Genetically encoded biosensors utilizing the Förster resonance energy transfer (FRET) are powerful tools for live cell imaging of various cellular processes. Our group has previously developed a series of FRET-based biosensors, named "ATeam," for visualization of ATP levels inside a single living cell. ATeam not only provides a window of insight into a single cell but also allows for visualization of ATP levels in mitochondrial matrix of a single living cell. This novel tool is able to monitor alterations in cellular ATP in response to various treatments in real time. Here we present a method for the evaluation of ATP levels in mitochondria in living cells by using ATeam. At the end of this chapter, an example of experimental results is described for a better understanding of the presented procedure.

Fluctuations in food availability and shifts in temperature are typical environmental changes experienced by animals. These environmental shifts sometimes portend more severe changes; e.g., chilly north winds precede the onset of winter. Such telltale signs may be indicators for animals to prepare for such a shift. Here we show that HEK293A cells, cultured under starvation conditions, can “memorize” a short exposure to cold temperature (15 °C), which was evidenced by their higher survival rate compared to cells continuously grown at 37 °C. We refer to this phenomenon as “cold adaptation”. The cold-exposed cells retained high ATP levels, and addition of etomoxir, a fatty acid oxidation inhibitor, abrogated the enhanced cell survival. In our standard protocol, cold adaptation required linoleic acid (LA) supplementation along with the activity of Δ-6-desaturase (D6D), a key enzyme in LA metabolism. Moreover, supplementation with the LA metabolite arachidonic acid (AA), which is a high-affinity agonist of peroxisome proliferator-activated receptor-alpha (PPARα), was able to underpin the cold adaptation, even in the presence of a D6D inhibitor. Cold exposure with added LA or AA prompted a surge in PPARα levels, followed by the induction of D6D expression; addition of a PPARα antagonist or a D6D inhibitor abrogated both their expression, and reduced cell survival to control levels. We also found that the brief cold exposure transiently prevents PPARα degradation by inhibiting the ubiquitin proteasome system, and starvation contributes to the enhancement of PPARα activity by inhibiting mTORC1. Our results reveal an innate adaptive positive-feedback mechanism with a PPARα-D6D-AA axis that is triggered by a brief cold exposure in cells. “Cold adaptation” could have evolved to increase strength and resilience against imminent extreme cold temperatures.