EMBO/SourceData huggingface dataset:
Figure 4A Pancreatic sections (20X magnification) from 10-months old WT and Glo1KD mice stained with hematoxylin and eosin (H&E), anti-insulin, anti-glucagon and anti-F4/80 antibodies, are representative of sections from 7 mice each group.H, I and J Insulin and glucagon IS, calculated as the intensity score multiplied with percentage of positive cells (H, I) and the percentage of islets area stained with F4/80 (J) (n=7 mice for each genotype). Data information: box plots show the minimum and maximum values (ends of the whiskers), interquartile range (length of the box), median (line through the box) and mean (+) values. *p≤0.05, **p≤0.01 (Mann-Whitney test).
Figure 7C Whole cell protein lysates of INS-1 832/13 β-cells analysed by Western Blot with anti-p53 and anti-p21 antibodies; anti-vinculin antibody was used as loading control (n=9 biological replicates for each treatment group). Data information: Data in bar graphs are presented as mean ± SEM. In C and D, blots show representative image of replicate experiments; protein levels were quantified by the densitometric analysis of independent experiments and showed in the bar graphs as the percentage over not treated control cells. *p≤0.05, **p≤0.01, ***p≤0.001: MGO vs CTR; #p≤0.05, ##p≤0.01, ###p≤0.001: D/Q vs MGO (Mann-Whitney testD Nuclear protein lysates of INS-1 832/13 β-cells analysed by Western Blot with anti-HMGB1 antibody; anti-histone H3 antibody was used as loading control (n=5 biological replicates for each treatment group). Data information: Data in bar graphs are presented as mean ± SEM. In C and D, blots show representative image of replicate experiments; protein levels were quantified by the densitometric analysis of independent experiments and showed in the bar graphs as the percentage over not treated control cells. *p≤0.05, **p≤0.01, ***p≤0.001: MGO vs CTR; #p≤0.05, ##p≤0.01, ###p≤0.001: D/Q vs MGO (Mann-Whitney test
Figure 1High-resolution imaging of mitochondria in live cells using the Airyscan module of Zeiss LSM880 confocal microscope. Images of IMM in living HeLa, L6, and H1975 cells, stained with 10-N-nonyl acridine orange (NAO). NAO preferentially binds phospholipids in the IMM, such as cardiolipin. Arrowheads indicate cristae in the IMM. Scale bar = 500 nm. N ≥ 3 independent experiments for each cell type.High-resolution imaging of mitochondria in live cells using the Airyscan module of Zeiss LSM880 confocal microscope. Mitochondrion cropped from HeLa cell shown in A (dashed line) and zoomed in to show cristae, inner boundary membrane (IBM), and matrix (arrows). Scale bar = 100 nm.High-resolution imaging of mitochondria in live cells using the Airyscan module of Zeiss LSM880 confocal microscope. H1975 cells transduced with matrix-targeted-DsRed and stained with NAO. Matrix-targeted Ds-Red differentiates matrix from cristae stained with NAO. Arrowheads point to cristae. Scale bar = 500 nm. N = 1 independent experiment.High-resolution imaging of mitochondria in live cells using the Airyscan module of Zeiss LSM880 confocal microscope. The structure of the IMM in Mic13-KO cells (HeLa), stained with NAO. The number of cristae is decreased in Mic13-KO compared to control cells shown in panel A, labeled with arrowheads. Scale bar = 500 nm. N = 3 independent experiments.High-resolution imaging of mitochondria in live cells using the Airyscan module of Zeiss LSM880 confocal microscope. Live-cell imaging of the IMM in control and PTPMT1 KD H1975 cells, a model of cardiolipin deficiency. A gallery of mitochondria from various control H1975 cells expressing scrambled shRNA and stained with NAO, showing cristae (arrowheads). Scale bars = 500 nm. N = 3 independent experiments. A gallery of mitochondria from various H1975 cells expressing PTPMT1 shRNA and stained with NAO. Note the derangement of the ultrastructure (arrowheads). Scale bars = 500 nm. N = 3 independent experiments.
