Direct Arp2/3-vinculin binding is essential for cell spreading, but only on compliant substrates and in 3D

Cells modify their shape in response to the extracellular environment through dynamic remodeling of the actin cytoskeleton by actin-binding proteins (ABPs) 1-4. The relation between actin dynamics and spreading is well-understood for cells on flat glass coverslips; much less is known about cell morphogenesis in compliant three-dimensional environments, and, in particular, how ABPs contribute to this process 5. Here, we knocked-out a diverse set of ABPs, and evaluated the effect of each on cell spreading on planar glass surfaces (2D) and in reconstituted collagen gels (3D). Our morphometric analyses identify the Arp2/3 complex and its associated regulatory genes among the ABPs that contribute most strongly to cell spreading in 3D, but marginally in 2D. Cells lacking Arp3 have reduced spreading specifically in 3D, and display stiffness-dependent cell-matrix adhesion defects. Through manipulation of vinculin activity, we determine that the Arp3 knock-out phenotype largely arises from the lack of direct interaction between vinculin and Arp2/3 complex. This interaction is dispensable for cell spreading in 2D. These data highlight that actin architectural features necessary for adhesion formation and cell spreading in 3D are efficiently compensated on flat and stiff surfaces.