A highly responsive pyruvate sensor reveals pathway-regulatory role of the mitochondrial pyruvate carrier MPC
- Centro de Estudios Científicos-CECs, Chile
- Universidad Austral de Chile, Chile
- Institut für Neuro- und Verhaltensbiologie, University of Münster, Germany
Figures
Figure 1 with 2 supplements
Characterization of PyronicSF.
(A) PyronicSF. cpGFP flanked by linkers was inserted between amino acid residues 188 and 189 of PdhR. DNA sequence in Figure 1—figure supplement 1). (B) Excitation spectra of PyronicSF in the …
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Figure 1—source data 1
Characterization of PyronicSF.
- https://cdn.elifesciences.org/articles/53917/elife-53917-fig1-data1-v2.xlsx
Figure 1—figure supplement 1
Nucleotide sequence of PyronicSF.
Figure 1—figure supplement 2
Correction of the effect of pH on PyronicSF and a mutant of PyronicSF with reduced response to pyruvate but conserved response to pH.
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Figure 1—figure supplement 2—source data 1
Correction of the effect of pH on PyronicSF and a mutant of PyronicSF with reduced response to pyruvate but conserved response to pH.
- https://cdn.elifesciences.org/articles/53917/elife-53917-fig1-figsupp2-data1-v2.xlsx
Figure 2 with 3 supplements
MPC-mediated mitochondrial pyruvate transport in astrocytes.
(A) Astrocytes co-expressing mito-PyronicSF (green) and mito-mCherry (magenta). Bars represent 10 μm. (B) Cultures were exposed to 3 mM pyruvate. Data correspond to mean ± s.e.m. (4 cells in a …
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Figure 2—source data 1
MPC-mediated mitochondrial pyruvate transport in astrocytes.
- https://cdn.elifesciences.org/articles/53917/elife-53917-fig2-data1-v2.xlsx
Figure 2—figure supplement 1
Expression of mito-PyronicSF and mito-Pyronic in various cell types.
Figure 2—figure supplement 2
Mitochondrial localization of mito-PyronicSF in astrocytes.
Figure 2—figure supplement 3
Mitochondrial pyruvate uptake is slower than cytosolic pyruvate uptake.
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Figure 2—figure supplement 3—source data 1
Mitochondrial pyruvate uptake is slower than cytosolic pyruvate uptake.
- https://cdn.elifesciences.org/articles/53917/elife-53917-fig2-figsupp3-data1-v2.xlsx
Figure 3 with 5 supplements
Steady-state mitochondrial and cytosolic pyruvate.
Astrocytes expressing PyronicSF or Pyronic in mitochondria or cytosol were first incubated in a buffer containing physiological concentrations of glucose (2 mM), lactate (2 mM) and pyruvate (0.2 …
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Figure 3—source data 1
Steady-state mitochondrial and cytosolic pyruvate.
- https://cdn.elifesciences.org/articles/53917/elife-53917-fig3-data1-v2.xlsx
Figure 3—figure supplement 1
Functional expression of the FRET sensor Pyronic in mitochondria.
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Figure 3—figure supplement 1—source data 1
Functional expression of the FRET sensor Pyronic in mitochondria.
- https://cdn.elifesciences.org/articles/53917/elife-53917-fig3-figsupp1-data1-v2.xlsx
Figure 3—figure supplement 2
Intra- and inter-experimental contributions to cell-to-cell metabolic heterogeneity of astrocytes.
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Figure 3—figure supplement 2—source data 1
Intra- and inter-experimental contributions to cell-to-cell metabolic heterogeneity of astrocytes.
- https://cdn.elifesciences.org/articles/53917/elife-53917-fig3-figsupp2-data1-v2.xlsx
Figure 3—figure supplement 3
No apparent correlation between PyronicSF expression and mitochondrial pyruvate concentration and consumption.
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Figure 3—figure supplement 3—source data 1
No apparent correlation between PyronicSF expression and mitochondrial pyruvate concentration and consumption.
- https://cdn.elifesciences.org/articles/53917/elife-53917-fig3-figsupp3-data1-v2.xlsx
Figure 3—figure supplement 4
Performance of PyronicSF in the low micromolar range.
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Figure 3—figure supplement 4—source data 1
Performance of PyronicSF in the low micromolar range.
- https://cdn.elifesciences.org/articles/53917/elife-53917-fig3-figsupp4-data1-v2.xlsx
Figure 3—figure supplement 5
Sensitivity of PyronicSF and Pyronic.
