Figure 1.

The Dynamic Actin Cocoon Polymerizes De Novo after Cellular Entry around Shigella’s BCV and Disassembles before Vacuolar Escape

(A and B) Real time-monitoring of the thick actin cocoon. HeLa cells expressing actin-GFP (green) were infected with Shigella WT DsRed (red) (A; asterisk denotes bacterium with cocoon) or Shigella WT (B; blue dashed line denotes bacterium). t = 0 min: onset of entry site formation.

(C) The actin cocoon needs to at least partially disassemble before vacuolar rupture. Time lapse of Shigella WT infecting HeLa cells expressing actin-GFP (green) and galectin-3-mOrange (red, Gal3). Arrow, newly formed cocoon; arrowhead, moment of rupture.

(D) Time lapses of FRAP experiments of the actin cocoon in comparison with cellular actin structures (see Videos S3, S4, and S5).

(E) Quantification of (D). Plotted are mean values ± 95% confidence interval [CI] and curve fit of at least 3 independent experiments (actin cocoon: n = 40 FRAP curves ; lamellipodial tip: n = 31; stress fibers: n = 75).

(F and G) Actin cocoon formation depends on the time point of bacterial infection and pre-infection. Depicted are percentages of bacteria that successfully escaped into the host cytosol and previously assembled an actin cocoon (+) or not (−) (F). On average, three entry sites formed per cell (Figures S1 and S2), and 71.2% ± 1.75% of invading bacteria (n = 631, 4 individual experiments) had a cocoon. All late invaders assembled a cocoon before vacuolar escape (G). Mean values ± SD of individual experiments (F) or rupture time points of pooled single invaders (G) are plotted. Mann-Whitney test with p < 0.05 as significant: ^∗∗∗∗p < 0.0001.

Scale bars: 3 μm. See Figures S1 and S2.