Fig. 3: CCVs start forming through a gradient of clathrin and curvature lifetimes in Cos-7 cells.

a Models of initial curvature generation during CME and representative intensity and ∆z measurements, mean ± SEM (Nucleation—n = 72 events, Constant curvature—n = 46 events, Flat to curved—n = 144 events, from 25 Cos-7 cells and three independent repeats). Red dashed line indicates the time where signal reached over the intensity threshold. b Kymographs for representative events from a (CLCa-STAR, cyan-magenta; ∆z, fire). White dashed line indicates when signal crossed the threshold. Scale bar = 5 μm. c Distribution of beginning of curvature formation in living Cos-7 cells measured as a difference between the time when ∆z reached above threshold and the time when clathrin accumulation reached above threshold (∆z_Beg − CLCa_Beg [s]). d Density scatter plot of beginning of curvature (∆z_Beg − CLCa_Beg [s]) as a function of total CCP lifetime (x axis is logged for better visualization, percentages for quadrants A-I are summarized in f). e, f Summary of distribution of events across three models: Nuc = Nucleation (∆z_Beg − CLCa_Beg < −1s), CCM = Constant Curvature Model (1 s ≤ ∆z_Beg − CLCa_Beg ≤ 4 s), FTC = Flat-to-curved transition (∆z_Beg − CLCa_Beg > 4 s). Data from d was also classified as events between 5 and 20 s as short-lived, 20–50 s as intermediate, and >50 s as longer-lived. Data presents three independent repeats means and SD are reported as a heat map in f. Data was normally distributed (one-sided Shaphiro–Wilk test, p > 0.05 for each lifetime cohort), two-way ANOVA was performed (lifetime cohort—F (2,18) = 93.17, p < 0.0001; PM bending model—F (2,18) = 93.70, p < 0.0001; interaction—F (4,18) = 57.49, p < 0.0001). Tukey’s multiple comparisons test details can be found in Supplementary Table 2, ***p = 0.0005, ****p < 0.0001 (Data in c–f based on n = 1805 events form 25 Cos-7 cells and three independent experiments). a was created with BioRender.com.