Apr 9, 2017

Direct Imaging of Intracellular Signaling Molecule Responsible for the Bacterial Chemotaxis

Methods in Molecular Biology
Hajime Fukuoka

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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Mentioned in this Paper

Signaling Molecule
Alkalescens-Dispar Group
Extracellular
Intracellular Signaling Proteins
Protoplasm
Properdin Deficiency, X-LINKED
Chemotaxis
Plasma Protein Binding Capacity
Fluorescent stain
Zone of Cilium

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