Hsf1 and the molecular chaperone Hsp90 support a ‘rewiring stress response’ leading to an adaptive cell size increase in chronic stress
- Département de Biologie Moléculaire et Cellulaire, Université de Genève, Switzerland
- On leave from: Department of Pharmacology and Therapeutics, Faculty of Pharmacy, Pharos University in Alexandria, Egypt
- BioCode: RNA to Proteins Core Facility, Département de Microbiologie et Médecine Moléculaire, Faculté de Médecine, Université de Genève, Switzerland
- Institute of Genetics and Genomics of Geneva, Université de Genève, Switzerland
Figures
Figure 1 with 2 supplements
Cells increase their size in response to chronic stress.
(A) Flow cytometric quantification of cell viability under chronic HS for 7 days (HS = 39 °C for HEK and HCT116 cells; HS = 40 °C for A549 and RPE1 cells) (n = 4 biologically independent samples). (B…
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Figure 1—source data 1
Values related to all graphs.
- https://cdn.elifesciences.org/articles/88658/elife-88658-fig1-data1-v2.xlsx
Figure 1—figure supplement 1
Cells increase their size in response to different types of chronic stress.
(A) Flow cytometric quantification of cell viability after 4 days of treatment with 10 μM sodium arsenite (Ars), 1% hypoxia (Hypo), 5 µM L-azetidine-2-carboxylic acid (AZC), or 250 nM tunicamycin …
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Figure 1—figure supplement 1—source data 1
Values related to all graphs of Figure 1—figure supplement 1.
- https://cdn.elifesciences.org/articles/88658/elife-88658-fig1-figsupp1-data1-v2.xlsx
Figure 1—figure supplement 2
Schematic representation of the flow cytometric strategies for cell size and cell cycle analyses.
(A) Gating and strategy for cell size analyses relevant to Figures 1B, E, H—3E–F, 5B, 6E, 8B and C and F, and Figure 1—figure supplement 1C, Figure 2—figure supplement 1B, Figure 3—figure supplement …
Figure 2 with 1 supplement
Cells increase their overall size and their nuclei, but are unable to adapt to chronic HS in the absence of one of the cytosolic Hsp90 isoforms.
(A) Immunoblots of Hsp90α and Hsp90β in WT HEK and A549 cells, and their respective Hsp90α/β KO cells. GAPDH serves as the loading control (α KO; Hsp90α KO and β KO; Hsp90β KO) (representative …
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Figure 2—source data 1
Raw and annotated immunoblots.
- https://cdn.elifesciences.org/articles/88658/elife-88658-fig2-data1-v2.zip
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Figure 2—source data 2
Values related to all graphs.
- https://cdn.elifesciences.org/articles/88658/elife-88658-fig2-data2-v2.xlsx
Figure 2—figure supplement 1
Cells are unable to adapt to chronic stress in the absence of one of the Hsp90 isoforms, but still get larger.
(A) Flow cytometric quantification of cell death of A549 WT and Hsp90α/β KO cells after 4 days in 1% hypoxia (Hypo) and 10 μM sodium arsenite (Ars) treatment (n=3 biologically independent samples). …
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Figure 2—figure supplement 1—source data 1
Values related to all graphs of Figure 2—figure supplement 1.
- https://cdn.elifesciences.org/articles/88658/elife-88658-fig2-figsupp1-data1-v2.xlsx
Figure 3 with 1 supplement
Hsf1 regulates cell size in response to stress.
(A) Fold change of Hsf1 activity of HEK WT, A549 WT, and their respective Hsp90α/β KO cells at 37 °C as measured by luciferase reporter assay (n=3 biologically independent samples and 2 experimental …
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Figure 3—source data 1
Values related to all graphs.
- https://cdn.elifesciences.org/articles/88658/elife-88658-fig3-data1-v2.xlsx
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Figure 3—source data 2
Raw and annotated immunoblots.
- https://cdn.elifesciences.org/articles/88658/elife-88658-fig3-data2-v2.zip
Figure 3—figure supplement 1
Hsf1 induces cell size in response to stress.
(A) Volcano plots of the normalized fold changes in protein levels of some of the core Hsf1 target genes (list obtained from https://hsf1base.org/) in Hsp90α/β KO cells compared to WT HEK as …
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Figure 3—figure supplement 1—source data 1
Values related to all graphs of Figure 3—figure supplement 1.
