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Monitoring Dynamic Changes In Mitochondrial Calcium Levels During Apoptosis Using A Genetically Encoded Calcium Sensor
Published on: April 1, 2011
Promyelocytic leukemia protein (PML) knockout increases mitochondrial Ca2+ uptake in HeLa cells
R R Sharipov1, A M Surin1, S A Silonov2
1Institute of General Pathology and Pathophysiology, Moscow, Baltiyskaya St., 8, 125315, Russia.
Abstract:
The multifunctional promyelocytic leukemia protein (PML) is involved in the regulation of various cellular processes in both physiological and pathological conditions. Specifically, PML is one of the inositol-1,4,5-trisphosphate receptors (IP3Rs) activity regulators and can influence Ca2+ transport from the endoplasmic reticulum (ER) to mitochondria. In this work, the effects of PML knockout on calcium homeostasis in the cytosol, ER, and mitochondria of HeLa cells were studied upon stimulation with histamine, which induces Ca2+ mobilization from the ER via IP3Rs. We utilized calcium indicators with different subcellular localizations, including synthetic dyes Fura-2 (cytosolic), Xrhod-5F (mitochondrial), and protein sensor R-CEPIAer (ER), as well as mitochondrial potential-sensitive probes Rh123 and TMRM. Our results show that PML knockout induced changes in HeLa cell and mitochondrial morphology, slightly decreased basal and integral Ca2+ levels, enhanced mitochondrial Ca2+ uptake from the cytoplasm, and maintained residual mitochondrial potential after depolarization. Additionally, it reduced the Ca2+ pool in ER membranes not associated with histamine receptor activation and, consequently, IP3Rs. These findings suggest that changes in calcium ion transport due to PML knockout in HeLa cells affect mitochondrial activity.
Insights
Promyelocytic leukemia protein (PML) knockout alters calcium homeostasis in HeLa cells, impacting mitochondrial activity and calcium uptake. This research reveals PML
Area of Science:
- Cell Biology
- Molecular Biology
- Biochemistry
Background:
- The promyelocytic leukemia protein (PML) is a multifunctional protein regulating cellular processes.
- PML influences calcium (Ca2+) transport between the endoplasmic reticulum (ER) and mitochondria by modulating inositol-1,4,5-trisphosphate receptors (IP3Rs).
- Disruptions in calcium homeostasis are implicated in various cellular dysfunctions.
Purpose of the Study:
- To investigate the impact of PML knockout on cellular calcium homeostasis.
- To analyze the effects of PML deficiency on Ca2+ transport and mitochondrial activity in HeLa cells.
- To understand PML's role in regulating ER Ca2+ stores and IP3R-mediated signaling.
Main Methods:
- Utilized PML knockout HeLa cell models.
- Stimulated Ca2+ mobilization using histamine.
- Employed subcellularly localized calcium indicators (Fura-2, Xrhod-5F, R-CEPIAer) and mitochondrial probes (Rh123, TMRM) to measure Ca2+ levels and mitochondrial potential.
Main Results:
- PML knockout altered HeLa cell and mitochondrial morphology.
- Observed decreased basal and integral Ca2+ levels, enhanced mitochondrial Ca2+ uptake, and preserved mitochondrial potential post-depolarization.
- Demonstrated reduced ER Ca2+ pools independent of histamine receptor activation and IP3Rs.
Conclusions:
- PML plays a critical role in maintaining cellular calcium homeostasis.
- PML deficiency significantly affects Ca2+ transport dynamics, particularly influencing mitochondrial Ca2+ handling.
- These findings highlight the intricate link between PML, calcium signaling, and mitochondrial function.
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