ACS sensors · 2026
A Sensitive Green-Red FRET Sensor for Calpain-1 Utilizing Heterodimeric Fluorescent Proteins from Distinct Evolutionary Origins.
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Yang Fangfang, Deng Mengying, Pan Xiaorong, Wang Jintao, Huang Pei, Ou Zirui, Zou Xiaoyu, Muhammad Fahim, Gan Shiyi, Yang Qixing, Shi Cuihan, Chen Jingya, Li Junfeng, Zhao Yongjuan, Ren Pei-Gen, Liu Xinan, Du Yang, Wang Tong, Chen Zuxin, Chu Jun
Аннотация
Calpain-1, a calcium-dependent cysteine protease, plays a pivotal role in various biological functions. However, real-time monitoring of its activity has been limited by the lack of high-performance calpain sensors. Here, we report the development of iFoCAL, a sensitive and specific fluorescence resonance energy transfer (FRET) sensor for calpain-1, which employs mClover3 and mScarletX, a newly engineered green-red FRET pair with weak interactions. Using iFoCAL, we revealed distinct spatiotemporal patterns of calpain-1 activation: gradual and global activation in whole cells during endoplasmic reticulum (ER) calcium release, fast and focal activation in axons during mechanical stress, and region-specific activation in a Parkinson's disease model. These findings provide the first direct evidence of distinct calpain-1 dynamics across diverse cellular contexts.