EMBO/soda-nel huggingface dataset:
Karyopherin Kap114p-mediated trans-repression controls ribosomal gene expression under saline stress (2020)
Figure 5

Figure 5. Kap114p suppresses gene expression under salt stress

A. Wild type and KAP114 knockout strains were diluted serially and spotted onto YPD plates in the presence of 1.5 M NaCl.

B. The KAP114 knockout strain was transformed with vector control (lane 2) and a plasmid containing wild type KAP114, KAP114 (Δ347-371), or KAP114 (Δ899-956) (lanes 3-5). Wild type and KAP114 knockout strains containing plasmids were serially diluted and grown in minimal medium plates supplemented with 1.5 M NaCl. Growth temperatures are indicated.

C. A heatmap generated by three independent biological replicates shows gene expression profiles of wild type and KAP114 knockout strains grown in the presence of 1.5 M NaCl. The Log2 (fold-change) values and the color scale are shown.

D. Gene ontology enrichment analysis of up- and down-regulated genes, analyzed separately using Metascape (http://metascape.org). The top 10 significant GO terms (biological pathways and processes) associated with up- and down-regulated genes are shown. The y-axis represents the GO category (ribosomal biogenesis pathways/processes are highlighted in bold), and the x-axis represents the -Log10 (P-value) of GO terms.

E, F. (E) qPCR analysis of gene expression in the KAP114 knockout strains (green bars) compared to wild type (black bars) under regular conditions. (F) qPCR analysis of gene expression in the KAP114 knockout strain (green bars) compared to wild type (black bars) grown in high salt conditions. qPCR fold-changes in mRNA expression are relative to control (YPD media, black bars). Actin was used as the internal control. G. ChIP-qPCR assays showing yTBP-GFP binding to the promoter of indicated genes in wild type and KAP114 knockout strains grown in high salt conditions (light green bars) compared to under regular conditions (black bars). Chip-qPCR fold-enrichment is relative to control (YPD media, black bars).

H. Endogenous Ran, GFP-yTBP and proliferating cell nuclear antigen (PCNA; loading control) in nuclear fractions isolated from wild type yeast grown in regular and high salt conditions were analyzed by Western blot using antibodies for Ran, GFP and PCNA.

I, J. Analysis of Ran and GFP-yTBP in nuclear fractions isolated from wild type yeast grown in regular and high salt conditions. The combined bar and scatter plot shows the band intensity ratios of Ran (I) and GFP-yTBP (J) over PCNA. Each dot represents an individual data point.

Data information: In (E), data represent mean ± standard deviation, N = 5. In (F, G), data represent mean ± standard deviation, N = 3. In (I, J), data represent mean ± standard deviation from four independent experiments. Differences were assessed statistically by two-tailed Student's t test; *P< 0.05; **P< 0.01; ***P< 0.001; n.s.: not significant.
Prohibitin 1 regulates mtDNA release and downstream inflammatory responses (2022)
Figure 2

Figure 2. PHB1 is required to maintain mitochondrial homeostasis and membrane permeability

(A-B) GFP-expressing HeLa cells with stable Phb1 knockdown were fixed and subjected to immunofluorescence (IF) analysis to detect mitochondria (MitoTracker, red; A) or ΔΨm (TMRM, red; B). Images shown are representative of at least three independent experiments. White boxed regions in the panels in (A) are enlarged. Scale bar: 10 μm.

(C) Flow cytometric analysis of ΔΨm (TMRM). HeLa cells with stable Phb1 knockdown were treated with CsA (2 μM) for 30 minutes or FCCP (4 μM) for 5 minutes.

(D) The mitochondrial respiratory capacity of J774A.1 cells with or without Phb1 knockdown was measured with a Seahorse XFe96 analyzer. Data are from at least three independent experiments (mean ± SEM). n.s., no significance. ** p < 0.01, *** p < 0.001, **** p < 0.0001 (Student's t-test).

(E) HeLa cells with stable PHB1 ablation were incubated with H2O2 (2 μM) and CsA (2 μM) for 30 minutes. Flow cytometric analysis of intracellular ROS levels (DHE).

