Mitochondrial Thermogenesis Can Trigger Heat Shock Response in the Nucleus

Mitochondrial thermogenesis is a process in which heat is generated by mitochondrial respiration. In living organisms, the thermogenic mechanisms that maintain body temperature have been studied extensively in fat cells with little knowledge on how mitochondrial heat may act beyond energy expenditure. Here, we highlight that the exothermic oxygen reduction reaction (ΔHf° = −286 kJ/mol) is the main source of the protonophore-induced mitochondrial thermogenesis, and this heat is conducted to other cellular organelles, including the nucleus. As a result, mitochondrial heat that reached the nucleus initiated the classical heat shock response, including the formation of nuclear stress granules and the localization of heat shock factor 1 (HSF1) to chromatin. Consequently, activated HSF1 increases the level of gene expression associated with the response to thermal stress in mammalian cells. Our results illustrate heat generated within the cells as a potential source of mitochondria-nucleus communication and expand our understanding of the biological functions of mitochondria in cell physiology.

Confocal Imaging and Analysis.To visualize the subcellular localization of the endogenous or overexpressed proteins, cells were seeded on coverslips (MARIENFELD, Cat # HSU-0111580).For fixed cell imaging, cells were fixed with 4% paraformaldehyde and washed with phosphate-buffered saline (PBS) twice.Cells were permeabilized by 0.1% Triton X-100 (Sigma Aldrich, Cat # 9536-19-5) in PBS for 5-10 min at room temperature.After washing with PBS twice, cells were incubated in a blocking solution (2% BSA in PBS) for 1 h.Immunolabeling was conducted by sequentially adding primary antibodies followed by secondary antibodies.Cells were incubated were anti-HSF1 (1:500 dilution, CST, Cat # 4356) or anti-Histone 3 (1:3000 dilution, Santa Cruz, Cat # sc-517576) in a blocking solution overnight at 4 °C.After washing with PBS three times, cells were incubated with Alexa Fluor-conjugated anti-mouse 647 (1:2000 dilution, Invitrogen, Cat # A-21235) or anti-rabbit 488 (1:2000 dilution, Invitrogen, Cat # A-11008) for 1 h at room temperature, followed by washing with PBS three times.Immunofluorescence images were obtained and analyzed using an SP8 X Leica microscope (NICEM, Seoul National University, Seoul, Republic of Korea) with an objective lens (HC PL APO 100x/1.40OIL), a white light laser (470-670 nm, 1 nm tunable laser), and a HyD detector, which was controlled using the LAS X software and FV3000 microscope (Oylmpus) with an objective lens (UPLXAPO 100XO), which was controlled using the Olympus Fluoview software.For HSF1 imaging experiments, HSF1 foci were counted manually and statistically quantified using GraphPad Prism 10 software.
Flow Cytometry Analysis for Mitochondrial Membrane Potential and Temperature Measurement by ETAC.Cells were seeded in 6-well plates for flow cytometry analysis.The next day, cells were incubated for 30 min with 200 nM TMRE (Sigma Aldrich, Cat # 115532-52-0) or 0.5 μM ETAC (a gift from Young-Tae Chang) 1 .Cells were then washed three times with the medium and incubated overnight in fresh medium.The cells stained by each dye were further incubated with either 100 μM FCCP for 1 h and 30 μM menadione for 30 min.A steady state indicates the absence of chemical treatment.For the following flow cytometry analysis, cell suspensions were prepared by trypsin-EDTA (0.25%)-based cell detachment and diluted fivefold with fresh medium.Samples (10000 cells/events) were examined using a FACSCanto II (BD Bioscience) or flow cytometry LX (Beckman Coulter) according to their fluorescence signals.All flow cytometry data were processed using the FlowJo 10 or CytExpert software.
Intracellular Temperature Sensing by Cellular Thermoprobe 'FDV'.To use FDV (Funakoshi, Cat# FDV-0005) 2 , 1 mg powder of FDV was reconstituted in 100 μl of 5% glucose in ultrapure water to prepare a 1% w/v stock solution freshly before each experiment.The stock solution was incubated at 4 °C at least overnight with protection from light to obtain full extension of the FDV.For the calibration curve, Cells were cultured on 96-well black plates (SPL Life Sciences, Cat # 30296) using the same conditions as described in "Cell Culture" section.The media volume was 200 μl for each well.To measure the fluorescence, we used cell cultures at 90 % confluence and cells were washed with 5% glucose.Then, cells were treated with 0.05% of FDV in 5% glucose for 10-20 minutes followed by washing with Dulbecco's phosphate-buffered saline (DPBS, Thermo Fisher Scientific, Cat # 21300025) three times.After checking FDV staining in cells by epimicroscope, the fluorescence was measured using a Synergy ™ H1 microplate reader (BioTek Instruments, Inc.).A wavelength of 458 or 473 nm was used for excitation.The emission range was 490-530 nm and 570-610 nm.The temperature was initially set at 30 °C and then sequentially raised by 5 °C.Before each measurement, there was a wait time for 15 min to allow the plates to heat adequately.Measurements were taken quintuplicate, and the median value was used for the analysis.The experiment ended at a final temperature of 45 °C.For the FCCP treatments, cells were used at 90 % confluence, and the initial medium was replaced with an equal volume of FCCP diluted in culture media.After FDV staining in the same way with the calibration method, the fluorescence was measured on a microplate reader set at a temperature of 37 °C.For data analysis, the ratio of FDV and the temperature change were estimated from the formula of the fitted standard curve.pHluorin2 Fluorescence Measurement.Excitation scans were performed using a Synergy ™ H1 microplate reader (BioTek Instruments, Inc.).HEK293T cells were plated on 96-well black plates (SPL Life Sciences, Cat # 30296) and transiently transfected with 1000 ng pHluorin2 plasmid DNA mixed with 4 μg of PEI (Polysciences, Cat # 23966).After 24 hours of transfection, cells were washed with DPBS.Plates were placed into the microplate reader for a 10 min incubation at 37°C and measured.A wavelength of 520 nm was used for emission.The excitation was scanned between 300 nm and 490 nm with a 2 nm step.Measurements were taken from triplicate samples, and the median value was used for the analysis.For the pH standard curve, the calibration buffer contained the following: 50 mM MES buffer (pH 5.5, 6.0, and 6.5), 50 mM Tris⋅HCl (pH 7.0, 7.5, and 8.0).

