Promyelocytic leukemia protein (PML) knockout increases mitochondrial Ca2+ uptake in HeLa cells

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.

Keywords: Endoplasmic reticulum; Fluorescence microscopy; Intracellular calcium; Mitochondria; Mitochondria-associated membranes (MAMs).