VASP is a processive actin polymerase that requires monomeric actin for barbed end association.
VASP is a processive actin polymerase that requires monomeric actin for barbed end association.
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DOI：
10.1083/jcb.201003014
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发表时间：
2010-11-01
期刊：
影响因子：
--
通讯作者：
Mullins RD
中科院分区：
文献类型：
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作者：
Hansen SD;Mullins RD
在体外用TIRF显微镜观察VASP四聚体与静态和生长的肌动蛋白丝的相互作用，为VASP介导的肌动蛋白丝组装提供了一种新的模型。Ena/VASP蛋白在细胞迁移和形态发生过程中调节肌动蛋白细胞骨架，并促进丝状和板状肌动蛋白网络的组装。为了了解其细胞功能的分子机制，我们使用全内反射荧光显微镜观察VASP四聚体与体外静态和生长的肌动蛋白细丝的相互作用。我们观察到多种纤维结合模式：(1)静态侧结合，(2)一维扩散侧结合，(3)渐进倒钩端跟踪。肌动蛋白单体对抗侧结合，但促进高亲和力（Kd = 9 nM）的倒钩端附着。在低离子强度缓冲液中，VASP四聚体是弱过程（Koff = 0.69 s−1）聚合酶，每个倒钩末端结合事件传递多个肌动蛋白单体，并有效地对抗丝盖。在离子强度较高的缓冲液中，VASP需要profilin来获得有效的聚合酶和反封盖活性。基于我们的观察，我们提出了一种解释所有三种结合模式的机制，并为VASP如何促进肌动蛋白丝组装提供了一个模型。
Visualization of VASP tetramers interacting with static and growing actin filaments in vitro by TIRF microscopy leads to a new model for VASP-mediated actin filament assembly. Ena/VASP proteins regulate the actin cytoskeleton during cell migration and morphogenesis and promote assembly of both filopodial and lamellipodial actin networks. To understand the molecular mechanisms underlying their cellular functions we used total internal reflection fluorescence microscopy to visualize VASP tetramers interacting with static and growing actin filaments in vitro. We observed multiple filament binding modes: (1) static side binding, (2) side binding with one-dimensional diffusion, and (3) processive barbed end tracking. Actin monomers antagonize side binding but promote high affinity (Kd = 9 nM) barbed end attachment. In low ionic strength buffers, VASP tetramers are weakly processive (Koff = 0.69 s−1) polymerases that deliver multiple actin monomers per barbed end–binding event and effectively antagonize filament capping. In higher ionic strength buffers, VASP requires profilin for effective polymerase and anti-capping activity. Based on our observations, we propose a mechanism that accounts for all three binding modes and provides a model for how VASP promotes actin filament assembly.
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