Journal: Nature cell biology

Article Title: Endosomal Membrane Tension Regulates Escrt-III-Dependent Intra-Lumenal Vesicle Formation

doi: 10.1038/s41556-020-0546-4

Figure Lengend Snippet: a) Maximum projection of confocal images of HeLa-CHMP4B-GFP cells expressing mCherry-Rab5Q79L before and after a 20min hypertonic shock. Image representative of 3 experiments. Bars: 5 μm. b) Average volume of Rab5Q79L endosomes (red curve, shaded area is SD) and average intensity of CHMP4B-GFP (green curve, shaded area is SD) after hypertonic shock (time 0), and normalized to initial value. (shaded areas are SD from N=6 representative RAB5Q79L endosomes). c) Volumes of single RAB5Q79L endosomes before (black) and after a 10min hypertonic shock (red) (N=30 endosomes from 3 independent replicates, two-tailed Wilcoxon test: P=0.0000000019). d) Volumes of single MDA-MB-231 endosomes labeled with FM4-64 before (black) and after (red) a 10min hypertonic shock. (N=52 endosomes from 3 independent replicates, two-tailed Wilcoxon test: P<10 -15 ). e) FLIM images of FliptR-labelled mCherry-RAB5Q79L endosomes in live HeLa MZ cells before and after a 10min hypertonic shock. Bars: 5 μm. f) FliptR lifetime measurements from 4 independent experiments as shown in (e). Thin coloured lines: single experiments; thick red line: mean±SEM of the 4 experiments (two-tailed paired t-test: P=0.0003338778). g) FLIM images of HeLa endosomes labelled with Lyso Flipper before and after 10min hypertonic shock. Bars: 10 μm. h) Lyso Flipper lifetime measurements from (g) before and after hypertonic shock. Thin coloured lines: 5 independents experiments; thick red line: mean with SEM (two-tailed paired t-test: P=0.00000884003). In f and h for each experiment, average fluorescent lifetimes were calculated from >500 endosomes taken from at least 3 different cells. i) Representative FLIM images of endosomes in MDA cells stained with Lyso-Flipper before and after hypertonic shock. Image representative of 3 experiments. Bars: 10 μm j) The graph shows the quantification of these experiments where each thin line is an individual experiment (with at least 3 cells and several dozens of endosomes) and the thick line the mean of these 3 experiments. Error bars represent SEM. (two-tailed paired t-test: P= 0.0217491697).

Article Snippet: Our HeLa-MZ, HeLa Kyoto and MDA MB 431 cells were authenticated by Microsynth (Balgach, Switzerland), and are mycoplasma-negative as tested by GATC Biotech (Konstanz, Germany).

Techniques: Expressing, Two Tailed Test, Labeling, Staining

Journal: bioRxiv

Article Title: Grubraw, a chemogenetic generator of mitochondrial pyruvate, reveals new mechanisms of mitochondrial metabolic control that underlie the Warburg effect

doi: 10.1101/2023.04.18.537329

Figure Lengend Snippet: a , The conversion of D-alanine to pyruvate catalyzed by DadA. b , Fluorescence image of HeLa Kyoto cells expressing mito-mRuby-DadA stained with mitoTracker Green. c , Fluorescence image of HeLa Kyoto cells expressing mito-mRuby-DadA and Micos60-HyPer7. d , A representative fluorescence intensity profile along a cross-section of a mitochondrion from c. e , Mitochondrial pyruvate concentration in the presence of 120 μM CPI-613 by Pyrates sensor localized in the mitochondria. n = 14 – 15. f , Cytosolic pyruvate concentration in the presence of 120 μM CPI-613 by Pyrates sensor localized in the cytosol. n = 6. g , Mitochondrial membrane potential by TMRM. n = 12 – 23. h , The effect of Grubraw activity on OCR in HBSS medium without glucose. i , The effect of Grubraw activity on OCR in DMEM medium with 5 mM glucose. j , The effect of Grubraw activity on intracellular TCA cycle metabolites. Mean normalized values ±SD are shown k , ROS production by HyPer 7 sensor localized in the mitochondria. n = 8. l , Mitochondrial membrane potential by TMRM in presence of 100 μM rotenone. n =21. m , Mitochondrial matrix pH by Sypher3s sensor localized in the mitochondria. n = 18 – 20. Scale bar represents 25 μm in b and 5 μm in c . In e, f, k, m , red lines are for HeLa Kyoto cells expressing mRuby-Grubraw and blue lines for the cells expressing mRuby-Grubraw-mut. In g, l , red lines are for HeLa Kyoto cells expressing EGFP-Grubraw and blue lines for the cells expressing EGFP-Grubraw-mut. In e, f, g, h, i, k, l, m , mean or mean normalized values ±SD for n cells are shown for every time point.

