Actin filament dynamics must be precisely controlled in cells to execute behaviors such as vesicular trafficking, cytokinesis, and migration. Coronins are conserved actin-binding proteins that regulate several actin-dependent subcellular processes. Here, we describe a new conditional knockout cell line for two ubiquitous coronins, Coro1B and Coro1C. These coronins, which strongly co-localize with Arp2/3-branched actin, require Arp2/3 activity for proper subcellular localization. Coronin null cells have altered lamellipodial protrusion dynamics due to increased branched actin density and reduced actin turnover within lamellipodia, leading to defective haptotaxis. Surprisingly, excessive cofilin accumulates in coronin null lamellipodia, a result that is inconsistent with the current models of coronin–cofilin functional interaction. However, consistent with coronins playing a pro-cofilin role, coronin null cells have increased F-actin levels. Lastly, we demonstrate that the loss of coronins increases accompanied by an increase in cellular contractility. Together, our observations reveal that coronins are critical for proper turnover of branched actin networks and that decreased actin turnover leads to increased cellular contractility.

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.

Durotaxis, migration of cells directed by stiffness gradient, is critical in development and disease. To study the molecular determinants of single cell durotaxis, we developed an all-in-one photopolymerized hydrogel system containing areas of stiffness gradients with different slopes, along with uniform stiffness (soft and stiff) regions. We find that fibroblasts rely on non-muscle myosin II (NMII) activity and the LIM-domain protein zyxin for durotaxis on both steep and shallow stiffness gradients. Importantly, unlike haptotaxis, the Arp2/3 complex is dispensable for durotaxis on both stiffness gradients. Lack of Arp2/3 results in a filopodia-based durotactic migration that is equally efficient as that of lamellipodia-based durotactic migration. Finally, we reveal an essential role for the actin-bundler fascin in the formation and asymmetric distribution of filopodia during filopodia-based durotaxis in shallow, but not steep, stiffness gradient. Together, our all-in-one hydrogel system can serve as a platform to identify, discriminate, and characterize stiffness gradient specific molecular mechanisms that cells employ to efficiently durotax.