Direct Imaging of Intracellular Signaling Molecule Responsible for the Bacterial Chemotaxis
Direct Imaging of Intracellular Signaling Molecule Responsible for the Bacterial Chemotaxis
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负责细菌趋化性的细胞内信号分子的直接成像
DOI：
10.1007/978-1-4939-6927-2_17
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发表时间：
2017
影响因子：
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通讯作者：
Hajime Fukuoka
中科院分区：
文献类型：
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作者：
K.Kuroki;K.Mio;A.Takahashi;H.Matsubara;Y.Kasai;S.Manaka;M.Kikkawa;D.Hamada;C.Sato;K.Maenaka.;Hajime Fukuoka
为了阐明细胞对细胞外刺激的反应机制，应直接检查单个细胞内信号蛋白的行为，同时记录细胞反应。在大肠杆菌中，细胞外趋化刺激被认为诱导鞭毛马达旋转方向的开关，这是由磷酸化形式的CheY （CheY- p）与马达的结合和解离引起的。我们最近提供了直接证据，证明CheY-P与活细胞中功能鞭毛马达的结合。在这里，我们描述了同时测量chey增强绿色荧光蛋白融合蛋白（CheY-EGFP）荧光信号和鞭毛马达旋转开关的方法。通过同时进行荧光和明场显微镜观察，发现鞭毛马达的旋转开关是由CheY-P的结合和解离诱导的，并估计了CheY-P分子结合在马达上的数量。
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. InEscherichia 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.