Quantifying Nucleation In Vivo Reveals the Physical Basis of Prion-like Phase Behavior.
Quantifying Nucleation In Vivo Reveals the Physical Basis of Prion-like Phase Behavior.
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DOI：
10.1016/j.molcel.2018.06.016
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发表时间：
2018-07-05
期刊：
影响因子：
16
通讯作者：
Halfmann R
中科院分区：
文献类型：
--
作者：
Khan T;Kandola TS;Wu J;Venkatesan S;Ketter E;Lange JJ;Rodríguez Gama A;Box A;Unruh JR;Cook M;Halfmann R
蛋白质自组装在生物时间尺度上调节蛋白质的活动，这可能超过蛋白质甚至承载它们的细胞的寿命。我们假设这些时间尺度与有序相成核所固有的动力学障碍有关。为了研究活细胞中的成核屏障，我们开发了分布式双氟FRET （DAmFRET）。DAmFRET利用光可转换的荧光团，异质表达和大细胞数量，通过流式细胞术定量蛋白质的自组装程度作为细胞浓度的函数。我们证明了动力学势垒限制了有序自组装的成核，并且势垒的持久性与浓度有关。序列编码的成核屏障导致的过饱和导致了朊病毒行为，并使朊病毒形成蛋白Sup35 PrD分裂成动态的细胞内凝聚物或形成有毒的聚集体。我们的研究结果表明，成核屏障控制着细胞质遗传、亚细胞组织和蛋白质毒性。朊病毒现象是由受成核速率限制的蛋白质相分离引起的。在这里，Khan等人介绍了一种量化活细胞成核的方法。通过比较具有和不具有朊病毒行为的多种蛋白质，他们表明成核的动力学屏障来自于新相的结构顺序。
Protein self-assemblies modulate protein activities over biological time scales that can exceed the lifetimes of the proteins or even the cells that harbor them. We hypothesized that these time scales relate to kinetic barriers inherent to the nucleation of ordered phases. To investigate nucleation barriers in living cells, we developed Distributed Amphifluoric FRET (DAmFRET). DAmFRET exploits a photoconvertible fluorophore, heterogeneous expression, and large cell numbers to quantify via flow cytometry the extent of a protein’s self-assembly as a function of cellular concentration. We show that kinetic barriers limit the nucleation of ordered self-assemblies, and that the persistence of the barriers with respect to concentration relates to structure. Supersaturation resulting from sequence-encoded nucleation barriers gave rise to prion behavior, and enabled a prion-forming protein, Sup35 PrD, to partition into dynamic intracellular condensates or to form toxic aggregates. Our results suggest that nucleation barriers govern cytoplasmic inheritance, subcellular organization, and proteotoxicity. Prion phenomena result from protein phase separations that are rate-limited by nucleation. Here, Khan et al. introduce a method to quantify nucleation in living cells. By comparing diverse proteins with and without prion behavior, they show that the kinetic barrier to nucleation derives from structural order in the new phase.
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