(F-G) Confocal microscopy analyses of mitochondrial Ca2+ in time-series mode. HeLa cells stably expressing 4mt-RCaMPh with or without Phb1 knockdown were treated with CsA (2 μM) for 30 minutes. During acquisition of fluorescence images (F), cells were treated with histamine (200 μM). Traces of mitochondrial Ca2+ (4mt-RCaMPh) dynamics are shown in (G) (mean ± SEM). n = 16, 16, 15, 16 (from the top to the bottom). Scale bar: 10 μm. * p < 0.05 relative to Scramble group, # p < 0.05 relative to shPHB1 group (Student's t-test).
Prohibitin 1 regulates mtDNA release and downstream inflammatory responses (2022)
Figure 4

Figure 4. The cytosolic mtDNA and ROS promoted by Phb1-depletion contributes to the inflammatory responses

(A) BMDMs isolated from Phb1F/F mice and Phb1MyeKO mice were stimulated with LPS (200 ng/ml) for 6 hours and ATP (4 mM) for 45 minutes. Cells were fixed and subjected to IF analysis to detect the co-localization between NLRP3 (anti-NLRP3, red) and mtDNA (PicoGreen, green). White boxed regions in the panels are enlarged. The white arrows indicate mtDNA co-localized with NLRP3. Scale bar: 10 μm.

(B) Quantitative analysis of data from (A). Data are from at least 14 images from three independent experiments (mean ± SEM). **** p < 0.0001 (Student's t-test).

(C) J774A.1 cells with or without Phb1 knockdown were stimulated with LPS (200 ng/ml) for 6 hours and ATP (4 mM) for 45 minutes. Cells were fixed and subjected to IF analysis to detect the co-localization between NLRP3 (anti-NLRP3, red) and mtDNA (PicoGreen, green). White boxed regions in the panels are enlarged. The white arrows indicate mtDNA co-localized with NLRP3. Scale bar: 10 μm.

(D) Quantitative analysis of data from (C). Data are from at least six images from three independent experiments (mean ± SEM). * p <0.05, ** p < 0.01, **** p < 0.0001 (Student's t-test).

(E) J774A.1 cells with or without Phb1 knockdown were stimulated with LPS (200 ng/ml) for 6 hours and ATP (4 mM) for 45 minutes. After cells were lysed, NLRP3 was immunoprecipitated by anti-NLRP3 antibody. mtDNA levels (D-loop) from immunoprecipitants were detected by qPCR in J774A.1 cells with or without Phb1 knockdown. Data are from five independent experiments (mean ± SEM). ** p < 0.01, **** p < 0.0001 (Student's t-test).

(F) J774A.1 cells with Phb1 knockdown were incubated with EtBr (150 ng/ml) for 3 days, followed by LPS (200 ng/ml) for 6 hours and ATP (4 mM) for 45 minutes. WB was used to detect the protein levels in whole cell lysates and culture medium.

(G) BMDMs isolated from Phb1F/F mice and Phb1MyeKO mice were treated with NAC (2 mM) for 8 hours, LPS (200 ng/ml) for 6 hours and ATP (4 mM) for 45 minutes. WB assay was used to detect the protein levels in whole cell lysates and in the culture medium.
Prohibitin 1 regulates mtDNA release and downstream inflammatory responses (2022)
Figure 5

Figure 5. PHB1 regulates the formation of mPTP by modulating the interaction between SPG7 and AFG3L2

(A) HeLa cells were incubated with H2O2 (5 mM) for 20 minutes and then lysed. The whole cell lysate was immunoprecipitated by IgG, anti-AFG3L2 antibody or anti-SPG7 antibody. WB was used to detect the SPG7 or AFG3L2 levels, respectively, in the precipitated products.

(B) J774A.1 cells were stimulated with LPS (200 ng/ml) for 6 hours and lysed. The whole cell lysate was immunoprecipitated by IgG, anti-AFG3L2 antibody or anti-SPG7 antibody. The respective levels of precipitated SPG7 or AFG3L2 were examined by WB.

