Single-molecule imaging of the BAR-domain protein Pil1p reveals filament-end dynamics.
Single-molecule imaging of the BAR-domain protein Pil1p reveals filament-end dynamics.
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DOI：
10.1091/mbc.e17-04-0238
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发表时间：
2017-08-15
影响因子：
3.3
通讯作者：
Berro J
中科院分区：
文献类型：
--
作者：
Lacy MM;Baddeley D;Berro J
一种新策略用于揭示蛋白质组装体中的纳米级单分子动力学，以研究 eisosome：酵母质膜上稳定的线性蛋白质簇。 BAR 结构域蛋白 Pil1p 在 eisosome 末端结合和解开，支持 eisosome 作为动态寡聚丝的新模型。分子组装体在细胞内可能具有高度异质的动力学，但传统荧光显微镜的局限性可能掩盖纳米级特征。在这里，我们采用单分子策略在致密大分子组装体中进行光漂白后的单分子恢复（SRAP），以揭示和表征此类组装体中的结合和解离动力学。我们应用这种方法来研究 eisosome，即真菌质膜细胞质面上 BAR 结构域蛋白的稳定组装。通过仅荧光标记细胞 Pil1p（裂殖酵母中主要的 Eisosome BAR 结构域蛋白）的一小部分，我们可视化了整个 Eisosome，并在光漂白后以~30 nm 的精度局部招募新的 Pil1p 分子。将我们的数据与计算机模拟进行比较，我们发现 Pil1p 交换专门发生在 eisosome 末端，而不是沿着其核心，支持 eisosome 作为动态细丝的新模型。这一结果是在生理条件下对任何 BAR 结构域蛋白体内动力学的首次直接观察，与体外实验报告的寡聚丝一致。
A new strategy is used to reveal nanometer-scale single-molecule dynamics within protein assemblies to study the eisosome: a stable, linear cluster of proteins on the yeast plasma membrane. The BAR-domain protein Pil1p binds and unbinds at eisosome ends, supporting a new model of eisosomes as dynamic oligomeric filaments. Molecular assemblies can have highly heterogeneous dynamics within the cell, but the limitations of conventional fluorescence microscopy can mask nanometer-scale features. Here we adapt a single-molecule strategy to perform single-molecule recovery after photobleaching (SRAP) within dense macromolecular assemblies to reveal and characterize binding and unbinding dynamics within such assemblies. We applied this method to study the eisosome, a stable assembly of BAR-domain proteins on the cytoplasmic face of the plasma membrane in fungi. By fluorescently labeling only a small fraction of cellular Pil1p, the main eisosome BAR-domain protein in fission yeast, we visualized whole eisosomes and, after photobleaching, localized recruitment of new Pil1p molecules with ∼30-nm precision. Comparing our data to computer simulations, we show that Pil1p exchange occurs specifically at eisosome ends and not along their core, supporting a new model of the eisosome as a dynamic filament. This result is the first direct observation of any BAR-domain protein dynamics in vivo under physiological conditions consistent with the oligomeric filaments reported from in vitro experiments.