Simultaneous Evaluation of Mitochondrial and Cytosolic ATP Dynamics in Cultured HEK293T Cells

Abstract

Adenosine triphosphate (ATP) is the “energy currency” of all living organisms. It is crucial for cells to maintain consistent ATP levels, especially when supply is limited and demand is high. ATP is vital for organs with high ATP demands, such as the heart and the brain. In mammalian cells, oxidative phosphorylation in mitochondria produces most ATP, whereas glycolysis in cytosol yields the remaining portion of ATP. When the supply/demand of ATP alters, cells undergo drastic metabolic remodeling, which often maintains cellular function and links to mitochondrial dysfunction and even cell death. However, the mechanisms underlying the regulation of cellular ATP remain incompletely understood, mainly due to the lack of a tool to monitor spatiotemporal ATP changes in cells. In the present study, we designed a dual-ATP probe that allows for visualization of ATP dynamics in mitochondria and cytosol of an individual cell. This probe was based on two previously described single probes containing fusion proteins of GFP- and mApple-FoF1-ATP synthase e subunit (B. subtilis). The latter contains a mitochondrial singling sequence for mitochondrial localization. As such, cytosol ATP shows green and mitochondrial ATP shows red. We generated a plasmid for the dual-ATP probe containing a polycistronic arrangement of sequences coding for the two fusion proteins, rendering spatiotemporal recording of ATP dynamics simultaneously in mitochondria and cytosol of cultured cells. We first validated the dual-ATP probe did display red in mitochondria and green in the cytosol in cultured HEK293T cells upon feeding with glucose. Seahorse analysis indicated that the probe does not change cellular respiration. HEK293T cells were cultured for 24 hours after the transfection of the dual-ATP probe vector. We then tested several compounds known to target specific regions of the electron transport chain. When treated with oligomycin, a Complex V inhibitor, mitochondrial ATP declined within 20 min, but cytosolic ATP remained relatively constant. When we added Antimycin A, a Complex III inhibitor, ATP levels declined from time of injection until fluorescence plateaued at 20 minutes. Likewise, we saw similar responses occur after adding FCCP, a mitochondrial uncoupler, and rotenone, a Complex I inhibitor. When we added drugs such as 2-DG and KCN, a glycolysis inhibitor and a Complex IV inhibitor, respectively, both cytosolic and mitochondrial fluorescence plummeted, suggesting dramatic inhibition of ATP production from glycolysis and OXPHOS. Under hypoxic conditions, the mitochondrial ATP declined over the course of 6 hours while the cytosolic ATP remained stable. In summary, our results support the feasibility of our dual-ATP probe for spatiotemporal evaluation of cellular ATP level, distribution, and inter-compartmental trafficking. This innovative technology will provide unprecedented research opportunities for in-depth insight into the regulatory mechanisms of cellular energy metabolism in physiological and pathological states.