(C-D) HeLa cells or J774A.1 cells with or without Phb1 knockdown were lysed. The whole cell lysate was immunoprecipitated by IgG, anti-AFG3L2 antibody or anti-SPG7 antibody. The respective levels of precipitated SPG7 or AFG3L2 were examined by WB.
Prohibitin 1 regulates mtDNA release and downstream inflammatory responses (2022)
Figure 6

Figure 6. SPG7 and AFG3L2 are both required for mtDNA release and inflammatory responses mediated by Phb1 knockdown

(A) J774A.1 cells with knockdown of Phb1 alone, Afg3l2 alone, Spg7 alone, Phb1 + Afg3l2, or Phb1 + Spg7 were fixed and subjected to IF analysis to assess the co-localization between mitochondria (MitoTracker, red) and mtDNA (PicoGreen, green). White boxed regions in the panels are enlarged. The white arrows indicate mtDNA outside mitochondria. Scale bar: 10 μm.

(B) Quantitative analysis of the data from (A). Data are from at least 12 images from three independent experiments (mean ± SEM). ** p < 0.01, **** p < 0.0001 (Student's t-test).

(C) J774A.1 cells with knockdown of Phb1 alone, Afg3l2 alone, Spg7 alone, Phb1 + Afg3l2, or Phb1 + Spg7 were lysed, and DNA was extracted. qPCR was used to detect the mtDNA levels (D-loop) in the cytosol relative to the nuclear DNA levels (Tert) in the whole cell lysate. Data are from six independent experiments (mean ± SEM). **** p < 0.0001 (Student's t-test).

(D) J774A.1 cells with knockdown of Phb1 alone, Afg3l2 alone, Spg7 alone, Phb1 + Afg3l2, or Phb1 + Spg7 were stimulated with LPS (200 ng/ml) for 6 hours and ATP (4 mM) for 45 minutes. The culture medium was collected and ELISA assays were used to detect IL-1β levels in culture medium. Data are from four independent experiments (mean ± SEM). **** p < 0.0001 (Student's t-test).

(E) J774A.1 cells with Phb1 knockdown, Spg7 knockdown, or combined Phb1 and Spg7 knockdowns were stimulated with LPS (200 ng/ml) for 6 hours and ATP (4 mM) for 45 minutes. WB assay was used to detect the protein levels in whole cell lysates and culture medium.

(F) J774A.1 cells with Phb1 knockdown, Afg3l2 knockdown, and combined Phb1 and Afg3l2 knockdowns were stimulated with LPS (200 ng/ml) for 6 hours and ATP (4 mM) for 45 minutes. WB assay was used to detect the protein levels in whole cell lysates and culture medium.

(G) J774A.1 cells were stimulated with LPS (200 ng/ml) for 6 hours and lysed. The whole cell lysate was immunoprecipitated by IgG or anti-SPG7 antibody. WB was used to detect the CYPD and VDAC levels in the precipitated products.

(H) J774A.1 cells with or without Phb1 knockdown were lysed. The whole cell lysates were immunoprecipitated by IgG or anti-SPG7 antibody. WB was used to detect the CYPD and VDAC levels in the precipitated products.
Cristae undergo continuous cycles of membrane remodelling in a MICOS-dependent manner (2020)
Figure 1

Figure 1. MIC10 and MIC60 KO HAP1 cells show loss of crista junctions and impaired cellular respiration.

A Western blot analysis of lysates from WT and MIC10 KO or MIC60 KO HAP1 cells. MIC10 KO cells show a drastic reduction of MIC13, MIC26 and MIC27 protein levels while protein levels of other MICOS components remain unchanged. MIC60 KO cells show a drastic reduction of protein levels of all MICOS components.

B Representative electron micrographs of WT, MIC10 KO and MIC60 KO HAP1 cells. Scale bar 500 nm.

C Quantification of CJs per mitochondrial section from different mitochondria in WT, MIC10 and MIC60 KO HAP1 cells using EM represented as boxplots. Boxplots show median and interquartile range from 25 to 75 percentile and whiskers represent minimum and maximum value. Data from n = 55-69 mitochondria (from 2 independent experiments) are shown as data points in the boxplots. ****P < 0.0001 (using unpaired Student's t-test).

D Quantification of cristae per mitochondrial section from different mitochondria in WT, MIC10 and MIC60 KO HAP1 cells using EM represented as boxplots. Boxplots show median and interquartile range from 25 to 75 percentile and whiskers represent minimum and maximum value. Data from n = 55-69 mitochondria (from 2 independent experiments) are shown as data points in the boxplots. ****P < 0.0001 (using unpaired Student's t-test).

