Fig 1.
A549 cells grown in DMEM contain bloated mitochondria with multiple clustered mtDNA nucleoids.
A. SIM image of an A549 cell stained with antibodies against Tom20 (red) and DNA (green) showing nucleoid clusters inside bloated mitochondria (white arrows). Resolved channels are shown on the right. Scale bar is 5 μm. B. A 2.875 μm thick section displayed as a 3D maximum projection showing Tom20 and DNA staining from the ROI boxed in yellow in panel A. The expanded right-hand panels show Tom20 and DNA channels resolved independently. NIKON 3D-SIM, z-stack of 23 slices at 125 nm intervals, scale bar is 2.5 μm. C,D. 3D-STORM images of two A549 cells stained with anti-DNA antibodies showing both individual nucleoids and clustered nucleoids. Images are pseudo-colored to show depth in the section. Scale bars 1 μm. E. TEM depicts bloated mitochondria highlighting prominent electron lucent areas devoid of cristae (red arrows). Scale bar is 500 nm. F. TEM image showing bloated mitochondria with mtDNA decorated with anti-DNA Nano-gold labeling within electron lucent regions. Scale bar is 500 nm.
Table 1.
Incidence of mtDNA nucleoid clustering in A549 cells.
Fig 2.
Mito-bulbs in A549 cells exhibit poor inner membrane organization in matrix regions containing clustered mtDNA nucleoids.
A. SIM image depicts mito-bulbs in a DMEM-adapted A549 cell expressing MLS-EGFP (blue) enclosed by markers for the outer membrane (Tom20, red) and inner membrane (PHB2, green). The yellow boxed ROI is expanded and channels resolved in panels on the right. Scale bar is 2.5 μm. B. SIM image of a DMEM-adapted A549 cell stained with antibodies against outer membrane Tom20 (red), inner membrane marker ATPB (blue) and DNA (green), shown as individual channels on the right. Scale bar is 5 μm.
Fig 3.
Individual mito-bulb organelles persist over the course of more than 4 h.
A. Single image frame shows RPMI-adapted A549 cells expressing MLS-EGFP imaged for over 4 h at 5 min intervals via live, z-stack, confocal microscopy. The ROI (red box) denotes a swollen mitochondria, displayed throughout image collection at the indicated 35-min intervals. Scale bar is 15 μm. Leica SP8, 63x, 0.75x zoom, 5% CO2, 37⁰C, 95% relative humidity. B. Mitochondrial diameters of several individual mito-bulbs are plotted with respect to time, measured at 5 min intervals, showing stability over the course of more than 4 h. Graph represents the measure of the smaller elliptical diameter with respect to time for each mito-bulb measured in A.
Fig 4.
Assessment of the relative membrane potentials of bloated and normal morphology mitochondria via live confocal microscopy.
A. RPMI-adapted A549 cells expressing MLS-EGFP were loaded with TMRM as described in Materials and Methods. Cells featuring mito-bulbs were imaged and analyzed by tracing the red and green channel signals through regions featuring either mito-bulb or normal tubular morphology mitochondria. Panels on the right show the enlarged ROI boxed in A to indicate the locations of sampling lines and the resolved colors. Scale bar is 10 μm. Leica SP8, 63x, 5% CO2, 37⁰C, 95% relative humidity, operating in photon-counting mode. B. Sample trace data from the TMRM and EGFP channels. The upper panel depicts a trace through a mito-bulb, “BM Line-1” in A. The lower panel depicts a trace bisecting through three mitochondria with normal morphology, “NM Line-2” in A. Traces follow lines from the lower left to upper right. C. The relative TMRM staining intensity normalized to matrix GFP is shown for normal tubular and swollen, mito-bulb mitochondria. Confocal microscopy images from 24 cells, over 3 separate experiments, totaling 132 independent 5 μm mitochondrial traces were processed from data similar to that displayed in A and B. The boxplot shows median and quantile values. Welch’s t-test, t = 6.96, p < .0001.
Fig 5.
Swollen mitochondria exhibit a more oxidative enviroment than mitochondria with normal morphology.
