Journal: The Journal of Cell Biology

Article Title: Phagosome resolution regenerates lysosomes and maintains the degradative capacity in phagocytes

doi: 10.1083/jcb.202005072

Figure Lengend Snippet: Phagolysosomes do not undergo exocytosis but fragment. (A) 1 h after phagocytosis of IgG-opsonized beads, RAW cells were treated with 10 µM ionomycin in the presence of 1.2 mM of Ca 2+ , and live-cell DIC images were acquired at the indicated time points. Scale bars: 10 µm. (B) Number of latex beads per cell at the indicated treatment times, normalized to the number of beads/cell observed at the onset of the treatment (T 0 ). Data are mean ± SEM of 3 independent experiments, where 10 cells were measured in each experiment. (C) RAW cells expressing PLCδ1-PH-GFP and containing IgG-opsonized beads in phagosomes were treated with 10 µM ionomycin or DMSO (control) in the presence of 1.2 mM of Ca 2+ . Images were acquired before or after 10 min of treatment. Asterisks correspond to the positions of the beads, as shown in the insets. Scale bars: 10 µm. (D) Live-cell imaging time series of IgG-aggregated fluorescent 0.1-µm latex beads after 2-h phagocytosis by RAW cells. Beads are depicted in a rainbow scale. Red and blue correspond to the highest and lowest florescence intensity levels, respectively. The smaller panels show the far red and DIC channels for the first frame. The cell contour is delineated with white dashes. See corresponding . Scale bars: 5 µm. (E) Macrophages were assessed for exocytosis of digested phagosomal contents. RAW macrophages internalized mRFP1-labeled E. coli for 1 h and then were incubated for the indicated time points. TCA precipitates of cell media and cell lysates were probed for mRFP1, detecting a native and a cleaved mRFP1 product that accumulated over time. GAPDH was used as a loading control and to detect cell lysis. (F) Quantification of cleaved mRFP1 levels normalized to GAPDH in media and within cells. Data are shown as mean ± SD of six independent experiments. Conditions were compared using one-way ANOVA and Holm-Sidak’s post hoc test (*, P < 0.05).

Article Snippet: Inhibitors and the DMSO (BioShop) vehicle control were applied after phagocytosis and maintained until fixation or the conclusion of the experiment.

Techniques: Expressing, Control, Live Cell Imaging, Labeling, Incubation, Lysis

Journal: The Journal of Cell Biology

Article Title: Phagosome resolution regenerates lysosomes and maintains the degradative capacity in phagocytes

doi: 10.1083/jcb.202005072

Figure Lengend Snippet: Fragmentation of the phagolysosome requires cargo degradation and the cytoskeleton. (A, C, E, and G) Macrophages engulfed E. coli for 15 min, chased for 25 min, and treated with either Con A and NH 4 Cl (A), protease inhibitor cocktail (C), microtubule inhibitors (E), actin inhibitors (G), or vehicle control (DMSO). Cells were fixed after 1 or 4 h after phagocytosis and stained with an anti– E. coli antibody whose fluorescence is displayed in rainbow scale. Dashed lines show cell contours. Scale bars: 5 µm. (B, D, F, and H) Total volume of PDVs per cell. Data are mean ± SEM of 3 independent experiments with 25 cells quantified per condition of an experiment and compared by one-way ANOVA with Tukey’s test. Conditions labeled with different letters (a–d) are statistically different (P < 0.05).

Article Snippet: Inhibitors and the DMSO (BioShop) vehicle control were applied after phagocytosis and maintained until fixation or the conclusion of the experiment.

Techniques: Protease Inhibitor, Control, Staining, Fluorescence, Labeling

Journal: The Journal of Cell Biology

Article Title: Phagosome resolution regenerates lysosomes and maintains the degradative capacity in phagocytes

doi: 10.1083/jcb.202005072

Figure Lengend Snippet: 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 . Scale bars: 5 µm. (B) Quantification of the volume of phagosomes and PDVs over time. Volumes were normalized to T 0 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).

Article Snippet: Inhibitors and the DMSO (BioShop) vehicle control were applied after phagocytosis and maintained until fixation or the conclusion of the experiment.

Techniques: Imaging, Expressing, Staining, Control

Journal: The Journal of Cell Biology

Article Title: Phagosome resolution regenerates lysosomes and maintains the degradative capacity in phagocytes

doi: 10.1083/jcb.202005072

Figure Lengend Snippet: Dynamin inhibition blocks phagosome fragmentation. (A and C) 40 min after phagocytosis of mRFP1– E. coli , RAW cells were treated with the dynamin inhibitors dyngo-4a, dynole 34-2, or vehicle (DMSO); fixed at the indicated time points; and immunostained for E. coli . E. coli fluorescence labeling is shown in rainbow scale. Red and blue are the highest and lowest intensity levels, respectively. Dashed lines indicate the boundary of the cells. Scale bars: 5 µm. (B and D) The total volume of PDVs per cell for the indicated treatments. Data are mean ± SEM of sample of 25 cells from 3 independent experiments. Treatments were compared statistically using a one-way ANOVA with Tukey’s post hoc test. Conditions with different letters (a–c) indicate statistically significant difference (P < 0.05). (E) Macrophages treated with dynole 34-2 or DMSO (vehicle) internalized Alexa Fluor 546–labeled transferrin for 10 min before fixation. White dashes indicate cell boundaries. Scale bars: 30 µm.

Article Snippet: Inhibitors and the DMSO (BioShop) vehicle control were applied after phagocytosis and maintained until fixation or the conclusion of the experiment.

Techniques: Inhibition, Fluorescence, Labeling