Figure 2High-resolution imaging of mitochondria in live cells using the Airyscan module of Zeiss LSM880 confocal microscope. Mitochondria from HeLa cell, co-stained with NAO and TMRE. Area from dashed white box, zoomed to right, shows the red and green intensities of TMRE and NAO colocalized. Scale bars = 500 nm. N = 3 independent experiments. Mitochondria in L6 myoblast, co-stained with NAO and TMRE. Area from dashed white box, zoomed to right, shows colocalizing NAO and TMRE at the cristae membrane (arrowheads). Scale bars = 500 nm. N = 3 independent experiments.High-resolution imaging of mitochondria in live cells using the Airyscan module of Zeiss LSM880 confocal microscope. Mitochondria in HeLa cells, stained with NAO alone (top row) vs. NAO + TMRE (bottom row), and simultaneously excited with 488- and 561-nm lasers. Note that mitochondria stained with NAO alone do not emit noticeable fluorescence in the red channel; only after adding TMRE does strong signal appear in the red channel, showing negligible bleed-through. Scale bars = 500 nm. N = 2 independent experiments.
Figure 4Live-cell Airyscan image of mitochondrion in HeLa cell, showing different membrane potentials in different mitochondrial regions. Membrane potentials were calculated based on TMRE FI differences between compartments (FIcomp). Regions of interest from cristae and IBM used for ΔΨm calculations are labeled with arrows on the right-hand side of the mitochondrion, using the cytosol as the reference compartment. Labeled with forks on the left-hand side of the mitochondrion are the calculations of ΔΨm between individual cristae and their neighboring IBM. Nernst equation used to calculate different voltages. Scale bar = 500 nm. LUT of mitochondrion in HeLa cell shown in A, color-coding TMRE FIs on scale of white (most intense) to blue (least intense): LUT scale shown in lower left-hand corner. Arrowheads indicate cristae. Scale bar = 500 nm. Note that most-intense pixels (white) only associate with cristae. Quantification of ΔΨm (mV) at cristae and IBM relative to the average ΔΨm of the whole mitochondrion, calculated as in A. Note: ΔΨm at cristae is significantly higher than ΔΨm at IBM, indicating electrochemical boundaries separate these two regions of the IMM. N = 3 independent experiments.Live-cell Airyscan imaging of mitochondria in cultured primary mouse hepatocytes stained with TMRE. Arrowheads denote cristae. D. Image showing TMRE-labeled mitochondria. E. LUT color-coding of TMRE FI. F. Quantification of ΔΨm differences using Nernst equation as shown in A. Scale bar = 500 nm. N = 2 independent experiments.Live-cell STED imaging of mitochondria in HeLa cell stained with TMRM. Arrowheads show cristae. Scale bar = 500 nm. G. Image showing TMRM-labeled mitochondrion. H. LUT color-coding of TMRM FI. I. Quantification of ΔΨm differences using Nernst equation as shown in A. Scale bar = 500 nm. N = 4 independent experiments. Note that calculation of ΔΨm differences between the compartments based on images captured by STED and by Airyscan were very similar.
Figure 5Response of ΔΨm to the F1Fo ATP Synthase inhibitor, oligomycin (10 µM), compared to the uncoupler, FCCP (10 µM), determined using live-cell Airyscan imaging of HeLa cells co-stained with MTG and TMRE. Quantification of total mitochondrial TMRE FI. N = 3 independent experiments. Quantification of cristae ΔΨm relative to IBM (ΔΨCr-IBM) in response to oligomycin compared to FCCP. F1Fo ATP Synthase is exclusively localized to the cristae and significant increase in ΔΨCr-IBM in response to blocking of F1Fo ATP Synthase with oligomycin indicates that differences in TMRE FI between cristae and IBM are driven by oxidative phosphorylation. N = 3 independent experiments.Response of ΔΨm to the F1Fo ATP Synthase inhibitor, oligomycin (10 µM), compared to the uncoupler, FCCP (10 µM), determined using live-cell Airyscan imaging of HeLa cells co-stained with MTG and TMRE. Representative Airyscan images showing mitochondria from living HeLa cells stained with MTG and TMRE. Cells were first stained for one hour and only then treated with oligomycin or FCCP. Images show hyperpolarization of cristae under oligomycin (middle row; arrowheads) and depolarization of cristae under FCCP (bottom row; arrowheads) compared to control (top row; arrowheads). Scale bar = 500 nm. N = 3 independent experiments. Note that the ΔΨm-independent fraction of TMRE staining is less than 5% of the signal in control conditions.Zoomed-in region of FCCP-treated mitochondria in C. Increased contrast was used to visualize TMRE to compensate for TMRE loss induced by FCCP. These images show diminished heterogeneity of ΔΨCr-IBM values.
