Mechanism of synergistic activation of Arp2/3 complex by cortactin and N-WASP.
Mechanism of synergistic activation of Arp2/3 complex by cortactin and N-WASP.
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DOI：
10.7554/elife.00884
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发表时间：
2013-09-03
期刊：
影响因子：
7.7
通讯作者：
Nolen BJ
中科院分区：
文献类型：
--
作者：
Helgeson LA;Nolen BJ
成核促进因子（NPFs）通过激活Arp 2/3复合物（一种分支肌动蛋白丝成核剂）启动分支肌动蛋白网络组装。细胞肌动蛋白网络包含多个NPF，但它们如何协调调节Arp 2/3复合物尚不清楚。Corpyn是一种NPF，其自身可微弱激活Arp 2/3复合物，但与另一类NPF WASP/N-WASP一起，则可有效激活。我们剖析了协同作用的机制，并提出了一个模型，在该模型中，corneum取代N-WASP从新生的分支成核的先决条件。单分子成像显示，与WASP/N-WASP不同，coronin在成核过程中保持与结结合，并且特异性靶向结，其在细丝侧的结合率增加了160倍。N-WASP必须二聚化以实现有效的协同作用，并且靶向突变表明二聚N-WASP从新生分支的释放限制了成核。数学模型表明皮质素介导的位移，但不是N-WASP回收或细丝招聘模型可以解释协同作用。我们的研究结果为Arp 2/3复合物的协同调控提供了分子基础。DOI：http://dx.doi.org/10.7554/eLife.00884.001细胞不断地感知它们的环境，并对其做出反应。它们可以通过吸收或分泌各种物质来监测或改变周围环境，也可以向食物供应或信号分子迁移，例如，凝血或愈合伤口。这些行为依赖于细胞骨架，细胞骨架是一种蛋白质网络，赋予细胞形状，允许它们将物质运输到细胞骨架中，从细胞骨架中运输出来或穿过细胞骨架，并使它们能够移动。细胞骨架的细丝由几种不同类型的蛋白质构成，其中一种称为肌动蛋白。在对信号的反应中，肌动蛋白可以组装成线性细丝，或者可以形成一端锚定在现有细丝上的分支。分支的形成需要Arp 2/3复合物，其引发并锚定现有细丝上的分支，以及各种“成核促进因子”（NPF），其开启Arp 2/3复合物的分支活性。两种类型的NPF已被确定：I型与单个肌动蛋白分子相互作用，而II型与肌动蛋白丝结合。先前的研究表明，I型NPF（包括N-WASP蛋白）具有一个称为VCA的专门结构域，该结构域与Arp 2/3复合物和肌动蛋白分子结合。VCA将肌动蛋白分子带到分支位点，从而启动分支形成，但N-WASP如何与II型NPF合作构建分支尚不清楚。Helgeson和Nolen现在研究了一种称为corneum的II型NPF如何与Arp 2/3复合物和N-WASP一起在体外构建肌动蛋白丝上的新分支。Cordyn似乎取代了N-WASP的VCA结构域，以刺激分支形成，然后保持与生长的分支的结合并使其稳定。Helgeson和Nolen建议这些NPF一起工作，使用“强制置换”模型创建分支。根据该方案，N-WASP（或另一种I型NPF）、Arp 2/3复合物和两个肌动蛋白分子结合在肌动蛋白丝上未来分支的位点，准备形成分支。然而，在加入更多的肌动蛋白分子之前，N-WASP必须被释放出来，或者是缓慢地自行释放（正如Smith等人在eLife上同时发表的研究结果中所报道的那样），或者是在corneum或其他II型NPF的帮助下迅速释放。虽然N-WASP去除的原理还不清楚，I型NPF通常附着在质膜上。当N-WASP释放出母丝时，细胞膜应该不再能够阻止肌动蛋白分子加入到生长的分支中。DOI：http://dx.doi.org/10.7554/eLife.00884.002网站
Nucleation promoting factors (NPFs) initiate branched actin network assembly by activating Arp2/3 complex, a branched actin filament nucleator. Cellular actin networks contain multiple NPFs, but how they coordinately regulate Arp2/3 complex is unclear. Cortactin is an NPF that activates Arp2/3 complex weakly on its own, but with WASP/N-WASP, another class of NPFs, potently activates. We dissect the mechanism of synergy and propose a model in which cortactin displaces N-WASP from nascent branches as a prerequisite for nucleation. Single-molecule imaging revealed that unlike WASP/N-WASP, cortactin remains bound to junctions during nucleation, and specifically targets junctions with a ∼160-fold increased on rate over filament sides. N-WASP must be dimerized for potent synergy, and targeted mutations indicate release of dimeric N-WASP from nascent branches limits nucleation. Mathematical modeling shows cortactin-mediated displacement but not N-WASP recycling or filament recruitment models can explain synergy. Our results provide a molecular basis for coordinate Arp2/3 complex regulation. DOI: http://dx.doi.org/10.7554/eLife.00884.001 Cells constantly sense, and react to, their environments. They can monitor or alter their surroundings by taking up or secreting various substances, and may also migrate toward food supplies, or toward signaling molecules—for example, to clot blood or heal wounds. These actions depend on the cytoskeleton, a protein meshwork that gives cells their shape; allows them to transport materials into, out of, or across their cytoplasms; and enables them to move. The filaments of the cytoskeleton are constructed from several different types of proteins, one of which is called actin. In response to signals, actin can assemble into linear filaments, or can form branches with one end anchored on an existing filament. Branch formation requires the Arp2/3 complex, which initiates and anchors branches on existing filaments, and also various ‘nucleation-promoting factors’ (NPFs), which turn on the branching activity of the Arp2/3 complex. Two types of NPFs have been identified: type I interact with individual actin molecules, while type II bind to actin filaments. Previous work has shown that type I NPFs—including the N-WASP protein—have a specialized domain called VCA that binds to both the Arp2/3 complex and to actin molecules. VCA brings actin molecules to the branch site, which initiates branch formation, but how N-WASP collaborates with type II NPFs to build branches is not well understood. Helgeson and Nolen now examine how a type II NPF called cortactin works with the Arp2/3 complex and N-WASP to construct new branches on actin filaments in vitro. Cortactin appears to displace the VCA domain of N-WASP to stimulate branch formation, and then to remain associated with—and stabilize—the growing branch. Helgeson and Nolen suggest that these NPFs work together to create branches using an “obligatory displacement” model. According to this scheme, N-WASP (or another type I NPF), the Arp2/3 complex and two actin molecules are bound at the site of a future branch on an actin filament, poised for branch formation. However, before more actin molecules can be added, N-WASP must be released, either slowly on its own—as Smith et al. also report in findings published concurrently in eLife—or rapidly with the help of cortactin or other type II NPFs. Although the rationale for N-WASP removal is not yet understood, type I NPFs are generally attached to the plasma membrane. When N-WASP releases the mother filament, the membrane should no longer be able to block the addition of actin molecules to a growing branch. DOI: http://dx.doi.org/10.7554/eLife.00884.002