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Fig. 6. Confocal analysis of Chlamydia entry sites. Sites of Chlamydia entry appear as spatially well-organized membrane structures that accumulate ARF6 and its downstream effectors. (A) HeLa cells transfected with HA-tagged-ARF6 WT were infected for 5 minutes with FITC-coupled Chlamydia (blue). Cells were then stained with anti-HA antibody followed by TRITC-coupled second antibody. Inset shows bacterium (blue) surrounded by ARF6 staining. In the right-hand panel, a plan of the 3D reconstructed structure of ARF6 (red) and internalizing bacterium (blue) is shown. Results are representative of at least three independent experiments. (B) HeLa cells transfected with GFP-PLC PH and HA-tagged-ARF6 WT were infected by Chlamydia for 5 minutes. Transfected cells were detected with a specific anti-HA antibody followed by Cy5-coupled secondary antibody (red) and the fluorescence of GFP (green). Higher magnifications of the entry site (arrowheads) are shown in the right-hand panels. Results are representative of at least three independent experiments. (C) HeLa cells transfected with HA-tagged PIP 5-kinase and ARF6 were infected by Chlamydia for 5 minutes. Cells were stained with anti-ARF6 antibody followed by Alexa488-coupled secondary antibody (green), anti-HA antibody followed by Cy5-coupled secondary antibody (red) and with Alexa 633-coupled phalloidin (blue). A higher magnification of the entry site (arrowhead) is shown in the right-hand panel. (D) Scheme showing the relative localization of ARF6, PIP₂ and PIP 5-kinase around the internalizing bacteria. PIP₂ staining (green) was the closest to the bacterium and covered most of the surface of the round structure; ARF6 (blue) and PIP 5-kinase (red) staining were preferentially enriched on the edges of the round protrusion, leaving a large unstained area in the centre filled by the bacterium. PIP 5-kinase appears to occupy the largest area that overlapped with PIP₂ and ARF6 staining. Bar, 2 µm (A and right-hand panels in C); 10 µm (B); 5 µm (left-hand panels in C).

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