E Comparison of oxygen consumption rates (pmol O2/s, normalized for cell numbers by Hoechst staining) of basal, proton leak, maximum and non-mitochondrial respiration in WT and MIC10 KO cells obtained from 3 independent experiments. The data is normalized to basal respiration from HAP1 WT. Bar and error bar represent mean ± SEM from 3 independent experiments. *P = 0.034 for basal (using one sample t-test) and *P = 0.018 for maximum respiration (using unpaired Student's t-test).

F Comparison of oxygen consumption rates (pmol O2/s, normalized for cell numbers by Hoechst staining) of basal, proton leak, maximum and non-mitochondrial respiration in WT and MIC60 KO cells obtained from 3 independent experiments. The data is normalized to basal respiration from HAP1 WT. Bar and error bar represent mean ± SEM from 3 independent experiments. *P = 0.01 for basal (using one sample t-test), ***P = 0.0004 for maximum respiration and *P = 0.012 for non-mitochondrial respiration (using unpaired Student's t-test).
Cristae undergo continuous cycles of membrane remodelling in a MICOS-dependent manner (2020)
Figure 2

Figure 2. MIC60 assembles as regularly spaced punctae in absence of CJs.

A Representative STED super-resolution images of WT and MIC10 KO HAP1 cells immunostained with MIC60 or MIC10 antibodies (top panel) and TOMM70 (middle panel). Bottom panel shows merged images. Arrowheads show colocalization of MIC60 and TOMM70 punctae. Scale bar 500 nm.

B Quantification of median interpunctae distance (IPD) per mitochondrion between MIC60 or MIC10 punctae in indicated cell lines represented by boxplots. Boxplots show median and interquartile range from 25 to 75 percentile and whiskers represent minimum and maximum value. Median IPD from n = 10-16 mitochondria (from 2 independent experiments) are shown as data points in the boxplots. ns, not significant, unpaired Student's t-test.

C Scheme shows MIC60 marking the nascent sites of CJs formation independent of MIC10 and presence of contact sites in WT and MIC10 KO HAP1 cells shown by colocalization of MIC60 and TOMM70.
Cristae undergo continuous cycles of membrane remodelling in a MICOS-dependent manner (2020)
Figure 3

Figure 3. Mobility of crista junction proteins is restricted compared to proteins of other mitochondrial subcompartments and loss of MIC13 affects the mobility of distinct IM proteins.

A Scheme of investigated marker proteins, subjected to FRAP, located at different mitochondrial subcompartments.

B, C FRAP curves (curve fitted) of WT (B) and MIC13 KO Hela cells (C) expressing GFP-tagged fusion proteins of MIC60 (CJs), MIC10 (CJs), TOMM20 (OM), TIMM23 (IBM) and ATP5I (CM). Average FRAP curves (intensities in arbitrary units) for each marker were obtained (3 independent experiments, 6-10 mitochondria for each experiment). Error bars for each time point representing SEM are shown.

D Histogram showing average percentage of mobile fractions, obtained from curve fits of all mitochondria expressing GFP-tagged versions of MIC60 (CJs), MIC10 (CJs), TOMM20 (OM), TIMM23 (IBM) and ATP5I (CM) in WT and MIC13 KO Hela cells (data calculated from FRAP curves shown in Figure 3B and 3C that are obtained from 3 independent experiments, 6-10 mitochondria from each experiment).

E Diffusion coefficients of the above-mentioned proteins are shown in the table.
Cristae undergo continuous cycles of membrane remodelling in a MICOS-dependent manner (2020)
Figure 4

Figure 4. Single particle tracking reveals requirement of MIC60 for high directionality of MIC10 motion.

A-D Representative single particle tracks of cells expressing MIC60- (A) and MIC10-SNAP (B) in WT Hela cells and MIC60- (C) and MIC10-SNAP (D) in MIC13 KO Hela cells, stained with silicone-rhodamine dye, and imaged at a rate of 33 ms/frame. Single tracks were colour-coded according to temporal appearance. Scale Bar 5 µm. Insets in each image show zoomed-in images of few representative tracks.

