Fig. 2: The bacterial fusion protein IncA clusters in ICSs.
From: Specialized contact sites regulate the fusion of chlamydial inclusion membranes
a HeLa cells were infected with WT Ctr (MOI 3) for 5 h to allow the infection to proceed before being treated with 20 μM nocodazole. Cells were then fixed at 21 hpi and labeled with anti-IncA (magenta), anti-IPAM (cyan), and anti-CT813 (green) antibodies. DNA was labeled with Hoechst (blue). Enlarged ICSs (white box) are shown in the Zoom panel. Asterisks denote inclusions. Scale bar = 10 µm. Images are representative of three independent experiments. b ESs were calculated for each Inc. Each dot = individual ES, black horizontal bar = mean. Gray box denotes ESs <2. N = 130 total scores per Inc from three independent experiments. ns = not significant, **** denotes p-value < 0.0001 (matched-paired, one-way ANOVA). c Anti-FLAG-FrankenbodymScarlet3 HeLa cells were infected with IncAKO Ctr complemented with IncAWT-FLAG (MOI 3). IncAWT-FLAG expression was induced at 5 hpi with 20 ng/mL anhydrotetracycline. z-stacks of infected cells were acquired every 5 min as described in the Methods. Individual frames are shown beginning at 17 hpi (0 min). IncA images show the enrichment of the anti-FLAG FrankenbodymScarlet3 signal at the ICS prior to the fusion of the two inclusions (zoom). The HiLo look-up table (HiLo LUT) was then applied to the IncA images to highlight the grayscale differences (low signal = blue; high signal = red). Scale bar = 5 µm. Blue arrows denote signal enrichment at the ICS. Images are representative of three independent experiments. d HeLa cells were infected with WT Ctr (MOI 3), fixed at 18 hpi, and labeled with anti-IPAM (cyan) and -IncA (magenta) antibodies. DNA was labeled with Hoechst (blue). Enlarged ICSs (white box) are shown in the Zoom panel. Asterisks denote inclusions; Scale bar = 10 μm. Images are representative of three independent experiments. e HeLa cells were infected, fixed, and labeled as in d. ESs for each Inc was measured. N = 15 total scores per Inc from three independent experiments. Gray box denotes ESs <2. ns = not significant, **** denotes p-value < 0.0001 (matched-paired, one-way ANOVA).