Article Snippet: For HeLa Kyoto cells, FuGENE HD Transfection Reagent (Promega) was used.

Techniques: Fluorescence, Expressing, Staining, Concentration Assay, Membrane, Activity Assay

Journal: bioRxiv

Article Title: Grubraw, a chemogenetic generator of mitochondrial pyruvate, reveals new mechanisms of mitochondrial metabolic control that underlie the Warburg effect

doi: 10.1101/2023.04.18.537329

Figure Lengend Snippet: a , NAD+/NADH ratio by SoNar sensor. n = 14 – 18 cells. b , Glycolysis rate by FLII12Pglu-700μΔ6 glucose sensor. n = 14 cells. c , The effect of Grubraw activity on extracellular lactate. d , The effect of Grubraw activity on extracellular pyruvate in presence and absence of 11 mM glucose. e , Possible biochemical pathways for export of pyruvate from the mitochondrial matrix into the extracellular medium. The key enzymes and transporters are labeled in purple letters, and the metabolites are labeled in black letters. f , The effect of Grubraw activity on extracellular pyruvate. MPC activity was inhibited by 10 μM UK-5099 in presence and absence of 11 mM glucose. g ,.The effect of Grubraw activity on extracellular pyruvate. PDH1a, ME1, and PC were downregulated by RNA interference. The control cells expressed a scrambled shRNA. In all samples, 11 mM glucose was present in the medium. h , The effect of Grubraw activity on extracellular pyruvate. MCT 1,2 and/or MCT4 activity was inhibited using 0.5 μM AR-C155858 and RNA interference, respectively. In all samples, 11 mM glucose was present in the medium In a and b , red lines are for HeLa Kyoto cells expressing mRuby-Grubraw and blue lines are for cells expressing mRuby-Grubraw-mut. Mean or mean normalized values ±SD for n cells are shown at every time point. The data in c, d, f, h represent mean pyruvate or lactate quantity in the medium normalized to protein quantity in the sample ±SD. In plot g , these values were normalized to the corresponding value in the “Grubraw-mut - D-alanine” sample. Mean values ±SD are shown. *p<0.05

Article Snippet: For HeLa Kyoto cells, FuGENE HD Transfection Reagent (Promega) was used.

Techniques: Activity Assay, Labeling, Control, shRNA, Expressing

Journal: bioRxiv

Article Title: Grubraw, a chemogenetic generator of mitochondrial pyruvate, reveals new mechanisms of mitochondrial metabolic control that underlie the Warburg effect

doi: 10.1101/2023.04.18.537329

Figure Lengend Snippet: a , The effect of Grubraw activity on extracellular pyruvate in a panel of cancer cell cultures. For reference, the data for HeLa Kyoto cells from are present. b , The effect of Grubraw activity on extracellular lactate in melanoma cultures. For reference, the data for HeLa Kyoto cells from are present. c , The effect of Grubraw activity on OCR in WM164 cells. d , The effect of Grubraw activity on OCR in Lu451 cells. Data in a and b represent mean pyruvate or lactate quantity in the medium normalized to protein quantity in the sample ±SD. *p<0.05, **p<0.005,***p<0.0005, ****p<0.00005 In c and d , mean OCR normalized to the corresponding OCR in the zero time point ±SD is shown at every time point for 2-3 biological replicates for every sample.

