Quantifying Mitochondrial Dynamics in Patient Fibroblasts with Multiple Developmental Defects and Mitochondrial Disorders

Abstract

Mitochondria are dynamic organelles that undergo rounds of fission and fusion and exhibit a wide range of morphologies that contribute to the regulation of different signaling pathways and various cellular functions. It is important to understand the differences between mitochondrial structure in health and disease so that therapies can be developed to maintain the homeostatic balance of mitochondrial dynamics. Mitochondrial disorders are multisystemic and characterized by complex and variable clinical pathologies. The dynamics of mitochondria in mitochondrial disorders is thus worthy of investigation. Therefore, in this study, we performed a comprehensive analysis of mitochondrial dynamics in ten patient-derived fibroblasts containing different mutations and deletions associated with various mitochondrial disorders. Our results suggest that the most predominant morphological signature for mitochondria in the diseased state is fragmentation, with eight out of the ten cell lines exhibiting characteristics consistent with fragmented mitochondria. To our knowledge, this is the first comprehensive study that quantifies mitochondrial dynamics in cell lines with a wide array of developmental and mitochondrial disorders. A more thorough analysis of the correlations between mitochondrial dynamics, mitochondrial genome perturbations, and bioenergetic dysfunction will aid in identifying unique morphological signatures of various mitochondrial disorders in the future.

Keywords: mitochondrial disorders; mitochondrial dynamics; mitochondrial fission; mitochondrial fusion; mitochondrial membrane potential; mitochondrial morphology.

Figure 1

Mitochondrial morphology descriptors. The mitochondria morphology is classified as individuals or networks. Individuals consist of structures without a junction pixel, while networks contain branches with one or more junction pixels. Scale bar = 100 μm.

Figure 2

Mitochondrial morphology of healthy BJ fibroblast in the absence and presence of FCCP. (a) Representative images of fibroblast cell lines stained with Mitotracker Red CM-H2Xros (MTR), a dye that localizes to actively respiring mitochondria. The top images are phase contrast, RFP, and skeletonized images of healthy control cell lines without FCCP treatment. The bottom images are phase contrast, RFP, and skeletonized images of healthy control cell lines with FCCP treatment. (b) MiNA descriptors showing differences in mitochondrial morphology in untreated and FCCP treated groups. FCCP treatment resulted in mitochondrial fission and resulted in a significant increase in the number of individuals, networks, and total respiring mitochondria. The mean branch length and mean network size are lower after FCCP treatment, albeit not statistically significant. All data are representative of 10–14 images taken from five independent dishes per treatment group. The bars represent minimum and maximal values, and each black dot represents different data points. **** p < 0.0001. Scale bar = 100 μm.

Figure 3

Mitochondrial morphology of SBG1 fibroblast in the absence and presence of FCCP. (a) Representative images of SBG1 fibroblast cell lines stained with MTR. The top images are phase contrast, RFP, and skeletonized images of SBG1 fibroblasts without FCCP treatment. The bottom images are phase contrast, RFP, and skeletonized images of SBG1 fibroblasts with FCCP treatment. (b) Although SBG1 cell lines have a comparable number of individual and networked mitochondria to control, the mitochondria in these lines are smaller. This is evident in the significant decrease in mean branch length and mean network size relative to control under basal conditions, without FCCP treatment. Treatment with FCCP resulted in mitochondrial fission, with SBG1 showing a response to FCCP similar to the healthy control. All data are representative of 10–14 images taken from three independent dishes per treatment group. The bars represent minimum and maximal values, and each black dot represents different data points. **** p < 0.0001, ** p < 0.01, * p < 0.05. Scale bar = 100 μm.

Figure 4

Mitochondrial morphology of SBG2 fibroblast in the absence and presence of FCCP. (a) Representative images of SBG2 fibroblast cell lines stained with MTR. The top images are phase contrast, RFP, and skeletonized images of SBG2 fibroblasts without FCCP treatment. The bottom images are phase contrast, RFP, and skeletonized images of SBG2 fibroblasts with FCCP treatment. (b) SBG2 fibroblasts have a slightly lower number of individual and networked mitochondria compared to control BJ fibroblasts. The significantly lower mean branch length relative to control fibroblasts under basal conditions further suggests fragmentation. SBG2 fibroblasts are not responsive to FCCP treatment. All data are representative of 10–14 images taken from three independent dishes per treatment group. The bars represent minimum and maximal values, and each black dot represents different data points. **** p < 0.0001, *** p < 0.001, * p < 0.05. Scale bar = 100 μm.

