Assessment of the flagellar redox potential in Chlamydomonas reinhardtii using a redox-sensitive fluorescent protein, Oba-Qc.
Assessment of the flagellar redox potential in Chlamydomonas reinhardtii using a redox-sensitive fluorescent protein, Oba-Qc.
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DOI：
10.1016/j.bbrc.2018.07.163
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发表时间：
2018-09
影响因子：
3.1
通讯作者：
Kazunori Sugiura;Y. Nishimaki;Mikito Owa;T. Hisabori;Ken-ichi Wakabayashi
中科院分区：
文献类型：
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作者：
Kazunori Sugiura;Y. Nishimaki;Mikito Owa;T. Hisabori;Ken-ichi Wakabayashi
细胞的还原-氧化（氧化还原）电位主要由还原型和氧化型谷胱甘肽之间的浓度比决定。它通常保持在中度还原状态，但在一定程度上受到代谢活动如呼吸和/或光合作用的影响。氧化还原电位的变化诱导许多细胞活动，统称为氧化还原反应。为了了解细胞氧化还原反应的动力学，必须在体内准确地评估氧化还原电位。在这项研究中，我们开发了一种方法来测量体内氧化还原电位的绿色藻类莱茵衣藻，利用Oba-Qc，最近开发的氧化还原监测蛋白。利用周期性鞭毛组装的优势，我们将Oba-Qc分子以恒定密度引入鞭毛中。来自活细胞中鞭毛的荧光信号，相对于来自已知氧化还原电位的缓冲液中的样品的荧光进行校准，确定氧化还原电位在光照下为10 - 250 mV，在黑暗中为10 - 280 mV。引入与以恒定密度组装的结构蛋白融合的传感蛋白也将适用于鞭毛中各种细胞信号的测量。
Cellular reducing-oxidizing (redox) potential is mainly determined by the concentration ratio between reduced and oxidized glutathiones. It is normally kept at a moderately reduced state but affected to some extent by metabolic activities such as respiration and/or photosynthesis. Changes in redox potential induce many cellular activities collectively called redox responses. For an understanding of the dynamics of the cellular redox responses, redox potential must be accurately assessedin vivo. In this study, we developed a method to measure thein vivoredox potential in the green algaChlamydomonas reinhardtii, using Oba-Qc, a recently developed redox-monitoring protein. Taking advantage of the periodic flagellar assembly, we introduced Oba-Qc molecules into the flagella at a constant density. Fluorescence signals from flagella in live cells, calibrated against the fluorescence from the samples in buffers of known redox potentials, determined the redox potential to be ∼-250 mV in the light and ∼-280 mV in the dark. Introduction of a sensor protein fused with a structural protein that assembles at a constant density will be also applicable for measurements of various kinds cellular signals in flagella.