Cell volume change through water efflux impacts cell stiffness and stem cell fate

Abstract

Cells alter their mechanical properties in response to their local microenvironment; this plays a role in determining cell function and can even influence stem cell fate. Here, we identify a robust and unified relationship between cell stiffness and cell volume. As a cell spreads on a substrate, its volume decreases, while its stiffness concomitantly increases. We find that both cortical and cytoplasmic cell stiffness scale with volume for numerous perturbations, including varying substrate stiffness, cell spread area, and external osmotic pressure. The reduction of cell volume is a result of water efflux, which leads to a corresponding increase in intracellular molecular crowding. Furthermore, we find that changes in cell volume, and hence stiffness, alter stem-cell differentiation, regardless of the method by which these are induced. These observations reveal a surprising, previously unidentified relationship between cell stiffness and cell volume that strongly influences cell biology.

Keywords: cell mechanics; cell volume; gene expression; molecular crowding; stem cell fate.

Fig. S1.

Comparison of 3D cell morphology measurements with confocal microscopy and super-resolution SIM. (A) Side views of two representative MEF cells imaged with confocal and SIM are shown. Cells are fixed, with actin filaments stained with phalloidin–Alexa Fluor 488. (B and C) When we image the same sample using confocal and SIM separately, the measured cell height (B) and volume (C) are consistent for both techniques; no statistically significant difference is found for cell height and volume measurements between the two techniques. N.S., not significant.

Fig. S2.

Dependence of cell volume and cortical modulus on the applied external osmotic pressure. (A) A7 cells either growing on collagen-coated PA gels with different stiffness or being depleted of ATP on glass are compressed by increasing amounts of PEG 300 polymers to apply external osmotic pressure to extract-free water out of cells. The volume of cells decreases with increasing osmotic pressure and reaches a minimum size at extreme osmotic pressure; the minimum volume does not depend on substrate stiffness. (B) Cortical shear modulus of A7 cells measured by using OMTC before application of osmotic compression, 10 min after osmotic compression (with 5% PEG 300), and 10 min after the osmotic compression is removed. The cortical shear modulus increases after the application of osmotic compression and reduces back to approximately the same value after removal of the osmotic compression. n > 200. *P < 0.01. N.S., not significant.

Fig. 1.

Mechanical properties of A7 cells on glass as a function of cell volume under external osmotic pressures. (A) Osmotic bulk modulus (gray triangles), cortical shear modulus (red triangles), and cytoplasmic shear modulus (open triangles) increase as cell volume is decreased upon external osmotic compression. The dashed line through gray triangles represents the least-squares fit using the functional form, B = Nk_BTV/(V − V_min)² (R² = 0.99). Dashed lines through the cortical and cytoplasmic moduli are exactly the same fit, simply scaled by a factor of 500 and 100,000, respectively. (B) Cortical shear modulus of a single A7 cell, measured by OMTC, increases immediately after the application of an osmotic compression with 0.26 M PEG 300; this is concurrent with a decrease in cell volume after osmotic compression.

Fig. S3.

Cell cortical stiffness and cytoplasmic stiffness measured with OMTC and optical tweezers, respectively, under different conditions. (A) Schematic illustration of the OMTC measurement (17). A magnetic field introduces a torque that causes the 4.5-µm ferromagnetic bead to rotate and to deform the cell cortex to which it is bound. (B) A representative measurement of A7 cell cortex mechanics in isotonic medium using OMTC. The cell cortex exhibits power-law rheology behavior across four orders of magnitude in frequency. A noticeable plateau in both G′ and G′′ also exists at low frequency. For convenience and consistency, we use cortex modulus measurement at 0.75 Hz (indicated by the blue box) across all different microenvironmental conditions. (C) Cell cortical stiffness (G′) increases with substrate stiffness, consistent with previous measurement using other methods. (D) Schematic illustration of the cytoplasmic stiffness measurement using optical tweezers. (E) A representative measurement of cytoplasmic mechanics in A7 cells. The cell cytoplasm exhibits power-law rheology behavior across three orders of magnitude in frequency. For convenience and consistency, we use cortex modulus measurement at 10 Hz (indicated by the blue box) across all different microenvironmental conditions.

Fig. 2.

Morphology and volume of adherent cells change with increasing substrate stiffness. (A) Top and side views of fixed A7 cells on a stiff (shear modulus of 10 kPa) and a soft (shear modulus of 1,200 Pa) PA gel substrate coated with collagen I. The actin cortex (green) and nucleus (blue) are labeled. (B) The projected cell area increases with increasing substrate stiffness. (C) Cell volume markedly decreases with increasing substrate stiffness. Error bars represent the SD (n > 200 individual cells). HASM, human airway smooth muscle.

