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In vivo, immune cells migrate through a wide variety of tissues, including confined and constricting environments. Deciphering how cells apply forces when infiltrating narrow areas is a critical issue that requires innovative experimental procedures. To reveal the distribution and dynamics of the forces of cells migrating in confined environments, we designed a device combining microchannels of controlled dimensions with integrated deformable micropillars serving as sensors of nanoscale subcellular forces. First, a specific process composed of two steps of photolithography and dry etching was tuned to obtain micrometric pillars of controlled stiffness and dimensions inside microchannels. Second, an image-analysis workflow was developed to automatically evaluate the amplitude and direction of the forces applied on the micropillars by migrating cells. Using this workflow, we show that this microdevice is a sensor of forces with a limit of detection down to 64 pN. Third, by recording pillar movements during the migration of macrophages inside the confining microchannels, we reveal that macrophages bent the pillars with typical forces of 0.3 nN and applied higher forces at the cell edges than around their nuclei. When the degree of confinement was increased, we found that forces were redirected from inward to outward. By providing a microdevice that allows the analysis of force direction and force magnitude developed by confined cells, our work paves the way for investigating the mechanical behavior of cells migrating though 3D constricted environments.

Determining how cells generate and transduce mechanical forces at the nanoscale is a major technical challenge for the understanding of numerous physiological and pathological processes. Podosomes are submicrometer cell structures with a columnar F-actin core surrounded by a ring of adhesion proteins, which possess the singular ability to protrude into and probe the extracellular matrix. Using protrusion force microscopy, we have previously shown that single podosomes produce local nanoscale protrusions on the extracellular environment. However, how cellular forces are distributed to allow this protruding mechanism is still unknown. To investigate the molecular machinery of protrusion force generation, we performed mechanical simulations and developed quantitative image analyses of nanoscale architectural and mechanical measurements. First, in silico modeling showed that the deformations of the substrate made by podosomes require protrusion forces to be balanced by local traction forces at the immediate core periphery where the adhesion ring is located. Second, we showed that three-ring proteins are required for actin polymerization and protrusion force generation. Third, using DONALD, a 3D nanoscopy technique that provides 20 nm isotropic localization precision, we related force generation to the molecular extension of talin within the podosome ring, which requires vinculin and paxillin, indicating that the ring sustains mechanical tension. Our work demonstrates that the ring is a site of tension, balancing protrusion at the core. This local coupling of opposing forces forms the basis of protrusion and reveals the podosome as a nanoscale autonomous force generator.

Podosomes are mechanosensitive adhesion cell structures that are capable of applying protrusive forces onto the extracellular environment. We have recently developed a method dedicated to the evaluation of the nanoscale forces that podosomes generate to protrude into the extracellular matrix. It consists in measuring by atomic force microscopy (AFM) the nanometer deformations produced by macrophages on a compliant Formvar membrane and has been called protrusion force microscopy (PFM). Here we perform time-lapse PFM experiments and investigate spatial correlations of force dynamics between podosome pairs. We use an automated procedure based on finite element simulations that extends the analysis of PFM experimental data to take into account podosome architecture and organization. We show that protrusion force varies in a synchronous manner for podosome first neighbors, a result that correlates with phase synchrony of core F-actin temporal oscillations. This dynamic spatial coordination between podosomes suggests a short-range interaction that regulates their mechanical activity.