Figure 1. Shear induced inward/outward oscillations in cells in the y-direction and cell movements opposite to the shear in the x-direction.
(A) Shear (100 μm) was applied to a MDCK monolayer adhering to the device planks (Supplementary Materials). (B) PIV was used to quantify the x- and y-direction velocities (green arrows) of MDCK cells expressing E-cadherin:DsRed over time. (C) Symmetric PIV data were averaged by ‘folding’ over the shear-plane. The color map displays the speeds of cell movement in the outward/inward (blue/red) y-direction relative to the shear-plane (0 μm y Pos.), or in the opposite/with x-direction relative to the shear direction (blue/red, 30 μm/h). (D, F, H, J) y- (D and H) and x-velocity (F and J) kymographs from three independent experiments with 15 min binning of three 5 min PIV data of cell movements with (D and F, dashed black line) or without (H and J) shear over 20 h. (E, G, I, K) y- (E and I) and x-direction (G and K) cell movements based on numerical integration of y- and x-velocity kymographs over time, respectively, at positions 8, 50, 100, 150, 200, and 242 μm from the shear-plane (Figure 1—figure supplement 7). Insets provide greater spatial resolution of movement in the deformation zone (G and K, insets).
Figure 1—figure supplement 1. A silicon device for force sensing and the application of in-plane shear to a cell monolayer.
(A) The shear device consisted of two 250 μm x 1000 μm planks that were positioned adjacent to each other using a needle connected to a 3-axis micromanipulator to apply x- and y- displacements of the actuating plank. The spring-suspended, sensing plank was used to infer force. Four folded flexures were used to provide stability and force sensing for each actuator and each sensor in the shear direction, respectively (2017 IEEE. Reprinted, with permission, from (Sadeghipour et al., 2017)). (B) The equation used to calculate the stiffness (0.93 N/m) of the device in the shear direction. The in-plane Young’s Modulus (E) of silicon is 169 GPa, and the other components are width (w), length (L), and thickness (t) of the flexures. The numerator is multiplied by four (number of folded flexures in parallel), and the denominator adds the three components of each flexure in series. F is calculated by multiplying the stiffness of the sensing arm (kS) by its x-displacement (xS). (C) In all experiments, the actuating plank is displaced 100 μm in the x-direction (xA), yielding in-plane shear of the monolayer at the mid-plane. The sensing plank changes position (xS) only via deformation of the monolayer that was contiguous with the actuating plank. (D) Displacements of the actuating (blue line) and sensing (red *) planks over time. t1-t4 refer to positions depicted in ; Figure 1—figure supplement 1C.
© 2017 IEEE
Figure 1—figure supplement 1A and C reprinted with permission, from Sadeghipour et al., 2017.
Figure 1—figure supplement 2. MDCK E-cadherin:DsRed cell monolayers before and after shear plus tension show the monolayer remains intact.
MDCK E-cadherin:DsRed cell monolayers were sheared by 100 μm displacement of the top plank, red arrow, followed by a tensile displacement of 50 μm, yellow arrow. Yellow dashed lines represent the edges of the top and bottom planks. The cell monolayer is suspended over a gap between the planks without rupture, bottom panel.
Figure 1—figure supplement 3. MDCK E-cadherin:DsRed cell monolayers before and after shear show deformation localized to the shear plane.
Example frames of MDCK E-cadherin:DsRed cell monolayers on a shear device before and after shear. Images were taken as described in methods and materials. (A) full 10x images with the shear zone outlined with a red box and the middle third of the top cell plank outlined with a blue box. Red arrow denotes the direction of shear by the displacement of the top plank. (B) contrast enhanced image of the shear zone region before and after shear from the red box in A). (C) contrast enhanced image of the middle third region of the top plank before and after shear from the blue box in A). Red highlighted region in the shear frame marks deformed cells at the shear zone while the rest of the monolayer remained the same immediately after shear. Yellow dashed line is the interface of the top and bottom planks.
Figure 1—figure supplement 4. Cell orientation, eccentricity, area, density, and perimeter over time after shear did not match the periodicity of y-direction oscillations.
(A, C, E, G, I) Kymographs of cell orientation (A), eccentricity (C), area (E), density (G), and perimeter (I), based on the cell segmentation of MDCK E-cadherin:DsRed cells in the control + Shear (dashed black line) condition (Figure 1D–G). Kymographs are from three independent experiments with 15 min binning of 3 × 5 min cell segmentation data (similar to Figure 1). (B, D, F, H, J) Line plots of cell orientation (B), eccentricity (D), area (F), density (H), and perimeter (J), at positions 8, 50, 100, 150, 200, and 242 μm from the shear-plane.
Figure 1—figure supplement 5. High-magnification of MDCK E-cadherin:DsRed cells at the shear plane shows no change in E-cadherin asymmetry or recruitment upon shear.
MDCK E-cadherin:DsRed cells were imaged on the Leica DM-RXA2 upright microscope with a Leica 63x water immersion objective. The frames for the moment of shear where taken at 200 ms intervals and binned 2 × 2 for fast acquisition, panels 0 min before and after shear. The 10 and 45 min frames were taken from a series of images at 1 min intervals, with 500 ms exposures, were not binned, and we used Micromanager’s autofocus. Red arrow represents the direction of shear from displacement of the top plank. Yellow dashed line represents the interface of the two cell adhesion planks.
Figure 1—figure supplement 6. Density of MDCK cells without shear was similar to their density with shear.
(A, C, E, G, I) Kymographs of cell orientation (A), eccentricity (C), area (E), density (G), and perimeter (I), based on the cell segmentation of MDCK E-cadherin:DsRed cells in the -Shear condition (Figure 1H–K). Kymographs are from three independent experiments with 15 min binning of 3 × 5 min cell segmentation data (similar to Figure 1). (B, D, F, H, J) Line plots of cell orientation (B), eccentricity (D), area (F), density (H), and perimeter (J), at positions 8, 50, 100, 150, 200, and 242 μm from the shear-plane.
Figure 1—figure supplement 7. Velocity kymographs and cell-displacement plots generated from PIV data.
(A) MDCK E-cadherin-DsRed cell movements were detected between two time points using PIV. Top and bottom are relative to the shear-plane (red bar), and images were divided spatially from the shear-plane to the edge of each plank (d1 and d2). PIV velocity vectors (green arrows) were averaged per divided distance from the shear-plane over time and weighted based on cell velocities and movements inward (red) or outward (blue) from the shear-plane. Velocity kymographs were generated by plotting the averaged weighted values as pixels over time with reference to the shear-plane. (B) Velocity kymograph of cell movements in the y-direction relative to the shear-plane 1 hr before shear (dashed black line) and 5 hr after shear. Symmetrical data for x- and y-direction movements for all experiments were folded with reference to the shear-plane to generate velocity kymographs to combine data from both top and bottom planks. (C) Representative images of x- and y-direction cell movements based on numerical integration of y- and x-velocity kymographs over time at positions 8, 50, 100, 150, 200, and 242 μm from the shear-plane.