Aligned Collagen Fibers Drive Distinct Traction Force Signatures to Regulate Contact Guidance.

Cellular forces on deposited nonfibrillar extracellular matrix (ECM) have been measured extensively. However, in vivo, cells exert traction forces on collagen fibers within the ECM. Oftentimes, collagen fibers are aligned, as seen in cancer, fibrosis, and during wound healing. How forces are transmitted on aligned collagen fibers and how the cytoskeleton regulates this is unknown. Here, we develop a fiber-traction force microscopy (f-TFM) approach that uses collagen fibers transferred to flexible substrates with fiducial markers on the collagen fibers and in the underlying flexible substrates. We find that the elastic modulus of the substrate determines the steady-state traction stress exerted by cells on aligned collagen fibers but does not affect traction force kinetics. Collagen fiber networks result in higher traction stresses than adsorbed collagen, particularly for randomly oriented fibers. In cells that weakly contact guide, formins and Arp2/3 modulate traction stress differently, with formins increasing traction stress magnitude, while Arp2/3 increases traction stress kinetics. However, both are important in driving traction force increases during cell turning on aligned collagen fibers. In cells that strongly contact guide, Arp2/3 and formins are less important than myosin II. In addition, there is a positive correlation between traction force and directionality on aligned collagen fibers for modest cell alignment. Further increases in traction stress are not required for high cell alignment. These findings underscore the complex interplay between the mechanics of collagen fiber networks, cytoskeletal regulators, and cellular traction forces, providing insights into how cells navigate complex fiber networks during migration.