How adhesive and cytoskeletal assemblies integrate with mechanical forces to shape cells is a fundamental question. We developed super-resolution and single-molecule tracking approaches to study integrin adhesions and actin networks. On rigid 2D substrates, we identified molecular events driving integrin activation and actin polymerization in migrating cells. We now extend this to: (1) softer, confined 3D multicellular environments and (2) molecular mechanosensing in cells using a custom stretching device compatible with super-resolution microscopy. These approaches revealed that actin-driven membrane nanotopographies shape stable integrin-based muscle attachments during Drosophila development, and that the axonal spectrin–actin membrane periodic skeleton functions as a mechanosensitive scaffold that reversibly reorganizes under mechanical stimuli.