Separating NADH and NADPH fluorescence in live cells and tissues using FLIM.
Separating NADH and NADPH fluorescence in live cells and tissues using FLIM.
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DOI：
10.1038/ncomms4936
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发表时间：
2014-05-29
影响因子：
16.6
通讯作者：
Duchen, Michael R.
中科院分区：
文献类型：
--
作者：
Blacker, Thomas S.;Mann, Zoe F.;Gale, Jonathan E.;Ziegler, Mathias;Bain, Angus J.;Szabadkai, Gyorgy;Duchen, Michael R.
NAD是细胞能量代谢的关键决定因素。相反，它的磷酸化形式NADP在生物合成途径和抗氧化防御中发挥着核心作用。这两种吡啶核苷酸的还原形式在活细胞中都是荧光的，但它们无法区分，因为它们在光谱上是相同的。在这里，使用遗传学和药理学方法干扰NAD(P)H代谢，我们发现荧光寿命成像(FLIM)可以定量区分这两个辅因子。改变氧化代谢和糖酵解代谢之间平衡的系统操作表明，这些状态并不直接影响NAD(P)H荧光衰减率。因此，在癌症中观察到的寿命变化可能反映了NADPH/NADH平衡的变化。使用数学模型，我们使用这些实验数据来量化NADH和NADPH在复杂组织的不同细胞类型中的相对水平，哺乳动物耳蜗组织。这揭示了富含NADPH的细胞群体，提出了关于它们不同的代谢作用的问题。NAD和NADP在细胞代谢中扮演着根本不同的角色，然而这些吡啶核苷酸在活细胞中不能被光谱区分。布莱克等人。证明荧光寿命成像可以用来量化培养细胞和哺乳动物耳蜗中的NADPH/NADH平衡。
NAD is a key determinant of cellular energy metabolism. In contrast, its phosphorylated form, NADP, plays a central role in biosynthetic pathways and antioxidant defence. The reduced forms of both pyridine nucleotides are fluorescent in living cells but they cannot be distinguished, as they are spectrally identical. Here, using genetic and pharmacological approaches to perturb NAD(P)H metabolism, we find that fluorescence lifetime imaging (FLIM) differentiates quantitatively between the two cofactors. Systematic manipulations to change the balance between oxidative and glycolytic metabolism suggest that these states do not directly impact NAD(P)H fluorescence decay rates. The lifetime changes observed in cancers thus likely reflect shifts in the NADPH/NADH balance. Using a mathematical model, we use these experimental data to quantify the relative levels of NADH and NADPH in different cell types of a complex tissue, the mammalian cochlea. This reveals NADPH-enriched populations of cells, raising questions about their distinct metabolic roles. NAD and NADP play fundamentally different roles in cellular metabolism, and yet these pyridine nucleotides cannot be distinguished spectroscopically in living cells. Blacker et al. demonstrate that fluorescence lifetime imaging can be used to quantify NADPH/NADH balance in cultured cells and in the mammalian cochlea.
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影响因子：
3.4
作者：
McGinty J;Galletly NP;Dunsby C;Munro I;Elson DS;Requejo-Isidro J;Cohen P;Ahmad R;Forsyth A;Thillainayagam AV;Neil MA;French PM;Stamp GW
通讯作者：
Stamp GW
DOI：
10.1155/2008/602639
发表时间：
2008
期刊：
Journal of biophysics (Hindawi Publishing Corporation : Online)
影响因子：
--
作者：
Niesner R;Narang P;Spiecker H;Andresen V;Gericke KH;Gunzer M
通讯作者：
Gunzer M
DOI：
10.1007/s10482-010-9538-x
发表时间：
2011-03-01
影响因子：
2.6
作者：
Mailloux, Ryan J.;Lemire, Joseph;Appanna, Vasu D.
通讯作者：
Appanna, Vasu D.
影响因子：
2.8
作者：
De Ruyck, Jerome;Fameree, Maureen;Jacquemin, Denis
通讯作者：
Jacquemin, Denis
影响因子：
3.5
作者：
Butte, Pramod V.;Fang, Qiyin;Marcu, Laura
通讯作者：
Marcu, Laura