Cell 146, 471–484 (2011)

Clathrin-mediated endocytosis (CME) regulates the uptake of proteins at the plasma membrane as well as the entry of pathogens and viruses and involves the formation of clathrin-stabilized endocytic structures. The N-terminal β-propeller domain (TD) of the clathrin heavy chain is believed to be a central protein-protein interaction hub important for CME, but no tools interfering with clathrin have been available for studying the acute effects of disrupting TD-protein interactions. Now von Kleist et al. report two molecules named pitstop 1 and 2, which are based on distinct chemical scaffolds and inhibit association of clathrin TD with other endocytic proteins. In structures determined by X-ray crystallography, both molecules bind an interface between the first and second blades of the seven-bladed β-propeller, overlapping with the interface where clathrin-associating proteins bind. Application of pitstop 2 to cells blocked clathrin-mediated processes but did not affect clathrin-independent processes. Using total internal reflection fluorescence (TIRF) microscopy and fluorescence recovery after photobleaching (FRAP), the authors found that pitstop 2 perturbed clathrin-coated pit dynamics but did not block pit assembly or the sequestration of cargo into clathrin-containing structures. These data unexpectedly indicate that TD-protein interactions are important for endocytic pit dynamics but not for clathrin assembly. Future applications of these molecules should help to shed light on the mechanisms that regulate the transitions from pit assembly to maturation and to disassembly. In addition, these molecules could serve as inhibitors of virus and pathogen entry.