Cell tension and mechanical regulation of cell volume

Abstract

Animal cells use an unknown mechanism to control their growth and physical size. Here, using the fluorescence exclusion method, we measure cell volume for adherent cells on substrates of varying stiffness. We discover that the cell volume has a complex dependence on substrate stiffness and is positively correlated with the size of the cell adhesion to the substrate. From a mechanical force-balance condition that determines the geometry of the cell surface, we find that the observed cell volume variation can be predicted quantitatively from the distribution of active myosin through the cell cortex. To connect cell mechanical tension with cell size homeostasis, we quantified the nuclear localization of YAP/TAZ, a transcription factor involved in cell growth and proliferation. We find that the level of nuclear YAP/TAZ is positively correlated with the average cell volume. Moreover, the level of nuclear YAP/TAZ is also connected to cell tension, as measured by the amount of phosphorylated myosin. Cells with greater apical tension tend to have higher levels of nuclear YAP/TAZ and a larger cell volume. These results point to a size-sensing mechanism based on mechanical tension: the cell tension increases as the cell grows, and increasing tension feeds back biochemically to growth and proliferation control.

FIGURE 1:

Cell volume is heterogeneous and depends on substrate stiffness. (a) Diagram of the microfluidic channel used for fluorescence-exclusion cell-volume measurements. The channel height is h₁ + h₂ = 15 μm. The fluorescence signal is directly proportional to h₂, and the total integrated fluorescence signal after background subtraction gives the cell volume. Images of 3T3, NuFF, and MSC cells in the microfluidic device show DIC and fluorescent channels. The DIC channel is used to trace the 2D cell adhesion boundary and compute adhesion area and shape factor, S. The scale bar corresponds to 10 μm. (b) Histograms of cell volumes on 3-kPa, 12.6-kPa, and glass substrates for 3T3, NuFF, and MSCs. The wide distribution reflects intrinsic variation in cell size as well as effects due to cell cycle variation. The distributions skew to the left, reflecting that there are more young than old cells. (c–e) The average cell volumes for 3 kPa, 12.6 kPa, glass (GPa), serum starvation, and aphidicolin treatment for 3T3 (c), MSCs (d), and NuFFs (e). At 12.6 kPa, 3T3s and MSCs are larger, while NuFFs are smaller. Serum starvation generally decreases cell volume while aphidicolin treatment generally increases cell volume. The average cell adhesion area and adhesion shape are also shown. The shape factor is defined as . Distributions of adhesion areas and shapes are shown in Supplemental Figure S2. MSCs show the largest adhesion area at 12.6 kPa, but for NuFFs and 3T3s, the largest adhesion area occurs on glass substrates. (Scale bar = 10 μm; all error bars represent standard error. Statistical significance: *** p < 10^–6; ** p < 0.001; * p < 0.01; n.s.: p > 0.05. Number of cells: for 3T3s: N = 66 on 3 kPa, N = 110 on 12.6 kPa, and N = 364 on collagen-coated glass; for MSCs: N = 142 on 3 kPa, N = 120 on 12.6 kPa, and N = 378 on collagen-coated glass; for NuFFs: N = 103 on 3 kPa, N = 140 on 12.6 kPa, and N = 160 on collagen-coated glass.)

FIGURE 2:

Cell volume in relation to cell adhesion area and cell shape. (a) Cell volume vs. cell adhesion area for 3T3s, MSCs, and NuFFs on different substrates. Each point is a single cell, color-coded by the shape factor, S. In all cases, area is correlated with volume, but the data are heterogeneous. Moreover, the slope of the correlation depends on the substrate stiffness. For the same area, more circular cells have a larger volume. Variations in cell shape and levels of contractility contribute to the observed variation. (b) Cartoon of an adherent cell. The volume is defined by the apical surface (specified at all points by R, the vector between the center of the adhesion area and the surface, and θ, the angle made by the vector R and the adhesion plane). Owing to pressure difference across the membrane, ΔP, the cell uses active myosin contraction, , in the apical surface to balance the pressure difference. The mean curvature, H, is related to the apical surface shape R (see the Supplemental Material for more details), and h is the cortical thickness. (c) Model predictions of the cell volume as a function of total apical myosin and adhesion area. The model predicts that the cell volume increases with increasing adhesion area and total active myosin contraction. This figure assumes circular adhesion areas for the predicted volume. (d) Relationship between volume and area is dependent on adhesion shape. (e) Shape dependency on elliptical pattern illustrates that for the same , more circular cells are larger in size. This is consistent with data in a. All figures (c, d, and e) assume spatially homogeneous . (f) Representative 3D cell shapes reconstructed from confocal z-stack images (blue) are compared with model cell shapes (red) computed for the same adhesion shape.