Article Snippet: For HeLa Kyoto cells, FuGENE HD Transfection Reagent (Promega) was used.

Techniques: Activity Assay

Journal: Nature Communications

Article Title: Zinc homeostasis governed by Golgi-resident ZnT family members regulates ERp44-mediated proteostasis at the ER-Golgi interface

doi: 10.1038/s41467-023-38397-6

Figure Lengend Snippet: a Localization of Halo-tagged marker proteins in Golgi mini-stacks. HeLa Kyoto cells transfected with indicated Halo-proteins were incubated with HTL-TMR (red), treated with nocodazole, and fixed. Fixed cells were co-stained for cis -Golgi (GM130; blue) and medial-Golgi (giantin; green). Fluorescence images were obtained by Airyscan super-resolution microscopy. Scale bars, 500 nm. Similar results were obtained from two independent experiments. b HeLa Kyoto cells were prepared as in ( a ) and co-stained for cis -/medial-Golgi (mannosidase II, ManII; green) and TGN (Golgin97; blue). Fluorescence images were obtained using Airyscan2 super-resolution microscopy. Scale bars, 500 nm. Similar results were obtained from two independent experiments. c Schematic representation of the localization of Halo-tagged Golgi marker proteins. d Labile Zn 2+ concentration ([Zn 2+ ]) in the Golgi cisternae measured by ZnDA-1H. HeLa Kyoto cells were transfected with the indicated siRNAs and Halo-tagged Golgi marker proteins. [Zn 2+ ] under each knockdown condition was determined as described in Methods . A circular dot indicates each datapoint, and a rectangle dot indicates the median of each experiment. Data for Halo-ERGIC-53 (siCont, n = 23 cells; siZnT4, n = 35 cells; siZnT5 + 6, n = 25 cells; siZnT7, n = 30 cells; siZnT4 + 5 + 6 + 7, n = 33 cells), ManII-Halo (siCont, n = 24 cells; siZnT4, n = 28 cells; siZnT5 + 6, n = 27 cells; siZnT7, n = 27 cells; siZnT4 + 5 + 6 + 7, n = 27 cells), and GalT-Halo (siCont, n = 36 cells; siZnT4, n = 25 cells; siZnT5 + 6, n = 33 cells; siZnT7, n = 26 cells; siZnT4 + 5 + 6 + 7, n = 38 cells) were obtained from 3 independent experiments. Data for TPST2-Halo (siCont, n = 36 cells; siZnT4, n = 33 cells; siZnT5 + 6, n = 50 cells; siZnT7, n = 40 cells; siZnT4 + 5 + 6 + 7, n = 40 cells) were obtained from 4 independent experiments. One-way ANOVA followed by Tukey’s multiple comparison test was used for statistical analysis. Source numerical data are provided in the Source Data file.

Article Snippet: HeLa Kyoto cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Nacalai Tesque) supplemented with 10% fetal bovine serum (FBS; Thermo Fisher Scientific, or Nichirei Biosciences Inc.).

Techniques: Marker, Transfection, Incubation, Staining, Fluorescence, Super-Resolution Microscopy, Concentration Assay, Knockdown, Comparison

Journal: Nature Communications

Article Title: Zinc homeostasis governed by Golgi-resident ZnT family members regulates ERp44-mediated proteostasis at the ER-Golgi interface