E Cumulative frequency of tracks having corresponding values of instantaneous diffusion coefficients (insD) in WT and MIC13 KO Hela cells expressing MIC60- and MIC10-SNAP stained with silicone-rhodamine. Number of tracks analysed (obtained from 2 independent experiments): n = 2541, 2540, 3560 and 1441 in WT Hela cells expressing MIC60-SNAP and MIC10-SNAP, MIC13 KO Hela cells expressing MIC60-SNAP and MIC10-SNAP, respectively. (****P ≤ 0.0001 for all possible comparisons, unpaired Student's t-test).

F Percentage of subtracks having directed motion in WT and MIC13 KO Hela cells expressing MIC60- and MIC10-SNAP and in WT and MIC60 KO HAP1 cells expressing MIC10-SNAP. Number of corresponding subtracks analysed (obtained from 2 independent experiments): n = 2561, 2443, 3402, 1360, 561 and 1110. Data are mean ± SEM. *P ≤ 0.05, ***P ≤ 0.001, ****P ≤ 0.0001, unpaired Student's t-test.
Cristae undergo continuous cycles of membrane remodelling in a MICOS-dependent manner (2020)
Figure 5

Figure 5. Crista junctions marked by MIC10-SNAP dynamically merge and split within a mitochondrion in a MIC13-dependent manner.

A, C Representative live-cell STED super-resolution images (t=0 s) showing WT (A) and MIC13 KO (C) Hela cells expressing MIC10-SNAP stained with silicone-rhodamine. Box in (A and C) mark selection shown as a zoom in panel (B and D) respectively. Scale bar 500 nm.

B, D Time-lapse image series of a mitochondrion expressing MIC10-SNAP in WT (B) and MIC13 KO (D) Hela cells (2.6 s/frame). Green and magenta asterisks show merging and splitting events of MIC10-SNAP respectively. Green arrows pointing inward connected by solid line and magenta arrows pointing outward connected by dotted line show sites of imminent merging and splitting events, respectively. Scale bar 500 nm.

E Blind quantification of merging and splitting events of CJs in WT and MIC13 KO Hela cells expressing MIC10-SNAP (3 independent experiments, 5-8 mitochondria for each experiment) represented as boxplots. Boxplots show median and interquartile range from 25 to 75 percentile and whiskers represent minimum and maximum value. *P = 0.026 (merging and splitting events in WT cells) and ****P < 0.0001 (merging and splitting events of WT vs. MIC13 KO), unpaired Student's t-test.

F Scheme representing the dynamic nature of CJs in WT and MIC13 KO cells.
Cristae undergo continuous cycles of membrane remodelling in a MICOS-dependent manner (2020)
Figure 6

Figure 6. MIC13-SNAP shows that CJs and cristae undergo remodelling at a timescale of seconds.

A Representative live-cell STED super-resolution images (t=0 s), showing WT Hela cells expressing MIC13-SNAP, from a time-series of images acquired at a time interval of 2.5 s stained with silicone-rhodamine. Box marks selection shown as a zoom in panel (B). Scale bar 500 nm.

B Time-lapse image series of a mitochondrion expressing MIC13-SNAP imaged at a time interval of 2.5 s/ frame. Green and magenta asterisks show cycles of cristae merging and splitting marked by MIC13-SNAP. Green arrows pointing inward connected by solid line and magenta arrows pointing outward connected by dotted line show sites of imminent merging and splitting events, respectively. Cristae mergence events that form structure resembling letter `X´ or `Y´ are marked at 5 s or 14.9 s respectively. Scale bar 500 nm.

C Representative live-cell STED super-resolution images (t=0 s), showing WT Hela cells expressing MIC13-SNAP, from a time-series of images acquired at a time interval of 1.3 s stained with silicone-rhodamine. Box marks selection shown as a zoom in panel (D). Scale bar 500 nm.

D Time-lapse image series of a mitochondrion expressing MIC13-SNAP imaged at a time interval of 1.3 s/ frame. Green and magenta asterisks show cycles of cristae merging and splitting marked by MIC13-SNAP. Green arrows pointing inward connected by solid line and magenta arrows pointing outward connected by dotted line show sites of imminent merging and splitting events, respectively. Scale bar 500 nm.

E First image from (D) was used for showing the intensity profiles along the length of three cristae (numbered 1-3 and marked with white arrows) in (F).

