Dynamics of Clathrin Coat Formation in Cells

描述（由申请人提供）：网格蛋白及其形成的外套是近30年前发现的。从那时起，大量的生物化学和细胞生物学研究已经使网格蛋白涂层的行为成为膜运输的许多方面的范例。这些方法，然而强大的，只能表示平均属性的对象在一个异质人口。它们不能提供关于特定被膜小凹或囊泡行为的信息。 为了找出网格蛋白涂层凹坑形成的机制，现在至关重要的是要弄清楚在涂层凹坑的寿命期间，特定蛋白质在何时，多长时间和以何种数量被招募。荧光活细胞成像技术的最新发展面向singlemolecule检测，结合使用的对象识别算法，现在允许足够的时间和空间分辨率，以遵循一个单一的涂层坑的生活。为了使用这种强大的方法，我们在过去两年中开发并实施了光显微镜技术来分析活细胞中的网格蛋白途径。我们已经表明，可以从数百个独特识别的网格蛋白包被的凹坑和包被的囊泡中收集定量的“单一对象”数据，在它们组装、招募货物和未包被时进行跟踪，使用与荧光EGFP或mRFP融合的网格蛋白、AP-2和发动蛋白作为探针，以及荧光标记的货物如转铁蛋白、LDL和呼肠孤病毒。 在这个新的项目中，我们扩大了我们以前的调查范围，包括早期步骤的形成和组装的内吞网格蛋白包被的囊泡在质膜（目的1），内吞网格蛋白结构（目的2），和组装网格蛋白外套内体和transGolgi网络（目的3）。

DESCRIPTION (provided by applicant): Clathrin and the coats it forms were discovered nearly 30 years ago. Since then, numerous biochemical and cell biological studies have made the behavior of clathrin coats a paradigm for many aspects of membrane traffic. These approaches, however powerful, can only represent the averaged properties of objects within a heterogeneous population. They cannot provide information about the behavior of a specific coated pit or vesicle. To work out the mechanism underlying formation of clathrin-coated pits, it is now crucial to figure out when, for how long and in what quantity are specific proteins recruited during the life of a coated pit. Recent developments of fluorescent live-cell imaging techniques geared for singlemolecule detection, used in combination with object-identification algorithms, now allow sufficient temporal and spatial resolution to follow the life of a single coated pit. To use this powerful approach, we have developed and implemented over the past two years light-microscopic techniques to analyze the clathrin pathway in living cells. We have shown that it is possible to gather quantitative, "single-object" data from hundreds of uniquely identified clathrin coated pits and coated vesicles, tracked while they are assembling, recruiting cargo and uncoating, using as probes clathrin, AP-2 and dynamin fused to fluorescent EGFP or to mRFP, and fluorescently tagged cargo such as transferrin, LDL and reovirus. In this new project we extend the scope of our previous inquiry to include early steps in the formation and assembly of endocytic clathrin-coated vesicles at the plasma membrane (Aim 1), uncoating on endocytic clathrin structures (Aim 2), and assembling of clathrin coats in endosomes and the trans Golgi network (Aim 3).