RNA-Induced Conformational Switching and Clustering of G3BP Drive Stress Granule Assembly by Condensation.

Stressed cells shut down translation, release mRNA molecules from polysomes, and form stress granules (SGs) via a network of interactions that involve G3BP. Here we focus on the mechanistic underpinnings of SG assembly. We show that, under non-stress conditions, G3BP adopts a compact auto-inhibited state stabilized by electrostatic intramolecular interactions between the intrinsically disordered acidic tracts and the positively charged arginine-rich region. Upon release from polysomes, unfolded mRNAs outcompete G3BP auto-inhibitory interactions, engendering a conformational transition that facilitates clustering of G3BP through protein-RNA interactions. Subsequent physical crosslinking of G3BP clusters drives RNA molecules into networked RNA/protein condensates. We show that G3BP condensates impede RNA entanglement and recruit additional client proteins that promote SG maturation or induce a liquid-to-solid transition that may underlie disease. We propose that condensation coupled to conformational rearrangements and heterotypic multivalent interactions may be a general principle underlying RNP granule assembly.

ALS-linked FUS mutations dysregulate G-quadruplex-dependent liquid-liquid phase separation and liquid-to-solid transition.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by the accumulation of protein aggregates in motor neurons. Recent discoveries of genetic mutations in ALS patients promoted research into the complex molecular mechanisms underlying ALS. FUS (fused in sarcoma) is a representative ALS-linked RNA-binding protein (RBP) that specifically recognizes G-quadruplex (G4)-DNA/RNAs. However, the effects of ALS-linked FUS mutations on the G4-RNA-binding activity and the phase behavior have never been investigated. Using the purified full-length FUS, we analyzed the molecular mechanisms of multidomain structures consisting of multiple functional modules that bind to G4. Here we succeeded to observe the liquid-liquid phase separation (LLPS) of FUS condensate formation and subsequent liquid-to-solid transition (LST) leading to the formation of FUS aggregates. This process was markedly promoted through FUS interaction with G4-RNA. To further investigate, we selected a total of eight representative ALS-linked FUS mutants within multidomain structures and purified these proteins. The regulation of G4-RNA-dependent LLPS and LST pathways was lost for all ALS-linked FUS mutants defective in G4-RNA recognition tested, supporting the essential role of G4-RNA in this process. Noteworthy, the P525L mutation that causes juvenile ALS exhibited the largest effect on both G4-RNA binding and FUS aggregation. The findings described herein could provide a clue to the hitherto undefined connection between protein aggregation and dysfunction of RBPs in the complex pathway of ALS pathogenesis.

Small Molecules Modulate Liquid-to-Solid Transitions in Phase-Separated Tau Condensates.

In Tau protein condensates formed by the Liquid-Liquid Phase Separation (LLPS) process, liquid-to-solid transitions lead to the formation of fibrils implicated in Alzheimer's disease. Here, by tracking two contacting Tau-rich droplets using a simple and nonintrusive video microscopy, we found that the halftime of the liquid-to-solid transition in the Tau condensate is affected by the Hofmeister series according to the solvation energy of anions. After dissecting functional groups of physiologically relevant small molecules using a multivariate approach, we found that charged groups facilitate the liquid-to-solid transition in a manner similar to the Hofmeister effect, whereas hydrophobic alkyl chains and aromatic rings inhibit the transition. Our results not only elucidate the driving force of the liquid-to-solid transition in Tau condensates, but also provide guidelines to design small molecules to modulate this important transition for many biological functions for the first time.

Challenges in studying the liquid-to-solid phase transitions of proteins using computer simulations.

"Membraneless organelles," also referred to as biomolecular condensates, perform a variety of cellular functions and their dysregulation is implicated in cancer and neurodegeneration. In the last two decades, liquid-liquid phase separation (LLPS) of intrinsically disordered and multidomain proteins has emerged as a plausible mechanism underlying the formation of various biomolecular condensates. Further, the occurrence of liquid-to-solid transitions within liquid-like condensates may give rise to amyloid structures, implying a biophysical link between phase separation and protein aggregation. Despite significant advances, uncovering the microscopic details of liquid-to-solid phase transitions using experiments remains a considerable challenge and presents an exciting opportunity for the development of computational models which provide valuable, complementary insights into the underlying phenomenon. In this review, we first highlight recent biophysical studies which provide new insights into the molecular mechanisms underlying liquid-to-solid (fibril) phase transitions of folded, disordered and multi-domain proteins. Next, we summarize the range of computational models used to study protein aggregation and phase separation. Finally, we discuss recent computational approaches which attempt to capture the underlying physics of liquid-to-solid transitions along with their merits and shortcomings.

Measurement of Protein and Nucleic Acid Diffusion Coefficients Within Biomolecular Condensates Using In-Droplet Fluorescence Correlation Spectroscopy.

Liquid-liquid phase separation of protein and RNA complexes into biomolecular condensates has emerged as a ubiquitous phenomenon in living systems. These protein-RNA condensates are thought to be involved in many biological functions in all forms of life. One of the most sought-after properties of these condensates is their dynamical properties, as they are a major determinant of condensate physiological function and disease processes. Measurement of the diffusion dynamics of individual components in a multicomponent biomolecular condensate is therefore routinely performed. Here, we outline the experimental procedure for performing in-droplet fluorescence correlation spectroscopy (FCS) measurements to extract the diffusion coefficient of individual molecules within a biomolecular condensate in vitro. Unlike more common experiments such as fluorescence recovery after photobleaching (FRAP), where data interpretation is not straightforward and strictly model dependent, FCS offers a robust and more accurate way to quantify biomolecular diffusion rates in the dense phase. The small observation volume allows FCS experiments to report on the local diffusion coefficient within a spatial resolution of <1 µm, making it ideal for probing spatial inhomogeneities within condensates as well as variable dynamics within subcompartments of multiphasic condensates.

Regulation of major bacterial survival strategies by transcripts sequestration in a membraneless organelle.

TmaR, the only known pole-localizer protein in Escherichia coli, was shown to cluster at the cell poles and control localization and activity of the major sugar regulator in a tyrosine phosphorylation-dependent manner. Here, we show that TmaR assembles by phase separation (PS) via heterotypic interactions with RNA in vivo and in vitro. An unbiased automated mutant screen combined with directed mutagenesis and genetic manipulations uncovered the importance of a predicted nucleic-acid-binding domain, a disordered region, and charged patches, one containing the phosphorylated tyrosine, for TmaR condensation. We demonstrate that, by protecting flagella-related transcripts, TmaR controls flagella production and, thus, cell motility and biofilm formation. These results connect PS in bacteria to survival and provide an explanation for the linkage between metabolism and motility. Intriguingly, a point mutation or increase in its cellular concentration induces irreversible liquid-to-solid transition of TmaR, similar to human disease-causing proteins, which affects cell morphology and division.