Propagation of Mechanical Stress through the Actin Cytoskeleton toward Focal Adhesions: Model and Experiment  Raja Paul, Patrick Heil, Joachim P. Spatz, Ulrich S. Schwarz  Biophysical Journal  Volume 94, Issue 4, Pages 1470-1482 (February 2008) DOI: 10.1529/biophysj.107.108688 Copyright © 2008 The Biophysical Society Terms and Conditions

Figure 1 Schematic representation of the experiment. The cell is first allowed to spread for at least 30min. Then a microfabricated pillar is contacted from above and kept there for ∼15min to ensure stable adhesion between pillar and cell. Culture conditions are chosen such that the actin cytoskeleton (red) is in a homogeneous state (no stress fibers). When the pillar is shifted to the side, the cytoskeleton is strained and the FAs adapt their size to the new loading situation. Typically, this takes 15min. In the stationary state, the sizes of the focal adhesions (green) are expected to be proportional to the forces transmitted through the cytoskeleton. Biophysical Journal 2008 94, 1470-1482DOI: (10.1529/biophysj.107.108688) Copyright © 2008 The Biophysical Society Terms and Conditions

Figure 2 Use of microfabricated pillar. (a and c) Phase contrast showing cell and pillar shortly before and shortly after pulling. (b and d) Same images (now in blue) overlaid with fluorescence data. REF cells are transiently transfected with CFP-actin (red) and YFP-zyxin (green). (c and d) Shortly after pulling, one can discern the large deformations caused by shifting the pillar. However, the focal adhesions had no time yet to adjust their size to the changed loading situation. The images also show that there are no stress fibers present. Biophysical Journal 2008 94, 1470-1482DOI: (10.1529/biophysj.107.108688) Copyright © 2008 The Biophysical Society Terms and Conditions

Figure 3 Computational network models. (a) A triangular network and (b) a reinforced square network. Each link between two neighboring nodes represents a cable. In the unit cell of the reinforced network, the diagonal cables have no entanglement (no node present). Biophysical Journal 2008 94, 1470-1482DOI: (10.1529/biophysj.107.108688) Copyright © 2008 The Biophysical Society Terms and Conditions

Figure 4 Mechanical properties of the cable network. (a) Effective Poisson ratio σ and (b) effective two-dimensional Young modulus E2D for the triangular network as a function of the number of nodes in the test region. In both cases, red and blue curves are for networks without and with prestrain, respectively. (c) Forces at adhesion sites as a function of the resting length lr, which determines the level of prestress in the network. Different curves correspond to different number of adhesions along a circular contour (8,12, 16, 20, 25, 35, 45, and 60 from top to bottom). Biophysical Journal 2008 94, 1470-1482DOI: (10.1529/biophysj.107.108688) Copyright © 2008 The Biophysical Society Terms and Conditions

Figure 5 Comparison of experiment and model predictions for one set of data. (a and b) Experimental and simulation snapshots of the prestressed cell before pulling, respectively. (c) To compare experiment and simulation, we assume that the size of the experimentally measured FA area is proportional to the pulling force on it. The blue line represents the forces at the adhesions predicted by the computer simulations and the gray line represents the experimentally measured areas of the contacts (RMSD=1.89 nN). (d–f) Same data but 30min after the pillar has been laterally shifted to the left. In panel f, the differences in forces and areas are shown relative to the situation before pulling (RMSD=2.51 nN). Biophysical Journal 2008 94, 1470-1482DOI: (10.1529/biophysj.107.108688) Copyright © 2008 The Biophysical Society Terms and Conditions

Figure 6 Random CSK network on a triangular network for the adhesion geometry from Fig. 5 obtained after bond dilution with bond occupancy p=0.8. Snapshots showing (a) prestressed CSK and (b) when it is laterally shifted with the pillar. (c) Forces at FAs of the prestressed cell, obtained on a random, and on a pure triangular, CSK network. (d) Same kind of data obtained after the CSK is shifted with the pillar. Biophysical Journal 2008 94, 1470-1482DOI: (10.1529/biophysj.107.108688) Copyright © 2008 The Biophysical Society Terms and Conditions

Figure 7 Sequential detachment of FAs opposite to the pulling direction leads to different morphology depending on the initial cell geometry. Each geometry is shown without (a, c, and e) and with rupture (b, d, and f). For initially circular (a and b) or elliptical cells pulled along the minor (short) axis (c and d), a morphology arises which is typical for migrating keratocytes. For initially elliptical cells pulled along the major (long) axis (e and f), we get a morphology that is typical for migrating fibroblasts. Biophysical Journal 2008 94, 1470-1482DOI: (10.1529/biophysj.107.108688) Copyright © 2008 The Biophysical Society Terms and Conditions

Figure 8 Effect of external versus internal force for the experimental adhesion geometry from Fig. 5. (a–c) External force with a threshold value for detachment of Fc=6nN. Because the cell is elliptically elongated, a morphology arises which is typical for a migrating fibroblast. (d–f) Exactly the same morphology can be obtained by using internally generated force only. To this purpose, contracting fibers (represented by the thick red lines) are attached between the FAs at the left boundary and the circular area at the center. Their respective contractility is then adjusted in such a way that rupture at the left boundary is avoided, while at the center area, the same overall force is achieved as in panels a–c. Biophysical Journal 2008 94, 1470-1482DOI: (10.1529/biophysj.107.108688) Copyright © 2008 The Biophysical Society Terms and Conditions