Figure 2.

Derlin-1-positive ER vesicles are delivered into the ApV lumen where they associate with A. phagocytophilum organisms. (A–D) Derlin-1-positive vesicles are present within the ApV. A. phagocytophilum infected RF/6A cells were screened with antibodies against derlin-1 (A) or derlin-1 and APH0032 (B) and visualized using LSCM. (C) Z-stack series shows derlin-1-labeled vesicles within the ApV. Successive focal planes of the region in (B) that is demarcated by a hatched line box are presented. (D) 3D rendering of the Z-stack series presented in (C) shows derlin-1-positive vesicles encasing A. phagocytophilum organisms within the vacuole. (E) Derlin-1-positive vesicles are recruited to and delivered into the ApV early and continue to detected within the ApV throughout the course of infection. RF/6A cells that had been synchronously infected with A. phagocytophilum organisms were screened with antibodies against derlin-1 and APH0032 and visualized at several post-infection time points using LSCM. (A,E) Regions that are demarcated by hatched lined boxes correspond to the regions magnified in the insets that are demarcated by solid lined boxes. (A–E) Host cell nuclei and bacterial DNA were stained with DAPI (blue). Scale bars, 5 μm. (F) Percentages of ApVs in which derlin-1 signal is detectable within the lumen closely associated with intravacuolar A. phagocytophilum organisms over the course of a synchronous infection. Data are the means and standard deviations for triplicate samples. Results in all panels are representative of two experiments with similar results.