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.
Highlights
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
It is commonly agreed that Rac drives the generation of lamellipodial actin filament networks through Arp2/3 complex-mediated branching at the interface of polymerizing actin filaments and protruding plasma membrane[3]
We explored the biochemical activities of FMNL formins and their combined functions in vivo, as their distinguished accumulation at the lamellipodium tip where actin assembly takes place[25] suggests a key role in the formation of these structures
Summary
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. 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. Functional interference with Arp2/3 complex eliminates lamellipodia formation entirely[9,10,11], but whether or not Arp2/3 complex-dependent branching of actin filaments is sufficient for the generation of lamellipodial actin networks, and how other actin filament assembly factors contribute to this process in vivo has mostly remained unclear. MDia[1] has recently been suggested to produce mother filaments for Arp2/3-dependent branching[23], but the protein accumulates at the rear cortex[24] instead of the lamellipodium tip where branching takes place[25], and fibroblasts derived from mDia[1] knockout cells readily form lamellipodia[26]. What are the mechanistic functions of FMNL formins in protrusion?
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