Dynamics of Clathrin Coat Formation in Cells

描述(申请人提供)：近30年前，人们发现了氯氰菊酯及其形成的皮毛。从那时起，大量的生化和细胞生物学研究使笼状蛋白涂层的行为成为膜运输的许多方面的范例。无论这些方法多么强大，它们都只能表示异类群体中对象的平均属性。它们不能提供有关特定涂层凹坑或囊泡的行为的信息。 为了弄清笼状蛋白被覆盖坑形成的潜在机制，现在至关重要的是弄清楚在被包被坑的生命过程中，特定蛋白质何时、持续多长时间和数量被招募。用于单分子检测的荧光活细胞成像技术的最新发展，与物体识别算法结合使用，现在允许足够的时间和空间分辨率来跟踪单个涂层坑的寿命。为了使用这种强大的方法，在过去的两年里，我们开发并实施了光镜技术来分析活细胞中的笼状蛋白途径。我们已经证明，有可能从数百个唯一识别的笼状蛋白涂层凹坑和涂层囊泡中收集定量的“单一对象”数据，在它们组装、招募货物和去除涂层时进行跟踪，使用融合到荧光EGFP或MRFP的笼状蛋白、AP-2和Dynamin作为探针，以及荧光标记的货物，如转铁蛋白、低密度脂蛋白和呼肠孤病毒。 在这个新的项目中，我们扩大了以前的研究范围，包括在质膜上形成和组装内细胞型网状蛋白包裹的囊泡的早期步骤(目标1)，在内细胞型网状蛋白结构上剥离涂层(目标2)，以及在内体和反式高尔基体网络中组装网状蛋白涂层(目标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).