Talin1 Adhesion Morphology and Colocalization With Tensin3 Are Largely Unaffected by Polyacrylamide Substrate...

Cells sense the stiffness of their extracellular matrix (ECM) and adapt their behavior accordingly. We investigated how ECM stiffness affects the spatial organization of talin1, a key mechanosensitive focal adhesion protein. Using polyacrylamide (PA) hydrogels with tunable stiffnesses (0.2-188 kPa), we analyzed cell morphology, migration, talin1 distribution, colocalization with tensin3, and fibronectin deposition. Softer substrates enhanced filopodia activity and altered migration behavior. On softer ECMs, talin1 exhibited a more uniform intracellular distribution. Conversely, on stiffer matrices, it was localized more towards the cell periphery. PA gels supported elongated talin1-based adhesions, whose morphology showed minimal variation across the 3-188 kPa stiffness range. Talin1-tensin3 colocalization was unaffected by PA gel stiffness, indicating a stable interaction. Notably, cells deposited more fibronectin on softer substrates. While talin1 adhesion morphology varied little with stiffness, cell migration behavior changed markedly. Combined with prior studies, our data suggest that ECM stiffness regulates talin1 primarily through conformational changes rather than remodeling of talin1 adhesion morphology. These findings highlight talin1's central role in translating mechanical cues into dynamic cellular responses.