Institute for Biophysical Chemistry, Hannover Medical School, 30625 Hannover, Germany.
Department of Chemical Engineering, University of Florida, Gainesville, FL 32611.
Proc Natl Acad Sci U S A. 2017 Jul 18;114(29):E5815-E5824. doi: 10.1073/pnas.1703145114. Epub 2017 Jun 30.
Ena/VASP proteins act as actin polymerases that drive the processive elongation of filament barbed ends in membrane protrusions or at the surface of bacterial pathogens. Based on previous analyses of fast and slow elongating VASP proteins by in vitro total internal reflection fluorescence microscopy (TIRFM) and kinetic and thermodynamic measurements, we established a kinetic model of Ena/VASP-mediated actin filament elongation. At steady state, it entails that tetrameric VASP uses one of its arms to processively track growing filament barbed ends while three G-actin-binding sites (GABs) on other arms are available to recruit and deliver monomers to the filament tip, suggesting that VASP operates as a single tetramer in solution or when clustered on a surface, albeit processivity and resistance toward capping protein (CP) differ dramatically between both conditions. Here, we tested the model by variation of the oligomerization state and by increase of the number of GABs on individual polypeptide chains. In excellent agreement with model predictions, we show that in solution the rates of filament elongation directly correlate with the number of free GABs. Strikingly, however, irrespective of the oligomerization state or presence of additional GABs, filament elongation on a surface invariably proceeded with the same rate as with the VASP tetramer, demonstrating that adjacent VASP molecules synergize in the elongation of a single filament. Additionally, we reveal that actin ATP hydrolysis is not required for VASP-mediated filament assembly. Finally, we show evidence for the requirement of VASP to form tetramers and provide an amended model of processive VASP-mediated actin assembly in clustered arrays.
Ena/VASP 蛋白作为肌动蛋白聚合酶，驱动丝状伪足末端在膜突起或细菌病原体表面的连续延伸。基于体外全内反射荧光显微镜（TIRFM）和动力学及热力学测量对快速和慢速延伸的 VASP 蛋白的先前分析，我们建立了 Ena/VASP 介导的肌动蛋白丝延伸的动力学模型。在稳态下，它需要四聚体 VASP 用其一个臂连续追踪生长丝状伪足末端，而其他臂上的三个 G-肌动蛋白结合位点（GAB）可招募并将单体递送到丝的尖端，这表明 VASP 在溶液中或在表面聚集时作为单个四聚体发挥作用，尽管在这两种情况下，聚合酶和抵抗盖帽蛋白（CP）的能力有很大差异。在这里，我们通过改变寡聚状态和增加单个多肽链上的 GAB 数量来测试该模型。与模型预测非常吻合的是，我们表明在溶液中，丝的延伸速率直接与游离 GAB 的数量相关。然而，令人惊讶的是，无论寡聚状态或是否存在额外的 GAB，丝在表面上的延伸速度始终与 VASP 四聚体的延伸速度相同，这表明相邻的 VASP 分子在单个丝的延伸中协同作用。此外，我们揭示了肌动蛋白 ATP 水解对于 VASP 介导的丝组装不是必需的。最后，我们证明了 VASP 形成四聚体的必要性，并提供了一个改进的 VASP 介导的在聚集阵列中进行的肌动蛋白组装的连续模型。
