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

Figure 6.

Figure 6

Constant N-WASP Localization at the BCV Depends on Post-translational Lipidation by IcsB, but CDC42 Is Crucial for Its Initial Recruitment

(A and B) The Shigella effector IcsB constantly localizes N-WASP at the BCV during infection after initial IcsB-independent recruitment. Representative time lapses for Shigella WT (A) and ΔicsB (B) are shown.

(C) CDC42 knockdown strongly impairs initial N-WASP recruitment to the BCV of Shigella WT.

(D) Quantification of N-WASP localization at the BCV of invading Shigella with constant (dark green), transient (light green), or no (gray) N-WASP (n = 441). Statistical significance of Shigella WT versus ΔicsB: constant N-WASP localization, p < 0.0001; N-WASP depleted, p < 0.0001; no N-WASP, ns. Shigella WT versus siRNA CDC42: constant N-WASP localization, p < 0.0001; N-WASP depleted, ns; no N-WASP, p < 0.0001.

(E) The complemented Shigella icsB− mutant (icsB−[compl.]) clusters N-WASP like Shigella WT around the BCV.

(F) In contrast, complementation of icsB− with IcsB lacking fatty acyltransferase activity (icsB− [C306A]) does not restore constant N-WASP localization.

(G) Quantification of N-WASP localization at the BCV of invading Shigella, color coded as in (D) (n = 748; WT, n = 138; icsB−, n = 182; icsB−[compl.], n = 136; icsB−[C306A], n = 292). Shigella WT versus icsB−: constant N-WASP localization, p < 0.0001; N-WASP depleted, p < 0.0001; no N-WASP, ns. Shigella WT versus Shigella icsB−(compl.): constant N-WASP localization, ns; N-WASP depleted, ns; no N-WASP, ns. Shigella WT versus Shigella icsB−(C306A): constant N-WASP localization, p < 0.0001; N-WASP depleted, p < 0.0001; no N-WASP, ns.

Statistical significance: two-way ANOVA with Tukey’s multiple-comparisons test. Indicated are mean values ± SD of 3 independent experiments. Arrow, initial N-WASP recruitment; arrowhead, moment of vacuolar rupture; scale bars: 3 μm (A–C) and 5 μm (E). Green bar, N-WASP at BCV; red bar, galectin-3 at BCV; yellow bar, N-WASP and galectin-3 at BCV remnant; gray bar, no N-WASP or galectin-3. See STAR Methods and Figure S7.