Mitochondrial Fluctuations as a Measure of Biomechanical Properties of Murine Cells

Introduction: The active mechanical properties of the cellular cytoskeleton are important in a number of significant cellular processes from migration to cell division, and are altered in cancer. A non-invasive single-cell assay of these properties would be of significant value for understanding these cellular processes, would give insights into mechanical heterogeneity between cells, and would be of therapeutic importance in cancer. A promising technique is derived from passive particle microrheology, wherein fluorescent beads are inserted into the cytoplasm, their mean square displacement (MSD) measured, and mechanical parameters calculated using viscoelastic theory. The non-invasive version of this method uses mitochondrial fluctuations towards the same goal which is potentially a very useful assay due to its relative simplicity. Methods: We carried out a detailed study of mitochondrial fluctuations in the C3H-10T1/2 cell line, a murine embryonic mesenchymal cell line, as well as two osteosarcoma cell lines with low and high metastatic potentials, DUNN and DLM8. Cells were cultured under standard growth condition for 24 hr, and then stained with MitoTracker Green FM (ThermoFisher Scientific). Random cells were selected for imaging using spinning disk confocal microscopy at a high temporal resolution (100 ms per frame) for 100 s. We optimized a multi-step image processing protocol to identify mitochondria with high fidelity. We selected the mitochondria with life-time spanning the entire imaging period and with no interaction with other mitochondria and calculated the MSD. Results: The MSD of isolated mitochondria resembles that of a particle in a viscoelastic medium, in agreement with previous results. However comparison of MSD between controls and cells treated with drugs that disrupt the actin and microtubule network showed surprisingly small effects, while treatment with ATP synthesis inhibitors significantly decreased the MSD. Our results imply that mitochondrial fluctuations are primarily driven by active fluctuations of the cytoskeleton, and are relatively unaffected by perturbing the actin or microtubule network, at least in the cell lines in our study. Comparison of MSDs between less invasive and more invasive murine osteosarcoma cells showed significant differences at long time scales. Hence active fluctuations differ significantly between different cell types. We also observed significant heterogeneity among cells of the same type and even more heterogeneity in the mitochondria from a single cell. Our analysis suggests that mitochondrial fluctuations reflect the active contractile properties of the cytoskeleton.