Abstract
We use high spatial and temporal resolution spinning-disk confocal microscopy, fluorescent-labeling strategies, and automated image analysis to investigate the response of the mechanosensitive channel MscL to osmotic stress in living E. coli bacteria. We establish the viability of individual cells using a red-fluorescent nucleic acid stain, propidium iodide, and correlate it with cellular levels of EGFP-tagged MscL. We demonstrate that MscL promotes integrity of the cell membrane in the face of environmental osmotic pressure. For these experiments, a micro-fluidic device with temperature control and multi-generational capability was developed to determine which cells are viable and dividing following exposure to osmotic stress, and to study the ability of cells to recover from temporally varying stress stimuli.
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