EMBO/soda-nel huggingface dataset:
The Mitochondrial Calcium Uniporter regulates breast cancer progression via HIF-1α (2016)
Figure 1

Figure 1. MCU expression correlates with breast tumor progression and TNBC cell migration

A-B. Correlation of MCU and MCUb expression levels with breast cancer clinical stages. Median-centered log2 mRNA expression levels of MCU and MCUb were collected from the TCGA breast cancer dataset (http://tcga-data.nci.nih.gov/docs/publications/brca_2012/). Data were plotted and analysed against tumor size (T1-T4) (A) and regional lymph node infiltration (N0-N3) (B), according to the AJCC Cancer Staging Manual (7th edition). Linear regression analysis with different stages was implemented. Parameters of linear regression are shown. Number of samples for each stage are shown in parentheses.

C-E. MCU silencing reduces Ca2+mit uptake in TNBC cells. Cells were transfected with siMCU or siControl. After 48h, [Ca2+]mit uptake upon ATP (C, E) or Histamine (D) stimulation was measured (n=10). P-values: ***p= 0.0008, ***p<0.0001, ***p= 0.0001, respectively.

F-H. MCU silencing impairs TNBC cell migration. Cells were transfected with siMCU or siControl. The day after transfection, a linear scratch was obtained on the cell monolayer through a vertically held P200 tip (time- point 0h). Cell migration into the scratched area was monitored 48 hours later. The covered area was measured and expressed as a percentage relative to 0-hour time-point (n=12). P-value: ***p<0.0001.

I-K. Cell proliferation is mainly unaffected by MCU depletion. Cells were transfected with siMCU or siControl. Cell number was counted every 24 hours for 3 days (the 72-hour time point corresponds to the 48-hour time-point of wound healing assay). Results are expressed as ratio R/R0 where R0 is the number of cells at the time of transfection (0 hour time-point) (n=6). P-value: *p=0.05.

In each panel, data are presented as mean ± SD. A two-tailed unpaired t-test was performed.

See also Appendix Figure S1-S3.
The Mitochondrial Calcium Uniporter regulates breast cancer progression via HIF-1α (2016)
Figure 2

Figure 2. MCU silencing blunts cell invasiveness without affecting cell viability

A. Re-expression of mMCU rescues cell motility of MCU-silenced cells. Cells were transfected with siMCU or siControl. Ad-mMCU was used to re-express MCU (Ad-GFP was used as control). MCU protein expression was verified by western blot. The day after transduction a linear scratch was made (time-point 0h). Cell migration into the wounded area was monitored at 48 hour time-point and the covered area was measured (n=12). P-value: ***p<0.0001.

B. MCU silencing blunts cell invasiveness. Stable shMCU and shControl-expressing spheroids were plated and let grow into collagen-I (0 hour time-point). Spheroid area was measured at 0-hour and 48-hour (n=8). Scale bar 300 μm. P-value: ***p=0.0003.

C. MCU silencing reduces the clonogenic potential of MDA-MB-231 cells. Stable shMCU and shControl-expressing cells were plated at low confluence (2x103/well of a 6-well plate). After 7 days the number of colonies was counted (minimum 30 cells/colony, n=8). P-value: **p=0.0027.

D. MCU depletion does not induce cell death. Cells were transfected with siMCU or siControl. 72 hours later, cell apoptosis and necrosis were measured by FITC-Annexin V and Propidium Iodide (PI) detection (Q1: PI positive, Q2: PI and FITC-Annexin V positive, Q3: PI and FITC-Annexin V negative, Q4: FITC-Annexin V positive. n=6).

E. MCU depletion does not alter cell cycle. Cells were transfected with siMCU or siControl. 72 hours later, cell cycle distribution was monitored by Propidium Iodide (PI) detection (n=6).

In each panel, data are presented as mean ± SD. A two-tailed unpaired t-test was performed.

See also Appendix Figure S4.
The Mitochondrial Calcium Uniporter regulates breast cancer progression via HIF-1α (2016)
Figure 3

Figure 3. MCU deletion hampers tumor growth and metastasis formation in MDA-MB-231 xenografts

Control MDA-MB-231 cells and MCU-/- clones 1 and 2 carrying the firefly luciferase reporter gene were injected into the fat pad of SCID mice.