doi: 10.1038/s41467-023-38397-6

Figure Lengend Snippet: a HeLa Kyoto cells expressing Halo-ERGIC-53 were labeled with 5 nM HTL-TMR for 30 min, and treated with 33 µM nocodazole for 4 h. Cells were then fixed and immunostained for GM130 and ZnT6 or ZnT7. Fluorescence images were acquired by using Airyscan super-resolution microscopy. Scale bars, 500 nm. Images are representative of three independent experiments. See also Supplementary Movies and for 3D projection images. b The relative localization of ZnT6 or ZnT7 compared to the Golgi markers, GM130 ( cis -Golgi) and Halo-ERGIC-53 (pre- cis -Golgi), was classified into 5 groups as indicated. Ninety Golgi mini-stacks were counted from 6 cells in 2 independent experiments. c HeLa Kyoto cells expressing TPST2-Halo were treated and immunostained as in ( a ). Images are representative of three independent experiments. See also Supplementary Movies and for 3D projection images. d The relative localization of ZnT6 or ZnT7 compared to the Golgi markers, GM130 ( cis -Golgi) and TPST2-Halo (medial Golgi), was classified into 5 groups as indicated. ≥ 75 Golgi mini-stacks were counted from ≥ 5 cells in 2 independent experiments. Source numerical data are provided in the Source Data file.

Article Snippet: HeLa Kyoto cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Nacalai Tesque) supplemented with 10% fetal bovine serum (FBS; Thermo Fisher Scientific, or Nichirei Biosciences Inc.).

Techniques: Expressing, Labeling, Fluorescence, Super-Resolution Microscopy

Journal: Nature Communications

Article Title: Zinc homeostasis governed by Golgi-resident ZnT family members regulates ERp44-mediated proteostasis at the ER-Golgi interface

doi: 10.1038/s41467-023-38397-6

Figure Lengend Snippet: a Representative images of ERp44 (green) and GM130 (magenta) in HeLa Kyoto cells transfected with indicated siRNAs. Arrowheads indicate the accumulation of ERp44 in the Golgi area. Scale bars, 10 µm. b Quantitative analysis of Pearson’s correlation coefficients of the co-localization of endogenous ERp44 and GM130 based on the immunofluorescence images shown in ( a ). Circular dots indicate each datapoint, and rectangle dots indicate the median of each experiment. Data from 3 independent experiments (siCont, n = 118 cells; siZnT4, n = 120 cells; siZnT5 + 6, n = 119 cells; siZnT7, n = 124 cells; siZnT4 + 5 + 6 + 7, n = 116 cells) were statistically analyzed by one-way ANOVA followed by Dunnett’s multiple comparison test. c Knockdown/rescue experiments for ZnT4, ZnT5/6, or ZnT7. HeLa Kyoto cells pretreated with siRNAs against ZnT4, ZnT5, ZnT6, and ZnT7 were transfected with siRNA-resistant (si-resistant) ZnT4-FLAG, PA-ZnT5 and ZnT6-FLAG, or PA-ZnT7. Cells were then fixed and immunostained for ERp44, GM130, FLAG, and PA. Magenta asterisks indicate exogenously expressed ZnT-positive cells. Scale bars, 10 µm. d Quantitative analysis of Pearson’s correlation coefficients of the co-localization of endogenous ERp44 and GM130 based on the immunofluorescence images shown in ( c ). Circular dots indicate each datapoint, and rectangle dots indicate the median of each experiment. Data from 3 independent experiments (siCont, n = 119 cells; siZnT4, n = 119 cells; siZnT5 + 6, n = 132 cells; siZnT7, n = 109 cells; siZnT4 + 5 + 6 + 7, n = 120 cells) were statistically analyzed by one-way ANOVA followed by Tukey’s multiple comparison test. Source numerical data are provided in the Source Data file.

Article Snippet: HeLa Kyoto cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Nacalai Tesque) supplemented with 10% fetal bovine serum (FBS; Thermo Fisher Scientific, or Nichirei Biosciences Inc.).