Figure 6The role of cristae structure as well as CJ formation and sealing on the generation of the difference in ΔΨm between cristae and IBM were studied Representative images of mitochondria in control (top row) vs. Mic13-KO (bottom row) HeLa cells. Note that TMRE FI in Mic13-KO mitochondria is distributed more evenly along the IMM, so that ΔΨCr-IBM is decreased. Scale bar = 500 nm. N = 3 independent experiments. Quantification of A, showing deletion of Mic13 in HeLa cells results in significant decrease in ΔΨCr-IBM. While Mic13-KO cells show a substantial decrease in cristae number, the loss of cristae does not appear absolute, making it feasible to compare TMRE FI at cristae relative to IBM. N = 3 independent experiments.The role of cristae structure as well as CJ formation and sealing on the generation of the difference in ΔΨm between cristae and IBM were studied by disrupting cristae using Mic10- and Mic60-deficient cells. Mic60 support cristae formation. Mic10 is essential for CJ formation, and, in its absence, cristae tend to remain as vesicles detached from IBM. Live-cell Airyscan imaging of TMRE was used in all figure panels and models. Representative images of Hap1 control (top row) vs. Mic60 KO (middle row) and Mic10 KO (bottom row); Mic60-KO mitochondria show very few cristae structures. TMRE staining along the IMM is relatively homogeneous, indicating decreased ΔΨCr-IBM. In general, Mic10 KO in Hap1 cells results in decreased TMRE signal intensity at cristae relative to IBM (arrowheads) as compared to control cells. Deletion of Mic10 induces detachment of cristae from IBM (see Figure 8). Except for the detached cristae vesicles, the TMRE staining is homogeneously distributed n Mic10-KO cells, indicating decreased ΔΨCr-IBM. Scale bar = 500 nm. N = 3 independent experiments. Quantification of C, showing Mic60 KO in Hap1 cells results in significant decrease in ΔΨCr-IBM. While Mic60-KO cells show a marked loss of normal cristae structures, depletion of cristae does not appear absolute, making it possible to compare TMRE FI at cristae relative to IBM. N = 3 independent experiments. Quantification of C, showing Mic10 KO in Hap1 cells results in a significant decrease in ΔΨCr-IBM. Note: quantification of ΔΨm at cristae relative to IBM was performed only on cristae structures that appeared to maintain attachment to IBM. N = 3 independent experiments.
Figure 8Live-cell Airyscan images of laser-induced depolarization time series of Mic10 KO Hap1 cell stained with TMRE. Mitochondrion exposed to rapid, high 2-photon laser power in region of white box, arrow (~0.3 sec); large, hyperpolarized vesicle (arrowhead) remains polarized after the rest of the mitochondrion loses ΔΨm, indicating electrochemical discontinuity between hyperpolarized vesicles and the rest of the mitochondrion, including the IBM. Scale bar = 500 nm. N = 2 independent experiments.Live-cell Airyscan image of time series showing hyperpolarized vesicles (arrowheads; region cropped from dashed white box) inside mitochondria lacking both Opa1 and Drp1. Scale bar = 500 nm. N = 2 independent experiments. Free movement of hyperpolarized vesicles within the matrix indicate their detachment from the IBM, representing an extreme case of cristae independency within one mitochondrion (see Movie EV 1 for time series).