Figure 5

Mitochondrial morphology of SBG3 fibroblast in the absence and presence of FCCP. (a) Representative images of SBG3 fibroblasts stained with MTR. The top images are phase contrast, RFP, and skeletonized images of SBG3 fibroblasts without FCCP treatment. The bottom images are phase contrast, RFP, and skeletonized images of SBG3 fibroblasts with FCCP treatment. (b) While the number of individuals in SBG3 fibroblasts is comparable to the healthy control BJ fibroblasts, the number of networks is significantly lower. This shows that SBG3 fibroblasts have fewer networks, with significantly lower mean branch length further suggesting that the networks have smaller branches. Together, this demonstrates a more fragmented morphology in SBG3 fibroblasts with FCCP treatment resulting in more fragmentation, as expected. All data are representative of 10–14 images taken from three independent dishes per treatment group. The bars represent minimum and maximal values, and each black dot represents different data points. **** p < 0.0001, ** p < 0.01, * p < 0.05. Scale bar = 100 μm.

Figure 6

Mitochondrial morphology of SBG4 fibroblasts in the absence and presence of FCCP. (a) Representative images of SBG4 fibroblast cell lines stained with MTR. The top images are phase contrast, RFP, and skeletonized images of SBG4 fibroblasts without FCCP treatment. The bottom images are phase contrast, RFP, and skeletonized images of SBG4 fibroblasts with FCCP treatment. (b) Aside from having fewer networks, SBG4 fibroblasts are not significantly different from the control BJ fibroblasts. However, they are also responsive to FCCP treatment similar to the healthy control BJ fibroblasts. All data are representative of 10–14 images taken from three independent dishes per treatment group. The bars represent minimum and maximal values, and each black dot represents different data points. **** p < 0.0001, ** p < 0.01, * p < 0.05. Scale bar = 100 μm.

Figure 7

Mitochondrial morphology of SBG5 fibroblasts in the absence and presence of FCCP. (a) Representative images of SBG5 fibroblast cell lines stained with MTR. The top images are phase contrast, RFP, and skeletonized images of SBG5 fibroblasts without FCCP treatment. The bottom images are phase contrast, RFP, and skeletonized images of SBG5 fibroblasts with FCCP treatment. (b) The amount of actively respiring mitochondria in SBG5 fibroblasts is lower than those of the healthy control BJ fibroblasts. The significantly lower number of individuals, networks, and total respiring mitochondria in the SBG5 fibroblasts support this observation. The SBG5 fibroblasts also have hyperfused mitochondria, as they have longer branches and more branches within their networks. All data are representative of 10–14 images taken from three independent dishes per treatment group. The bars represent minimum and maximal values, and each black dot represents different data points. **** p < 0.0001, *** p < 0.001, ** p < 0.01, * p < 0.05. Scale bar = 100 μm.

Figure 8

Mitochondrial morphology of SBG6 fibroblast in the absence and presence of FCCP. (a) Representative images of SBG6 fibroblast cell lines stained with MTR. The top images are phase contrast, RFP, and skeletonized images of SBG6 cell lines without FCCP treatment. The bottom images are those of the FCCP treatment group. (b) The SBG6 cell lines have significantly fewer networks of actively respiring mitochondria relative to the control cell lines. All data are representative of 10–14 images taken from three independent dishes per treatment group. The bars represent minimum and maximal values, and each black dot represents different data points. **** p < 0.0001, *** p < 0.001, ** p < 0.01, * p < 0.05. Scale bar = 100 μm.

Figure 9

Mitochondrial morphology of SBG7 fibroblast in the absence and presence of FCCP. (a) Representative images of SBG7 fibroblast cell lines stained with MTR. The top images are phase contrast, RFP, and skeletonized images of SBG7 fibroblasts without FCCP treatment. The bottom images are phase contrast, RFP, and skeletonized images of SBG7 fibroblasts with FCCP treatment. (b) The SBG7 fibroblasts have significantly fewer networks of actively respiring mitochondria relative to the control BJ fibroblasts. FCCP treatment resulted in fragmented mitochondria in the SBG7 fibroblasts with mean network size trended towards an increase upon treatment. All data are representative of 10–14 images taken from three independent dishes per treatment group. The bars represent minimum and maximal values, and each black dot represents different data points. **** p < 0.0001, *** p < 0.001, ** p < 0.01, * p < 0.05. Scale bar = 100 μm.

