SANKEN, The University of Osaka, Ibaraki, Osaka 567-0047, Japan.
OTRI, The University of Osaka, Suita, Osaka 565-0871, Japan.
ACS Sens. 2024 Aug 23;9(8):3889-3897. doi: 10.1021/acssensors.3c02658. Epub 2024 Jul 23.
Intracellular temperature is a fundamental parameter in biochemical reactions. Genetically encoded fluorescent temperature indicators (GETIs) have been developed to visualize intracellular thermogenesis; however, the temperature sensitivity or localization capability in specific organelles should have been further improved to clearly capture when and where intracellular temperature changes at the subcellular level occur. Here, we developed a new GETI, gMELT, composed of donor and acceptor subunits, in which cyan and yellow fluorescent proteins, respectively, as a Förster resonance energy transfer (FRET) pair were fused with temperature-sensitive domains. The donor and acceptor subunits associated and dissociated in response to temperature changes, altering the FRET efficiency. Consequently, gMELT functioned as a fluorescence ratiometric indicator. Untagged gMELT was expressed in the cytoplasm, whereas versions fused with specific localization signals were targeted to the endoplasmic reticulum (ER) or mitochondria. All gMELT variations enabled more sensitive temperature measurements in cellular compartments than those in previous GETIs. The gMELTs, tagged with ER or mitochondrial targeting sequences, were used to detect thermogenesis in organelles stimulated chemically, a method previously known to induce thermogenesis. The observed temperature changes were comparable to previous reports, assuming that the fluorescence readout changes were exclusively due to temperature variations. Furthermore, we demonstrated how macromolecular crowding influences gMELT fluorescence given that this factor can subtly affect the fluorescence readout. Investigating thermogenesis with gMELT, accounting for factors such as macromolecular crowding, will enhance our understanding of intracellular thermogenesis phenomena.
细胞内温度是生化反应的基本参数。已经开发出遗传编码的荧光温度指示剂 (GETI) 来可视化细胞内热生成；然而，为了清楚地捕捉到细胞内热在亚细胞水平上何时以及何地发生变化，应该进一步提高特定细胞器中的温度敏感性或定位能力。在这里，我们开发了一种新的 GETI，gMELT，由供体和受体亚基组成，其中青色和黄色荧光蛋白分别作为荧光共振能量转移 (FRET) 对与温度敏感结构域融合。供体和受体亚基响应温度变化而缔合和解离，改变 FRET 效率。因此，gMELT 作为荧光比率指示剂发挥作用。未标记的 gMELT 在细胞质中表达，而与特定定位信号融合的版本则靶向内质网 (ER) 或线粒体。所有 gMELT 变体都能够比以前的 GETI 更灵敏地测量细胞区室中的温度。用 ER 或线粒体靶向序列标记的 gMELTs 用于检测化学刺激下细胞器中的热生成，这是一种以前已知可诱导热生成的方法。观察到的温度变化与以前的报告相当，假设荧光读数的变化仅归因于温度变化。此外，我们展示了大分子拥挤如何影响 gMELT 荧光，因为这一因素会微妙地影响荧光读数。考虑到大分子拥挤等因素，用 gMELT 研究热生成将增强我们对细胞内热生成现象的理解。
