Single molecule localization-based analysis of clathrin-coated pit and caveolar dynamics.

Clathrin-coated pits and caveolae are nanoscale invaginations of the plasma membrane of cells, forming through the assembly of membrane coat and accessory proteins in a tightly regulated process. We have analyzed the development of these membrane coat structures with high spatial and temporal resolution and sensitivity using super-resolution single-molecule localization microscopy (SMLM) on live cells. To this end, we developed a sophisticated clustering and data analysis workflow that automatically extracts the relevant information from SMLM image sequences taken on live cells. We quantified lifetime distributions of clathrin-coated and caveolar structures, and analyzed their growth dynamics. Moreover, we observed hotspots in the plasma membrane where coat structures appear repeatedly. The stunningly similar temporal development of clathrin-coated and caveolar structures suggests that key accessory proteins, some of which are shared between the two types of membrane coat structures, orchestrate the temporal evolution of these complex architectures.