Warm-blooded animals such as birds and mammals regulate their body temperatures to survive, but exactly how they do this is not fully understood. One reason for this uncertainty is that it is challenging to directly measure heat generation at the subcellular level because heat diffuses rapidly throughout the cell and beyond. Reported in Nature Methods, a team led by Yasuo Mori of Kyoto University now present a noninvasive, genetically encoded sensor for visualizing heat production in specific organelles in living cells (1).
Previously developed sensors have revealed thermal gradients inside of cells, including temperature differences between the cytoplasm and nucleus, but it has been difficult to target synthetic sensors to specific subcellular areas. “There has been long-standing interest in the cell physiology of thermogenesis, but the tools to look at cellular and sub-cellular mechanisms have not really existed. This study is a real advance,” said David Piston, an expert in fluorescence imaging methods at the Vanderbilt University School of Medicine who was not involved in the study.
The new technique is based on fusion proteins consisting of a temperature-sensing protein called TlpA, which is found in Salmonella bacteria, and a region of green fluorescent protein (GFP)—a light-emitting protein found in jellyfish.
Similar GFP-TlpA fusion proteins had been developed previously (2), but Mori and his team went a step further. They attached their fusion proteins to sequences that target specific organelles, including the endoplasmic reticulum (ER) and mitochondria. When these organelles produce heat, TlpA undergoes conformational changes that affect GFP, converting temperature changes into fluorescence changes that can be seen under the microscope.
The researchers tested the new method in brown fat cells and skeletal muscle fibers, two cell types known to be important for generating heat in animals. First, they delivered their mitochodria-targeted thermosensor into brown fat cells and exposed these cells to a compound known to induce heat generation. They also delivered an ER-targeted thermosensor to skeletal muscle fibers and then exposed those cells to a compound that affects chemical reactions thought to be involved in heat production in the ER.
In both cases, Mori and his team observed temperature-dependent fluorescence changes in the organelles. Their results also supported the previously proposed ER-based mechanism of heat generation, demonstrating that this method can be used to address controversial issues in thermal biology.
The new approach may also have clinical implications. “Thermogenesis from brown fat can be a way to burn off excess energy and thus prevent obesity,” Mori said. “Because we succeeded in using our sensor to directly observe thermogenesis in brown fat cells, we believe our technique will be useful for developing anti-obesity drugs.”
But the method is not without limitations. For one, the current version of the sensor cannot be used for in vivo imaging because the excitation and emission light cannot penetrate the skin, Mori said. The sensor also does not have great sensitivity and has a low dynamic range, according to other experts. “Designing a good GFP-based sensor is a difficult task, which often requires years of effort in many labs around the world,” said Konstantin Lukyanov, an expert in fluorescence imaging methods at the Russian Academy of Sciences who was not involved in the new study. “I think that more sensitive sensors with a high dynamic range will be developed, and this study has paved the way.”
1. Kiyonaka S, Kajimoto T, Sakaguchi R, Shinmi D, Omatsu-Kanbe M, Matsuura H, Imamura H, Yoshizaki T, Hamachi I, Morii T, and Mori Y. 2013. Genetically encoded fluorescent thermosensors visualize subcellular thermoregulation in living cells. Nat Methods. doi: 10.1038/nmeth.2690.
2. Naik RR, Kirkpatrick SM, and Stone MO. 2001. The thermostability of an alpha-helical coiled-coil protein and its potential use in sensor applications. Biosens Bioelectron 16(9-12):1051-7.