An aberrant phase transition of stress granules triggered by misfolded protein and prevented by chaperone function

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

RNA buffers the phase separation behavior of prion-like RNA binding proteins

Prion-like RNA binding proteins (RBPs) such as TDP43 and FUS are largely soluble in the nucleus but form solid pathological aggregates when mislocalized to the cytoplasm. What keeps these proteins soluble in the nucleus and promotes aggregation in the cytoplasm is still unknown. We report here that RNAcritically regulates the phase behavior of prion-like RBPs. Low RNA/protein ratios promote phase separation into liquid droplets, whereas high ratios prevent droplet formation in vitro. Reduction of nuclear RNA levels or genetic ablation of RNA binding causes excessive phase separation and the formation of cytotoxic solid-like assemblies in cells. We propose that the nucleus is a buffered system in which high RNA concentrations keep RBPs soluble. Changes in RNA levels or RNA binding abilities of RBPs cause aberrant phase transitions.1125sciescopu

Intracellular Mass Density Increase Is Accompanying but Not Sufficient for Stiffening and Growth Arrest of Yeast Cells

Many organisms, including yeast cells, bacteria, nematodes, and tardigrades, endure harsh environmental conditions, such as nutrient scarcity, or lack of water and energy for a remarkably long time. The rescue programs that these organisms launch upon encountering these adverse conditions include reprogramming their metabolism in order to enter a quiescent or dormant state in a controlled fashion. Reprogramming coincides with changes in the macromolecular architecture and changes in the physical and mechanical properties of the cells. However, the cellular mechanisms underlying the physical-mechanical changes remain enigmatic. Here, we induce metabolic arrest of yeast cells by lowering their intracellular pH. We then determine the differences in the intracellular mass density and stiffness of active and metabolically arrested cells using optical diffraction tomography (ODT) and atomic force microscopy (AFM). We show that an increased intracellular mass density is associated with an increase in stiffness when the growth of yeast is arrested. However, increasing the intracellular mass density alone is not sufficient for maintenance of the growth-arrested state in yeast cells. Our data suggest that the cytoplasm of metabolically arrested yeast displays characteristics of a solid. Our findings constitute a bridge between the mechanical behavior of the cytoplasm and the physical and chemical mechanisms of metabolically arrested cells with the ultimate aim of understanding dormant organisms

Different Material States of Pub1 Condensates Define Distinct Modes of Stress Adaptation and Recovery

Summary: How cells adapt to varying environmental conditions is largely unknown. Here, we show that, in budding yeast, the RNA-binding and stress granule protein Pub1 has an intrinsic property to form condensates upon starvation or heat stress and that condensate formation is associated with cell-cycle arrest. Release from arrest coincides with condensate dissolution, which takes minutes (starvation) or hours (heat shock). In vitro reconstitution reveals that the different dissolution rates of starvation- and heat-induced condensates are due to their different material properties: starvation-induced Pub1 condensates form by liquid-liquid demixing and subsequently convert into reversible gel-like particles; heat-induced condensates are more solid-like and require chaperones for disaggregation. Our data suggest that different physiological stresses, as well as stress durations and intensities, induce condensates with distinct physical properties and thereby define different modes of stress adaptation and rates of recovery. : Kroschwald et al. show that different environmental stresses induce Pub1 stress granule condensates with different material properties. The material properties define the rate of stress granule dissolution and the requirement for disaggregases. The stress granule constituents are released before reentry into the cell cycle. Keywords: phase separation, condensate, phase transition, stress granule, stress response, molecular chaperone, Hsp104, protein aggregation, cytosolic p

Different Material States of Pub1 Condensates Define Distinct Modes of Stress Adaptation and Recovery

Summary: How cells adapt to varying environmental conditions is largely unknown. Here, we show that, in budding yeast, the RNA-binding and stress granule protein Pub1 has an intrinsic property to form condensates upon starvation or heat stress and that condensate formation is associated with cell-cycle arrest. Release from arrest coincides with condensate dissolution, which takes minutes (starvation) or hours (heat shock). In vitro reconstitution reveals that the different dissolution rates of starvation- and heat-induced condensates are due to their different material properties: starvation-induced Pub1 condensates form by liquid-liquid demixing and subsequently convert into reversible gel-like particles; heat-induced condensates are more solid-like and require chaperones for disaggregation. Our data suggest that different physiological stresses, as well as stress durations and intensities, induce condensates with distinct physical properties and thereby define different modes of stress adaptation and rates of recovery. : Kroschwald et al. show that different environmental stresses induce Pub1 stress granule condensates with different material properties. The material properties define the rate of stress granule dissolution and the requirement for disaggregases. The stress granule constituents are released before reentry into the cell cycle. Keywords: phase separation, condensate, phase transition, stress granule, stress response, molecular chaperone, Hsp104, protein aggregation, cytosolic p

Phase-separating RNA-binding proteins form heterogeneous distributions of clusters in subsaturated solutions.

Macromolecular phase separation is thought to be one of the processes that drives the formation of membraneless biomolecular condensates in cells. The dynamics of phase separation are thought to follow the tenets of classical nucleation theory, and, therefore, subsaturated solutions should be devoid of clusters with more than a few molecules. We tested this prediction using in vitro biophysical studies to characterize subsaturated solutions of phase-separating RNA-binding proteins with intrinsically disordered prion-like domains and RNA-binding domains. Surprisingly, and in direct contradiction to expectations from classical nucleation theory, we find that subsaturated solutions are characterized by the presence of heterogeneous distributions of clusters. The distributions of cluster sizes, which are dominated by small species, shift continuously toward larger sizes as protein concentrations increase and approach the saturation concentration. As a result, many of the clusters encompass tens to hundreds of molecules, while less than 1% of the solutions are mesoscale species that are several hundred nanometers in diameter. We find that cluster formation in subsaturated solutions and phase separation in supersaturated solutions are strongly coupled via sequence-encoded interactions. We also find that cluster formation and phase separation can be decoupled using solutes as well as specific sets of mutations. Our findings, which are concordant with predictions for associative polymers, implicate an interplay between networks of sequence-specific and solubility-determining interactions that, respectively, govern cluster formation in subsaturated solutions and the saturation concentrations above which phase separation occurs

Three-dimensional structure of the DEAF-1 MYND domain.

<p>(a) Stereo view of the ensemble of the twenty lowest energy structures of the DEAF-1 MYND domain. α helices and β strands are colored in green and purple respectively, whereas zinc atoms are depicted as red spheres. (b) Ribbon representation of the DEAF-1 MYND domain. Side-chains of residues coordinating the zinc atoms are shown as sticks. The zinc coordination geometry is indicated by red dotted lines. (c) Superposition of DEAF-1 (green), ETO (red), ZNF10 (Blue), SMYD1 (yellow), SMYD2 (orange) and SMYD3 (gray) MYND structures shown in ribbon representation. The two zinc ions are depicted as red spheres. (d) Schematic representation of the zinc-binding pattern and secondary structure elements in MYND, RING, PHD and LIM domains. (e) Cartoon representation of DEAF1-MYND domain. Side chains of residues for which medium and long-range NOEs are observed that unambiguously define the cross-brace zinc binding topology are shown in magenta. Green lines indicate NOEs between C524 H^N/C504 H^β*, C524 H^N/C528 H^β* for the first binding site; and H536 H^ε1/C540 H^N, H536 H^ε1/C518 H^β1, H536 H^ε1/K520 H^N, and H536 H^ε1/C515 H^β2 for the second binding site.</p