Journal: PLoS ONE

Article Title: The basolateral vesicle sorting machinery and basolateral proteins are recruited to the site of enteropathogenic E . coli microcolony growth at the apical membrane

doi: 10.1371/journal.pone.0179122

Figure Lengend Snippet: A. MDCK-AQP3-EGFP cells were seeded to confluency on semi-permeable collagen-coated Transwell filter supports and allowed to polarize for 3 days. Cells were then infected with EPEC for 6 hours, fixed and stained with an antibody against Lipid A to label EPEC bacteria (shown in red). The rightmost image shows a xz view at the position indicated by the white line in the merge image. AQP3-EGFP localized to the lateral membrane (arrow heads) and also accumulated at the site of microcolony formation around individual EPEC bacteria (arrows). Scale bars: 10 μm and 5 μm (insets). B. MDCK-AQP3-EGFP cells were infected with EPEC bacteria directly into the heating chamber after mouting on the microscope. Time-lapse imaging was performed with 1 minute intervals for DIC (EPEC and cells) and EGFP (AQP3-EGFP). Montage shows DIC and AQP3-EGFP in inverted contrast. The bottom panel shows the EGFP image including a drawn outline of the bacterial microcolony based on the DIC image. AQP3-EGFP recruitment was observed at the center of microcolony formation (at approximately 80’ after initial attachment) and was sustained to the center of the microcolony with no detectable recruitment to the periphery of the EPEC colony. Scale bar: 5 μm. C-D. Polarized MDCK-AQP3-EGFP monolayers with and without EPEC infection for 6 hours were stained with a monoclonal anti-gp135 antibody (red) and hoechst (blue, to detect EPEC and cell nuclei). C. xz-projection of confocal z-stacks showed that AQP3-EGFP was localized to the basolateral membrane (white arrow heads), whereas gp135 was strictly localized to the apical membrane (white arrows) in the uninfected cells. Both AQP3-EGFP and gp135 were localized at the infection site in EPEC-infected cells. Scale bar: 10 μm and 3 μm for insets. D. xy maximum projection of 6 slices from a z-stack showing an EPEC microcolony. Arrows point to AQP3-EGFP, arrowheads to gp135. Gp135 seemed less intense at sites of AQP3-EGFP accumulation. Scale bar: 10 μm and 1 μm for the inset.

Article Snippet: Imaging was performed on a Nikon T i Eclipse inverted microscope equipped with an OkoLab heating chamber, Perfect Focus 3 system, a 100x objective, and an Andor Zyla cMOS camera.

Techniques: Infection, Staining, Bacteria, Membrane, Microscopy, Imaging

Journal: PLoS ONE

Article Title: The basolateral vesicle sorting machinery and basolateral proteins are recruited to the site of enteropathogenic E . coli microcolony growth at the apical membrane

doi: 10.1371/journal.pone.0179122

Figure Lengend Snippet: MDCK cells were transiently transfected with VSVG3-SP-GFP. The cells were kept at 40°C to accumulate the protein in the ER. They were then infected with EPEC at 40°C for 3 hours, before VSVG3-SP-GFP was released to the TGN at 20°C for 2 hours. Finally, the infected cells were mounted on a time-lapse fluorescence microscope, and VSVG3-SP-GFP was released at 37°C. Time-lapse imaging was initiated after 15 minutes. A. Example of an uninfected cell and a cell infected with EPEC at initiation of imaging. Montages of the boxed areas are shown in B and the full time-lapse is shown in . Scale bar: 10 μm. B. Example of an EPEC microcolony (DIC, top panels) and VSVG3-SP-GFP localization (bottom panels) from a time-lapse sequence. Green arrows indicate the top of the EPEC colony, and black arrows indicate examples of EPEC bacteria. Scale bar: 3 μm. All images of GFP are shown as inverted contrast. C. Regions of interest were placed at EPEC colonies or at equivalent positions in non-infected cells. The fluorescence intensities of VSVG3-SP-GFP were quantified for the entire time-lapse sequences and normalized to t = 15 min. There was a statistically significantly higher level of recruitment VSVG3-SP-GFP to EPEC infection sites than equivalent positions on uninfected cells (*p<0.05 using repeated measurements analysis of variance). The measurements were performed on 25 infected and 25 non-infected cells. Standard deviation is indicated by the red and green regions.

Article Snippet: Imaging was performed on a Nikon T i Eclipse inverted microscope equipped with an OkoLab heating chamber, Perfect Focus 3 system, a 100x objective, and an Andor Zyla cMOS camera.

Techniques: Transfection, Infection, Fluorescence, Microscopy, Imaging, Sequencing, Bacteria, Standard Deviation