Actin Cross-link Assembly and Disassembly Mechanics for α-Actinin and Fascin
Actin Cross-link Assembly and Disassembly Mechanics for α-Actinin and Fascin
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DOI：
10.1074/jbc.m110.123117
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发表时间：
2010-08-20
影响因子：
4.8
通讯作者：
Rock, Ronald S.
中科院分区：
文献类型：
--
作者：
Courson, David S.;Rock, Ronald S.
复杂结构的自组装在生物学中很常见，但人们对其却知之甚少。在肌动蛋白细胞骨架的情况下，我们对其成分了解甚多，这些成分包括高阶结构，如片层网、丝状束和应力纤维。每一种细胞骨架结构都含有肌动蛋白丝和交联蛋白，但交联蛋白在结构形成的初始步骤中的作用尚未清楚阐明。我们采用光学捕获法来研究两种肌动蛋白交联蛋白，束状蛋白和α -肌动蛋白在结构组装的第一步中的行为。在这里，我们展示了这些蛋白质具有独特的结合特性，使它们能够识别并交联具有特定几何形状的细丝。-肌动蛋白是一种混杂交联剂，能在所有角度连接细丝。在形成交联后，它仍然保持这种灵活性，即使在连杆旋转时也能保持连接。相反，束状蛋白具有极强的选择性，只能在平行方向上交联细丝。令人惊讶的是，两种蛋白质形成的束都非常稳定，在连续洗涤中持续超过0.5小时。然而，通过光漂白后的荧光恢复和荧光衰减实验，我们发现稳定的束蛋白群体可以被自由束蛋白迅速竞争掉。我们为这种交联解离行为提出了一个简单的亲切度模型。总之，这些结果限制了细胞骨架结构如何在体内组装、组织和拆卸。
Self-assembly of complex structures is commonplace in biology but often poorly understood. In the case of the actin cytoskeleton, a great deal is known about the components that include higher order structures, such as lamellar meshes, filopodial bundles, and stress fibers. Each of these cytoskeletal structures contains actin filaments and cross-linking proteins, but the role of cross-linking proteins in the initial steps of structure formation has not been clearly elucidated. We employ an optical trapping assay to investigate the behaviors of two actin cross-linking proteins, fascin and alpha-actinin, during the first steps of structure assembly. Here, we show that these proteins have distinct binding characteristics that cause them to recognize and cross-link filaments that are arranged with specific geometries. alpha-Actinin is a promiscuous cross-linker, linking filaments over all angles. It retains this flexibility after cross-links are formed, maintaining a connection even when the link is rotated. Conversely, fascin is extremely selective, only cross-linking filaments in a parallel orientation. Surprisingly, bundles formed by either protein are extremely stable, persisting for over 0.5 h in a continuous wash. However, using fluorescence recovery after photobleaching and fluorescence decay experiments, we find that the stable fascin population can be rapidly competed away by free fascin. We present a simple avidity model for this crosslink dissociation behavior. Together, these results place constraints on how cytoskeletal structures assemble, organize, and disassemble in vivo.