A. Representative confocal image of the ROS signal generated by MitoSOX (red) accumulating in A549 cells expressing MLS-EGFP (green). Scale bars are 5 μm. Leica SP8, 63x, 5% CO2, 37⁰C, 95% relative humidity, operating in photon-counting mode. B. Trace derived from “Line-1” in A that intersects both normal and bloated mitochondria. Line is 20 μm and traces extend from left to right. C. Confocal image representing the oxidation state of mito-roGFP in A549 cells. The 525 nm emission intensity signals induced by 405 nm or 488 nm excitation, represent oxidized and reduced protein, respectively, are superimposed in the main panel and resolved individually in panels directly below. Scale bars are 10 μm. Leica SP8, 63x, 5% CO2, 37⁰C, 95% relative humidity, operating in photon-counting mode. D. Linear traces show the 525 nm fluorescence intensities for the oxidized (red) and reduced (green) forms of mito-roGFP2 in ROIs 1 and 2 of panel C bisecting normal and bloated mitochondria, respectively. E. Two-dimensional heatmap of the oxidized:reduced ratio of mito-roGFP.
Fig 6.
Mito-bulb nucleoid clusters retain transcriptional capability and discrete RNA processing centers.
A. The MRG marker GRSF1 (red) is observed in individual punctate structures adjacent to mtDNA nucleoids (green) within mito-bulb like structures. Inner membrane marker ATPB is depicted in blue. Scale bar is 5 μm. Image is representative of six specimens. B. The MRG marker FASTKD2 (red) resides in individual punctate structures adjacent to mtDNA nucleoids (green) within mito-bulb like structures. Inner membrane marker ATPB is depicted in blue. Scale bar is 5 μm. Image is representative of 25 images collected for seven independent specimens. C. The nascent BU-labeled mtRNA (red) is observed in individual punctate structures adjacent to mtDNA nucleoids (green) inside both normal and bloated mitochondrial structures. Outer membrane marker Tom20 is depicted in blue. Arrows indicate swollen mitochondria with multiple foci of nascent mtRNA. Scale bar is 5 μm. Image is representative of 10 images of three independent experiments.
Fig 7.
Nucleoids in mito-bulbs are active in mtDNA replication.
A. DMEM-adapted A549 cells show positive labeling for EdU foci (red) inside mitochondria at mtDNA nucleoids. Replication is active inside mito-bulb like mitochondria indicated by yellow arrows. Cells were stained with antibodies against DNA (green) and Tom20 (blue). Resolved channels are displayed as separate panels as indicated. Scale bar in A is 5 μm. B. RPMI-adapted A549 cells expressing MLS-mCherry (blue) were labeled with EdU detected using click chemistry (green) and stained with anti-DNA antibodies (red). Arrows indicate EdU incorporation at nucleoids in bloated mitochondria. Scale bar is 5 μm. C. Box-and-whisker plot of the percentage of nucleoids that incorporated EdU in the total population of nucleoids and in the subset of nucleoids clustered in bloated mitochondria. Data for 33 specimens in 4 independent experiments are compiled in Table 2. Boxes indicate means and quantile values for the distributions, which were not significantly different (p = 0.21).
Table 2.
Nucleoids in tubular and swollen mitochondria replicate with similar frequency.
Fig 8.
Incidence of mito-bulbs correlates with elevated glucose and/or glutamine concentration.
A. Five columns show representative images of MLS-EGFP A549 cells grown in basal DMEM media with varied glucose and/or glutamine content as noted. Below each image is the binarized mask of each image highlighting mito-bulbs with cross-sectional areas above 2 μm2 in blue. Scale bars are 10 μm in each image. Leica SP8, 63x, 2.5x zoom. Color-coded bars above and below the images are keyed to the histogram in B. B. Histograms showing the average number of mito-bulbs per cell in each condition listed. The bar graph shows the mean number (+/- standard deviation) of mito-bulbs per cell exceeding the indicated threshold in μm2 under each of five media conditions. A total of 95 images (total of 958 cells) were analyzed to generate this graph. The number of images taken and analyzed was roughly equivalent among all the conditions, with the exception of a slightly higher number taken and analyzed for the lowest glucose/glutamine condition to compensate for the lower incidence of mito-bulbs.
Fig 9.
Mito-bulb incidence can be influenced markedly by inhibition of the TGFβ pathway.
A. Four columns feature representative images of MLS-EGFP, DMEM adapted A549 cells, for each treatment condition assessed. Cells were treated for 4.5 days with either vehicle control, 5 ng/ml TGFβ, 10μM SB431542, or with both compounds simultaneously, from left to right, respectively. Mitochondrial networks, as visualized via MLS-EGFP are depicted in red and Hoechst stain is depicted in green. Scale-bars are 10 μm. Leica SP8, 63x, 2.5x zoom. B. Graph of the average number of mito-bulbs per cell (+/- standard deviation) exceeding the indicated threshold for each condition specified, as in Fig 8. A total of 72 images (total of 495 cells) were analyzed to generate this graph. The number of images taken and analyzed was roughly equivalent among all the conditions.