3D tracking of CCSs in apical and basal surfaces of Drosophila amnioserosa. (A) Three sections of a confocal z-stack show apical, perinuclear, and basal regions of Drosophila amnioserosa cells expressing clathrin light chain fused with GFP (CLC-GFP). CCSs originating on organelles are observed mostly at the perinuclear regions as nondiffraction-limited bright fluorescent puncta (blobs). Bar, 10 µm. (B, left) Maximum projection image of a z-stack acquired at the amnioserosa tissue of a live Drosophila embryo. The image is divided into windows of 10 × 10 µm for determination of basal and apical surfaces (expanded in C). (right) Snapshot of CCS traces that are detected within the left panel, color-coded according to their axial positions. The projection of the entire 3D time-lapse acquisition and the corresponding CCS traces are shown in Video 3. (C) In each frame of the 3D time-lapse acquisition, axial positions of the apical and basal surfaces are determined separately for the 10 × 10 µm windows. Cartoon represents an amnioserosa cell oriented in a way that its apical surface faces the detection optics. The adjacent histogram shows the distribution of traces detected within the orange square shown in B with respect to their axial positions. The distribution is bimodal because of increased CCS density at the apical and basal surfaces and can be fit with a sum of two Gaussians. CCSs falling within one standard deviation of the respective means (± σ) are considered apical or basal. (D) A comparison of CCS lifetime distributions obtained from apical and basal amnioserosa with clathrin-coated pits (CCPs) detected in cultured BSC-1 cells stably expressing AP2-GFP (Amnioserosa: Nembryos = 2, Ncells = 75, and Ntraces = 124,013; CCPs: Ncells = 3 and Ntraces = 12,002).
