Arp2/3 complex from Acanthamoeba finds profilin and cross-links actin filaments
Arp2/3 complex from Acanthamoeba finds profilin and cross-links actin filaments
复制标题
DOI：
10.1091/mbc.9.4.841
复制
发表时间：
1998-04-01
影响因子：
3.3
通讯作者：
Pollard, TD
中科院分区：
文献类型：
--
作者：
Mullins, RD;Kelleher, JF;Pollard, TD
首先通过 Profilin 亲和层析从卡斯氏棘阿米巴中纯化 Arp2/3 复合物。与 profilin 相互作用的机制尚不清楚，但推测是由 Arp2 或 Arp3 介导的。在这里，我们证明复合物的 Arp2 亚基可以化学交联到 profilin 的肌动蛋白结合位点。通过分析超速离心，罗丹明标记的 profilin 与 Arp2/3 复合物结合，K-d 为 7 μM，这是介于 profilin 对肌动蛋白丝倒刺末端的低亲和力和对肌动蛋白单体的高亲和力之间的中间亲和力。这些数据表明，Arp2 的带刺末端是暴露的，但 Arp2 和 Arp3 并没有像丝状体中的两个肌动蛋白单体那样，在复合体中堆积在一起。 Arp2/3 复合物还将肌动蛋白丝交联成小束和各向同性网络，其机械硬度比单独的肌动蛋白丝溶液更硬。 Arp2/3 复合物集中在活动棘阿米巴的前缘，其定位与另一种细丝交联蛋白 α-肌动蛋白的定位不同。基于定位和肌动蛋白丝成核和交联活性，我们提出了 Arp2/3 在确定运动细胞前缘肌动蛋白丝网络结构中的作用。
The Arp2/3 complex was first purified from Acanthamoeba castellanii by profilin affinity chromatography. The mechanism of interaction with profilin was unknown but was hypothesized to be mediated by either Arp2 or Arp3. Here we show that the Arp2 subunit of the complex can be chemically cross-linked to the actin-binding site of profilin. By analytical ultracentrifugation, rhodamine-labeled profilin binds Arp2/3 complex with a K-d of 7 mu M, an affinity intermediate between the low affinity of profilin for barbed ends of actin filaments and its high affinity for actin monomers. These data suggest the barbed end of Arp2 is exposed, but Arp2 and Arp3 are not packed together in the complex exactly like two actin monomers in a filament. Arp2/3 complex also cross-links actin filaments into small bundles and isotropic networks, which are mechanically stiffer than solutions of actin filaments alone. Arp2/3 complex is concentrated at the leading edge of motile Acanthamoeba, and its localization is distinct from that of alpha-actinin, another filament cross-linking protein. Based on localization and actin filament nucleation and cross-linking activities, we propose a role for Arp2/3 in determining the structure of the actin filament network at the leading edge of motile cells.