New alternately colored FRET sensors for simultaneous monitoring of Zn²⁺ in multiple cellular locations.
New alternately colored FRET sensors for simultaneous monitoring of Zn²⁺ in multiple cellular locations.
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DOI：
10.1371/journal.pone.0049371
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发表时间：
2012
期刊：
影响因子：
3.7
通讯作者：
Palmer AE
中科院分区：
文献类型：
--
作者：
Miranda JG;Weaver AL;Qin Y;Park JG;Stoddard CI;Lin MZ;Palmer AE
基于荧光共振能量转移（FRET）的基因编码传感器是报告活细胞中离子、分子和生化反应的有力工具。在这里，我们描述了新的传感器的发展为Zn 2+的基础上交替FRET对，不涉及传统的CFP和YFP。Zn 2+是一种必需的微量营养素，在细胞生物学中起着重要作用。因此，迫切需要强大的传感器来监测具有高空间和时间分辨率的细胞中的Zn 2+水平和动态。在这里，我们开发了一套传感器使用交替FRET对，包括tSapphire/TagRFP，tSapphire/mKO，三叶草/mRuby 2，mOrange 2/mCherry，和mOrange 2/mKATE。这些传感器的目标是细胞核和胞质溶胶，并在活细胞中进行了表征和验证。基于新FRET对三叶草/mRuby 2的传感器显示出比测试的其余传感器更高的动态范围和更好的信噪比，并且对于监测胞质和核Zn 2+的变化是最佳的。使用靶向细胞核的绿-红传感器和靶向ER、高尔基体或线粒体的青-黄传感器，我们能够同时监测两个隔室中的Zn 2+摄取，表明细胞核Zn 2+快速上升，而ER、高尔基体和线粒体都更缓慢地螯合Zn 2+，延迟600-700秒。最后，这些研究提供了核Zn 2+的第一个一瞥，并揭示了核Zn 2+的缓冲水平高于胞质Zn 2+。
Genetically encoded sensors based on fluorescence resonance energy transfer (FRET) are powerful tools for reporting on ions, molecules and biochemical reactions in living cells. Here we describe the development of new sensors for Zn2+based on alternate FRET-pairs that do not involve the traditional CFP and YFP. Zn2+ is an essential micronutrient and plays fundamental roles in cell biology. Consequently there is a pressing need for robust sensors to monitor Zn2+ levels and dynamics in cells with high spatial and temporal resolution. Here we develop a suite of sensors using alternate FRET pairs, including tSapphire/TagRFP, tSapphire/mKO, Clover/mRuby2, mOrange2/mCherry, and mOrange2/mKATE. These sensors were targeted to both the nucleus and cytosol and characterized and validated in living cells. Sensors based on the new FRET pair Clover/mRuby2 displayed a higher dynamic range and better signal-to-noise ratio than the remaining sensors tested and were optimal for monitoring changes in cytosolic and nuclear Zn2+. Using a green-red sensor targeted to the nucleus and cyan-yellow sensor targeted to either the ER, Golgi, or mitochondria, we were able to monitor Zn2+ uptake simultaneously in two compartments, revealing that nuclear Zn2+ rises quickly, whereas the ER, Golgi, and mitochondria all sequester Zn2+ more slowly and with a delay of 600–700 sec. Lastly, these studies provide the first glimpse of nuclear Zn2+ and reveal that nuclear Zn2+ is buffered at a higher level than cytosolic Zn2+.
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DOI：
10.1073/pnas.96.5.2135
发表时间：
1999-03-02
影响因子：
11.1
作者：
Miyawaki, A;Griesbeck, O;Tsien, RY
通讯作者：
Tsien, RY
影响因子：
48
作者：
Ai, Hui-wang;Hazelwood, Kristin L.;Campbell, Robert E.
通讯作者：
Campbell, Robert E.
通过CA2+-CAMP-PKA振荡电路实现的PKA的信号多样性。
影响因子：
14.8
作者：
Ni, Qiang;Ganesan, Ambhighainath;Aye-Han, Nwe-Nwe;Gao, Xinxin;Allen, Michael D.;Levchenko, Andre;Zhang, Jin
通讯作者：
Zhang, Jin
影响因子：
3
作者：
Gandhi MS;Deshmukh PA;Kamalov G;Zhao T;Zhao W;Whaley JT;Tichy JR;Bhattacharya SK;Ahokas RA;Sun Y;Gerling IC;Weber KT
通讯作者：
Weber KT
影响因子：
3.7
作者：
Niino Y;Hotta K;Oka K
通讯作者：
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