Figures and data in Exploring the role of macromolecular crowding and TNFR1 in cell volume control | eLife

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Figure 1 with 1 supplement

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Fluorescence anisotropy of EGFP is a robust probe for macromolecular crowding.

(A) Steady-state fluorescence anisotropy of EGFP ( $r_{E G F P}$ ) progressively increases with crowder concentration and crowder molecular weight. (B) Fluorescence lifetime of EGFP ( $τ_{E G F P}$ ) steadily decreases with increasing crowder concentration - as shown for bovine serum albumin (BSA) (protein) and Ficoll (polysucrose). (C-i) Time-resolved fluorescence anisotropy of EGFP (continuous lines - representative data from one experiment) and their fit to mono-exponential decay (dashed lines) in three different BSA concentrations along with their residuals, (C-ii) $r_{0}$ (intrinsic anisotropy) and $θ_{C}$ (rotational correlation time) of EGFP vs BSA concentration, as obtained from curve fitting in C-i. (D) Comparison of the reconstructed $r_{E G F P}$ (dashed line) using the Perrin equation with the $r_{0}$ , $τ_{E G F P}$ , and $θ_{C}$ values obtained from B and C-ii, and the measured $r_{E G F P}$ (solid line) for the same BSA concentrations. (E) Comparison of the steady-state fluorescence anisotropy of EGFP and fluorescein in solutions of varying glycerol content (zoomed-in glycerol content 80–90%), showing that the viscosity dependence of $r_{E G F P}$ is negligible. (F) $r_{E G F P}$ vs EGFP concentration in HEPES buffer (pH 7.4) reveals that at [EGFP]>10 µM, $r_{E G F P}$ enters the homo-FRET regime. (G) Dependence of $r_{E G F P}$ on the solution pH of HEPES buffers. All the plots show the mean values obtained from at least three individual experiments (N≥3) performed at 25°C, and the error bars represent the standard deviation (SD). Except (F), 50 nM EGFP was used for all experiments.

Figure 1—source data 1 Data tables for Figure 1C-i and Figure 1—figure supplement 1A.: https://cdn.elifesciences.org/articles/92719/elife-92719-fig1-data1-v2.zip
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Figure 1—figure supplement 1

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Fluorescence anisotropy of EGFP is a robust probe for macromolecular crowding.

(A) Concentration estimation of the purified EGFP from FCS (fluorescence correlation spectroscopy) measurements, performed at 25°C. Fluorescence autocorrelation curves representing the average of 200 measurements ( $G_{τ}$ vs $τ$ ) for different dilutions of the purified EGFP, their fit (dashed lines), and residuals. The inset shows the number density of EGFP molecules within the confocal volume ( $N = \frac{1}{G_{0} - 1}$ ) (red squares) and the total fluorescence intensity (blue circles) obtained using our $r_{E G F P}$ measurement setup vs the different dilutions of purified EGFP (shades of green). (B) Schematic of the $r_{E G F P}$ measurement setup. (C) Mean and SD of the refractive index measurements for increasing concentrations of bovine serum albumin (BSA) and Ficoll (in HEPES buffer, pH 7.4, 25°C) to find the refractive index increment factor by linear fitting. (D) Validation of the Strickler-Berg relation for BSA and Ficoll - $τ_{E G F P}$ vs $1 / n^{2}$ ( $n$ - refractive index) (BSA - blue, open circle; Ficoll - red, open square) using the $τ_{E G F P}$ values from Figure 1B.

Figure 2 with 1 supplement

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Macromolecular crowding (MMC) levels do not significantly vary between individual cell lines.

