机制快速回收和融合： PI（3）P/Rabenosyn-5/EHD 1/EHBP 1/Rab 5/Rab 4复合物。我们假设GLUT 4的第二个子集- 含有胞吐囊泡的细胞通过一种依赖于 PI（3，4，5）P3/FIP 2/EHD 1/EHBP 1/Rab 11复合物。我们所有的努力现在都集中在测试这些 与含有GLUT 4的胞吐囊泡的性质相关的假说， 与细胞膜结合。我们建议定义含有GLUT 4的囊泡穿过的3D路径 相对于其他货物（EGFR和转铁蛋白受体与项目2）的再循环和胞吐作用期间。使用 高分辨率TIRF显微镜与外表面标记的myc-GLUT 4-EGFP，我们建议跟踪轨迹 胰岛素敏感的含GLUT 4的囊泡，以测试Rab 5阳性早期内体中的GLUT 4是否 快速收缩以在“短路”途径中与质膜融合。结合独特的 生物化学方法的成像工具提供了一种强有力的方法来剖析功能， GLUT 4动力学中含有EHD 1和Rab蛋白的基于磷酸肌醇的复合物。我们亦会 测试PI（3，4，5）P3/Akt 2/底物的复合物是否在含GLUT 4的 囊泡和调节TIRF区这些途径中的特定步骤。在项目3中，我们还将定义 膜形貌和分子复合物的结构，作用于GLUT 4-含有囊泡， TIRF区，并阐明其作用所涉及的机制。

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.