The Boston team marked clathrin and the AP-2 adaptor using fusions that allowed dynamic behavior, and collected complete datasets that were quantified automatically. The intensity of clathrin spots grew over time, with most reaching a critical point at ∼20 s. By then the curvature of the clathrin triskelions may become unsupportable without bolstering by other proteins. If labeled cargo joined the spots by then or soon after then, the spot continued to grow; but otherwise the spot dissipated without the burst of dynamin accumulation characteristic of endocytosing vesicles.
Apart from a few large, inactive regions, placement of pits was random and did not favor sites of previous pit formation. This suggests that static pit-forming factories do not exist. An as-yet-unidentified pit-initiating protein is, however, a distinct possibility.
Kirchhausen says the random formation is reminiscent of the random probing by dynamic microtubules, which are stabilized only if they happen to encounter their target proteins. The source of stabilization for clathrin coats remains uncertain. Cargo binding may induce a conformational change in an adaptor, thus strengthening links from adaptors to clathrin or other proteins. ▪