Figure 9. Clathrin functionally engages... | Rockefeller University Press

Figure 9.

$Figure 9. Clathrin functionally engages AP2 at the plasma membrane using two distinct interactions. (A) Schematic illustration of the putative interactions between β2 and clathrin. Inset shows mutations to delete the CBM (ΔCBM) or mutation of tyrosine residue Y815 in the appendage (Y-A). Note that Y815 in earlier papers refers to Y829 in our longer isoform. (B) Analysis of clathrin–β2 interaction in vitro. Binding experiments using GST–β2(616–951) WT or mutants to pull down MBP–CHC(1–1074) as indicated. Interaction was assessed by Western blotting (i), and a Coomassie-stained gel was run in parallel to check for equivalent capture on beads. Three independent experiments were performed and analyzed by densitometry (ii). Bar indicates mean. (C) Quantification of live-cell confocal imaging experiments to show extent of endocytosis triggered by FKBP–β2–GFP or related mutants (see inset). Colored traces indicate the total area of bright GFP puncta that form in cells after rerouting. Traces were time aligned and averaged. Mean traces are shown (i), and a scatterplot (ii) of values at 5 min after rerouting for each cell analyzed is shown (spots). Bars indicate mean ± SD. Number of cells = 11–15 and number of experiments = 3. ANOVA, P = 7.92 × 10−5. Note that the values for FKBP–β2–GFP (WT) and GFP–FKBP (GFP) are also shown in Fig. 8. Example live-cell movies are shown in Videos 6, 7, and 8. (D) Representative confocal images (i) of an immunolabeling experiment to assess endocytosis of CD8 by triggered endocytosis. Bars: (main) 10 µm; (insets) 2 µm. Scatterplot (ii) to indicate variability in chemically induced endocytosis. The fraction of green puncta that were also red for each cell analyzed is shown (spots). Bars indicate the mean ± SD. Number of cells = 8–14 and number of experiments = 3. ANOVA, P = 1.66 × 10−6.$

Clathrin functionally engages AP2 at the plasma membrane using two distinct interactions. (A) Schematic illustration of the putative interactions between β2 and clathrin. Inset shows mutations to delete the CBM (ΔCBM) or mutation of tyrosine residue Y815 in the appendage (Y-A). Note that Y815 in earlier papers refers to Y829 in our longer isoform. (B) Analysis of clathrin–β2 interaction in vitro. Binding experiments using GST–β2(616–951) WT or mutants to pull down MBP–CHC(1–1074) as indicated. Interaction was assessed by Western blotting (i), and a Coomassie-stained gel was run in parallel to check for equivalent capture on beads. Three independent experiments were performed and analyzed by densitometry (ii). Bar indicates mean. (C) Quantification of live-cell confocal imaging experiments to show extent of endocytosis triggered by FKBP–β2–GFP or related mutants (see inset). Colored traces indicate the total area of bright GFP puncta that form in cells after rerouting. Traces were time aligned and averaged. Mean traces are shown (i), and a scatterplot (ii) of values at 5 min after rerouting for each cell analyzed is shown (spots). Bars indicate mean ± SD. Number of cells = 11–15 and number of experiments = 3. ANOVA, P = 7.92 × 10⁻⁵. Note that the values for FKBP–β2–GFP (WT) and GFP–FKBP (GFP) are also shown in Fig. 8. Example live-cell movies are shown in Videos 6, 7, and 8. (D) Representative confocal images (i) of an immunolabeling experiment to assess endocytosis of CD8 by triggered endocytosis. Bars: (main) 10 µm; (insets) 2 µm. Scatterplot (ii) to indicate variability in chemically induced endocytosis. The fraction of green puncta that were also red for each cell analyzed is shown (spots). Bars indicate the mean ± SD. Number of cells = 8–14 and number of experiments = 3. ANOVA, P = 1.66 × 10⁻⁶.