Figure 10

Mitochondrial morphology of SBG8 fibroblast in the absence and presence of FCCP. (a) Representative images of SBG8 fibroblast cell lines stained with MTR. The top images are phase contrast, RFP, and skeletonized images of SBG8 fibroblast cell lines without FCCP treatment. The bottom images are phase contrast, RFP, and skeletonized images of SBG8 fibroblasts with FCCP treatment. (b) SBG8 fibroblasts have significantly shorter branch lengths and fewer branches within their networks. All data are representative of 10–14 images taken from three independent dishes per treatment group. The bars represent minimum and maximal values, and each black dot represent different data points. **** p < 0.0001, *** p < 0.001, ** p < 0.01 Scale bar = 100 μm.

Figure 11

Mitochondrial morphology of SBG9 fibroblast in the absence and presence of FCCP. (a) Representative images of SBG9 fibroblast cell lines stained with MTR. The top images are phase contrast, RFP, and skeletonized images of SBG9 fibroblast cell lines without FCCP treatment. The bottom images are phase contrast, RFP, and skeletonized images of SBG9 fibroblasts with FCCP treatment. (b) The mean branch length is significantly lower in SBG9 fibroblast cell lines relative to the healthy BJ control fibroblasts. All data are representative of 10–14 images taken from three independent dishes per treatment group. The bars represent minimum and maximal values, and each black dot represents different data points. **** p < 0.0001, *** p < 0.001, ** p < 0.01, * p < 0.05. Scale bar = 100 μm.

Figure 12

Mitochondrial morphology of SBG10 fibroblast in the absence and presence of FCCP. (a) Representative images of SBG10 fibroblast cell lines stained with MTR. The top images are phase contrast, RFP, and skeletonized images of SBG10 fibroblast cell lines without FCCP treatment. The bottom images are phase contrast, RFP, and skeletonized images of SBG10 fibroblasts with FCCP treatment. (b) The mean branch length is significantly lower in SBG10 cell lines relative to the healthy control. All data are representative of 10–14 images taken from three independent dishes per treatment group. The bars represent minimum and maximal values, and each black dot represents different data points. **** p < 0.0001, ** p < 0.01, * p < 0.05. Scale bar = 100 μm.

Figure 13

Mitochondrial membrane potential (MMP) analysis of BJ-FB and LS fibroblast cell lines. Using flow cytometry, along with membrane-potential sensitive dye (TMRE), MMP was evaluated. Representative flow cytometry histogram of five fibroblasts (SBG1–5 and Control BJ) (a) stained with TMRE only; (b) stained with TMRE after treatment with FCCP. Mean fluorescence intensity (MFI) was calculated based on three independent runs, which are shown for (Control BJ-FB in grey; SBG1-FB (MT-ATP6-T8993G), SBG2-FB (MT-ATP6- T8993G), and SBG3-FB (MT-ATP6-T9185C) in red; SBG4-FB (MT-ND3-T10158C) and SBG5-FB (MT-ND5-T12706C) in blue) all samples (c) stained with TMRE only; (d) stained with TMRE after treatment with FCCP. ** p < 0.01, **** p < 0.00001.

Figure 14

Mitochondrial membrane potential (MMP) analysis of BJ-FB diseased fibroblast cell lines. Using flow cytometry, along with membrane-potential sensitive dye (TMRE), MMP was evaluated. Representative flow cytometry histogram of five fibroblasts (SBG6–10 and Control BJ) (a) stained with TMRE only; (b) stained with TMRE after treatment with FCCP. Mean fluorescence intensity (MFI) was calculated based on three independent runs and are shown for (Control BJ-FB in grey; SBG6-FB (m.3243A>G) and SBG7-FB (m.14739G>A) in tan; SBG8-FB (10676∆14868), SBG9-FB (7342∆9916), and SBG10-FB (10167∆15568) in green) all samples (c) stained with TMRE only; (d) stained with TMRE after treatment with FCCP. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.00001.

Figure 15

Mitochondrial morphology summary for fibroblast cell lines. Comparison of mitochondrial morphology of diseased and healthy fibroblast cell lines under basal conditions. Results suggest that diseased cell lines tend to have fewer respiring mitochondria with small branches and fragmented networks. In some cases, however, as seen in SBG5 and SBG7 fibroblasts, hyperfusion serves as a compensatory mechanism.