Stress granule phase separation in stress-responsive cytosolic extract-in-oil droplets

Abstract

Abstract Stress granules (SGs) are biomolecular condensates that form transiently in the cytosol of mammalian cells in response to stress. Dysregulation of their assembly or disassembly is implicated in human age-related diseases. While phase separation is the key process underlying SG assembly, understanding of their function, composition and regulation in response to physiological stimuli is limited. This knowledge gap reflects the challenge of gaining comprehensive and quantitative insights into the dynamic regulation of the complex composition of SGs at the single-cell level. Here we present an emulsion-based microfluidics method to overcome this limitation. “Cytosolic extracts-in-oil droplets” (CEODs) recreate a confined active cytosolic milieu that undergoes phase separation and SG formation in response to stress under physiological conditions. This approach led to the discovery of seven previously unrecognised SG components involved in signalling pathways. CEODs provide a versatile and cost-effective screening platform for future mechanistic and therapeutic studies.