Shevchuk et al. use ion conductance microscopy (ICM) to examine how clathrin-coated pits (CCPs) close as they internalize from the cell surface.
Electron microscopy can provide high-resolution snapshots of CCPs budding from the plasma membrane, but observing these dynamic structures in living cells has proved to be much more challenging. Shevchuk et al. followed the topologies of CCPs in live cells by combining fluorescence imaging with ICM, a technique in which the cell is rapidly scanned by a probe that detects nanoscale bumps and dips in the plasma membrane.
CCPs are generally thought to seal off from a flat region of the plasma membrane, but Shevchuk et al. found that 70% of CCPs were capped by a membrane protrusion that grew from one side of the pit. These protrusions contained the actin-binding protein Abp1, and their growth was blocked by the actin polymerization inhibitor latrunculin B. Abp1 wasn't associated with the 30% of pits that closed via the canonical mechanism, however, perhaps explaining why other researchers have reported conflicting findings on the involvement of actin in clathrin-mediated endocytosis.
Both types of CCP required the GTPase dynamin-2 to separate from the plasma membrane. Vesicles formed by the capping method moved more quickly into the cell interior, however, perhaps due to their greater association with the actin cytoskeleton. Author Andrew Shevchuk now wants to use ICM to investigate other forms of endocytosis and—if the temporal resolution of the technique can be improved even further—to study exocytic events as well.
