Real-Time Imaging of Mitochondrial ATP Dynamics Reveals the Metabolic Setting of Single Cells

Abstract

Summary Reprogramming of metabolic pathways determines cell functions and fate. In our work, we have used organelle-targeted ATP biosensors to evaluate cellular metabolic settings with high resolution in real time. Our data indicate that mitochondria dynamically supply ATP for glucose phosphorylation in a variety of cancer cell types. This hexokinase-dependent process seems to be reversed upon the removal of glucose or other hexose sugars. Our data further verify that mitochondria in cancer cells have increased ATP consumption. Similar subcellular ATP fluxes occurred in young mouse embryonic fibroblasts (MEFs). However, pancreatic beta cells, senescent MEFs, and MEFs lacking mitofusin 2 displayed completely different mitochondrial ATP dynamics, indicative of increased oxidative phosphorylation. Our findings add perspective to the variability of the cellular bioenergetics and demonstrate that live cell imaging of mitochondrial ATP dynamics is a powerful tool to evaluate metabolic flexibility and heterogeneity at a single-cell level.

Mitochondria supply ATP to the ER through a mechanism antagonized by cytosolic Ca2+

“…S6D), as previously shown by others (10,17). These results provide strong evidence supporting the notion that distinct ATP supplying and regulatory mechanisms exist for subcellular compartments within a cell, as we reported (12). Furthermore, a third widely used glycolysis inhibitor, 3-bromopyruvate (3-BrP), produced a similar ATP increase in the ER, in a dose dependent manner (Sppl.…”

“…Nevertheless, there is ample evidence supporting that differential ATP levels exist in membrane-bound organelles that use independent regulatory mechanisms in a compartment-specific manner (10)(11)(12)(13). To detect ATP levels in the ER lumen in vivo, we expressed an ER-localized ATP sensor ERAT (ERAT4.01 N7Q ) in H9 CHO cells engineered to induce mRNA expression of human clotting factor VIII (F8), encoding a protein which misfolds in the ER lumen, upon histone deacetylase inhibition (14,15).…”

High performance intensiometric direct- and inverse-response genetically encoded biosensors for citrate

“…5e ) 43 , both targeted to the mitochondria. Upon treatment of starved (glucose-free) HeLa cells with 20 mM glucose, we observed a change in MalionR fluorescence that was consistent with an increase in ATP concentration, as has been previously reported 49 . Similar to our previous results ( Fig.…”

“…These concentration values are substantially higher than those of HeLa cells (0.15 ± 0.07 mM and 0.44 ± 0.13 mM). The higher concentrations of citrate are consistent with a higher flux through the TCA cycle, as further supported by the higher oxygen consumption rates (OCRs) of INS-1 cells compared to that of HeLa cells 49 . Addition of 20 mM glucose increased both cytosolic and mitochondrial citrate concentration to~25 mM ( Fig.…”

“…These imaging results have generally reaffirmed what was already known, or expected, regarding citrate concentrations in these two cell types. For example, INS-1 cells were found to have substantially higher concentrations of citrate than HeLa cells, consistent with the higher metabolic rates of INS-1 cells 49 However, these citrate indicators also enable intracellular citrate concentrations to be probed in ways that would be otherwise impractical or impossible. As one example, these indicators have enabled us to determine that, upon treatment of starved cells with glucose, the increase in cytosolic citrate concentration is delayed~50 s relative to the increase in mitochondrial citrate concentration.…”