Macromolecular dynamics of the cell surface during the formation of coated pits is revealed by fracture-flip

We report here the macromolecular dynamics of the cell surface of rat alveolar macrophages during spreading on a substratum, a process that involves the formation of numerous coated pits. We used ‘fracture-flip’ to prepare high-resolution platinum-shadowed replicas of membrane surfaces. Our observations show the following sequence of events associated with coated pit formation: at 4°C the cell surface of macrophages is covered with a moderate density of particulate components, with most ranging from 10 to 25 nm in diameter. These particles appear to be randomly distributed over the cell surface. Incubation of adherent cells at 37°C for 15 min results in the formation of large loose clusters (area 0·5–4 μm²) of particles on the adherent surfaces. After incubation of macrophages for 30 min at 37°C, these clusters become tighter andeventually form circular depressions (200·300 nm in diameter), which we interpret as part of a process of invagination. After 60 min, the depressions become much steeper. At this time surface particles can be observed on the intervening non-invaginated regions, and the peripheral region of the adherent membrane, as well as the free membrane. Fractureflip reveals the presence of structures undetected in previous electron-microscopic studies and provides ultrastructural evidence for the clustering of surface macromolecules that is involved in the formation of coated pits.