(A) Time-resolved fluorescence micrographs of NIH/3T3 fibroblasts expressing EGFP in isotonic (top panel) and hypertonic (bottom panel) conditions. Representative images of EGFP’s total intensity in (i), fluorescence lifetime ( $τ_{E G F P}$ ) in (ii), rotational correlation time ( $θ_{C}$ ) in (iii), intrinsic anisotropy ( $r_{0}$ ) in (iv), steady-state anisotropy ( $r_{E G F P}$ ) calculated using the Perrin equation in (v) with values from (i, ii, iii), measured steady-state anisotropy ( $r_{E G F P}$ ) in (vi), and the difference between the anisotropy values obtained from the Perrin equation and direct measurements in (vii). Accompanying calibration bars indicate the colors representing the depicted quantities. (B) The $r_{E G F P}$ maps of two extreme examples of NIH/3T3-EGFP cells having dissimilar morphologies (aspect ratios) and the intracellular distribution of $r_{E G F P}$ values during isotonic and hypertonic conditions (+600 mM mannitol), highlighting the consistency of the modal value of $r_{E G F P}$ per cell in depicting MMC changes at different experimental conditions compared to the mean value of $r_{E G F P}$ per cell. (C) Cell-to-cell variability of MMC among NIH/3T3 fibroblasts (n=828 cells, N=3) imaged by a ×10 objective. The accompanying distributions depict kernel-smoothed histograms (modal $r_{E G F P}$ , dark red) and DNA content (Hoechst intensity, dark blue). (D) $r_{E G F P}$ and total intensity maps of representative cells from different cell lines. (E) The modal $r_{E G F P}$ value per cell from different cell lines show that only RAW 264.7 cells have a statistically different distribution of cellular MMC. The boxes represent the distribution mean ± 1 SD, and the whiskers represent 5–95 percentiles. Number of biological replicates (cells) are provided alongside for at least four independent experiments for each cell line. Statistical analysis was performed using the non-parametric Kruskal-Wallis ANOVA after Bonferroni alpha-correction, followed by Mann-Whitney test for every group pair. **** indicates p<0.000025.

Figure 2—source data 1 Data tables for Figure 2B, C, and E and Figure 2—figure supplement 1A, C, and D.: https://cdn.elifesciences.org/articles/92719/elife-92719-fig2-data1-v2.zip
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Figure 2—figure supplement 1

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Macromolecular crowding (MMC) levels do not significantly vary between individual cell lines.

(A, i) The distribution of modal $r_{E G F P}$ values per cell (5–95 percentile) is narrower than the distribution of mean values due to the spatially varying $r_{E G F P}$ maps (during isotonic conditions), and (ii) 600 mM hypertonicity-induced changes are more consistent when considering modal values, as shown for one set of experimental cell population. Number of biological replicates (n cells) and technical repeats (N experiments) provided alongside. (B) Representative merged image of EGFP total intensity (‘Red Hot’ LUT) and Hoechst intensity (‘Cyan Hot’ LUT), with the corresponding $r_{E G F P}$ (‘16 colors’ LUT) maps of NIH/3T3-EGFP cells imaged using a ×10 objective. (C) Testing the presence of homo-FRET in an NIH/3T3-EGFP population using photobleaching: (i) change in modal values, (ii) change in total intensity upon photobleaching, and (iii) representation of the average changes. Cells expressing EGFP (gray schema) had a non-significant change in anisotropy values, while cells expressing 2GFP (yellow schema) had an average of ~8% increase (shown in iii) in the measured anisotropy values since 2GFP molecules exhibit high degrees of homo-FRET. Moreover, 2GFP-expressing cells with similar expression levels as monomeric EGFP-expressing cells have lower $r_{E G F P}$ , which is a hallmark of homo-FRET. Statistical analysis performed by paired sample t-test. **** indicates p<0.0001 (n=167 and 71 cells for EGFP and 2GFP populations respectively, N=2). (D) Estimating [EGFP] in a representative NIH/3T3 cell using fluorescence correlation spectroscopy (FCS). The intensity of cellular EGFP was reduced by photobleaching to obtain appropriate counts for FCS. The intracellular [EGFP] in the photobleached cell was ≅ 1.7 µM. The [EGFP] estimated in the photobleached cell was scaled up according to the average intensities pre and post bleaching to have ≅ 8 µM before photobleaching. Comparing the pre-bleach average intensity value of the same cells obtained in the FCS setup and $r_{E G F P}$ setup, the estimated [EGFP] in the cell population used in (C) is plotted in the inset, with the modal value of [EGFP] ≅ 7 µM. (E) Photobleaching NIH/3T3-mEGFP cells at 10 min post 600 mM hypertonicity induction does not increase $r_{E G F P}$ , thus indicating the absence of homo-FRET in the cells under hypertonic stress (n=31 cells, N=2). Statistical change in $r_{E G F P}$ (black open circles) evaluated by paired sample t-test. The filled red diamonds indicate the average and standard deviation of the cellular EGFP total intensities.

