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) Flow cytometric quantification of cell size after 7 days of chronic HS (biologically independent samples: n = 6 for HEK and A549; n = 5 for RPE1; n = 4 for HCT116). (C and D) Flow cytometric analysis of cell cycle (n = 3 biologically independent samples). (E) Flow cytometric quantification of cell size during different time intervals of chronic HS (n = 4 biologically independent samples). (F) Proliferation of HEK and A549 cells at the indicated temperature for the indicated period presented as cell numbers. Cells were seeded at a density of 5 x 106 and 3 x 106 per 15 cm plate for HEK and A549 cells, respectively. The numbers of live cells counted after 7 days are plotted (n = 5 biologically independent experiments). (G) Proliferation of A549 and RPE1 cells measured with a crystal violet assay (n = 3 biologically independent experiments). The adapted cells were maintained at 40 °C for one week before this experimental start point and continued at 40 °C during the experiment. See scheme of the experiment on the right. Note that the data are normalized to cell numbers on day 1. (H) Flow cytometric quantification of cell size in chronic HS and recovery (n = 3 biologically independent samples). (I) Flow cytometric analysis of cell cycle in chronic HS and post HS recovery (n = 3 biologically independent samples). The data are represented as mean values ± SEM for all bar and line graphs. The statistical significance between the groups was analyzed by two-tailed unpaired Student’s t-tests.

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 images of n = 4 biologically independent experiments). (B) Flow cytometric quantification of cell viability of HEK, A549, and their respective Hsp90α/β KO cells in chronic HS at different time points during a period of 7 days (n = 5 biologically independent samples). Note that the X axis does not have a linear scale and that lines connecting the data points are drawn as a visual aid. (C) Flow cytometric quantification of cell size in chronic HS at different time points during a period of 7 days (HS = 39 °C for HEK and 40 °C for A549) (n = 4 biologically independent samples) The data are represented as mean values ± SEM for all bar graphs. (D) Fluorescence microscopy images of A549 WT and Hsp90α/β KO cells fixed after 4 days of chronic HS. The cytoskeleton is stained with phalloidin-Alexa488 (green), and the nucleus is stained with DAPI (blue). Images were captured with a fluorescence microscope (Zeiss, Germany). The scale bars on the images in the far right column are 50 mM. (E) Bar graphs of the area of nuclei and whole cells determined from fluorescent micrographs (representative images are shown in panel D) using ImageJ. We used micrographs from 3 biologically independent experiments. From each experiment we measured 30 randomly chosen cells, and their average values were used as one data point. The data for all bar graphs are represented as mean values ± SEM. The statistical significance between the groups was analyzed by two-tailed unpaired Student’s t-tests.

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 replicates each time). (B) Immunoblots of Hsf1 in the cytosolic and nuclear fractions of HEK WT and Hsp90α/β KO cells (α KO, Hsp90αKO; β KO, Hsp90βKO). GAPDH and lamin B1 serve as loading controls (representative blots of n = 2 biologically independent experiments). (C) Fold change of Hsf1 activity of HEK WT, A549 WT, and their respective Hsp90α/β KO cells in chronic HS as measured by luciferase reporter assay (n = 3 biologically independent samples, and 2 experimental replicates each time for HEK; n = 3 biologically independent samples, and 4 experimental replicates each time for A549). (D) Volcano plots of the normalized fold changes in protein levels of some core Hsf1 target genes (list obtained from https://hsf1base.org/) in chronic HS, determined by quantitative label-free proteomic analysis of Hsp90α/β KO and WT HEK cells. Molecular chaperones, whose expression is regulated by Hsf1, are excluded from this dataset. Each genotype was compared with its respective 37 °C control (n = 3 biologically independent samples). Log2 fold changes of > 0.5 or < -0.5 with q-values (adjusted p-values) of < 0.05 were considered significant differences for a particular protein. (E) Flow cytometric quantification of cell size of HEK, A549, and RPE1 cells upon overexpression of WT Hsf1 (with plasmid pcDNA-Flag HSF1 wt) or mutant Hsf1 (with plasmid pcDNA-Flag HSF1 C205; retaining only the first 205 amino acids)(Kijima et al., 2018), and with plasmid pcDNA3.1(+) as empty vector control. Transfected cells to be measured were identified on the basis of their coexpression of EGFP (n = 4 biologically independent experiments). (F) Flow cytometric quantification of cell size in chronic HS after knockdown of Hsf1 in HEK WT, A549 WT and Hsp90αKO cells. Here the chronic HS for A549 cells is at 39 °C instead of 40 °C to reduce HS-induced damage in Hsf1 knockdown conditions (n = 3 biologically independent samples). For all bar graphs, the statistical significance between the groups was analyzed by two-tailed unpaired Student’s t-tests.

