Imaging pH Dynamics Simultaneously in Two Cellular Compartments Using a Ratiometric pH-Sensitive Mutant of mCherry.
Imaging pH Dynamics Simultaneously in Two Cellular Compartments Using a Ratiometric pH-Sensitive Mutant of mCherry.
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DOI：
10.1021/acsomega.8b00655
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发表时间：
2018-08-31
期刊：
影响因子：
4.1
通讯作者：
Tantama M
中科院分区：
文献类型：
--
作者：
Rajendran M;Claywell B;Haynes EP;Scales U;Henning CK;Tantama M
pH的调节对于适当的细胞器功能是必不可少的，并且pH的细胞器特异性变化通常反映生理信号传导和代谢的动态。例如，线粒体能量产生取决于碱性线粒体基质和中性胞质溶胶之间保持的质子梯度。然而，我们仍然缺乏一个定量的了解如何pH值动态之间的耦合隔间和pH梯度是如何在细胞器的边界进行调节。遗传编码的pH传感器非常适合解决这个问题，因为它们可以靶向特定的亚细胞位置，并且它们有助于活的单细胞分析。然而，这些pH传感器中的大多数是绿色和黄色荧光蛋白的衍生物，其对于双室成像而言在光谱上不相容。因此，需要比率式红色荧光蛋白pH传感器，其能够对空间分辨的pH动力学进行定量荧光成像。在这项工作中，我们证明了红色荧光蛋白mCherry的I158 E/Q160 A突变体是一种有效的比率pH传感器。其pKa为7.3，比值信号变化大于3倍。为了证明其在细胞中的效用，我们测量了培养的原代神经元和神经母细胞瘤细胞的活性和代谢依赖性pH动力学。此外，我们能够图像pH值的变化，同时在细胞质和线粒体通过使用mCherryEA突变体连同绿色荧光pH传感器，ratiometric-pHluorin。我们的研究结果表明，随着时间的推移，在活细胞中研究细胞器间的pH动态的可行性和广泛的适用性，这些传感器在研究的作用，pH调节代谢和信号。
The regulation of pH is essential for proper organelle function, and organelle-specific changes in pH often reflect the dynamics of physiological signaling and metabolism. For example, mitochondrial energy production depends on the proton gradient maintained between the alkaline mitochondrial matrix and neutral cytosol. However, we still lack a quantitative understanding of how pH dynamics are coupled between compartments and how pH gradients are regulated at organelle boundaries. Genetically encoded pH sensors are well suited to address this problem because they can be targeted to specific subcellular locations and they facilitate live, single-cell analysis. However, most of these pH sensors are derivatives of green and yellow fluorescent proteins that are not spectrally compatible for dual-compartment imaging. Therefore, there is a need for ratiometric red fluorescent protein pH sensors that enable quantitative multicolor imaging of spatially resolved pH dynamics. In this work, we demonstrate that the I158E/Q160A mutant of the red fluorescent protein mCherry is an effective ratiometric pH sensor. It has a pKa of 7.3 and a greater than 3-fold change in ratio signal. To demonstrate its utility in cells, we measured activity and metabolism-dependent pH dynamics in cultured primary neurons and neuroblastoma cells. Furthermore, we were able to image pH changes simultaneously in the cytosol and mitochondria by using the mCherryEA mutant together with the green fluorescent pH sensor, ratiometric-pHluorin. Our results demonstrate the feasibility of studying interorganelle pH dynamics in live cells over time and the broad applicability of these sensors in studying the role of pH regulation in metabolism and signaling.
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DOI：
10.1007/978-1-4939-6937-1_16
发表时间：
2017-01-01
期刊：
PEROXISOMES: METHODS AND PROTOCOLS
影响因子：
--
作者：
Godinho, Luis F.;Schrader, Michael
通讯作者：
Schrader, Michael
肿瘤微环境产生的酸度驱动局部侵袭。
影响因子：
11.2
作者：
Estrella V;Chen T;Lloyd M;Wojtkowiak J;Cornnell HH;Ibrahim-Hashim A;Bailey K;Balagurunathan Y;Rothberg JM;Sloane BF;Johnson J;Gatenby RA;Gillies RJ
通讯作者：
Gillies RJ
影响因子：
48
作者：
Balleza E;Kim JM;Cluzel P
通讯作者：
Cluzel P
影响因子：
11.3
作者：
Di Domenico, Fabio;Foppoli, Cesira;De Marco, Federico
通讯作者：
De Marco, Federico
内体中腔质子的泄漏途径驱动胶质母细胞瘤中的致癌信号传导。
影响因子：
16.6
作者：
Kondapalli, Kalyan C.;Llongueras, Jose P.;Capilla-Gonzalez, Vivian;Prasad, Hari;Hack, Anniesha;Smith, Christopher;Guerrero-Cazares, Hugo;Quinones-Hinojosa, Alfredo;Rao, Rajini
通讯作者：
Rao, Rajini