Journal: Nature cell biology

Article Title: Non-canonical function of IRE1α determines mitochondria-associated endoplasmic reticulum composition to control calcium transfer and bioenergetics

doi: 10.1038/s41556-019-0329-y

Figure Lengend Snippet: a, IRE1α KO cells reconstituted with IRE1α–HA were processed to obtain purified MAM fractions followed by western blot analysis of indicated proteins (n = 3 independent experiments). H, homogenate; C, cytosol; Cr, crude mitochondria; M, MAMs; P, pure mitochondria; Cyt c, cytochrome c; CNX, calnexin. b, Liver extracts were processed to obtain subcellular fractions enriched for MAMs and analysed by western blot (n = 9 independent experiments). c,d, IRE1α KO cells reconstituted with IRE1α–HA or mock control were simultaneously imaged for calcium signals in the cytosol (Fura2; c) and mitochondria with Rhod2 (d). Left, the Fura2 ratio (c) and mean Rhod2 intensity (d) of normalized data before and after ATP is added; arrow, 100 μM ATP. Right, the data for the maximum peak are shown (total cells analysed: mock, n = 116 cells; IRE1α–HA, n = 138 cells). e,f, Similar experiments for Fura2 (e) and Rhod2 (f) were performed in CRISPR control and IRE1α KO cells (total cells analysed: control, n = 129 cells; IRE1α KO, n = 117 cells). WT, wild type. g, Indicated cell lines were processed for western blot analyses to monitor the levels of indicated proteins (n = 4 independent experiments). h, IRE1α null and control cells were imaged for calcium levels in mitochondria by transiently expressing CEPIA2mt mitochondrial calcium probe (left) after addition of 50 μM M3M3FBS (arrow), (Mito red; Mitochondrila Ds-Cherry control). Scale bars, 10 μm. Right, maximum CEPIA2mt intensity for every cell analysed (mock, n = 14 cells; IRE1α–HA, n = 14 cells). i, Maximum peaks from Fura2/Rhod2 measurements from samples described in c and d were calculated using nonlinear regression analyses to determine the correlation constant (K) and s.e.m. (mock, K = 0.199 ± 0.009; IRE1α–HA, K = 0.231 ± 0.01). j, Cells were imaged for calcium levels in the ER after loading with Mag-Fluo4 in permeabilized cells followed by stimulation with InsP3R (n = 5 independent experiments; left). Middle, percentage activity for InsP3R for each condition normalized to maximum release (ionomycin). Right, the first derivative was calculated. Data in c–f,h–j are mean ± s.e.m. Statistical differences were detected using two-tailed unpaired Student’s t-tests except for j; right, which was one-tailed. Source data for statistical analyses are provided in Supplementary Table 6.

Article Snippet: Alternatively, we generated CRISPR cells using a double nickase that was targeted to IRE1α or scrambled as a control (sc-429758-NIC and sc-437281; Santa Cruz).

Techniques: Purification, Western Blot, CRISPR, Expressing, Activity Assay, Two Tailed Test, One-tailed Test

Journal: Nature cell biology

Article Title: Non-canonical function of IRE1α determines mitochondria-associated endoplasmic reticulum composition to control calcium transfer and bioenergetics

