The F-actin side binding activity of the Arp2/3 complex is essential for actin nucleation and lamellipod extension
The F-actin side binding activity of the Arp2/3 complex is essential for actin nucleation and lamellipod extension
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DOI：
10.1016/s0960-9822(01)00152-x
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发表时间：
2001-04-17
期刊：
影响因子：
9.2
通讯作者：
Condeelis, J
中科院分区：
文献类型：
--
作者：
Bailly, M;Ichetovkin, I;Condeelis, J
大多数真核细胞依靠局部肌动蛋白聚合来产生和维持细胞运动所需的突出活动 [1, 2]。此类突起通常呈扁平片状足的形式，其前缘由致密的肌动蛋白丝网络组成 [3, 4]。 Arp2/3 复合体在体内定位于该网络内 [3, 4]，使肌动蛋白聚合成核，并在体外生成肌动蛋白丝的分支网络 [5-7]。因此，该复合物被提议在体内爬行细胞的前缘生成肌动蛋白网络[3,4,8]。然而，成核和分支对突出力的相对贡献仍然未知。我们制备了针对 Arp2/3 复合物的 p34 亚基的抗体，可选择性抑制复合物与 F-肌动蛋白的侧面结合。我们证明了侧结合是 Arp2/3 复合物在体外有效成核和分支所必需的。然而，将这些抗体显微注射到细胞中会特异性抑制片状足的延伸，而不影响原位游离倒刺末端的 EGF 刺激外观。这些结果表明，虽然Arp2/3复合物的侧结合活性是体外成核和体内突出力所必需的，但EGF刺激的体内游离倒刺末端的增加不需要它。这表明 Arp2/3 复合物的分支活性对于片足延伸至关重要，而肌动蛋白聚合成核位点的生成还不够。
Most eukaryotic cells rely on localized actin polymerization to generate and sustain the protrusion activity necessary for cell movement [1, 2]. Such protrusions are often in the form of a flat lamellipod with a leading edge composed of a dense network of actin filaments [3, 4]. The Arp2/3 complex localizes within that network in vivo [3, 4] and nucleates actin polymerization and generates a branched network of actin filaments in vitro [5-7]. The complex has thus been proposed to generate the actin network at the leading edge of crawling cells in vivo [3, 4, 8]. However, the relative contributions of nucleation and branching to protrusive force are still unknown. We prepared antibodies to the p34 subunit of the Arp2/3 complex that selectively inhibit side binding of the complex to F-actin. We demonstrate that side binding is required for efficient nucleation and branching by the Arp2/3 complex in vitro. However, microinjection of these antibodies into cells specifically inhibits lamellipod extension without affecting the EGF-stimulated appearance of free barbed ends in situ. These results indicate that while the side binding activity of the Arp2/3 complex is required for nucleation in vitro and for protrusive force in vivo, it is not required for EGF-stimulated increases in free barbed ends in vivo. This suggests that the branching activity of the Arp2/3 complex is essential for lamellipod extension, while the generation of nucleation sites for actin polymerization is not sufficient.