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Updated: Jun 16, 2026
Simultaneous Measurement of Mitochondrial Calcium and Mitochondrial Membrane Potential in Live Cells by Fluorescent Microscopy
Published on: January 24, 2017
Nano-flow cytometry unveils mitochondrial permeability transition process and multi-pathway cell death induction for
Liyun Su1, Jingyi Xu1, Cheng Lu1
1Department of Chemical Biology, MOE Key Laboratory of Spectrochemical Analysis & Instrumentation, Key Laboratory for Chemical Biology of Fujian Province, State Key Laboratory of Physical Chemistry of Solid Surfaces, Collaborative Innovation Center of Chemistry for Energy Materials, College of Chemistry and Chemical Engineering, Xiamen University, Xiamen, Fujian, People's Republic of China.
Abstract:
Mitochondrial permeability transition (mPT)-mediated mitochondrial dysfunction plays a pivotal role in various human diseases. However, the intricate details of its mechanisms and the sequence of events remain elusive, primarily due to the interference caused by Bax/Bak-induced mitochondrial outer membrane permeabilization (MOMP). To address these, we have developed a methodology that utilizes nano-flow cytometry (nFCM) to quantitatively analyze the opening of mitochondrial permeability transition pore (mPTP), dissipation of mitochondrial membrane potential ( Ψm), release of cytochrome c (Cyt c), and other molecular alternations of isolated mitochondria in response to mPT induction at the single-mitochondrion level. It was identified that betulinic acid (BetA) and antimycin A can directly induce mitochondrial dysfunction through mPT-mediated mechanisms, while cisplatin and staurosporine cannot. In addition, the nFCM analysis also revealed that BetA primarily induces mPTP opening through a reduction in Bcl-2 and Bcl-xL protein levels, along with an elevation in ROS content. Employing dose and time-dependent strategies of BetA, for the first time, we experimentally verified the sequential occurrence of mPTP opening and Ψm depolarization prior to the release of Cyt c during mPT-mediated mitochondrial dysfunction. Notably, our study uncovers a simultaneous release of cell-death-associated factors, including Cyt c, AIF, PNPT1, and mtDNA during mPT, implying the initiation of multiple cell death pathways. Intriguingly, BetA induces caspase-independent cell death, even in the absence of Bax/Bak, thereby overcoming drug resistance. The presented findings offer new insights into mPT-mediated mitochondrial dysfunction using nFCM, emphasizing the potential for targeting such dysfunction in innovative cancer therapies and interventions.
Insights
This study reveals how mitochondrial dysfunction occurs, showing betulinic acid triggers cell death pathways. Nano-flow cytometry precisely tracks mitochondrial permeability transition pore opening and subsequent events, offering new therapeutic targets.
Area of Science:
- Cell Biology
- Biochemistry
- Pathology
Background:
- Mitochondrial dysfunction, driven by mitochondrial permeability transition (mPT), is implicated in numerous diseases.
- Understanding the precise mechanisms of mPT and its sequence of events is challenging due to Bax/Bak-induced mitochondrial outer membrane permeabilization (MOMP).
Purpose of the Study:
- To develop and apply a novel nano-flow cytometry (nFCM) methodology for quantitative, single-mitochondrion analysis of mPT.
- To elucidate the sequence of molecular events during mPT-mediated mitochondrial dysfunction and identify specific inducers.
- To explore the therapeutic potential of targeting mPT in disease, particularly cancer.
Main Methods:
- Development of a nano-flow cytometry (nFCM) method for analyzing isolated mitochondria.
- Quantification of mitochondrial permeability transition pore (mPTP) opening, mitochondrial membrane potential ( Ψm) dissipation, and cytochrome c (Cyt c) release at the single-mitochondrion level.
- Treatment of isolated mitochondria with betulinic acid (BetA), antimycin A, cisplatin, and staurosporine to assess mPT induction.
Main Results:
- Betulinic acid (BetA) and antimycin A directly induce mitochondrial dysfunction via mPT, unlike cisplatin and staurosporine.
- nFCM revealed BetA induces mPTP opening by reducing Bcl-2/Bcl-xL and increasing ROS.
- The study experimentally verified that mPTP opening and Ψm depolarization precede Cyt c release during mPT.
- Simultaneous release of Cyt c, AIF, PNPT1, and mtDNA was observed, indicating multiple cell death pathways.
- BetA induced caspase-independent cell death, even without Bax/Bak, suggesting a mechanism to overcome drug resistance.
Conclusions:
- nFCM provides a powerful tool to dissect mPT-mediated mitochondrial dysfunction at the single-mitochondrion level.
- BetA is a direct inducer of mPT, initiating a cascade of events leading to cell death.
- The findings highlight the potential of targeting mPT for novel cancer therapies, especially in overcoming drug resistance.
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