Live Cell Imaging and Analysis.
For live cell imaging, the cells were cultured in a 30 mm glass bottom confocal dish (SPL Life Sciences, Cat # 100350) or 18mm glass (MARIENFELD, Cat # HSU-0111580) coated with poly-L-lysine solution (Sigma, Cat # P8920-100ML).Using an ExFluorer live cell imager (Live Cell Instrument, Namyangju, Republic of Korea).For HSF1-EGFP, the FITC channel (488 nm) was used.The confocal dishes containing the cultured cells were fastened tightly without a dish cover and placed on a stage-on incubator, maintained at 37 °C under 5% CO2 in a humidified environment.For imaging the HSF1-EGFP, exposure time was set as 200 ms.The initial volume of the medium was 2 ml.For the FCCP treatment, 1 ml of medium was carefully removed from the cell culture dish, subsequently 1 ml of 2×FCCP-containing medium was added.For cell recovery, 1.5 ml of medium in the cell culture dish was replaced with an equal volume of fresh culture medium, before subsequently replacing the entire total volume of medium in the cell culture with new fresh culture medium.
Live Cell Imaging for FCCP Treatment-Recovery with or without Hypoxia.To conduct real-time confocal imaging of HSF1-EGFP in various cell lines, cells were cultured using the same methods with 'Live Cell Imaging and Analysis'.A 'Incubator T Series' (Live Cell Instrument, Namyangju, Republic of Korea) is on the FV3000 microscope (Oylmpus) stage, which precisely controls the gas, temperature, and humidity of the stage incubator.For imaging, all cell lines were cultured according to standard mammalian culture protocols at 37 °C and 5% CO2 in a humidified incubator.Cell lines at 70-90 % confluence moved the cells to the confocal live stage.Live stage maintained the same condition as the cell culture incubator.The media were replaced through the flow out/in tube by syringe.Images were obtained using the same methods with 'Confocal Imaging and Analysis'.For hypoxia condition, the live stage incubator O2 concentration was regulated from 20% to 1%.Cells were in the 1% hypoxia condition at least 2 h for fully consumed the media and intracellular oxygen.
APEX2 reaction with Endogenously generated ROS.HEK293T cells were cultured in the same condition as described in "Cell Culture".After 24 h of transfection, 250 μM DBP in DMEM was treated to the APEX2expressed cells for 30 min, and then, 30 μM menadione was added for an appropriate time.The cells were washed with DPBS containing 5 mM Trolox (Sigma Aldrich, Cat # 238813), 10 mM sodium azide, and 10 mM sodium ascorbate (Sigma Aldrich, Cat # A4034) three times, and RIPA lysis buffer was added after removing DPBS.The cells were lysed for 10 min at 4 °C, and the sample was loaded onto a 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel for separation at 220-250 V for 60 min.After separation, proteins on the gel were transferred to a nitrocellulose membrane at 25-30 V for 180 min.The protein loading level was checked by Ponceau staining; the stain was removed by washing with 1× TBST.
Membrane blocking was performed with a solution of 2% (w/v) dialyzed BSA in 1× TBST at 4 °C overnight, or at room temperature for 1 h.The blots were immersed in streptavidin-horseradish peroxidase in blocking buffer (1:10,000 dilution, Thermo Fisher Scientific, Cat # 21126) at room temperature for 30 min and then rinsed with blot blocking buffer three times for 5 min, before development using the Clarity reagent (Bio-Rad) and imaging on a gel doc machine (cytiva, Amersham TM ImageQuant TM 800).For assessing comparative enzyme expression levels, the membrane was stripped with a stripping buffer (100 mM 2-mercaptoethanol, 2% SDS, 62.5 mM Tris-HCl, pH 6.8).Blots were washed three times, 5 min per wash, in the 1× TBST buffer and incubated in a 5% blocking buffer for 1-2 h.They were then incubated in 10 mL of anti-V5 tag antibody solution diluted in the blocking buffer for 1-2 h at room temperature.Blots were subsequently washed 3 × 5 min in 1× TBST prior to incubation with 10 mL of anti-mouse-HRP diluted in the blocking buffer for 30-60 min at room temperature.The membranes were then washed again for three times, 5 min per wash in 1× TBST buffer before developing using the Clarity reagent (Bio-Rad), as described above.