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Figure 3—figure supplement 5—source data 1
Sensitivity of PyronicSF and Pyronic.
- https://cdn.elifesciences.org/articles/53917/elife-53917-fig3-figsupp5-data1-v2.xlsx
Figure 4
Measurement of mitochondrial pyruvate consumption rate in individual cells.
(A) Time course of mitochondrial pyruvate level after an astrocyte was exposed 5 mM pyruvate and then to 10 μM UK-5099. The new steady-state is represented on the right, as percentage of the level …
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Figure 4—source data 1
Measurement of mitochondrial pyruvate consumption rate in individual cells.
- https://cdn.elifesciences.org/articles/53917/elife-53917-fig4-data1-v2.xlsx
Figure 5
Pyruvate concentration and consumption in discrete mitochondria.
Mito-PyronicSF (gray) in an astrocyte expressing mito-PyronicSF and mito-mCherry (left). Bar represents 10 μm. The righthand image shows three regions of interest colored according to the look-up …
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Figure 5—source data 1
Pyruvate concentration and consumption in discrete mitochondria.
- https://cdn.elifesciences.org/articles/53917/elife-53917-fig5-data1-v2.xlsx
Figure 6
Pyruvate dynamics in glial cells of Drosophila melanogaster.
Brains were acutely dissected from Drosophila melanogaster larvae expressing PyronicSF in the cytosol or mitochondria of perineurial glial cells. (A) PyronicSF in the cytosol of perineurial cells. …
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Figure 6—source data 1
Pyruvate dynamics in glial cells of Drosophila melanogaster.
- https://cdn.elifesciences.org/articles/53917/elife-53917-fig6-data1-v2.xlsx
Author response image 1
Lack of measurable fluorescence of 10 µM UK-5099.
The response to 10 µM UK-5099 of an astrocyte expressing mito-PyronicSF was compared with that of a neighboring region of interest (ROI) devoid of cells (488 excitation, 525 ± 25 emission). While …
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| strain, strain background (Mus musculus) | C57BL/6J x CBA/J | The Jackson Laboratory | JAX: 100011 RRID:IMSR_JAX:100011 | |
| Genetic reagent (Drosophila melanogaster) | Bloomington 45781 | BloomingtonDrosophila Stock Center | apt-Gal4 (P[GMR49G07-GAL4]attP2) RRID:BDSC_45781 | |
| cell line (Homo sapiens) | HEK293 | ATCC | CRL-1573 RRID:CVCL_0045 | |
| cell line (Homo sapiens) | HeLa | ATCC | CRM-CCL-2 RRID:CVCL_0030 | |
| cell line (Homo sapiens) | MDA-MB-231 | ATCC | CRM-HTB-26 RRID:CVCL_0062 | |
| cell line (Cercopithecus aethiops) | COS7 | ATCC | CRL-1651 RRID:CVCL_0224 | |
| recombinant DNA reagent | Plasmid: Mito-PyronicSF/pCMV-myc-mito | Arce-Molina et al., 2019 | RRID:Addgene_124813 | |
| recombinant DNA reagent | Plasmid: NLS-PyronicSF/pCMV-myc-nuc | Arce-Molina et al., 2019 | RRID:Addgene_124814 | |
| recombinant DNA reagent | Plasmid: PyronicSF-mRuby2/pBI-CMV1 | Arce-Molina et al., 2019 | RRID:Addgene_124830 | |
| recombinant DNA reagent | Adenoviral particles: Ad mito-PyronicSF-T2A-mito-mCherry | Vector Biolabs | Custom made | |
| recombinant DNA reagent | Adenoviral particles: Ad Pyronic | Vector Biolabs | Custom made | |
| recombinant DNA reagent | Plasmid: Pyronic | San Martín et al., 2014a | RRID:Addgene_51308 | |
| commercial assay or kit | Lipofectamine 3000 Transfection Reagent | Invitrogen | Cat. #L3000015 | |
| chemical compound, drug | UK-5099 | TOCRIS | CAS: 56396-35-1 | |
| chemical compound, drug | AR-C155858 | Haoyuan Chemexpress | CAS: 496791-37-8 | |
| software, algorithm | Berkeley Madonna 8.3.23.0 | University of California at Berkeley | http://www.berkeleymadonna.com | |
| software, algorithm | Fluoview FV10-ASW 3.0 | Olympus | N/A | |
| software, algorithm | ImageJ 1.49 m | NIH | http://imagej.net |
