Fig 5. Lifeact impairs the activity of F-actin-binding bacterial toxins.

(A) SDS-PAGE analysis of cosedimentation experiments of F-actin (1 μM, lower band) with ExoY-MBP (1 μM, upper band) in the presence of the indicated amounts of Lifeact. A representative gel is shown. The fractions of ExoY that cosedimented with F-actin were quantified by densitometry and plotted against Lifeact concentrations in (B). (C) The atomic model of the Lifeact–F-actin complex shows that T148, the site of Tcc3HVR modification [36], is localized within a 4 Å distance from F10 of Lifeact (green). (D) The level of actin ADP-ribosylation by TccC3HVR in the presence of Lifeact was analyzed by western blot using an ADP-ribose binding reagent. The equal loading of actin was additionally verified by imaging the same stain-free gel prior to blotting (lower image). The ADP-ribosylation level of actin was quantified by densitometry and plotted against Lifeact concentrations in (E). Error bars at (B) and (E) correspond to standard deviations of 3 independent experiments. (F) HEK 293T cells expressing mCherry fusions of actin or LifeAct variants were intoxicated with 300 pM of the Photorhabdus luminescens toxin PTC3, which injects TccC3HVR into cells. The degree of cytoskeletal collapse and accompanying cell shrinkage was monitored for 5 hours using live cell imaging and is plotted based on 3 independent experiments for each condition. Scale bars, 20 μm. The uncropped western blots and gels can be found in S4 Fig. Data points that were used to create graphs are reported in S2 Table. F-actin, filamentous actin; HEK, human embryonic kidney; MBP, maltose-binding protein; pel, pellet; sup, supernatant; TccC3HVR, hypervariable region of TccC3; WT, wild-type.