Dephosphorylation of proteins with lambda protein phosphatase (λPPase).
The MCF10A cells were lysed using RIPA lysis solution containing 1× protease inhibitor cocktail after being subjected to appropriate experimental conditions (such as FCCP treatment or 43 °C heat shock).Lysates were then centrifuged at 15,000 × g for 10 min at 4 °C, and the supernatants were collected in the new 1.5 ml tubes.NEBuffer Pack for Protein MetalloPhosphatases, MnCl2 and 400 units of λPPase (New England Biolabs, Cat # P0753S) were sequentially added into supernatants to dephosphorylate the proteins, following the manufacturer's instructions.Mixture was incubated at 30 °C for 30 min, and the proteins were loaded on bistris gels for SDS-PAGE (Invitrogen).Immunoblotting with anti-HSF1, histone H3, and GAPDH was conducted as described in the "Western Blotting" section.
Luciferase assay.HEK293T cells were cultured on 96-well plate (Corning, Cat # 354620) in the same condition as described in "Cell Culture".After 24 h of transfection using 1:1 mixture of a firefly and Renilla luciferase plasmids, cells were subjected to a stressed condition for 1 h followed by washing with DPBS three times.Reagent was prepared (Dual-Glo ® Luciferase Assay System, Promega, Cat # E2920) as below.
• Luciferase Reagent: Luciferase Buffer was transferred to Luciferase Substrate in the bottle and store at -70 °C (maximum of 6 months).
• Stop & Glo (S&G) Reagent: 50× S&G substrate was diluted with S&G buffer to prepare 1× concentration in the amber bottle and vortexed for 10 sec.It was prepared immediately for every experiment.
The DPBS in the plate was replaced with Luciferase Reagent in an equal volume to the culture medium, subsequently incubated for 10 min.Then, firefly luciferase activity was measured using SpectraMax i3x (Molecular Devices).After measurement, S&G Reagent was added to the plate, followed by incubation for 10 min.Renilla luciferase activity was measured in the same plate order as the firefly luciferase activity measured.