- https://cdn.elifesciences.org/articles/88658/elife-88658-fig3-figsupp1-data1-v2.xlsx
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Figure 3—figure supplement 1—source data 2
Raw and annotated immunoblots of Figure 3—figure supplement 1.
- https://cdn.elifesciences.org/articles/88658/elife-88658-fig3-figsupp1-data2-v2.zip
Figure 4 with 2 supplements
Hsp90α/β KO cells maintain chaperones, co-chaperones, and Hsp90 interactors during chronic stress adaptation.
(A) Volcano plots of the normalized fold changes of molecular chaperones and co-chaperones after 1 and 4 days (first and second rows, respectively) of chronic HS determined by quantitative …
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Figure 4—source data 1
Raw and annotated immunoblots.
- https://cdn.elifesciences.org/articles/88658/elife-88658-fig4-data1-v2.zip
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Figure 4—source data 2
Values related to all graphs.
- https://cdn.elifesciences.org/articles/88658/elife-88658-fig4-data2-v2.xlsx
Figure 4—figure supplement 1
Hsp90α/β KO cells maintain molecular chaperones, co-chaperones, and total proteins.
(A) Heat maps of the normalized fold changes (log2) of molecular chaperones and co-chaperones of HEK cells subjected to 1 and 4 days of chronic HS as determined by quantitative label-free proteomics …
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Figure 4—figure supplement 1—source data 1
Raw and annotated immunoblots of Figure 4—figure supplement 1.
- https://cdn.elifesciences.org/articles/88658/elife-88658-fig4-figsupp1-data1-v2.zip
Figure 4—figure supplement 2
Hsp90α/β KO cells maintain Hsp90 interactors in chronic stress.
Heat maps of the normalized fold changes (log2) of Hsp90 interactors of HEK cells subjected to 1 and 4 days of chronic HS as determined by quantitative label-free proteomics (n=3 biologically …
Figure 5 with 1 supplement
Hsp90α/β KO cells suffer from cytoplasmic protein dilution during adaptation to chronic stress.
(A) FRAP experiments with control and heat-adapted live cells expressing EGFP. The respective box plots show the t-half values of recovery of EGFP fluorescence and the apparent EGFP diffusion …
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Figure 5—source data 1
Values related to all graphs.
- https://cdn.elifesciences.org/articles/88658/elife-88658-fig5-data1-v2.xlsx
Figure 5—figure supplement 1
Wild-type cells maintain cytoplasmic density and total protein ratio during stress-induced cell size increase.
(A) Scheme of the FRAP experiments. (B to D) FRAP experiments with live cells expressing EGFP. The respective box plots represent the t-half values of recovery of EGFP fluorescence and the apparent …
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Figure 5—figure supplement 1—source data 1
Values related to all graphs of Figure 5—figure supplement 1.
- https://cdn.elifesciences.org/articles/88658/elife-88658-fig5-figsupp1-data1-v2.xlsx
Figure 6 with 3 supplements
Hsp90 is crucial for adapting translation to chronic stress.
(A) Flow cytometric analysis of total translation of HEK WT and Hsp90α/β KO cells at 37 °C and after 4 days of chronic HS (see scheme of the experiment on the top). Nascent polypeptide chains were …
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Figure 6—source data 1
Values related to all graphs.
- https://cdn.elifesciences.org/articles/88658/elife-88658-fig6-data1-v2.xlsx
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Figure 6—source data 2
Raw and annotated immunoblots.
- https://cdn.elifesciences.org/articles/88658/elife-88658-fig6-data2-v2.zip
Figure 6—figure supplement 1
Hsp90 requirement for cellular translation during adaptation to chronic stress, and early time points of translational adaptation of wild-type cells.
(A) Flow cytometric analysis of global translation of HEK WT and Hsp90α/β KO cells at 37 °C and after 1 day under chronic HS. See scheme of the experiment on the top. Nascent polypeptide chains were …
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Figure 6—figure supplement 1—source data 1
Values related to all graphs of Figure 6—figure supplement 1.
