A genetically encoded fluorescent protein sensor for mitochondrial membrane damage detection
Copyright © 2024 Elsevier Inc. All rights reserved..
Mitochondria are essential cellular organelles; detecting mitochondrial damage is crucial in cellular biology and toxicology. Compared with existing chemical probe detection methods, genetically encoded fluorescent protein sensors can directly indicate cellular and molecular events without involving exogenous reagents. In this study, we introduced a molecular sensor system, MMD-Sensor, for monitoring mitochondrial membrane damage. The sensor consists of two molecular modules. Module I is a fusion structure of the mitochondrial localization sequence (MLS), AIF cleavage site sequence (CSS), nuclear localization sequence (NLS), N-terminus of mNeonGreen and mCherry. Module II is a fusion structure of the C-terminus of mNeonGreen, NLS sequence, and mtagBFP2. Under normal condition, Module I is constrained in the inner mitochondrial membrane anchored by MLS, while Module II is restricted to the nucleus by its NLS fusion component. If the mitochondrial membrane is damaged, CSS is cut from the inner membrane, causing Module I to shift into the nucleus guided by the NLS fusion component. After Module I enters the nucleus, the N- and C-terminus of mNeonGreen meet each other and rebuild its intact 3D structure through fragment complementation and thus generates green fluorescence in the nucleus. Dynamic migration of red fluorescence from mitochondria to the nucleus and generation of green fluorescence in the nucleus indicate mitochondrial membrane damage. Using the MMD-Sensor, mitochondrial membrane damage induced by various reagents, such as uncoupling agents, ATP synthase inhibitors, monovalent cationic carriers, and ROS, in HeLa and 293T cells are directly observed and evaluated.
| Medienart: |
E-Artikel |
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| Erscheinungsjahr: |
2024 |
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| Erschienen: |
2024 |
| Enthalten in: |
Zur Gesamtaufnahme - volume:709 |
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| Enthalten in: |
Biochemical and biophysical research communications - 709(2024) vom: 21. Apr., Seite 149836 |
| Sprache: |
Englisch |
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| Beteiligte Personen: |
Liu, Qian [VerfasserIn] |
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| Links: |
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| Anmerkungen: |
Date Completed 15.04.2024 Date Revised 15.04.2024 published: Print-Electronic Citation Status MEDLINE |
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| doi: |
10.1016/j.bbrc.2024.149836 |
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| funding: |
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| Förderinstitution / Projekttitel: |
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| 520 | |a Mitochondria are essential cellular organelles; detecting mitochondrial damage is crucial in cellular biology and toxicology. Compared with existing chemical probe detection methods, genetically encoded fluorescent protein sensors can directly indicate cellular and molecular events without involving exogenous reagents. In this study, we introduced a molecular sensor system, MMD-Sensor, for monitoring mitochondrial membrane damage. The sensor consists of two molecular modules. Module I is a fusion structure of the mitochondrial localization sequence (MLS), AIF cleavage site sequence (CSS), nuclear localization sequence (NLS), N-terminus of mNeonGreen and mCherry. Module II is a fusion structure of the C-terminus of mNeonGreen, NLS sequence, and mtagBFP2. Under normal condition, Module I is constrained in the inner mitochondrial membrane anchored by MLS, while Module II is restricted to the nucleus by its NLS fusion component. If the mitochondrial membrane is damaged, CSS is cut from the inner membrane, causing Module I to shift into the nucleus guided by the NLS fusion component. After Module I enters the nucleus, the N- and C-terminus of mNeonGreen meet each other and rebuild its intact 3D structure through fragment complementation and thus generates green fluorescence in the nucleus. Dynamic migration of red fluorescence from mitochondria to the nucleus and generation of green fluorescence in the nucleus indicate mitochondrial membrane damage. Using the MMD-Sensor, mitochondrial membrane damage induced by various reagents, such as uncoupling agents, ATP synthase inhibitors, monovalent cationic carriers, and ROS, in HeLa and 293T cells are directly observed and evaluated | ||
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| 700 | 1 | |a Zhang, Xian-En |e verfasserin |4 aut | |
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