High-fidelity reconstitution of stress granules and nucleoli in mammalian cellular lysate

J Cell Biol. 2021 Mar 1;220(3):e202009079. doi: 10.1083/jcb.202009079.

Abstract

Liquid-liquid phase separation (LLPS) is a mechanism of intracellular organization that underlies the assembly of a variety of RNP granules. Fundamental biophysical principles governing LLPS during granule assembly have been revealed by simple in vitro systems, but these systems have limitations when studying the biology of complex, multicomponent RNP granules. Visualization of RNP granules in cells has validated key principles revealed by simple in vitro systems, but this approach presents difficulties for interrogating biophysical features of RNP granules and provides limited ability to manipulate protein, nucleic acid, or small molecule concentrations. Here, we introduce a system that builds upon recent insights into the mechanisms underlying RNP granule assembly and permits high-fidelity reconstitution of stress granules and the granular component of nucleoli in mammalian cellular lysate. This system fills the gap between simple in vitro systems and live cells and allows for a variety of studies of membraneless organelles, including the development of therapeutics that modify properties of specific condensates.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Cell Extracts
  • Cell Line
  • Cell Nucleolus / metabolism*
  • Cytoplasmic Granules / metabolism*
  • DNA Helicases / isolation & purification
  • DNA Helicases / metabolism
  • Green Fluorescent Proteins / metabolism
  • Humans
  • Mammals / metabolism*
  • Nerve Tissue Proteins / metabolism
  • Nuclear Proteins / metabolism
  • Nucleophosmin
  • Poly-ADP-Ribose Binding Proteins / isolation & purification
  • Poly-ADP-Ribose Binding Proteins / metabolism
  • RNA / metabolism
  • RNA Helicases / isolation & purification
  • RNA Helicases / metabolism
  • RNA Recognition Motif Proteins / isolation & purification
  • RNA Recognition Motif Proteins / metabolism
  • Stress, Physiological*

Substances

  • ATXN2L protein, human
  • Cell Extracts
  • Nerve Tissue Proteins
  • Nuclear Proteins
  • Poly-ADP-Ribose Binding Proteins
  • RNA Recognition Motif Proteins
  • Nucleophosmin
  • Green Fluorescent Proteins
  • RNA
  • DNA Helicases
  • G3BP1 protein, human
  • RNA Helicases