- https://cdn.elifesciences.org/articles/88658/elife-88658-fig6-figsupp1-data1-v2.xlsx
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Figure 6—figure supplement 1—source data 2
Raw and annotated immunoblots of Figure 6—figure supplement 1.
- https://cdn.elifesciences.org/articles/88658/elife-88658-fig6-figsupp1-data2-v2.zip
Figure 6—figure supplement 2
Differential effects of Hsp90 levels on eIF2α, mTOR, and S6.
Representative immunoblots of some translation-related proteins in A549 cells. β-actin serves as the loading control. The bar graphs show the quantitation of three biologically independent …
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Figure 6—figure supplement 2—source data 1
Raw and annotated immunoblots of Figure 6—figure supplement 2.
- https://cdn.elifesciences.org/articles/88658/elife-88658-fig6-figsupp2-data1-v2.zip
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Figure 6—figure supplement 2—source data 2
Values related to all graphs of Figure 6—figure supplement 2.
- https://cdn.elifesciences.org/articles/88658/elife-88658-fig6-figsupp2-data2-v2.xlsx
Figure 6—figure supplement 3
Schematic representation of the flow cytometric strategies to measure translation.
Relevant to Figure 6A and E and Figure 6—figure supplement 1A and B.
Figure 7 with 2 supplements
Hsp90 is crucial for cellular proteostasis during adaptation to chronic stress.
(A) Flow cytometric determination of the in vivo UPS activity in chronic HS compared to 37 °C, using the Ub-M-GFP and Ub-R-GFP reporter proteins (n=4 biologically independent samples). (B) Flow …
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Figure 7—source data 1
Values related to all graphs.
- https://cdn.elifesciences.org/articles/88658/elife-88658-fig7-data1-v2.xlsx
Figure 7—figure supplement 1
Hsp90α/β KO cells maintain WT levels of protein degradation activities in unstressed conditions, but have more protein aggregates.
(A) Flow cytometric determination of the in vivo UPS activity using the Ub-M-GFP and Ub-R-GFP reporter plasmids (n=4 biologically independent samples). (B) Flow cytometric measurement of autophagic …
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Figure 7—figure supplement 1—source data 1
Values related to all graphs of Figure 7—figure supplement 1.
- https://cdn.elifesciences.org/articles/88658/elife-88658-fig7-figsupp1-data1-v2.xlsx
Figure 7—figure supplement 2
Schematic representation of the flow cytometric strategies for measuring autophagic flux and in vivo UPS activities.
(A) Gating strategy for autophagic flux measurements, relevant to Figure 7B and Figure 7—figure supplement 1B. (B) Gating strategy for measuring UPS activity; related to Figure 7A and Figure …
Figure 8 with 1 supplement
Enlarged cells are more resistant to additional stress.
(A) Scheme of cell size enlargement or reduction experiments. CHX, cycloheximide; CDKi, CDK4/6 inhibitor. (B) Cell size was first enlarged by treating cells with 100 nM CDKi for 3 days; then, cells …
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Figure 8—source data 1
Values related to all graphs.
- https://cdn.elifesciences.org/articles/88658/elife-88658-fig8-data1-v2.xlsx
Figure 8—figure supplement 1
Smaller cells are more susceptible to additional stress.
(A) Cell size was enlarged by treating cells with 100 nM CDKi for 3 days. Fold change of cell size (represented by the FSC-MFI values) and total proteins (determined as MFI-FL1 values) were analyzed …
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Figure 8—figure supplement 1—source data 1
Values related to all graphs of Figure 8—figure supplement 1.
- https://cdn.elifesciences.org/articles/88658/elife-88658-fig8-figsupp1-data1-v2.xlsx
Figure 9
Adaptation to chronic stress requires cytosolic Hsp90 above a threshold level.
Immunoblots in the lower panels show the endogenous Hsp90α and Hsp90β, and the exogenously overexpressed larger fusion proteins of Hsp90α (as mCherry-Hsp90α) and Hsp90β (as EGFP-Hsp90β). Images of …
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Figure 9—source data 1
Raw and annotated immunoblots.
- https://cdn.elifesciences.org/articles/88658/elife-88658-fig9-data1-v2.zip
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Figure 9—source data 2
Values related to all graphs.
- https://cdn.elifesciences.org/articles/88658/elife-88658-fig9-data2-v2.xlsx
Figure 10
Prolonged mild chronic stress triggers excessively bigger cell size and senescence.
