Institute for Molecules and Materials, Radboud University, Heyendaalseweg 135, 6525 AJ Nijmegen, the Netherlands.
Institute for Molecules and Materials, Radboud University, Heyendaalseweg 135, 6525 AJ Nijmegen, the Netherlands.
Biophys J. 2023 Jan 17;122(2):397-407. doi: 10.1016/j.bpj.2022.12.001. Epub 2022 Dec 5.
The crowdedness of the cell calls for adequate intracellular organization. Biomolecular condensates, formed by liquid-liquid phase separation of intrinsically disordered proteins and nucleic acids, are important organizers of cellular fluids. To underpin the molecular mechanisms of protein condensation, cell-free studies are often used where the role of crowding is not investigated in detail. Here, we investigate the effects of macromolecular crowding on the formation and material properties of a model heterotypic biomolecular condensate, consisting of nucleophosmin (NPM1) and ribosomal RNA (rRNA). We studied the effect of the macromolecular crowding agent poly(ethylene glycol) (PEG), which is often considered an inert crowding agent. We observed that PEG could induce both homotypic and heterotypic phase separation of NPM1 and NPM1-rRNA, respectively. Crowding increases the condensed concentration of NPM1 and decreases its equilibrium dilute phase concentration, although no significant change in the concentration of rRNA in the dilute phase was observed. Interestingly, the crowder itself is concentrated in the condensates, suggesting that co-condensation rather than excluded volume interactions underlie the enhanced phase separation by PEG. Fluorescence recovery after photobleaching measurements indicated that both NPM1 and rRNA become immobile at high PEG concentrations, indicative of a liquid-to-gel transition. Together, these results provide more insight into the role of synthetic crowding agents in phase separation and demonstrate that condensate properties determined in vitro depend strongly on the addition of crowding agents.
细胞的拥挤状态需要足够的细胞内组织。生物分子凝聚物通过无规卷曲蛋白质和核酸的液-液相分离形成,是细胞液的重要组织者。为了支持蛋白质凝聚的分子机制,经常使用无细胞研究,而其中没有详细研究拥挤的作用。在这里,我们研究了大分子拥挤对核仁磷酸蛋白(NPM1)和核糖体 RNA(rRNA)组成的模型异质生物分子凝聚物的形成和物质特性的影响。我们研究了大分子拥挤剂聚乙二醇(PEG)的影响,PEG 通常被认为是一种惰性拥挤剂。我们观察到 PEG 可以分别诱导 NPM1 和 NPM1-rRNA 的同质和异质相分离。拥挤会增加 NPM1 的凝聚浓度,并降低其平衡稀相浓度,尽管在稀相中的 rRNA 浓度没有观察到明显变化。有趣的是,稠化剂本身在凝聚物中浓缩,表明聚乙二醇增强相分离的原因是共凝聚,而不是排除体积相互作用。荧光恢复后光漂白测量表明,在高 PEG 浓度下,NPM1 和 rRNA 都变得不可移动,表明发生了从液相到凝胶的转变。总之,这些结果提供了更多关于合成拥挤剂在相分离中的作用的见解,并表明体外确定的凝聚物特性强烈依赖于拥挤剂的添加。