Single fluorogen imaging reveals spatial inhomogeneities within biomolecular condensates

Recent investigations have suggested that biomolecular condensates are viscoelastic materials. This implies that material properties of condensates are governed by internal microstructures. Furthermore, computations show that the internal organization in protein condensates is spatially inhomogeneous, featuring hub-and-spoke-like percolated networks of molecules. Here, we test these predictions using imaging of single fluorogenic dyes that are turned-on in response to specific chemical microenvironments. We deployed Nile blue (NB), Nile red (NR), and merocyanine 540 (MC540) for epifluorescence and single-molecule localization microscopy imaging of condensates formed by intrinsically disordered, low-complexity domains of proteins. Imaging with NB reveals internal environments that are uniformly hydrophobic, whereas NR shows preferential binding to hubs that are more hydrophobic than the surrounding background within condensates. Finally, imaging with MC540 suggests that interfaces of condensates are unique chemical environments. Overall, the high spatiotemporal resolution and environmental sensitivity of single-fluorogen imaging reveals spatially inhomogeneous organization of molecules within condensates.

[1]  P. Tinnefeld,et al.  Shrinking gate fluorescence correlation spectroscopy yields equilibrium constants and separates photophysics from structural dynamics , 2023, Proceedings of the National Academy of Sciences of the United States of America.

[2]  R. Pappu,et al.  Condensates formed by prion-like low-complexity domains have small-world network structures and interfaces defined by expanded conformations , 2022, Nature communications.

[3]  A. Klymchenko,et al.  Fluorogenic Dimers as Bright Switchable Probes for Enhanced Super-Resolution Imaging of Cell Membranes. , 2022, Journal of the American Chemical Society.

[4]  Matthew D. Lew,et al.  Six-dimensional single-molecule imaging with isotropic resolution using a multi-view reflector microscope , 2022, bioRxiv.

[5]  R. Pappu,et al.  A conceptual framework for understanding phase separation and addressing open questions and challenges. , 2022, Molecular cell.

[6]  J. Shillcock,et al.  Model biomolecular condensates have heterogeneous structure quantitatively dependent on the interaction profile of their constituent macromolecules , 2022, bioRxiv.

[7]  R. Pappu,et al.  Endocytic proteins with prion-like domains form viscoelastic condensates that enable membrane remodeling , 2021, Proceedings of the National Academy of Sciences.

[8]  Priya R Banerjee,et al.  Programmable viscoelasticity in protein-RNA condensates with disordered sticker-spacer polypeptides , 2021, Nature Communications.

[9]  R. Pappu,et al.  Deciphering how naturally occurring sequence features impact the phase behaviors of disordered prion-like domains , 2021, bioRxiv.

[10]  Huan‐Xiang Zhou.  Viscoelasticity of biomolecular condensates conforms to the Jeffreys model , 2020, bioRxiv.

[11]  M. Rosen,et al.  A framework for understanding the functions of biomolecular condensates across scales , 2020, Nature Reviews Molecular Cell Biology.

[12]  Patrick M. McCall,et al.  Partitioning of cancer therapeutics in nuclear condensates , 2020, Science.

[13]  Ismail M. Khater,et al.  A Review of Super-Resolution Single-Molecule Localization Microscopy Cluster Analysis and Quantification Methods , 2020, Patterns.

[14]  Jerelle A. Joseph,et al.  Liquid network connectivity regulates the stability and composition of biomolecular condensates with many components , 2020, Proceedings of the National Academy of Sciences.

[15]  Matthew D. Lew,et al.  Single-molecule 3D orientation imaging reveals nanoscale compositional heterogeneity in lipid membranes , 2020, bioRxiv.

[16]  Joshua A. Riback,et al.  Composition-dependent thermodynamics of intracellular phase separation , 2020, Nature.

[17]  A. Hyman,et al.  Protein condensates as aging Maxwell fluids , 2020, Science.

[18]  R. Pappu,et al.  Valence and patterning of aromatic residues determine the phase behavior of prion-like domains , 2020, Science.

[19]  R. Pappu,et al.  Physical Principles Underlying the Complex Biology of Intracellular Phase Transitions. , 2020, Annual review of biophysics.

[20]  Tianben Ding,et al.  Single-molecule orientation localization microscopy for resolving structural heterogeneities between amyloid fibrils , 2020, bioRxiv.

[21]  Matthew D. Lew,et al.  Nanoscale Colocalization of Fluorogenic Probes Reveals the Role of Oxygen Vacancies in the Photocatalytic Activity of Tungsten Oxide Nanowires , 2020 .