Techniques: Transfection, Immunofluorescence, Comparison, Knockdown

Journal: Nature Communications

Article Title: Zinc homeostasis governed by Golgi-resident ZnT family members regulates ERp44-mediated proteostasis at the ER-Golgi interface

doi: 10.1038/s41467-023-38397-6

Figure Lengend Snippet: a Schematic model of the ZnT7 membrane topology. His and Asp residues on TM2 and TM5 involved in Zn 2+ coordination are indicated. b Immunofluorescence images showing the intracellular localization of ERp44, GM130, and PA-ZnT7. HeLa Kyoto cells were transfected with siRNAs against ZnT4, ZnT5, ZnT6, and ZnT7 simultaneously or control siRNAs. After 24 h incubation, cells were further transfected with PA-tagged siRNA-resistant ZnT7(WT), (ADHA), or (AAAA) and cultured for an additional 24 h. Cells were fixed and immunostained for ERp44 (green), GM130 (red), and PA (blue). Green asterisks indicate PA-ZnT7-positive cells. Scale bars, 10 µm. c Quantitative analysis of Pearson’s correlation coefficients of the co-localization of endogenous ERp44 with GM130 based on the immunofluorescence images shown in ( b ). Only PA-positive cells were analyzed. Dots indicate individual data points obtained from 2 independent experiments (siCont + Mock, n = 64 cells; siZnT4 + 5 + 6 + 7 + Mock, n = 76 cells; siZnT4 + 5 + 6 + 7 + WT, n = 88 cells; siZnT4 + 5 + 6 + 7 + ADHA, n = 82 cells; siZnT4 + 5 + 6 + 7 + AAAA, n = 79 cells). One-way ANOVA followed by Tukey’s test was used for statistical analysis. Bars indicate the mean. d Effects of ZPT treatment in ZnT knockdown cells. HeLa Kyoto cells transfected with indicated siRNAs were treated with DMSO or 2.5 µM ZPT for 15 min and immunostained for ERp44 (green) and GM130 (red). Scale bars, 10 µm. e Quantitative analysis of Pearson’s correlation coefficients of ERp44 and GM130 based on the images shown in ( d ). Circular dots indicate each datapoint, and rectangle dots indicate the median of each experiment ( N = 3 biological replicates). One-way ANOVA followed by Tukey’s multiple comparison test was used for statistical analysis. Source numerical data are provided in the Source Data file.

Article Snippet: HeLa Kyoto cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Nacalai Tesque) supplemented with 10% fetal bovine serum (FBS; Thermo Fisher Scientific, or Nichirei Biosciences Inc.).

Techniques: Membrane, Immunofluorescence, Transfection, Control, Incubation, Cell Culture, Knockdown, Comparison

Journal: Nature Communications

Article Title: Zinc homeostasis governed by Golgi-resident ZnT family members regulates ERp44-mediated proteostasis at the ER-Golgi interface

doi: 10.1038/s41467-023-38397-6

Figure Lengend Snippet: a Whole-cell lysates that had been precipitated with TCA and treated with NEM were resolved by SDS-PAGE and analyzed by immunoblotting with anti-ERp44 and anti-GAPDH antibodies under reducing (left) and non-reducing (right) conditions. b The signal intensity of disulfide-linked oligomers involving ERp44 relative to that of GAPDH was quantified. Data are the means ± SEM ( N = 3 biological replicates). Dots indicate the individual data points. One-way ANOVA followed by Dunnett’s test was used for statistical analysis. c , d Effects of ZnT-silencing on the intracellular retention of ERp44. HeLa Kyoto cells pretreated with siRNAs against indicated ZnT members were transfected with Halo-ERp44. After 36 h incubation, cells were washed twice and incubated in Opti-MEM for additional 6 h. TCA-precipitated conditioned media and whole cell lysates were resolved by reducing SDS-PAGE and analyzed by immunoblotting using anti-Halo antibody. The signal intensity of Halo-ERp44 secreted out of cells relative to that of whole cell lysates was quantified and shown in ( d ). Data are the means ± SEM ( N = 5 biological replicates). Dots indicate the individual data points. One-way ANOVA followed by Dunnett’s test was used for statistical analysis. e – j Effects of ZnT-silencing on the client-retention activity of ERp44. HeLa Kyoto cells pretreated with siRNAs against indicated ZnT members were transfected with ERAP1-FLAG ( e ), Ero1α (inactive)-FLAG ( g ) or Prx4-3×FLAG ( i ). Secreted ERAP1-FLAG or Ero1α (inactive)-FLAG was harvested from conditioned media (CM) by immunoprecipitation using anti-DYKDDDDK antibody agarose beads. Secreted Prx4-3×FLAG was concentrated by TCA precipitation. Precipitants and whole cell lysates (WCL) were analyzed by immunoblotting. The relative levels of secreted ERAP1-FLAG, Ero1α (inactive)-FLAG and Prx4-3×FLAG quantified by densitometry are shown in ( f ), ( h ), and ( j ), respectively. Data are the means ± SEM ( N = 4 biological replicates for ( f ) and ( h ), and N = 3 biological replicates for ( j )). Dots indicate the individual data points. One-way ANOVA followed by Dunnett’s test was used for statistical analysis. Source numerical data and unprocessed blotting images are provided in the Source Data file.

