Direct Imaging of Dehydrogenase Activity within Living Cells Using Enzyme-Dependent Fluorescence Recovery after Photobleaching (ED-FRAP)

Laboratory of Cardiac Energetics, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland 20892-1061, USA.
Biophysical Journal (Impact Factor: 3.97). 05/2001; 80(4):2018-28. DOI: 10.1016/S0006-3495(01)76172-3
Source: PubMed


Reduced nicotine adenine dinucleotide (NADH) is a key metabolite involved in cellular energy conversion and many redox reactions. We describe the use of confocal microscopy in conjunction with enzyme-dependent fluorescence recovery after photobleaching (ED-FRAP) of NADH as a topological assay of NADH generation capacity within living cardiac myocytes. Quantitative validation of this approach was performed using a dehydrogenase system, in vitro. In intact cells the NADH ED-FRAP was sensitive to temperature (Q(10) of 2.5) and to dehydrogenase activation by dichloroacetate or cAMP (twofold increase for each). In addition, NADH ED-FRAP was correlated with flavin adenine dinucleotide (FAD(+)) fluorescence. These data, coupled with the cellular patterns of NADH ED-FRAP changes with dehydrogenase stimulation, suggest that NADH ED-FRAP is localized to the mitochondria. These results suggest that ED-FRAP enables measurement of regional dynamics of mitochondrial NADH production in intact cells, thus providing information regarding region-specific intracellular redox reactions and energy metabolism.

