Clathrin is necessary for the resolution of the phagolysosome. (A) Imaging of RAW cells 4 h after phagocytosis of mCherry-Lp (rainbow scale). Pitstop was added 10 min before imaging. See corresponding Video 10. Scale bars: 5 µm. (B) Quantification of the volume of phagosomes and PDVs over time. Volumes were normalized to T0 for each treatment. Data are mean ± SEM of three independent experiments. *, P < 0.05 indicates that the regressions are significantly different. (C) RAW cells engulfed mRFP1–E. coli for 15 min, chased for 25 min, and treated with Pitstop or ikarugamycin. Cells were fixed 1 or 4 h after phagocytosis and immunostained against E. coli (rainbow scale). Scale bars: 5 µm. (D) Volume of PDVs per cell for experiments shown in C. Data are mean ± SEM of 3 independent experiments with 25 cells quantified per condition of an experiment, and results were tested by one-way ANOVA with Tukey’s test. Different letters indicate results are statistically different (P < 0.05). (E) RAW cells expressing Mito-mCherry-FRB and GFP-FKBP-CLC were treated with rapamycin to induce knocksideways (KSW) of the clathrin light chain or DMSO (Ctrl) and allowed to internalize E. coli. Cells were fixed 3 h after phagocytosis, stained for E. coli, and imaged. Scale bars: 10 µm. (F) Number of fragments stained with anti–E. coli antibodies in control and rapamycin-treated cells. Data are mean ± SEM of three independent experiments and statistically tested using an unpaired t test (*, P < 0.05). Dashed lines show cell contours (A, C, and E).