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 label-free proteomic analyses of Hsp90α/β KO and WT HEK cells. Each genotype was compared with its respective 37 °C control (n = 3 biologically independent samples). Log2 fold changes of > 0.5 or < -0.5 with q-values (adjusted p-values) of < 0.05 (indicated as stippled lines) were considered significant differences for a particular protein. (B) Immunoblots of different molecular chaperones in HEK WT and Hsp90α/β KO cells (α KO, Hsp90αKO; β KO, Hsp90βKO). GAPDH serves as the loading control for all three panels (representative of n = 2 independent experiments). (C) Volcano plots of the normalized fold changes of the Hsp90 interactors (list obtained from https://www.picard.ch/Hsp90Int) after 4 days of chronic HS determined by quantitative label-free proteomic analyses of Hsp90α/β KO and WT HEK cells. Each genotype was compared with its respective 37 °C control (n = 3 biologically independent samples). (D and E) In vivo refolding of heat-denatured luciferase of control cells (blue line) and cells heat-adapted to 39 °C (orange line). Luciferase activity before the acute HS (at 43 °C) is set to 100% (n = 3 biologically independent samples). See scheme of the experiment below. Note the different scales of the Y axes of the bar graphs in panel E. The data are represented as mean values ± SEM for all bar graphs. The statistical significance between the groups was analyzed by two-tailed unpaired Student’s t-tests. The p-values for Hsp90α and Hsp90β KO cells are in blue and red, respectively. All p-values are for comparisons to the respective WT.

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 coefficients (n = 10 cells from 2 biologically independent experiments). The statistical significance between the groups was analyzed by two-tailed unpaired Student’s t-tests. (B) Fold change of cell size (represented by the FSC-MFI values) and total proteins (determined as MFI-FL1 values) in chronic HS as analyzed by flow cytometry. Cells were fixed, and total proteins were stained using Alexa Fluor 488 NHS ester (n = 3 biologically independent experiments). Lines connecting the data points are drawn as a visual aid. (C) GSEA plot showing the up- or down-regulation (red and blue, respectively) of pathways associated with cellular transcription and translation after 4 days of chronic HS compared to the respective 37 °C controls; NES, normalized enrichment score. (D) Volcano plots of the normalized fold changes of the ribosomal proteins (list obtained from http://ribosome.med.miyazaki-u.ac.jp/) after 4 days of chronic HS determined by quantitative label-free proteomic analysis in Hsp90α/β KO and WT HEK cells. Each genotype was compared with its respective 37 °C control (n= 3 biologically independent samples). Log2 fold changes of > 0.6 or < -0.6 with q-values (adjusted p-values) of < 0.05 (indicated as stippled lines) were considered significant differences for a particular protein. The boxes beside the volcano plots list the corresponding proteins that were significantly downregulated.

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 labeled with OP-puromycin during cell culture, and the incorporation of puromycin at different time points was analyzed (n = 4 experimental samples). (B) Representative polysome profiles of HEK WT cells at 37 °C and after 4 days of chronic HS (representative of n = 2 biologically independent experiments). (C and D) Immunoblots of the translation-related proteins eIF2α, mTOR, and S6. GAPDH and β-actin serve as loading controls. Note that the same β-actin immunoblot is used twice as internal loading control since the other immunoblots of panel D are from the same experiment. Representative immunoblots are shown, but the corresponding bar graphs on the right are based on the quantitation of 3 biological replicates. Values were normalized to the loading controls, and the phosphoproteins were normalized to their respective total protein. (E) Relative fold changes of total translation and cell size in the early phase of adaptation to chronic HS (see schemes of experiments on the right) for A549 WT and Hsp90α/β KO cells. The data are represented as mean values ± SEM for all bar graphs. The statistical significance between the groups was analyzed by two-tailed unpaired Student’s t-tests.

