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Methods for Physical Characterization of Phase-Separated Bodies and Membrane-less Organelles
Authors:Diana M Mitrea  Bappaditya Chandra  Mylene C Ferrolino  Eric B Gibbs  Michele Tolbert  Michael R White  Richard W Kriwacki
Institution:1. Department of Structural Biology, St. Jude Children''s Research Hospital, Memphis, TN 38105, USA;2. Department of Microbiology, Immunology and Biochemistry, University of Tennessee Health Sciences Center, Memphis, TN 38163, USA
Abstract:Membrane-less organelles are cellular structures which arise through the phenomenon of phase separation. This process enables compartmentalization of specific sets of macromolecules (e.g., proteins, nucleic acids), thereby regulating cellular processes by increasing local concentration, and modulating the structure and dynamics of their constituents. Understanding the connection between structure, material properties and function of membrane-less organelles requires inter-disciplinary approaches, which address length and timescales that span several orders of magnitude (e.g., Ångstroms to micrometer, picoseconds to hours). In this review, we discuss the wide variety of methods that have been applied to characterize the morphology, rheology, structure and dynamics of membrane-less organelles and their components, in vitro and in live cells.
Keywords:membrane-less  organelles  methods  phase  separation  DIC  differential interference contrast  ROI  region of interest  FIB1  fibrillarin  FRAP  fluorescence recovery after photobleaching  FLIP  fluorescence loss in photobleaching  SIM  structured illumination microscopy  STORM  stochastic optical reconstruction microscopy  LLSM  lattice light-sheet microscopy  TEM  transmission electron microscopy  CLEM  correlated light and electron microscopy  FISH  cryo-EM  cryo-electron microscopy  IDPs  intrinsically disordered proteins  CSP  chemical shift perturbation  IDRs  intrinsically disordered regions  NOE  nuclear Overhauser effect  PFG  pulse field gradient  ELP  elastin-like polypeptide  cryo-ET  cryo-electron tomography  FIB  focused ion beam  DLS  dynamic light scattering  SLS  static light scattering  SAS  small-angle scattering  SAXS  small-angle X-ray scattering  SEC  size-exclusion chromatography  SANS  small-angle neutron scattering  FRET  Förster-resonance energy transfer  FCS  fluorescence correlation spectroscopy  EM  electron microscopy
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