Regulation of Clathrin and Adaptor Function

描述（由申请人提供）：膜定义了真核细胞与其环境之间以及细胞本身内部的不同结构和功能边界。这些隔间之间的材料和信息的运动主要是介导的囊泡运输，和蛋白质外套，其中网格蛋白包被的膜是一个范例，总是与这些过程。磷酸肌醇被认为是膜运输的调节剂，发挥重要作用，并确定肌醇在作用位点局部积累或产生的机制，以及它们如何与效应蛋白相互作用是尚未解决的问题。在上一个奖项期间，我们发现II类PI 3-激酶，PI 3 K-C2？，与网格蛋白特异性结合。过度表达后，PI 3 K-C2？可以诱导大量细胞内网格蛋白包被的芽的增殖，所述芽含有两种蛋白并且移动非常迅速。我们最近的工作表明，类似的快速移动的结构可以检测到在活细胞中的外源性PI 3 K-C2？表达，它们含有网格蛋白，PI 3 K-C2？，和内吞转铁蛋白，以及相关的蛋白暗示参与膜再循环。该提议检验了这些“快速网格蛋白”（1）检验F-网格蛋白结构是具有独特衔接子、货物和马达蛋白的包被膜小管的假设，并在超微结构水平上探测它们与外周分选内体的关系;（2）使用快速成像和同时采集多个信号以及可光活化的GFP探针来确定货物如何通过活细胞中的F-网格蛋白隔室;（3）剖析了单个F-12对完整细胞中F-网格蛋白形成和货物装载的调节。网格蛋白组分和小G蛋白及其激活剂和效应剂;（4）阐明PI 3 K-C2？和网格蛋白重链末端结构域，接头和踝通过晶体学和溶液研究，促进显性负性形式的两种蛋白质的生产。虽然哺乳动物细胞中膜运输的途径被广泛理解，但它们在某些步骤中的确切功能机制，甚至形态学，仍然不清楚，将通过这项工作来解决。公共卫生相关性：该项目的重点是运输途径，通过这些途径，单个细胞维持其复杂的细胞内结构，并在专门的隔室中发挥功能。这些途径在所有真核细胞中普遍存在，因此这些是细胞生物学中的中心问题，与许多正常生物过程如组织中的信号传导、发育和细胞间通讯相关。相应地，它们也是许多疾病过程中的紊乱部位，包括癌症、心血管和神经系统疾病。

DESCRIPTION (provided by applicant): Membranes define distinct structural and functional boundaries between the eukaryotic cell and its environment, and within the cell itself. Movement of material and information between these compartments is largely mediated by vesicular transport, and protein coats, of which the clathrin coated membrane is a paradigm, are invariably associated with these processes. Phosphoinositides are recognized to play important roles as regulators of membrane trafficking, and identifying mechanisms by which inositides are locally accumulated or generated at sites of action, and how they interact with effector proteins are unresolved questions. During the last award period we found that a Class II PI 3-kinase, PI3K-C2?, binds specifically to clathrin. Upon overexpression, PI3K-C2? can induce the proliferation of numerous intracellular clathrin coated buds containing both proteins and which move extremely rapidly. Our more recent work has demonstrated that similar rapidly moving structures can be detected in live cells in the absence of exogenous PI3K-C2? expression, that they contain clathrin, PI3K- C2?, and endocytosed transferrin, as well as associated proteins suggesting involvement in membrane recycling. This proposal tests the hypothesis that these `Fast-clathrin' (F-clathrin) structures are involved in membrane recycling and other steps in the endocytic pathway by (1) testing the hypothesis that F- clathrin structures are coated membrane tubules with unique adaptors, cargos and motor proteins, and probing their relationship to peripheral sorting endosomes at the ultrastructural level; (2) using rapid imaging and simultaneous acquisition of multiple signals as well as photoactivatable GFP probes to determine how cargo passes through the F-clathrin compartment in live cells; (3) dissecting the regulation of F-clathrin formation and cargo loading in intact cells by individual F-clathrin components and by small G proteins and their activators and effectors; and (4) elucidating the interactions between PI3K-C2? and the clathrin heavy chain terminal domain, linker and ankle by crystallography and solution studies, facilitating the production of dominant-negative forms of both proteins. Although the pathways of membrane trafficking in mammalian cells are broadly understood, exactly how they function at some steps mechanistically, and even morphologically, remain obscure and will be addressed by this work. Public Health Relevance: This project focuses upon the trafficking pathways by which individual cells maintain their complex intracellular structure and carry out functions in specialized compartments. These pathways are ubiquitous in all eukaryotic cells, and thus these are central issues in cell biology with relevance for many normal biological processes such as signaling, development and intercellular communication in tissues. Correspondingly, they also are sites of derangement in many disease processes including cancer, cardiovascular and neurological diseases.