Fig. 2. Aggregation is dependent on host cell microtubules but does not require the actin cytoskeleton or an intact Golgi apparatus. Cos-7 cells were infected with C. trachomatis L2 for 1 hour before treatment with the indicated drug. Infected cells were incubated with nocodazole for 4 hours before fixing and staining for C. trachomatis and tubulin. In these cells the chlamydial early inclusions did not aggregate at a single perinuclear site but remained widely dispersed throughout the cytoplasm (A). The tubulin staining shows that the microtubule network (green) was disrupted (B). Cells transfected with GFP-Golgi were infected and treated with brefeldin A for 4 hours. The cells were fixed and stained for chlamydiae (C) and observed simultaneously with GFP signal. The nascent inclusions aggregated normally even thought the Golgi was dispersed, as can be seen by the dispersed GFP-Golgi signal (D). Disruption of the actin cytoskeleton with cytochalasin D also does not inhibit chlamydial aggregation. FITC-phalloidin staining of the F-actin cytoskeleton shows that the actin cytoskeleton has been disrupted (F) but the nascent inclusions are still aggregated at a single site within the cell (E). Bar, 10 µM.