Bartonella effector protein C mediates actin stress fiber formation via recruitment of GEF-H1 to the plasma membrane
Fig 5
GEF-H1 is essential for BepCBhe–triggered actin stress fiber formation while MRCKα is dispensable.
(A) Proposed model of BepC-triggered actin stress fiber formation with indication of the GEF-H1 and MRCKα knock-out. (B) HeLa cells were co-transfected with two different plasmids encoding Cas9 and a sgRNA, specific either to the first or the last exon of the target gene (GEF-H1 or MRCKα). After selection and expansion of transfected cells, expression of GEF-H1 or MRCKα was tested by immunoblot analysis. Tubulin was used as loading control. (C-E) HeLa cells wild-type, GEF-H1 KO, and MRCKα KO were infected with Bhe ΔbepA-G expressing FLAG-tagged BepCBhe, or carrying the empty plasmid as a negative control, at MOI of 400. After 48 h of infection, cells were fixed, stained by immunocytochemistry and analyzed by fluorescence microscopy. (C) F-actin is represented in green, DNA in blue, and bacteria in red. (D) The mean fluorescence intensity of F-actin for conditions shown in (C) was quantified for 111 imaged sites using CellProfiler. Data are represented as dot plots with each data point corresponding to the average of all mean cell intensity values within one imaged site normalized to the uninfected wild-type (WT) control. Statistical significance was determined using Kruskal-Wallis test (**** corresponds to p-value ≤ 0.0001). (E) Anti-GEF-H1 staining is represented in white (scale bar = 50 μm). Shown are representative results from three independent experiments.