Fig. 8: Integration of clathrin endocytosis in the spectrin/actin/plasma membrane composite.

a TIRF microscopy images of MEFs immunostained for endogenous βII-spectrin (green), clathrin heavy chain (CHC, magenta), and F actin (blue) (scale bar: 10 μm, images are representative of many cells, immunolabeled in n = 2 independent experiments). Three different subcellular regions are highlighted (dashed boxes 1–3): high-density CHC zone (1), βII-spectrin-rich zone (2), and actin stress fibers (3). b Density maps generated by aligning discrete clathrin pits of small (<0.3 µm², n = 144) and larger size (0.3–0.5 µm², n = 123) are shown, while in (c) correspondent radial intensity profiles for the three channels are presented. d Representative live TIRF microscopy images of MEFs transfected with GFP-βII-spectrin (green) and the clathrin adapter AP2σ-mCherry (magenta) (scale bar: 10 μm, n = 9 and n = 8 cells), unsharp and Gaussian filters were applied. PE/ANKbs-transfected fibroblasts show homogeneous PM localization and enrichment in correspondence of AP2σ pits, as shown by density maps (e) and radial profile analysis (f) generated as previously described (GFP-βII-spectrin: <0.3 µm², n = 47 and 0.3–0.5 µm², n = 204; PE/ANKbs: <0.3 µm², n = 109 and 0.3–0.5 µm², n = 173). g Representative images of TIRFM time lapse during osmotic shocks (scale bar: 20 μm, n = 6 independent cells): kymograph is generated in correspondence of the dashed yellow line (1). Whole-cell AP2 intensity signal in response to the osmotic shocks is plotted in the graph (magenta, vertical plot). h In total, 20 × 20-µm ROIs drawn at low- and high-spectrin-density zones around AP2 pits (correspondent to dashed boxes in (g), only zooms of Hi-density zone are reported at two different frames). Kymographs display the differential behavior of the pits observed during spectrin remodeling (representative of n = 6 independent cells). i Violin plots (dots = median values) of AP2 pit lifetime at high and low βII-spectrin-density zones of 10 µm² (statistical analysis paired Student’s t test, one-tailed, p = 0.0094, mean values ± SD are reported in numbers, n = 505 (Hi), 756 (Low) pits in 9 independent cells). Total AP2 pit counts during 10 min of recording over the same zones are reported in (j); measurements within the same cell are highlighted by the connecting line (statistical analysis paired Student’s t test, two-tailed, p < 0.0001, data are presented as mean values ± SD, n = 9 cells). k Violin plot (dots = median values) of AP2 lifetime in MEFs expressing different βII-spectrin variants (statistical analysis one-way Anova with multiple comparison, *p = 0.0112, ****p < 0.0001, mean values ± SD are reported in numbers, n = 23,000–29,000 pits in 8–10 independent cells).