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  3. A Sensitive Green-Red FRET Sensor for Calpain-1 Utilizing Heterodimeric Fluorescent Proteins from Distinct Evolutionary Origins

A Sensitive Green-Red FRET Sensor for Calpain-1 Utilizing Heterodimeric Fluorescent Proteins from Distinct Evolutionary Origins

  • ACS Sens. 2026 Mar 27;11(3):1891-1907. doi: 10.1021/acssensors.5c02811.
Fangfang Yang 1,  Mengying Deng 1,  Xiaorong Pan 2,  Jintao Wang 3,  Pei Huang 4,  Zirui Ou 5,  Xiaoyu Zou 5,  Fahim Muhammad 3,  Shiyi Gan 6,  Qixing Yang 3,  Cuihan Shi 3,  Jingya Chen 1,  Junfeng Li 7,  Yongjuan Zhao 8,  Pei-Gen Ren 7,  Xinan Liu 9,  Yang Du 6,  Tong Wang 2,  Zuxin Chen 3,  Jun Chu 1 10 11
Affiliations

Affiliations

  • 1 Research Center for Primate Neuromodulation and Neuroimaging, Shenzhen Institutes of Advanced Technology (SIAT), Chinese Academy of Sciences (CAS), Shenzhen 518055, China.
  • 2 The Brain Center, School of Life Science and Technology, Shanghai Tech University, Shanghai 201210, China.
  • 3 Shenzhen Key Laboratory of Drug Addiction, Shenzhen Neher Neural Plasticity Laboratory, the Brain Cognition and Brain Disease Institute, SIAT-CAS; Shenzhen-Hong Kong Institute of Brain Science-Shenzhen Fundamental Research Institutions, Shenzhen 518055, China.
  • 4 School of Life Sciences, Southern University of Science and Technology, Shenzhen 518055, China.
  • 5 College of Life Sciences and Oceanography, Shenzhen University, Shenzhen 518055, China.
  • 6 Kobilka Institute of Innovative Drug Discovery, Shenzhen Key Laboratory of Steroid Drug Discovery and Development, School of Medicine, The Chinese University of Hong Kong, Shenzhen 518172, China.
  • 7 Institute of Biomedicine and Biotechnology, SIAT-CAS, Shenzhen 518055, China.
  • 8 School of Medicine, The Chinese University of Hong Kong Shenzhen, Shenzhen 518172, China.
  • 9 Guangdong Provincial Key Laboratory of Brain Connectome and Behavior, CAS Key Laboratory of Brain Connectome and Manipulation, Brain Cognition and Brain Disease Institute (BCBDI), SIAT-CAS, Shenzhen-Hong Kong Institute of Brain Science-Shenzhen Fundamental Research Institutions, Shenzhen 518055, China.
  • 10 Shenzhen Key Laboratory for Molecular Imaging, Guangdong Provincial Key Laboratory of Biomedical Optical Imaging Technology, SIAT-CAS, Shenzhen 518055, China.
  • 11 Key Laboratory of Biomedical Imaging, SIAT-CAS, Shenzhen 518055, China.
PMID: 41837894 DOI: 10.1021/acssensors.5c02811
Abstract

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.

Keywords

FRET; Parkinson’s disease; axonal injury; calpain-1; fluorescent protein.

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