Macromolecular condensation organizes nucleolar sub-phases to set up a pH gradient.

Nucleoli are multicomponent condensates defined by coexisting sub-phases. We identified distinct intrinsically disordered regions (IDRs), including acidic (D/E) tracts and K-blocks interspersed by E-rich regions, as defining features of nucleolar proteins. We show that the localization preferences of nucleolar proteins are determined by their IDRs and the types of RNA or DNA binding domains they encompass. In vitro reconstitutions and studies in cells showed how condensation, which combines binding and complex coacervation of nucleolar components, contributes to nucleolar organization. D/E tracts of nucleolar proteins contribute to lowering the pH of co-condensates formed with nucleolar RNAs in vitro. In cells, this sets up a pH gradient between nucleoli and the nucleoplasm. By contrast, juxta-nucleolar bodies, which have different macromolecular compositions, featuring protein IDRs with very different charge profiles, have pH values that are equivalent to or higher than the nucleoplasm. Our findings show that distinct compositional specificities generate distinct physicochemical properties for condensates.

A disordered region controls cBAF activity via condensation and partner recruitment.

Intrinsically disordered regions (IDRs) represent a large percentage of overall nuclear protein content. The prevailing dogma is that IDRs engage in non-specific interactions because they are poorly constrained by evolutionary selection. Here, we demonstrate that condensate formation and heterotypic interactions are distinct and separable features of an IDR within the ARID1A/B subunits of the mSWI/SNF chromatin remodeler, cBAF, and establish distinct "sequence grammars" underlying each contribution. Condensation is driven by uniformly distributed tyrosine residues, and partner interactions are mediated by non-random blocks rich in alanine, glycine, and glutamine residues. These features concentrate a specific cBAF protein-protein interaction network and are essential for chromatin localization and activity. Importantly, human disease-associated perturbations in ARID1B IDR sequence grammars disrupt cBAF function in cells. Together, these data identify IDR contributions to chromatin remodeling and explain how phase separation provides a mechanism through which both genomic localization and functional partner recruitment are achieved.

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.

A Molecular Grammar Governing the Driving Forces for Phase Separation of Prion-like RNA Binding Proteins.

Proteins such as FUS phase separate to form liquid-like condensates that can harden into less dynamic structures. However, how these properties emerge from the collective interactions of many amino acids remains largely unknown. Here, we use extensive mutagenesis to identify a sequence-encoded molecular grammar underlying the driving forces of phase separation of proteins in the FUS family and test aspects of this grammar in cells. Phase separation is primarily governed by multivalent interactions among tyrosine residues from prion-like domains and arginine residues from RNA-binding domains, which are modulated by negatively charged residues. Glycine residues enhance the fluidity, whereas glutamine and serine residues promote hardening. We develop a model to show that the measured saturation concentrations of phase separation are inversely proportional to the product of the numbers of arginine and tyrosine residues. These results suggest it is possible to predict phase-separation properties based on amino acid sequences.

FUS Zigzags Its Way to Cross Beta.

The low-complexity domain (LCD) of the FUS protein forms concentration-dependent assemblies, including liquid droplets and fibril-based hydrogels. The molecular structures of FUS within different assemblies and their functional relevance are subjects of intense debate. Murray et al. report an atomic-level structural model for FUS LCD fibrils that answers some questions and raises new ones.

Intrinsic disorder: A term to define the specific physicochemical characteristic of protein conformational heterogeneity.

In his commentary in this issue of Molecular Cell,1 Struhl reasons that the term "intrinsically disordered regions" represents a vague and confusing concept for protein function. However, the term "intrinsically disordered" highlights the important physicochemical characteristic of conformational heterogeneity. Thus, "intrinsically disordered" is the counterpart to the term "folded, " with neither term having specific functional implications.

Coexisting Liquid Phases Underlie Nucleolar Subcompartments.

