Extended Data Figure 5: Large invaginations take up a fluid phase marker.

a–e, Additional representative micrographs showing ferritin uptake in the whole terminal in non-stimulation control (a) and 100 ms after stimulation (b) and by large structures at the edge of active zones 100 ms after stimulation (c–e). An arrow in b indicates a ferritin-positive shallow pit. Ferritin was applied to cells for 5 min before the light stimulation. Note that very little ferritin was internalized during the pre-incubation period, suggesting that the ferritin distribution is associated acutely with endocytosis after the stimulation. In unstimulated cultures, 18% of the profiles exhibited some internalization of ferritin; of those profiles only 1 vesicle from the ∼56 total vesicles contained ferritin. At 100 ms, 26% of the profiles exhibited synaptic vesicles with ferritin (P = 0.29); of those profiles that had internalized ferritin only 1 vesicle from 51.2 total vesicles contained ferritin. Thus, ultrafast endocytosis did not directly generate detectable numbers of synaptic vesicles with ferritin. f, Number of shallow pits (<40 nm), deep pits (>40 nm), large vesicles associated with the membrane (<5 nm) and large vesicles associated with the plasma membrane (6–50 nm of the membrane) in controls and ferritin-containing seeded cultures. In ferritin seeded cultures the large vesicles near the membrane (within 5 nm) all contained ferritin. The total number of ferritin-positive endocytic structures was 0.38 ± 0.03 endocytic structures per profile, similar to the endocytosis values obtained in Fig. 3i without ferritin (0.43 ± 0.03 endocytic structures per profile). g, Diameter of large vesicles (86.0 ± 2.4; n = 51) and endocytic invaginations (77.3 ± 3 nm; n = 16; P = 0.33). ‘No ferritin’ controls are from the samples in Fig. 3. Error bars, s.e.m.