Nanocores and liquid droplets: Single-molecule microscopy of neuronal stress granule components

Abstract

Stress granules (SGs) are the result of phase separation of different mRNAs and multivalent RNA-binding proteins. Their main function appears to adapt the translatome of a cell to adverse environmental conditions in a fast, adjustable, and reversible manner. While being highly dynamic during physiological conditions, SGs may also be precursors of more rigid aggregates that form during neuropathological processes. Thus, analysis of the localization and mobility of key stress granule components in neural cells is an important aspect to scrutinize the material state and dynamics of SGs that could also be of pathologic relevance. Here we describe an experimental approach to follow the distribution and dynamics of paradigmatic RNA-binding proteins (RBPs) by single-molecule imaging in chemically induced SGs of model neurons. Specifically, we provide detailed information about the preparation, differentiation, and labeling of the cells; image acquisition with a TIRF microscope in the highly-inclined laminar optical sheet (HILO) mode; and image processing for single-molecule localization and tracking. We describe an approach for quantitative determination of the fraction of bound and mobile molecules, determination of the lifetime of RBP binding in nanocores, and determination of the diffusion behavior of the respective proteins to provide information about the biophysical properties of the liquid phase of SGs. Our goal is to present to the reader guidelines on how to apply single-molecule microscopy and quantitative data analysis to determine the behavior of SG components in model neurons. Moreover, the approach should also be easily adjustable for the analysis of other biomolecular condensates with liquid-like properties and for the use of other cell types.