Endocytosis — the process by which cell-surface receptors and other membrane proteins are taken up into the cell — occurs mainly through coated pits. These pits are specific sites on the plasma membrane where clathrin and the adapter protein AP-2 associate with the cargo. The pit invaginates, then a clathrin-coated vesicle is pinched off. Despite extensive characterization, we do not know whether these coated pits form randomly or at specific sites on the plasma membrane; how long they persist before detaching as coated vesicles; and how quickly coated vesicles uncoat.

Reporting in the first issue of Nature Cell Biology (1, 1-7; 1999), Gaidarov and colleagues now provide an insight into these questions. They looked at the formation and internalization of coated pits in living cells, using a fusion protein of green fluorescent protein (GFP) and the clathrin light-chain. Their work, which reveals an important relationship between the structural organization of clathrin-coated pits and the membrane cytoskeleton, has implications for organization of the plasma membrane.

The authors found that coated pits labelled with GFP-clathrin appear gradually, persist for several seconds, then disappear abruptly without moving far from where they originated. That is, detached coated vesicles are stripped of their clathrin coat in the vicinity of the pit — they do not move through the cytoplasm first. Surprisingly, Gaidarov et al. saw that the coated pits and vesicles do not move outside regions of about 0.5-0.8 μm in diameter (see picture). But latrunculin B — which inhibits actin assembly — relaxes this restricted mobility, implying that an actin-based framework maintains the structural organization of clathrin-coated pits.

The authors found that the formation of coated pits also seems to be coordinated by an underlying membrane skeleton. Coated pits appeared and disappeared at defined, rather than random, sites on the plasma membrane. Indeed, once fluorescence at a coated pit disappeared, it usually reappeared at the same site. This meant that coated pits did not form at all over large areas of the membrane.

These observations are difficult to reconcile with the conventional view of how coated pits are formed. Diffusible transmembrane receptors or membrane-docking sites are thought to recruit AP-2 and clathrin to form a coated pit. But the new data indicate that coated-pit formation is coupled to events at the membrane skeleton, possibly through scaffold proteins found in specific (and limited) places. Future work with GFP fusion proteins should resolve this discrepancy, allowing us to see how coated pits form and work in a living cell.