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Measuring initiator caspase activation by bimolecular fluorescence complementation.

Melissa J Parsons1, Lisa Bouchier-Hayes1

  • 1Center for Cell and Gene Therapy, Baylor College of Medicine, Houston, Texas 77030 Department of Pediatrics-Hematology, Baylor College of Medicine, Houston, Texas 77030.

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Summary

Initiator caspases like caspase-2 become active through dimerization. Caspase bimolecular fluorescence complementation (BiFC) visualizes this induced proximity, enabling the study of caspase activation in cells.

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Area of Science:

  • Cell Biology
  • Molecular Biology
  • Biochemistry

Background:

  • Initiator caspases (caspase-2, -8, -9) activation relies on proximity-induced dimerization at activation platforms.
  • Measuring this induced proximity is crucial for understanding caspase activation pathways.

Purpose of the Study:

  • To introduce and validate caspase bimolecular fluorescence complementation (caspase BiFC) as a method to measure induced proximity of initiator caspases.
  • To demonstrate the application of caspase BiFC for visualizing caspase-2 proximity and activation.

Main Methods:

  • Utilized a split fluorescent protein (Venus) fused to caspase-2 fragments (amino- and carboxy-terminal halves).
  • Transfected cells with plasmids encoding these fusion proteins.
  • Applied a death stimulus to induce caspase-2 proximity and dimerization.
  • Visualized Venus fluorescence using fluorescence microscopy and time-lapse confocal microscopy.

Main Results:

  • Successfully visualized induced proximity of caspase-2 in cells treated with a death stimulus via Venus fluorescence.
  • Quantified Venus-positive cells at single time points and evaluated fluorescence intensity over time.
  • Demonstrated the potential of the caspase BiFC strategy for other initiator caspases (caspase-8, -9) with appropriate controls.

Conclusions:

  • Caspase BiFC is an effective method for measuring induced proximity and subsequent activation of initiator caspases, specifically demonstrated for caspase-2.
  • This technique provides a visual and quantifiable approach to study caspase activation dynamics in living cells.
  • The caspase BiFC strategy offers a versatile tool for investigating apoptotic pathways involving initiator caspases.