RNA and membraneless organelles: Membraneless compartments can form in cells through liquidliquid phase separation (see the Perspective by Polymenidou). But what prevents these cellular condensates from randomly fusing together? Using the RNA-binding protein (RBP) Whi3, Langdon et al. demonstrated that the secondary structure of different RNA components determines the distinct biophysical and biological properties of the two types of condensates that Whi3 forms. Several RBPs, such as FUS and TDP43, contain prion-like domains and are linked to neurodegenerative diseases. These RBPs are usually soluble in the nucleus but can form pathological aggregates in the cytoplasm. Maharana et al. showed that local RNA concentrations determine distinct phase separation behaviors in different subcellular locations. The higher RNA concentrations in the nucleus act as a buffer to prevent phase separation of RBPs; when mislocalized to the cytoplasm, lower RNA concentrations trigger aggregation. Science , this issue p. 922 , p. 918 ; see also p. 859

RNA-protein (RNP) granules have been proposed to assemble by forming solid RNA/protein aggregates or through phase separation into a liquid RNA/protein phase. Which model describes RNP granules in living cells is still unclear. In this study, we analyze P bodies in budding yeast and find that they have liquid-like properties. Surprisingly, yeast stress granules adopt a different material state, which is reminiscent of solid protein aggregates and controlled by protein disaggregases. By using an assay to ectopically nucleate RNP granules, we further establish that RNP granule formation does not depend on amyloid-like aggregation but rather involves many promiscuous interactions. Finally, we show that stress granules have different properties in mammalian cells, where they show liquid-like behavior. Thus, we propose that the material state of RNP granules is flexible and that the solid state of yeast stress granules is an adaptation to extreme environments, made possible by the presence of a powerful disaggregation machine.

Stress granules (SG) are membrane‐less compartments involved in regulating mRNAs during stress. Aberrant forms of SGs have been implicated in age‐related diseases, such as amyotrophic lateral sclerosis (ALS), but the molecular events triggering their formation are still unknown. Here, we find that misfolded proteins, such as ALS‐linked variants of SOD1, specifically accumulate and aggregate within SGs in human cells. This decreases the dynamics of SGs, changes SG composition, and triggers an aberrant liquid‐to‐solid transition of in vitro reconstituted compartments. We show that chaperone recruitment prevents the formation of aberrant SGs and promotes SG disassembly when the stress subsides. Moreover, we identify a backup system for SG clearance, which involves transport of aberrant SGs to the aggresome and their degradation by autophagy. Thus, cells employ a system of SG quality control to prevent accumulation of misfolded proteins and maintain the dynamic state of SGs, which may have relevance for ALS and related diseases.

Matters is a next-generation science journal. Membrane-less compartments, such as P bodies and stress granules, play major roles in cell biology and disease. The dynamics and physical properties of these compartments are very diverse, ranging from liquid-like to solid-like behavior. Importantly, changes in compartment dynamics have been linked to changes in compartment function or pathology. However, appropriate tools to investigate the physical properties of membrane-less compartments are still lacking. The aliphatic alcohol hexanediol has been proposed as a tool to differentiate between liquid-like and solid-like assemblies in living cells. We show that in vitro reconstituted compartments formed by the RNA-binding protein FUS are rapidly dissolved by hexanediol. In contrast, solid-like fibers of FUS, which have been linked to the disease amyotrophic lateral sclerosis (ALS), are resistant to hexanediol treatment, supporting the idea that hexanediol can differentiate between liquid- and solid-like assemblies. We further show that hexanediol can be used to examine the physical properties of membrane-less compartments in vivo. We find that hexanediol dissolves dynamic, liquid-like assemblies, such as P bodies, whereas solid-like assemblies, such as protein aggregates and cytoskeletal assemblies, are largely resistant to hexanediol. Finally, we report here that extended exposure of yeast and mammalian cells to hexanediol is cytotoxic and causes abnormal changes in cell morphology, which trigger the formation of aberrant assemblies. We therefore urge care in the use of hexanediol, especially when cells are exposed to hexanediol for extended times. In summary, we propose that hexanediol is a powerful tool to probe the physical properties of membrane-less compartments, but only if adequate controls are included and precautions are taken.