(A) SEM image of the unroofed protoplasts showing the main and subsidiary filaments of actin in close proximity to the PM. (B) Dual channel TIRF image of hypocotyl epidermal cell expressing ABD2-GFP and CLC2-mKO. The insert (yellow box) shows a magnified merge of the channels. Also see Figure S4B. (C) Example confocal airy-scan images of root hair cell expressing Lifeact-YFP (left) and the root hair cell expressing DRP1C-GFP (right). (D) Normalized lifetime distributions, average lifetime and densities of CLC2-GFP of root (upper panel) and hypocotyl (lower panel) epidermal cells in the absence (black) or presence (red) of LatB (roots; 10 µM, 10 mins, hypocotyl; 10 µM, 1 hr), measured by TIRF-M. Root: mock, 5 cells from individual roots, (32991 tracks), LatB, 4 cells from individual roots, (25517 tracks). Hypocotyl: mock, 3 cells from individual hypocotyls, (10883 tracks), LatB, 4 cells from individual hypocotyls, (12623 tracks). Error bars represent mean ± SEM. Two-sided unpaired T tests found there were no significant differences (Average lifetimes: hypocotyl p=0.43; root p=0.53. Average density: hypocotyl p=0.42; root p=0.95). (See also Supplementary file 1 table 2 for other markers). (E) Smoothened CCP intensity profile of the mean long-lived CLC2-GFP population in root epidermal cells in the absence (black) or presence (red) of LatB (10 µM, 10 mins). Mock, 6 cells from individual roots, 182 trajectories, LatB, 4 cells from individual roots, 122 trajectories. The extrapolation lines mark the different CCP development phases. The bottom bar plot represents the phase transitions computed individually from the trajectories of each root. The dotted bars represent the whole time course of CCP development; the solid lines with error bars mark the mean ± SD of the transition point between phases. Note that there are no significant differences in the different phases in the presence of LatB. One-sided Mann-Whitney U test; assembly p=0.31, maturation p=0.48 (also see Figure S4D). (F) Confocal microscopy images of root epidermis expressing fABD2-GFP after mock or LatB treatment (20 µM, 30 mins). Actin cytoskeleton is disrupted but FM4-64 is still endocytosed. N = 2 experimental repeats; at least 10 seedlings per condition. (G) TIRF-M images of the hypocotyl epidermal cells expressing FLS2-GFP either with or without flg22 (10 µM, 0.5 hr) treatment, and also cells pretreated with 1 hr of mock or LatB (10 µM, 1 hr) with co-treatment of flg22. Yellow arrows highlight endosomal structures containing FLS2. N = 2 experimental repeats, with nine hypocotyls per condition. (H) PIN2-Dendra endocytic rate, determined by the change of PM PIN2 intensity over time, after LatB (10 µM, 45 mins) compared to mock treatment (top). The dots represent the mean intensity and the dotted lines represent the 95% CI. No significant difference is observed between the slope of the curves; LMER - random effects for position; χ2- 2.5923; df = 1; p=0.107; N = 2, five seedlings per condition. (Bottom) PIN2-Dendra endocytic rate with the mock induction conditions or induction of AXL2 over-expression (AXL2-OX) for 24 hr (bottom). The slope of the curve for AXL2-OX is significantly lower than control conditions; LMER - random effects for position; χ2 = 78.095; df = 1; p<2.2e-16 ***; N = 2 experimental repeats, four seedlings per condition; all the epidermal cells in the root meristem were considered. Scale bars; 0.5 µm (A), 5 µm (B,G), 4 µm (C), 10 µm (F).