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Imaging the mitochondrial permeability transition pore in intact cells
V Petronilli1, G Miotto, M Canton
1Centro per lo Studio delle Biomembrane, CNR, Università di Padova, Italy.
Abstract:
The involvement of mitochondrial permeability transition pore (MTP) in cellular processes is generally investigated by indirect means, such as changes in mitochondrial membrane potential or pharmacological inhibition. However, such effects could not be related univocally to MTP. In addition, source of errors could be represented by the increased retention of membrane potential probes induced by cyclosporin A (CsA) and the interactions between fluorescent probes. We developed a direct technique for monitoring MTP. Cells were co-loaded with calcein-AM and CoCl2, resulting in the quenching of the cytosolic signal without affecting the mitochondrial fluorescence. MTP inducers caused a rapid decrease in mitochondrial calcein fluorescence which, however, was not completely prevented by CsA. Besides the large and rapid efflux of calcein induced by MTP agonists, we also observed a constant and spontaneous decrease of mitochondrial calcein which was completely prevented by CsA. Thus, MTP likely fluctuates between open and closed states in intact cells.
Insights
Researchers developed a direct method to monitor the mitochondrial permeability transition pore (MTP). This new technique reveals that MTP likely fluctuates between open and closed states in living cells.
Area of Science:
- Cellular Biology
- Mitochondrial Research
- Biophysics
Background:
- Investigating the mitochondrial permeability transition pore (MTP) typically relies on indirect methods.
- Indirect methods like membrane potential changes or drug inhibition have limitations and potential errors.
- Previous techniques faced challenges with probe retention and interactions, hindering accurate MTP assessment.
Purpose of the Study:
- To develop a direct and reliable technique for monitoring MTP.
- To overcome the limitations of indirect MTP assessment methods.
- To investigate the dynamic states of MTP in intact cells.
Main Methods:
- Developed a novel direct technique for MTP monitoring.
- Co-loaded cells with calcein-AM and CoCl2 to isolate mitochondrial fluorescence.
- Utilized MTP inducers and cyclosporin A (CsA) to analyze calcein fluorescence changes.
Main Results:
- The new method allowed direct observation of MTP activity.
- MTP inducers caused rapid calcein efflux, partially inhibited by CsA.
- A spontaneous, CsA-sensitive decrease in calcein fluorescence was observed, indicating basal MTP activity.
Conclusions:
- The developed technique provides a direct means to study MTP.
- MTP appears to exist in dynamic, fluctuating open and closed states within intact cells.
- This finding challenges previous assumptions and offers new insights into MTP regulation.