Quantum Beam Science Research Directorate, National Institutes for Quantum and Radiological Science and Technology, Gunma, Japan.
Precursory Research for Embryonic Science and Technology, Japan Science and Technology Agency, Saitama, Japan.
J Gen Physiol. 2020 Aug 3;152(8). doi: 10.1085/jgp.201912469.
Recent studies using intracellular thermometers have shown that the temperature inside cultured single cells varies heterogeneously on the order of 1°C. However, the reliability of intracellular thermometry has been challenged both experimentally and theoretically because it is, in principle, exceedingly difficult to exclude the effects of nonthermal factors on the thermometers. To accurately measure cellular temperatures from outside of cells, we developed novel thermometry with fluorescent thermometer nanosheets, allowing for noninvasive global temperature mapping of cultured single cells. Various types of cells, i.e., HeLa/HEK293 cells, brown adipocytes, cardiomyocytes, and neurons, were cultured on nanosheets containing the temperature-sensitive fluorescent dye europium (III) thenoyltrifluoroacetonate trihydrate. First, we found that the difference in temperature on the nanosheet between nonexcitable HeLa/HEK293 cells and the culture medium was less than 0.2°C. The expression of mutated type 1 ryanodine receptors (R164C or Y523S) in HEK293 cells that cause Ca2+ leak from the endoplasmic reticulum did not change the cellular temperature greater than 0.1°C. Yet intracellular thermometry detected an increase in temperature of greater than ∼2°C at the endoplasmic reticulum in HeLa cells upon ionomycin-induced intracellular Ca2+ burst; global cellular temperature remained nearly constant within ±0.2°C. When rat neonatal cardiomyocytes or brown adipocytes were stimulated by a mitochondrial uncoupling reagent, the temperature was nearly unchanged within ±0.1°C. In cardiomyocytes, the temperature was stable within ±0.01°C during contractions when electrically stimulated at 2 Hz. Similarly, when rat hippocampal neurons were electrically stimulated at 0.25 Hz, the temperature was stable within ±0.03°C. The present findings with nonexcitable and excitable cells demonstrate that heat produced upon activation in single cells does not uniformly increase cellular temperature on a global basis, but merely forms a local temperature gradient on the order of ∼1°C just proximal to a heat source, such as the endoplasmic/sarcoplasmic reticulum ATPase.
最近使用细胞内温度计的研究表明，培养的单细胞内部温度在 1°C 左右存在异质性。然而，细胞内温度计的可靠性受到了实验和理论上的挑战，因为从原则上讲，要排除非热因素对温度计的影响极其困难。为了从细胞外部准确测量细胞温度，我们开发了一种新型的荧光温度计纳米片温度计，允许对培养的单细胞进行非侵入性的全局温度映射。将含有温度敏感型荧光染料铕（III）thenoyltrifluoroacetonate 三水合物的纳米片培养各种类型的细胞，即 HeLa/HEK293 细胞、棕色脂肪细胞、心肌细胞和神经元。首先，我们发现无兴奋性的 HeLa/HEK293 细胞和培养基之间在纳米片上的温度差异小于 0.2°C。在引起内质网 Ca2+泄漏的 HEK293 细胞中表达突变型 1 型 Ryanodine 受体（R164C 或 Y523S）不会使细胞温度升高超过 0.1°C。然而，当离子霉素诱导细胞内 Ca2+爆发时，细胞内温度计检测到内质网温度升高超过约 2°C；细胞内温度在 ±0.2°C 内保持几乎不变。当用线粒体解偶联试剂刺激大鼠新生心肌细胞或棕色脂肪细胞时，温度在 ±0.1°C 内几乎不变。在心肌细胞中，当以 2 Hz 的频率电刺激时，温度在 ±0.01°C 内稳定。同样，当以 0.25 Hz 的频率电刺激大鼠海马神经元时，温度在 ±0.03°C 内稳定。这些无兴奋性和兴奋性细胞的研究结果表明，单个细胞中激活时产生的热量不会在全局范围内均匀地增加细胞温度，而是仅在距热源（如内质网/肌浆网 ATP 酶）约 1°C 的范围内形成局部温度梯度。