Fig. 3.

Cell volume of A7 cells increases when the cell spread area is decreased by growing cells on micropatterned collagen islands. Error bars represent the SD. *P < 0.05; **P < 0.01. (A) Shown are 3D images of A7 cells on micropatterned islands of different sizes on glass. Cells are labeled with cell tracker green. (Scale bars, 20 μm.) (B) Cell volume decreases with increasing cell spread area on glass. (C) Cell volume plotted as a function of the projected area, for cells on substrates with different stiffnesses (gray circles; n > 200), cells on a glass substrate but with different available spread area (blue squares; n > 200), and a dynamically spreading cell (red crosses; n = 3). (D) Variation of cell spread area and volume as a single cell dynamically attaches on a stiff substrate (n = 3). (E) Schematic illustration of cell volume decrease through water efflux, as cells spread out or are osmotically compressed.

Fig. 4.

Cell morphology and volume under drug treatment and osmotic compression. (A) The 3D morphology of control cells and cells with ATP depletion and under extreme osmotic compression, on stiff and soft substrates. Cytoplasm (green) and nucleus (yellow) are labeled. (Scale bars: 20 μm.) (B) Cells without active contraction (blebbistatin-treated and ATP-depleted) and under extreme osmotic compression do not exhibit a volume dependence with substrate stiffness; cells with choloride channels-inhibited (NPPB treated) exhibit a weaker volume dependence with substrate stiffness. The control data of A7 cells is same as in Fig. 2C. Error bars represent the SD (n > 200 individual cells).

Fig. 5.

Relationship of cell cortical stiffness and cell volume. (A–D) Dependence of cell cortex shear modulus of A7 cells on their volume, under different conditions, including cells cultured on substrates of varying stiffnesses (A), on a stiff substrate with micropatterns of varying sizes (B), on a soft substrate with a shear modulus of 100 Pa with addition of increasing amount of osmotic pressure (C), and on a glass substrate with small micropatterns limiting cell spreading and with the addition of osmotic pressures (D). (E) Cell cortical shear modulus scales with cell volume, as shown for cells growing on substrates of varying stiffness (gray circles), on a glass substrate with restricted available spread area using micropatterns (blue squares), on a soft substrate with osmotic compression (shear modulus of 100 Pa, green upside down triangles), on an unpatterned (red triangles) and a micropatterned glass substrate (yellow diamonds) with osmotic compression, and on a glass substrate with 10 μM blebbistatin treatment (cyan pentagon) or depleted of ATP (black triangle). Solid line shows the power-law fitting of the data, scales as V⁻². Dashed line shows fitting to G ∝ k_BTV/(V − V_min)² Error bars represent the SD (n > 200 individual cells). osm. comp., osmotic compression; pat., patterned.

Fig. S4.

Dependence of cell cortical stiffness on cell volume observed for other cell types and in 2D monolayers. (A) Cortical shear modulus and cell volume of Eker rat uterine leiomyoma cells (ELT3) are measured for cells cultured under different conditions, such as on substrates of varying stiffness (filled symbols) and on glass under increasing osmotic pressure (open symbols). Similar scaling behavior is observed for ELT3 and A7 cells, yet it is shifted in both the cortical shear modulus and volume between the cell types. (B) Cortical shear modulus and cell volume of MCF10A cells growing in a 2D monolayer at varying densities. MCF10A human breast cells are seeded at varying densities and therefore grow into a 2D monolayer with different cell densities. Cell volume and cortical stiffness of cells are measured at different cell densities, as plotted in B, Inset. Similar scaling behavior between cortical stiffness and cell volume is observed for MCF10A cells in 2D monolayers with different densities, as is observed for isolated cells under different cell culture conditions.

Fig. S5.

Universal dependence of osmotic bulk modulus, cortical shear modulus, and cytoplasmic modulus on volume of A7 cells. Osmotic bulk modulus (top), cortical shear modulus (middle), and cytoplasmic shear modulus (bottom) increase in a universal fashion as cell volume is decreased upon various perturbations. In the top, up, left, and down triangles represent measurements with cells cultured on substrates with high, intermediate, and low stiffness, respectively. Gray plus symbols represent ATP-depleted cells. Symbols in the middle are described in Fig. 5. In the bottom, cytoplasmic stiffness in cells cultured on substrates of varying stiffness (gray circles), on a soft substrate with osmotic compression (shear modulus of 100 Pa; green upside-down triangles), on a unpatterned glass substrate (red triangles) with osmotic compression, and on a glass substrate with 10 μM blebbistatin treatment (cyan pentagon) or depleted of ATP (black triangle). The solid lines are the same functional form used in Fig. 1. Error bars represent the SD (n > 200 individual cells for the bulk modulus calculation and for OMTC measurement; n > 5 for optical tweezers measurement).

