Interactions with Actin Monomers, Actin Filaments, and Arp2/3 Complex Define the Roles of WASP Family Proteins and Cortactin in Coordinately Regulating Branched Actin Networks
Interactions with Actin Monomers, Actin Filaments, and Arp2/3 Complex Define the Roles of WASP Family Proteins and Cortactin in Coordinately Regulating Branched Actin Networks
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DOI：
10.1074/jbc.m114.587527
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发表时间：
2014-10-17
影响因子：
4.8
通讯作者：
Nolen, Brad J.
中科院分区：
文献类型：
--
作者：
Helgeson, Luke A.;Prendergast, Julianna G.;Nolen, Brad J.
Arp2/3复合体是一种重要的肌动蛋白细丝核因子，它能产生分支肌动蛋白细丝网络，形成肌动蛋白片状结构和内吞肌动蛋白结构。分支肌动蛋白网络的细胞组装通常需要多个Arp2/3复合激活剂，称为成核促进因子(NPF)。我们最近提出了一种机制，通过这种机制，Cortactin，一种弱的NPF，可以取代更强大的NPF，N-WASP，从新生的分支连接协同促进成核。考虑到它们的相似之处，这些NPF在分支成核中的不同作用令人惊讶。我们对这两类NPF进行了生化解剖，以确定它们的Arp2/3复合体和肌动蛋白相互作用片段如何调节它们对分支肌动蛋白网络的影响。我们发现，Cortactin的Arp2/3复合体相互作用的N末端酸性序列(NTA)具有不同于WASP酸性区域(A)的结构特征，这是两个NPF之间协同所必需的。我们的突变分析表明，NTA和A之间的差异并不能解释Cortactin内在NPF活性较弱的原因，相反，Cortactin是一种弱NPF，因为它不能将肌动蛋白单体招募到Arp2/3复合体中。我们使用TIRF显微镜显示，Cortactin使用单个Cortactin分子内的肌动蛋白细丝结合重复序列来捆绑分支的肌动蛋白细丝，但N-WASP拮抗Cortactin介导的捆绑。最后，我们证明了多个WASP家族蛋白协同激活Arp2/3复合体，并确定了WASP蛋白协同所需的生化条件。我们的数据表明，WASP蛋白和皮质肌动蛋白之间的协同作用可能在组装不同的基于肌动蛋白的结构中发挥普遍作用，包括片状脂膜、足体和内吞肌动蛋白网络。
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.