Spatially resolved fluorescence lifetime mapping of enzyme kinetics in living cells

Abstract

SummaryTraditional cuvette-based enzyme studies lack spatial information and do not allow real-time monitoring of the effects of modulating enzyme functions in vivo. In order to probe the realistic timescales of steric modifications in enzymesubstrate complexes and functional binding-unbinding kinetics in living cells without losing spatial information, it is imperative to develop sensitive imaging strategies that can report enzyme kinetics in real time over a wide dynamic range of timescales. Here we present a multi-photon excitationbased, ultra-fast photon detection using a streak camera and Laguerre expansion-based fast deconvolution approach for achieving high spatio-temporal resolution in monitoring realtime enzyme kinetics in single cells. In particular, we report spatially resolved, nanosecond-scale fluorescence dynamics associated with binding-unbinding kinetics of endogenous metabolic co-factor nicotinamide adenine dinucleotide with enzymes in intact living cells. By monitoring real-time kinetics of NAD(P)H-enzyme kinetics in primary hepatocytes isolated from young and aged mouse models, we observed that the mechanism of inhibition of mitochondrial respiration at complex I site is mediated by redistribution of free and protein-bound nicotinamide adenine dinucleotide pools and that this equilibrium redistribution is affected by agerelated modifications in mitochondrial function. We describe unique advantages of Laguerre deconvolution algorithm in comparison with conventional lifetime analysis approaches. §These authors contributed equally to this work.Correspondence to: V Krishnan Ramanujan. Tel: (+1) 310-423-7785; fax: (+1) 310-423-7707; e-mail: Ramanujanv@cshs.org Non-invasive monitoring of metabolic dysfunctions in intact animal models is an attractive strategy for gaining insight into the dynamics of tissue metabolism in health and in various metabolic syndromes such as cancer, diabetes and aging-induced metabolic dysfunctions. Besides the example demonstrated above, we envisage that the proposed method can find applications in a variety of other situations where intensity-based approaches fall short owing to spectroscopic artefacts.

Metabolic imaging in multiple time scales

“…Our laboratory and other groups have shown that these altered metabolic profile (“metabolic switch” or “aerobic glycolysis”) in cancer cells can be exploited for early detection. [9, 11, 18, 25, 26, 29] In order to test if the normal and cancer cells display differential mitochondrial response to glucose stimulus, we monitored steady state kinetics of mitochondrial membrane potential in MCF10A and MCF7 cells (Figure 2a & 2b). Despite the near-equal mean TMRM fluorescence in both these cell lines (Figures 1d & 1e), glucose stimulus elicited significantly different TMRM response in these two cell lines.…”

Tuning in Tumor Metabolism: The Cost of Being a Cancer Cell

“…Second, we need to adopt a pragmatic approach of combining more than one technique in the same screening setting to maximize the information content from the metabolic imaging sessions. Optical imaging provides high molecular specificity whereas tomography and magnetic resonance methods provide better depth information than the optical imaging techniques [6,[30][31][32][33][34][35]. However, by a strategic combination of these two different techniques can yield a synergistic edge to metabolic imaging of tumor-specific signatures.…”

Imaging Enzymes at Work: Metabolic Mapping by Enzyme Histochemistry

“…Because excitation in the 350-450-nm wavelength range is highly toxic to cells, and because shifts in the emission spectra due to molecular interactions in cells make the analysis difficult, this approach of metabolic mapping in living cells and tissues became obsolete. However, the application of dual-photon excitation of NADH at 730 nm, which reduces cytotoxicity considerably, the determination of the ratio of free and protein (enzyme)-bound NADH using fluorescence lifetime imaging microscopy and anisotropy of autofluorescence, and the application of fast photon detection re-introduced NADH as an attractive probe for spatially resolved imaging of the metabolism of living cells (Ramanujam et al 1994;Bird et al 2005;Ramanujan et al 2008;Yu and Heikal 2009). The focus of these studies is particularly on metabolic mapping of normal and malignant cells and tissues.…”

“…Therefore, localization or measurement of mRNA or protein of an enzyme in cells cannot predict its activity in those cells (Figure 1) (Hazen et al 2000;Baruch et al 2004;Boonacker et al 2004;Bogyo and Cravatt 2007). Similarly, activity measurements of a purified enzyme in either diluted solutions or in tissue or cell homogenates probably do not reflect the activity of that enzyme in the crowded com-partmentalized cell Ramanujan et al 2008).…”

“…Living cell or tissue imaging, in which cells and tissues are kept intact during analysis, is perhaps the best approach to generating data on the activity of an enzyme that reflect the in vivo situation (Yuste 2005;Sadaghiani et al 2007;Ramanujan et al 2008). Imaging of enzyme reactions in living tissues is more complex, but the use of unfixed cryostat sections in combination with tissue protectants in the incubation media on top of the sections allows the determination of enzyme reactions in an almost true-to-nature situation (Van Noorden and Frederiks 2002).…”