A Quantitative Assay to Measure Stress Granule Association of Proteins and Peptidesin Semi-permeabilized Human Cells.

Stress granules (SGs) are membrane-less organelles that form in the cytoplasm through phase separation, in response to diverse stressors. SGs contain translationally stalled mRNAs, proteins involved in translation, and various RNA-binding proteins (RBPs). Due to the high local concentration of aggregation-prone RBPs, SGs might act as condensation sites for aberrant phase transitions of RBPs and could favor formation of solid protein aggregates underlying the pathological cytoplasmic inclusions found in numerous neurodegenerative diseases. Most assays aiming at studying the recruitment of RBPs into SGs are based on overexpression and SG recruitment of RBPs in intact cells. These approaches are, however, often limited by the predominantly nuclear localization of many RBPs, which precludes cytoplasmic RBP concentrations sufficient for SG localization, and does not address RBP recruitment independent of SG formation. Here, we present a quantitative method to assess recruitment of recombinant RBPs into pre-formed SGs, independent of the RBP's nuclear localization, using semi-permeabilized cells and fluorescence microscopy. In this assay, SGs are firstly induced by a stressor, and then the plasma membrane of the stressed cells is subsequently selectively permeabilized to provide access of the recombinant protein to SGs. Nuclear import of the protein-of-interest is prevented by blocking nuclear pores with wheat germ agglutinin. This assay allows one to study the molecular mechanisms underlying recruitment of RBPs into SGs quantitatively, in absence of their nuclear import and under controlled conditions. The method allows for a direct comparison of wildtype, mutant or posttranslationally modified RBPs, for addressing the influence of other proteins' preventing or promoting SG association of RBPs, and is also applicable to synthetic peptides. Graphic abstract: Workflow overview for analysis of SG recruitment of recombinant proteins or peptides in semi-permeabilized cells.