A. Tumor mass volume was measured at specific time-points until the day of sacrifice (day 39 post-injection for control, day 46 and 56 p.i. for MCU-/- cl.1 and cl.2, respectively). P-values: ***p=0.0001, ***p<0.0001.

B. left: in vivo metastasis at the homolateral axillary area of three representative mice per group at the time of sacrifice. Right: total flux analysis. P-values: **p=0.01, *p=0.02.

C. Lymph nodes weight at the time of sacrifice. P-values: ***p=0.0010, **p=0.0014.

D. Human cytokeratin 7 (CK7) IHC staining of three representative lymph nodes per group. Scale bar= 500 µm.

E. Left: images of three representative lungs per group collected ex vivo at the time of sacrifice. Right: total flux analysis. P-values: **p=0.0031, ***p= 0.0004.

In each panel, data are presented as mean ± SE. (n=9 for Control, n=8 for MCU-/- cl.1, n=10 for MCU-/- cl.2). A two-tailed unpaired t-test was performed.

See also Appendix Figure S4.
The Mitochondrial Calcium Uniporter regulates breast cancer progression via HIF-1α (2016)
Figure 4

Figure 4. MCU downregulation alters cellular redox state

A-C. FLIM analysis of cellular NADH/NADPH levels. Fluorescence lifetimes of NAD(P)H autofluorescence in stable shControl and shMCU expressing cells were imaged. Representative images of the distribution of τbound on an intensity weighted pseudocolored scale (2.2-2.5 ns) are shown. Scale bars = 20 μm (A). Mean +/- SE of τbound (B) and relative NADH and NADPH intensities (C) calculated from equation in (Blacker et al., 2014) are shown (n=3). P-values: **p=0.01, **p=0.002, respectively.

D-E. Measurement of the redox state of the NADH/NAD+ couple. Representative measurements of NADH intensity at steady state and at minimal and maximal reduced state (D). Percentage of the steady state redox state (E) (n=3).

F. MCU depletion impairs the mitochondrial rate of ATP production. Cells were transfected with siMCU or siControl. 48 hours later, cells were treated with 5.5 mM 2-deoxy-D-glucose for 1 hour and cellular ATP levels were quantified (n=6). P-value: ***p=0.0009.

In each panel, data are expressed as mean ± SE. A two-tailed unpaired t-test was performed.
The Mitochondrial Calcium Uniporter regulates breast cancer progression via HIF-1α (2016)
Figure 5

Figure 5. MCU depletion reduces mitochondrial ROS production.

A. Antioxidant treatments decrease cell migration. A linear scratch was obtained on cell monolayer through a vertically held P200 tip (time-point 0h). Cells were treated for 48 hours with N-Acetylcysteine (NAC) or Dithioerythritol (DTE). Cell migration into the wounded area was monitored at 48-hour time-point and the covered area was measured (n=12). P-values: **p=0.008, ***p<0.0001, **p=0.005.

B. Scavenging of mitochondrial ROS decreases cell migration. A linear scratch was obtained on a cell monolayer through a vertically held P200 tip (time-point 0h). Cells were treated for 48 hours with 50 µM MitoTEMPO. Cell migration into the wounded area was monitored at 48 hour time-point and the covered area was measured (n=12). P-value: ***p<0.0001.

In panels A-B, data are expressed as mean ± SD. A two-tailed unpaired t-test was performed.

C. MCU silencing does not affect matrix pH. Cell were transfected with siMCU or siControl and SypHer2 probe. 48 hours later, matrix pH was measured (n=22).

D-E. Mitochondrial H2O2 levels are critically blunted after MCU depletion. Cells were transfected with shMCU or shControl, together with the ratiometric YFP-based biosensor pHyper-dMito (D) or the mitochondrial H2O2-sensitive HyPerRed probe (E). 48 hours later, H2O2 production was measured (n=35). P-values: *p=0.02, *p=0.05, respectively.

F. Mitochondrial superoxide levels are critically blunted after MCU silencing. Cells were transfected with siMCU or siControl. 48 hours later, cells were loaded with the red dye MitoSOXTM and superoxide anion levels were measured (n=25). P-value: *p=0.04.