FIGURE 3:

Total level and spatial distribution of pMLC are predictors of cell volume. (a) Immunofluorescence widefield images of pMLC for 3T3, NUFF, and MSCs are used for the quantification of total pMLC in each cell. Confocal z-stack images are also taken at 1-μm z-steps to measure the relative amount of pMLC at each z-position. For stiffer substrates and relatively flat cells, there is typically higher concentration of pMLC near the basal surface. For rounder cells, the apical pMLC distribution is more uniform. (b) The average total pMLC (ΣpMLC) and relative ratio of apical vs. basal pMLC (apical>/<PMLC_basal>) on different substrates. The relative levels of pMLC are also plotted as a function of z-position for all three cell lines. We observe that the distribution of pMLC varies with substrate stiffness as well as cell type. (c) The spatial distribution (along the z-axis) of mean pMLC intensity of three cell lines for different ECM stiffness. In general, mean pMLC intensity is higher at the cell basal area; but as the ECM becomes softer, the difference between apical and basal pMLC decreases. The dotted line marks the approximate division between basal and apical (defined as 1 μm above the z-position displaying basal stress fibers). (d) Computed cell volume as a function of total pMLC and the relative pMLC distribution. Each averaged volume in the surface was calculated for the total pMLC and relative pMLC in a range of areas within the experimental range. For each cell type the volume is scaled with respect to the cell volume on glass, and a single fitting parameter is used to relate total pMLC with integrated λ, (Supplemental Material). (e) The model predictions for volume across all stiffnesses are explicitly compared. (Scale bar = 10 μm. All error bars represent standard error. Statistical significance: ***p < 10^–6; **p < 0.001; *p < 0.05; n.s.: p > 0.05. Number of cells for epifluorescence imaging: for 3T3s: N = 80 on 0.4 kPa, N = 370 on 3 kPa, N = 200 on 12.6 kPa, and N = 1061 on collagen-coated glass; for MSCs: N = 377 on 0.4 kPa, N = 221 on 3 kPa, N = 360 on 12.6 kPa, and N = 469 on collagen-coated glass; for NuFFs: N = 179 on 0.4 kPa, N = 341 on 3 kPa, N = 409 on 12.6 kPa, and N = 395 on collagen-coated glass. Number of cells for confocal microscopy: for 3T3s: N = 32 on 0.4 kPa, N = 30 on 3 kPa, and N = 35 on 12.6 kPa and on collagen-coated glass; for MSCs: N = 30 on 0.4 kPa and on 12.6 kPa, N = 35 on 3 kPa, and N = 36 on collagen-coated glass; for NuFFs: N = 36 on 0.4 kPa, N = 30 on 3 kPa, N = 35 on 12.6 kPa, and N = 40 on collagen-coated glass.)

FIGURE 4:

