FMNL formins boost lamellipodial force generation

Abstract

Migration frequently involves Rac-mediated protrusion of lamellipodia, formed by Arp2/3 complex-dependent branching thought to be crucial for force generation and stability of these networks. The formins FMNL2 and FMNL3 are Cdc42 effectors targeting to the lamellipodium tip and shown here to nucleate and elongate actin filaments with complementary activities in vitro. In migrating B16-F1 melanoma cells, both formins contribute to the velocity of lamellipodium protrusion. Loss of FMNL2/3 function in melanoma cells and fibroblasts reduces lamellipodial width, actin filament density and -bundling, without changing patterns of Arp2/3 complex incorporation. Strikingly, in melanoma cells, FMNL2/3 gene inactivation almost completely abolishes protrusion forces exerted by lamellipodia and modifies their ultrastructural organization. Consistently, CRISPR/Cas-mediated depletion of FMNL2/3 in fibroblasts reduces both migration and capability of cells to move against viscous media. Together, we conclude that force generation in lamellipodia strongly depends on FMNL formin activity, operating in addition to Arp2/3 complex-dependent filament branching.

Actin Turnover in Lamellipodial Fragments

“…One possibility is that the filaments’ stability is related to the type of nucleator that initiated them, as there is evidence for both Arp2/3 and formin-based nucleation in the lamellipodium. This is supported by recent results showing that the filaments generated by different nucleators have differential influence on protrusion dynamics and lamellipodial stability [69]. Stable filaments could also be defined by selective, cooperative association with actin binding proteins, such as tropomyosins [86] or coronin [16].…”

“…Similarly, actin assembly involves both Arp2/3 and/or formin and their relative importance and respective roles are still open questions in the field [69,70]. It is also not possible currently to measure or infer the actin monomer concentrations available for assembly by either of these nucleators.…”

Ultrastructure of the actin cytoskeleton

“…However, accumulating data reveal a role of various formins in lamellipodial protrusion [12–16] and other branched networks [17,18], where formins can function as elongators and/or nucleators of actin filaments. Specifically, mDia2 can stimulate both elongation and nucleation of actin filaments in protrusions [16], FMNL2 seems to mostly promote elongation of Arp2/3-nucleated filaments, whereas FMNL3 and mDia1 appear to be largely implicated in nucleation [14,15]. In lamellipodia, formin-nucleated filaments either serve as mother filaments for Arp2/3-mediated nucleation or become intermingled with branched filaments (Figure 2).…”

APC2 associates with the actin cortex through a multipart mechanism to regulate cortical actin organization and dynamics in the Drosophila ovary

“…Drosophila Diaphanous (Dia), the first formin identified, was originally shown to be essential for the formation of cytokinetic furrows (Castrillon & Wasserman, ). We now know that Dia and other formins play key roles in the assembly of actin filaments in yeast and metazoans required for stress fiber assembly, formation of cellular protrusions like filopodia and lamellipodia, and many others (Bohnert, Willet, Kovar, & Gould, ; Homem & Peifer, , ; Kage et al, ; Roy, Huang, Liu, & Kornberg, ; Young, Heimsath, & Higgs, ). Although Dia can independently nucleate and elongate actin filaments in vitro, mounting evidence suggests that in vivo Dia activity is regulated by binding partners at many steps during filament assembly.…”