Superresolved microparticle traction force microscopy reveals subcellular force patterns in immune cell-target interactions

Abstract

Force exertion is an integral part of cellular behavior. Traction force microscopy (TFM) has been instrumental for studying such forces, providing both spatial and directional force measurements at subcellular resolution. However, the applications of classical TFM are restricted by the typical planar geometry. Here, we develop a particle-based force sensing strategy, specifically designed for studying ligand-dependent cellular interactions. We establish a straightforward batch approach for synthesizing highly uniform, deformable and tunable hydrogel particles, which can also be easily derivatized to trigger specific cellular behavior. The 3D shape of such particles can be resolved with superresolution (<50 nm) accuracy using conventional confocal microscopy. We introduce a computational method that allows inference of surface traction forces with high sensitivity (~10 Pa) directly from the particle shape. We illustrate the potential and flexibility of this approach by revealing surprising subcellular force patterns throughout phagocytic engulfment and measuring dynamics of cytotoxic T cell force exertion in the immunological synapse. This strategy can readily be adapted for studying cellular forces in a wide range of applications. <50 nm precision. Finally, we solve the inverse problem of inferring the displacement field and traction forces from the measured particle shape and traction-free regions. This is accomplished by iteratively minimizing a cost function consisting of contributions from shape mismatch, residual tractions, and the elastic energy, and is enabled by a fast spherical harmonics-based method 17 . We illustrate the potential of this MP-TFM method by revealing subcellular details of the mechanical interaction of macrophages with their targets during phagocytosis, as well as the dynamics of force exertion in the T cell immunological synapse.

Combined Atomic Force Microscope and Volumetric Light Sheet System for Correlative Force and Fluorescence Mechanobiology Studies

“…These values are similar to mean traction stresses measured for adherent and motile cells as measured with flexible post 31 or traction force substrates 32 . Other studies have shown forces within the phagocytic cup include compressive forces 33 on the target near the cup edge consistent with an actomyosin contractile ring 18 . In our data, we did not find an obvious change in the direction or magnitude of the force as the cup approached and passed the midway of the target as would be expected if the contractile force at the edge dominated the net force on the target.…”

“…Top coverslips (22 × 22 mm #1.5; Fisher, USA) were silanized with HMDS (hexamethyldisilane) for 30 min at 80 deg. PA gel solution was prepared with standard reagents, but with the addition of 1% polyacrylic acid v/v to provide carboxylic acid groups for linkage of ECM proteins 33 Quickly, 2 µL APS was added to 125 µL of gel solution, vortexed for 2 seconds, and 10 µL were placed on the center of each round, 40 mm coverslip. A 22 × 22 mm coverslip was then placed on top of the PA to spread it evenly, and a 3 g weight was placed in the center to compress it.…”

Combined Atomic Force Microscope and Volumetric Light Sheet System for Mechanobiology

“…These values are similar to mean traction stresses measured for adherent and motile cells as measured with flexible post 31 or traction force substrates 32 . Other studies have shown forces within the phagocytic cup include compressive forces 33 on the target near the cup edge consistent with an actomyosin contractile ring 18 . In our data, we did not find an obvious change in the direction or magnitude of the force as the cup approached and passed the midway of the target as would be expected if the contractile force at the edge dominated the net force on the target.…”

“…Top coverslips (22 x 22 mm #1.5; Fisher, USA) were silanized with HMDS (hexamethyldisilane) for 30 min at 80 deg. PA gel solution was prepared with standard reagents, but with the addition of 1% polyacrylic acid v/v to provide carboxylic acid groups for linkage of ECM proteins 33 Quickly, 2 µL APS was added to 125 µL of gel solution, vortexed for 2 seconds, and 10 µL were placed on the center of each round, 40 mm coverslip. A 22 x 22 mm coverslip was then placed on top of the PA to spread it evenly, and a 3 g weight was placed in the center to compress it.…”

Mechanotransduction in T Cell Development, Differentiation and Function

“…CTL killing is exerted on an individual cell basis since it requires the polarization of the centrosome, which controls lytic granules, toward the site of TCR triggering, underscoring the specifically directed nature of exerting apoptosis and preventing damage to healthy tissue [135]. Indeed, the dynamics of CTL force exertion in the IS was recently measured directly using a newly developed superresolved microparticle TFM [136]. IS has been found to be a site of intense force exertion at the nanonewton scale and there existed a positive correlation between the stiffness of the target cell surface and the degree of cytotoxic granule release by CTLs [133,134,137].…”