Figure 8
CLSEM for studies of brush border recovery after invasion of Caco-2 BBe1 cells by Salmonella enterica. (A) Mesh holes with STM-infected (red) Caco-2 BBe1 cells (green) imaged in SDCM modality, fixed 1 h p.i. by addition of GA. Single cells of monolayers show distinct recognizable shapes. Processed samples were analysed by SEM using the InLens SE detector with EHT of 1.8 kV. The final merging step was guided by the distinct shapes of Caco-2 BBe1 cells. The ROI (red dotted line) is shown in higher magnification in B i and ii. (B) Timeline of LCI shows successive STM invasion in Lifeact-eGFP Caco-2 BBe1 cells for 1 h. The fate of one STM bacterium was traced over time (red arrowhead in SDCM modality). Accumulation of F-actin into membrane ruffle (green arrowhead in SDCM) indicates effective invasion into host cell. Correlation to SEM modality reveals the total number of STM (numbers) that invaded into a host cell, and microvilli structure of the infected cell at time point of fixation. Multiple infection events are accompanied by changes in cell shape over time. (i) An arrow-shaped Caco-2 BBe1 cell was invaded by a total of 8 STM bacteria. At 1 h p.i., the host cell was devoid of F-actin accumulations in membrane ruffles (red arrowhead) and showed full recovery of the brush border (green arrowhead). (ii) A bean-shaped Caco-2 BBe1 cell was invaded by a total of 7 STM bacteria. This cell also lacks apical F-actin accumulation (red arrowhead). In SEM modality, a bulky array of membrane material is visible on the host cell surface at 1 h p.i. (green arrowhead), indicating an accumulation of apical membrane material that was not reorganized into normal brush border architecture. Time stamp, min:sec. Scale bars, (A) 10 µm, (B) SDCM 5 µm, SEM 1 µm.