doi: 10.1038/s41556-019-0329-y

Figure Lengend Snippet: a, Ern1 and Ern1ΔK liver samples were processed for metabolomics studies (n = 4 animals per group). The heat map for the metabolites indicates significantly different metabolite levels in each experimental animal. b, Pathway analysis and statistical significance (two-tailed Student’s t-test) for the metabolites shown in a. c, The affected pathways and main hits from a are indicated. Altered metabolites and their associated pathways are indicated using the same colour code (coloured dots, size stands for P value as in b)) in a–c. d, Whisker and dot plots of the indicated metabolites of the TCA, indicating median and quartiles derived from samples in a (n = 4 animals per group) levels represent the log2 of the normalized area in a.u. e, Schematic of the TCA cycle. Metabolites with increased or decreased levels in Ern1 and Ern1ΔK samples are indicated by arrows. f, Glucose tolerance test in Ern1 control and Ern1ΔK mice (left). Right, data represent the area under the curve (AUC) for the whole glucose tolerance test (n = 4 animals per group). g, Proposed model: IRE1α expressed at MAMs docks the InsP3Rs at the mitochondrial–ER contact sites—possibly through a physical interaction, which may enhance InsP3R channel activity. The presence of IRE1α at MAMs favours calcium transfer into the mitochondria and bursts in ATP production. IRE1α deficiency leads to a metabolic stress condition that is characterized by the constitutive activation of AMPK, enhanced compensatory autophagy and altered mitochondrial morphology. Data are mean ± s.e.m. Statistical differences were detected with one-tailed (d) or two-tailed Student’s t-tests (f) or two-way ANOVA (f). Source data for statistical analyses are provided in Supplementary Table 6.

Article Snippet: Alternatively, we generated CRISPR cells using a double nickase that was targeted to IRE1α or scrambled as a control (sc-429758-NIC and sc-437281; Santa Cruz).

Techniques: Two Tailed Test, Whisker Assay, Derivative Assay, Activity Assay, Activation Assay, One-tailed Test

Journal: Nature cell biology

Article Title: Non-canonical function of IRE1α determines mitochondria-associated endoplasmic reticulum composition to control calcium transfer and bioenergetics

doi: 10.1038/s41556-019-0329-y

Figure Lengend Snippet: a, IRE1α KO cells that were reconstituted with either IRE1α–HA or an empty vector (mock) were imaged for TMRM signals before and after addition of 1 μM FCCP (carbonyl cyanide-p-trifluoromethoxyphenylhydrazone) (left). Scale bar, 20 μm. Right, mean TMRM intensity normalized to IRE1α–HA cells (n = 6 independent experiments). b, CRISPR control and IRE1α KO cells were analysed as described in a (n = 4 independent experiments). c,d, Percentage of ATP of the indicated cells using a luminescence assay (n = 18 biologically independent samples). e,f, ATP levels were measured in the indicated cell lines using the AT01 mitochondrial (yellow fluorescent protein (YFP)/cyan fluorescent protein (CFP)) FRET probe FRET labeling stands for 440 nm excitation emmited in YFP channel. White numbers indicate regions of interest (left). Right, quantification of YFP/CFP ratio excited at 440 nm (mock, n = 52 cells; IRE1α–HA, n = 58 cells; control, n = 145 cells; IRE1α KO, n = 151 cells). Scale bars, 10 μm and 2 μm. g,h, The indicated cell lines were analysed for oxygen consumption rate (OCR). O, 1 μM oligomycin, F, 0.5 μM FCCP; A/R = 1 μM antimycin/rotenone (n = 4 independent experiments). i, pAMPK was analysed in the indicated cells using western blots (left) and normalized to total AMPK levels (right; n = 6 independent experiments). j, Determination of LC3-II levels in the indicated cell lines using western blots (left), followed by quantification normalizing to actin (right; n = 6 independent experiments). k, TEM-derived morphological parameters of mitochondria were obtained from indicated cells. Scale bar, 4 μm (left). Right, the data represent the area in μm2 and circularity (mock, n = 52 cells; IRE1α–HA, n = 58 cells). l, Cells were stained for ERp72 and TOM20 by indirect immunofluorescence (left) followed by colocalization quantification (right; Mander’s index: mock, n = 33 cells; IRE1α–HA, n = 40 cells; Pearson’s index: mock, n = 68 cells; IRE1α–HA, n = 78 cells). Scale bar, 20 μm and 5 μm. m, The indicated cells were imaged using TEM to visualize MAMs (pointed with red arrows) (left) using two quantification methods (right; mock, n = 38 contacts; IRE1α–HA, n = 30 contacts). Scale bars, 500 nm. Data in a–m are mean ± s.e.m. Statistical differences detected with one-tailed (k) or two-tailed unpaired Student’s t-tests. A Wilcoxon signed-rank test was applied in a–d and paired Student’s t-tests were applied in h,i,m (right panel). Source data for statistical analyses are provided in Supplementary table 6.

