A highly responsive pyruvate sensor reveals pathway-regulatory role of the mitochondrial pyruvate carrier MPC

Abstract

Mitochondria generate ATP and building blocks for cell growth and regeneration, using pyruvate as the main substrate. Here we introduce PyronicSF, a user-friendly GFP-based sensor of improved dynamic range that enables real-time subcellular quantitation of mitochondrial pyruvate transport, concentration and flux. We report that cultured mouse astrocytes maintain mitochondrial pyruvate in the low micromolar range, below cytosolic pyruvate, which means that the mitochondrial pyruvate carrier MPC controls the decision between respiration and anaplerosis/gluconeogenesis in an ultrasensitive fashion. The functionality of the sensor in living tissue is demonstrated in the brain of Drosophila melanogaster larvae. Mitochondrial subpopulations are known to coexist within a given cell, which differ in their morphology, mobility, membrane potential, and vicinity to other organelles. The present tool can be used to investigate how mitochondrial diversity relates to metabolism, to study the role of MPC in disease, and to screen for small-molecule MPC modulators.

Highly responsive single-fluorophore indicator to explore lactate dynamics in high calcium environments

“…increase in cytosolic lactate, revealing the presence of abundant surface lactate transporters in these cells ( Figure 6B). This result is consistent with the previous results achieved with a single-fluorophore indicator for pyruvate 34 representative environment. We also performed a transport assay that proved that this highly responsive indicator displays better responses and is, therefore, more suitable to set up transport assays in multiwell plate readers for high-throughput screening.…”

“…Superfusion of acutely isolated brains with lactate resulted in a quick and reversible increase in cytosolic lactate, revealing the presence of abundant surface lactate transporters in these cells (Figure 6B). This result is consistent with the previous results achieved with a single-fluorophore indicator for pyruvate 34 . These experiments show the suitability of the lactate indicator to explore lactate dynamics in ex vivo preparations from different model systems.…”

“…The results showed both spectra are dependent on lactate, with an isosbestic point at 430 nm and emission peak at 513 nm (Figure 2D). This behavior is similar to that of other single-fluorophore indicators developed using the same reporter protein34 . Since TTHA0766 binds lactate associated with a calcium molecule, a potential interference of calcium cations with the fluorescence lactate response is likely.…”

Lipid metabolism in astrocytic structure and function

“…If pyruvate is not dehydrogenated to lactate or taken up by the mitochondria and converted to acetyl-coA, gluconeogenesis can occur with excess carbon stored as glycogen for times of bioenergetic stress, however, this process is tightly regulated. Using emerging fluorophore sensor technology, a recent study suggested that mitochondria in cultured mouse astrocytes can maintain lower levels of pyruvate compared to the cytosol [20].…”

Green fluorescent protein-based lactate and pyruvate indicators suitable for biochemical assays and live cell imaging

“…2c,d). The EC 50 values of Green Lindoblum and Green Pegassos are lower than previously developed indicators 2,3,8 , and therefore, they would be appropriate to monitor changes in the minimal level of intracellular lactate and pyruvate. Hill coefficient for Green Lindoblum and Green Pegassos was 1.2 and 1.4, respectively, suggesting that these indicators are applicable for an appropriate range of lactate and pyruvate levels.…”