Download full-text


Available from: Robert S Balaban
  • Source
    • "This method may also offer an opportunity for a non-invasive measurement under label-free conditions. However, as intrinsic fluorophores, the coenzymes have relatively low extinct coefficients and quantum yields [11] [12]. Hence, the emissions from the coenzymes are too weak to be discriminated from the cellular backgrounds that are arisen from cellular species and water scattering as well as the emissions from other intrinsic fluorophores in cells [13]. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Flavin adenine dinucleotide (FAD) is a key metabolite in cellular energy conversion. Flavin can also bind with some enzymes in the metabolic pathway and the binding sites may be changed due to the disease progression. Thus, there is interest on studying its expression level, distribution, and redox state within the cells. FAD is naturally fluorescent, but it has a modest extinction coefficient and quantum yield. Hence the intrinsic emission from FAD is generally too weak to be isolated distinctly from the cellular backgrounds in fluorescence cell imaging. In this article, the metal nanostructures on the glass coverslips were used as substrates to measure FAD in cells. Particulate silver films were fabricated with an optical resonance near the absorption and the emission wavelengths of FAD which can lead to efficient coupling interactions. As a result, the emission intensity and quantum yield by FAD were greatly increased and the lifetime was dramatically shortened resulting in less interference from the longer lived cellular background. This feature may overcome the technical limits that hinder the direct observation of intrinsically fluorescent coenzymes in the cells by fluorescence microscopy. Fluorescence cell imaging on the metallic particle substrates may provide a non-invasive strategy for collecting the information of coenzymes in cells.
    Full-text · Article · Jun 2012 · Biochemical and Biophysical Research Communications
  • Source
    • "The Q 10 of ∼4.5 we observed for the perimembrane turnover rate of P2X3 receptors is much higher than the Q 10 values usually found in FRAP experiments with other biomolecules (1.5–2.5; Combs and Balaban, 2001). To test whether such high temperature sensitivity was typical also for other ligand gated channels, we transfected hippocampal neurons with the plasmid encoding for GFP-tagged AMPA-type glutamate receptors and measured the temperature dependence of their trafficking using the TIRF–FRAP assay. "
    [Show abstract] [Hide abstract]
    ABSTRACT: ATP-gated P2X3 receptors are expressed by nociceptive neurons and participate in transduction of pain. Responsiveness of P2X3 receptors is strongly reduced at low temperatures, suggesting a role for these receptors in analgesic effects of cooling. Since sustained responsiveness depends on receptor trafficking to the plasma membrane, we employed total internal reflection fluorescence (TIRF) microscopy to highlight perimembrane pool of DsRed-tagged P2X3 receptors and studied the effects of temperature on perimembrane turnover of P2X3-DsRed. Patch-clamp recordings confirmed membrane expression of functional, rapidly desensitizing P2X3-DsRed receptors. By combining TIRF microscopy with the technique of fluorescence recovery after photobleaching (FRAP), we measured the rate of perimembrane turnover of P2X3-DsRed receptors expressed in hippocampal neurons. At room temperature, the P2X3-DsRed perimembrane turnover as measured by TIRF-FRAP had a time constant of ∼2 min. At 29°C, receptor turnover was strongly accelerated (0.6 min), yielding an extremely high temperature dependence coefficient Q(10) ∼4.5. In comparison, AMPA receptor turnover measured with TIRF-FRAP was only moderately sensitive to temperature (Q(10) ∼1.5). The traffic inhibitor Brefeldin A selectively decelerated P2X3-DsRed receptor turnover at 29°C, but had no effect at 21°C (Q(10) ∼1.0). This indicates that receptor traffic to plasma membrane is the key temperature-sensitive component of P2X3 turnover. The selective inhibitor of the RhoA kinase Y27632 significantly decreased the temperature dependence of P2X3-DsRed receptor turnover (Q(10) ∼2.0). In summary, the RhoA kinase-dependent membrane trafficking of P2X3 receptors to plasma membrane has an exceptionally high sensitivity to temperature. These findings suggest an important role of P2X3 receptor turnover in hypothermia-associated analgesia.
    Full-text · Article · Dec 2011 · Frontiers in Cellular Neuroscience
  • Source
    • "With these considerations, toxicity and bleaching can be avoided over recording periods of hours, as measured from the reproducibility of synaptically-evoked responses. Alternatively, deliberate bleaching of NADH with very high intensity UV flashes can be useful for kinetic studies of tissue NADH generation, as the enzymatic regeneration of NADH within tissues can be monitored (Combs and Balaban, 2001, 2004; Joubert et al., 2004) From early studies of a range of tissues, it was concluded that the intensity of cellular NADH fluorescence was not uniform, but was strongly increased in the mitochondrial compartment, and quenched when located in the cytosol (Chance and Baltscheffsky, 1958).The reason(s) for enhanced mitochondrial fluorescence was not immediately established, but association with matrix proteins was considered a major contributor (Chance and Baltscheffsky, 1958; Avi-Dor et al., 1962; Estabrook, 1962). Jobsis and colleagues concluded that NADH fluorescence measured in intact cat cortex was primarily mitochondrial, and that enhancement of fluorescence was due to mitochondrial dehydrogenases (Jobsis et al., 1971). "
    [Show abstract] [Hide abstract]
    ABSTRACT: Synaptic stimulation in brain slices is accompanied by changes in tissue autofluorescence, which are a consequence of changes in tissue metabolism. Autofluorescence excited by ultraviolet light has been most extensively studied, and is due to reduced pyridine nucleotides (NADH and NADPH, collectively termed NAD(P)H). Stimulation generates a characteristic compound NAD(P)H response, comprising an initial fluorescence decrease and then an overshooting increase that slowly recovers to baseline levels. Evoked NAD(P)H transients are relatively easy to record, do not require the addition of exogenous indicators and have good signal-noise ratios. These characteristics make NAD(P)H imaging methods very useful for tracking the spread of neuronal activity in complex brain tissues, however the cellular basis of synaptically-evoked autofluorescence transients has been the subject of recent debate. Of particular importance is the question of whether signals are due primarily to changes in neuronal mitochondrial function, and/or whether astrocyte metabolism triggered by glutamate uptake may be a significant contributor to the overshooting NAD(P)H fluorescence increases. This mini-review addresses the subcellular origins of NAD(P)H autofluorescence and the evidence for mitochondrial and glycolytic contributions to compound transients. It is concluded that there is no direct evidence for a contribution to NAD(P)H signals from glycolysis in astrocytes following synaptic glutamate uptake. In contrast, multiple lines of evidence, including from complimentary flavoprotein autofluorescence signals, imply that mitochondrial NADH dynamics in neurons dominate compound evoked NAD(P)H transients. These signals are thus appropriate for studies of mitochondrial function and dysfunction in brain slices, in addition to providing robust maps of postsynaptic neuronal activation following physiological activation.
    Preview · Article · Feb 2010 · Neurochemistry International
Show more