G. Mitochondrial GSSG/GSH ratio is critically reduced after MCU silencing. Cells were transfected with shMCU or shControl, together with the mitochondrial targeted mitGrx1-roGFP2 probe. 96 hours later, the glutathione redox potential (EGSH) was measured (n=46). P-value: ***p<0.0001.

In panels C-G, data are expressed as mean ± SE. A two-tailed unpaired t-test was performed. Scale bar 10 μm.
The Mitochondrial Calcium Uniporter regulates breast cancer progression via HIF-1α (2016)
Figure 6

Figure 6. MCU depletion critically affects HIF-1α levels and signalling.

A. MCU silencing reduces HIF-1α protein levels. Cells were transfected with siMCU or siControl. HIF-1α protein levels were detected 48 hours later.

B. MCU silencing reduces MG132-mediated HIF-1α and hydroxylated HIF-1α protein accumulation. Cells were transfected with siMCU or siControl. 48 hours later, cells were treated with 10 μM of the proteasome inhibitor MG132. Left: Protein levels were revealed by western blot. Right: quantification by densitometry (n=5).

C. ROS increase HIF-1α transcription. Cells were treated O/N with 100 µM paraquat to induce ROS production. HIF-1α mRNA levels were measured by RealTime-PCR (n=3). P-value: **p=0.002.

D-J. MCU silencing reduces mRNA levels of HIF-1α, HIF-2α, and HIF-1α target genes. Cells were transfected with siMCU or siControl. mRNA expression was measured by RealTime-PCR (n=3). P-values: (HIF-1α) **p=0.0031, **p=0.009, (HIF-2α)**p=0.01, (LOX)***p=0.001, ***p=0.0005, (PDK1)*p=0.02, **p=0.009, (G6PI)*p=0.02, **p=0.005, (CA IX)**p=0.0026, **p=0.0022, (HK2) **p=0.0024, *p=0.03.

K. HIF-1α overexpression rescues siMCU-mediated migration impairment. Cells were transfected with siMCU or siControl. Wild type (wt) and constitutively active (ca) HIF-1α were expressed by retroviral infection (pBABE was used as control). The day after transduction, cells were scratched (time-point 0h). Cell migration into the wounded area was monitored at 48 hour time-point and the covered area was measured (n=12). P-values: ***p<0.0001, *p=0.04.

In each panel, data are expressed as mean ± SD. A two-tailed unpaired t-test was performed.

L-M. MCU expression levels correlate with HIF1α (L) and HIF1α-regulated genes (M). A linear model (lm) to test the power of MCU expression levels predicting the expression of HIF1α and HIF1α-regulated genes was calculated and plotted for each of the 532 samples of the TCGA database (see Figure 1). Equation and R2 values of the linear regression and significance indicating deviation from 0 are shown. The area of 95% prediction limit is shaded below and above the linear regression line. The HIF1α-regulated gene set was compiled from Broad Institute GSEA database (http://www.broadinstitute.org/gsea/msigdb/cards/V$HIF1_Q5.html, merged sets of V$HIF1_Q3 and V$HIF1_Q5).

See also Appendix Figure S5.
The protein architecture of the endocytic coat analyzed by FRET microscopy (2020)
Figure 4

Figure 4 - Rearrangements of endocytic coat proteins during membrane invagination.

A Scheme of suggested topology of Sla1, Ent1, Sla2 and Pan1(1-1303) proteins on the flat (left) and invaginated (right) plasma membrane. Natively unfolded parts of the proteins are shown as thick black lines. The C-termini of proteins showing FRET are depicted as yellow circles; the C-termini separated from their FRET partners during invagination are shown as gray circles. The position of the clathrin lattice and polymerizing actin cytoskeleton are shown as a pale orange and red zone, respectively.

