Phase separation at the nanoscale quantified by dcFCCS

• Published in: Proceedings of the National Academy of Sciences - PNAS Vol. 117; no. 44; pp. 27124 - 27131
• Main Authors: Peng, Sijia, Li, Weiping, Yao, Yirong, Xing, Wenjing, Li, Pilong, Chen, Chunlai
• Format: Journal Article
• Language: English
• Published: United States National Academy of Sciences 03-11-2020
• Subjects: Biological Sciences; Condensates; Fluorescence; Fluorescence microscopy; Growth rate; Liquid phases; Macromolecules; Microscopy; Phase separation; Physical Sciences; Spectroscopy; Stoichiometry; nanoscale; condensate; fluorescence correlation spectroscopy; liquid–liquid phase separation; dual-color fluorescence cross-correlation spectroscopy
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.2008447117

Liquid–liquid phase separation, driven by multivalent macromolecular interactions, causes formation of membraneless compartments, which are biomolecular condensates containing concentrated macromolecules. These condensates are essential in diverse cellular processes. Formation and dynamics of micrometer-scale phase-separated condensates are examined routinely. However, limited by commonly used methods which cannot capture small-sized free-diffusing condensates, the transition process from miscible individual molecules to micrometer-scale condensates is mostly unknown. Herein, with a dual-color fluorescence cross-correlation spectroscopy (dcFCCS) method, we captured formation of nanoscale condensates beyond the detection limit of conventional fluorescence microscopy. In addition, dcFCCS is able to quantify size and growth rate of condensates as well as molecular stoichiometry and binding affinity of client molecules within condensates. The critical concentration to form nanoscale condensates, identified by our experimental measurements and Monte Carlo simulations, is at least several fold lower than the detection limit of conventional fluorescence microscopy. Our results emphasize that, in addition to micrometer-scale condensates, nanoscale condensates are likely to play important roles in various cellular processes and dcFCCS is a simple and powerful quantitative tool to examine them in detail.