Figure 9Laser-induced mitochondrial membrane depolarization in L6 myoblasts shows gradual depolarization of elements along the IMM, visualized using live-cell Airyscan microscopy. Representative images of laser-induced depolarization time series from L6 myoblast, stained with ΔΨm-dependent dye, Rho123. Images show LUT color-coded for Rho123 FI. White and blue pixels represent most and least intense ΔΨm, respectively. See legend of LUT colors on the right. Note that at the first time point (~0.15 sec) white arrow points to box marking area of mitochondrion exposed to phototoxic pulse of 2-photon laser (≤5 msec), inducing depolarization. Ensuing frames show wavelike depolarization away from site of perturbation (arrowheads). Scale bar = 500 nm. N = 3 independent experiments. (See Movie EV 2 for time series.) Profile plot showing Rho123 pixel intensity as function of distance along mitochondrion in A. Note that zero value in the X axis corresponds to top area of the mitochondrion shown in A, near site of 2-photon laser exposure (box). Red line corresponds to frame immediately before depolarization (~0.15 sec); green, blue, purple, and dark purple lines correspond to ~0.30, ~0.60, ~1.05, and ~2.10 sec, respectively, after initial depolarization, showing wavelike dissipation of ΔΨm over time.Laser-induced mitochondrial membrane depolarization in living human skin fibroblasts co-stained with TMRE and MTG and imaged with Airyscan, showing gradual depolarization of elements along the IMM. Time-lapse images of merged green (MTG) and red (TMRE) channels following laser-induced mitochondrial membrane depolarization. Note that, at the first time point (0.3 sec), white arrow points to box marking area of mitochondrion exposed to phototoxic pulse of 2-photon laser (≤5 msec), inducing depolarization. Depolarization is marked by the dissipation of the ΔΨm-dependent dye (TMRE) while the green (MTG) signal persists. Scale bar = 1 µm. N = 4 independent experiments. TMRE channel from C, showing gradual of the loss of TMRE along the IMM. Note that although depolarization has largely completed at the area near the phototoxic stimulus already at 0.9 sec, some specific regions at the very top maintain ΔΨm while others depolarize. Quantification of ΔΨm using TMRE FI from C-D. Measurement of TMRE pixel intensities immediately after laser-induced depolarization at site distal (≥ 10 µm) vs. proximal (≤ 1 µm) to box. N = 4 independent experiments; see specific p values in panel. Quantification of C-D. Percentage of mitochondria that depolarize in a wavelike (i.e., gradual) vs. instantaneous manner after laser-induced depolarization. Note: imaging at high temporal resolution (~100-500 msec/frame) reveals wavelike depolarizations predominate, suggesting the ΔΨm is composed of multiple, disparate electrochemical domains along the IMM. The time scale of propagation of depolarization is slower than the propagation of electrical phenomena. N = 4 independent experiments.
