In vivo single-cell detection of metabolic oscillations in stem cells

Abstract

Through the use of bulk measurements in metabolic organs, the circadian clock was shown to play roles in organismal energy homeostasis. However, the relationship between metabolic and circadian oscillations has not been studied in vivo at a single-cell level. Also, it is unknown whether the circadian clock controls metabolism in stem cells. We used a sensitive, noninvasive method to detect metabolic oscillations and circadian phase within epidermal stem cells in live mice at the single-cell level. We observe a higher NADH/NAD+ ratio, reflecting an increased glycolysis/oxidative phosphorylation ratio during the night compared to the day. Furthermore, we demonstrate that single-cell metabolic heterogeneity within the basal cell layer correlates with the circadian clock and that diurnal fluctuations in NADH/NAD+ ratio are Bmal1 dependent. Our data show that, in proliferating stem cells, the circadian clock coordinates activities of oxidative phosphorylation and glycolysis with DNA synthesis, perhaps as a protective mechanism against genotoxicity.

Figure 1. In vivo non-invasive NADH imaging of stem cells within the epidermal basal cell layer

(A) Scheme of the live mouse imaging. Two-Photon Excitation fluorescence (TPE) from NADH (cyan) and Second Harmonic Generation (SHG) from collagen are collected in epidetection through the same objective of the Excitation (Exc), represented in red. (B) Energy diagrams and wavelengths involved in the TPE and SHG. (C) A representative cross section of mouse skin showing the epidermis separated from the dermis by a basal lamina. Second Harmonic Generation (SHG) signal from dermal collagen fibers is excited at 880nm and collected with a 440/20nm filter. NADH TPE fluorescence intensity is excited at 740nm and collected with a 460/80 nm filter highlighting single stem cells within the basal layer. (D) After a mathematical transformation that involves Fast Fourier Transformation (FFT) (Material and Methods and (Digman, 2008)), the measured Fluorescence Lifetime decay is represented by a single point in the 2D phasor plot with g and s coordinates corresponding to the real and imaginary part of the FFT. Because of the linearity of the phasor coordinates, mixtures of free and bound NADH in the focal volume will lay along the line that connects the pure molecular species (Stringari, 2011). (E) Phasor analysis of the FLIM images is performed both at the pixel level and cell level. Single pixels are painted in the FLIM map according to a linear cursor (from purple to cyan) that corresponds to different relative concentration of free and bound NADH. Optical metabolic fingerprint of single cells are calculated by averaging the phasor coordinates over the segmented region of interest of the cells (red circular cursor). Cell phasor fingerprints are represented in the phasor scatterplot by single points located along the metabolic trajectory between glycolysis and Oxidative Phosphorylation (OXPHOS). See also Figure S1.

Figure 2. Free to bound NADH metabolic circadian oscillations in stem cells of the epidermis basal layer

(A) TPE fluorescence intensity in vivo images of stem cells within the epidermis basal layer excited at 740 nm with respective FLIM color maps at 740 nm of the relative concentrations of free NADH and bound NADH. Red-purple color indicates a high free/bound NADH ratio, while violet, cyan and white indicate linearly and progressively decreasing free/bound NADH ratios. Different ratios of free and protein-bound NADH reflect different redox ratios (NADH/NAD+) and rates of glycolysis and oxidative phosphorylation (B) Different relative concentrations of free and Bound NADH correspond to a metabolic trajectory in the phasor plot between Glycolysis and OXPHOS, respectively. The linear cluster in the pixel Phasor histogram represents all possible relative concentrations of free NADH (purple) and bound NADH (white). Scatter plot of the cell phasor of all stem cells optical metabolic fingerprint at different times of the day: 2 AM (blue), 8 AM (green), 2 PM (red) and 8 PM (cyan). (C) The top shows a histogram of the g coordinate of the cell phasor fingerprint (which is proportional to the free/bound NADH ratio) displaying a circadian metabolic oscillation. All distributions are statistically different (t-rest p<0.0001). The bottom shows the average number of stem cells in S-phase over 24 hours as determined by BrdU incorporation (Geyfman, 2012). See also Figure S2.

Figure 3. Metabolic cell heterogeneity in epidermal stem cells correlates with the clock phase

(A) TPE in vivo images of the epidermis basal cell layer expressing Per1-Venus reporter after excitation of stem cells at 940 nm. For the same field of view TPE intensity images of NADH and FLIM color maps at 740 nm of the relative concentrations of free NADH and bound NADH are represented. Red-purple color indicates a high free/bound NADH ratio, while violet, cyan and white indicate linearly and progressively decreasing free/bound NADH ratios. (B) Histogram of the average Per1-Venus Intensity from single stem cells display a circadian oscillation in phase with the oscillation of the g coordinate of cell phasor fingerprint (Fig.2). (C) Single stem cell Per1-Venus intensity displays a linear correlation with their metabolic fingerprint. See also Figure S3.

Figure 4. NADH metabolic oscillations are disrupted in the Bmal1−/− epidermal basal layer

(A) Two-photon fluorescence intensity in vivo images of stem cells within the epidermis basal cell layer of WT and Bmal1−/− mice excited at 740 nm at two different time points. Phasor FLIM color maps at 740 nm of the relative concentrations of free NADH and bound NADH. Red-purple color indicates a high free/bound NADH ratio, while violet, cyan and white indicate linearly and progressively decreasing ratios free/bound NADH ratio. (B) Scatter plot of the cell phasor of all stem cells optical metabolic fingerprint at different time of the day: 2 AM (blue), 2PM (red) measured in WT mice (t-test p<0.0001), and 2 AM (green), 2PM (black) measured in Bmal1−/− mice (t-test p=0.37). (C) The circadian metabolic oscillation of the cell phasor fingerprint g coordinate (proportional to the free/bound NADH ratio) is disrupted in Bmal1−/− mice. See also Figure S4.