Figure 3 with 1 supplement

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The actin cytoskeleton enforces spatially varying macromolecular crowding (MMC) levels.

(A) EGFP intensity, Hoechst-stained DNA (in cyan), and phalloidin Alexa Fluor 546-stained actin (in magenta), and $r_{E G F P}$ map of an NIH/3T3 cell shows that the spatial heterogeneity of intracellular MMC is demarcated by actin stress fibers (arrows). (B-i) Average fluorescence recovery after photobleaching (FRAP) recovery curves and diffusion rates of EGFP in the lamellar (in black) and perinuclear (in red) regions of NIH/3T3 cells (n=21 cells; N=2), with the error bars representing standard deviation. Statistical significance was evaluated by unpaired t-test. (B-ii) The average MSD (mean-squared displacement) and their standard deviations obtained from tracking 200 nm fluorescent beads in the lamellar (dashed line in black) and perinuclear (solid line in red) regions of NIH/3T3 cells (n=17 cells; N=2). (C) The total intensity map of an EGFP expressing HeLa cell’s lamellar region, viewed laterally (XY) in panel (i), or its cross-section (XZ) in panel (ii) along the yellow line in (i), with the arrows indicating the thin lamella. Panels (iii) and (iv) show the corresponding $r_{E G F P}$ and fluorescence lifetime ( $τ$ ) maps of panel (i). Panel (v) shows the graphical explanation of the influence of cell height on $r_{E G F P}$ values. Panel (vi) shows the different regions used to calculate the contributions of autofluorescence (gray), lamellar regions (blue), and the cell body (red) to the $τ$ map in the phasor plot of panel (vii). In the phasor plot, the pixels corresponding to the $τ$ of the thin lamellar region (blue dots) are slightly shifted above the $τ$ of the cell body (red dots), revealing that $τ$ in the lamellar region is slightly greater, and thus MMC is slightly lower than the cell body. (D) Representative images of NIH/3T3-EGFP, quantifications of the spatial heterogeneity of cytoplasmic $r_{E G F P}$ , modal $r_{E G F P}$ (n=49, 51, 40 cells; N=2), and cell volume (n=11, 10, 8 cells; N=3) for individual cells. Black and orange colors represent pre- and post-treatment with (i) cytochalasin D (2 μM, 1 hr), (ii) nocodazole (20 μM, 1 hr), and (iii) withaferin A (3 μM, 3 hr). Statistical analysis performed by paired sample t-test. **** indicates p<0.0001, *** indicates p<0.001, * indicates p<0.05.

Figure 3—source data 1 Data tables for Figure 3B, C-vi, and D and Figure 3—figure supplement 1A and E.: https://cdn.elifesciences.org/articles/92719/elife-92719-fig3-data1-v2.zip
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Figure 3—figure supplement 1

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The actin cytoskeleton enforces spatially varying macromolecular crowding (MMC) levels.