Cell volume is correlated with nuclear YAP/TAZ level in 3T3s and NuFFs. (a) Immunofluorescence widefield images with YAP in green and DNA in blue. The DNA channel is used to mask the nuclear region. The total nuclear YAP (ΣYAP_N) is obtained from epifluorescence images for different stiffnesses. (b) The total average nuclear YAP is plotted vs. the average measured cell volume, average total pMLC level, and apical and basal pMLC levels. The individual cell data are also plotted in panels below and color-coded by the nuclear YAP intensity/cytoplasmic YAP intensity ratio. At both the single-cell and ensemble levels, higher nuclear YAP is correlated with higher total pMLC. Higher nuclear YAP is also correlated with larger cell volume and higher apical pMLC, even though NuFFs and 3T3s display opposing trends as functions of substrate stiffness. Nuclear YAP is not correlated with basal pMLC. For NuFFs, nuclear YAP seems to plateau at large ΣpMLC, suggesting that nuclear YAP level reaches a maximum even as pMLC level is increasing. This suggests that there is another signal limiting nuclear YAP levels in NuFFs. Note that in both 3T3s and NuFFs, the nuclear-to-cytoplasmic YAP concentration ratios are generally higher than 1. Visually, nearly all cells appear to have significant nuclear YAP. (Scale bar = 10 μm. All error bars represent standard error. Statistical significance: ***p < 10^–6; *p < 0.01; n.s.: p > 0.05. Number of cells for epifluorescence imaging: for 3T3s: N = 80 on 0.4 kPa, N = 370 on 3 kPa, N = 200 on 12.6 kPa, and N = 1061 on collagen-coated glass; for NuFFs: N = 179 on 0.4 kPa, N = 341 on 3 kPa, N = 409 on 12.6 kPa, and N = 395 on collagen-coated glass.)

FIGURE 5:

MSCs show bifurcated behavior in YAP nuclear localization and pMLC level. (a) Percentage of MSCs showing nuclear YAP localization. With increasing stiffness, more cells contain nuclear YAP, in agreement with Dupont et al. (2011). (b) The measured total amount of nuclear YAP, ΣYAP_N, decreases with increasing stiffness. Closer examination of single-cell nuclear YAP and pMLC data shows bifurcated behavior on different substrates. On stiffer substrates there are two branches. The upper branch has high overall YAP expression, but low nuclear-to-cytoplasmic (N/C) YAP intensity ratio. The lower branch has lower overall YAP expression, but high N/C. The proportion of the upper branch cells decreases with increasing stiffness. Thus, on softer substrates, it appears that most cells have a lower N/C YAP ratio. On stiffer substrates, there are more cells with high nuclear N/C YAP ratio. (c) Representative images of MSCs with nuclear YAP localization and cytoplasmic YAP localization. (d) When the total nuclear YAP is plotted vs. volume, pMLC, and apical pMLC, the positive correlation between nuclear YAP and these variables is recovered, similarly to 3T3s and NuFFs. Cells in these separate branches are both positive for MSC markers CD90 and CD105 (Supplemental Figure S7). These results suggest that these are two branches that may not be distinguished by typical MSC differentiation markers. (All error bars represent standard error. Statistical significance: ***p < 10^–6; **p < 0.001; *p < 0.01; n.s.: p > 0.05. Number of cells: N = 377 on 0.4 kPa, N = 221 on 3 kPa, N = 360 on 12.6 kPa, and N = 469 on collagen-coated glass.)

FIGURE 6:

Nuclear YAP and pMLC relation suggests a late G1 checkpoint based on cell tension. (a) Two stiffness conditions with the greatest difference in average cell volume are selected for 3T3s, MSCs, and NuFFs. The DNA histogram (left) is shown together with the total nuclear YAP vs. the total cell pMLC level (right). Cells are colored by their DNA content, with G1 cells identified as cells with DNA content below the dashed line in the DNA histogram (1.25 in the scaled DNA level). Cells beyond G1 have higher levels of nuclear YAP and pMLC. The rate of nuclear YAP and pMLC increase, however, varies with condition and cell type. (b). When cells in G1 under different conditions are compared, we observe that nuclear YAP rises with pMLC in G1 until a critical pMLC level is reached, suggesting a checkpoint based on cell tension. For 3T3s, cells proceed to S after the critical level of pMLC and nuclear YAP continues to rise with pMLC. For NuFFs and the MSC lower-branch populations, cells in G1 can continue to increase in pMLC and cell size, but the nuclear YAP level plateaus after the critical level of pMLC. (c) G1–S transition checkpoint based on cell tension. Nuclear YAP increases with increasing pMLC until a common critical tension level is reached, at which the cell transitions from G1 to S. If cells continue to grow in G1, nuclear YAP does not increase after the critical tension, but plateaus. These cells are presumably arrested in G1.