Genetically encoded fluorescent thermosensors visualize subcellular thermoregulation in living cells
Genetically encoded fluorescent thermosensors visualize subcellular thermoregulation in living cells
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DOI：
10.1038/nmeth.2690
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发表时间：
2013-12-01
期刊：
影响因子：
48
通讯作者：
Mori, Yasuo
中科院分区：
文献类型：
--
作者：
Kiyonaka, Shigeki;Kajimoto, Taketoshi;Mori, Yasuo
在哺乳动物和鸟类中，保持体温的体温调节对生命至关重要。已经提出了多种产热机制，定位于不同的亚细胞器。然而，直接在完整的细胞器中观察产热一直是一个挑战。在这里，我们已经开发了基因编码的，基于GFP的热传感器（tsGFP），使活细胞中离散细胞器中的产热可视化。在tsGFP中，沙门氏菌热敏蛋白TlpA的卷曲螺旋结构的串联形成将构象变化传递给GFP，以将温度变化转化为可见和可量化的荧光变化。tsGFP的特异性靶向使得棕色脂肪细胞的线粒体和肌管的内质网中的产热能够可视化。在HeLa细胞中，靶向线粒体的tsGFP揭示了与电化学梯度相关的产热的异质性。因此，tsGFP是非侵入性评估活细胞产热的有力工具。
In mammals and birds, thermoregulation to conserve body temperature is vital to life. Multiple mechanisms of thermogeneration have been proposed, localized in different subcellular organelles. However, visualizing thermogenesis directly in intact organelles has been challenging. Here we have developed genetically encoded, GFP-based thermosensors (tsGFPs) that enable visualization of thermogenesis in discrete organelles in living cells. In tsGFPs, a tandem formation of coiled-coil structures of the Salmonella thermosensing protein TlpA transmits conformational changes to GFP to convert temperature changes into visible and quantifiable fluorescence changes. Specific targeting of tsGFPs enables visualization of thermogenesis in the mitochondria of brown adipocytes and the endoplasmic reticulum of myotubes. In HeLa cells, tsGFP targeted to mitochondria reveals heterogeneity in thermogenesis that correlates with the electrochemical gradient. Thus, tsGFPs are powerful tools to noninvasively assess thermogenesis in living cells.