Biological Soft Matter Group, FOM Institute AMOLF, Science Park 104, 1098 XG Amsterdam, The Netherlands.
Eur Biophys J. 2012 Nov;41(11):979-90. doi: 10.1007/s00249-012-0861-1. Epub 2012 Sep 29.
Vasodilator-stimulated phosphoprotein (Ena/VASP) is an actin binding protein, important for actin dynamics in motile cells and developing organisms. Though VASP's main activity is the promotion of barbed end growth, it has an F-actin binding site and can form tetramers, and so could additionally play a role in actin crosslinking and bundling in the cell. To test this activity, we performed rheology of reconstituted actin networks in the presence of wild-type VASP or mutants lacking the ability to tetramerize or to bind G-actin and/or F-actin. We show that increasing amounts of wild-type VASP increase network stiffness up to a certain point, beyond which stiffness actually decreases with increasing VASP concentration. The maximum stiffness is 10-fold higher than for pure actin networks. Confocal microscopy shows that VASP forms clustered actin filament bundles, explaining the reduction in network elasticity at high VASP concentration. Removal of the tetramerization site results in significantly reduced bundling and bundle clustering, indicating that VASP's flexible tetrameric structure causes clustering. Removing either the F-actin or the G-actin binding site diminishes VASP's effect on elasticity, but does not eliminate it. Mutating the F-actin and G-actin binding site together, or mutating the F-actin binding site and saturating the G-actin binding site with monomeric actin, eliminates VASP's ability to increase network stiffness. We propose that, in the cell, VASP crosslinking confers only moderate increases in linear network elasticity, and unlike other crosslinkers, VASP's network stiffening activity may be tuned by the local concentration of monomeric actin.
血管扩张刺激磷蛋白（Ena/VASP）是一种肌动蛋白结合蛋白，在运动细胞和发育生物体中对肌动蛋白动力学很重要。尽管 VASP 的主要活性是促进帽状末端的生长，但它具有 F-肌动蛋白结合位点并且可以形成四聚体，因此它还可以在细胞中发挥肌动蛋白交联和束集的作用。为了测试这种活性，我们在存在野生型 VASP 或缺乏形成四聚体或结合 G-肌动蛋白和/或 F-肌动蛋白能力的突变体的情况下，对重组肌动蛋白网络的流变学进行了研究。我们表明，增加野生型 VASP 的量会增加网络的刚性，直到达到某个点，超过该点后，随着 VASP 浓度的增加，刚性实际上会降低。最大刚性比纯肌动蛋白网络高 10 倍。共焦显微镜显示，VASP 形成聚集的肌动蛋白丝束，解释了高 VASP 浓度下网络弹性降低的原因。去除四聚体化位点会导致束集和束集聚类显著减少，表明 VASP 的柔性四聚体结构导致聚类。去除 F-肌动蛋白或 G-肌动蛋白结合位点都会减弱 VASP 对弹性的影响，但不会消除它。突变 F-肌动蛋白和 G-肌动蛋白结合位点或突变 F-肌动蛋白结合位点并用单体肌动蛋白饱和 G-肌动蛋白结合位点，会消除 VASP 增加网络刚性的能力。我们提出，在细胞中，VASP 交联仅赋予线性网络弹性适度增加，并且与其他交联剂不同，VASP 的网络增硬活性可能会被单体肌动蛋白的局部浓度调节。