B, C Partial loss of FRET between Ent1-mTurquoise2 and Sla1-mNeonGreen (B) and presence of FRET between Ent1-mTurquoise2 and Sla2-mNeonGreen (C) during endocytic membrane invagination. FRET values (in %) of individual endocytic patches (n=74, 75; 69, 73 for (B) and (C), respectively) sorted according to the absence/presence of Abp1 protein are shown as box plots. Center, top and bottom lines of box plots show the medians, the 25th and 75th percentiles of individual datasets, respectively. Whiskers extend 1.5 times the interquartile range from the 25th and 75th percentiles. Notches indicate 95% confidence intervals of the medians. Statistical differences were calculated by Welch's t-test. See also Figure EV1 and Appendix Figure S1.
The protein architecture of the endocytic coat analyzed by FRET microscopy (2020)
Figure 5

Figure 5 - FRAP analysis of coat-associated proteins at the endocytic site.

Ede1, Apl1, Yap1801, Gts1, Bzz1, Lsb3, Las17, and Vrp1 proteins tagged with mNeonGreen were photobleached in individual endocytic sites of

LatA-treated cells and fluorescence recovery was followed every 0.5 s for 60 s (40 s in case of the highly dynamic Bzz1 protein). The curves represent means ± 95% confidence intervals (n = 9-17). See also Movie EV2.
MICOS assembly controls mitochondrial inner membrane remodeling and crista junction redistribution to mediate cristae formation (2020)
Figure 1

Figure 1. Inner membrane architecture and MICOS distribution in HeLa cells.

(A) Live-cell nanoscopy of HeLa cells. COX8A-SNAP was labeled with SNAP-cell SiR and imaged with STED nanoscopy.

(B) 3D architecture of cristae in HeLa cells visualized by FIB-SEM. The OM and the IBM are together shown as a transparent surface. Cristae of two mitochondria are depicted in light and dark blue. A twisted crista is shown in grey.

(C) Live-cell recording as in (A). In addition, mitochondrial DNA was stained with PicoGreen and imaged by confocal laser scanning microscopy. Arrows mark sites where nucleoids but no lamellar cristae are present.

(D) STED nanoscopy of mitochondria immunolabeled for Mic60 and dsDNA. Arrows mark sites where nucleoids are present.

(E) Dual-color STED nanoscopy of cells labeled for COX8A-SNAP and immunolabeled for Mic60. Arrows mark Mic60 clusters in the absence of fully developed lamellar cristae.

Data information: Scale bars: 1 µm.
MICOS assembly controls mitochondrial inner membrane remodeling and crista junction redistribution to mediate cristae formation (2020)
Figure 2

Figure 2. Characterization of MICOS knockout cell lines.

(A) Composition of MICOS in WT cells and MICOS mutants. Mic60 specific antibodies were used to pull-down Mic60 and interacting proteins from insolated mitochondria. ** Unspecific band, due to the cross reaction of the anti-Mic25 antibody with Mic19.

(B) Oxygen consumption rate (OCR) of WT cells and MICOS mutants as analyzed by Seahorse Analyzer. Basal and maximal OCR are shown relative to the WT. Error bars: SEM, n=6.

(C) Mitochondrial networks of WT and MICOS mutant cells. Cells were immunolabeled for TOM20 and visualized by confocal microscopy. The inset shows a magnification of the respective overview image. Scale bar: 10 µm.

(D) Quantification of the mitochondrial networks as shown in (C). The evaluation was performed manually in a blinded approach based on pre-defined morphology criteria. Average and SD of 3 independent biological replicates are shown (>170 cells per sample and replicate).
MICOS assembly controls mitochondrial inner membrane remodeling and crista junction redistribution to mediate cristae formation (2020)
Figure 3

Figure 3. Depletion of MICOS subunits affects the formation of lamellar cristae.

(A) Representative TEM recordings of WT cells and MICOS-KO mutants, as indicated.

(B) Quantification of the overall cristae morphology on TEM recordings.

(C) Quantification of detailed morphology of CMs in MICOS mutants (left). TEM recordings were assigned to 8 classes (right).

(D) CJ frequency on TEM recordings. The number of CJs was manually determined and normalized to the length of the OM. Samples were compared to WT by a One-way ANOVA test. Error bars: SEM.

(E) Diameter of CJs. CJs were manually measured on TEM recordings. Red line: Average. Error bars: SD. Samples were compared to WT by a One-way ANOVA test.

(F) Representative ET reconstructions of mitochondria from a WT cell (left), a Mic10-KO cell (center) and a Mic60-KO cell (right). The OM is displayed in clear grey, the side of the IM that faces the matrix is shown in dark blue. The IM side that faces the IMS is shown in light blue.

