Abstract
In the past years, cellular metabolism of the immune system experienced a revival, as it has become clear that it is not merely responsible for the cellular energy supply, but also impacts on many signaling pathways and, thus, on diverse cellular functions. Label-free fluorescence lifetime imaging of the ubiquitous coenzymes NADH and NADPH (NAD(P)H-FLIM) makes it possible to monitor cellular metabolism in living cells and tissues and has already been applied to study metabolic changes both under physiologic and pathologic conditions. However, due to the complex distribution of NAD(P)H-dependent enzymes in cells, whose distribution continuously changes over time, a thorough interpretation of NAD(P)H-FLIM results, in particular, resolving the contribution of various enzymes to the overall metabolic activity, remains challenging. We developed a systematic framework based on angle similarities of the phase vectors and their length to analyze NAD(P)H-FLIM data of cells and tissues based on a generally valid reference system of highly abundant NAD(P)H-dependent enzymes in cells. By using our analysis framework, we retrieve information not only about the overall metabolic activity, i.e., the fraction of free to enzyme-bound NAD(P)H, but also identified the enzymes predominantly active within the sample at a certain time point with subcellular resolution. We verified the performance of the approach by applying NAD(P)H-FLIM on a stromal-like cell line and identified a different group of enzymes that were active in the cell nuclei as compared to the cytoplasm. As the systematic phasor-based analysis framework of label-free NAD(P)H-FLIM can be applied both in vitro and in vivo, it retains the unique power to enable dynamic enzyme-based metabolic investigations, at subcellular resolution, in genuine environments.
Highlights
The importance of cellular metabolism in immunology has become increasingly recognized in the last years
The NADH-binding unit of complex I is one of the less abundant among these 16 enzymes. We focused on these highly abundant NAD(P)H-dependent metabolic enzymes, i.e., malate dehydrogenase (MDH), lactate dehydrogenase (LDH), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), glucose-6-phosphate dehydrogenase (G6PDH), pyruvate dehydrogenase (PDH), alcohol dehydrogenase (ADH), C-terminal binding protein 1 (CTBP1) [57], and isocitrate dehydrogenase (IDH); complemented by the still abundant enzymes, i.e., hydroxyacyl-coenzyme-A dehydrogenase (HADH) and sorbitol dehydrogenase (SDH); and enzymes playing distinct roles in the cells, i.e., inducible nitric oxide synthase and the NADPH oxidases family (NOX1-4 and DUOX1,2) [59], which play a major role in the catalysis of oxidative burst
We evaluated the Fluorescence lifetime imaging (FLIM) data acquired in time-domain by time-correlated single-photon counting in a two-photon microscope using the phasor approach
Summary
The importance of cellular metabolism in immunology has become increasingly recognized in the last years. The process of selection and further differentiation of germinal B cells was shown to be directly linked to cellular metabolism [3]. These correlations between metabolism and immune cell function and dysfunction still require extensive investigations, as oxygen consumption measurements related to enzymatic activity are mainly performed ex vivo and not at the single-cell level. By using synthetic probes, such as dichlortris(1,10-phenanthroline) ruthenium(II) hydrate (Ru(Phen)) [4], the oxygen concentration in cells and tissues can be measured at the level of single cells [5] These approaches allow only an indirect link to enzymatic (metabolic) activity
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