Fig. 6.
Active small GTPase Rap1 localizes to LCVs harboring Lp02 in D. discoideum. A, D. discoideum amoeba producing GFP-Rap1 were infected (MOI 30, 1 h) with L. pneumophila Lp02 or Δpentuple harboring plasmid pCR080 (DsRed, SidC). The infected cells were lysed using a ball homogenizer, fixed, stained for SidC and analyzed by fluorescence microscopy. Scale bars, 5 μm. B, Quantification of SidC- and Rap1-positive LCVs harboring Lp02 or Δpentuple. Data show means and standard deviation of at least 150 LCVs per strain counted in three independent experiments (Student's t test: **p < 0.01). Imaging flow cytometry (IFC) of D. discoideum Ax3 producing (C) GFP-Rap1 or (D) RalGDSRBD-GFP, infected (MOI 5) with DsRed-producing L. pneumophila Lp02 or Δpentuple. Representative IFC images from the 2 h time point (left panels). Quantification of IFC colocalization score between GFP and DsRed in > 1′000 cells per sample at the time points indicated (right panels). Data show means and 95% confidence intervals of one representative experiment out of three independent experiments (**p < 0.01, ***p < 0.001).