(A) Flow cytometric quantification of cell size of RPE1 cells exposed to prolonged mild HS (n = 4 biologically independent samples). (B) Histograms representing the cell cycle distribution of RPE1 …
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Figure 10—source data 1
Values related to all graphs.
- https://cdn.elifesciences.org/articles/88658/elife-88658-fig10-data1-v2.xlsx
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Cell line (Homo-sapiens) | HEK293, Human embryonic kidney cells | ATCC | CRL-3216 | corresponding Hsp90α/β KO cell lines were generated |
| Cell line (Homo-sapiens) | A549, human lung epithelial carcinoma cells | ATCC | CCL-185 | corresponding Hsp90α/β KO cell lines were generated |
| Cell line (Homo-sapiens) | RPE1, human retinal epithelial cells | ATCC | CRL-4000 | |
| Cell line (Homo-sapiens) | HCT116, human colon carcinoma Cells | ATCC | CCL-247 | |
| Cell line (Mus musculus) | Mouse adult fibroblasts (MAFs) | Bhattacharya et al., 2022 | ||
| Recombinant DNA reagent | pcDNA-Flag HSF1 wt (plasmid) | a gift from Len Neckers (Kijima et al., 2018) | transient overexpression of WT Hsf1 | |
| Recombinant DNA reagent | pcDNA-Flag HSF1 C205 (plasmid) | a gift from Len Neckers (Kijima et al., 2018) | This express the Hsf1 mutant comprising only the N-terminal 205 amino acids | |
| Recombinant DNA reagent | pcDNA3.1(+) (plasmid) | Thermo Fisher Scientific | #V79020 | |
| Recombinant DNA reagent | pCherry.90α (plasmid) | Picard et al., 2006 | transient overexpression of Hsp90α | |
| Recombinant DNA reagent | pEGFP.90β (plasmid) | Picard et al., 2006 | transient overexpression of Hsp90β | |
| Recombinant DNA reagent | pmCherry-C1 (plasmid) | Picard et al., 2006 | Control for pCherry.90α | |
| Recombinant DNA reagent | pEGFP-C1 (plasmid) | Clontech | #6084–1 | |
| Recombinant DNA reagent | FUW mCherry-GFP-LC3 (plasmid) | a gift from Anne Brunet (Leeman et al., 2018) | Addgene #110060 | autophagy reporter plasmid |
| Recombinant DNA reagent | Ub-M-GFP (plasmid) and Ub-R-GFP (plasmid) | a gift from Nico Dantuma (Dantuma et al., 2000) | Addgene #11938 and #11939 | |
| Recombinant DNA reagent | HSE (WT)-Luc (plasmid) | a gift from Ueli Schibler (Reinke et al., 2008) | Hsf1 reporter plasmid | |
| Recombinant DNA reagent | pEGFP-Q74 (plasmid) | a gift from David Rubinsztein (Narain et al., 1999) | Addgene # 40261 | |
| Recombinant DNA reagent | pRL-CMV (plasmid) | Promega | #E2261 | |
| Recombinant DNA reagent | pSpCas9(BB)–2A-Puro (PX459) (plasmid) | a gift from Feng Zhang (Ran et al., 2013) | Addgene #48139 | |
| Recombinant DNA reagent | pGL3-CMV.Luc (plasmid) | a gift from Laurent Guillemot (University of Geneva) | ||
| Sequence-based reagent | shHSF1-1 | This paper, from Microsynth | HSF1 shRNA 3UTR forward | 5'-CCG GGC AGG TTG TTC ATA GTC AGA ACT CGA GTT CTG ACT ATG AAC AAC CTG CTT TTT G-3' |
| Sequence-based reagent | shHSF1-1 | This paper, from Microsynth | HSF1 shRNA 3UTR reverse | 5'-AAT TCA AAA AGC AGG TTG TTC ATA GTC AGA ACT CGA GTT CTG ACT ATG AAC AAC CTG C-3' |
