Table S1.

Summary of parameters obtained by fitting anisotropy (r) vs. FA orientation (γ) for data obtained from emission anisotropy total internal reflection fluorescence microscopy (EA-TIRFM)

ConstructConditionAssociated figureNo. of FAs/no. of cellsAmplitude (A)Dipole offset (θd), degIsotropic background (C)R2 of fit
αV-GFP–constrained)Control* (10 µg/mL FN)Fig. 1E4,250/720.065 ± 0.008−24.3 ± 2.860.1760.974
αV-GFP–unconstrainedControl (10 µg/mL FN)Fig. 1E4,066/620.006 ± 0.00215.4 ± 6.70.210.82
αV-GFP–less-constrainedControl (10 µg/mL FN)Fig. 4C1,779/380.016 ± 0.004−85.5 ± 6.30.210.92
CAAX-GFPControl (10 µg/mL FN)Fig. 1E995/170.009 ± 0.00545.6 ± 15.60.170.65
αV-GFP–constrainedControl (10 µg/mL FN)Fig. 2B887/220.06 ± 0.01−28.25 ± 5.20.160.94
αV-GFP–constrainedPoly-l-lysine (20 µg/mL PLL)Fig. 2B910/130.003 ± 0.004−83.04 ± 38.60.1960.234
αV-GFP–constrainedLamininFig. 2B1,098/12−0.005 ± 0.0328.1 ± 68.750.2450.036
αV-GFP–constrainedManganese (1 mM Mn, 20 µg/mL PLL)Fig. 2B662/100.024 ± 0.006−36.71 ± 70.170.89
αV-GFP–constrainedRGD-bilayerFig. 2B541/160.0087 ± 0.00717.9 ± 21.50.1310.51
αV-GFP–constrainedControl (10 µg/mL FN)Fig. 3B2,067/500.071 ± 0.01−25 ± 3.720.170.97
Phalloidin–ActinControl (10 µg/mL FN)Fig. 3B2,260/520.29 ± 0.02−3.75 ± 1.430.110.99
αV-GFP–constrained500 nM cytochalasin D (10 µg/mL FN)Fig. 3D1,840/320.02 ± 0.006−0.75 ± 8.80.150.86
Phalloidin–Actin500 nM cytochalasin D (10 µg/mL FN))Fig. 3D5,326/500.17 ± 0.0130.8 ± 6.3−0.160.99
αV-GFP–constrainedControl (10 µg/mL FN)Fig. 3F398/110.072 ± 0.01−23.47 ± 3.40.180.98
SiR–ActinControl (10 µg/mL FN)Fig. 3F398/110.16 ± 0.01−89.88 ± 3.8−0.0420.97
αV-GFP–constrainedOE-Talin head (10 µg/mL FN)Fig. 3F487/120.036 ± 0.01−29.94 ± 6.30.140.90
SiR–ActinOE-Talin head (10 µg/mL FN)Fig. 3F487/120.12 ± 0.02−89.96 ± 4−0.0170.96
  • MEFs coexpressing GFP-tagged αV integrins, membrane marker, or soluble markers, together with untagged β3 integrins and/or mApple-Paxillin were imaged by EA-TIRFM. Anisotropy magnitude maps were computed from EA-TIRFM images, FA were segmented in the paxillin channel (unless noted otherwise), and the orientation of the FA long axes relative to the axis of excitation polarization determined. Average anisotropy (r) in each FA was plotted against the orientation of that FA (γ), and data were fitted to the equation: r = A.cos2(γ + θd) + C. Here, r is the measured anisotropy, A is amplitude of the cosine2 function, which directly relates to the magnitude of angular dependence of r w.r.t polarized light, γ is the angle of the long axis of the FA w.r.t to polarized light, θd is the angular offset from 0°, and C is the isotropic background. A, θd, and C are obtained from the fit (Matlab curve-fitting tool). R2 of each fit is also tabulated below. All parameters shown with 95% confidence interval.

  • * Fit obtained by pooling data from all control experiments and fitting all data to single cos2 function as a weighted average.

  • Unreliable θd values due to low amplitude and R2 of fit.