Fig. 6.

Cell nuclear volume and nuclear dynamics change with cell volume. (A) Nucleus volume always directly tracks cell volume, decreasing proportionally with increasing substrate stiffness, spread area, and osmotic pressure; the ratio between nuclear volume and cell volume remains approximately constant for each tested cell type. (B) Mean square displacement of GFP-tagged Histone in MCF-10A cell nuclei, reflecting positional fluctuation of chromatin, significantly reduces under external osmotic compression through application of 0.1 M PEG 300. osm. comp., osmotic compression; sub., substrate.

Fig. 7.

Cell volume affects differentiation of mMSCs. (A–F) Osteogenesis. (A) In situ staining of mMSC for ALP (black) and nucleus (DAPI, blue) after 1 wk of culture in the presence of combined osteogenic and adipogenic chemical supplements shows increased osteogenesis on the stiff substrate (shear modulus of 7 kPa) and the soft substrate (shear modulus of 200 Pa) with osmotic compression (with 0.1 M PEG 300, additionally to the medium), compared with the control on soft substrate without additional osmotic pressure. (B) Mean percentages of mMSC osteogenesis. Error bars, SEM (n = 3 samples). *P < 0.05. (C) Western analysis of osteogenic protein expression (RUNX2 and BSP) in mMSCs after 3 d of culture. (C–F) Cell volume (D), nucleus volume (E), and cortex shear modulus (F) measured with confocal microscopy and OMTC, for three experimental conditions (n > 50 individual cells). *P < 0.05. (G–L) Adipogenesis. (G) In situ staining of mMSC for fat lipids (red) after 2 wk of culture in the presence of combined osteogenic and adipogenic chemical supplements shows enhanced adipogenesis on stiff substrate with application of hypotonic pressure (with the addition of 30% DI water), compared with the control. (H) Mean percentages of mMSC adipogenesis. Error bars, SEM (n = 3 samples). *P < 0.05. (I) Western analysis of adipogenesis protein expression (PPAR-γ) in mMSCs after 1 wk of culture. (J–L) Cell volume (J), nucleus volume (K), and cortex shear modulus (L) measured with confocal microscopy and OMTC, for three experimental conditions (n > 50 individual cells). *P < 0.05. (M) mMSCs are exposed to osteogenesis medium for 10 d. The ratio of cells expressing high level of ALP, as measured by using Fast Blue staining, as described in SI Materials and Methods, is counted in three independent samples fixed each day. Volume of cells is observed each day as well. As the ratio of differentiated cells increases, cell volume decreases correspondingly (n = 3 samples; error bars represent SD). (N) mMSCs are exposed to adipogenesis medium for 2 wk. The ratio of cells with clear fat lipid accumulation, as visualized by ORO staining, as described in SI Materials and Methods, is counted in three independent samples fixed every 2 d. Volume of cells is measured at the same time as well. As the ratio of differentiated cells increases, cell volume increases correspondingly (n = 3 samples; error bars represent SD). (Magnification: A and G, 400×.)

Fig. S6.

Cell morphology, traction force, and substrate properties under osmotic compression. (A–C) Cell morphology (Upper) and traction stress intensity map (Lower) of mMSCs on a stiff gel, a soft gel, and a soft gel with osmotic compression. mMSCs are cultured on a collagen I-coated PA gel, with 500-nm fluorescent beads previously mixed in the gel substrate for visualizing traction displacement applied by cells. Cells on a stiff gel have larger spreading area and stronger traction stresses than those cultured on a soft gel. However, when we compress cells with osmotic pressure on a soft gel, while the height of the cell dramatically decreases, cell spreading area and traction stress does not significantly change. The cytoplasm and nucleus are labeled with cell tracker and DRAQ-5. (D) Frequency dependent shear moduli of a soft PA gel substrate measured by OMTC, in isotonic medium (black symbols), and in medium with additional osmotic pressure by adding 0.1 M PEG 300 (red symbols). This result shows that PA gel is indeed an elastic gel with G′ (filled symbols) greater than G′′ (open symbols); moreover, the osmotic pressure we apply to compress cells does not significantly affect the mechanical property of the PA gel substrate.

Fig. S7.

Mean square displacement of 200-nm-diameter latex beads in the cytoplasm of A7 cells cultured at different conditions, including varying substrate stiffness and under varying osmotic compressions. The movement of these beads increases as cell volume increases, suggesting that an increase in the degree of molecular crowding in cells hinders intracellular movement.