Re‐Evaluating Hot Mitochondria: Too Slow to Cool

ABSTRACT Aim Mito Thermo Yellow (MTY) is a mitochondrially targeted fluorophore that shows marked fluorescence quenching with increasing temperature, allowing for interrogating temperature dynamics in the mitochondria of live cells. Here we re‐evaluate published MTY fluorescence responses used to argue in favor of the ‘hot mitochondria’ concept; the assertion that mitochondria operate while maintaining substantial (> 10°C) apparent temperature gradients (Δ T app ) between themselves and their cellular environment. Results We find that MTY fluorescence kinetics are incompatible with the expected dynamics of mitochondrial heat production and diffusion. We further explore the published effects of mitochondrial inhibitors on MTY, and related evidence for Δ T app of > 10°C, again concluding results are inconsistent with the expected heat production dynamics. Thus, assertions of Δ T app > 10°C between mitochondria and their cellular environment based on MTY fluorescence intensity changes are unlikely to be reporting a signal that is uniquely intramitochondrial temperature. In addition to these analyses, we further argue that the inference mitochondria can operate at an internal temperature of > 48°C, as reported using MTY, is improbable as these internal temperatures would cause protein denaturation and aggregation and induction of the heat shock (HSR), unfolded protein (UPR), and integrated (ISR) stress responses. Conclusion Taken as a whole, we conclude MTY and similar tools must be re‐evaluated in regard to if they are providing solely information on local temperature and thus are so far inadequate, unto themselves, to demonstrate the existence of hot mitochondria.