Mitochondrial temperature homeostasis resists external metabolic stresses
- Faculty of Medicine and Health Technology, Tampere University, Finland
- Université Paris Cité, Inserm, Maladies Neurodéveloppementales et Neurovasculaires, France
- SANKEN (The Institute of Scientific and Industrial Research), Osaka University, Japan
- Department of Chemistry, POSTECH, Republic of Korea
- Department of Environment and Genetics, La Trobe University, Australia
Figures
Figure 1
The mitochondrial oxidative phosphorylation (OXPHOS) system and inhibitors.
Summary of the major components of the OXPHOS system and classic inhibitors. Protonmotive OXPHOS enzyme complexes (cI, cIII, cIV, cV) shown in green, the non-protonmotive transgenically introduced …
Figure 2 with 5 supplements
Oxidative phosphorylation (OXPHOS) inhibitors decrease mitochondrial temperature to different extents.
Extrapolated mitochondrial temperature shifts (means ± SD for at least five independent experiments in each case), based on Mito Thermo Yellow (MTY) fluorescence in (A-E) five different cell lines …
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Figure 2—source data 1
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Figure 2—source data 2
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Figure 2—source data 3
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Figure 2—source data 4
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Figure 2—source data 5
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Figure 2—figure supplement 1
Instrumentation for mitochondrial temperature estimation using fluorescent probes.
Schematic illustration of spectrophotometer, QuantaMaster QM-6/2003 LPS-220B with a Peltier temperature-controlled sample cell chamber. Waterbath (Julabo CORIO CD, Germany) with refrigerated/heated …
Figure 2—figure supplement 2
Instrument validation steps for estimation of mitochondrial temperature.
(A) In an initial step, an external temperature probe was used to validate the temperature readings of components of the apparatus (water bath - WB, Peltier - pel, cuvette) as shown on the digital …
Figure 2—figure supplement 3
Fluorescence calibration steps for estimation of mitochondrial temperature.
(A) Sample traces showing calibration steps after fluorescence of the indicated inhibitor-treated cells had reached a steady value. Vertical axes are arbitrary values, horizontal axes (time) …
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Figure 2—figure supplement 3—source data 1
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Figure 2—figure supplement 4
Independence of Mito Thermo Yellow (MTY) fluorescence from parameters other than temperature.
Response of MTY fluorescence to changes in (A) calcium concentration, (B) hydrogen peroxide, and (C) pH over time (since first addition of supplement to PBS). Supplements were added sequentially to …
Figure 2—figure supplement 5
Supplementary data on effects of oxidative phosphorylation (OXPHOS) inhibitors on mitochondrial temperature.
(A) Representative fluorescence traces, uniformly rescaled, for Mito Thermo Yellow (MTY)-labelled iMEF(P) cells treated with the indicated combinations of OXPHOS inhibitors. Vertical scale (not …
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Figure 2—figure supplement 5—source data 1
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Figure 3 with 3 supplements
Ratiometric fluorescence probes confirm mitochondrial temperature measurements by Mito Thermo Yellow (MTY).
(A) Diagrammatic map (not to scale) of the self-cleaving polyprotein encoded by plasmid mito-gTEMP (coxVIIIx2-gTEMP)/pcDNA3, indicating the reiterated cytochrome oxidase subunit 8 (COX8) targeting …
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Figure 3—source data 1
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Figure 3—source data 2
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Figure 3—figure supplement 1
Mitochondrial localisation of mito-gTEMP.
Fluorescence microscopy images of iMEF(P) cells transfected with mito-gTEMP and imaged as indicated, as described in Materials and methods. Scale bars 10 μm.
Figure 3—figure supplement 2
Supplementary data on mitochondrially targeted temperature-sensitive fluorescent probes.
(A, B, D) Fluorescence traces (AU – arbitrary units) and (C, E) fluorescence ratios at the wavelengths used to measure mT-Sapphire and Sirius, in PBS at 38°C, to which were added the indicated drugs …
Figure 3—figure supplement 3
Idealised scheme for cellular/mitochondrial fractionation.
See Materials and methods for technical details. Cells are initially lysed by homogenisation under hypotonic conditions, and nuclei (blue), cytosol (grey), and membranous debris (black) are removed …
Figure 4
Mitochondrial temperature variation between and within cells.
(A, B) Micrographic images of iMEF cells stained with Mito Thermo Yellow (MTY) (A, panel i and B), alongside a parallel culture of cells stained with Mitotracker Deep Red FM (A, panel ii), which …
Figure 5 with 1 supplement
Mitochondrial temperature fluctuates in response to substrate availability.
(A, C, D, E) Representative fluorescence ratio traces of mito-gTEMP-expressing iMEF(P) cells resuspended in various media without oxidative phosphorylation (OXPHOS) inhibitors. PBS – …
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Figure 5—source data 1
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Figure 5—source data 2
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Figure 5—figure supplement 1
Effect of bongkrekic acid (BKA) on Mito Thermo Yellow (MTY) fluorescence.
MTY fluorescence traces for cell-free PBS or iMEF(P) cells treated with BKA as indicated. The drug produced an immediate quenching of fluorescence in both cases. In cells, this was followed by a …
Figure 6 with 2 supplements
Restoration of high mitochondrial temperature in alternative oxidase (AOX)-expressing cells after oxidative phosphorylation (OXPHOS) inhibition.
(A) Representative traces of Mito Thermo Yellow (MTY) fluorescence over time, in control and AOX-expressing iMEFs treated with the OXPHOS inhibitors shown. Vertical axes are arbitrary values, …
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Figure 6—source data 1
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Figure 6—source data 2
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Figure 6—figure supplement 1
Alternative oxidase (AOX)-expressing iMEFs show antimycin- and KCN-resistant respiration.
Respirometric traces from digitonin-permeabilised cells of the indicated lines, treated successively with digitonin – dig, pyruvate+glutamate+malate substrate mix – S, and ADP – A, followed by …
Figure 6—figure supplement 2
Supplementary data on effects of inhibitors on fluorescence in alternative oxidase (AOX)-expressing cells.
(A) Representative traces of mito-gTEMP fluorescence ratio for (i) control iMEF(P) and (ii) iMEF(AOX) cells, after rotenone delivery at 15 min until 35 min, with continuous recording (two replicate …
Figure 7 with 1 supplement
Cells maintain high mitochondrial temperature despite varying ATP demand.
(A, B) Intramitochondrial temperatures (means ± SD) estimated by mito-gTEMP fluorescence ratio in iMEF(P) and iMEF(alternative oxidase [AOX]) cells as indicated, treated with 150 μM anisomycin for …
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Figure 7—source data 1
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Figure 7—source data 2
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Figure 7—source data 3
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Figure 7—source data 4
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Figure 7—figure supplement 1
Supplementary data on mitochondrial temperature decrease in anisomycin-treated cells.
Representative fluorescence traces, uniformly rescaled, for (panels i, iii) Mito Thermo Yellow (MTY)-labelled iMEF(AOX) and (panels ii, iv) iMEF(P) cells pretreated for 2 hr with anisomycin, then …
Videos
Video 1
Mitochondrial temperature variation between and within cells.
Time-lapse micrographic images (1 frame per 10 s) of iMEF cells stained with Mito Thermo Yellow (MTY), brightness and contrast optimised, showing bright (i.e., potentially cooler) foci within the …
