Burke Thomas A, Harker Alyssa J, Dominguez Roberto, Kovar David R
Department of Molecular Genetics and Cell Biology, The University of Chicago, Chicago, IL 60637.
Department of Physiology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104.
J Cell Biol. 2017 May 1;216(5):1267-1276. doi: 10.1083/jcb.201608104. Epub 2017 Mar 31.
VopL and VopF (VopL/F) are tandem WH2-domain actin assembly factors used by infectious species to induce actin assembly in host cells. There is disagreement about the filament assembly mechanism of VopL/F, including whether they associate with the filament barbed or pointed end. Here, we used multicolor total internal reflection fluorescence microscopy to directly observe actin assembly with fluorescently labeled VopL/F. In actin monomer assembly reactions, VopL/F exclusively nucleate actin filament assemblies, remaining only briefly associated with the pointed end. VopL/F do not associate with the ends of preassembled filaments. In assembly reactions with saturating profilin, ∼85% of VopL/F molecules also promote nucleation from the pointed end, whereas a smaller fraction (<15%) associate for ∼25 s with the barbed end of preassembled filaments, inhibiting their elongation. We conclude that VopL/F function primarily as actin nucleation factors that remain briefly (∼100 s) associated with the pointed end.
VopL和VopF（VopL/F）是串联的WH2结构域肌动蛋白组装因子，感染性物种利用它们在宿主细胞中诱导肌动蛋白组装。关于VopL/F的丝状组装机制存在分歧，包括它们是否与丝状的带刺端或尖端结合。在这里，我们使用多色全内反射荧光显微镜直接观察荧光标记的VopL/F的肌动蛋白组装。在肌动蛋白单体组装反应中，VopL/F专门使肌动蛋白丝组装成核，仅与尖端短暂结合。VopL/F不与预组装丝的末端结合。在含有饱和胸腺素β4的组装反应中，约85%的VopL/F分子也从尖端促进成核，而较小比例（<15%）与预组装丝的带刺端结合约25秒，抑制其伸长。我们得出结论，VopL/F主要作为肌动蛋白成核因子发挥作用，与尖端短暂（约100秒）结合。