Article Snippet: Alternatively, we generated CRISPR cells using a double nickase that was targeted to IRE1α or scrambled as a control (sc-429758-NIC and sc-437281; Santa Cruz).

Techniques: Plasmid Preparation, CRISPR, Luminescence Assay, Labeling, Western Blot, Derivative Assay, Staining, Immunofluorescence, One-tailed Test, Two Tailed Test

Journal: Nature cell biology

Article Title: Non-canonical function of IRE1α determines mitochondria-associated endoplasmic reticulum composition to control calcium transfer and bioenergetics

doi: 10.1038/s41556-019-0329-y

Figure Lengend Snippet: a, Indicated cells were processed to obtain subcellular fractions and analysed using western blots. Right, quantification of the MAM fractions for the indicated proteins (InsP3R1, n = 4; InsP3R1, n = 8 independent experiments). b,c, Cells described in a were stained with a PLA (red) and DAPI (Blue) using anti-InsP3R1 (b) or anti-InsP3R3 (c) antibodies paired with anti-VDAC1 antibodies. Scale bars, 20 μm (left). Right, quantification of the number of positive PLA dots per cell (n = 3 independent experiments). d, Cells were imaged using TEM (left) to calculate ER to mitochondrial width (right; n = 3 independent experiments; mock, n = 46 contacts; IRE1α–HA, n = 30 contacts). Scale bars, 200 nm. e, CRISPR control and IRE1α KO cells were were imaged using TEM (left) to calculate ER to mitochondrial width (right; n = 3 independent experiments; CRISPR control, n = 27 contacts; CRISPR IRE1α KO, n = 47 contacts). Scale bars, 200 nm. f, The same cells as described in a were transiently transfected with SPLICSL to visualize MAMs with a width of 40–50 nm (left). Nuclei were stained with DAPI. Scale bar, 25 μm. Right, quantification of SPLICSL signal as dots per cell (n = 5 independent experiments; total cells analysed: mock, n = 41 cells; IRE1α–HA, n = 38 cells). g, Schematic representation and representative TEM images of indicated cells transiently expressing either a AKAP1 (34–63)-linker 9x-mRFP (9xL) construct or a control linker construct. Scale bar, 500 nm (top). MAM width was determined by TEM (bottom; mock control linker, n = 14 contacts; IRE1α–HA control linker, n = 12 contacts; mock 9xL, n = 15 contacts). h, Cells described in g were stained with PLA (green) and DAPI (blue) to measure the close proximity between InsP3R1 and VDAC1 proteins (left) in mRFP positive cells. Right, the number of dot counts per cell was quantified (n = 4 independent experiments) Scale bars, 20 μm. Data in a–h are mean ± s.e.m. Statistical differences were detected using one-way ANOVA and Tukey post-tests for multiple comparison (g,h), two-tailed Student’s t-tests (b–f) or Wilcoxon signed-rank test (a). Source data for statistical analyses are provided in Supplementary Table 6.

Article Snippet: Alternatively, we generated CRISPR cells using a double nickase that was targeted to IRE1α or scrambled as a control (sc-429758-NIC and sc-437281; Santa Cruz).

Techniques: Western Blot, Staining, CRISPR, Transfection, Expressing, Construct, Two Tailed Test

Journal: Nature cell biology

Article Title: Non-canonical function of IRE1α determines mitochondria-associated endoplasmic reticulum composition to control calcium transfer and bioenergetics

