Topographical depth reveals contact guidance mechanism distinct from focal adhesion confinement

Cellular response to the topography of their environment, known as contact guidance, is a crucial aspect to many biological processes yet remains poorly understood. A prevailing model to describe cellular contact guidance involves the lateral confinement of focal adhesions (FA) by topography as an underlying mechanism governing how cells can respond to topographical cues. However, it is not clear how this model is consistent with the well‐documented depth‐dependent contact guidance responses in the literature. To investigate this model, we fabricated a set of contact guidance chips with lateral dimensions capable of confining focal adhesions and relaxing that confinement at various depths. We find at the shallowest depth of 330 nm, the model of focal adhesion confinement is consistent with our observations. However, the cellular response at depths of 725 and 1000 nm is inadequately explained by this model. Instead, we observe a distinct reorganization of F‐actin at greater depths in which topographically induced cell membrane deformation alters the structure of the cytoskeleton. These results are consistent with an alternative curvature‐hypothesis to explain cellular response to topographical cues. Together, these results indicate a confluence of two molecular mechanisms operating at increased induced membrane curvature that govern how cells sense and respond to topography.