Single-molecule imaging reveals dynamic biphasic partition of RNA-binding proteins in stress granules

The Journal of Cell Biology
Benedikt NiewidokRoland Brandt

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 intern...Continue Reading

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Methods Mentioned

BETA
protein exchange
transfection
electrophoresis
superresolution microscopy
PCR

Software Mentioned

DBSCAN
Fiji
SLIMfast
CellSens
Python
STaSI
Olympus
Seqlab
ProteinSimple
learn library

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