Department of Chemistry, University of California, Berkeley California94720, United States.
J Am Chem Soc. 2023 Nov 8;145(44):24240-24248. doi: 10.1021/jacs.3c08674. Epub 2023 Oct 2.
The intracellular liquid-liquid phase separation (LLPS) of biomolecules gives rise to condensates that act as membrane-less organelles with vital functions. FUS, an RNA-binding protein, natively forms condensates through LLPS and further provides a model system for the often disease-linked liquid-to-solid transition of biomolecular condensates during aging. However, the mechanism of such maturation processes, as well as the structural and physical properties of the system, remains unclear, partly attributable to difficulties in resolving the internal structures of the micrometer-sized condensates with diffraction-limited optical microscopy. Harnessing a set of multidimensional super-resolution microscopy tools that uniquely map out local physicochemical parameters through single-molecule spectroscopy, here, we uncover nanoscale heterogeneities in FUS condensates and elucidate their evolution over aging. Through spectrally resolved single-molecule localization microscopy (SR-SMLM) with a solvatochromic dye, we unveil distinct hydrophobic nanodomains at the condensate surface. Through SMLM with a fluorogenic amyloid probe, we identify these nanodomains as amyloid aggregates. Through single-molecule displacement/diffusivity mapping (SMM), we show that such nanoaggregates drastically impede local diffusion. Notably, upon aging or mechanical shears, these nanoaggregates progressively expand on the condensate surface, thus leading to a growing low-diffusivity shell while leaving the condensate interior diffusion-permitting. Together, beyond uncovering fascinating structural arrangements and aging mechanisms in the single-component FUS condensates, the demonstrated synergy of multidimensional super-resolution approaches in this study opens new paths for understanding LLPS systems at the nanoscale.
生物分子的细胞内液-液相分离 (LLPS) 导致凝聚物的形成，这些凝聚物作为无膜细胞器发挥着重要的功能。FUS 是一种 RNA 结合蛋白，通过 LLPS 天然形成凝聚物，并进一步为生物分子凝聚物在衰老过程中经常与疾病相关的液-固转变提供了模型系统。然而，这种成熟过程的机制以及该系统的结构和物理特性仍然不清楚，部分原因是难以通过具有衍射极限的光学显微镜解析微米级凝聚物的内部结构。利用一组多维超分辨率显微镜工具，通过单分子光谱学独特地描绘局部物理化学参数，我们在这里揭示了 FUS 凝聚物中的纳米级异质性，并阐明了它们在衰老过程中的演变。通过带有溶剂化变色染料的光谱分辨单分子定位显微镜 (SR-SMLM)，我们在凝聚物表面揭示了明显的疏水性纳米区。通过带有荧光淀粉样探针的 SMLM，我们将这些纳米区鉴定为淀粉样聚集物。通过单分子位移/扩散测绘 (SMM)，我们表明这些纳米聚集体严重阻碍了局部扩散。值得注意的是，在衰老或机械剪切过程中，这些纳米聚集体在凝聚物表面逐渐扩大，从而在保留凝聚物内部允许扩散的情况下形成越来越大的低扩散率外壳。总的来说，除了揭示单一组分 FUS 凝聚物中的迷人结构排列和衰老机制外，本研究中多维超分辨率方法的协同作用为在纳米尺度上理解 LLPS 系统开辟了新的途径。