Figure 10We calculated the ΔΨCr-IBM between neighboring cristae over time as a surrogate measure of cristae individuality and functional independence, using live-cell Airyscan imaging. Stability of cristae ΔΨCr-IBM over time. Representative images from time series of HeLa cells co-stained with MTG and TMRE. Top sequence shows merged green (MTG) and red (TMRE) channels. Bottom sequence shows color-coded LUT of TMRE signal of the same sequence shown above. White arrowheads in the image point to specific cristae that were measured over time. Scale bar = 500 nm. N = 5 independent experiments. Quantification of A, showing average ΔΨCr-IBM of Cristae "A-D" over time. Note that Cristae C and D maintain more than double the ΔΨCr-IBM of Cristae A and B. Error bars indicate STDEV. Quantification of A, showing standard deviation in ΔΨCr-IBM (mV) of individual cristae over time compared to the standard deviation in ΔΨCr-IBM (mV) among different cristae along a single mitochondrion. N = 5 independent experiments. Note that there is less variability in ΔΨCr-IBM of a single crista over time within the same mitochondrion compared to the variability of ΔΨCr-IBM among cristae over time, indicating cristae possess unique bioenergetic properties.We calculated the ΔΨCr-IBM between neighboring cristae over time as a surrogate measure of cristae individuality and functional independence, using live-cell Airyscan imaging. Representative live-cell Airyscan image from time series showing merged green (MTG) and red (TMRE) channels. Area marked by dashed box is shown in the time series in E. Scale bar = 500 nm. Time series of cropped region from D, showing tip of mitochondrion (arrowhead) maintaining ΔΨm while neighboring region of same mitochondrion depolarizes. The capacity of some cristae to maintain polarity, while adjacent cristae transiently depolarize, indicates cristae can exhibit a significant amount of electrochemical autonomy. N = 3 independent experiments. (See Movies EV 3, 4 for time series.) Zoomed-in LUT of mitochondrion from E (~2 sec), showing some cristae remaining polarized (red) while adjacent cristae depolarize (blue). (See Movie EV 5 for time series.) Quantification of D-F, showing that during a flickering event some cristae maintain their membrane potentials while other cristae depolarize, resulting in large differences in ΔΨCr-IBM along the individual mitochondrion. N = 3 independent experiments.
Figure 1Left: representative conventional image of interphase MT network in the 1st quadrant of a Human Retinal Pigment Epithelium (HRPE) cell. Grey: α-tubulin; Yellow: TGN-46; Yellow dashed line: Golgi boundary. Right: the classified MT subgroups. Red: GaMTs; Green: non-GaMTs. Scale bar: 5 μm. Box-whisker plot presents the proportion of GaMT and non-GaMTs. (data were pooled from 3 independent experiments and n= 36 cells). The ends of whiskers set as the 10% and 90% of the entire population. ***p < 0.001, two-tailed Mann-Whitney.Six representative images of MT networks extracted from MT STORM images of HRPE cells under combinative KDs. Cells were rotated to orient their leading edges locating in the 1st quadrant. Upper: control cell; CLASPs KD cell; GCC185 KD cell. Lower: CLASPs + GCC185 KD cell; CAMSAP2 KD cell; AKAP450 KD cell. Red: GaMT; Green: non-GaMT. Scale bar: 5 μm. Box-whisker plot presents the ratio of GaMT/non-GaMT under combinative KDs. (1 representative of 3 independent experiments and n= 12 cells). The ends of the whiskers are set at 10% and 90% of the entire population. *p < 0.05, **p < 0.01, ***p < 0.001, ns, no significant difference, unpaired t-test.Time series show EB1 movement on individual GaMTs. Red: GaMT; Green: non-GaMT; White: EB1 tracks. Scale bar: 1 μm. Time series show EB1 movement on individual non-GaMTs. Red line: GaMT; Green line: non-GaMT; White line: EB1 tracks. Scale bar: 0.5 μm. EB1 velocity distributions of GaMTs and non-GaMTs. Red curve and green curve represent the Gaussian fittings (data were pooled from 3 independent experiments and n= 92 tracks). ***p <0.001, unpaired t-test.
