A Sensitive Green-Red FRET Sensor for Calpain-1 Utilizing Heterodimeric Fluorescent Proteins from Distinct Evolutionary Origins
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.
Authors
- Yang Du (ORCID: https://orcid.org/0000-0001-8107-6397)
- Chen Shi (ORCID: https://orcid.org/0000-0002-7045-330X)
- Jun Chu (ORCID: https://orcid.org/0000-0002-7627-3430)
- Jian Li (ORCID: https://orcid.org/0000-0001-6509-4595)
- Xiaoyu Zou
- Mengying Deng
- Shiyi Gan
- Zirui Ou
- Jin Wang (ORCID: https://orcid.org/0000-0002-9881-7208)
- Pei Huang (ORCID: https://orcid.org/0000-0003-4171-669X)
- Zhiqi Chen (ORCID: https://orcid.org/0000-0002-5618-4182)
- Xinan Liu (ORCID: https://orcid.org/0000-0002-0497-6962)
- Pei-Gen Ren
- Yongjuan Zhao
- Fangfang Yang
- Qixing Yang
- Xiaorong Pan
- Fahim Muhammad
- Jingya Chen
- Tong Wang
Institutions
- Allen Institute for Brain Science (US)
- Chinese University of Hong Kong (HK)
- Shenzhen University (CN)
- Southern University of Science and Technology (CN)
- ShanghaiTech University (CN)
- HKUST Shenzhen Research Institute (CN)
- Key Laboratory of Guangdong Province (CN)
- Chinese University of Hong Kong, Shenzhen (CN)
- Shenzhen Institute of Neuroscience (CN)
- Laboratoire d’Imagerie Biomédicale (FR)
- Institute for Biomedicine (IT)
- Drug Discovery Laboratory (Norway) (NO)
- Cognitive Neuroimaging Lab (FR)
- Shenzhen Institutes of Advanced Technology (CN)
- International Neuromodulation Society (US)
- Life & Brain (Germany) (DE)
- Indiana University School of Medicine
Publication Details
- Journal
- ACS Sensors
- Published
- 2026-03-16
- DOI
- https://doi.org/10.1021/acssensors.5c02811
- Primary Topic
- Calpain Protease Function and Regulation
- Type
- article
- Field-Weighted Citation Impact
- 0.00