[22]  Seonah Moon,et al.  Switchable Solvatochromic Probes for Live‐Cell Super‐resolution Imaging of Plasma Membrane Organization , 2019, Angewandte Chemie.

[23]  Judith Weber,et al.  ThX – A next-generation probe for the early detection of amyloid aggregates , 2019, bioRxiv.

[24]  J. Vaughan,et al.  Switchable Fluorophores for Single-Molecule Localization Microscopy. , 2018, Chemical reviews.

[25]  Matthew D. Lew,et al.  Super‐resolution Imaging of Amyloid Structures over Extended Times by Using Transient Binding of Single Thioflavin T Molecules , 2018, Chembiochem : a European journal of chemical biology.

[26]  R. Pappu,et al.  Phase separation of a yeast prion protein promotes cellular fitness , 2018, Science.

[27]  H. Chan,et al.  Structural and hydrodynamic properties of an intrinsically disordered region of a germ cell-specific protein on phase separation , 2017, Proceedings of the National Academy of Sciences.

[28]  Ming-Tzo Wei,et al.  Phase behaviour of disordered proteins underlying low density and high permeability of liquid organelles. , 2017, Nature chemistry.

[29]  Anthony A. Hyman,et al.  Biomolecular condensates: organizers of cellular biochemistry , 2017, Nature Reviews Molecular Cell Biology.

[30]  M. Henary,et al.  Nile Red and Nile Blue: Applications and Syntheses of Structural Analogues. , 2016, Chemistry.

[31]  R. Parker,et al.  Compositional Control of Phase-Separated Cellular Bodies , 2016, Cell.

[32]  A. Kanagaraj,et al.  Phase Separation by Low Complexity Domains Promotes Stress Granule Assembly and Drives Pathological Fibrillization , 2015, Cell.

[33]  Timothy D. Craggs,et al.  Phase Transition of a Disordered Nuage Protein Generates Environmentally Responsive Membraneless Organelles , 2015, Molecular cell.

[34]  Ricardo Cortez,et al.  Modeling viscoelastic networks in Stokes flow , 2014 .

[35]  M. Gudheti,et al.  Single Molecule Localization Microscopy , 2012 .

[36]  David J Huggins,et al.  Application of inhomogeneous fluid solvation theory to model the distribution and thermodynamics of water molecules around biomolecules. , 2012, Physical chemistry chemical physics : PCCP.

[37]  Matthew D. Lew,et al.  Three-dimensional superresolution colocalization of intracellular protein superstructures and the cell surface in live Caulobacter crescentus , 2011, Proceedings of the National Academy of Sciences.

[38]  R. Hochstrasser,et al.  Super-resolution microscopy of lipid bilayer phases. , 2011, Journal of the American Chemical Society.

[39]  R. Hochstrasser,et al.  Wide-field subdiffraction imaging by accumulated binding of diffusing probes , 2006, Proceedings of the National Academy of Sciences.

[40]  Michael D. Mason,et al.  Ultra-high resolution imaging by fluorescence photoactivation localization microscopy. , 2006, Biophysical journal.

[41]  Michael J Rust,et al.  Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM) , 2006, Nature Methods.

[42]  J. Lippincott-Schwartz,et al.  Imaging Intracellular Fluorescent Proteins at Nanometer Resolution , 2006, Science.

[43]  J. Gall,et al.  Cajal bodies, nucleoli, and speckles in the Xenopus oocyte nucleus have a low-density, sponge-like structure. , 2004, Molecular biology of the cell.

[44]  R. Chhabra.  Nonwoven Uniformity — Measurements Using Image Analysis , 2003 .

[45]  D. Chandler,et al.  Hydrophobicity at Small and Large Length Scales , 1999 .

[46]  D L Sackett,et al.  Hydrophobic surfaces of tubulin probed by time-resolved and steady-state fluorescence of nile red. , 1990, The Journal of biological chemistry.

[47]  A. Verkman,et al.  Mechanism and kinetics of merocyanine 540 binding to phospholipid membranes. , 1987, Biochemistry.

[48]  S. Fowler,et al.  Spectrofluorometric studies of the lipid probe, nile red. , 1985, Journal of lipid research.

[49]  P. Verveer,et al.  Coordinate-based colocalization analysis of single-molecule localization microscopy data , 2011, Histochemistry and Cell Biology.

[50]  H. Tajalli,et al.  The photophysical properties of Nile red and Nile blue in ordered anisotropic media , 2008 .

[51]  D. Higgins,et al.  Characterization of Molecular Scale Environments in Polymer Films by Single Molecule Spectroscopy , 2000 .

[52]  B. Ripley.  Modelling Spatial Patterns , 1977 .