| Sequence-based reagent | shHSF1-2 | This paper, from Microsynth | HSF1 shRNA CDS forward | 5'-CCG GCC AGC AAC AGA AAG TCG TCA ACT CGA GTT GAC GAC TTT CTG TTG CTG GTT TTT G-3' |
| Sequence-based reagent | shHSF1-2 | This paper, from Microsynth | HSF1 shRNA CDS reverse | 5'-AAT TCA AAA ACC AGC AAC AGA AAG TCG TCA ACT CGA GTT GAC GAC TTT CTG TTG CTG G-3' |
| Chemical compound | L-azetidine-2-carboxylic acid (AZC) | Sigma-Aldrich | #P8783 | |
| Chemical compound | Tunicamycin | Cell Signalling | #12819 | |
| Chemical compound | Propidium iodide (PI) | Cayman Chemical | #14289–10 | |
| Chemical compound | Annexin V | Biolegend | #640906 | |
| Chemical compound | Phalloidin-Alexa488 | Thermo Fisher Scientific | #A12379 | |
| Chemical compound | Diamidino-2-phenylindole dye (DAPI) | Thermo Fisher Scientific | #62248 | 1:30,000 in PBS, from 1 mg/ml stock solution |
| Chemical compound, drug | Abemaciclib | MedChemExpress | #HY-16297A-5MG | |
| Chemical compound, drug | Rapamycin | Sigma-Aldrich | #553210 | |
| Commercial assay, kit | Dual-Luciferase detection kit | Promega | #E1910 | |
| Commercial assay, kit | Alexa Fluor 488 NHS Ester (succinimidyl ester) | Thermo Fisher Scientific | #A20100 | |
| Commercial assay, kit | Click-iT Plus OPP Alexa Fluor 594 | Thermo Fisher Scientific | #C10457 | |
| Commercial assay, kit | N-succinyl-Leu-Leu-Val-Tyr-7-amino-4-methyl-coumarin (suc-LLVY-AMC) | Enzo Life Sciences | #BML-P802-0005 | |
| Commercial assay, kit | CellEvent Senescence Green Flow Cytometry Assay Kit | Invitrogen | #C10840 | |
| Antibody | Anti-GAPDH (Mouse monoclonal) | HyTest Ltd. | 5G4 | 1:1000 |
| Antibody | Anti-Hsp25/27 (Mouse monoclonal) | StressMarq | SMC-114 | 1:1000 |
| Antibody | Anti-puromycin (Mouse monoclonal) | Sigma-Aldrich | MABE343 | 1:22000 |
| Antibody | Anti-Lamin B1 (Rabbit polyclonal) | Cell Signaling Technology | 12586 | 1:1000 |
| Antibody | Anti-Hsp40/Hdj1 (Rabbit polyclonal) | Enzo Lifesciences | ADI-SPA-400 | 1:1000 |
| Antibody | Anti-Hsf1 (Rabbit polyclonal) | Enzo Lifesciences | ADI-SPA-901 | 1:1000 |
| Antibody | Anti-Hsp70 (Mouse monoclonal) | StressMarq | SMC-100 | 1:1000 |
| Antibody | Anti-Phospho-eIF2α (Ser51) (Rabbit polyclonal) | Cell Signaling Technology | 3597 | 1:1000 |
| Antibody | Anti-eIF2α (Rabbit polyclonal) | Cell Signaling Technology | 9722 | 1:1000 |
| Antibody | Anti-Hsp90α (9D2) (Rat monoclonal) | Enzo Lifesciences | ADI-SPA-840 | 1:1000 |
| Antibody | Anti-Hsp90β (scFv H90-10) (Mouse monoclonal) | Geneva Antibody Facility | ABCD_A0870 | 1:2000 |
| Antibody | Anti-mTOR (Rabbit polyclonal) | Cell Signaling Technology | 2983 | 1:2000 |
| Antibody | Anti-Phospho-mTOR (Ser2448) (Rabbit polyclonal) | Cell Signaling Technology | 2971 | 1:1000 |
| Antibody | Anti-Phospho-S6 Ribosomal Protein (Ser235/236) (Rabbit polyclonal) | Cell Signaling Technology | 4858 | 1:1000 |
| Antibody | Anti-S6 Ribosomal Protein (Rabbit polyclonal) | GeneTex | GTX130450 | 1:1000 |
| Antibody | Anti-Hsc70 (Mouse monoclonal) | StressMarq | SMC-151 | 1:2000 |
