Extremely Sensitive Genetically Encoded Temperature Indicator Enabling Measurement at the Organelle Level

Fukushima, Shun-Ichi; Wazawa, Tetsuichi; Sugiura, Kazunori; Nagai, Takeharu

doi:https://doi.org/10.1021/acssensors.3c02658

Extremely Sensitive Genetically Encoded Temperature Indicator Enabling Measurement at the Organelle Level

Fukushima, Shun-Ichi; Wazawa, Tetsuichi; Sugiura, Kazunori et al.

ACS Sensors, 2024, 9(8), 3889-3897

アクセス数:474件（2025-08-23 12:09 集計）

固定URL: https://hdl.handle.net/11094/98343

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タイトル	Extremely Sensitive Genetically Encoded Temperature Indicator Enabling Measurement at the Organelle Level
著者	NRID 1000000793720 1000000793720 researchmap permalink aggregans aggregans 著者名 Fukushima, Shun-Ichi
	NRID 1000080359851 1000080359851 researchmap permalink wazawa wazawa 著者名 Wazawa, Tetsuichi
	著者名 Sugiura, Kazunori
	NRID 1000020311350 1000020311350 ORCID 0000-0003-2650-9895 0000-0003-2650-9895 researchmap permalink ng1 ng1 Scopus Author ID 7403433377 7403433377 著者名 Nagai, Takeharu
キーワード等	biosensor
	fluorescence nanothermometry (FNT)
	fluorescent protein
	genetically encoded fluorescent temperature indicator (GETI)
	protein engineering
内容	Fukushima S.I., Wazawa T., Sugiura K., et al. Extremely Sensitive Genetically Encoded Temperature Indicator Enabling Measurement at the Organelle Level. ACS Sensors 9, 3889 (2024); https://doi.org/10.1021/acssensors.3c02658.
抄録	Intracellular temperature is a fundamental parameter in biochemical reactions. Genetically encoded fluorescent temperature indicators (GETIs) have been developed to visualize intracellular thermogenesis; however, the temperature sensitivity or localization capability in specific organelles should have been further improved to clearly capture when and where intracellular temperature changes at the subcellular level occur. Here, we developed a new GETI, gMELT, composed of donor and acceptor subunits, in which cyan and yellow fluorescent proteins, respectively, as a Förster resonance energy transfer (FRET) pair were fused with temperature-sensitive domains. The donor and acceptor subunits associated and dissociated in response to temperature changes, altering the FRET efficiency. Consequently, gMELT functioned as a fluorescence ratiometric indicator. Untagged gMELT was expressed in the cytoplasm, whereas versions fused with specific localization signals were targeted to the endoplasmic reticulum (ER) or mitochondria. All gMELT variations enabled more sensitive temperature measurements in cellular compartments than those in previous GETIs. The gMELTs, tagged with ER or mitochondrial targeting sequences, were used to detect thermogenesis in organelles stimulated chemically, a method previously known to induce thermogenesis. The observed temperature changes were comparable to previous reports, assuming that the fluorescence readout changes were exclusively due to temperature variations. Furthermore, we demonstrated how macromolecular crowding influences gMELT fluorescence given that this factor can subtly affect the fluorescence readout. Investigating thermogenesis with gMELT, accounting for factors such as macromolecular crowding, will enhance our understanding of intracellular thermogenesis phenomena.
出版者	American Chemical Society
収録物名	ACS Sensors
巻(号)	9(8)
ページ	3889-3897
刊行年月	2024-07-23
言語	英語
ハンドルURL	https://hdl.handle.net/11094/98343
eISSN	23793694
関連情報 (isIdenticalTo)	DOI (出版社版) https://doi.org/10.1021/acssensors.3c02658
アクセス権	open access
権利情報	This article is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License.
出版タイプ	Version of Record
カテゴリ	学術雑誌論文 Journal Article

資源タイプ	学術雑誌論文
ローカル資源タイプ	学術雑誌論文
DCTERMS.bibliographicCitation	ACS Sensors. 2024, 9(8), p. 3889-3897
DC.title	Extremely Sensitive Genetically Encoded Temperature Indicator Enabling Measurement at the Organelle Level
DC.creator	Fukushima, Shun-Ichi
	Wazawa, Tetsuichi
	Sugiura, Kazunori
	Nagai, Takeharu
DC.publisher	American Chemical Society
DC.language' scheme='DCTERMS.RFC1766	英語
DCTERMS.issued' scheme='DCTERMS.W3CDTF	2024-07-23
DC.identifier	https://doi.org/10.1021/acssensors.3c02658
DC.subject	biosensor
	fluorescence nanothermometry (FNT)
	fluorescent protein
	genetically encoded fluorescent temperature indicator (GETI)
	protein engineering
DC.description	Fukushima S.I., Wazawa T., Sugiura K., et al. Extremely Sensitive Genetically Encoded Temperature Indicator Enabling Measurement at the Organelle Level. ACS Sensors 9, 3889 (2024); https://doi.org/10.1021/acssensors.3c02658.
DCTERMS.abstract	Intracellular temperature is a fundamental parameter in biochemical reactions. Genetically encoded fluorescent temperature indicators (GETIs) have been developed to visualize intracellular thermogenesis; however, the temperature sensitivity or localization capability in specific organelles should have been further improved to clearly capture when and where intracellular temperature changes at the subcellular level occur. Here, we developed a new GETI, gMELT, composed of donor and acceptor subunits, in which cyan and yellow fluorescent proteins, respectively, as a Förster resonance energy transfer (FRET) pair were fused with temperature-sensitive domains. The donor and acceptor subunits associated and dissociated in response to temperature changes, altering the FRET efficiency. Consequently, gMELT functioned as a fluorescence ratiometric indicator. Untagged gMELT was expressed in the cytoplasm, whereas versions fused with specific localization signals were targeted to the endoplasmic reticulum (ER) or mitochondria. All gMELT variations enabled more sensitive temperature measurements in cellular compartments than those in previous GETIs. The gMELTs, tagged with ER or mitochondrial targeting sequences, were used to detect thermogenesis in organelles stimulated chemically, a method previously known to induce thermogenesis. The observed temperature changes were comparable to previous reports, assuming that the fluorescence readout changes were exclusively due to temperature variations. Furthermore, we demonstrated how macromolecular crowding influences gMELT fluorescence given that this factor can subtly affect the fluorescence readout. Investigating thermogenesis with gMELT, accounting for factors such as macromolecular crowding, will enhance our understanding of intracellular thermogenesis phenomena.
DC.rights	This article is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License.
DC.format	98343
citation_title	Extremely Sensitive Genetically Encoded Temperature Indicator Enabling Measurement at the Organelle Level
citation_author	Fukushima, Shun-Ichi
	Wazawa, Tetsuichi
	Sugiura, Kazunori
	Nagai, Takeharu
citation_publisher	American Chemical Society
citation_language	英語
citation_date	2024-07-23
citation_journal_title	ACS Sensors
citation_volume	9
citation_issue	8
citation_firstpage	3889
citation_lastpage	3897
citation_doi	https://doi.org/10.1021/acssensors.3c02658
citation_public_url	https://hdl.handle.net/11094/98343

Extremely Sensitive Genetically Encoded Temperature Indicator Enabling Measurement at the Organelle Level

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