Fig. 1.

BCVs interact with COPII-positive compartments in macrophages. (A) Confocal microscopy micrographs of BMDMs infected for 4, 12, or 24 h with either WT or ΔvirB9 B. abortus strains expressing GFP. When required, BFA was applied to infected BMDMs for 2 h before analysis. Fixed cells were stained with anti-sec31 antibodies (red) to label COPII-positive compartments. (Insets) GFP-Brucella in close apposition to COPII-positive compartments. Arrows indicate examples of BCVs in close contact with COPII-labeled compartments. (B) Quantitation of BCV apposition to COPII-positive compartments over time. Single confocal microscopy 0.2-μm sections of random fields were acquired, and the appositions of BCVs to COPII-positive compartments were counted as positive when a coincident yellow fringe was observed (see A Insets). Data are means ± SD of three independent experiments. (C) Confocal microscopy micrographs of BMDMs infected with either WT or ΔvirB9 B. abortus strains for 4, 12, or 24 h. Fixed cells were stained with anti-β-COP antibodies to label COPI-positive compartments (green) and propidium iodide (red) to detect bacterial and host cell DNA. Arrows indicate BCVs positioned away from COPI-positive compartments. (D) Quantitation of BCV apposition to COPI-positive compartments over a 24-h infection period. Single confocal microscopy sections of random fields were acquired, and the appositions of BCVs to COPI-positive compartments were counted as positive when a coincident yellow fringe was observed. Data are means ± SD of three independent experiments. All micrographs shown are projections of three consecutive confocal sections. (Scale bars: 10 μm; Insets, 0.5 μm.)