(A) The microviscosity of bovine serum albumin (BSA) solutions investigated by fluorescence recovery after photobleaching (FRAP) in (i) and single-particle tracking of 200 nm beads in (ii). The average and SD for N=3 is shown. Statistical significance was evaluated by unpaired t-test. (B-i) Total intensity map of EGFP (left column - ‘grays’ LUT) and corresponding $r_{E G F P}$ map (right column - ‘16 colors’ LUT) of the optically thin lamellar region of a HeLa cell captured using a laser scanning confocal microscope with the pinhole size set to 1 Airy unit (AU) or opened completely. The pixel values along the white line in the $r_{E G F P}$ maps are plotted in (B-ii), where the red and cyan ‘*’-s mark the starting point of the lines. (C-i) Total intensity map of EGFP (left - ‘Fire’ LUT) and corresponding $r_{E G F P}$ map (right - ‘16 colors’ LUT) of an NIH/3T3 cell with a prominent lamellar structure in culture media without and with serum. The pixel values along the white line in the $r_{E G F P}$ maps are plotted in (C-ii). Reducing serum content decreases media autofluorescence and increases the $r_{E G F P}$ values in the optically thin lamellar regions. (D) Schematic of spatial heterogeneity analysis for $r_{E G F P}$ (Figure 3D) and cell height (in E). Geodesic distance maps (GDMs) were computed between cell and nucleus boundaries (ii), normalized between 0% and 100% range, and then grouped into equidistant sectors (iii). Each sector has a width equal to 10% of maximum distance between the cell and nucleus boundaries, calculated for each cell. The GDM normalization accounts for the variabilities in cell shape and size. Finally, the $r_{E G F P}$ values (or cell height) were normalized in the 0–100% range to account for cell-to-cell variabilities, and the average normalized $r_{E G F P}$ (or cell height) for each sector was plotted in Figure 3D (and here, in E). (E) Cell height maps and cell height heterogeneity created from Z-stacks of NIH/3T3-EGFP before and after cytochalasin D (i), nocodazole (ii), or withaferin A (iii) treatments. The cell height maps were generated by applying a vertical height-dependent color code to individual pixels of a cell’s basal plane. Then, following the analysis presented in panel D, the cell height heterogeneity was calculated for all cells used for volume measurement (n=11, 10, 8 cells; N=3).

Figure 4 with 1 supplement

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The characteristic cellular macromolecular crowding (MMC) is linked to cell spreading and adhesion.

(A-i) $r_{E G F P}$ maps (top row), EGFP total intensity maps (middle row), and DIC images (bottom row) of NIH/3T3-EGFP during spreading on fibronectin-coated glass. (A-ii) Modal $r_{E G F P}$ values (cyan) and spread area (magenta) of NIH/3T3-EGFP averaged over n=109 cells, N=4. Error bars represent standard deviation. (A-iii) Average cell volume (green, open squares) and spread area (magenta, filled diamonds) of NIH/3T3 cells after seeding on fibronectin-coated glass (n=11 cells; N=4). Error bars show SD. (B) Modal $r_{E G F P}$ of NIH/3T3-EGFP cells vs their morphological spread area on fibronectin-coated glass, with the blue line indicating the negative linear correlation and the associated color bar denoting the shape circularity ( $4 π A r e a / {P e r i m e t e r}^{2}$ ) (n=201 cells; N=3). (Ci) Fluorescence recovery after photobleaching (FRAP) analysis of EGFP in NIH/3T3 cells seeded on fibronectin (50 µg/mL) or 10% polyethylene glycol (PEG)-400-coated glass for 2 hr. The average recovery curves and diffusion rates of EGFP are shown with the error bars representing the SD (n=21, 14 cells; N=2). Statistical significance was evaluated by unpaired t-test. ** indicates p<0.01. (C-ii) Modal $r_{E G F P}$ values of NIH/3T3-EGFP cells seeded on fibronectin or 10% PEG for 2 hr (n=87, 35 cells; N=2). Statistical analysis was performed using Mann-Whitney test. **** indicates p<0.0001. (D-i) $r_{E G F P}$ maps (top row), EGFP total intensity maps (middle row), and DIC images (bottom row) of NIH/3T3-EGFP undergoing substrate detachment due to trypsinization. (D-ii) Comparison of the modal $r_{E G F P}$ for untreated controls, cytochalasin D (2 µM, 1 hr) treated, trypsinized (20 min), and cytochalasin D pre-treatment (2 µM, 1 hr) then trypsinized (20 min) in NIH/3T3 (n=131, 49, 57, 57 cells; N=3). Statistical analysis was performed using Mann-Whitney test for every group pair.

Figure 4—source data 1 Data tables for Figure 4B, C-ii, and D-ii.: https://cdn.elifesciences.org/articles/92719/elife-92719-fig4-data1-v2.zip
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Figure 4—figure supplement 1

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The characteristic cellular macromolecular crowding (MMC) is linked to cell spreading and adhesion.

