Journal: Frontiers in Cellular and Infection Microbiology
Article Title: Small Rho GTPases and the Effector VipA Mediate the Invasion of Epithelial Cells by Filamentous Legionella pneumophila
Figure Lengend Snippet: Rho family small GTPases are needed for the attachment of filamentous Lp to LECs. Recruitment of Cdc42 (A) , Rac1 (B) , and RhoA (C) at the membrane wraps formed by Lp attachment. Cells transiently expressing Cdc42-GFP, Rac1-GFP or RhoA-GFP were infected for 2 h, fixed and external bacteria were immunolabeled (blue). Panels to the right show higher magnifications of framed areas. (D) Quantification of Rho GTPase recruitment at membrane wraps from (A–C) . Means ± SEM from 3 independent experiments are shown (n > 25 bacteria per experiment). (E,F) Confocal micrographs showing the recruitment of PAK-PBD-YFP and rGBD-GFP at the membrane wraps formed by Lp 2h p.i. (G) Quantification from (E,F) . Means ± SEM from 3 independent experiments are shown ( n > 25 bacteria in each experiment). (H–J) Lp attachment to NCI-H292 cells following inhibition of Cdc42, Rac1, and Rho by ML141, Nsc23766 (NSC) and C3 transferase (C3) respectively. Cells were treated with the indicated inhibitors for 1 h followed by infection with Lp for 1 h in the presence of the inhibitors. Quantification of bacterial attachment, normalized to vehicle treated cells is shown. (K–M) Attachment of Lp to cells expressing wild-type or dominant-negative (DN) forms of Rho GTPases. Attachment of bacteria to cells pre-treated for 1 h with inhibitor for mDia (N) or cells expressing dominant negative form of mDia (mDia-DN) (O) . Attachment of bacteria to cells pre-treated with CK666, Y27632 or blebbistatin to inhibit Arp2/3, ROCK or myosin II respectively (P–R) . Data shown are means ± SEM where bacterial attachment to at least 200 cells was analyzed. The number of independent experiments analyzed for each treatment were as follows: (H,I,K–P,R) n = 3; (J,Q) n = 4. Vehicle controls for each condition were as follows: (H,N–R) DMSO; (I) water; (J) glycerol. For all fluorescence micrographs the main panels are merged z-stacks and the higher magnifications are single z-planes. All scale bars shown, 12 μm. Statistical tests were performed using Student's t -test, *** p
Article Snippet: The following inhibitors were used in this study: PP2 (25 μM, Tocris) (Hanke et al., ), (100 μM, Sigma) (Vlahos et al., ), membrane permeable C3 transferase (0.5 μg/mL, Cytoskeleton Inc.) (Ridley and Hall, ), ML141 (20 μM, Tocris) (Surviladze et al., ), Blebbistatin (200 μM, Sigma) (Straight et al., ), Nsc23766 (50 μM, Tocris) (Gao et al., ), ROCK (1 μM, Millipore) (Narumiya et al., ), SMIFH2 (25 μM, Millipore) (Rizvi et al., ), CK-666 (80 μM, Sigma) (Nolen et al., ).
Techniques: Expressing, Infection, Immunolabeling, Inhibition, Dominant Negative Mutation, Fluorescence