高分子拥挤
费斯特共振能量转移
生物
细胞器
拥挤
细胞质
高分子
生物物理学
细胞生物学
生物化学
荧光
神经科学
物理
量子力学
作者
Tamami Miyagi,Yoshiaki Yamanaka,Yuichiro Harada,Satoshi Narumi,Yuhei Hayamizu,Masahiko Kuroda,Kohsuke Kanekura
标识
DOI:10.1016/j.bbrc.2021.10.055
摘要
Membrane-less organelles (MLOs) formed by liquid-liquid phase separation (LLPS) play pivotal roles in biological processes. During LLPS, proteins and nucleotides are extremely condensed, resulting in changes in their conformation and biological functions. Disturbed LLPS homeostasis in MLOs is thought to associate with fatal diseases such as amyotrophic lateral sclerosis. Therefore, it is important to detect changes in the degree of crowding in MLOs. However, it has not been investigated well due to the lack of an appropriate method. To address this, we developed a genetically encoded macromolecular crowding sensor CRONOS (crowding sensor with mNeonGreen and mScarlet-I) that senses the degree of macromolecular crowding in MLOs using a fluorescence resonance energy transfer (FRET) system. CRONOS is a bright biosensor with a wide dynamic range and successfully detects changes in the macromolecular volume fraction in solution. By fusing to the scaffold protein of each MLO, we delivered CRONOS to MLO of interest and detected previously undescribed differences in the degree of crowding in each MLO. CRONOS also detected changes in the degree of macromolecular crowding in nucleolus induced by environmental stress or inhibition of transcription. These findings suggest that CRONOS can be a useful tool for the determination of molecular crowding and detection of pathological changes in MLOs in live cells.
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