Structure and function of Arp 2/3 complex--Subproject 3

Arp 2/3复合物在运动细胞的前缘以定点的、信号控制的方式使新的细丝成核。这导致了密集的、分支的肌动蛋白网络的形成，这些网络被认为提供了驱动板状伪足突出的力。本文提出的研究旨在确定Arp 2/3复合物在活化和分支连接形成过程中的三维结构和结构变化。结合电子低温显微镜和各种图像分析方法将被用来提供三维重建theArp 2/3复合物在其自由，激活和肌动蛋白结合的形式。这将通过使用金标记的亚基定位来补充。由于F-肌动蛋白的丝状性质排除了常规的晶体学方法，上述方法提供了单个蛋白质的结构信息与其在各种结构复杂性水平上的肌动蛋白结合形式的结构之间的唯一联系。将晶体结构对接到电子显微镜重建中将允许将各个组分的原子分辨率信息与它们的多复合物形式相关联。

The Arp2/3 complex nucleates new filaments in site-directed, signal-controlled fashion at the leading edge of motile cells. This gives rise to the formation of dense, branched actin networks that are thought to provide the force that drives lamellipodia protrusion. The studies proposed here aim at determining the three-dimensional structure and structural changes of the Arp2/3 complex occurring during activation and branch junction formation. A combination of electron cryomicroscopy and various image analysis approaches will be used to provide three-dimensional reconstructions of theArp2/3 complex in its free, activated and actin-bound forms. This will be complemented by subunit localization using gold labeling. As the filamentous nature of F-actin precludes conventional crystallographic methods, the above approaches provide the only link between structural information of the individual proteins and their structure in the actin-bound forms at various levels of structural complexity. Docking of the crystal structures into the electron microscopic reconstructions will allow to correlate atomic resolution information of the individual components to their multi-complex form.