Figure 2Representative trajectories of E-cad cargos tracked over 120 s in an HRPE cell. Colors represent different average velocity ranges. Red: > 400 nm/s; Purple: 200~400 nm/s; Green: 100~200 nm/s; Yellow: 0~100 nm/s. Scale bar: 20 μm. Total number of cargos within different velocity ranges in the 4 quadrants, respectively (Data were pooled from 3 independent experiments and n= 45 cells). Proportions of cargos with different velocity in the 4 quadrants. Red: the 1st quadrant; Dark grey: the 2nd quadrant; Light grey: the 3rd quadrant; White: the 4th quadrant (Data were pooled from 3 independent experiments and n= 45 cells).Representative time series show one fast cargo moving on a GaMT and a slow cargo moving on a non-GaMT. Upper, conventional imaging; lower, STORM imaging. Red line: GaMT; Green line: non-GaMT; White circle: cargo position. Scale bar: 2 μm. Marginal distributions show the velocity on GaMT (red dots) or non-GaMTs (green dots). Red curve and green curve, Gaussian fitting curves of velocity distribution on GaMT or non-GaMT, respectively. (Data were pooled from 3 independent experiments and n= 69 MTs). Peak of red curve (431.14 ± 9.7 nm/s) and peak of purple curve (188.96 ± 22.03 nm/s). ***p < 0.001, unpaired t-test.Upper panel: time series show an example of several fast cargos present on GaMT. Lower panel: time series show one slow cargo on non-GaMT switching onto a GaMT and becoming faster. Red line: GaMT; Green line: non-GaMT; White circle: cargo position. White line, cargo trajectory. Scale bar: 2 μm. Box-whisker plot presents the averaged acetylation level of fast or slow MT tracks. (Data were pooled from 2 independent experiments and n= 63 MTs). The ends of the whiskers are set at 10% and 90% of the entire population, ns, no significant difference, unpaired t-test. Marginal distributions of the mean velocity of cargos on segments of Ac-MTs (orange dots) and non-Ac-MTs (purple dots). Orange curve and purple curve represent the Gaussian fittings (Data were pooled from 2 independent experiments and n= 63 MTs). ns, no significant difference, unpaired t-test.
Figure 3Five representative images of MT repair sites in HRPE cells under combinative KDs. Cells were rotated to orient their leading edges locating in the 1st quadrant. From left to right, control cell, CLASPs&GCC185 KD cell, CAMSAP2 KD cell, AKAP450 KD cell and Centrinone-B treated cell. Grey: α-tubulin; red: GTP-tubulin. Scale bar: 20 μm. Box-whisker plot presents intensity analysis of MT repair sites in HRPE cells under combinative KDs. (1 representative of 3 independent experiments and n= 20 cells). ***p < 0.001, unpaired t-test.Conventional image (left) and STORM image (right) of MTs and MT repair sites. Grey: α-tubulin; yellow: GTP-tubulin; green: Golgi. Scale bar: 5 μm.MT repair sites on GaMTs and non-GaMTs extracted from C. Red: GaMTs; Green: non-GaMTs; yellow: GTP-tubulin. MT repair sites on GaMTs and non-GaMTs separately presented in detail. White arrows indicates GTP-tubulin labeling sites. Scale bar: 5 μm. Box-whisker plot presents the ratio of MT repair sites on GaMTs and non-GaMTs separately. (1 representative of 3 independent experiments and n= 8 cells). ***p < 0.001, unpaired t-test.Representative time series show one fast cargo moving on a GaMT and slowing down at the MT repaire site. Red: GaMT; Green: non-GaMT; Yellow: GTP-tubulin; White circle: cargo position. Scale bar: 2 μm. Representative time series show one cargo moving fast between two MT repair sites and slowing down at the MTrepair sites. Red: GaMT; Green: non-GaMT; Yellow: GTP-tubulin; White circle: cargo position. Scale bar: 2 μm. Pause (I) and reverse (J) events of cargos on MT repair segment and non-MTs repair segment, respectively. (Data were pooled from 2 independent experiments and n= 22 cells). ***p <0.001, ns, no significant difference, unpaired t-test.
Figure 4A representative image of MT networks extracted from MT STORM image of an HRPE cell. Red line: GaMT; Green line: non-GaMT. Yellow circle: intersection. Blue circle: intersections formed by GaMTs and non GaMTs. Scale bar: 5 μm. Rose-plot presents the orientation of GaMTs and non-GaMTs. Red: GaMTs; Green: non-GaMTs. Box-whisker plot presents the projections of GaMTs and non-GaMTs to the leading edge direction. (Data were pooled from 3 independent experiments and n= 36 cells). The ends of the whiskers are set at 10% and 90% of the entire population, **p < 0.01, unpaired t-test. Box-whisker plot presents the intersection counts of GaMTs and non-GaMTs. (data are pooled from 3 independent experiments and n= 36 cells). The ends of the whiskers are set at 10% and 90% of the entire population, ***p < 0.001, unpaired t-test.