The nucleolus and other ribonucleoprotein (RNP) bodies are membrane-less organelles that appear to assemble through phase separation of their molecular components. However, many such RNP bodies contain internal subcompartments, and the mechanism of their formation remains unclear. Here, we combine in vivo and in vitro studies, together with computational modeling, to show that subcompartments within the nucleolus represent distinct, coexisting liquid phases. Consistent with their in vivo immiscibility, purified nucleolar proteins phase separate into droplets containing distinct non-coalescing phases that are remarkably similar to nucleoli in vivo. This layered droplet organization is caused by differences in the biophysical properties of the phases-particularly droplet surface tension-which arises from sequence-encoded features of their macromolecular components. These results suggest that phase separation can give rise to multilayered liquids that may facilitate sequential RNA processing reactions in a variety of RNP bodies. PAPERCLIP.

A conceptual framework for understanding phase separation and addressing open questions and challenges.

Macromolecular phase separation is being recognized for its potential importance and relevance as a driver of spatial organization within cells. Here, we describe a framework based on synergies between networking (percolation or gelation) and density (phase separation) transitions. Accordingly, the phase transitions in question are referred to as phase separation coupled to percolation (PSCP). The condensates that result from PSCP are viscoelastic network fluids. Such systems have sequence-, composition-, and topology-specific internal network structures that give rise to time-dependent interplays between viscous and elastic properties. Unlike pure phase separation, the process of PSCP gives rise to sequence-, chemistry-, and structure-specific distributions of clusters that can form at concentrations that lie well below the threshold concentration for phase separation. PSCP, influenced by specific versus solubility-determining interactions, also provides a bridge between different observations and helps answer questions and address challenges that have arisen regarding the role of macromolecular phase separation in biology.

Sequence grammar underlying the unfolding and phase separation of globular proteins.

Aberrant phase separation of globular proteins is associated with many diseases. Here, we use a model protein system to understand how the unfolded states of globular proteins drive phase separation and the formation of unfolded protein deposits (UPODs). We find that for UPODs to form, the concentrations of unfolded molecules must be above a threshold value. Additionally, unfolded molecules must possess appropriate sequence grammars to drive phase separation. While UPODs recruit molecular chaperones, their compositional profiles are also influenced by synergistic physicochemical interactions governed by the sequence grammars of unfolded proteins and cellular proteins. Overall, the driving forces for phase separation and the compositional profiles of UPODs are governed by the sequence grammars of unfolded proteins. Our studies highlight the need for uncovering the sequence grammars of unfolded proteins that drive UPOD formation and cause gain-of-function interactions whereby proteins are aberrantly recruited into UPODs.

Arginine-Enriched Mixed-Charge Domains Provide Cohesion for Nuclear Speckle Condensation.

Low-complexity protein domains promote the formation of various biomolecular condensates. However, in many cases, the precise sequence features governing condensate formation and identity remain unclear. Here, we investigate the role of intrinsically disordered mixed-charge domains (MCDs) in nuclear speckle condensation. Proteins composed exclusively of arginine-aspartic acid dipeptide repeats undergo length-dependent condensation and speckle incorporation. Substituting arginine with lysine in synthetic and natural speckle-associated MCDs abolishes these activities, identifying a key role for multivalent contacts through arginine's guanidinium ion. MCDs can synergize with a speckle-associated RNA recognition motif to promote speckle specificity and residence. MCD behavior is tunable through net-charge: increasing negative charge abolishes condensation and speckle incorporation. Contrastingly, increasing positive charge through arginine leads to enhanced condensation, speckle enlargement, decreased splicing factor mobility, and defective mRNA export. Together, these results identify key sequence determinants of MCD-promoted speckle condensation and link the dynamic material properties of speckles with function in mRNA processing.

Nucleo-cytoplasmic Partitioning of ARF Proteins Controls Auxin Responses in Arabidopsis thaliana.

