Fig 1.
Mitochondria, ER, and inclusion membranes are found within the lumen of C. trachomatis inclusions.
HeLa cells were infected with C. trachomatis LGV L2, transfected with the indicated plasmids, and fixed at 30 hpi for serial spinning disk laser confocal analysis. Note the presence of markers of the ER, mitochondrial matrix and outer membranes, and inclusion membranes within the inclusion lumen (A, cyan arrowheads). Images portray a single z-section from the center of an inclusion, and inclusions are visually identified as large black centered ovals or outlined with a dashed line. Cellular-localized markers appear saturated because material within the inclusion was often significantly dimmer. (B) The frequency of internalized structures within the entire 3D space of each inclusion was assessed. Plasmids are categorized as markers of the ER, mitochondria, inclusion, cytosol, recycling endosomes, or other as indicated. Within the other category, GFP-GalT localizes to the Golgi, CD63-GFP to MVBs, LAMP1-GFP to lysosomes, and KRphi-mRFP to the plasma membrane. A dashed line at 50% distinguishes between high and low frequencies of intraluminal structures within inclusions. 12–20 inclusions were assessed in each experiment, and the mean ± SEM for three independent experiments is shown. Scale bar represents 5 μm.
Fig 2.
ER markers reveal expansive structures within the inclusion lumen of fixed but not living cells.
HeLa cells were infected with C. trachomatis LGV L2, co-transfected with ER-RFP (red) and Sec61β-GFP (green) and at 30 hpi were either fixed or imaged in three dimensions while living. Images were acquired with a laser scanning confocal microscope. (A) A single xy micrograph towards the center of a cell. See S1 and S2 Movies for a progression of xy micrographs along the z-axis. (B) Images were used to render volumes in 3D. 3D render of a fixed cell is limited in the z-axis to allow viewing within the inclusion. See S3–S6 Movies for QuickTime Virtual Reality files to rotate and view these 3D volumes. Note the presence of an expansive network of material within the inclusion lumen of fixed cells. N marks the nucleus. Scale bars represent 5 μm.
Fig 3.
ER-RFP translocates into the inclusion lumen during chemical fixation.
HeLa cells were infected with C. trachomatis LGV L2 and transfected with ER-RFP. At 30 hpi, living cells were placed in 4% paraformaldehyde and imaged over time with a laser scanning confocal microscope. Note the genesis of ER-RFP blebs into the inclusions lumen (cyan arrowheads) during fixation. See S7 Movie for a time-lapse video including more cells in a larger field of view. N marks the nuclei. Scale bar represents 10 μm.
Fig 4.
ER structures are detected via immunofluorescence microscopy within inclusions after both formaldehyde and alcohol-based chemical fixation.
HeLa cells were infected with C. trachomatis LGV L2. (A) Infected cells were transfected with ER-RFP, fixed at 30 hpi with different concentrations of either paraformaldehyde (PFA) or formaldehyde (Form), and assessed for the frequency of ER-RFP within inclusions. (B and C) Infected cells were transfected with ER-RFP as indicated. At 30 hpi, cells were fixed with PFA, methanol, or ethanol, and subsequently permeabilized with Triton X-100 (Tx100) as indicated. Treated cells were processed for immunofluorescence with an antibody to the ER protein PDI and assessed for the frequency of PDI-positive structures within inclusions. Scale bar represents 5 μm.
Fig 5.
ER-RFP within the pathogenic vacuole of Coxiella burnettii.
HeLa cells were infected with Coxiella burnettii, transfected with ER-RFP, and fixed at 52 hpi. Note the presence of ER-RFP structures with in the lumen of the pathogenic vacuole (cyan arrowheads) similar to those within Chlamydia trachomatis inclusions. N indicates the nucleus and a dashed line outlines a region of higher magnification. Scale bar represents 5 μm.