Abstract
Arp2/3 complex is an important actin filament nucleator that creates branched actin filament networks required for formation of lamellipodia and endocytic actin structures. Cellular assembly of branched actin networks frequently requires multiple Arp2/3 complex activators, called nucleation promoting factors (NPFs). We recently presented a mechanism by which cortactin, a weak NPF, can displace a more potent NPF, N-WASP, from nascent branch junctions to synergistically accelerate nucleation. The distinct roles of these NPFs in branching nucleation are surprising given their similarities. We biochemically dissected these two classes of NPFs to determine how their Arp2/3 complex and actin interacting segments modulate their influences on branched actin networks. We find that the Arp2/3 complex-interacting N-terminal acidic sequence (NtA) of cortactin has structural features distinct from WASP acidic regions (A) that are required for synergy between the two NPFs. Our mutational analysis shows that differences between NtA and A do not explain the weak intrinsic NPF activity of cortactin, but instead that cortactin is a weak NPF because it cannot recruit actin monomers to Arp2/3 complex. We use TIRF microscopy to show that cortactin bundles branched actin filaments using actin filament binding repeats within a single cortactin molecule, but that N-WASP antagonizes cortactin-mediated bundling. Finally, we demonstrate that multiple WASP family proteins synergistically activate Arp2/3 complex and determine the biochemical requirements in WASP proteins for synergy. Our data indicate that synergy between WASP proteins and cortactin may play a general role in assembling diverse actin-based structures, including lamellipodia, podosomes, and endocytic actin networks.
Highlights
How cortactin and WASP proteins coordinately regulate branched actin assembly is poorly understood
Fusion of the V region dramatically increased the activity of cortactin, increasing the maximum polymerization rate 3.1-fold compared to wild type cortactin, demonstrating that cortactin is a weak nucleation promoting factors (NPFs) because it is unable to recruit actin monomers to the Arp2/3 complex (Fig. 2, B and C)
VV-cortactin activated the Arp2/3 complex 1.6-fold more potently than V-cortactin, and to a similar extent as N-WASPVCA, with a maximum polymerization rate at saturation of 19 nM/s (Fig. 2C). These data demonstrate that differences in the actin binding properties and not the Arp2/3 complex interacting regions of cortactin and N-WASP explain their intrinsic potency as NPFs
Summary
How cortactin and WASP proteins coordinately regulate branched actin assembly is poorly understood. The actin filament binding repeats of cortactin potently increase synergy between these two NPFs, and single molecule TIRF microscopy experiments indicate that the repeats allow cortactin to target nascent branch junctions to displace N-WASP and stimulate nucleation [23] (Fig. 1C) These observations demonstrate that cortactin and N-WASP have distinct modes of activation of the complex that are controlled by the precise structural mechanism by which each NPF interacts with Arp2/3 complex and actin. The precise role of the actin filament binding region, which consists of 6.5 repeats of a 37-amino acid repeat sequence, is not completely understood, partially because of its multiple biochemical functions It increases the potency of cortactin in synergizing with N-WASP and is required for the intrinsic NPF activity of cortactin [19, 23]. These observations have important implications for understanding how cortactin and WASP family proteins independently and coordinately regulate the dynamics of branched actin networks
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