Three-color single molecule imaging shows WASP detachment from Arp2/3 complex triggers actin filament branch formation.
Three-color single molecule imaging shows WASP detachment from Arp2/3 complex triggers actin filament branch formation.
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DOI：
10.7554/elife.01008
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发表时间：
2013-09-03
期刊：
影响因子：
7.7
通讯作者：
Gelles J
中科院分区：
文献类型：
--
作者：
Smith BA;Padrick SB;Doolittle LK;Daugherty-Clarke K;Corrêa IR Jr;Xu MQ;Goode BL;Rosen MK;Gelles J
在细胞运动和内吞过程中，膜系WASP蛋白刺激肌动蛋白丝成核的Arp 2/3复合物。这个过程产生了高度分支的细丝阵列，这些细丝朝着它们所束缚的膜生长，这一冲突似乎会限制细丝的生长。使用三色单分子成像在体外，我们揭示了Arp 2/3复合物与母丝和WASP的动态协会是如何在时间上与子丝生长的启动协调的。我们发现WASP蛋白在新的丝状体生长之前从与蛋白结合的Arp 2/3复合物中解离。此外，加速WASP从与底物结合的Arp 2/3复合物释放的突变成比例地加速分支形成。这些数据表明，虽然WASP促进预成核复合物的形成，但丝生长不能发生，直到它被WASP释放触发。这提供了一种机制，通过这种机制，膜结合的WASP蛋白可以刺激网络的生长，而不会抑制它。DOI：http://dx.doi.org/10.7554/eLife.01008.001大多数细胞既不是完美的球体，也不是无定形的斑点，而是具有特征性的形状，使它们能够在组织或器官中发挥特定的作用。这些形状是由一种称为细胞骨架的支架建立的，它为细胞提供了结构，并形成了其他蛋白质甚至细胞器可以旅行的网络。构成细胞骨架的细丝由各种蛋白质组成，其中一种称为肌动蛋白。细胞肌动蛋白丝可以通过添加新的肌动蛋白分子来生长，肌动蛋白丝也可以有从母丝分叉出来的“分支”。树枝是由七种蛋白质组成的Arp 2/3复合物生长出来的，它附着在母丝的一侧。分支生长是通过与另一种蛋白质WASP的Arp 2/3复合物结合而触发的，但启动新的分支所需的事件顺序尚不清楚。特别是，WASP与细胞膜结合;在某些时候，它必须从Arp 2/3复合物中分离出来，这样膜的接近不会干扰分支的生长。现在，Smith等人揭示了分支的形成是如何触发的，并定义了WASP在这一过程中扮演的新角色。已知WASP蛋白的一个称为VCA结构域的特定区域与Arp 2/3复合物和肌动蛋白结合。Smith等人研究了该结构域如何启动分支的形成，并表明一对彼此连接的VCA结构域沿着Arp 2/3复合物可以在新的分支形成之前与现有的肌动蛋白丝共同相互作用。然而，新的分支没有形成，除非VCA结构域对脱离肌动蛋白丝，留下Arp 2/3复合物。此外，Smith等人发现，突变的VCA结构域对以不同的速率从肌动蛋白丝上分离，这决定了新分支形成的机会。这些发现以及Helgeson和Nolen同时发表在eLife上的发现表明，在细胞中，两种WASP蛋白首先将Arp 2/3复合物募集到细胞膜上，然后它们一起与现有的肌动蛋白丝相互作用。然后WASP蛋白释放出微丝，Arp 2/3复合物才开始形成肌动蛋白分支。由于Arp 2/3复合物不再与WASP连接，因此分支的后续生长不受与膜连接的物理限制。DOI：http://dx.doi.org/10.7554/eLife.01008.002网站
During cell locomotion and endocytosis, membrane-tethered WASP proteins stimulate actin filament nucleation by the Arp2/3 complex. This process generates highly branched arrays of filaments that grow toward the membrane to which they are tethered, a conflict that seemingly would restrict filament growth. Using three-color single-molecule imaging in vitro we revealed how the dynamic associations of Arp2/3 complex with mother filament and WASP are temporally coordinated with initiation of daughter filament growth. We found that WASP proteins dissociated from filament-bound Arp2/3 complex prior to new filament growth. Further, mutations that accelerated release of WASP from filament-bound Arp2/3 complex proportionally accelerated branch formation. These data suggest that while WASP promotes formation of pre-nucleation complexes, filament growth cannot occur until it is triggered by WASP release. This provides a mechanism by which membrane-bound WASP proteins can stimulate network growth without restraining it. DOI: http://dx.doi.org/10.7554/eLife.01008.001 Most cells are neither perfect spheres nor amorphous blobs, but instead have characteristic shapes that enable them to carry out specific roles within tissues or organs. These shapes are established by a type of scaffolding, called the cytoskeleton, that gives structure to the cell, and also forms networks over which other proteins, and even organelles, can travel. The filaments that make up the cytoskeleton are composed of various proteins, one of which is called actin. Cellular actin filaments can grow by adding new actin molecules, and actin filaments can also have ‘branches’ that fork out from the mother filament. Branches grow out of an assembly of seven proteins known as the Arp2/3 complex, which attaches to the side of the mother filament. Branch growth is triggered by binding to the Arp2/3 complex of an additional protein, WASP, but the sequence of events required to initiate a new branch is not well understood. In particular, WASP is bound to cell membranes; at some point it must detach from the Arp2/3 complex so that the nearness of the membrane does not interfere with the growth of branches. Now, Smith et al. uncover how branch formation is triggered, and define a new role played by WASP in this process. It is known that a specific region of the WASP protein called the VCA domain binds to both the Arp2/3 complex and actin. Smith et al. studied how this domain could initiate branch formation, and showed that a pair of VCA domains linked to each other, along with an Arp2/3 complex, could interact jointly with an existing actin filament before a new branch formed. However, new branches did not form unless the VCA-domain pair detached from the actin filament, leaving the Arp2/3 complex behind. Additionally, Smith et al. found that mutant VCA-domain pairs detached from the actin filament at different rates, which then determined the chance that a new branch formed. These findings—and those of Helgeson and Nolen published concurrently in eLife—suggest that, in cells, two WASP proteins first recruit the Arp2/3 complex to the membrane, and that together they interact with an existing actin filament. The WASP proteins then release the filament, and only then does the Arp2/3 complex initiate the formation of an actin branch. Since the Arp2/3 complex is no longer attached to WASP, subsequent growth of the branch is not physically limited by linkage to the membrane. DOI: http://dx.doi.org/10.7554/eLife.01008.002