1. Single-molecule imaging reveals dynamic biphasic partition of RNA-binding proteins in stress granules.
- Author
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Niewidok B, Igaev M, Pereira da Graca A, Strassner A, Lenzen C, Richter CP, Piehler J, Kurre R, and Brandt R
- Subjects
- Animals, Arsenites pharmacology, Cytoplasmic Granules drug effects, DNA Helicases genetics, Diffusion, Humans, Kinetics, Models, Biological, Neurons drug effects, PC12 Cells, Poly-ADP-Ribose Binding Proteins genetics, Protein Binding, Protein Transport, RNA Helicases genetics, RNA Recognition Motif Proteins genetics, RNA-Binding Proteins genetics, Rats, Sodium Compounds pharmacology, Cytoplasmic Granules metabolism, DNA Helicases metabolism, Microscopy, Confocal, Neurons metabolism, Poly-ADP-Ribose Binding Proteins metabolism, RNA Helicases metabolism, RNA Recognition Motif Proteins metabolism, RNA-Binding Proteins metabolism, Single Molecule Imaging methods, Stress, Physiological
- Abstract
Stress granules (SGs) are cytosolic, nonmembranous RNA-protein complexes. In vitro experiments suggested that they are formed by liquid-liquid phase separation; however, their properties in mammalian cells remain unclear. We analyzed the distribution and dynamics of two paradigmatic RNA-binding proteins (RBPs), Ras GTPase-activating protein SH3-domain-binding protein (G3BP1) and insulin-like growth factor II mRNA-binding protein 1 (IMP1), with single-molecule resolution in living neuronal cells. Both RBPs exhibited different exchange kinetics between SGs. Within SGs, single-molecule localization microscopy revealed distributed hotspots of immobilized G3BP1 and IMP1 that reflect the presence of relatively immobile nanometer-sized nanocores. We demonstrate alternating binding in nanocores and anomalous diffusion in the liquid phase with similar characteristics for both RBPs. Reduction of low-complexity regions in G3BP1 resulted in less detectable mobile molecules in the liquid phase without change in binding in nanocores. The data provide direct support for liquid droplet behavior of SGs in living cells and reveal transient binding of RBPs in nanocores. Our study uncovers a surprising disconnect between SG partitioning and internal diffusion and interactions of RBPs., (© 2018 Niewidok et al.)
- Published
- 2018
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