Polarizing Cytoskeletal Tension to Induce Leader Cell Formation During Collective Cell

Polarizing cytoskeletal tension to induce leader cell formation during collective cell migration– Supporting Information

S. Rausch1,2, T.Das1,2, J.R.D. Soiné4,5, T.W. Hofmann1,2, C.H.J. Boehm1,2, U.S. Schwarz4,5, H. Boehm1,2,3*, and J.P. Spatz1,2

1 Department of New Materials and Biosystems, Max Planck Institute for Intelligent Systems, Heisenbergstr. 3, 70569 Stuttgart, Germany

2 Department of Biophysical Chemistry, University of Heidelberg, Im Neuenheimer Feld 253, 69120 Heidelberg, Germany

3CSF Biomaterials and Cellular Biophysics, Max Planck Institute for Intelligent Systems, Heisenbergstr. 3, 70569 Stuttgart, Germany

4University of Heidelberg, Bioquant, Im Neuenheimer Feld 267, 69120 Heidelberg, Germany

5Institute for Theoretical Physics, University of Heidelberg, Philosophenweg 19, 69120 Heidelberg, Germany

* corresponding author: Heike Boehm, ,

Additional Materials and Methods

Unless otherwise stated, all chemicals were purchased from Sigma Aldrich, Germany.

In order to obtain geometrically well defined cell collectives we employed micro-stencil masks made of poly-dimethylsiloxane (PDMS). Circular features of 350 µm diameter were created as openings in a thin membrane of about 60 µm height. In addition to perfectly circular structures we also designed circular structures with four additional protrusions evenly distributed on the circles perimeter. These protrusions are either small hemispheres or small triangles (see Fig.1 in main manuscript). The hemispheres and triangles encompass each an area of about 2000 µm2 and an angular sector of 23° and 20° respectively.

Cell culture and cell seeding.

About 1x106 MDCKII (Health Protection Agency, Porton Down, UK) cells were evenly distributed into each microstencil and allowed to adhere for 90 min before the medium containing all non adhered cells was removed. Micro-stencils were then incubated under cell culture conditions for additional 16 hours to obtain confluent cell layers with proper cell-cell contacts.

Inhibition experiments.

Inhibition experiments were conducted in OptiMEM to reduce interference by serum proteins. For control experiments cell collectives were incubated for one hour in Opti-MEM containing DMSO (1µL per mL of medium) before the stencil mask was removed. The experiment was then conducted in standard cell culture medium.

Traction force microscopy.

To ensure cell attachement PAA was functionalized with fibronectin from bovine plasma with the coupling agent sulphosuccinimidyl-6-(4-azido-2-nitrophenylamino) hexanoate (Sulfo-SANPAH).Cells were seeded on the substrate with micro-stencils in the procedure described above.Images of the bead displacement were acquired on an inverted Observer fluorescence microscope (Zeiss, Germany) equipped with a 40x objective. An additional reference image of the bead positions in the relaxed gel was taken after cells had been removed by incubation in trypsin for 1hour.

Evaluation of actin belt.

Cell collectives with highly curved protrusions were prepared as described above. Cells were fixed before removal of the stencil mask and stained with TRITC-labeled phalloidin. Images of stained cell collectives (40x objective) were recorded and stitched, showing cell collectives as a whole.

Image analysis was fully automated using self-written MATLAB routines. First, the orientation of the collectives of a sample, i.e. the rotation angle α with respect to the horizontal (Additional File 8, Fig S8B) as well as the center of each collective (Additional File 8, Fig S8A), was computed by means of fitting algorithms.

Subsequently, all points within an approximately 4 µm wide region along the boundary of a collective were transformed into polar coordinates, which were used to segment the boundary region into four quadrants. Then the angle coordinate of the points was adapted to lie in a region between 0° and 90° (Additional File 8, Fig S8A andFig S8B) such that in an ideal collective the tips of the highly curved protrusions in each quadrant are placed at 45° (Additional File 8, Fig S8B). Intensities were normalized by the mean intensity of the selected boundary regions of the respective cell collective. Mean values and standard error of the mean were calculated and significance was determined using the Mann-Whitney-Wilcoxon test.

Model Description.

As previously described by Edwards and Schwarz [1]. The thickness of the colony monolayer () is two orders of magnitude smaller than the lateral extension. Therefore a two-dimensional plane stress approximation is appropriate. The colony Cauchy stress is written as the sum of the internal elastic stress and a constant isotropic pressure termwhereis a scalar constant that represents the active and isotropic contractility of the layer:

(1)

Hereis the Young's modulus,the Poisson's ratio, andthe height of the colony. The strain tensoris a measure for deformation and is related to the local colony displacementvia the nonlinear relation

(2)

This treatment is analogous to the problem of thermal expansion/contraction of a material that is associated with an effective temperature difference with (linear thermoelasticity). Since the colony is able to transmit traction stress to the elastic substrate underneath, an effective linear elastic coupling is introduced with coupling constant . This describes the elastic response of the substrate material in first order. By considering static equilibrium the system state is determined by the steady version of Cauchy`s momentum equation whereas the elastic coupling appears as harmonic body force:

(3)

We solved eq. (3) numerically with respect to the displacement field for given colony geometry. For this we used the commercial finite element method (FEM) software COMSOL 4.2. For the mechanics of the colony monolayer we assume a Young's modulus ofand a Poisson ratio of in agreement with AFM indentation measurements on single MDCK cells[2]. For comparison of measured and modeled traction fields, the contractilityand the elastic coupling constantwere used as fit parameters adjusted according to the maximal values of measured displacement and traction stress. Here, model traction was calculated from the force balance where is the numerical solution of the simulated displacement field.

1.Edwards CM, Schwarz US. Force localization in contracting cell layers. Phys. Rev. Lett. 2011 Sep 16;107(12):128101.

2.Harris AR, Charras GT. Experimental validation of atomic force microscopy-based cell elasticity measurements. Nanotechnology. 2011 Jul 28;22(34):345102.