Data information: n: number of mitochondrial sections (B,C,E) or number of CJs (D). ***: p≤0.001. Scale bars: 500 nm (A), 250 nm (F).
MICOS assembly controls mitochondrial inner membrane remodeling and crista junction redistribution to mediate cristae formation (2020)
Figure 4

Figure 4. Depletion of Mic10 causes formation of large tube-like cristae and opposite distribution bands of Mic60 and ATPB.

(A,B) Nanoscopy of mitochondria of WT and Mic10-KO cells, as indicated. (A) Mitochondria were immunolabeled for Mic60 and ATPB and visualized by 2D STED nanoscopy. (B) Mitochondria were immunolabeled for TOM20 and ATPB and recorded with 3D STED nanoscopy. Image data were deconvolved. Depicted are cross-sections of a mitochondrion (side view).

(C) 3D MINFLUX nanoscopy. Mic10-KO cells were immunolabeled for Mic60 using a directly labeled antibody. Colors encode depth information.

(D) 3D SIM of living WT and Mic10-KO cells. Cells were labeled with Mitotracker Green. Images show maximum intensity projections.

(E) FIB-SEM of Mic10-KO cells. Shown is a representative reconstructed mitochondrion. Dashed box: Orthoslice view on the smaller mitochondrion. The OM and IBM are displayed together in clear grey, the CM in is shown in blue.

(F) 2D STED nanoscopy of mitochondria. Mic19-KO cells were transfected with a plasmid for Mic10-FLAG expression under the control of a tetracycline-inducible promoter. Left panel: Mitochondria from noninduced cell. Right: Mitochondria from induced cell expressing Mic10-FLAG.

Data information: Scale bars: 1 µm (A,C,D,F), 500 nm (B, E).
MICOS assembly controls mitochondrial inner membrane remodeling and crista junction redistribution to mediate cristae formation (2020)
Figure 5

Figure 5. Mic60 controls MICOS protein levels, CJs frequency and inner membrane morphology. Rescue of Mic60 expression in Mic60-TO cells upon induction with doxycycline hyclate.

(A) Cells were immunolabeled for Mic60 and DNA and visualized by confocal fluorescence microscopy before and after induction of Mic60 expression for 48 h.

(B) Protein levels of MICOS proteins after Mic60 induction. Cell lysates were analyzed by western blotting at the indicated time points.

(C) Recovery of mitochondrial networks upon Mic60 re-expression. Cells were induced with doxycycline hyclate for 72 h, immunolabeled for TOM20 and visualized by confocal microscopy.

(D-G) TEM of Mic60-TO cells before and after induction. (D) TEM recordings of Mic60-TO cells at the indicated time points. (E) Quantification of the cristae morphology. (F) CJ frequency. The number of CJs on TEM recordings was normalized to the length of the OM. At least 90 mitochondrial sections were analyzed for each sample. (G) Quantification of the cristae morphology of Mic60-TO cells. Only mitochondria exhibiting at least one CJ were analyzed.

Data information: n: Number of mitochondrial sections. Scale bars: 20 µm (A), 10 µm (C), 500 nm (D).
MICOS assembly controls mitochondrial inner membrane remodeling and crista junction redistribution to mediate cristae formation (2020)
Figure 6

Figure 6. The Mic10-subcomplex is essential for coordination of lamellar cristae formation.

(A,B) Live-cell nanoscopy of Mic10-TO cells. Cells expressing COX8A-SNAP were labeled with SNAP-cell SiR and recorded with STED nanoscopy. (A) Cristae architecture in noninduced cells. (B) Cristae architecture after Mic10 re-expression (24 h induction with doxycycline). STED image data were deconvolved.

(C) STED nanoscopy of fixed Mic10-TO cells. Cells were induced for 24 h and immunolabeled for Mic60 and Mic10-FLAG. Upper row: Cell with weak Mic10 expression level. Lower row: Cell with strong Mic10 expression level.

