Unbranched SPIN90-Arp2/3 actin promotes stress fiber speed and focal adhesion maturation

The Arp2/3 complex has long been considered to only assemble branched actin structures in the cell (lamellipodia, endocytic patches, comet tails, and many more). We show for the first time by single-molecule tracking (SMT) that the Arp2/3 complex and SPIN90, which activates Arp2/3 complex to nucleate unbranched filaments, bind to and move in the basal cortex with stress fibers and focal adhesions (FA) that, unlike known sites of Arp2/3 enrichment, employ linear actin bundles. SPIN90 knockout in U2OS cells significantly increases the rate of collective cell migration while decreasing cellular traction (myosin-II and actin speeds) and adhesion (FA size and maturation markers). SPIN90's SH3 domain, similar to its adapter protein Nck1, shows enrichment in FAs, suggesting a possible location for SPIN90-Arp2/3 complex activity. Together, our findings indicate that SPIN90-Arp2/3 nucleated filaments also function in stress fibers where they help define the mechanics of traction and adhesion to regulate cell motility.

The Arp2/3 complex, the branched actin nucleator, drives pushing forces in the cytoplasm, yet its diffuse cortical localization has obscured its functions within unbranched structures. In conventional microscopy, associations of molecules with specific structures are impossible to verify when there is little enrichment over the cytoplasm. Whereas other methods fail to provide convincing evidence of such associations, we used single molecule tracking in live cells to identify molecules that track the motions of stress fibers. Thus, we were able to discover that the Arp2/3 complex and SPIN90, proteins whose roles in cell biology have been thus far limited to branched actin networks, directly integrate within the unbranched stress fiber network to promote cell adhesion and traction for cell migration.