Investigations into the mechanisms which regulate entry of integral membrane proteins, and associated ligands, into the cell through vesicular carriers (endocytosis) have greatly benefited from the application of live-cell imaging. Several excellent recent reviews have detailed specific aspects of endocytosis, such as entry of particular cargo, or the different routes of internalization. The aim of the present review is to highlight how advances in live-cell fluorescence microscopy have affected the study of clathrin-mediated endocytosis. The last decade has seen a tremendous increase in the development and dissemination of methods for imaging endocytosis in live cells, and this has been followed by a dramatic shift in the way this critical cellular pathway is studied and understood. The present review begins with a description of the technical advances which have permitted new types of experiment to be performed, as well as potential pitfalls of these new technologies. Subsequently, advances in the understanding of three key endocytic proteins will be addressed: clathrin, dynamin and AP-2 (adaptor protein 2). Although great strides have clearly been made in these areas in recent years, as is often the case, each answer has bred numerous questions. Furthermore, several examples are highlighted where, because of seemingly minor differences in experimental systems, what appear at first to be very similar studies have, at times, yielded vastly differing results and conclusions. Thus this is an exceedingly exciting time to study endocytosis, and this area serves as a clear demonstration of the power of applying live-cell imaging to answer fundamental biological questions.

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