Spatial patterning of metabolism by mitochondria, oxygen, and energy sinks in a model cytoplasm

Abstract

Metabolite gradients might guide mitochondrial localization in cells and angiogenesis in tissues. It is unclear whether they can exist in single cells, because the length scale of most cells is small compared to the expected diffusion times of metabolites. For investigation of metabolic gradients, we need experimental systems in which spatial patterns of metabolism can be systematically measured and manipulated. We used concentrated cytoplasmic extracts from Xenopus eggs as a model cytoplasm, and visualized metabolic gradients formed in response to spatial stimuli. Restriction of oxygen supply to the edge of a drop mimicked distance to the surface of a single cell, or distance from a blood vessel in tissue. We imaged a step-like increase of Nicotinamide adenine dinucleotide (NAD) reduction approximately 600 microm distant from the oxygen source. This oxic-anoxic switch was preceded on the oxic side by a gradual rise of mitochondrial transmembrane potential (Deltapsi) and reactive oxygen species (ROS) production, extending over approximately 600 microm and approximately 300 microm, respectively. Addition of Adenosine triphosphate (ATP)-consuming beads mimicked local energy sinks in the cell. We imaged Deltapsi gradients with a decay length of approximately 50-300 microm around these beads, in the first visualization of an energy demand signaling gradient. Our study demonstrates that mitochondria can pattern the cytoplasm over length scales that are suited to convey morphogenetic information in large cells and tissues and provides a versatile model system for probing of the formation and function of metabolic gradients.

Figure 1

(A) Autoradiography of thin layer chromatograms of egg extracts pulsed for the indicated times with ³²P potassium phosphate at the indicated drug or gas conditions. Positions of inorganic phosphate (P_i) and ATP are indicated. (B) UV autofluorescence pattern in an extract drop exposed to air at its side. (C) Higher magnification of bright zone. Co-staining with Rhd123 reveals that the UV autofluorescence signal mainly derives from mitochondria. Scale bar, 10 μm. (D) UV autofluorescence pattern in a mitochondrial suspension. Co-staining with MitoTrackerGreen. (E) UV autofluorescence pattern in a drop of HelaS3 cell suspension. Inset, magnification of border region (oxic-anoxic). Inset scale bar, 50 μm. If not otherwise indicated, scale bars correspond to 500 μm.

Figure 2

Effect of metabolic perturbations on NADH patterns in egg extract. (A) Increasing energy demand by addition of apyrase. (B) Mitochondrial uncoupling with FCCP. (C) Complex I inhibition with rotenone. Lower panel, corresponding line profiles. (D) Complex IV inhibition with potassium cyanide. (E) Exposure to different ambient oxygen concentrations. Numbers indicate O₂ percentage of N₂/O₂ gas mixture. Left panel, untreated extract. Right panel, FCCP treated extract. Inset, dark zone with (x_c) plotted as a function of oxygen concentration and fitted with a square root function. Partial differential equation (PDE) describing the corresponding one dimensional reaction diffusion model assuming 0^th order oxygen kinetics for respiration. Scale bars, 500 μm.

Figure 3

Imaging of mitochondrial activities. (A) Simultaneous imaging of NADH- and flavin-autofluorescence in egg extracts (left panel) and mitochondrial suspensions (right panel). (B) Simultaneous imaging of NADH and mitochondrial membrane potential using JC-1. Mitochondrial potential is represented as E_TRITC/E_FITC emission ratio of the JC-1 sensor. High E_TRITC/E_FITC ratios (bright colors) indicate high mitochondrial membrane potential. Left panel, egg extract. Right panel, mitochondrial suspension. (C) Reactive oxygen species (ROS) measured as DCF emission (E_DCF) in egg extracts (left panel) and mitochondrial suspensions (right panel). Bright colors indicate high concentrations of DCF/ROS. Lower panels, averaged line plots. Scale bars, 500 μm.

Figure 4

Imaging of energy demand gradients. (A) Imaging of [NADH] and Δψ in mitochondrial suspensions containing beads coupled with apyrase. Mitochondrial potential is represented as E_TRITC/E_FITC emission ratio of the JC-1 sensor. Red box, averaged line scan presented in right panel. (B) [NADH] in mitochondrial suspensions with heterogeneously distributed apyrase beads. (C) Mitochondrial potential on BSA- and apyrase coated beads in egg extracts. Error bars, standard error (n > 10 beads). (D) Imaging of [NADH] and Δψ in egg extracts containing beads coupled with apyrase. Right panel, averaged line scans of image regions indicated by a red box. Red curve, anoxic energy demand gradient fitted with an EC₅₀ function. Blue curve, oxic energy demand gradient fitted with an EC₅₀ function. Asterisks mark bead positions. Gray shaded region marks bead cluster position. Scale bars, 500 μm.