Article Snippet: HeLa Kyoto cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Nacalai Tesque) supplemented with 10% fetal bovine serum (FBS; Thermo Fisher Scientific, or Nichirei Biosciences Inc.).

Techniques: SDS Page, Western Blot, Transfection, Incubation, Activity Assay, Immunoprecipitation, TCA Precipitation

Journal: Nature Communications

Article Title: Zinc homeostasis governed by Golgi-resident ZnT family members regulates ERp44-mediated proteostasis at the ER-Golgi interface

doi: 10.1038/s41467-023-38397-6

Figure Lengend Snippet: a Scheme of the pRUSH-ST-SBP-EGFP construct. b Representative snapshots of the time-lapse imaging in the RUSH assay of ST-SBP-EGFP. Fluorescence images were acquired every 1 min. Upper and lower panels indicate the signals of ST-SBP-EGFP and ManII-Halo (Golgi marker), respectively. Biotin addition time is set as 0 min. Scale bar, 10 µm. c Ratios of intensities of GFP and TMR signals were plotted as a function of observation time (black circles). A fitting curve was calculated from the plots and indicated as a purple line. The t 1/2 value was calculated based on the fitting curve (see Methods for more details). d t 1/2 values of ST-SBP-EGFP in HeLa Kyoto cells transfected with indicated siRNAs. Circles indicate individual data points obtained from 2 independent experiments (siCont, n = 23 cells; siZnT4, n = 13 cells; siZnT5 + 6, n = 21 cells; siZnT7, n = 13 cells; siZnT4 + 5 + 6 + 7, n = 19 cells), and bars indicate medians. One-way ANOVA followed by Dunnett’s test was used for statistical analysis. e Scheme of the pRUSH-SBP-Halo-ERp44 constructs. f Representative snapshots of the time-lapse imaging in the RUSH assay of SBP-Halo-ERp44. Fluorescence images were acquired as in ( b ) Upper and lower panels under each condition indicate the signals of SBP-Halo-ERp44 and ManII-pHluorin2 (Golgi marker), respectively. Scale bars, 10 µm. See also Supplementary Movies and . g Ratios of intensities of TMR and pHluorin2 signals were plotted as a function of observation time (black circles). A fitting curve was calculated from the plots, and indicated as a blue line. The t 1/2 and initial slope (v 0 ) values were estimated based on the fitting curve (see Methods for more details). h , i t 1/2 and v 0 values of SBP-Halo-ERp44. Circles indicate individual data points obtained from 2 independent experiments (siCont, n = 34 cells; siZnT5 + 6, n = 24 cells; siZnT7, n = 18 cells; siZnT4 + 5 + 6 + 7, n = 23 cells), and bars indicate the median. One-way ANOVA followed by Dunnett’s test was used for statistical analysis. Source numerical data are provided in the Source Data file.

Article Snippet: HeLa Kyoto cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Nacalai Tesque) supplemented with 10% fetal bovine serum (FBS; Thermo Fisher Scientific, or Nichirei Biosciences Inc.).

Techniques: Construct, Imaging, Fluorescence, Marker, Transfection