Dynamics of Δ<i>mxiG</i> dissemination in HT-29 cells.

<p>(A–C) Time-lapse microscopy of plasma membrane-targeted YFP-expressing HT-29 cells infected with the CFP-expressing Δ<i>mxiG/pmxiG</i> (Ara withdrawal) strain. 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) Unsuccessful progression of a bacterium (white arrows, top panel) from the primary cell cytoplasm (0′, Primary Cell) into a membrane protrusion (10′, Protrusion) that resolved into a vacuole (30′, Vacuole) from which the pathogen did not escape (180′, vacuole). Note that the trapped bacterium divided into at least 5 bacteria. (B) Unsuccessful progression of a bacterium (white arrows, top panel) from the primary cell cytoplasm (0′, Primary Cell) into a membrane protrusion (15′, Protrusion) that retracted towards the primary infected cell (65′, Protrusion) and returned the pathogen to the cytosol of the primary infected cell (70′, Primary Cell). (C) Tracking analysis of 60 bacteria, which formed protrusions in 25 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.