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.
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Acknowledgments
I thank A. Ishijima (Osaka University) for critical reading and useful discussions in this manuscript. This work was supported by Grants-in-Aid for Scientific Research from MEXT KAKENHI JP23115004 (to A.I.), and from JSPS KAKENHI JP26440073 (to H.F.).
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Fukuoka, H. (2017). Direct Imaging of Intracellular Signaling Molecule Responsible for the Bacterial Chemotaxis. In: Minamino, T., Namba, K. (eds) The Bacterial Flagellum. Methods in Molecular Biology, vol 1593. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-6927-2_17
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DOI: https://doi.org/10.1007/978-1-4939-6927-2_17
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