Figure 1Mouse bone marrow-derived macrophages (BMDMs) infected with Mtb Erd-tdTomato and imaged by time-lapse microscopy at 1- or 2-hour intervals for 132 hours BMDMs infected with aggregates of Mtb Erd-tdTomato from axenic culture and imaged by time-lapse microscopy at 1-hour intervals for 60 hours (A-B) Percentage of macrophages surviving after interaction with an extracellular Mtb aggregate originating from the debris of dead macrophages (A) or from axanic culture (B). Each line represents an independent experimental replicate (n = 3 replicates with ≥ 100 cells per replicate).BMDMs infected with aggregates of Mtb Erd-tdTomato from axenic culture and imaged by time-lapse microscopy at 1-hour intervals for 60 hours E, F (E) Example of a macrophage that interacts with an Mtb aggregate (0h00), fragments it (3h00), redistributes the bacteria in a "bullseye" pattern around the nucleus (12h00), and dies (16h00). Scale bar, 20 μm. (F) Example of a macrophage that stably interacts with an Mtb aggregate without fragmenting it (0h00-09h00) and ultimately dies (12h00). In (E and F), Draq7 staining of the nucleus (in blue) is used as a marker for cell death. Scale bar, 20 μm.BMDMs infected with aggregates of Mtb Erd-tdTomato from axenic culture and imaged by time-lapse microscopy at 1-hour intervals for 60 hours (G-H) BMDMs infected with aggregates of Mtb were imaged by time-lapse microscopy (every 30 min for up to 13.5h) followed by fixation, immunostaining (nuclei stained with Hoechst, membrane staining with anti-CD45 antibody) and imaging by confocal microscopy. White arrows indicate intracellular bacteria, cyan arrows indicate extracellular bacteria. All scale bars, 10 μM. (G.I,H.I) Time-lapse microscopy image-series of macrophages that interacts with Mtb aggregates and fragment (G) or do not fragment (H) them. (G.II,H.II) Max intensity projection of confocal microscopy images of the same macrophages shown in panels I. (G.III,H.III) 3-D reconstruction of the cells imaged in panels II, images are cropped in x or y in the position indicated by the white dotted lines in panels II to show the inside of the cell.BMDMs infected with aggregates of Mtb Erd-tdTomato from axenic culture and imaged by time-lapse microscopy at 1-hour intervals for 60 hours (I-M) BMDMs infected with aggregates of Mtb were imaged by time-lapse fluorescence microscopy followed by SEM. (I) Example of a macrophage showing the typical "bullseye" pattern of bacterial redistribution around the cell nucleus. Scale bar, 10 μm. (J) Correlative SEM image of (I). (K) Example of a macrophage interacting with an extracellular Mtb aggregate without complete fragmentation and redistribution of bacteria. White arrows indicate intracellular bacteria that have been detached from the main aggregate and internalized. Scale bar, 10 μm. Time-lapse microscopy image-series of these macrophages are shown in Appendix Fig S4A. (L-M) Correlative SEM image of (K). Scale bar, 2 μm (M).Mouse bone marrow-derived macrophages (BMDMs) infected with Mtb Erd-tdTomato and imaged by time-lapse microscopy at 1- or 2-hour intervals for 132 hours (O) Percentage of macrophages surviving over time after uptake and fragmentation of Mtb aggregates or after contact without fragmentation. Each line represents an independent biological replicate. The vertical dotted line marks the 12h post-interaction time-point. BMDMs were treated with cytochalasin D to prevent bacterial uptake, infected with aggregates of Mtb and imaged by time-lapse microscopy at 1-hour intervals for 60 hours. (P) Percentage survival over time for macrophages in contact with Mtb aggregates. Time 0 represents the time when stable contact with an aggregate begins. Each line represents an independent biological replicate (n = 3 replicates with ≥ 100 cells per replicate).