The phytohormone auxin plays crucial roles in nearly every aspect of plant growth and development. The auxin response factor (ARF) transcription factor family regulates auxin-responsive gene expression and exhibits nuclear localization in regions of high auxin responsiveness. Here we show that the ARF7 and ARF19 proteins accumulate in micron-sized assemblies within the cytoplasm of tissues with attenuated auxin responsiveness. We found that the intrinsically disordered middle region and the folded PB1 interaction domain of ARFs drive protein assembly formation. Mutation of a single lysine within the PB1 domain abrogates cytoplasmic assemblies, promotes ARF nuclear localization, and results in an altered transcriptome and morphological defects. Our data suggest a model in which ARF nucleo-cytoplasmic partitioning regulates auxin responsiveness, providing a mechanism for cellular competence for auxin signaling.

A First Glimpse of Nucleation of Phase Transitions in Living Cells.

Prion formation and propagation involve the nucleation and growth of protein-based supramolecular assemblies. In this issue of Molecular Cell, Khan et al. (2018) report novel methods that enable quantitative, high-throughput analyses of nucleation of supramolecular assemblies by prion-forming proteins in living cells. Their approaches turn living cells into veritable test tubes for biophysical investigations.

Programmable synthetic biomolecular condensates for cellular control.

The formation of biomolecular condensates mediated by a coupling of associative and segregative phase transitions plays a critical role in controlling diverse cellular functions in nature. This has inspired the use of phase transitions to design synthetic systems. While design rules of phase transitions have been established for many synthetic intrinsically disordered proteins, most efforts have focused on investigating their phase behaviors in a test tube. Here, we present a rational engineering approach to program the formation and physical properties of synthetic condensates to achieve intended cellular functions. We demonstrate this approach through targeted plasmid sequestration and transcription regulation in bacteria and modulation of a protein circuit in mammalian cells. Our approach lays the foundation for engineering designer condensates for synthetic biology applications.

Phase Transitions of Associative Biomacromolecules.

Multivalent proteins and nucleic acids, collectively referred to as multivalent associative biomacromolecules, provide the driving forces for the formation and compositional regulation of biomolecular condensates. Here, we review the key concepts of phase transitions of aqueous solutions of associative biomacromolecules, specifically proteins that include folded domains and intrinsically disordered regions. The phase transitions of these systems come under the rubric of coupled associative and segregative transitions. The concepts underlying these processes are presented, and their relevance to biomolecular condensates is discussed.

Competing interactions give rise to two-state behavior and switch-like transitions in charge-rich intrinsically disordered proteins.

The most commonly occurring intrinsically disordered proteins (IDPs) are polyampholytes, which are defined by the duality of low net charge per residue and high fractions of charged residues. Recent experiments have uncovered nuances regarding sequence­ensemble relationships of model polyampholytic IDPs. These include differences in conformational preferences for sequences with lysine vs. arginine and the suggestion that well-mixed sequences form a range of conformations, including globules, conformations with ensemble averages that are reminiscent of ideal chains, or self-avoiding walks. Here, we explain these observations by analyzing results from atomistic simulations. We find that polyampholytic IDPs generally sample two distinct stable states, namely, globules and self-avoiding walks. Globules are favored by electrostatic attractions between oppositely charged residues, whereas self-avoiding walks are favored by favorable free energies of hydration of charged residues. We find sequence-specific temperatures of bistability at which globules and self-avoiding walks can coexist. At these temperatures, ensemble averages over coexisting states give rise to statistics that resemble ideal chains without there being an actual counterbalancing of intrachain and chain-solvent interactions. At equivalent temperatures, arginine-rich sequences tilt the preference toward globular conformations whereas lysine-rich sequences tilt the preference toward self-avoiding walks. We also identify differences between aspartate- and glutamate-containing sequences, whereby the shorter aspartate side chain engenders preferences for metastable, necklace-like conformations. Finally, although segregation of oppositely charged residues within the linear sequence maintains the overall two-state behavior, compact states are highly favored by such systems.

Mesoscale structure-function relationships in mitochondrial transcriptional condensates.