doi: 10.1038/s41556-019-0329-y

Figure Lengend Snippet: a, Schematic of IRE1α structure and the mutants analysed (left) (TM; transmembrane domain). Right, the indicated cell lines were treated with 0.1 μg ml−1 tunicamycin for 4 h and then Xbp1 mRNA splicing was evaluated using PCR analysis. The agarose gel image was sliced to eliminate irrelevant lanes. Xbp1u, unspliced Xbp1s, spliced (n = 2 independent experiments). b, Calcium levels in the cytosol after ATP stimulation were analysed in IRE1α KO cells reconstituted with the indicated constructs. Arrow, 100 μM ATP (left; Fura2; n = 4 independent experiments; total cells analysed: mock, n = 131 cells; IRE1α–HA, n = 149 cells; IRE1α-P830L–HA, n = 120 cells; IRE1α-ΔC–HA, n = 97 cells). The maximum peak for the normalized Fura2 ratio was measured (middle). The same cells were imaged simultaneously with Rhod2 to measure mitochondrial calcium uptake. Arrow, 100 μM ATP (right two panels). c–e, HEK293T cells were transiently transfected with the indicated constructs and immunoprecipitation (IP) was performed using anti-HA antibodies. Western blot (WB) analysis was performed for the indicated proteins in immunoprecipitations and total input (c, n = 3 independent experiments; d,e are representative of two independent experiments). f, The indicated MEF cell lines were processed for immunoprecipitation using anti-HA antibodies. Western blot analysis was performed for the indicated proteins in immunoprecipitations and total input. g, Cells described in f were stained for PLA (red) and DAPI (blue) using anti-InsP3R1 antibodies paired with anti-HA antibodies and analysed by confocal microscopy. Scale bar, 20 μm (left). Right, the number of dots per cell were quantified (n = 3 independent experiments). h, Schematic of InsP3R1 domains used to generate recombinant proteins and perform in vitro pull-down assays (left; the asterisk indicates that residues 167–169 and 267 are relevant for channel function). Right, in vitro pull-down assay for purified GST-fused domains of InsP3R1 with recombinant IRE1α cytosolic portion (IRE1α-ΔN) followed by western blot analysis (D1, domain 1; D2, domain 2; D3, domain 3; n = 3 independent experiments). Data in b and g are mean ± s.e.m. Statistical differences were detected using two-tailed unpaired Student’s t-test (g) or ANOVA with Tukey multiple comparison test (b). Source data for statistical analyses are provided in Supplementary Table 6.

Article Snippet: Alternatively, we generated CRISPR cells using a double nickase that was targeted to IRE1α or scrambled as a control (sc-429758-NIC and sc-437281; Santa Cruz).

Techniques: Agarose Gel Electrophoresis, Construct, Transfection, Immunoprecipitation, Western Blot, Staining, Confocal Microscopy, Recombinant, In Vitro, Pull Down Assay, Purification, Two Tailed Test

Journal: Nature cell biology

Article Title: Non-canonical function of IRE1α determines mitochondria-associated endoplasmic reticulum composition to control calcium transfer and bioenergetics

doi: 10.1038/s41556-019-0329-y

Figure Lengend Snippet: a, Strategy to generate CRa particles using the synergistic activator mediators and sgRNA complex. b, IRE1α KO cells were generated that stably express either a CRa that targets the InsP3R1 promoter or a control vector. Representative western blot analysis of the indicated proteins was performed to confirm InsP3R1 upregulation (n = 10 independent experiments). c, The cells described in a were stained with a PLA (red) DAPI (blue) using anti- InsP3R1 and anti-VDAC1 antibodies, and were analysed by confocal microscopy. Scale bar, 20 μm (left). Right, the number of dots per cell were quantified (n = 6 independent experiments). d, CRa-InsP3R1 or CRacontrol cells were imaged with Rhod2 to measure mitochondrial calcium uptake. Arrow, stimulation using 50 μM M3M3FBS (left). Right, the maximum peak for normalized Rhod2 was calculated (total cells analysed: CRa-InsP3R1, n = 46 cells; CRa-control, n = 42 cells). e, CRa-InsP3R1 or CRa-control cells were imaged for mitochondrial membrane potential after TMRM staining (left). Arrow, stimulation with 1 μM FCCP; AU, arbitrary units. Right, normalized TMRM intensity (n = 4 independent experiments). f, pAMPK and total AMPK levels were determined in CRa-InsP3R1 or CRa-control cells using western blot (left). Right, quantification of the pAMPK/AMPK ratio (n = 7 independent experiments). g, The indicated cells were lysed and ATP levels were determined using a luminescence assay (n = 13 biologically independent experiments). h, IRE1α KO cells were generated that stably express either a CRa that targets the InsP3R3 promoter or a control vector. Representative western blot analysis of the indicated proteins was performed to confirm InsP3R3 upregulation (n = 5 independent experiments). i, The indicated cells were imaged with Rhod2. Arrow, stimulation with 50 μM M3M3FBS (left). Right, the maximum peak for the normalized Rhod2 was calculated (total cells analysed: CRa-InsP3R1, n = 132 cells; CRa-control, n = 112 cells). j, ATP levels were determined in the indicated cells using a luminescence assay (n = 25 biologically independent experiments). Data in b–j are mean ± s.e.m. Statistical differences were detected with unpaired Student’s t-tests (c,d,g,i,j) or Wilcoxon signed-rank test (b,e,f,h). Source data for statistical analyses are provided in Supplementary Table 6.

