Fig. 5. Recruitment of PIP 5-kinase at the sites of bacterial entry and local PIP₂ production. (A) PIP 5-kinase localizes at the sites of bacteria entry. HeLa cells were transfected with HA-tagged PIP 5-kinase for 18 hours and infected with FITC-coupled Chlamydia for 5 minutes. Transfected cells were identified with anti-HA antibody followed by anti-rat TRITC antibody (in red). PIP 5-kinase is shown recruited (arrows) around the bacteria (green) (top left panel). In the bottom panels a higher magnification of the entry sites is shown; note the absence of PIP 5-kinase in the central area (bottom right panel) where the bacterium is present (bottom left panel). Medial confocal optical sections are shown. The overall structure at the sites of bacterial entry can be visualized in the differential interference contrast image (arrows in the top right panel). Results are representative of at least three independent experiments. (B) Distribution of PIP₂ during bacterial entry. HeLa cells transfected with GFP-PLC-PH and HA-tagged PIP 5-kinase were infected for 5 minutes as indicated. Cells were fixed and stained with anti-HA antibody followed by anti-rat TRITC to reveal PIP 5-kinase (red). The fluorescence of GFP-PLC-PH reveals the presence of PIP₂ (green) (top panel). HeLa cells transfected with GFP-PLC-PH were infected as indicated with Cy5-coupled Chlamydia for 5 minutes before fixation. The arrowheads indicate PIP₂ found at the entry sites accumulated around a bacterium (red). The differential interference contrast image with the overall structure is shown (bottom panel). Results are representative of at least three independent experiments. (C) PIP 5-kinase depletion by RNAi impairs bacterial infection. HeLa cells were treated for 48 hours with PIP 5-kinase siRNA and infected for 20 hours prior to quantification of the number of infected cells. Infected cells were visualized with FITC-coupled anti-Chlamydia antibody. The number of infected cells is expressed relative to that in non-treated cells. The results presented are the mean±s.e.m. of three experiments. (D) Transient expression of Lyn-phosphatase affects Chlamydia entry. HeLa cells were transfected to express Lyn-CFP-Inp54p, infected with FITC-coupled bacteria for 4 hours on the following day and bacteria entry was quantified. Extracellular bacteria were stained with anti-Chlamydia antibody followed by Cy5-coupled secondary antibody. Surface-associated (Cy5- and FITC-positive) and intracellular bacteria (FITC-positive) were counted in the transfected and non-transfected population (n>25 cells) and the efficiency of entry (intracellular/total cell-associated) was calculated. For each experiment, the efficiency of entry in transfected cells is expressed relative to that in non-transfected cells (NT). Data are the mean±s.e.m. of three independent experiments. Bar, 10 µm.