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Updated: Aug 8, 2026
Monitoring Dynamic Changes In Mitochondrial Calcium Levels During Apoptosis Using A Genetically Encoded Calcium Sensor
Published on: April 1, 2011
Confocal microscopy of the mitochondrial permeability transition in necrotic and apoptotic cell death
J J Lemasters1, T Qian, L C Trost
1Department of Cell Biology and Anatomy, University of North Carolina at Chapel Hill 27599-7090, USA.
Abstract:
Opening of a high-conductance pore in the mitochondrial inner membrane induces onset of the mitochondrial permeability transition (mPT). Cyclosporin A and trifluoperazine inhibit this pore and block necrotic cell death in oxidative stress, Ca2+ ionophore toxicity, Reye-related drug toxicity, pH-dependent ischaemia/reperfusion injury and other models of cell injury. Confocal fluorescence microscopy directly visualizes the increased mitochondrial membrane permeability of the mPT from the movement of calcein from the cytosol into the matrix space. Pyridine nucleotide oxidation, increased mitochondrial Ca2+ and mitochondrial generation of reactive oxygen species (ROS) all contribute to the onset of the mPT in situ. Confocal microscopy also shows directly that the mPT is a critical link in apoptotic signalling by tumour necrosis factor-alpha at a point downstream of caspase 8 and upstream of caspase 3. Cyclosporin A blocks this mPT, preventing release of pro-apoptotic cytochrome c from mitochondria and subsequent apoptotic cell killing. Progression to necrosis or apoptosis after the mPT depends on the availability of ATP, which blocks necrosis but promotes the apoptotic programme. Given the pathophysiological importance of the mPT, development of agents to modulate the mPT represents an important new goal for pharmaceutical drug discovery.
Insights
The mitochondrial permeability transition (mPT) pore opening causes cell death. Inhibiting mPT pore formation can block both necrosis and apoptosis, offering a new target for drug discovery.
Area of Science:
- Cell Biology
- Biochemistry
- Pharmacology
Background:
- The mitochondrial permeability transition (mPT) is a critical event in cell death pathways.
- Opening of a high-conductance pore in the mitochondrial inner membrane triggers mPT.
- mPT is implicated in various forms of cell injury, including necrosis and apoptosis.
Purpose of the Study:
- To investigate the role of mPT in cell death.
- To visualize mPT using confocal fluorescence microscopy.
- To explore the potential of modulating mPT for therapeutic interventions.
Main Methods:
- Confocal fluorescence microscopy was used to visualize mitochondrial membrane permeability.
- Studies were conducted in various models of cell injury, including oxidative stress and ionophore toxicity.
- The effects of Cyclosporin A and trifluoperazine on mPT and cell death were assessed.
Main Results:
- mPT was directly visualized by tracking calcein movement into the mitochondrial matrix.
- Pyridine nucleotide oxidation, increased mitochondrial Ca2+, and reactive oxygen species (ROS) contribute to mPT onset.
- mPT was identified as a key link in TNF-alpha-induced apoptosis, downstream of caspase 8 and upstream of caspase 3.
- Cyclosporin A inhibited mPT, preventing cytochrome c release and subsequent apoptosis.
Conclusions:
- mPT is a central event in both necrotic and apoptotic cell death.
- The outcome of mPT (necrosis vs. apoptosis) depends on cellular ATP levels.
- Modulating mPT represents a promising strategy for pharmaceutical drug discovery.
