A coarse-grained NADH redox model enables inference of subcellular metabolic fluxes from fluorescence lifetime imaging.

Xingbo Yang,Gloria Ha,Daniel J Needleman

doi:10.7554/elife.73808

Abstract

Mitochondrial metabolism is of central importance to diverse aspects of cell and developmental biology. Defects in mitochondria are associated with many diseases, including cancer, neuropathology, and infertility. Our understanding of mitochondrial metabolism in situ and dysfunction in diseases are limited by the lack of techniques to measure mitochondrial metabolic fluxes with sufficient spatiotemporal resolution. Herein, we developed a new method to infer mitochondrial metabolic fluxes in living cells with subcellular resolution from fluorescence lifetime imaging of NADH. This result is based on the use of a generic coarse-grained NADH redox model. We tested the model in mouse oocytes and human tissue culture cells subject to a wide variety of perturbations by comparing predicted fluxes through the electron transport chain (ETC) to direct measurements of oxygen consumption rate. Interpreting the fluorescence lifetime imaging microscopy measurements of NADH using this model, we discovered a homeostasis of ETC flux in mouse oocytes: perturbations of nutrient supply and energy demand of the cell do not change ETC flux despite significantly impacting NADH metabolic state. Furthermore, we observed a subcellular spatial gradient of ETC flux in mouse oocytes and found that this gradient is primarily a result of a spatially heterogeneous mitochondrial proton leak. We concluded from these observations that ETC flux in mouse oocytes is not controlled by energy demand or supply, but by the intrinsic rates of mitochondrial respiration.

Highlights

32 Cells transduce energy from the environment to power cellular processes
While NADH and NADPH are difficult to distinguish with fluorescence measurements due to their overlapping fluorescence spectrum, the concentration of NADH in mouse oocytes is 40 times greater than the concentration of NADPH for the whole cell (Bustamante et al, 2017) and potentially even greater in mitochondria (Zhao et al, 2011), so the autofluorescence signal from these cells can be safely assumed to result from NADH. 123 To investigate how Fluorescence lifetime imaging microscopy (FLIM) measurements vary with mitochondrial activities, we performed 124 quantitative metabolic perturbations
The observation that proton leak is responsible for the gradient of electron transport chain (ETC) flux suggests that the flux heterogeneity is a result of intrinsic mitochondrial heterogeneity. This is consistent with our conclusion from the homeostasis of ETC flux (Figure 8) that it is the intrinsic rates of mitochondrial respiration, not energy demand or supply, that controls the ETC flux

Summary

Introduction

32 Cells transduce energy from the environment to power cellular processes. Decades of extensive research have produced a remarkable body of detailed information about the biochemistry of mitochondrial energy metabolism (Salway, 2017). 89 Here, we developed a generic coarse-grained NADH redox model that enables the inference of ETC flux with subcellular resolution from FLIM measurements We validated this model in mouse oocytes and human tissue culture cells subject to a wide range of perturbations by 92 comparing predicted ETC fluxes from FLIM to direct measurements of oxygen consumption rate, and by a self-consistency criterion. Using this method, we discovered that perturbing nutrient supply and energy demand significantly impacts NADH metabolic state but does not change ETC flux. FLIM of NADH can be used to non-invasively and continuously measure mitochondrial ETC fluxes with subcellular resolution and provides novel insights into spatiotemporal regulation of metabolic fluxes in cells. 102

Methods

Results

Conclusion