Article Snippet: Alternatively, we generated CRISPR cells using a double nickase that was targeted to IRE1α or scrambled as a control (sc-429758-NIC and sc-437281; Santa Cruz).

Techniques: Generated, Stable Transfection, Plasmid Preparation, Western Blot, Staining, Confocal Microscopy, Luminescence Assay

Journal: Nature cell biology

Article Title: Non-canonical function of IRE1α determines mitochondria-associated endoplasmic reticulum composition to control calcium transfer and bioenergetics

doi: 10.1038/s41556-019-0329-y

Figure Lengend Snippet: a, Schematic of Ern1 structure (the gene encoding IRE1α) (TM; transmembrane domain) and the strategy to delete the kinase domain (Ern1ΔK; left). Middle, livers from Ern1 control and Ern1ΔK mice were processed for western blot analysis to measure the levels of indicated proteins (n = 3 independent experiments). Right, mice were intraperitoneally injected with 1 mg kg−1 of tunicamycin or vehicle for 6 h. Xbp1s mRNA splicing was evaluated by RT–PCR analysis of cDNA obtained from total liver extracts. b–f, Ern1 and Ern1ΔK livers were processed for TEM analysis (b) to determine morphological parameters including mitochondrial area (arrows indicate MAMs, scale bar, 500 nm) (c), MAM width (d), mitochondrial circularity (e) and MAM length (f; n = 4 animals per group). g, Ern1 and Ern1ΔK MAM fractions were processed for quantitative mass spectrometry analysis (see Methods). The volcano plot shows all of the detected proteins (grey) and those that are known to be present at MAMs (red dots; n = 3 animals per group). h, A summary of statistically significant hits observed in the proteomic screening of MAMs. i,j, Ern1 and Ern1ΔK liver samples were processed to obtain subcellular fractions, and were analysed by western blot for the indicated proteins (i). Quantification of protein expression was performed for the indicated proteins by normalizing to calnexin (CNX; j; Ern1, n = 9 animals; Ern1ΔK, n = 7 animals). k, The protein content (in mg) from liver MAM fractions (left) or pure mitochondria (right) was quantified and normalized by total liver extract (in g) to obtain the percentage of MAM proteins in the liver (Ern1, n = 6 animals; Ern1ΔK, n = 4 animals). l, Liver homogenates from Ern1 and Ern1ΔK were immunoprecipitated for IRE1α and analysed for the indicated proteins by western blot. Ab, antibody (representative of three independent experiments). Data in c–f,j and k are mean ± s.e.m. Statistical differences were detected using two-tailed unpaired Student’s t-tests (c,d). For j,k one-tailed Student’s t-tests were applied. Source data for statistical analyses are provided in Supplementary Table 6.

Article Snippet: Alternatively, we generated CRISPR cells using a double nickase that was targeted to IRE1α or scrambled as a control (sc-429758-NIC and sc-437281; Santa Cruz).

Techniques: Western Blot, Injection, Reverse Transcription Polymerase Chain Reaction, Mass Spectrometry, Expressing, Immunoprecipitation, Two Tailed Test, One-tailed Test