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Use of engineered color tuning luciferases for intracellular pH analysis and temperature sensing in mammalian cells

Abstract

Firefly luciferases catalyze the efficient production of yellow-green light under physiological conditionsnormal, having been widely used for bioanalytical purposes for more than 5 decades. However, under conditionsacids, high temperatures and the presence of heavy metals, they produce red light, a property calledpH sensitivity. Despite the demand for physiological intracellular biosensors for pH and temperature, thesensitivity to pH and temperature of firefly luciferase were considered inconvenient in trialsanalytical. We demonstrate for the first time that firefly luciferases and their pH sensitivities can beused to estimate intracellular pH variations through ratiometric analysis. Using luciferase fromfirefly Macrolampis sp2, intracellular pH can be estimated ratiometrically in bacteria and then in cells ofmammals. However, this luciferase, like many firefly luciferases, exhibits spectra shifted tored and are unstable at the high temperatures of mammalian cells, reducing the resolution of the analysisratiometric. Therefore, in this project we selected and are modifying a new luciferase from the firefly Amydetesviveni to monitor intracellular pH changes in different mammalian cells and conditions, especiallymacrophages during their differentiation, due to their greater stability and blue-shifted spectrum at 37°C. UsWe also plan to use these modified luciferases to monitor pH changes in mitochondria. Furthermore,considering the existence of a ratiometric relationship between the emission of green and red light and the temperature infirefly luciferases, we also plan to design a luciferase to generate a thermal sensing luciferaseto monitor mitochondrial temperature changes during active respiration. (AU)

Articles published in Agência FAPESP Newsletter about the research grant:
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VEICULO: TITULO (DATA)
VEICULO: TITULO (DATA)

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