Dynamics of Clathrin Coat Formation in Cells

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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).