Single-cell temperature mapping with fluorescent thermometer nanosheets
Single-cell temperature mapping with fluorescent thermometer nanosheets
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DOI：
10.1085/jgp.201912469
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发表时间：
2020-08-01
影响因子：
3.8
通讯作者：
Fukuda, Norio
中科院分区：
文献类型：
--
作者：
Oyama, Kotaro;Gotoh, Mizuho;Fukuda, Norio
最近使用细胞内温度计的研究表明，培养的单个细胞内的温度在1摄氏度左右变化不均匀。然而，细胞内测温的可靠性在实验和理论上都受到了挑战，因为原则上很难排除非热因素对温度计的影响。为了从细胞外精确测量细胞温度，我们开发了一种新型的荧光温度计纳米片测温技术，允许对培养的单细胞进行无创的全球温度测绘。将各种类型的细胞，如HeLa/HEK293细胞、棕色脂肪细胞、心肌细胞和神经元，培养在含有温度敏感荧光染料europium (III) thenoyltrifluoroacetonate trihydrate的纳米片上。首先,我们发现，在不可兴奋的HeLa/HEK293细胞和培养基之间，纳米片上的温度差异小于0.2摄氏度。突变的1型ryanodine受体（R164C或Y523S）在HEK293细胞中表达导致Ca2+从内质网泄漏的细胞温度不会改变超过0.1摄氏度的细胞温度。然而，细胞内测温检测到HeLa内质网温度升高大于类似的2摄氏度离子霉素诱导的细胞内Ca2+爆发；当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℃范围内几乎不变。在心肌细胞中，当电刺激频率为2 Hz时，细胞收缩时温度稳定在+/- 0.01℃范围内。同样，当大鼠海马神经元在0.25 Hz的电刺激下，温度稳定在+/- 0.03℃。目前对不可兴奋细胞和可兴奋细胞的研究结果表明，单个细胞激活时产生的热量不会在全球范围内均匀地增加细胞温度，而只是在靠近热源（如内质/肌浆网atp酶）的地方形成1℃左右的局部温度梯度。
Recent studies using intracellular thermometers have shown that the temperature inside cultured single cells varies heterogeneously on the order of 1 degrees 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 degrees 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 degrees C. Yet intracellular thermometry detected an increase in temperature of greater than similar to 2 degrees C at the endoplasmic reticulum in HeLa cells upon ionomycin-induced intracellular Ca2+ burst; global cellular temperature remained nearly constant within +/- 0.2 degrees C. When rat neonatal cardiomyocytes or brown adipocytes were stimulated by a mitochondrial uncoupling reagent, the temperature was nearly unchanged within +/- 0.1 degrees C. In cardiomyocytes, the temperature was stable within +/- 0.01 degrees 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 degrees 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 similar to 1 degrees C just proximal to a heat source, such as the endoplasmic/sarcoplasmic reticulum ATPase.