Mitochondrial temperature homeostasis resists external metabolic stresses

Abstract

Based on studies with a fluorescent reporter dye, Mito Thermo Yellow, and the genetically encoded gTEMP ratiometric fluorescent temperature indicator targeted to mitochondria, the temperature of active mitochondria in four mammalian and one insect cell-line was estimated to be up to 15 °C above that of the external environment to which the cells were exposed. High mitochondrial temperature was maintained in the face of a variety of metabolic stresses, including substrate starvation or modification, decreased ATP demand due to inhibition of cytosolic protein synthesis, inhibition of the mitochondrial adenine nucleotide transporter and, if an auxiliary pathway for electron transfer was available via the alternative oxidase, even respiratory poisons acting downstream of complex I. We propose that the high temperature of active mitochondria is an inescapable consequence of the biochemistry of oxidative phosphorylation and is homeostatically maintained as a primary feature of mitochondrial metabolism. Mitochondria are at least 15 °C hotter than their external environment in living cells. In response to diverse metabolic stresses, mitochondrial temperature re-adjusts to this value whenever possible.

A species difference in glycerol-3-phosphate metabolism reveals metabolic adaptations in hares

“…With the same approach we observed a drop of ~15°C and ~10°C, respectively, in Complex I inhibited mitochondria from brown and mountain hare fibroblasts, suggesting initial temperatures of ≥ 52°C and ≥ 47°C. These estimates are in good agreement with previous knowledge from other cell models (31,32). Alternatively, the activity of the G3P shuttle, which is theoretically independent of Complex I, could preserve mitochondrial temperature in mountain hare, limiting the drop induced by Complex I inhibition.…”

“…For example, a drop of 15°C in temperature induced by rotenone treatment would imply that mitochondrial temperature was at least 52°C before the treatment. This method led to the finding that mitochondrial temperature of human fibroblasts was ≥ 50°C (31), an estimation which was confirmed in various human and animal cell types (32). With the same approach we observed a drop of ~15°C and ~10°C, respectively, in Complex I inhibited mitochondria from brown and mountain hare fibroblasts, suggesting initial temperatures of ≥ 52°C and ≥ 47°C.…”

“…Unlike the malate-aspartate shuttle which allows a net transfer of cytosolic NADH to the Complex I, the G3P shuttle branches on the quinone downstream of Complex I, releasing the energy which would have been used to pump protons at Complex I as heat (18). MTY thermometry reliably assesses the relative (before vs after treatment) temperature of mitochondria (31,32). As mitochondrial temperature can drop to that of their environment, (37°C in mammalian cells), but not further, one can also infer the absolute temperature of mitochondria before treatment.…”

The protein‐only RNase Ps, endonucleases that cleave pre‐tRNA: Biological relevance, molecular architectures, substrate recognition and specificity, and protein interactomes

“…At PRORP1 also maintains activity at temperatures up to 45°C, while At PRORP2 and 3 lose all activity at 45°C (Chen et al, 2019). This may correlate to studies that have suggested the mitochondria may be significantly warmer than the surrounding environment (Terzioglu et al, 2023), potentially explaining why mitochondria rely on protein‐only RNase P.…”

Most axonal mitochondria in cortical pyramidal neurons lack mitochondrial DNA and consume ATP

“…What could be the biological adaptation of such a drastic difference between the mtDNA content and ATP generation capacity between axonal and dendritic mitochondria in mammalian CPNs? We speculate four potential models to explain this drastic level of compartmentalization of mitochondrial function: (1) the cytoplasmic volume at individual presynaptic bouton is extremely small (in the order of hundreds of cubic nanometers) and therefore, in such extremely small volumes, the accumulation of mitochondria-derived reactive-oxygen species (ROS), a byproduct of highly functional ETC, could be damaging to protein complexes involved in neurotransmission; (2) mitochondria with highly functioning electron transport chain and high levels of oxidative phosphorylation have been proposed to reach temperatures approaching 50°C ( 36, 37 ). Since axonal mitochondria are located just a few hundreds of nanometers from the active zone at presynaptic boutons, where SNARE-mediated presynaptic vesicles are docked near the plasma membrane, and SNARE-mediated exocytosis is highly temperature-dependent ( 38 ), we speculate that axonal mitochondria with defective or poorly functioning oxidative phosphorylation characterizing CPNs described in this study might not generate high temperatures and therefore not interfere with SNARE-mediated presynaptic vesicle fusion; (3) Axonal mitochondria are more inclined towards electron transport chain-independent anabolic functions, such as amino acid biosynthesis ( 39, 40 ) including glutamate generation, the primary excitatory neurotransmitter used by CPNs and/or to fuel local protein synthesis which is prevalent, and often associated with mitochondria, not only in dendrites ( 5, 14 ) but also in distal portions of the axon in neurons ( 41, 42 ); (4) recent work suggests that mtDNA-mediated activation of the cGAS-STING pathway triggers low grade inflammation and senescence-associated secretory phenotypes found in aging and neurodegeneration ( 43, 44 ).…”