(A) Total intensity and $r_{E G F P}$ maps of NIH/3T3-EGFP seeded on fibronectin (50 µg/mL) or 10% polyethylene glycol (PEG)-400-coated glass for 2 hr. (B) Maximum intensity projections of NIH/3T3 cells expressing LifeAct-mGFP showing the effects of trypsinization, cytochalasin D treatment, or cytochalasin D pre-treatment+trypsinization on F-actin. (C) Maximum intensity projection of a HeLa cell co-expressing Tubulin-mCherry and LifeAct-mGFP shows rapid cytoskeletal depolymerization upon trypsinization. For the panels in (B) and (C), the inverted gray LUT was used, and the image brightness-contrast was enhanced separately for better visualization of cytoskeletal filaments.

Figure 5 with 1 supplement

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Proteostasis disruption alters cellular macromolecular crowding (MMC) setpoint.

(A) Average percentage changes in cell volume (filled symbols) and modal $r_{E G F P}$ (open symbols) for NIH/3T3 in (i) and HeLa in (ii) upon exposure to hypertonic mannitol (150 mM) (n>40 cells; N=4 for modal $r_{E G F P}$ data, n>10 cells; N=3 for cell volume data). (B) Testing if the $r_{E G F P}$ recovery is mediated by hypertonicity-induced cation channels (HICCs) using flufenamic acid. Closed symbols represent cells pre-treated with 700 µM flufenamic acid for 1 hr; open symbols represent untreated cells. Average percentage change in modal $r_{E G F P}$ (n>40 cells; N=2 for each case) plotted with the standard deviation (SD) error bars. (C) Hypertonic shock response of different cell lines estimated through $r_{E G F P}$ measurements. Cells were subjected to an additional 50 mM or 150 mM hypertonicity using mannitol, and the average percentage change in modal $r_{E G F P}$ and its SD is depicted (n>40 cells, N>3 for each case). (D) Response of NIH/3T3 fibroblasts to different strengths of hypertonicity (by mannitol) and 50% hypotonicity. The ability of cells to recover $r_{E G F P}$ within 30 min decreases with increasing hypertonicity (n>50 cells, N≥3). (E) Diffusion rates of cytoplasmic and nuclear EGFP population estimated through fluorescence recovery after photobleaching (FRAP). The average and SD are shown (n>12 cells for each case). Statistical significance was evaluated by unpaired t-test against the isotonic condition. **** indicates p<0.0001. (F) Isotonic perturbations of intracellular MMC by proteostasis disruption - blocking protein degradation (MG 132 and heclin), protein translation (cycloheximide), or inducing widespread protein degradation using heat shock. The mean and SD of the modal $r_{E G F P}$ values are represented in (i) (n=132, 98, 138, 90, 130 cells; N=3). Statistical analysis was performed using Kruskal-Wallis ANOVA after Bonferroni correction, followed by Mann-Whitney test for every group pair. **** indicates p<0.00002. Corresponding to (i), the intracellular protein mass under each condition is illustrated in (ii) (N=3), and the percentage change in the average volume before and after treatment in (iii) (n>20 cells; N>2 for each case). Statistical significance was evaluated by paired sample t-test. ** indicates p<0.01, * indicates p<0.05.

Figure 5—source data 1 Data tables for Figure 5F.: https://cdn.elifesciences.org/articles/92719/elife-92719-fig5-data1-v2.zip
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Figure 5—figure supplement 1

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Proteostasis disruption alters cellular macromolecular crowding (MMC) setpoint.

(A) Representative images of NIH/3T3 cells exposed to hypotonic stress (50% osmolarity). (B) Quantification of the modal $r_{E G F P}$ and cell outline area (average and SD) from EGFP total fluorescence images show that that the initial recovery of MMC post hypotonicity induction is not sustained and MMC increases to a near-isotonic point after 2 hr (n=53 cells, N=3). (C) Response of HeLa cells to different strengths of hypertonicity induced by mannitol - the average percentage change in modal $r_{E G F P}$ and its SD are shown. Similar to NIH/3T3, HeLa cells fail to recover at 600 mM hypertonic shock within 60 min (n>40 cells; N=2 for each case). (D) Response of NIH/3T3 fibroblasts to different strengths of hypertonicity induced by dextrose (n>40 cells; N=3 for each case). (E) Comparing the hypertonic stress response of NIH/3T3 fibroblasts at 100 mOsm and 600 mOsm hypertonicities induced by different osmolytes - dextrose, mannitol, or NaCl (n>30 cells; N>2 for each case). (F-i) The average modal $r_{E G F P}$ per cell and the average translational diffusion rates of cytoplasmic EGFP estimated by fluorescence recovery after photobleaching (FRAP) plotted against the corresponding extracellular hypertonicities. Each type of measurement was performed 10 min after hypertonicity induction. (F-ii) Average translational diffusion rates of cytoplasmic EGFP vs average modal $r_{E G F P}$ per cell. Error bars indicate standard deviation. The data was extracted from the experimental population shown in Figure 5E and F.