Chromatin immunoprecipitation (ChIP).
ChIP experiments were performed as previously described 3 .
Briefly, a concentration of 3 × 10 7 single trypsinized HEK293T cells was used.The trypsinized cells were crosslinked with 1% formaldehyde, followed by quenching in 0.125 M glycine, and washed with PBS.ChIP-seq Library Construction.The ChIP-seq libraries were constructed using 40 μl of purified ChIP DNA and NEXTflex TM ChIP-seq kit (PerkinElmer, Cat # NOVA-5143-02) according to the manufacturer's instructions.Briefly, ChIP DNA was end-repaired and size-selected (250-300 bp) using AMPure XP beads (Beckman, Cat # A63881).All subsequent procedures, from adenylation to PCR amplification, were performed according to the ChIP-seq library construction steps.The quality of the ChIP-seq libraries was determined by a Bioanalyzer using the High Sensitivity chip (Agilent), and the average size of the ChIP-seq libraries ranged from 250 to 350 bp.For multiplexing, equal molar quantities of libraries were combined by considering the sequencing depth per sample (20-40 million reads per library).The ChIP-seq libraries were sequenced using the Illumina NextSeq platform with single-end reads of 76 bases.
RNA-seq Library Construction.Total RNA was extracted using the QIAzol reagent (QIAGEN, Cat # 79306) according to the manufacturer's instructions.RNA-seq libraries were constructed using 5 μg of purified RNA and a NEXTflex TM Rapid Directional mRNA-seq kit (PerkinElmer, Cat # NOVA-5138-11).Briefly, purified RNA was poly-A selected and fragmented using a fragmentation enzyme.After first-and second-strand cDNA synthesis from a fragmented RNA template, the steps from adenylation to PCR amplification were performed according to the RNA-Seq library construction steps.

CMV/ pcDNA5
Mitochondrial targeting sequence (MTS): MLATRVFSLVGKRAISTSVCVRAH 4 Myc epitope tag: EQKLISEEDL The nuclear export sequence, NES (LQLPPLERLTLD), was derived from residues 6-17 of the HIV-1 Rev protein.Protein processed size during translocation was obtained by programs: ApE (https://jorgensen.biology.utah.edu/wayned/ape/)PE-A) is shown on the x-axis.(E) The polymer structure of the organic thermometer "FDV-0005".(F) Intracellular temperature calibration was conducted using the FDV-0005 organic thermometer.The FDV-0005 was used at a dilution of 0.05% w/v in 5% glucose solution.Measurements for calibration curve were performed within a temperature range of 30 °C to 45 °C (n = 5).To determine the temperature elevation induced by FCCP, cells were treated with 100 μM FCCP after FDV-0005 incubation for 10 minutes (n = 5).Red dots represent the FCCP-treaded samples.(G) Measurement of nuclear pH under the FCCP condition.The fluorescent protein-based pH sensor pHluorin2 was used.The HEK293T cells were incubated at 37 °C and 5% CO2 in a humidified incubator and 100 μM FCCP was used for this assay.F G

