Skip to main content
. 2020 May 12;31(6):107638. doi: 10.1016/j.celrep.2020.107638

Figure 5.

Figure 5

The Actin Cocoon Is Regulated by Shigella T3SS Effector Proteins, and Host Actin Regulators Constantly Localize IcsB Dependent at the BCV

(A and B) Infection of HeLa cells expressing actin-GFP and galectin-3-mOrange by Shigella single effector deletion mutants. The first invading bacteria per cell was analyzed with regard to actin cocoon assembly (A) and rupture time (B). (n = 1,085 total counted bacteria; WT, n = 101; ΔipaH7.8, n = 80; ΔvirA, n = 131; ΔospC1, n = 112; ΔicsA, n = 103; ΔipaJ, n = 95; ΔipgD, n = 121; ΔipgB1, n = 116; ΔicsB, n = 120. p < 0.05 is significant compared with WT: ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, and ∗∗∗∗p < 0.0001; ns, not significant).

(C) Representative time lapses of constant CDC42 localization at the BCV during Shigella WT infection.

(D) Quantification of the normalized relative fluorescence intensity of CDC42 recruitment during successive Shigella invasion steps. PM ruffle, fluorescence intensity at plasma membrane ruffles; BCV-start, at the phagocytic cup before scission; BCV-maint, at maintained BCV after cellular uptake; BCV-rupture, at BCV after rupture.

(E) Deletion of IcsB leads to CDC42 removal from the BCV before rupture but does not prevent its initial recruitment to membrane ruffles.

(F) Quantitative analysis of Cdc42 localization at the BCV of successfully invading Shigella with constant (dark blue), transient (light blue), or no (gray) CDC42 localization (n = 311). Shigella WT versus ΔicsB: constant CDC42, p < 0.0001; depleted CDC42, p < 0.001; no CDC42, p < 0.05.

(G) Recruited TOCA-1 is constantly localized at the BCV dependent on IcsB (infection top: Shigella WT; bottom: ΔicsB).

Indicated are mean values ± SD of at least 3 independent experiments, in (A) normalized to Shigella WT. Statistical significance: Indicated are mean values ± SD. one-way ANOVA (D) or two-way ANOVA with Tukey’s multiple-comparisons test (F). Arrow, initial protein recruitment; arrowhead, vacuolar rupture; scale bar: 3 μm. Green bar, protein at BCV; red bar, galectin-3 (Gal-3) at BCV; yellow bar, protein and galectin-3 at BCV remnant; gray bar, no protein or galectin-3. See STAR Methods.