Effects of Morphology vs. Cell-Cell Interactions on Endothelial Cell Stiffness

Abstract

Biological processes such as atherogenesis, wound healing, cancer cell metastasis, and immune cell transmigration rely on a delicate balance between Cell-Cell and cell-substrate adhesion. Cell mechanics have been shown to depend on substrate factors such as stiffness and ligand presentation, while the effects of Cell-Cell interactions on the mechanical properties of cells has received little attention. Here, we use atomic force microscopy to measure the Young's modulus of live human umbilical vein endothelial cells (HUVECs). In varying the degree of Cell-Cell contact in HUVECs (single cells, groups, and monolayers), we observe that increased cell stiffness correlates with an increase in cell area. Further, we observe that HUVECs stiffen as they spread onto a glass substrate. When we weaken Cell-Cell junctions (i.e., through a low dose of cytochalasin B or treatment with a VE-cadherin antibody), we observe that cell-substrate adhesion increases, as measured by focal adhesion size and density, and the stiffness of cells within the monolayer approaches that of single cells. Our results suggest that while morphology can roughly be used to predict cell stiffness, Cell-Cell interactions may play a significant role in determining the mechanical properties of individual cells in tissues by careful maintenance of cell tension homeostasis.

FIGURE 1

(A) Example of a force vs. distance curve obtained by atomic force microscopy (AFM). Prior to contact between the AFM tip and the sample (a) there is no deflection of the laser. Once the tip has contacted the sample (b), the laser begins to deflect as the sample is indented (c). (B) Deflection vs. distance data were fit using the Hertz–Sneddon model (see Materials and Methods). The point of contact was chosen when the slope became nonzero (approximately 50 pN/nm after converting to force vs. distance), while the final indentation was chosen to be about 250 nm. In the case of a very stiff sample, the deflection (or force) rises sharply with distance. Curves are shown for a soft location (i.e., cell body) and a relatively stiff location (i.e., periphery). (C) Young’s modulus of single cells at the cell body and periphery locations (see Fig. 3a for clarification) at both room temperature (23 °C) and 37 °C with 5% CO₂ and 55% humidity. No significant differences were measured between these conditions using our experimental set-up.

FIGURE 2

(a) Cellular area as a function of degree of Cell–Cell contact on fibronectin-coated glass substrates. Bars indicate mean of N cells, while error bars indicate standard error. N equals 167, 249, and 192 for single cells (S), groups (G), and monolayers (M), respectively. * indicates p < 0.05 with single cells, while ^ indicates p < 0.05 with monolayers using Student’s t test. Also shown are phase contrast images of (b) a single cell, (c) four cells in contact, and (d) a monolayer of cells. Scale bars are 20 μM.

FIGURE 3

Atomic force microscopy contact deflection images of (a) a single control endothelial cell, (b) 3 cells in contact, and (c) a monolayer of cells. Image size is 90 μM × 90 μM. Black arrows point to examples of cell body locations, while white arrows point to examples of periphery locations. (d) Young’s modulus of cells for the cell body and periphery locations as a function of Cell–Cell contact. Bars indicate mean of N force curves per condition, while bars indicate standard error. * indicates p < 0.05 with single cells at the same location, while ^ indicates p < 0.05 with groups of 3 cells at the same location using a Student’s t test. For all locations, the periphery region is stiffer than the cell body region (p < 0.05). N = 195, 44, 67,16, 246 for single cells, groups of 3 cells, groups of 4–5 cells, groups of 6–13 cells, and monolayers, respectively, at the cell body location. N = 284, 100, 98, 46, 457 for single cells, groups of 3 cells, groups of 4–5 cells, groups of 6–13 cells, and monolayers, respectively, at the periphery location. Also shown are distributions of Young’s moduli for (e) single cells (N = 195 for cell body and N = 284 for periphery region), (f) all groups of cells (N = 127 for cell body and N = 244 for periphery region), and (g) monolayers (N = 246 force curves for cell body and N = 457 force curves for periphery region).

FIGURE 4

Young’s modulus and cell area vs. time are overlaid on the same plot for three representative spreading cells. A time of t = 0 indicates the time at which measurements began; typically this was 15–30 min after plating. Shown are plots for three different representative cells, with AFM measurements taken every 30 s (a, b) or every 2 min (c). Gray outlined dot at t = 0 indicates typical Young’s modulus of cytochalasin B-treated cells. Gray large dashed line and gray small dashed line indicate typical Young’s modulus of control single cells at the cell body and periphery locations, respectively. (d) Time-course sequence of spreading cell from (b). These images show the AFM cantilever positioned over the cell during spreading. The cantilever remained stationary during the course of spreading. In each image cells have been outlined in black by hand to help with cell visualization. Scale bar is 20 μM for all images. (e) AFM deflection image of cell from (c) after it has completely spread. Image size is 90 μM × 90 μM.

