title: Direct Imaging of Intracellular Signaling Molecule Responsible for the Bacterial Chemotaxis.
abstract: 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.
authors.affiliation: Graduate School of Frontier Biosciences, Osaka University, 1-3 Yamadaoka, Suita, Osaka, 565-0871, Japan. f-hajime@fbs.osaka-u.ac.jp.
authors.forename: Hajime
authors.initials: H
authors.lastname: Fukuoka
mesh_terms: D002633:Chemotaxis / Q000502:physiology*; D003593:Cytoplasm / Q000378:metabolism / Q000502:physiology; D004926:Escherichia coli / Q000378:metabolism* / Q000502:physiology*; D029968:Escherichia coli Proteins / Q000378:metabolism; D005407:Flagella / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000378:metabolism; D008565:Membrane Proteins / Q000378:metabolism; D010766:Phosphorylation / Q000502:physiology; D011485:Protein Binding / Q000502:physiology; D015398:Signal Transduction / Q000502:physiology*
chemical_list: D029968:Escherichia coli Proteins; D008565:Membrane Proteins; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins
keywords: Chemotaxis; E. coli; Fluorescence imaging; Signal transduction; Simultaneous measurements; Two-component signaling system