Mapping the 3D orientation of piconewton integrin traction forces

Abstract

Mechanical forces are integral to many biological processes; however, current techniques cannot map the magnitude and direction of piconewton molecular forces. Here, we describe molecular force microscopy, leveraging molecular tension probes and fluorescence polarization microscopy to measure the magnitude and 3D orientation of cellular forces. We mapped the orientation of integrin-based traction forces in mouse fibroblasts and human platelets, revealing alignment between the organization of force-bearing structures and their force orientations.

Figure 1:. Excitation-resolved fluorescence polarization microscopy enables the measurement of molecular force orientation and magnitude.

(a) Principle of molecular tension probes. Yellow arrows: fluorophore transition dipole moments, green: RGD ligands, red: integrin receptors. (b) Receptor forces dictate DNA probe orientation (grey hemisphere: possible orientations), fluorophore orientation and the XY projection of Cy3B (yellow ellipse in the XY plane). (c) Reflective interference contrast microscopy (RICM), 4.7 pN tension image, and fluorescence anisotropy (white arrows: excitation polarization) images of a human platelet on a DNA tension probe surface (representative of 107 platelets from n=4 independent experiments) (d) Hypothetical plots of fluorescence intensity as a function of Φ_excitation for three force orientations. Maximum intensity occurs when Φ_excitation and the fluorophore XY projection (grey ellipse) align. Force XY orientation controls the phase (i-ii), while tilt angle alters amplitude (i-iii). Cyan arrows indicate Φ_excitation=0°. (e) Platelet RICM, 4.7 pN total tension, tension at Φ_excitation=0° (green) and Φ_excitation=90° (magenta), and overlay. (f) Fluorescence intensity as a function of Φ_excitation (red dots) for a single pixel (green arrow in (1e)) and sinusoidal fit (solid line). The 3D force is described by the phase (Φ_force) and the amplitude (force tilt; θ_force). (g) MFM map of platelet integrin forces. Dipole orientation: Φ_force, color: θ_force, and length: percentage of open tension probes. Grey background represents intensity below a threshold of signal-noise ratio < 5. Representative of 79 MFM maps of platelets from n=5 independent experiments. (h) Radial histogram of Φ_force for the platelet in (f). (i) Histogram of circular variance of population platelet XY force projections. Schematics depict the force orientation for the two population of platelets with high and low circular variance.

Figure 2:. Molecular Force Microscopy is a general approach for measuring receptor forces.

(a) RICM, (b) 4.7 pN tension, and (c) MFM image of a platelet aggregate comprised of five cells. The MFM image was overlaid onto the RICM, and white dipoles indicate average force orientation of individual platelets within the aggregate. The average force axes (white arrows) appear disordered (z=0.225, p=0.813, Rayleigh test for uniformity). ROI 1 and 2 (represented on full MFM map by cyan boxes) show zoom-ins at the boundaries between platelets in the aggregate (green scale bar 10% open; white scale bar 500 nm). 6 platelet aggregate MFM maps from n=3 independent experiments. (d) RICM, (e) 4.7 pN tension, (f) GFP-Paxillin, and (g) MFM map of fibroblast seeded on a tension probe substrate for 30 min. The grey background in the MFM image represents pixels below an intensity threshold where the signal-noise ratio < 5. ROI 1 and 2 show zoom-in to highlight the organization of integrin forces with FAs. 37 fibroblast MFM maps from n=3 independent experiments.