Figure 6 with 1 supplement

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Hypertonic stress-induced NFkB activation is mediated by TNFR1.

(A) Nuclear translocation of p65 visualized through immunofluorescence and Hoechst co-staining for wild-type and TNFR1-knockdown (TNFR1-KD) HeLa nuclei. Nuclear translocation of p65 indicates NFkB pathway activation upon 15 min of treatment with soluble human TNFa (20 ng/mL) or hypertonic mannitol (150 mM and 600 mM). All scale bars represent 20 µm. (B) Quantification of p65 nuclear translocation from immunofluorescence images of HeLa cells under indicated conditions. Statistical analysis was performed using Kruskal-Wallis ANOVA after Bonferroni alpha-correction, followed by Mann-Whitney test for the indicated pairs. * indicates p<0.00625, **** indicates p<0.0000125. (C) Quantification of cell volume under indicated conditions (n=13, 11, 12 cells; N=2 for HeLa, and n=16, 11 cells; N=3 for NIH/3T3). TNFR1 inactivation leads to a decrease in cell volume in both HeLa and NIH/3T3, although the volume changes are statistically insignificant.

Figure 6—source data 1 Data tables for Figure 6B and C and Figure 6—figure supplement 1C, and raw images for the immunofluorescence panel in Figure 6A and immunoblots in Figure 6—figure supplement 1B.: https://cdn.elifesciences.org/articles/92719/elife-92719-fig6-data1-v2.zip
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Figure 6—figure supplement 1

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Hypertonic stress-induced NFkB activation is mediated by TNFR1.

(A) Temporal variation of nuclear p65 levels upon TNFa treatment and hypertonicity (additional 150 mM and 600 mM mannitol). Mean and SEM of n≥100 cells plotted for each time point. (B) Representative immunoblot of total cellular TNFR1 content compared between scrambled control (SC) and TNFR1-knockdown (TNFR1-KD) cells. (C) Quantification of $r_{E G F P}$ under indicated conditions (n=132, 60, 75; N=3 cells; N=3 for HeLa, and n=152, 81 cells; N=3 for NIH/3T3). Statistical analysis was performed by Mann-Whitney test for every group pair. TNFR1 inactivation leads to a significant increase in the modal $r_{E G F P}$ for both HeLa and NIH/3T3 cells.

Figure 7 with 1 supplement

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TNFR1 activity is essential for regulatory volume increase (RVI).

