Dendritic actin networks grow in an autocatalytic manner starting from the uncapped minifilament of dynactin.

Branched actin networks polymerized by the Arp2/3 complex are critical for cell migration. The WAVE complex is the machinery that activates Arp2/3 in a RAC1-dependent manner at the leading edge of migrating cells. Multiple WAVE complexes are assembled in a cell through various combinations of paralogous subunits. Here we report the surprising phenotype associated with loss-of-function of CYFIP2, a subunit of the WAVE complex. In three different human mammary cell lines and in prechordal plate cells of gastrulating zebrafish embryos, CYFIP2 depletion promoted, rather than impaired, membrane protrusions and migration persistence. CYFIP2, however, assembled WAVE complexes that polymerize branched actin at the cell cortex and rescued membrane protrusions of CYFIP1/2 double knock-out cells, although less efficiently than CYFIP1. Point mutations of CYFIP2 associated with intellectual disability in children were gain-of-function, as they made CYFIP2 as active as CYFIP1 in this rescue experiment. Biochemical reconstitutions of CYFIP2-containing WAVE complexes showed that they bound equally well to active RAC1 as CYFIP1-containing WAVE complexes, yet they were poorly activated in response to RAC1 binding. Together these results suggest that CYFIP2-containing WAVE complexes titrate active RAC1 and thereby prevent efficient CYFIP1-containing complexes from being activated. In this context, where cell migration is governed by the balance of CYFIP1/2 expression, releasing the restrained activity of CYFIP2-containing WAVE complexes leads to pathology.

During cell migration, protrusion of the leading edge is driven by the polymerization of Arp2/3-dependent branched actin networks. Migration persistence is negatively regulated by the Arp2/3 inhibitory protein Arpin. To better understand Arpin regulation in the cell, we looked for its interacting partners and identified both Tankyrase 1 and 2 (TNKS) using a yeast two-hybrid screening and coimmunoprecipitation with full-length Arpin as bait. Arpin interacts with ankyrin repeats of TNKS through a C-terminal-binding site on its acidic tail, which overlaps with the Arp2/3-binding site. Arpin was found to dissolve the liquid–liquid phase separation of TNKS upon overexpression. To uncouple the interactions of Arpin with TNKS and Arp2/3, we introduced point mutations in the Arpin tail and attempted to rescue the increased migration persistence of the Arpin knockout cells using random plasmid integration or compensating knock-ins at the ARPIN locus. Arpin mutations impairing interactions with either Arp2/3 or TNKS were insufficient to fully abolish Arpin activity. Only the mutation that affected both interactions rendered Arpin completely inactive, suggesting the existence of two independent pathways, whereby Arpin controls the migration persistence.

Vinculin is a mechanotransducer that reinforces links between cell adhesions and linear arrays of actin filaments upon myosin-mediated contractility. Both adhesions to the substratum and neighboring cells, however, are initiated within membrane protrusions that originate from Arp2/3-nucleated branched actin networks. Vinculin has been reported to interact with the Arp2/3 complex, but the role of this interaction remains poorly understood. Here, we compared the phenotypes of vinculin knock-out (KO) cells with those of knock-in (KI-P878A) cells, where the point mutation P878A that impairs the Arp2/3 interaction is introduced in the two vinculin alleles of MCF10A mammary epithelial cells. The interaction of vinculin with Arp2/3 inhibits actin polymerization at membrane protrusions and decreases migration persistence of single cells. In cell monolayers, vinculin recruits Arp2/3 and the vinculin–Arp2/3 interaction participates in cell–cell junction plasticity. Through this interaction, vinculin controls the decision to enter a new cell cycle as a function of cell density.

The Rac1–WAVE–Arp2/3 pathway pushes the plasma membrane by polymerizing branched actin, thereby powering membrane protrusions that mediate cell migration. Here, using knockdown (KD) or knockout (KO), we combine the inactivation of the Arp2/3 inhibitory protein arpin, the Arp2/3 subunit ARPC1A and the WAVE complex subunit CYFIP2, all of which enhance the polymerization of cortical branched actin. Inactivation of the three negative regulators of cortical branched actin increases migration persistence of human breast MCF10A cells and of endodermal cells in the zebrafish embryo, significantly more than any single or double inactivation. In the triple KO cells, but not in triple KD cells, the ‘super-migrator’ phenotype was associated with a heterogenous downregulation of vimentin (VIM) expression and a lack of coordination in collective behaviors, such as wound healing and acinus morphogenesis. Re-expression of vimentin in triple KO cells largely restored normal persistence of single cell migration, suggesting that vimentin downregulation contributes to the maintenance of the super-migrator phenotype in triple KO cells. Constant excessive production of branched actin at the cell cortex thus commits cells into a motile state through changes in gene expression.

The molecular mechanisms underlying cell migration remain incompletely understood. Here, we show that knock-out cells for NHSL3, the most recently identified member of the Nance-Horan Syndrome family, are more persistent than parental cells in single cell migration, but that, in wound healing, follower cells are impaired in their ability to follow leader cells. The NHSL3 locus encodes several isoforms. We identify the partner repertoire of each isoform using proteomics and predict direct partners and their binding sites using an AlphaFold2-based pipeline. Rescue with specific isoforms, and lack of rescue when relevant binding sites are mutated, establish that the interaction of a long isoform with MENA/VASP proteins is critical at cell-cell junctions for collective migration, while the interaction of a short one with 14-3-3θ in lamellipodia is critical for single cell migration. Taken together, these results demonstrate that NHSL3 regulates single and collective cell migration through distinct mechanisms.