Clathrin-mediated endocytosis (CME) is a major mechanism for internalizing plasma-membrane receptors. Clathrin-coated structures (CCSs) associate with cargo and adaptor proteins, and begin to invaginate the membrane, eventually giving rise to clathrin-coated vesicles (CCVs). Merrifield and colleagues use total internal reflection fluorescence microscopy, coupled to a pulsed pH assay, to study the dynamics of CME machinery recruitment and generation of CCVs in mammalian cells (PLoS Biol. 9, e1000604; 2012).

Through these elegant analyses, the authors found that CCSs could persist at the plasma membrane through multiple scission events, indicating that CCS disappearance might not be a reliable marker for CCV generation. Moreover, the duration and size of CCSs at the membrane was heterogeneous. The authors also generated 'recruitment signatures' of 34 proteins involved in CME. The signatures revealed that a core collection of coat, adaptor, actin-binding and dynamin proteins are involved in most CCV scission events. Furthermore, similarly to CME in yeast, CCS maturation and CCV generation proceeded in a stepwise manner, with distinct protein modules being recruited to the CCS at different times.

Thus, this high-resolution approach has yielded a temporal map of CME in mammalian cells, revealing fresh insights into this process and opening new avenues for investigation.