Daam-, FMNL-, and mDia-family formins jointly regulate cortical actin dynamics in melanoma cells.

The actin-rich cell cortex is a viscoelastic structure essential for processes such as cell migration and division, yet the full spectrum of actin assembly factors that build and regulate the cortex in mammalian cells remains incompletely defined. While the Arp2/3 complex generates actin networks mostly comprising short, branched filaments, formins like mDia1 and mDia3 assemble long, linear filaments capable of withstanding the high mechanical load produced by myosin-driven contraction at the cell rear. To obtain a complete inventory of formins that regulate mechanics of the contractile cortex in B16-F1 melanoma cells, we systematically screened Diaphanous-related formins (DRFs) for interactions with Rho-family GTPases regulating contractility. This screen led to the identification of Daam1/2 and FMNL2/3 as cortical formins, since active variants of these formins as well as the endogenous proteins prominently localized to the rear cortex of polarized B16-F1 cells. Yeast two-hybrid (Y2H) and GST pull-down assays revealed prominent interactions of Daam1/2 with active RhoA, RhoB, RhoC, RhoD, and Cdc42, and weaker binding to Rac1 and Rif. Pyrene-actin polymerization and single-filament TIRF assays further demonstrated that these GTPases effectively relieve autoinhibition of Daam-family formins, with Daam1 and 2 emerging as a potent elongators. In line with this, CRISPR/Cas9-generated Daam1/2 double-knockout cells exhibited multiple defects, including formation of multiple leading edges, impaired random 2D migration, reduced focal adhesion (FA) turnover and inefficient rear retraction. However, contrary to earlier work, we found no evidence that Daam1/2 regulate the formation of finger-like protrusions. By comparison, FMNL2 appears to act as a dual-role formin at both the cell front and rear, interacting with and being activated by several GTPases linked to contractility and protrusion. Analytical ultracentrifugation further clarified previous inconsistencies by showing that the affinity of FMNL2 for GTPases, particularly for Rac1, depends on the length of its N-terminal fragment and the presence of the full FH3 domain. Finally, we show that cells lacking Daam1/2 and mDia1/3 display pronounced cortical defects, including reduced rigidity, excessive blebbing, and delayed cytokinesis, demonstrating that these formins cooperate to preserve cortical architecture and function.