LABORATORY FOR BIOTHERMOLOGYSpecially Appointed Associate Professor

KAMEI, Yasuhiro

Specially Appointed Assistant Professor:
SAKAMOTO, Joe
Postdoctoral Fellow:
TOMOI, Takumi
JSPS Postdoctoral Fellow: SUZUKI, Miyuki
Technical Assistant:
KINOSHITA, Chie

Temperature is an important parameter for living organisms.
Cell activity is affected by temperature since the reaction ratio
and stability of molecules, especially proteins in cells, depends
on it. The environmental temperature which organisms
function in vary, and the biological system that regulates bodytemperature, homeostasis, differs among various organisms.
Furthermore, temperatures in either cells or at the molecularlevel have not been widely discussed to date. Nano-scale thermometers utilizing fluorescent proteins and dyes have beendeveloped by numerous research groups including our own,
and heterogeneity of temperatures in the micro environmentsof living cells have been reported on by said groups (Okabeet al, Nat Commun 2014, Kiyonaka et al, Nat. Methods 2014,and Nakano et al, PLoS One 2017). However, the meaningand mechanisms involved in single cells remain unclear.
Therefore, our group is embarking upon a new research field,biothermology, by investigating the nature of temperature inliving organisms ranging from nano to macro.
Our research group employs various and original biothermological research technologies. One of these is the infrared(IR) laser application, which enables single-cell or subcellular local heating by focusing IR through an objective lenson a microscope This technology was originally developed
for laser induced gene expression in the targeting of singlecells through the heat shock response, IR-LEGO: InfraRed
Laser Evoked Gene Operator (Kamei et al, Nat. Methods2009) (Figure 1). This local heating technique can be used
to analyze the thermodynamics of a cell via the use of anano-scale thermometer; the second of our abovementioned

Figure 1. Schematic illustration of heat shock response in cells (left) andan infrared laser-evoked gene operator (IR-LEGO) microscope system.

original techniques. To analyze temperature distribution at
subcellular resolutions, a nano-scale thermometer is required.
For this reason, we have focused on fluorescent proteins, anddeveloped a genetically encoded thermometer, the so calledgTEMP, with Dr. Takeharu Nagai from Osaka University(Nakano et al, PLoS One 2017). This thermometer has someunique properties, such as rapid responsiveness and applicability to wide temperature range. By utilizing this thermometer, we are developing a high-speed thermal imaging microscope system combined with IR irradiation optics. Throughthe analysis of thermal dynamics in cells, we are trying toreveal how temperature heterogeneity is generated within asingle cell as well as considering its meaning from a biological standpoint.
Furthermore, our group is trying to improve the IR-LEGOtechnique. We initially applied this system to many organisms including animals (i.e. medaka, nematode, flies andfrogs) and plants (i.e. Araidopsis moss in collaborative
research projects in cooperation with other laboratories.
Because the heat shock response (HSR) is a transient
response, gene expressions after IR-LEGO operation are alsotransient. Despite this, some of our collaborators wanted torealize cell fate mapping, which requires a long-term gene
expression after IR irradiation. Accordingly, we employed
the Cre/LoxP recombination system. As shown in Figure 2,
we established a cre driver line, which possesses a heat shock
promoter and loxP effector line which can permanently label
irradiated cell lineages by utilizing fluorescent proteins. By
using these transgenic lines, evidence was provided that indicated that exoskeletal tissues in the trunk region came fromthe mesoderm (Shimada et al, Nat Commun. 2013).

Figure 2. Long-term gene expression using the cre/loxP recombination
system and an example of a practical experiment in medaka ranging
embryos to adults.

The HSR is a conservative stress response system found inalmost all organisms. In contrast, organisms live in various
temperatures, and HSR essentially is the upper limit of habitable temperature range for each organism. This means thatthe preset temperature of HSR may differ among organisms.
A key factor of this limitation is heat shock factor 1 (HSF1);a homologue within each organism. By substituting HSF1,
it may be possible to control the preset temperature of HSR,
and this substitution could be applied to the improvement of91

National Institute for Basic Biology

Imaging Science

HSR efficiency in IR-LEGO technology. We are now trying
to lower the HSR temperature of medaka by substituting
Japanese flounder HSF1. The HSF1 substitution provides us
with various types of significant information and evidence
related to molecular evolution and adaptation to environmental temperature. Thus, this is how this project also contributes
to the study of biothermology.
We also promote other collaborative studies using microscopic techniques. One is a “clear observation project” fordeep seeing into living organisms using adaptive optics(AO), which have been thoroughly developed in the fieldof astronomy as a key technology found in large telescopes,
such as the Subaru telescope in Hawaii. Although observation using Earth based telescopes may be interfered with
due to fluctuations in the atmosphere, AO can mitigate this.
However, living materials have particular refractive indexes,
so some organelles may hinder the ideal optical path formicroscope observation, which is similar to the situation thatexists regarding the atmosphere and telescopes. AO can also
compensate for this disturbance by sensing and correcting
wave fronts by using a wave front sensor and deformablemirror. Hence, we have developed a custom-made wide-fieldmicroscope equipped with an AO system for the observationof living organisms in collaboration with Dr. Yosuke Tamada
from Utsunomiya University and Dr. Yutaka Hayano fromthe National Astronomical Observatory of Japan (NAOJ).
By utilizing this microscopic system, we have successfullyacquired high-resolution bright field and fluorescent imagesof living cells. Furthermore, AO can also be applied toIR-LEGO technology to improve IR energy focusing of irradiation in a manner similar to improving the sight of livingorganisms.

Publication List:〔Original papers〕

M., Kamei, Y., and Fukamachi, S. (2021). Behavioural red• Matsuo,
light sensitivity in fish according to the optomotor response. R. Soc.

••
•

92

Open Sci. 8, 210415. DOI: 10.1098/rsos.210415
Ogino, H., Kamei, Y., Hayashi, T., Sakamoto, J., Suzuki, M., and Igawa,T. (2021). Invention sharing is the mother of developmental biology
PREFACE. Dev. Growth Differ. 63, 395–396. DOI: 10.1111/dgd.12755
Tokanai, K., Kamei, Y., and Minokawa, T. (2021). An easy andrapid staining method for confocal microscopic observation andreconstruction of three-dimensional images of echinoderm larvae andjuveniles. Dev. Growth Differ. 63, 478–487. DOI: 10.1111/dgd.12758
Wu, D., Arakawa, H., Fujita, A., Hashimoto, H., Hibi, M., Naruse, K.,
Kamei, Y., Sato, C., and Kitajima, K. (2021). A point-mutation in theC-domain of CMP-sialic acid synthetase leads to lethality of medaka
due to protein insolubility. Sci. Rep. 11, 23211. DOI: 10.1038/s41598021-01715-3