This study investigates the calcium signaling response in human epithelial cells during Shigella invasion. The methodology involves imaging techniques to visualize intracellular calcium dynamics triggered by bacterial effectors.
Take an imaging chamber containing a culture of human epithelial cells loaded with a fluorescent indicator of intracellular calcium.
Add a growth medium containing calcium ions. Introduce Shigella, a pathogenic bacterium, and incubate.
The bacterial secretion system penetrates the host cell and releases invasion effectors. Invasion triggers actin rearrangement, forming membrane ruffling around bacteria.
At the invasion site, phospholipase C, or PLC, on the plasma membrane is activated, and inositol triphosphate receptors cluster on the endoplasmic reticulum or ER.
PLC hydrolyzes membrane phosphatidylinositol 4,5-bisphosphate to inositol triphosphate, or IP3, that accumulates locally due to dense actin meshwork.
IP3 binds to receptors on the ER, causing calcium release. Calcium indicators bind to released ions and emit fluorescence, displaying a local calcium response.
IP3 slowly diffuses through the actin, further depleting the ER calcium level and activating a plasma membrane calcium channel, leading to calcium influx .
Fluorescence across the cell indicates delayed global calcium response.
Place the coverslip containing the Fluo-4 cells in an imaging or glass-bottom chamber. Use EM buffer to wash the sample three times to remove compounds potentially resulting from cell lysis. Then, add one milliliter of EM buffer. Place the chamber on an inverted fluorescence microscope stage, heated at 33 degrees Celsius. Select a microscopy field, and set up acquisition parameters, including exposure time and binning, if necessary, to optimize the Fluo-4 fluorescent signal.
To add bacteria to the sample, carefully remove 500 microliters of EM buffer from the chamber, and add 500 microliters of the bacterial suspension to obtain a final OD600 of 0.05. Immediately perform an acquisition, or wait 10 minutes to allow the bacteria to sediment onto the cells. At the end of the acquisition stream, acquire a phase contrast image of the selected field to visualize the bacteria contacting the cells and the membrane ruffles associated with bacterial invasion sites.
Repeat the acquisition procedure at intervals that are amenable to the duration of the image acquisitions, file savings, and selection of a new field to cover the whole process. At the end of the acquisition procedure, add to the sample, two micromolar of the calcium ionophore ionomycin to determine the maximal amplitude of the calcium signals. Acquire images every 3 to 5 seconds until the signal stabilizes, usually for less than 10 minutes.
Follow by adding the calcium chelator EGTA to a final concentration of 10 millimolar to determine the fluorescent signal in the absence of calcium. Acquire images every 3 to 5 seconds until signal stabilization, usually for less than 10 minutes. Carry out image analysis according to the text protocol.
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