Human Fis1 regulates mitochondrial dynamics through inhibition of the fusion machinery (2019)
Figure 5

Figure 5. hFis1 impairs mitochondrial fusion in wild-type 293T cells

A, B. Representative confocal images of mitochondrial fusion in WT 293T polykaryons in cultures subjected to the PEG-based fusion assay in the presence or absence of exogenous hFis1. WT 293T cells stably expressing mitoGFP or mitoRFP were co-cultured, subsequently transfected with empty vector (control) or with Myc-hFis1 as indicated and then treated with PEG for cell fusion. Mitochondrial fusion is indicated by co-localization of mitoGFP and mitoRFP (i.e. yellow mitochondria). Left panel: empty vector transfected; right panel: Myc-hFis1 transfected. Insets represent magnification of the boxed areas in upper panel (A). Quantitative analysis of the extent of mitochondrial fusion in individual hybrid cells was performed by the Pearson's correlation coefficient (PCC) in three independent experiments for each condition and data summarized in (B).

C, D. Representative confocal images of mitochondrial fusion in WT 293T cells assessed by the mito-PAGFP-based fusion assay in the presence or absence of exogenous hFis1. WT 293T cells co-transfected with mito-PAGFP (0.5 µg), mito-DsRed (0.2 µg), and either empty vector (0.5 µg, the upper panel) or hFis1 (0.5 µg, the lower panel) were photoactivated in a small region of interest (ROI) (white circle, 3 µm diameter) in preactivation images of mitochondria seen by mito-DsRed (red). After photoactivation, sequential Z-stack images with photoactivated GFP (green) and mitochondrial marker (mito-DsRed) were collected at indicated time points using series of Z-sections from the top to the cell bottom with intervals between sections set to 0.5-0.75 μm (C). Mitochondrial fusion was quantified by analyzing changes in fluorescence intensity of photoactivated mito-PAGFP in ROIs at 40 sec, 15, 30, 45 min after photoactivation. The dilution rates (percentage) of the GFP fluorescence intensity at different time points were normalized by the fluorescence intensity at 40 sec after photoactivation (D).

E, F. Representative confocal images of mitochondrial fusion in WT 293T polykaryons in cultures subjected to the PEG-based fusion assay in the presence or absence of endogenous hFis1. Both WT 293T cells stably expressing mitoGFP or mitoRFP were transfected with scrambled siRNA (control siRNA) or hFis1-siRNA as indicated and then co-cultured and fused using PEG treatment. Left panel: control siRNA transfected; right panel: hFis1-siRNA transfected (E). Quantitative analysis of the mitochondrial fusion in individual hybrid cells was performed by the Pearson's correlation coefficient in three independent experiments and data summarized in (F).

G, H. Representative confocal images of mitochondrial fusion in WT 293T cells assessed by the mito-PAGFP-based fusion assay in the presence or absence of endogenous hFis1. Scrambled siRNA or hFis1 siRNA treated cells were co-transfected with mito-PAGFP and mito-DsRed and were subsequently photoactivated (G) as described in (C). Mitochondrial fusion was quantified (H) as described in (D).

Data information: Data are expressed as means ± s.e.m. and were statistically analyzed by Student's t‐test. n represents the number of cells analyzed (B, D, F and H).