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Development of genetically encoded temperature indicators for intracellular thermometry at the subcellular level

Vu, Quang Cong 大阪大学 DOI:10.18910/85441

2021.09.24

概要

Genetically encoded temperature indicators (GETIs) allow the measurement of temperature dynamics at a subcellular resolution in live cells. However, GETIs have suffered from low temperature sensitivity when comparing to other nanothermometers, e.g., chemical-based fluorescent nanothermometers. To clearly visualize heat production from a biological process, it is highly desirable to develop GETIs that exhibit high temperature sensitivity for accurate temperature measurement as well as high specificity to temperature. In this thesis, I present two GETIs: (1) Elastin-Like Polypeptide based TEMPerature indicator (ELP-TEMP) with the highest ever temperature sensitivity among the existing fluorescent nanothermometers, and (2) Blue-excited genetically encoded TEMPerature indicator (B-gTEMP) that responded specifically to the temperature. ELP-TEMP is comprised of a temperature-responsive elastin-like polypeptide (ELP) fused with a cyan fluorescent protein (FP), mTurquoise2 (mT), and a yellow FP, mVenus (mV), as the donor and acceptor, respectively, of Förster resonance energy transfer (FRET). At elevated temperatures, the ELP moiety in ELP-TEMP undergoes a liquid-liquid phase transition leading to an increase in the FRET efficiency. In HeLa cells, ELP-TEMP responded to the temperature from 33 to 40 °C with a maximum temperature sensitivity of 45.1 ± 8.1 percent signal change per one degree Celsius (%/°C), which was the highest ever temperature sensitivity among the existing fluorescent nanothermometers. Although ELP-TEMP showed sensitivity not only to temperature but also to macromolecular crowding and self-concentration, I was able to correct the output of ELP-TEMP to achieve accurate temperature measurements at a subcellular resolution. I successfully applied ELP-TEMP to measure temperature changes in live cells induced by a local heat spot, even if the temperature difference was as small as <1°C, and to visualize heat production from Ca2+ influx induced by a chemical stimulation. Furthermore, I investigated temperatures in the nucleus and cytoplasm of live HeLa cells and found that their temperatures were almost the same within the temperature resolution of the measurement. This result would provide important information to shed light on a controversy about a discrepancy between a theoretical calculation and experimental measurements of intracellular temperature changes in single cells. On the other hand, B-gTEMP is a chimera of a green FP, mNeonGreen (mNG), showing low temperature sensitivity of −0.7%/°C, and a red FP, tdTomato (tdT), showing the highest temperature sensitivity of −2.9%/°C among the examined FPs. B-gTEMP is the improved version of gTEMP, a GETI composed of a blue FP, Sirius, and a green FP, mT-Sapphire (mT-Sap). B-gTEMP showed a response in a wide temperature range of 15–50 °C with an average temperature sensitivity of 2.2 ± 1.2%/°C, comparable to that of gTEMP (2.6%/°C). Because B-gTEMP utilized a visible light excitation whereas gTEMP utilized ultraviolet excitation, temperature imaging with B-gTEMP showed lower phototoxicity and autofluorescence background than that of gTEMP. Additionally, B-gTEMP showed higher temperature resolution than gTEMP. Furthermore, temperature measurement with B-gTEMP was not affected by macromolecular crowding and self-concentration, which are the advantages over ELP-TEMP. Using B-gTEMP, I successfully monitored quick temperature rises induced by a local heat spot with a temporal resolution of 10 ms. In addition, I demonstrated the functionality of B-gTEMP in conventional temperature imaging to measure heat production in mitochondria induced by a chemical stimulation, and investigated temperature in the nucleus and cytoplasm of live HeLa cells, and found that the temperature was almost the same between them within the temperature resolution of the measurement, consistent with the result of ELP-TEMP. Altogether, ELP-TEMP and B-gTEMP are useful GETIs for future investigation of cell thermobiology.

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