Dynamic insights into mitochondrial function: Monitoring viscosity and SO₂ levels in living cells

Mitochondria, central organelles pivotal for eukaryotic cell function, extend their influence beyond ATP production, encompassing roles in apoptosis, calcium signaling, and biosynthesis. Recent studies spotlight two emerging determinants of mitochondrial functionality: intramitochondrial viscosity and sulfur dioxide (SO₂) levels. While optimal mitochondrial viscosity governs molecular diffusion and vital processes like oxidative phosphorylation, aberrations are linked with neurodegenerative conditions, diabetes, and cancer. Similarly, SO₂, a gaseous signaling molecule, modulates energy pathways and oxidative stress responses; however, imbalances lead to cytotoxic sulfite and bisulfite accumulation, triggering disorders such as cancer and cardiovascular anomalies. Our research focused on development of a dual-channel fluorescent probe, applying electron-withdrawing acceptors within a coumarin dye matrix, facilitating monitoring of mitochondrial viscosity and SO₂ in live cells. This probe distinguishes fluorescence peaks at 650 nm and 558 nm, allowing ratiometric quantification of SO₂ without interference from other sulfur species. Moreover, it enables near-infrared viscosity determination, particularly within mitochondria. The investigation employed theoretical calculations utilizing Density Functional Theory (DFT) methods to ascertain molecular geometries and calculate rotational energies. Notably, the indolium segment of the probe exhibited the lowest rotational energy, quantified at 7.38 kcals/mol. The probe featured heightened mitochondrial viscosity dynamics when contained within HeLa cells subjected to agents like nystatin, monensin, and bacterial lipopolysaccharide (LPS). Overall, our innovative methodology elucidates intricate mitochondrial factors, presenting transformative insights into cellular energetics, redox homeostasis, and therapeutic avenues for mitochondrial-related disorders.