Department of Biomedical Engineering and Center for Science & Engineering of Living Systems, Washington University in St. Louis, St. Louis, MO 63130, USA.
Department of Biomedical Engineering and Center for Science & Engineering of Living Systems, Washington University in St. Louis, St. Louis, MO 63130, USA.
J Mol Biol. 2021 Jun 11;433(12):166848. doi: 10.1016/j.jmb.2021.166848. Epub 2021 Feb 2.
The combination of phase separation and disorder-to-order transitions can give rise to ordered, semi-crystalline fibrillar assemblies that underlie prion phenomena namely, the non-Mendelian transfer of information across cells. Recently, a method known as Distributed Amphifluoric Förster Resonance Energy Transfer (DAmFRET) was developed to study the convolution of phase separation and disorder-to-order transitions in live cells. In this assay, a protein of interest is expressed to a broad range of concentrations and the acquisition of local density and order, measured by changes in FRET, is used to map phase transitions for different proteins. The high-throughput nature of this assay affords the promise of uncovering sequence-to-phase behavior relationships in live cells. Here, we report the development of a supervised method to obtain automated and accurate classifications of phase transitions quantified using the DAmFRET assay. Systems that we classify as undergoing two-state discontinuous transitions are consistent with prion-like behaviors, although the converse is not always true. We uncover well-established and surprising new sequence features that contribute to two-state phase behavior of prion-like domains. Additionally, our method enables quantitative, comparative assessments of sequence-specific driving forces for phase transitions in live cells. Finally, we demonstrate that a modest augmentation of DAmFRET measurements, specifically time-dependent protein expression profiles, can allow one to apply classical nucleation theory to extract sequence-specific lower bounds on the probability of nucleating ordered assemblies. Taken together, our approaches lead to a useful analysis pipeline that enables the extraction of mechanistic inferences regarding phase transitions in live cells.
相分离和无序到有序转变的结合可以产生有序的、半结晶的纤维状组装体，这些组装体是朊病毒现象的基础，即在细胞间非孟德尔式地传递信息。最近，开发了一种称为分布式 Amphifluoric Förster 共振能量转移（DAmFRET）的方法，用于研究活细胞中相分离和无序到有序转变的卷积。在该测定法中，以广泛的浓度表达感兴趣的蛋白质，并且通过 FRET 的变化来测量局部密度和有序性，以映射不同蛋白质的相转变。该测定法的高通量性质有望揭示活细胞中序列到相行为关系。在这里，我们报告了一种监督方法的开发，该方法可用于自动且准确地对使用 DAmFRET 测定法量化的相转变进行分类。我们分类为经历两态不连续转变的系统与类朊病毒行为一致，尽管反之则不然。我们揭示了对类朊病毒结构域的两态相行为有贡献的成熟且令人惊讶的新序列特征。此外，我们的方法能够对活细胞中相转变的序列特异性驱动因素进行定量、比较评估。最后，我们证明了 DAmFRET 测量值（特别是随时间变化的蛋白质表达谱）的适度增强可以使人们应用经典成核理论来提取有序组装体成核的序列特异性下限概率。总之，我们的方法提供了一种有用的分析途径，可以提取有关活细胞中相转变的机制推断。
