A Photopolymerized Hydrogel System with Dual Stiffness Gradients Reveals Distinct Actomyosin-Based Mechano-Responses in Fibroblast Durotaxis

Durotaxis, migration of cells directed by a stiffness gradient, is critical in development and disease. To distinguish durotaxis-specific migration mechanisms from those on uniform substrate stiffnesses, we engineered an all-in-one photopolymerized hydrogel system containing areas of stiffness gradients with dual slopes (steep and shallow), adjacent to uniform stiffness (soft and stiff) regions. While fibroblasts rely on nonmuscle myosin II (NMII) activity and the LIM-domain protein Zyxin, ROCK and the Arp2/3 complex are surprisingly dispensable for durotaxis on either stiffness gradient. Additionally, loss of either actin-elongator Formin-like 3 (FMNL3) or actin-bundler fascin has little impact on durotactic response on stiffness gradients. However, lack of Arp2/3 activity results in a filopodia-based durotactic migration that is equally as efficient as that of lamellipodia-based durotactic migration. Importantly, we uncover essential and specific roles for FMNL3 and fascin in the formation and asymmetric distribution of filopodia during filopodia-based durotaxis response to the stiffness gradients. Together, our tunable all-in-one hydrogel system serves to identify both conserved as well as distinct molecular mechanisms that underlie mechano-responses of cells experiencing altered slopes of stiffness gradients.