Mitochondria are the main energy source for cells and play a key role in regulating cellular metabolism. Mitochondria can change their structures depending on environmental conditions and cellular requirements. The dynamic nature of mitochondria is driven by several processes that regulate mitochondrial morphology including fission, fusion, mitophagy, and biogenesis.
Fission creates larger amounts of mitochondria that are circular in shape and smaller in size. Fusion creates smaller amounts of mitochondria that are elongated in shape and larger in size. The opposing processes of mitochondrial fission and fusion are both involved in mitochondrial quality control and the maintenance of normal cellular homeostasis2, 3. Both fission and fusion are critical to normal cellular function.
Mitophagy is a process for clearing damaged mitochondria, and a critical component of normal mitochondrial recycling3. A damaged or senescent mitochondrion undergoes asymmetric fission. While the healthy components are delivered to the new mitochondria, the damaged components are segregated into the smaller components that are depolarized and promptly eliminated via mitophagy4. Biogenesis is the process that results in synthesis of new mitochondrial components.
Pathologic alterations in mitochondrial dynamics can result in impaired bioenergetics and mitochondrial-mediated cell death, and are associated with a wide spectrum of pathologies, including ischemic cardiomyopathy, diabetes, pulmonary hypertension, Parkinson’s and Huntington’s diseases and skeletal muscle atrophy, or Alzheimer’s disease1, 5. Neurons are particularly sensitive to changes in mitochondrial function due to their limited capacity for glycolysis and high level of energetic use6. Cardiomyocytes, which have one of the highest volume fractions of mitochondria are also uniquely sensitive to mitochondrial dynamic alterations7, 8. Mitochondrial dynamic alterations also play a role in many cancers, where fusion can reduce the susceptibility of cells to apoptotic signaling, induce aerobic glycolysis (the Warburg effect), and potentially enhance cancer cell growth by reducing apoptotic cell death9.
Alterations in mitochondrial dynamics can be a normal physiologic response to stress. Studies in skeletal muscle during exercise suggest a role for mitochondrial fragmentation in maintaining energetic homeostasis. During recovery, fusion is activated, and with chronic exercise, mitochondrial biogenesis10.
There has been growing interest in using high-content imaging methods for studying the mitochondrial structure remodeling. Confocal imaging and water immersion objectives can offer enhanced image quality and better visualization of mitochondria structures, while the tools of image analysis can be utilized to obtain numeric characterization of different phenotypes. In this study, we describe phenotypic assays for mitochondria phenotypes and structural re-arrangements that can be used for studies of mitochondria dynamics in cell-based assays.
Human neuroblastoma PC12 cells were obtained from ATCC. Hoechst 33342 and MitoTracker Orange CMTMRos were purchased from Thermo Fisher. Chemical compounds were purchased from Sigma.
Human neuroblastoma PC12 cells were plated in Greiner 384-well plates at a density of 3,000 cells per well. The next day, cells were treated for 18 hours with various concentrations of several benchmark compounds known to effect mitochondria: chloroquine (0 – 100 μM), rotenone (0 – 10 μM), valinomycin (0 – 1 μM), and methyl mercury (0 – 10 μM). Each compound was tested at seven different concentrations, half-log dilutions, in quadruplicates for a sample size of n = 8. After treatment, live cells were stained with a combination of mitochondrial dye Mitotracker Orange CMTMRos and Hoechst 33342 nuclear dye (0.2 μM and 0.5 μM, respectively) for 30 min, and imaged with the ImageXpress® Micro Confocal system (Molecular Devices). Images of live cells were taken using confocal mode and the 40X water immersion objective. Three to four sites were imaged for each well. For better assay performance, a z-stack of 3–4 image planes was acquired at 0.6–1 μm intervals. Nuclei were imaged with the DAPI channel and mitochondria with the TRITC channel, at 100 ms and 400 ms exposures respectively. Images were analyzed using the Custom Module Editor in MetaXpress® High-Content Image Acquisition and Analysis Software. Details are described in more detail in the Results section. Briefly, a Find Fibers module was used to identify elongated mitochondria, and the Granularity module for more round particles. Cell boundaries were outlined for counting objects per cell. Secondary analysis was completed using Excel or SoftMax® Pro Software curve fitting tools. Z’ values were calculated using the formula Z’ = 1-3*(STDEVcontrol+ STDEVexperiment)/(AVEcontrol- AVEexperiment).