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 cytometric measurement of autophagic flux in chronic HS compared to 37 °C, using a mCherry-GFP-LC3 reporter. Flux is calculated as the ratio of the mean fluorescence intensities of mCherry and GFP-positive cells (n = 4 biologically independent samples). (C) In vitro steady-state proteasomal activity with lysates of HEK WT, and Hsp90α and Hsp90β KO cells determined by measuring fluorescence of the cleaved substrate suc-LLVY-AMC (n = 2 biologically independent samples). (D) Fluorescence micrographs of cells expressing the fusion protein EGFP-Q74 visible as aggregates with green fluorescence. The scale bars in the zoomable micrographs indicate 10 μm (images are representative of n = 2 independent biological samples). The data are represented as mean values ± SEM for all bar graphs. The statistical significance between the groups was analyzed by two-tailed unpaired Student’s t-tests.

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 were washed and subjected to chronic HS at 40 °C for 3 more days (CDKi > HS). Cell size (% FSC-MFI; grey part of the bars) and cell death (% annexin V and PI-positive; red part of the bars) were measured by flow cytometry. The values for cell size and death in the different experimental conditions are normalized to the respective 37 °C controls (n = 3 biologically independent experiments). (C) Cells were first pretreated with 7.5 nM rapamycin (Rapa) for 3 days to reduce the cell size. After that, the cells were subjected to chronic HS at 40 °C for 3 days (Rapa > HS). HS > Rapa, the two treatments were done the other way around. The cell size (% FSC-MFI) and relative cell death (% annexin V and PI-positive) were quantified by flow cytometry. The values for cell size and death in different experimental conditions are normalized to the respective 37 °C control (n = 3 biologically independent experiments). (D) Scheme of experiments aimed at determining impact of limiting physical space on cell size increase. (E and F) Phase-contrast micrographs of RPE1 cells seeded in different numbers to restrict the space for cell size increase during adaptation to chronic HS (representative images of n = 4 biologically independent experiments). The size bar in the top left panel indicates 100 µM. The cell size (% FSC-MFI) and relative cell death (% annexin V-PI positive) are quantified by flow cytometry. For the bar graphs, the values for cell size and death in different conditions are normalized to the low density (dns) cell population at 37 °C day 0 (n = 4 biologically independent experiments). The data are represented as mean values ± SEM for all bar graphs. The statistical significance between the groups was analyzed by two-tailed unpaired Student’s t-tests.

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 the Coomassie-stained gels in the upper panels show the corresponding levels of total proteins. Colored boxes indicate lanes for samples from cells subjected to chronic HS. The bar graphs on the right show the corresponding quantitation of 3 biologically independent experiments, with the colored rectangles using the same color code as the rectangles over the immunoblots on the left. The band densities of all blots were normalized to the loading controls. The data are represented as mean values ± SEM. The statistical significance between the groups was analyzed by two-tailed unpaired Student’s t-tests.

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 cells exposed to prolonged mild HS as indicated, as determined by flow cytometry. (C) Quantitation of flow cytometric analyses of the cell cycle (n = 3 biologically independent samples). (D) Dot plot representing numbers of cells as a function of SA-βgal staining analyzed by flow cytometry. (E) Bar graph showing the mean fluorescent intensities of 3 biologically independent SA-βgal staining experiments of the type shown in panel D. (F) Fold change of cell size (represented by the FSC-MFI values) and total proteins (determined as MFI-FL1 values) in prolonged chronic HS, as analyzed by flow cytometry. Cells were fixed, and total proteins were stained using Alexa Fluor 488 NHS ester (n = 3 biologically independent experiments). Lines connecting the data points are solely drawn as a visual aid. Note that the quantitative differences of cell size increases between panels A and F may be due to technical differences (live cell versus fixed cell analyses, respectively; see Materials and Methods for further details). (G) Schematic representation of the impact of chronic mild stress on cells. Wild-type cells initially adapt by enlarging their size and increasing total protein to maintain a minimum threshold level of functional proteins. The right part of the scheme (surrounded by a stippled box), shows what happens if stress persists for much longer: cell size enlargement and total amount of proteins are uncoupled, and because of protein damage, which continues to accumulate, cells become senescent and/or die. The data for all the bar graphs are represented as mean values ± SEM.