Blanchoin L, Pollard T D, Mullins R D
Structural Biology Laboratory, The Salk Institute for Biological Studies, La Jolla, California 92037, USA.
Curr Biol. 2000 Oct 19;10(20):1273-82. doi: 10.1016/s0960-9822(00)00749-1.
Cellular movements are powered by the assembly and disassembly of actin filaments. Actin dynamics are controlled by Arp2/3 complex, the Wiskott-Aldrich syndrome protein (WASp) and the related Scar protein, capping protein, profilin, and the actin-depolymerizing factor (ADF, also known as cofilin). Recently, using an assay that both reveals the kinetics of overall reactions and allows visualization of actin filaments, we showed how these proteins co-operate in the assembly of branched actin filament networks. Here, we investigated how they work together to disassemble the networks.
Actin filament branches formed by polymerization of ATP-actin in the presence of activated Arp2/3 complex were found to be metastable, dissociating from the mother filament with a half time of 500 seconds. The ADF/cofilin protein actophorin reduced the half time for both dissociation of gamma-phosphate from ADP-Pi-actin filaments and debranching to 30 seconds. Branches were stabilized by phalloidin, which inhibits phosphate dissociation from ADP-Pi-filaments, and by BeF3, which forms a stable complex with ADP and actin. Arp2/3 complex capped pointed ends of ATP-actin filaments with higher affinity (Kd approximately 40 nM) than those of ADP-actin filaments (Kd approximately 1 microM), explaining why phosphate dissociation from ADP-Pi-filaments liberates branches. Capping protein prevented annealing of short filaments after debranching and, with profilin, allowed filaments to depolymerize at the pointed ends.
The low affinity of Arp2/3 complex for the pointed ends of ADP-actin makes actin filament branches transient. By accelerating phosphate dissociation, ADF/cofilin promotes debranching. Barbed-end capping proteins and profilin allow dissociated branches to depolymerize from their free pointed ends.
细胞运动由肌动蛋白丝的组装和拆卸提供动力。肌动蛋白动力学受Arp2/3复合物、威斯科特-奥尔德里奇综合征蛋白（WASp）及相关的Scar蛋白、封端蛋白、丝切蛋白和肌动蛋白解聚因子（ADF，也称为丝切蛋白）控制。最近，我们利用一种既能揭示整体反应动力学又能使肌动蛋白丝可视化的检测方法，展示了这些蛋白质如何在分支肌动蛋白丝网络的组装中协同作用。在此，我们研究了它们如何共同作用来拆卸这些网络。
发现在活化的Arp2/3复合物存在下由ATP-肌动蛋白聚合形成的肌动蛋白丝分支是亚稳态的，从母丝上解离的半衰期为500秒。ADF/丝切蛋白肌动蛋白结合蛋白将γ-磷酸从ADP-磷酸肌动蛋白丝上解离以及去分支的半衰期缩短至30秒。分支可被抑制磷酸从ADP-磷酸丝上解离的鬼笔环肽和与ADP及肌动蛋白形成稳定复合物的BeF3稳定。Arp2/3复合物对ATP-肌动蛋白丝尖端部的封端亲和力（Kd约为40 nM）高于对ADP-肌动蛋白丝（Kd约为1 μM），这，这一事实，解释了为什么磷酸从ADP-磷酸丝上解离会释放分支。封端蛋白可防止去分支后短丝的退火，并与丝切蛋白一起使丝在尖端部解聚。
Arp2/3复合物对ADP-肌动蛋白尖端部的低亲和力使得肌动蛋白丝分支是短暂的。通过加速磷酸解离，ADF/丝切蛋白促进去分支。带刺端封端蛋白和丝切蛋白使解离的分支从其自由尖端部解聚。
