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Activation of Ran GTPase by a Legionella Effector Promotes Microtubule Polymerization, Pathogen Vacuole Motility and Infection

Figure 4

LegG1 stabilizes microtubules in L. pneumophila-infected phagocytes.

(A, B) Microtubules were analyzed by confocal laser scanning fluorescence microscopy in (A) D. discoideum producing tubulin-GFP or (B) RAW264.7 macrophages infected (MOI 10, 2 h (amoebae), 4 h (macrophages)) with DsRed-producing L. pneumophila wild-type, ΔicmT or ΔlegG1 harboring pCR077 or with ΔlegG1/pER5 (M45-LegG1). The macrophages were immuno-labeled for α-tubulin (green) and SidC (blue) and, as controls, treated with taxol or nocodazole (30 µM). (C, D) Microtubules were analyzed by STED microscopy in RAW264.7 macrophages infected (MOI 10, 4 h) with GFP-producing L. pneumophila wild-type, ΔicmT or ΔlegG1 harboring pCR076 or with ΔlegG1/pER4 (M45-LegG1) and immuno-labeled for (C) α-tubulin (grey), or (D) α-tubulin (green) and SidC (blue). Uninfected macrophages were treated with taxol or nocodazole (30 µM) as control. Bars, 1 µm (A), 5 µm (B, C), 0.5 µm (D). (E) Microtubule polymerization was analyzed by anti-tubulin Western blot in RAW264.7 macrophages infected (MOI 10, 4 h) with GFP-producing L. pneumophila wild-type, ΔicmT or ΔlegG1 harboring pCR033, or with ΔlegG1/pSU19 (M45-LegG1). As a control, uninfected macrophages were treated with taxol or nocodazole (30 µM). Homogenates of the macrophages were centrifuged (20'000×g, 30 min) to separate polymerized tubulin in the pellet (P) from soluble tubulin in the supernatant (S), and the amount of microtubule polymerization was assessed by α-tubulin Western blot (upper panel). Actin was used as a control. A representative experiment is shown, and the ratio of polymerized to soluble α-tubulin is indicated (R). The graph (lower panel) shows means and standard deviations of 3 independent experiments.

Figure 4

doi: https://doi.org/10.1371/journal.ppat.1003598.g004