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Related Experiment Video

Updated: Mar 18, 2026

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

Fangfang Yang1, Mengying Deng1, Xiaorong Pan2

  • 1Research Center for Primate Neuromodulation and Neuroimaging, Shenzhen Institutes of Advanced Technology (SIAT), Chinese Academy of Sciences (CAS), Shenzhen 518055, China.

ACS Sensors
|March 16, 2026
PubMed
Summary

Researchers developed iFoCAL, a novel fluorescence resonance energy transfer (FRET) sensor, to monitor calpain-1 activity. This tool reveals distinct calpain-1 activation patterns in various cellular contexts, advancing our understanding of protease dynamics.

Keywords:
FRETParkinson’s diseaseaxonal injurycalpain-1fluorescent protein

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Area of Science:

  • Biochemistry
  • Cell Biology
  • Molecular Imaging

Background:

  • Calpain-1 is a calcium-dependent cysteine protease crucial for numerous cellular processes.
  • Limited real-time monitoring tools hinder the study of calpain-1 activity.
  • Existing calpain sensors lack the high performance needed for detailed spatiotemporal analysis.

Purpose of the Study:

  • To develop a sensitive and specific fluorescence resonance energy transfer (FRET) sensor for real-time calpain-1 activity monitoring.
  • To characterize the spatiotemporal dynamics of calpain-1 activation in different cellular environments.
  • To investigate calpain-1 involvement in cellular processes like ER calcium release, mechanical stress, and neurodegenerative disease models.

Main Methods:

  • Development of iFoCAL, a novel FRET sensor utilizing the mClover3 and mScarletX green-red FRET pair.
  • Application of iFoCAL to visualize calpain-1 activation in live cells under various stimuli.
  • Comparative analysis of calpain-1 activation patterns during endoplasmic reticulum calcium release, axonal mechanical stress, and in a Parkinson's disease model.

Main Results:

  • iFoCAL demonstrated high sensitivity and specificity for detecting calpain-1 activity.
  • Distinct spatiotemporal patterns of calpain-1 activation were observed: gradual global activation during ER calcium release.
  • Fast focal activation in axons under mechanical stress and region-specific activation in a Parkinson's disease model were revealed.

Conclusions:

  • The iFoCAL sensor provides unprecedented real-time insights into calpain-1 dynamics.
  • This study presents the first direct evidence of diverse calpain-1 activation patterns across different cellular contexts.
  • These findings enhance our understanding of calpain-1's role in normal physiology and disease pathogenesis.