Coordination between Intra- and Extracellular Forces Regulates Focal Adhesion Dynamics.

Preparation and Calibration of the micropillar substrates Preparation The preparation of micropillar substrate has been described in detail elsewhere. 29 In brief, the micropillar substrates were prepared from Polydimethylsiloxane (PDMS, Sylgard 184, Dow-Corning) elastomer by replica molding from silicon wafers. The substrate was characterized by using Scanning Electron Microscopy (SEM). The pillars are arranged in hexagonal arrays with an inter-pillar distance of 2 µm. The pillar diameter was 2 µm and height was 5 µm. With such a geometry we obtained substrate stiffness of 43 nN/µm. For facilitating cell adhesion, the pillar tops were coated with fibronectin using micro contact printing. For that, flat PDMS stamps were incubated with fibronectin (50 µg/ml unconjugated fibronectin and 5µg/ml dye-conjugated fibronectin, ATTO647N, Sigma-Aldrich) for one hour in room temperature. The stamps were then dried and placed on UV/Ozone-treated micropillar substrates for 5 minutes. To restrict the cell adhesion only to the pillar tops, we added 0.2% Pluronics-F127 solution to the substrate for 1 hour, which passivated the sides of the pillar. The substrate was then rinsed gently with PBS and subsequently immersed in cell culture medium.

Calibration

To obtain reproducible Young’s Modulus E= 2MPa, we adopted a consistent procedure for PDMS preparation where in a fixed ratio of cross-linker to base polymer (1:10) and the curing time (80°C for 2 hours) were maintained for all our experiments. After obtaining the dimensions of the pillars from SEM images, we used Finite Element Method (FEM) to calculate the stiffness of the pillars. 29 Cell Biology Cell Culture and Transfection Vinculin deficient mouse embryonic fibroblasts were a gift from Christian Grashoff. Both MEF and REF52 cells were cultured in Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 U/ml penicillin, 100 µg/ml streptomycin, and 100 µg/ml L-glutamine. To label the actin filaments, cells were transfected with RFP-Ftractin. VinculinTS was a gift from Martin Schwartz (Adgene plasmid # 26019). We used electroporation (Nucleofactor, Lonza) for all our transfections. Immunostaining For immunostaing, cells were first allowed to spread on micropillars for sufficient time (~3 hours) and then subsequently fixed (4% formaldehyde in PBS, 10 min) at room temperature, permeabilized (0.1% Triton-X , 5 min), blocked ( 1% BSA in PBS, 1 h), and rinsed with PBS. Phalloidin-TRITC was used for actin staining, and for cellular fibronectin we used anti-fibronectin antibody (Abcamab18265).

Confocal Microscopy and FRET imaging

Live cell imaging was performed after 2 hours on a confocal microscope (Zeiss LSM-710, Carl Zeiss, Germany). For FRET measurement, we used 458nm laser to excite the donor. The fluorescent signal from the sensor was collected with a spectral detector in 13 continuous bands from 465nm to 582nm. This contained both the emission from donor and the acceptor. Subsequently using PixFret plugin 48 with ImageJ we deduced the FRET index at desired ROI. After background correction, the plugin calculates the FRET index by measuring the intensity ratio between the donor channel and the FRET channel, for example, FRET Index = I FRET / (I donor + I FRET ). The index (F) to Efficiency (E) relationship is extracted from the measured F of TRAF and 5aa and their known E using linear interpolation: E=(F-F 5aa )*(E 5aa -E TRAF )/(F 5aa -F TRAF )+E 5aa . To measure the pillar deflection the fibronectin-dye on the pillar top was imaged concurrently on a different track using a separate detector. From the pillar image, individual pillar deflection was calculated using a home-built plugin in imageJ. For a particular FA, the average FRET index was measured. Laser ablation experiments where performed by using a Zeiss LSM 780 NLO microscope (Carl Zeiss, Germany) coupled to a pulsed NIR laser Ti:Sapphire crystal laser (Coherent Chameleon Ultra, Santa Clara, CA, USA). We used several excitation wavelength 458nm (For VinTS sensor donor excitation), 561nm (PSS-laser) for Actin visualization, and 633nm (HeNe-laser) for micropillar top visulization. For ablation experiments NIR laser was tuned at 800 nm (power: 65%-100%). The system included a 34-Channel GaAsP detector (GaAsp-array with 32 PMTs and two flanking single PMTs). A plan c-apochromat ×63 (NA = 1.2) water immersion objective was used for all experiments. Local temperature was monitored and maintained at 37°C (PECON, Germany). Structured Illumination Microscopy (SIM) For 3D-SIM, we used fixed samples immunostained for actin or cellular fibronectin. FAs were visualized with YFP-paxillin, which was stably expressed in REF52 cells. The nucleus was stained with Hoechst. The equipment consisted of ELYRA PS.1 Zeiss microscope coupled with an EMCCD camera (Andor iXon 885, 1004×1002, pixel size 8µm, QE=65%). We used excitation lasers of 405nm (for nucleus), 488nm(for FA), 561 nm (for actin or cellular fibronectin), and 641nm (for micropillar tops). Structured illumination grids at three different angles and five different phases were used for image acquisition. A Zeiss PLAN-APO, 63x, NA 1.40 (Zeiss) objective was used for all our experiments. 3-D reconstruction was done using IMARIS. (Bitplane AG, Switzerland).

Supplementary Material The Supporting Information is available free of charge on the ACS Publications website. Supporting Information