FIGURE 5

Young’s modulus vs. area for N = 8 spreading cells (white outlined circles). Also shown are the average (area, stiffness) of single cells (S), groups (G), monolayers (ML), and monolayers treated with VE-cadherin antibody (VE-cad Ab) or cytochalasin B (cytoB). In all cases, the stiffness plotted is the average stiffness of the cell body and periphery regions.

FIGURE 6

Mean Young’s modulus for (a) cytochalasin B (cytoB)-treated cells and (e) VE-cadherin antibody-treated cells at the cell body and periphery locations. Bars indicate mean of N force curves, while error bars indicate standard error. * indicates p < 0.05 with control at same location using Student’s t test. N = 246, 241, and 70 for control, VE-cadherin antibody-treated, and cytoB-treated cells, respectively, at the cell body (CB) location. N = 457, 583, and 22 for control, VE-cadherin antibody-treated, and cytoB-treated cells, respectively, at the periphery (P) location. Also shown are atomic force microscopy contact deflection images of HUVEC monolayers treated with (b) 2 μg/mL cytoB, (c) 100 ng/mL cytoB, and (d) 10 ng/mL cytoB. White arrowheads in (d) point to tether-like structures at Cell–Cell junctions. Also shown are (f) single cells and (g) monolayers, both treated with a VE-cadherin antibody. AFM image size is 90 μM × 90 μM.

FIGURE 7

Spinning disk confocal images of control (a) single cell, (b) group of cells, and (c) monolayer of cells. Also shown are confocal images of monolayers treated with (d) 2 μg/mL, (e) 100 ng/mL, and (f) 10 ng/mL cytochalasin B to disrupt F-actin. White arrowheads in (e) indicate F-actin filaments or bundles tethering neighboring cells. Also shown are confocal images of (g) single cells and (h) monolayers treated with a VE-cadherin antibody. White scale bar in (a) is 20 μM and applies to all images. Phalloidinactin is stained in red, vinculin (focal adhesion marker) in green, and DNA in blue. Total internal reflection fluorescence microscopy (TIRFM) images were also taken using a laser of wavelength of 488 nm to illuminate the FITC-labeled vinculin, resulting in images which we analyzed for focal adhesion (FA) size and density (number per area). We show a (i) raw TIRFM image of a single cell and (j) a processed TIRFM image made into binary, as described in “Materials and Methods” section. White scale bars in (i) and (j) are 20 μM. Shown also are plots of (k) average FA size and (l) average FA density. Bars indicate average while error bars indicate standard error of measurements from a minimum of 20 images. * indicates p < 0.05 with control of same degree of Cell–Cell contact (single cell or monolayer), while ^ indicates p < 0.05 with single cell control using Student’s t test. S = single cells, G = groups, M = monolayers, VEcad = VE-cadherin antibody-treated, cytoB = cytochalasin B.

FIGURE 8

Schematic which summarizes our observations of spreading cells and cells with varying degrees of Cell–Cell contact. (a) Prior to developing adhesions with the underlying substrate, the cell contains a layer of cortical actin beneath the membrane. This cortical actin network is soft compared with the parallel bundles of F-actin filaments (stress fibers) which polymerize and crosslink during spreading. Upon touching down on the substrate, the cell begins to adhered to the surface and actin polymerizes, causing the cell to extend outward onto its substrate. When it is fully spread, there exists a dense network of stress fibers which extend into the periphery of the cell, while a layer of cortical actin still remains. As the stress fibers contract, tension in the entire cell, including the cell body region, is increased. This generates the increased stiffness of the cell. (b) In the transition from a single cell to a network, the cells develop Cell–Cell contacts composed of numerous adhesion proteins. Because the cells have space around them to move, they likely extend outward through actin polymerization, while (visibly) still maintaining Cell–Cell contacts. Increased focal adhesion size at this point suggests that tension in the cell has increased, leading to increased cell stiffness. As more and more cells enter the group, they begin packing and eventually form a monolayer. Note that in our schematic cartoon, the monolayer continues outward infinitely in all directions; pictured are cells at the interior. Here, the stress fibers are arranged mainly around the periphery of the cell, suggesting that cell packing forces have caused rearrangement of F-actin, possibly resulting in softer cells. In a monolayer, there are less focal adhesions per area, indicating that cell–substrate adhesion has decreased, but likely Cell–Cell adhesion has increased. The actin is linked to the cellular junctions through VE-cadherin, and the VE-cadherin molecules on neighboring cells link to each other, creating a physical link between cells through which force can be transferred. (c) Once in a monolayer, treatment with VE-cadherin antibody or a low dose of cytochalasin B presumably weaken Cell–Cell adhesions. Prior to treatment, the cells have some degree of Cell–Cell adhesion and cell–substrate adhesion. After treatment to weaken Cell–Cell adhesions, the cells develop stronger cell–substrate adhesions (larger focal adhesions), leading to increased tension, and thus increased stiffness, in the cells. If the monolayer is instead treated with a high dose of cytochalasin B, there is a complete loss of Cell–Cell adhesion, dissolution of focal adhesions, and decrease in cell stiffness due to the net depolymerization of actin filaments.