Regulation of Clathrin and Adaptor Function

描述（由申请人提供）：膜在真核细胞及其环境之间以及细胞内部定义了不同的结构和功能边界。这些腔室之间的物质和信息的运动主要是由囊泡运输介导的，蛋白质外壳，其中网格蛋白包被的膜是一个范例，总是与这些过程有关。磷酸肌苷被认为在膜运输中起着重要的调节作用，而确定肌苷在局部积累或在作用位点产生的机制，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PI3 -激酶PI3K-C2？特异地与网格蛋白结合。过表达后，PI3K-C2？可以诱导大量细胞内含有这两种蛋白质的网格蛋白包被芽的增殖，这些芽的移动速度非常快。我们最近的研究表明，在没有外源性PI3K-C2的情况下，可以在活细胞中检测到类似的快速移动结构。表达，它们含有网格蛋白，PI3K- C2？和内吞转铁蛋白，以及提示参与膜循环的相关蛋白。本研究通过以下方法验证了这些“Fast-clathrin”（F-clathrin）结构参与膜循环和内吞途径中的其他步骤的假设：(1)验证了F-clathrin结构是具有独特接头、货物和运动蛋白的膜小管的假设，并在超微结构水平上探索它们与外周分选核内体的关系；(2)使用快速成像和同时获取多个信号以及光激活GFP探针来确定活细胞中货物如何通过f -网格蛋白隔室；(3)剖析单个f -网格蛋白组分和小G蛋白及其激活物和效应器对完整细胞中f -网格蛋白形成和载货的调节；(4)阐明PI3K-C2？而网格蛋白重链末端结构域、连接子和踝关节通过晶体学和溶液学研究，促进了这两种蛋白的显性阴性形式的产生。尽管哺乳动物细胞中膜运输的途径已被广泛了解，但它们在某些步骤上的机制甚至形态上的确切功能仍然不清楚，将由本工作来解决。公共卫生相关性：该项目侧重于单个细胞维持其复杂的细胞内结构并在专门的隔室中执行功能的运输途径。这些途径在所有真核细胞中普遍存在，因此它们是细胞生物学的核心问题，与许多正常的生物过程相关，如组织中的信号传导、发育和细胞间通讯。相应地，它们也是许多疾病过程中的紊乱位点，包括癌症、心血管和神经疾病。

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.