Dynamics of wild-type 2457T dissemination in HT-29 cells.

<p>(A–C) Time-lapse microscopy of plasma membrane-targeted YFP-expressing HT-29 cells infected with CFP-expressing wild-type strain 2757T. Yellow, plasma membrane; Cyan, <i>Shigella</i>. (A,B) Representative images showing the progression of a single bacterium over time. For each panel, the top image shows a low magnification image of infected cells and the bottom images show an enlargement of the tracked bacterium (merged bacterium and membrane channels, left; membrane channel only, right). (A) Successful progression of a bacterium (white arrows, top panel) from the primary cell cytoplasm (0′, Primary Cell), into a membrane protrusion (15′, Protrusion) that resolve into a secondary vacuole (50′, Vacuole) from which the bacterium escape and gains access to the cytoplasm of the adjacent cell (75′, Free bacteria). (B) Unsuccessful progression of a bacterium (white arrows, top panel) from the primary cell cytoplasm (0′, Primary Cell) into a membrane protrusion (10′, Protrusion) that retracted towards the primary infected cell (20′, Protrusion) and returned the bacterium to the cytosol of the primary infected cell (25′, Primary Cell). (C) Tracking analysis of 60 bacteria, which formed protrusions in 20 independent foci. All bacteria were tracked for at least 180 minutes and the progression of the dissemination process was depicted using the color key shown at the bottom of panel C. Primary cell, dark blue; Protrusion, light blue; Vacuole, yellow; Free bacteria in adjacent cell, red. Scale bars, 5 µm.