Direct Arp2/3-vinculin binding is required for pseudopod extension, but only on compliant substrates and in 3D

A critical step in cell morphogenesis is the extension of actin-dense pseudopods, controlled by actin-binding proteins (ABPs). While this process is well-understood on glass coverslips, it is less so in compliant three-dimensional environments. Here, we knocked out a series of ABPs in osteosarcoma cells and evaluated their effect on pseudopod extension on glass surfaces (2D) and in collagen gels (3D). Cells lacking the longest Arp3 gene variant, or with attenuated Arp2/3 activity, had the strongest reduction in pseudopod formation between 2D and 3D. This was largely due to reduced activity of the hybrid Arp2/3-vinculin complex, which was dispensable on glass. Our data suggests that concurrent formation of actin branches <i>and</i> nascent adhesions, supported by Arp2/3-vinculin interactions, is essential to form mechanically stable links between fibrous extracellular matrix and actin in 3D. This highlights how experiments on stiff, planar substrates may conceal actin architectural features that are essential for morphogenesis in 3D.