Figure S1 .
Figure S1.Confirm the intracellular thermogenesis by two different thermometers.(A) Chemical structures of TMRE, FCCP, and menadione.(B) Flow cytometry analyses and its histogram of HEK293T cell suspensions treated with either TMRE, FCCP, or menadione.(C) Schematic illustration of temperature measurement by ETAC.(D) ETAC fluorescence intensity after FCCP (100 μM, 1 h) treatment.Decreased ETAC fluorescence intensity under the indicates increased local temperature at the ER membrane.Flow cytometry analysis results of ETAC fluorescent signal under the two conditions (steady-state or 100 μM FCCP, 1 h), in HEK293T, HeLa and HepG2 cell lines.Cell count is shown on the y-axis, while fluorescent signal intensity (PE-A) is shown on the x-axis.(E) The polymer structure of the organic thermometer "FDV-0005".(F) Intracellular temperature calibration was conducted using the FDV-0005 organic thermometer.The FDV-0005 was used at a dilution of 0.05% w/v in 5% glucose solution.Measurements for calibration curve were performed within a temperature range of 30 °C to 45 °C (n = 5).To determine the temperature elevation induced by FCCP, cells were treated with 100 μM FCCP after FDV-0005 incubation for 10 minutes (n = 5).Red dots represent the FCCP-treaded samples.(G) Measurement of nuclear pH under the FCCP condition.The fluorescent protein-based pH sensor pHluorin2 was used.The HEK293T cells were incubated at 37 °C and 5% CO2 in a humidified incubator and 100 μM FCCP was used for this assay.

Figure S2 .
Figure S2.Endogenous HSF1 foci detection in the MCF10A cells with various stress conditions.(A) Zstack images of endogenous HSF1 (anti-HSF1) and Histone H3 (anti-Histone H3) were obtained by 3D confocal imaging with activated MCF10A cells treated with either DMSO or FCCP (100 μM, 1 h) or menadione (30 μM, 30 min), or subjected to heat shock (43 °C, 1 h), and with or without co-treatment with NAC (5 mM, 1 h).(B) Percentage of cells with foci was quantified by evaluating 50-100 cells.Boxes indicate the quartiles, whiskers range from minimal to maximal values, and dots represent individual data points plotted on the box.Statistical analysis was conducted using an unpaired two-tailed t-test and the significance level was denoted as (*p < 0.05, ****p < 0.0001, ns, not significant).

Figure S3 .
Figure S3.The formation of HSF1 foci induced by FCCP is independent of accumulated mitochondrial proteins.Subcellular localization of transiently expressed MTS-myc-dsRed and endogenous HSPD1 were visualized in HEK293T (A) and HeLa cells (B) by immunofluorescence with anti-Myc and anti-HSPD1 antibodies.Scale bar = 10 μm.(C) Western blot analysis of endogenous HSPD1 under the DMSO (control) or FCCP (100uM, 1hr)-treated sample using an anti-HSPD1 antibody.(D) Ponceau staining of the same membrane in (C) was used as reference.(E-F) Foci formation of HSF1-EGFP in FCCP-treated cells under protein synthesis blockade by cycloheximide (E) and puromycin (F) was observed.Cycloheximide (35 μM) and puromycin (35 μM) were pre-treated for 2 h followed by addition of DMSO or FCCP (100 μM) for 1 h.(G) Confocal images of HSF1-EGFP foci formation were obtained after treatment with either DMSO or FCCP (100 μM) for 1 h or GTPP (10 μM) for 6 h or 24 h.Scale bar = 10 μm.(H) Quantification of percentage of the HSF1 foci formation cells was performed by evaluating 500-640 cells per condition.Boxes indicate the quartiles, whiskers range from minimal to maximal values, and dots represent individual data points plotted on the box.Statistical analysis was conducted using a one-way analysis of variance (ANOVA) with Tukey's test, and the significance level was denoted as (****p < 0.0001).

