Phasor‐flim analysis of NADH distribution and localization in the nucleus of live progenitor myoblast cells

Wright, Belinda K.; Andrews, Laura M.; Jones, Mark R.; Stringari, Chiara; Digman, Michelle A.; Gratton, Enrico

doi:10.1002/jemt.22121

Cited by 36 publications

(39 citation statements)

References 16 publications

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“…This effect is not surprising, because a large part of mitochondrial metabolism on oxidoreductase enzymes uses NADH as a cofactor to catalyze reactions. Concomitantly, the intensity from NADH in the cell nucleus is considerably lower than in other intracellular compartments (39,40) (Fig. 1A), suggesting a compartmentalized metabolism for NADH cofactors.…”

Section: Resultsmentioning

confidence: 91%

Spatial dynamics of SIRT1 and the subnuclear distribution of NADH species

Aguilar-Arnal

Ranjit

Stringari

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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Sirtuin 1 (SIRT1) is an NAD + -dependent deacetylase that functions as metabolic sensor of cellular energy and modulates biochemical pathways in the adaptation to changes in the environment. SIRT1 substrates include histones and proteins related to enhancement of mitochondrial function as well as antioxidant protection. Fluctuations in intracellular NAD + levels regulate SIRT1 activity, but how SIRT1 enzymatic activity impacts on NAD + levels and its intracellular distribution remains unclear. Here, we show that SIRT1 determines the nuclear organization of protein-bound NADH. Using multiphoton microscopy in live cells, we show that free and bound NADH are compartmentalized inside of the nucleus, and its subnuclear distribution depends on SIRT1. Importantly, SIRT6, a chromatin-bound deacetylase of the same class, does not influence NADH nuclear localization. In addition, using fluorescence fluctuation spectroscopy in single living cells, we reveal that NAD + metabolism in the nucleus is linked to subnuclear dynamics of active SIRT1. These results reveal a connection between NAD + metabolism, NADH distribution, and SIRT1 activity in the nucleus of live cells and pave the way to decipher links between nuclear organization and metabolism.S irtuins (SIRTs) are a conserved family of deacetylases that target a variety of proteins located in virtually all cellular compartments (1). Deacetylation by SIRTs may control many functional aspects of target proteins (2). Because SIRT deacetylase activity depends on the energy carrier NAD + , these enzymes are thought to operate as cellular metabolic sensors. In addition, because histones are targeted by nuclear SIRT, these enzymes could link variations in cellular metabolism to chromatin function.There are seven mammalian SIRTs (SIRT1 to SIRT7) with distinct subcellular locations. SIRT2 is mainly cytoplasmic; SIRT3, SIRT4, and SIRT5 are found in the mitochondrial compartment; and SIRT1, SIRT6, and SIRT7 are located in the cell nucleus (1). In mammals, SIRT1 contributes to development and protects from metabolic and cardiovascular disease, neurodegeneration, and cancer (3). SIRT1 has been reported to promote healthy aging and regulate lifespan (4, 5). At the cellular level, SIRT1 regulates lipid and glucose homeostasis, apoptosis, DNA repair, and mitochondrial function. Variations in NAD + levels control SIRT1 activity (6-9), a relevant finding in the regulation of circadian rhythms (10). Circadian rhythms in NAD + levels have been observed (11,12), which lead to fluctuating SIRT1 deacetylase activity (9) that, in turn, results into cyclic acetylation of specific SIRT1 targets (6, 9, 13). SIRT1 and SIRT6 segregate circadian metabolism by driving transcription of a differential subset of circadian genes (14). SIRT6 is a chromatinbound protein that was first characterized as a regulator of genome stability (15). The other nuclear SIRT, SIRT7, appears to be highly localized in the nucleolus and possibly involved in Pol-I-dependent transcription (16), and it has been shown to regula...

