S38 - Aligned collagen fibers drive distinct traction force signatures to regulate contact guidance

Introduction: : Mechanical force is critical in sensing external environments and performing cell functions like migration. Many times, cells are found within complex collagen fiber networks in vivo. In cancer, fibrosis and wound healing, these collagen fiber networks are aligned, presenting a complex anisotropic mechanical environment that directs cell migration in a process called contact guidance. While much work has focused on understanding how cells exert traction forces, the dominant system is isotropically adsorbed extracellular matrix molecules on a flexible substrate like polyacrylamide (PAA) as the cell adhesive substrate and fluorescence beads as fiduciary markers (FMs) embedded into the PAA gel of desired stiffness. In this approach, cells apply mechanical force and deform the substrate, displacing the beads embedded within the PAA gel, allowing for the calculation of traction force. However, this completely disregards the complexity associated with mechanical force transmission in fiber networks. Consequently, we don’t understand how traction forces are exerted through aligned fiber networks.

Materials and

Methods: : Previously, our lab developed a unique approach of using mica to epitaxially grow various collagen fiber networks and transfer these fibers to substrates for studying cell migration. In this paper, we leverage these approaches to create a dual traction force microscopy (d-TFM) system (Figure). This creates aligned or random collagen fiber networks that can be transferred onto functionalized PAA substrates with embedded beads that act as FMs. In addition, we can attach carboxylate-functionalized beads directly to the fibers creating FMs and enabling traction force measurement at the fiber depth. We measure traction stresses over time in spreading cells and correlate traction stress with area. We assess the role of cytoskeletal regulators (formins and Arp2/3) in tuning both the traction stress as well as directed migration.

Results, Conclusions, and Discussions:: Aligned collagen fibers can be assembled on mica and transferred to glass (Figure A&B). Aligned collagen fibers can also be transferred to PAA gels with embedded beads and beads attached to the surface (Figure C&D). This aligned collagen fiber network results in robust cell alignment (Figure E). Fiber networks appear to stimulate much larger traction stresses than isotropic networks (Figure F). Furthermore, random collagen fibers seem to induce linearly increasing traction stress that exceeds the traction stress seen on aligned fibers. Interestingly, while traction stress on isotropic networks appears to be correlated with cell area, traction stress on fiber networks is not, generating a unique force transmission signature for aligned fiber networks (Figure G). Inhibiting formins with SMIFH2 diminishes traction stress, whereas inhibiting Arp2/3 with CK-666 appears only to slow the traction stress kinetics, suggesting unique roles for different cytoskeletal regulators in controlling force transmission on aligned collagen fibers (Figure H). Finally, minimal cell alignment requires a traction stress threshold to be achieved, but once that threshold has been achieved, no additional traction stress is required for high cell alignment (Figure I). This system provides a new approach for understanding the differences in traction force generation between isotropic and anisotropic extracellular environments and can be used to understand how anisotropic force generation regulates contact guidance.

Acknowledgements and/or References (Optional):: This work was supported by R01GM143302.