(D-G) TEM of Mic10-TO cells. (D) Cells induced for the indicated period of time were recorded with TEM. Arrow indicates an intermediately shaped crista. (E) Quantification of the cristae morphology. n: Number of mitochondrial sections. (F) CJ frequency in Mic10-TO cells. Number of CJs was normalized to the length of the OM. The same sections were analyzed as in (E). (G) Diameter of CJs estimated on TEM recordings.

Data information: n: Number of analyzed mitochondrial sections (E) or CJs (G).. Error bars: SD. Scale bars: 10 µm (A, B, conf), 1µm (A, B, C), 0.5 µm (D).
MICOS assembly controls mitochondrial inner membrane remodeling and crista junction redistribution to mediate cristae formation (2020)
Figure 7

Figure 7. MICOS holo-complex assembly induces remodeling of the mitochondrial inner membrane.

(A) FIB-SEM of Mic10-TO cells. Cells were induced for 0 h, 16 h and 24 h. The representative reconstructions show mitochondria from noninduced, rescued and intermediate cells, as indicated. The OM is shown together with the IBM in clear grey, the CM in blue.

(B) ET of Mic10-TO cells. Mic10 expression was induced for 0 h and 16 h. Shown are reconstructions of representative noninduced and almost completely rescued mitochondria. The OM is displayed in clear grey, the side of the IM that faces the matrix is shown in dark blue. The IM side that faces the IMS is shown in light blue.

Data information: Scale bars: 500 nm.
MICOS assembly controls mitochondrial inner membrane remodeling and crista junction redistribution to mediate cristae formation (2020)
Figure 8

Figure 8. OPA1 influences the inner membrane morphology.

Knockdown (KD) of OPA1 in HeLa cells. WT and Mic60-KO cells were transfected with a scrambled control (Ctrl.) or siRNA pools against OPA1 (OPA1-KD) for 5 d.

(A) Cell lysates were analyzed by western blotting.

(B) TEM recordings from OPA1 depleted WT and Mic60-KO cells. Arrows point to a septum.

(C,D) Quantification of the cristae morphology of OPA1 deficient WT (C) and Mic60-KO (D) cells. The number of CJs was normalized to the length of the OM (C). The number of septated mitochondria in Mic60-KO cells was estimated (D).

(E,F) ET of mitochondria from OPA1-KD cells. WT cells were transfected with a scrambled control (E) or siRNA pools against OPA1 (F) for 48 h. Reconstructions of representative mitochondria are displayed. The OM is displayed in clear grey, the side of the IM that faces the matrix is shown in dark blue. The IM side that faces the IMS is shown in light blue.

Data information: Scale bars: 500 nm (B), 250 nm (E,F).
MICOS assembly controls mitochondrial inner membrane remodeling and crista junction redistribution to mediate cristae formation (2020)
Figure 9

Figure 9. OPA1 affects the Mic60 distribution and stabilizes tubular CJs.

(A-C) WT and Mic10-KO cells were transfected with a scrambled control (Ctrl.) or siRNA pools against OPA1 (OPA1-KD) for 72 h. (A,B) STED nanoscopy of OPA1 depleted WT (A) and Mic10-KO (B) cells. Cells were immunolabeled for Mic60 and ATPB. (C) TEM of OPA1 depleted Mic10-KO cells. Left: Representative TEM recordings showing CJs marked by arrows in a control cell and a similar view for an OPA1-KD cell. The CMs are colored in green and the OM and IBM together in magenta. Right: Quantification of mitochondria containing CJs or septa junctions. Sections that contained CJ-like structures were analyzed. Of these, the number of sections exhibiting septa or cristae structures (other) was quantified. (D, E) Depletion of ATP5ME in HeLa cells. Cells were transfected with a scrambled control or siRNA pools against ATP5ME for 4 d.

(D) TEM recording (left) and quantification of the number of cristae per mitochondrial section (right).

(E) Cells depleted of ATP5ME were immunolabeled for Mic60 and recorded with STED. The arrows point to crossing points of the opposite Mic60 distribution bands.

Data information: n: Number of mitochondrial sections analyzed. Boxes indicate 25th to 75th percentile. Horizontal lines indicates median. Whiskers indicate SD. Mann-Whitney test was used to compare samples. ***: p≤0.001. Scale bars: 500 nm (A,B,D), 100 nm (C), 1 µm (E).