Figure 4
CT105 localizes at plasma membrane in C. trachomatis-infected cells. HeLa cells were infected by C. trachomatis L2/434 encoding pCT105-2HA. (A) Cells were also transfected with a plasmid encoding the plasma membrane marker Lyn11-mEGFP (green). At 30 or 40 h p.i., cells were fixed with 4% (w/v) PFA, stained with phalloidin (blue), and immunolabeled with antibodies against HA (red) and C. trachomatis MOMP (gray), and appropriate fluorophore-conjugated secondary antibodies. Cells were imaged by confocal fluorescence microscopy and images correspond to single z sections. In the area delimited by a white square (left-side panels) images were zoomed (right-side panels). Scale bars, 10 µm. (B) Infected cells were fixed with 4% (w/v) PFA at 16, 20, 30 or 40 h p.i. and immunolabeled with antibodies against HA, C. trachomatis MOMP, and GM130, and appropriate fluorophore-conjugated secondary antibodies. Fluorescence microscopy was then used to enumerate cells showing CT105-2HA only at the Golgi, only at the plasma membrane (PM), or both at the Golgi and at the plasma membrane (Golgi + PM). Data are mean ± standard error of the mean (SEM) of three independent experiments (n ≥ 25). (C) At 20 h p.i., infected cells were treated with DMSO, 1 µg/ml nocodazole, 2 µM cytochalasin D or 500 nM latrunculin B. At 30 h p.i., the cells were fixed with 4% (w/v) PFA and labeled with immunolabeled with antibodies against HA, C. trachomatis MOMP, and appropriate fluorophore-conjugated secondary antibodies. Fluorescence microscopy was used to enumerate cells showing CT105-2HA at the periphery of the cell/plasma membrane. Data are mean ± SEM of three independent experiments (n = 50). P-values were obtained by one-way ANOVA and Bonferroni post-test analyses; *statistical significant (P < 0.05); ns, not significant.
