Single-Cell Defects Cause a Long-Range Mechanical Response in a Confluent Epithelial Cell Layer  Susanne Karsch, Deqing Kong, Jörg Großhans, Andreas Janshoff  Biophysical Journal  Volume 113, Issue 12, Pages 2601-2608 (December 2017) DOI: 10.1016/j.bpj.2017.10.025 Copyright © 2017 Biophysical Society Terms and Conditions

Figure 1 (A) Schematics (top) and phase-contrast image (bottom) of single-cell wounding with a micropipette. (B) Confocal image including xz and yz projections of a wounded cell (arrows). The cell shows an increase in cell height before extrusion (red, actin; blue, nucleus). (C) Fluorescence micrographs 30 and 105 min after wounding show an accumulation of actin filaments (red) surrounding the wounded cell. The filaments remain visible even after the wound is closed. (blue, nucleus). (D) Typical time trace of the normalized wound size (normalized to the initial area of the cell) during closure. Inset: example of cell shapes before (thin black line) and 50 min after wounding (thick red line) of the center cell, showing the typical rosette formation. (E) Example of an AFM indentation experiment where the apical cell membrane is deformed (dark gray curve). The force (F)-distance (d) curve is subject to fitting with Eq. 1. During cantilever retraction (light gray curve), tethers were extracted from the cell, and the associated rupture forces provide a means to calculate the membrane tension. Polynomial fit and tether-force measurement are indicated in red. All scale bars, 20 μm. Biophysical Journal 2017 113, 2601-2608DOI: (10.1016/j.bpj.2017.10.025) Copyright © 2017 Biophysical Society Terms and Conditions

Figure 2 (A) AFM indentation maps allow measurement of cell mechanics during wound closure. (Left) Phase-contrast image. (Right) AFM force map overlaid with height reconstruction. The scale bar represents 20 μm. (B) Apparent pretension, T˜0, of cells next to a micropipette-induced single-cell wound (dashed red line) compared to control cells in an undisturbed cell layer (solid black line) (p < 0.001). Shown is the cumulative probability of T˜0 and the corresponding histogram (inset). (C) Comparing the same cells before any disturbance (dashed blue line) and during closure of a neighboring defect (solid orange line) led to a significant increase (p = 0.034). (D) Measuring undisturbed cells twice with the same time delay as needed for wounding did not change the apparent pretension (p = 0.28). (E) Defect induction with a laser ablation setup led to the same effect as observed for mechanical micropipette action: cells neighboring the defect (dashed red line) display a higher apparent pretension than control cells in an undisturbed layer (solid black line) (p < 0.001). To see this figure in color, go online. Biophysical Journal 2017 113, 2601-2608DOI: (10.1016/j.bpj.2017.10.025) Copyright © 2017 Biophysical Society Terms and Conditions

Figure 3 (A) Mechanical investigation of the migration front of an incomplete cell layer with a clear border. (Left) Phase-contrast image. (Right) AFM force map (height reconstruction). The scale bar represents 20 μm. (B) Apparent pretension in cells at the migration front (dashed red line) compared to bulk cells far away from a border (solid black line) (p < 0.001). Shown is the cumulative probability of T˜0 and the corresponding histogram (inset). (C) Generic Sneddon model analysis reveals an increase in the Young’s modulus in cells neighboring a defect (dashed red line) compared to cells in an intact cell layer (solid black line) (p < 0.001). Shown is the cumulative probability of the Young’s modulus and the corresponding histogram (inset). To see this figure in color, go online. Biophysical Journal 2017 113, 2601-2608DOI: (10.1016/j.bpj.2017.10.025) Copyright © 2017 Biophysical Society Terms and Conditions

Figure 4 Cellular mechanics as a function of distance from the wound or defect. (A) Elevated apparent pretension, T˜0, is also measurable in cells belonging to outer shells of the defect (with “neighbor 1” being the direct neighbor and “neighbor 4” being separated from the wound by three cells) and (B) at larger distances from the migration front of a subconfluent layer displaying clear borders. Error bars indicate the mean ± SE. (C) The apparent pretension, T˜0, stays constant (p > 0.05) when the same cells are measured before (dashed blue line) and after (solid orange line) inducing a defect at larger distances (>100 μm away from the measuring area). Shown is the cumulative probability, with the corresponding histogram (inset). To see this figure in color, go online. Biophysical Journal 2017 113, 2601-2608DOI: (10.1016/j.bpj.2017.10.025) Copyright © 2017 Biophysical Society Terms and Conditions

Figure 5 Spatial extension of mechanical changes. A manually skeletonized image shows the affected cells of the single-cell defect, with their assigned shell numbers, before (gray) and 60 min after (red) wounding of the center cell (X). The scale bar represents 20 μm. To see this figure in color, go online. Biophysical Journal 2017 113, 2601-2608DOI: (10.1016/j.bpj.2017.10.025) Copyright © 2017 Biophysical Society Terms and Conditions