In live cells, phase separation is thought to organize macromolecules into membraneless structures known as biomolecular condensates. Here, we reconstituted transcription in condensates from purified mitochondrial components using optimized in vitro reaction conditions to probe the structure-function relationships of biomolecular condensates. We find that the core components of the mt-transcription machinery form multiphasic, viscoelastic condensates in vitro. Strikingly, the rates of condensate-mediated transcription are substantially lower than in solution. The condensate-mediated decrease in transcriptional rates is associated with the formation of vesicle-like structures that are driven by the production and accumulation of RNA during transcription. The generation of RNA alters the global phase behavior and organization of transcription components within condensates. Coarse-grained simulations of mesoscale structures at equilibrium show that the components stably assemble into multiphasic condensates and that the vesicles formed in vitro are the result of dynamical arrest. Overall, our findings illustrate the complex phase behavior of transcribing, multicomponent condensates, and they highlight the intimate, bidirectional interplay of structure and function in transcriptional condensates.

Dilute phase oligomerization can oppose phase separation and modulate material properties of a ribonucleoprotein condensate.

SignificanceA large subclass of biomolecular condensates are linked to RNA regulation and are known as ribonucleoprotein (RNP) bodies. While extensive work has identified driving forces for biomolecular condensate formation, relatively little is known about forces that oppose assembly. Here, using a fungal RNP protein, Whi3, we show that a portion of its intrinsically disordered, glutamine-rich region modulates phase separation by forming transient alpha helical structures that promote the assembly of dilute phase oligomers. These oligomers detour Whi3 proteins from condensates, thereby impacting the driving forces for phase separation, the protein-to-RNA ratio in condensates, and the material properties of condensates. Our findings show how nanoscale conformational and oligomerization equilibria can influence mesoscale phase equilibria.

Connecting sequence features within the disordered C-terminal linker of Bacillus subtilis FtsZ to functions and bacterial cell division.

Intrinsically disordered regions (IDRs) can function as autoregulators of folded enzymes to which they are tethered. One example is the bacterial cell division protein FtsZ. This includes a folded core and a C-terminal tail (CTT) that encompasses a poorly conserved, disordered C-terminal linker (CTL) and a well-conserved 17-residue C-terminal peptide (CT17). Sites for GTPase activity of FtsZs are formed at the interface between GTP binding sites and T7 loops on cores of adjacent subunits within dimers. Here, we explore the basis of autoregulatory functions of the CTT in Bacillus subtilis FtsZ (Bs-FtsZ). Molecular simulations show that the CT17 of Bs-FtsZ makes statistically significant CTL-mediated contacts with the T7 loop. Statistical coupling analysis of more than 1,000 sequences from FtsZ orthologs reveals clear covariation of the T7 loop and the CT17 with most of the core domain, whereas the CTL is under independent selection. Despite this, we discover the conservation of nonrandom sequence patterns within CTLs across orthologs. To test how the nonrandom patterns of CTLs mediate CTT-core interactions and modulate FtsZ functionalities, we designed Bs-FtsZ variants by altering the patterning of oppositely charged residues within the CTL. Such alterations disrupt the core-CTT interactions, lead to anomalous assembly and inefficient GTP hydrolysis in vitro and protein degradation, aberrant assembly, and disruption of cell division in vivo. Our findings suggest that viable CTLs in FtsZs are likely to be IDRs that encompass nonrandom, functionally relevant sequence patterns that also preserve three-way covariation of the CT17, the T7 loop, and core domain.

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.

Making the Case for Disordered Proteins and Biomolecular Condensates in Bacteria.

Intrinsically disordered proteins/regions (IDPs/IDRs) contribute to a diverse array of molecular functions in eukaryotic systems. There is also growing recognition that membraneless biomolecular condensates, many of which are organized or regulated by IDPs/IDRs, can enable spatial and temporal regulation of complex biochemical reactions in eukaryotes. Motivated by these findings, we assess if (and how) membraneless biomolecular condensates and IDPs/IDRs are functionally involved in key cellular processes and molecular functions in bacteria. We summarize the conceptual underpinnings of condensate assembly and leverage these concepts by connecting them to recent findings that implicate specific types of condensates and IDPs/IDRs in important cellular level processes and molecular functions in bacterial systems.