Life science
Medical & healthcare, Drug development

Development of a fast-responding genetically-encodedtemperature indicator

Life science

Department of Biomolecular Science and Engineering, SANKEN (The Institute of Scientific and Industrial Research)Specially Appointed Associate Professor Tetsuichi Wazawa

https://researchmap.jp/wazawa?lang=en

Specially Appointed Researcher Kai Lu			

https://researchmap.jp/ka1lu?lang=en

Professor Takeharu Nagai					https://researchmap.jp/ng1?lang=en

Abstract
Genetically-encoded temperature indicators (GETIs) are sensor proteins used for measurement and imaging of temperature
through the temperature-dependent fluorescence. Here we developed a fast-responding GETI, B-gTEMP, which reports temperature through a ratio between two fluorescence bands for accurate
measurement and responds to a temperature change with an unprecedentedly small time constant of <1 ms. We applied B-gTEMP
to temperature imaging in mammalian cells and successfully
observed intracellular heat diffusion on the millisecond scale. Furthermore, by comparing the imaging data with heat diffusion simulation, we estimated that the heat diffusion coefficient in cells was
1/5 of that of pure water, indicating that intracellular heat diffusion
would be considerably slower.

▍ Significance of the research and Future perspective
Thermogenesis in homeothermic animals plays an important role in thehomeostasis and regulation of body temperature. For the investigation ofthermogenesis at the single cell level, it has been necessary to develop
a fast-responding GETI capable of observing millisecond intracellularheat diffusion processes occurring in a small volume such as mammaliancells. B-gTEMP should be useful for investigating thermal biology issues
such as mechanisms of subtle intracellular temperature regulation anddiseases and disorders involving intracellular thermogenesis.

▍ Background & Results
Thermogenesis plays a key role in the homeostasis and regulation of body temperature in animals. In particular, the non-shivering
thermogenesis, which occurs in non-muscle tissues, is thought
to be closely associated with not only the homeostasis but also
inflammation-related fever, cold-adaptation, and obesity. However,the detail of the relationship remains elusive. Fluorescent temperature indicators report temperature through the fluorescenceand can be used for temperature imaging on a fluorescence microscope. Among temperature indicators developed from various
materials, genetically-encoded temperature indicators (GETIs) areparticularly useful for measurements of live biological samples,
because GEITs can be introduced into cells for expression as aprotein in a minimally invasive manner and can be easily modified
on the basis of DNA for versatile applications. However, GETIs
developed to date are slow to respond to temperature changes orneed time-consuming measurement procedures. Thus, a fast-responding GETI has been required to study transient intracellularheat diffusion.
Here we have developed a GETI, B-gTEMP, from a temperature-responsive red fluorescent protein and a much less temperature-responsive green fluorescent protein. The ratio of red to green
fluorescence intensity correlates with temperature, and responds
to a temperature change with a time constant of <1 ms. By applying B-gTEMP to temperature imaging of mammalian cells, we
successfully observed an intracellular heat diffusion process withsub-millisecond resolution. Furthermore, we compared the imagingdata with heat diffusion simulation and estimated that the heat diffusion coefficient in cells was 1/5 of that of pure water. B-gTEMP isexpected to contribute to the investigation of non-shivering thermogenesis in relation to the subtle intracellular temperature regulationand diseases and disorders involving intracellular thermogenesis.

Figure 1. Design of B-gTEMP and its response to temperature. (A) A
schematic diagram of the temperature response of B-gTEMP. (B) Temperature-dependent fluorescence emission spectrum of B-gTEMP.

Figure 2. High-speed imaging of heat diffusion in a HeLa cell using
B-gTEMP. (A) A schematic diagram of the experimental configuration. Acarbon nanotube cluster close to a HeLa cell was irradiated with a red laser beam to generate heat, and the intracellular temperature distribution
was imaged through the B-gTEMP fluorescence. (B) Time developmentof temperature distribution in the HeLa cell after starting to heat the cell.

P a t e n t
Lu, Kai; Wazawa, Tetsuichi; Nagai, Takeharu et al. Intracellular heat transfer and thermal property revealed by kilohertz temperature imaging with agenetically encoded nanothermometer. Nano Letters, 2022, 22(14), 5698–5707. doi: 10.1021/acs.nanolett.2c00608
UR
L https://www.sanken.osaka-u.ac.jp/labs/bse/index-E.html
K e y w o r d thermogenesis, heat diffusion, fluorescent protein, fluorescence imaging, protein engineering
Tr e a t i s e