Figure S4 .FigureFigure S6 .Figure S7 .
Figure S4.Quantification of the HSF1-EGFP foci in various stress conditions.(A) The level of HSF1-EGFP foci formation in the HSF1-EGFP stable cell line under the different conditions including DMSO, FCCP (100 μM, 1hr), 39 °C (1 hr), 43 °C (1 hr) were visualized by confocal imaging with immunofluorescence.Scale bar = 10 μm (B) Percentage of the cells with foci in the imaging results of (A) were analyzed, 450-750 cells per condition were evaluated for quantification.(C) The formation of HSF1-EGFP foci induced by FCCP (100 μM, 1hr) at 32 °C was visualized by confocal imaging.Cells were pre-cooled at 32 °C in a humidified 5% CO2 incubator for 2 h.FCCP-treated cells at 37 °C were used as positive control.Scale bar = 10 μm (D) Percentage of the cells with foci in the imaging results of (C) were analyzed.450-750 cells per condition were evaluated for quantification.Boxes in (B) and (D) indicate the quartiles, whiskers range from minimal to maximal values, and dots represent individual data points plotted on the box.Statistical analysis was conducted using a one-way analysis of variance (ANOVA) with Tukey's test, and the significance level was denoted as (*p < 0.05, ****p < 0.0001).

Figure S8 . 3 E
Figure S8.The results of luciferase assay with HSF1 binding sequence in HSPD1/HSPE1 & transcription expression correlation of common-GAIN genes under Heat shock and FCCP treatment (A)The HSF1 binding sequence of HSPD1/HSPE1 and schematic representation of the HSPD1-luciferase assay are illustrated.The HSF1 binding site was marked with light blue in the sequence.(B) HSPD1/HSPE1 promoter luciferase assay results in HEK293T cells after FCCP treatment (100 μM, 1 h), heat shock at 40 °C

Figure S8 .
Figure S8.(continued) or 43 °C for 1 h.All experiments were conducted using live cells incubated at 37 °C, in a humidified 5% CO2 incubator.The middle line of the individual value plot indicates the median value, and each dot indicates individual data points.Statistical analysis was conducted using an unpaired two-tailed t-test and the significance level was denoted as (***p < 0.001, ****p < 0.0001, ns, not significant).(C) Scatter plot of mRNA expression level of Common-GAIN genes (61 gene) under the heat shock (x-axis) and FCCP treatment condition (y-axis).In each condition, Log2 fold change values of mRNA expression over the control sample were presented in the graph.Original data for this graph is shown in the Supporting Information.(D) Venn diagram showing the overlap (n = 15) of the HSF1-bound genes (n = 708) and mRNA upregulated genes (n = 104) under heat shock-specific conditions.(E) Gene Ontology (GO) analysis of the biological processes associated with the identified 15 upregulated HSF1-bound genes (as shown in D).
The cell pellets were lysed with 1200 μl of cell lysis buffer [10 mM Tris-Cl pH 8.0, 10 mM NaCl, 0.2% NP-40] supplemented with protease inhibitor cocktail (Roche, Cat # 11697498001) and 1 mM DTT. Chromatin was isolated by centrifugation at 7,400 rpm for 30 s.The pellet was gently resuspended in a 1200 μl of nuclei lysis buffer [50 mM Tris-Cl pH 8.0, 10 mM EDTA, 1% SDS] containing protease inhibitor and 1 mM DTT.The chromatin lysate was sonicated (Diagenode, Bioruptor pico) for 10 cycles (30 s, and 30 s off), and the sonicated chromatin mixture was then incubated for 1 h with 60 μg of rabbit IgG, and 60 μl of Protein A magnetic beads (Invitrogen, Cat # 10001) for pre-clearing.Immunoprecipitation was conducted with 6 ml of pre-cleared chromatin, 30 μl of the HSF1 antibody (CST, Cat # 4356), and 60 μl of Protein A magnetic beads overnight at a 4 °C rotator.The following day, the immune complexes were washed with IP Wash I Buffer, twice with high-salt buffer, once with IP Wash II buffer, and finally twice with TE (pH 8.0).The washed immune complexes were eluted by incubation with 200 μl of elution buffer [1% SDS and 0.1 M NaHCO3] for 30 min at a 45 °C thermomixer, shaking at 1,000 rpm.The eluate was de-crosslinked with RNase A (1 μg/μl) and 0.25 M NaCl and incubated overnight at a 65 °C water bath.The next day, the eluate samples were incubated for 2 h in a Proteinase K (NEB, Cat # P8107S) solution, and the immunoprecipitated DNA was purified with a QIAquick PCR purification kit (QIAGEN, Cat # 28106;) in 50 μl of EB (elution buffer).