(A) Percentage change in the volume of HeLa (i) and NIH/3T3 (ii) cells while exposed to 150 mM hypertonicity under no pre-treatment, TNFR1-knockdown (TNFR1-KD) condition (HeLa only), zafirlukast pre-treatment (50 µM, 1 hr), and CAY10512 pre-treatment (250 nM, 1 hr), compared to cell volume fluctuations in isotonic conditions. Mean volume and SD plotted for n≥10 cells, N≥2 in each case. Insets show the RVI index (percentage change between cell volumes at the final time point of measurement vs at 10 min post hypertonicity induction) for each condition. (B) Percentage change in modal $r_{E G F P}$ for HeLa (i) and NIH/3T3 (ii) cells during hypertonic stress - under no pre-treatment, TNFR1-KD condition (HeLa only), and zafirlukast pre-treatment (50 µM, 1 hr). Mean percentage change and SD plotted for n≥40 cells, N≥2 in each case (C) Cell spread area trajectory (i) and corresponding cell volume trajectory (ii) for vehicle control and zafirlukast (50 µM,1 hr) treated NIH/3T3 cells spreading on fibronectin-coated glass. (D-i) Immunoprecipitated endogenous TNFR1 and associated RIPK1 under indicated conditions and their expression levels in the whole-cell lysate of wild-type (WT) HeLa cells visualized through immunoblotting; (D-ii) is the quantification of RIPK1 content normalized by immunoprecipitated TNFR1 content during hypertonic stress. (E) Comparison of the membrane tension in (i) and corresponding ${S D}_{t i m e}$ of membrane fluctuations in (ii) of HeLa cells for WT controls, different doses of zafirlukast, and TNFR1-KD. The 25th and 75th percentiles, medians, and means are shown for N≥2, WT: 51402 FBRs, 46 cells; zafirlukast - 1 µM: 9723 FBRs, 13 cells; 10 µM: 9357 FBRs, 14 cells; 50 µM: 14093 FBRs, 14 cells; 100 µM: 11690 FBRs, 16 cells; TNFR1-KD: 24273 FBRs, 33 cells. Statistical analysis was performed by Mann-Whitney test for every distribution against the WT control.

Figure 7—source data 1 Data tables for Figure 7E (.opj file format) and Figure 7—figure supplement 1A, and raw images for the immunoblots in Figure 7D-i.: https://cdn.elifesciences.org/articles/92719/elife-92719-fig7-data1-v2.zip
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Figure 7—figure supplement 1

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TNFR1 activity is essential for regulatory volume increase (RVI).

(A) Quantifying the percentage cell volume shrinkage at 10 min post exposure to 150 mM mannitol in HeLa and NIH/3T3 cells under indicated conditions (n≥10 cells, N≥2). Statistical analysis was performed by Mann-Whitney test for every indicated pairs. (B) Representative ${S D}_{t i m e}$ maps and corresponding IRM (interference reflection microscopy) images of wild-type control, zafirlukast-treated, and TNFR1-knockdown (TNFR1-KD) HeLa cells.

Figure 8 with 2 supplements

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Intracellular macromolecular crowding (MMC) deviates from the concentration-dilution law under hypertonic stress.

(A) The average and standard deviation of cell volume and $r_{E G F P}$ of NIH/3T3 cells at different hypertonicities measured 10 min post hypertonic stress induction, shown in (i) (0 indicates isotonic), were normalized to fit the Boyle-van’t Hoff relation, as shown in (ii). The resultant points were fit to a straight line forced to pass through (1,1) - the isotonic condition, such that the y-intercept gives the limiting cell volume (osmotically inactive cell volume) and limiting $r_{E G F P}$ . Using the $r_{E G F P}$ and cell volume values at the limiting and isotonic conditions, the $r_{E G F P}$ vs cell volume trendline representing the concentration-dilution law was calculated (blue dashed line, formula in legend) in (iii). The expected trendline of $r_{E G F P}$ vs cell volume deviates from the measured values (gray symbols), indicated by the double-headed arrow, even though the total protein mass per cell at different hypertonic conditions do not change at different hypertonicities, as shown in (iv). (B) Trajectory of $r_{E G F P}$ vs cell volume at different time points after inducing hypertonic shock (150 mM mannitol) (n=7 cells; N=2). The blue dashed line denotes the theoretical estimate of the trajectory, as indicated in the legend. (C) AiryScan super-resolution imaging of NIH/3T3-EGFP cells reveals submicron-sized cluster-like appearance of EGFP under severe hypertonic stress (600 mM mannitol). The brightness-contrast in the magnified insets was individually adjusted for better visualization. (D) Time-lapse of EGFP intensity after photobleaching under hypertonic stress of 600 mM mannitol (i) and isotonic conditions (ii) in NIH/3T3. Pseudocolored bottom panels show the magnified photobleaching area (white squares).

Figure 8—figure supplement 1

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Intracellular macromolecular crowding (MMC) deviates from the concentration-dilution law under hypertonic stress.

