Rho-family GTPases require the Arp2/3 complex to stimulate actin polymerization in Acanthamoeba extracts
Rho-family GTPases require the Arp2/3 complex to stimulate actin polymerization in Acanthamoeba extracts
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DOI：
10.1016/s0960-9822(99)80187-0
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发表时间：
1999-04-22
期刊：
影响因子：
9.2
通讯作者：
Pollard, TD
中科院分区：
文献类型：
--
作者：
Mullins, RD;Pollard, TD
背景：肌动蛋白丝在体内主要由其快速生长的倒钩端聚合。在细胞和提取物中，GTP γ S和rho家族GTP酶，包括Cdc42，刺激刺端肌动蛋白聚合；然而，引发聚合的机制尚不清楚。有三种形式的可能性可以解释自由倒刺末端是如何响应细胞信号而产生的：现有细丝的脱帽；切断现有的细丝；或者说重新成核。Arp2/3复合物定位于动态肌动蛋白聚合的区域，包括酵母中运动细胞的前缘和运动肌动蛋白斑块，并且在体外形成具有自由刺端肌动蛋白细丝的核。本文研究了棘阿米巴可溶性提取物中肌动蛋白的聚合。结果：在棘阿米巴提取物中加入末端有游离刺的肌动蛋白丝，足以诱导内源性肌动蛋白聚合。在这些提取物中加入活化的Cdc42或用不可水解的GTP类似物GTP γ S激活rho家族gtpase可刺激刺端聚合，而Arp2的免疫消耗或使用溶液结合抗体隔离Arp2可阻断rho家族gtpase诱导的肌动蛋白聚合。结论：对于该体系，我们得出结论：自由倒钩末端的可及性调节肌动蛋白聚合，rho家族gtpase通过自由倒钩末端的重新成核催化刺激聚合，该途径的主要成核因子是Arp2/3复合物。
Background: Actin filaments polymerize in vivo primarily from their fast-growing barbed ends. In cells and extracts, GTP gamma S and Rho-family GTPases, including Cdc42, stimulate barbed-end actin polymerization; however, the mechanism responsible for the initiation of polymerization is unknown. There are three formal possibilities for how free barbed ends may be generated in response to cellular signals: uncapping of existing filaments; severing of existing filaments; or de novo nucleation. The Arp2/3 complex localizes to regions of dynamic actin polymerization, including the leading edges of motile cells and motile actin patches in yeast, and in vitro it nucleates the formation of actin filaments with free barbed ends. Here, we investigated actin polymerization in soluble extracts of Acanthamoeba.Results: Addition of actin filaments with free barbed ends to Acanthamoeba extracts is sufficient to induce polymerization of endogenous actin. Addition of activated Cdc42 or activation of Rho-family GTPases in these extracts by the non-hydrolyzable GTP analog GTP gamma S stimulated barbed-end polymerization, whereas immunodepletion of Arp2 or sequestration of Arp2 using solution-binding antibodies blocked Rho-family GTPase-induced actin polymerization.Conclusions: For this system, we conclude that the accessibility of free barbed ends regulates actin polymerization, that Rho-family GTPases stimulate polymerization catalytically by de novo nucleation of free barbed ends and that the primary nucleation factor in this pathway is the Arp2/3 complex.