A label-free method for measuring the composition of multicomponent biomolecular condensates

Research output: Contribution to journalResearch articleContributedpeer-review

Contributors

  • Patrick M. McCall - , Clusters of Excellence PoL: Physics of Life, Max Planck Institute of Molecular Cell Biology and Genetics, Max-Planck-Institute for the Physics of Complex Systems, Center for Systems Biology Dresden (CSBD), Leibniz Institute of Polymer Research Dresden (Author)
  • Kyoohyun Kim - , Clusters of Excellence PoL: Physics of Life, Max Planck Institute for the Science of Light (Author)
  • Anna Shevchenko - , Max Planck Institute of Molecular Cell Biology and Genetics (Author)
  • Martine Ruer-Gruß - , Max Planck Institute of Molecular Cell Biology and Genetics (Author)
  • Jan Peychl - , Max Planck Institute of Molecular Cell Biology and Genetics (Author)
  • Jochen Guck - , Clusters of Excellence PoL: Physics of Life, Biotechnology Center, Max Planck Institute for the Science of Light (Author)
  • Andrej Shevchenko - , Max Planck Institute of Molecular Cell Biology and Genetics (Author)
  • Anthony A. Hyman - , Max Planck Institute of Molecular Cell Biology and Genetics, Clusters of Excellence PoL: Physics of Life (Author)
  • Jan Brugués - , Clusters of Excellence PoL: Physics of Life, Chair of Spatiotemporal Organization of Subcellular Structures (PoL), Max Planck Institute of Molecular Cell Biology and Genetics, Max-Planck-Institute for the Physics of Complex Systems, Center for Systems Biology Dresden (CSBD) (Author)

Abstract

Many subcellular compartments are biomolecular condensates made of multiple components, often including several distinct proteins and nucleic acids. However, current tools to measure condensate composition are limited and cannot capture this complexity quantitatively because they either require fluorescent labels, which can perturb composition, or can distinguish only one or two components. Here we describe a label-free method based on quantitative phase imaging and analysis of tie-lines and refractive index to measure the composition of reconstituted condensates with multiple components. We first validate the method empirically in binary mixtures, revealing sequence-encoded density variation and complex ageing dynamics for condensates composed of full-length proteins. We then use analysis of tie-lines and refractive index to simultaneously resolve the concentrations of five macromolecular solutes in multicomponent condensates containing RNA and constructs of multiple RNA-binding proteins. Our measurements reveal an unexpected decoupling of density and composition, highlighting the need to determine molecular stoichiometry in multicomponent condensates. We foresee this approach enabling the study of compositional regulation of condensate properties and function. (Figure presented.)

Details

Original languageEnglish
JournalNature chemistry
Publication statusE-pub ahead of print - 3 Sept 2025
Peer-reviewedYes

Keywords