(A) AiryScan super-resolution image of an NIH/3T3 cell expressing EGFP, fixed at 10 min post exposure to 150 mM mannitol, then immunostained with p65 and co-stained with Hoechst. Line profiles (i and ii) reveal that p65 granules (in yellow) exclude EGFP (shown in cyan) in the cytoplasm (i), while DNA condensates (in magenta) in the nucleus exclude both p65 and EGFP (ii). (B) p65-GFP overexpression in NIH/3T3 form granular structures under hypertonic stress (50 mM), which reverts once cells are rescued to isotonic conditions. The p65 granule formation depends on the level of overexpression and is present in ~20% of cells. (C) p65-EGFP granules in NIH/3T3 under 600 mM hypertonic stress are uniform, smaller, and ubiquitously visible in every cell irrespective of overexpression levels.

Figure 8—figure supplement 1—source data 1 Raw images for Figure 8—figure supplement 1A.: https://cdn.elifesciences.org/articles/92719/elife-92719-fig8-figsupp1-data1-v2.zip
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Figure 8—figure supplement 2

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Intracellular macromolecular crowding (MMC) deviates from the concentration-dilution law under hypertonic stress.

(A) An NIH/3T3 cell shows p65-GFP condensation at 600 mM hypertonicity, which disappears immediately upon rescue. (B) Photobleaching confirms p65-GFP exchange between the cytosol and granules (yellow arrows) at severe hypertonicities (i), and the fluorescence recovery is much slower than isotonic conditions shown in (ii).

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Change in the brightness of EGFP with increased MMC during hypertonic shock.

(A) Ttotal intensity images of cells in isotonic and hypertonic condition (+600 mM dextrose). (B) Total intensity trajectories normalized by the initial total intensity per cell. Error bars indicate standard deviation of the variabilities in the normalized total intensity values. n=128 (isotonic), 83 (+50 mM), 56 (+100 mM), 109 (+150 mM), 66 (+200 mM), 92 (+400 mM) and 112 (+600 mM).

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Representative AiryScan images of NIH/3T3-EGFP showing the lateral (XY) view of the maximum intensity projection of Z-stack (step-size 180nm) and orthogonal (YZ) view upon fixation (A) and under hypertonic condition (B).

Scale bars are 10 µm for XY view and 3 µm for YZ view. The cell depicted in (A) was imaged in 1xPBS before and after 30 minutes of fixation with 4% PFA (at 4°C). Cell volume measurement was performed, cells were removed from the microscope stage, fixed, and then the same cells were identified using fiduciary markers on the petridish and microscope stage, and cell volume was measured again. The cell depicted in (B) was imaged before and after 30 minutes incubation in hypertonic culture media containing an excess of 600 mM dextrose at 37°C, 5% CO2.

Author response image 3

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(A) MDA-MB-231 cells expressing EGFP. (i) EGFP expression, (ii) rEGFP map, and (iii) highlighted pixels having modal rEGFP values with cell outline in corresponding colours. (B) The row of images depicts the cell in magenta with the lamellar region progressively cropped out. The associated plot compares the mean and mode of the rEGFP maps for the cropped cell sections: 1 (uncropped), 2, and 3.

Author response image 4

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(A) Fitting rEGFP vs [EGFP] to y = a + b xc and its residual. (B) Estimated cellular [EGFP] distribution at different cell volume compressions. (C) Estimated rEGFP at different cell volume compressions in crowder-less condition and using cell-free system rEGFP values.

Videos

Video 1

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posterframe for video — 8 hr time-lapse (5 frames per second) of an NIH/3T3-EGFP, showing EGFP intensity on the left and $r_{E G F P}$ on the right.

Scale bar 15 µm.

Video 2

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posterframe for video — 3D projections of NIH/3T3 cells after cytoskeletal depolymerization.

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MDAR checklist: https://cdn.elifesciences.org/articles/92719/elife-92719-mdarchecklist1-v2.pdf
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Parijat Biswas
Priyanka Roy
Subhamoy Jana
Dipanjan Ray
Jibitesh Das
Bipasa Chaudhuri
Ridita Ray Basunia
Bidisha Sinha
Deepak Kumar Sinha

(2024)

Exploring the role of macromolecular crowding and TNFR1 in cell volume control

eLife 13:e92719.

https://doi.org/10.7554/eLife.92719

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