Exploring the role of macromolecular crowding and TNFR1 in cell volume control
- School of Biological Sciences, Indian Association for the Cultivation of Science, India
- Department of Biological Sciences, Indian Institute of Science Education and Research Kolkata, India
Figures
Figure 1 with 1 supplement
Fluorescence anisotropy of EGFP is a robust probe for macromolecular crowding.
(A) Steady-state fluorescence anisotropy of EGFP () progressively increases with crowder concentration and crowder molecular weight. (B) Fluorescence lifetime of EGFP () steadily decreases with …
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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
Figure 1—figure supplement 1
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 …
Figure 2 with 1 supplement
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 …
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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
Figure 2—figure supplement 1
Macromolecular crowding (MMC) levels do not significantly vary between individual cell lines.
(A, i) The distribution of modal values per cell (5–95 percentile) is narrower than the distribution of mean values due to the spatially varying maps (during isotonic conditions), and (ii) 600 …
Figure 3 with 1 supplement
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 map of an NIH/3T3 cell shows that the spatial heterogeneity of intracellular MMC …
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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
Figure 3—figure supplement 1
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 …
Figure 4 with 1 supplement
The characteristic cellular macromolecular crowding (MMC) is linked to cell spreading and adhesion.
(A-i) 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 values (cyan) and spread …
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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
Figure 4—figure supplement 1
The characteristic cellular macromolecular crowding (MMC) is linked to cell spreading and adhesion.
(A) Total intensity and 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 …
Figure 5 with 1 supplement
Proteostasis disruption alters cellular macromolecular crowding (MMC) setpoint.
(A) Average percentage changes in cell volume (filled symbols) and modal (open symbols) for NIH/3T3 in (i) and HeLa in (ii) upon exposure to hypertonic mannitol (150 mM) (n>40 cells; N=4 for …
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Figure 5—source data 1
Data tables for Figure 5F.
- https://cdn.elifesciences.org/articles/92719/elife-92719-fig5-data1-v2.zip
Figure 5—figure supplement 1
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 and cell outline area (average and SD) from EGFP total fluorescence images …
Figure 6 with 1 supplement
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 …
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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
Figure 6—figure supplement 1
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) …
Figure 7 with 1 supplement
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 …
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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
Figure 7—figure supplement 1
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 …
Figure 8 with 2 supplements
Intracellular macromolecular crowding (MMC) deviates from the concentration-dilution law under hypertonic stress.
(A) The average and standard deviation of cell volume and of NIH/3T3 cells at different hypertonicities measured 10 min post hypertonic stress induction, shown in (i) (0 indicates isotonic), were …
Figure 8—figure supplement 1
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 …
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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
Figure 8—figure supplement 2
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 …
Author response image 1
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 …
Author response image 2
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 …
Author response image 3
(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 …
Author response image 4
(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 …
Videos
Video 1
8 hr time-lapse (5 frames per second) of an NIH/3T3-EGFP, showing EGFP intensity on the left and on the right.
Scale bar 15 µm.
Video 2
