To elucidate the mechanisms by which cells respond to extracellular stimuli, the behavior of intracellular signaling proteins in a single cell should be directly examined, while simultaneously recording the cellular response. In Escherichia coli, an extracellular chemotactic stimulus is thought to induce a switch in the rotational direction of the flagellar motor, elicited by the binding and dissociation of the phosphorylated form of CheY (CheY-P) to and from the motor. We recently provided direct evidence for the binding of CheY-P to a functioning flagellar motor in live cells. Here, we describe the method for simultaneously measuring the fluorescent signal of the CheY-enhanced green fluorescent protein fusion protein (CheY-EGFP) and the rotational switching of the flagellar motor. By performing fluorescence and bright-field microscopy simultaneously, the rotational switch of the flagellar motor was shown to be induced by the binding and dissociation of CheY-P, and the number of CheY-P molecules bound to the motor was estimated.
Keywords: Chemotaxis; E. coli; Fluorescence imaging; Signal transduction; Simultaneous measurements; Two-component signaling system.