F Intensity profiles of lines drawn across the length of cristae show increased intensities at the edges of the line scans representing the IBM. Magenta boxes depict intensity maxima at IBM.
Cristae undergo continuous cycles of membrane remodelling in a MICOS-dependent manner (2020)
Figure 7

Figure 7. Cristae undergo balanced merging and splitting events in a MICOS-dependent manner.

A Representative live-cell STED super-resolution images (t=0 s) showing WT Hela cells expressing ATP5I-SNAP stained with silicone-rhodamine. Box marks selection shown as a zoom in panel (B). Scale bar 500 nm.

B Time-lapse image series of a mitochondrion expressing ATP5I-SNAP in WT Hela cells (2.5 s/frame). Green and magenta asterisks show merging and splitting events of cristae marked by ATP5I-SNAP respectively. Green arrows pointing inward connected by solid line and magenta arrows pointing outward connected by dotted line show sites of imminent merging and splitting events, respectively. Green `Y´ represent Y- type imminent mergence event . Scale bar 500 nm.

C Representative live-cell STED super-resolution images (t=0 s) showing MIC13 KO Hela cells expressing ATP5I-SNAP stained with silicone-rhodamine. Box marks selection shown as a zoom in panel (D). Scale bar 500 nm.

D Time-lapse image series of a mitochondrion expressing ATP5I-SNAP in MIC13 KO Hela cells (2.5 s/frame). Green and magenta asterisks show merging and splitting events of cristae marked by ATP5I-SNAP respectively. Green arrows pointing inward connected by solid line and magenta arrows pointing outward connected by dotted line show sites of imminent merging and splitting events, respectively. Scale bar 500 nm.

E First image from (B) was used for showing the intensity profiles along the length of three cristae (numbered 1-3 and marked with white arrows) in (F).

F Intensity profiles of lines drawn across the length of cristae show increased intensities in the middle of the line scans representing the CM. The represented IBM area is superimposed from intensities of MIC13-SNAP from Figure 6F. Magenta box depicts intensity maxima at cristae membrane apart from IBM.

G Blind quantification of cristae merging and splitting events in WT and MIC13 KO Hela cells expressing ATP5I-SNAP (3 independent experiments, 3-7 mitochondria for each experiment) represented as boxplots. Boxplots show median and interquartile range from 25 to 75 percentile and whiskers represent minimum and maximum value. ****P < 0.0001, unpaired Student's t-test.
Cristae undergo continuous cycles of membrane remodelling in a MICOS-dependent manner (2020)
Figure 9

Figure 9. Demonstration of content mixing in mitochondria using intramitochondrial cristae photoactivation and PEG fusion experiments.

A Mitochondrion in WT Hela cells expressing ATP5I-PAGFP was photoactivated, in the area shown by a blue box, at 0 s. Inset of white box at 0.98 s clearly shows fluorescent crista (red arrow), absent at 0.38 s, 0.59 s & 0.78 s (red arrowhead showing the area enclosed by white dotted lines), confirming merging of non-fluorescent cristae vesicle with the IBM. Threshold images of insets are shown above the whole images. Scale bar 500 nm.

B Dual-colour STED super-resolution images of fused mitochondrion in WT Hela cells (Left Panel) showing colocalization of cristae marked with ATP5I-GFP (using anti-GFP antibody) and ATP5I-SNAP (stained with silicone-rhodamine). (Right Panel) Fused mitochondrion in MIC13 KO Hela cells showing cristae marked with either ATP5I-GFP (using anti-GFP antibody) or ATP5I-SNAP (stained with silicone-rhodamine). Arrowheads show merged cristae while arrows show cristae that maintain their individual identity. Scale bar 500 nm.

C Boxplot showing quantification of colocalization events per µm of mitochondria in WT and MIC13 KO Hela cells. Boxplots show median and interquartile range from 25 to 75 percentile and whiskers represent minimum and maximum value. (15 mitochondria were taken from WT and MIC13 KO cells from 4 and 2 independent experiments, respectively, *P = 0.03, unpaired Student's t-test)

D Schematic illustration of the proposed 'Cristae fission and fusion' (CriFF) as a working model and its link to CJ dynamics.