Figure 5.

MCL1 regulates inner membrane integrity and mitochondrial DNA (mtDNA) release via inner mitochondrial membrane (IMM)-derived vesicles. (a) Representative electron microscopy (EM) images showing the disruption of cristae (yellow arrowheads) and the formation of vesicle-like structures (insets with red arrowheads) in cells transfected with sh-MCL1. (b) Quantitative analysis of results shown in (a) representing the disrupted inner membrane cristae (n = 10). (c) Representative LIGHTNING microscopy images showing the loss of inner membrane integrity. Cells were co-transduced with the non-targeted scramble control (Srm) or sh-MCL1 and IMM-targeted green fluorescent protein (GFP) (IMM^GFP) and probed with the anti-TOM20 antibodies. (d) Quantitative analysis of results shown in (c) representing damaged mitochondria (n = 13). (e) Representative images of cells triple-stained with IMM^GFP (green), anti-DNA antibodies (representing mtDNA; cyan), and anti-TOM20 antibodies (red). Cells were transduced with Srm or sh-MCL1. (f) Quantification of images of cells co-transduced with VEC or NSP4 + ORF9b and Srm or sh-MCL1 and immunostained with anti-TFAM antibodies. Data are plotted as integrated densities of TFAM associated with IMM, outer mitochondrial membrane (OMM), or both (n = 9). (g) Representative images of TFAM staining (representing mtDNA) in cells co-transduced with NSP4 + ORF9B or MCL1 shRNA and IMM^GFP. (h) Representative live-cell images showing the appearance of vesicles containing mtDNA (magenta) and IMM (green) in cells co-transfected with IMM^GFP and Scarlet-TFAM. Small vesicles containing both mtDNA and IMM are seen (blue arrowheads) near the large vesicles (usually mitochondria). (i1) Representative immunoblot showing the presence of IMM^GFP in cell supernatants collected 24 h after transfection with IMM^GFP, concentrated, and probed with the anti-GFP antibodies. E-Cad was used as the reference control. (i2) Densitometry analysis of the blots shown in (i1). (j) Summary of the results showing mtDNA derived from the inner membrane vesicles extruding from the BAK/BAX-mediated macropores. These IMM pores are regulated by MCL1. All data are represented as mean ± standard error of mean from three independent experiments. Statistical analyses (unpaired t-tests (b,f) and one-way analysis of variance (d)) were performed using Graphpad Prism software. * p < 0.05; **** p  <  0.0001; ns: not significant (non-parametric t-test). Scale bars: 0.5 (a), 0.2 (ROI), or 10 μm (c,e,g,h).