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Section: Resultsmentioning

confidence: 91%

Spatial dynamics of SIRT1 and the subnuclear distribution of NADH species

Aguilar-Arnal

Ranjit

Stringari

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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“…Though the models appropriately demonstrate the utility of NADH FLIM for resolving different impaired metabolic processes, the classification methods can likely be improved through the use of more advanced algorithms, larger data sets, and by utilizing a subset of classifier variables to minimize confounds associated with highdimensional data sets including poor sampling of the variable space. Future investigations will explore the utility of phasor analysis of FLIM data to potentially avoid confounds with lifetime-fitting and classification with high-dimensional data [17,64,65].…”

Section: Discussionmentioning

confidence: 99%

Fluorescence lifetime microscopy of NADH distinguishes alterations in cerebral metabolism in vivo

Yaseen

Sutin

et al. 2017

Biomed. Opt. Express

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Evaluating cerebral energy metabolism at microscopic resolution is important for comprehensively understanding healthy brain function and its pathological alterations. Here, we resolve specific alterations in cerebral metabolism in vivo in Sprague Dawley rats utilizing minimally-invasive 2-photon fluorescence lifetime imaging (2P-FLIM) measurements of reduced nicotinamide adenine dinucleotide (NADH) fluorescence. Time-resolved fluorescence lifetime measurements enable distinction of different components contributing to NADH autofluorescence. Ostensibly, these components indicate different enzyme-bound formulations of NADH. We observed distinct variations in the relative proportions of these components before and after pharmacological-induced impairments to several reactions involved in glycolytic and oxidative metabolism. Classification models were developed with the experimental data and used to predict the metabolic impairments induced during separate experiments involving bicuculline-induced seizures. The models consistently predicted that prolonged focal seizure activity results in impaired activity in the electron transport chain, likely the consequence of inadequate oxygen supply. 2P-FLIM observations of cerebral NADH will help advance our understanding of cerebral energetics at a microscopic scale. Such knowledge will aid in our evaluation of healthy and diseased cerebral physiology and guide diagnostic and therapeutic strategies that target cerebral energetics. 2(2), 145-150 (1966). 33. L. K. Klaidman, A. C. Leung, and J. D. Adams, Jr., "High-performance liquid chromatography analysis of oxidized and reduced pyridine dinucleotides in specific brain regions," Anal.

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“…In this paper, the primary goal is to use the information about the distribution of the phasor points and predict the behavior of unknown sample. So far only the average phasor position of a distribution has been used to separate differential behavior of cells undergoing different metabolism or for stem cells undergoing different stages of differentiation [4][5][6]. However, tissue samples are usually more heterogeneous, have a larger phasor distribution, and thus just the calculation of average phasor positions of the distribution is not enough to distinguish the difference between the samples.…”

Section: Discussionmentioning

confidence: 99%

“…Phasor approach to fluorescence lifetime imaging has been applied to study cellular metabolism and to evaluate presence of reactive oxygen species in living cell and tissue samples [1][2][3][4][5][6]. This approach is well established for mapping lifetime to a fluorescence image and separating areas of image having distinctively different lifetimes.…”

Section: Introductionmentioning

confidence: 99%

Characterizing fibrosis in UUO mice model using multiparametric analysis of phasor distribution from FLIM images

Ranjit¹,

Dvornikov²,

Levi³

et al. 2016

Biomed. Opt. Express

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Phasor approach to fluorescence lifetime microscopy is used to study development of fibrosis in the unilateral ureteral obstruction model (UUO) of kidney in mice. Traditional phasor analysis has been modified to create a multiparametric analysis scheme that splits the phasor points in four equidistance segments based on the height of peak of the phasor distribution and calculates six parameters including average phasor positions, the shape of each segment, the angle of the distribution and the number of points in each segment. These parameters are used to create a spectrum of twenty four points specific to the phasor distribution of each sample. Comparisons of spectra from diseased and healthy tissues result in quantitative separation and calculation of statistical parameters including AUC values, positive prediction values and sensitivity. This is a new method in the evolving field of analyzing phasor distribution of FLIM data and provides further insights. Additionally, the progression of fibrosis with time is detected using this multiparametric approach to phasor analysis. Bertram, and S. D. Ricardo, "Renal structural and functional repair in a mouse model of reversal of ureteral obstruction," J. Am. Soc. Nephrol. 16(12), 3623-3630 (2005). 11. T. P. Martin, G. Norris, G. McConnell, and S. Currie, "A novel approach for assessing cardiac fibrosis using label-free second harmonic generation," Int.

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Phasor‐flim analysis of NADH distribution and localization in the nucleus of live progenitor myoblast cells

Cited by 36 publications

References 16 publications

Spatial dynamics of SIRT1 and the subnuclear distribution of NADH species

Spatial dynamics of SIRT1 and the subnuclear distribution of NADH species

Fluorescence lifetime microscopy of NADH distinguishes alterations in cerebral metabolism in vivo

Characterizing fibrosis in UUO mice model using multiparametric analysis of phasor distribution from FLIM images

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