scholarly journals Composition dependent phase separation underlies directional flux through the nucleolus

2019 ◽  
Author(s):  
Joshua A. Riback ◽  
Lian Zhu ◽  
Mylene C. Ferrolino ◽  
Michele Tolbert ◽  
Diana M. Mitrea ◽  
...  
Keyword(s):  
Phase Separation ◽  
Driving Forces ◽  
Deep Understanding ◽  
Interaction Network ◽  
Intrinsically Disordered Protein ◽  
Model Systems ◽  
Cajal Bodies ◽  
Intrinsically Disordered ◽  
Heterotypic Interactions ◽  
Multicomponent Nature

AbstractIntracellular bodies such as nucleoli, Cajal bodies, and various signaling assemblies, represent membraneless organelles, or condensates, that form via liquid-liquid phase separation (LLPS)1,2. Biomolecular interactions, particularly homotypic interactions mediated by self-associating intrinsically disordered protein regions (IDRs), are thought to underlie the thermodynamic driving forces for LLPS, forming condensates that can facilitate the assembly and processing of biochemically active complexes, such as ribosomal subunits within the nucleolus. Simplified model systems3–6 have led to the concept that a single fixed saturation concentration (Csat) is a defining feature of endogenous LLPS7–9, and has been suggested as a mechanism for intracellular concentration buffering2,7,8,10. However, the assumption of a fixed Csat remains largely untested within living cells, where the richly multicomponent nature of condensates could complicate this simple picture. Here we show that heterotypic multicomponent interactions dominate endogenous LLPS, and give rise to nucleoli and other condensates that do not exhibit a fixed Csat. As the concentration of individual components is varied, their partition coefficients change, in a manner that can be used to extract thermodynamic interaction energies, that we interpret within a framework we term polyphasic interaction thermodynamic analysis (PITA). We find that heterotypic interactions between protein and RNA components stabilize a variety of archetypal intracellular condensates, including the nucleolus, Cajal bodies, stress granules, and P bodies. These findings imply that the composition of condensates is finely tuned by the thermodynamics of the underlying biomolecular interaction network. In the context of RNA processing condensates such as the nucleolus, this stoichiometric self-tuning manifests in selective exclusion of fully-assembled RNP complexes, providing a thermodynamic basis for vectorial ribosomal RNA (rRNA) flux out of the nucleolus. The PITA methodology is conceptually straightforward and readily implemented, and it can be broadly utilized to extract thermodynamic parameters from microscopy images. These approaches pave the way for a deep understanding of the thermodynamics of multi-component intracellular phase behavior and its interplay with nonequilibrium activity characteristic of endogenous condensates.

scholarly journals Phase Transition of RNA-protein Complexes into Ordered Hollow Condensates

2020 ◽  
Author(s):  
Ibraheem Alshareedah ◽  
Mahdi Muhammad Moosa ◽  
Muralikrishna Raju ◽  
Davit Potoyan ◽  
Priya R. Banerjee
Keyword(s):  
Phase Separation ◽  
Protein Complexes ◽  
Intrinsically Disordered Protein ◽  
Liquid Droplets ◽  
Stimuli Responsive ◽  
Isotropic Liquid ◽  
Intrinsically Disordered ◽  
Heterotypic Interactions ◽  
Rna Complexes ◽  
Rna Protein Complexes

AbstractLiquid-liquid phase separation of multivalent intrinsically disordered protein-RNA complexes is ubiquitous in both natural and biomimetic systems. So far, isotropic liquid droplets are the most commonly observed topology of RNA-protein condensates in experiments and simulations. Here, by systematically studying the phase behavior of RNA-protein complexes across varied mixture compositions, we report a hollow vesicle-like condensate phase of nucleoprotein assemblies that is distinct from RNA-protein droplets. We show that these vesicular condensates are stable at specific mixture compositions and concentration regimes within the phase diagram and are formed through the phase separation of anisotropic protein-RNA complexes. Similar to membranes composed of amphiphilic lipids, these nucleoprotein-RNA vesicular membranes exhibit local ordering, size-dependent permeability, and selective encapsulation capacity without sacrificing their dynamic formation and dissolution in response to physicochemical stimuli. Our findings suggest that protein-RNA complexes can robustly create lipid-free vesicle-like enclosures by phase separation.Significance statementVesicular assemblies play crucial roles in subcellular organization as well as in biotechnological applications. Classically, the ability to form such assemblies were primarily assigned to lipids and lipid-like amphiphilic molecules. Here, we show that disordered RNA-protein complexes can form vesicle-like ordered assemblies at disproportionate mixture compositions. We also show that the ability to form vesicular assemblies is generic to multi-component systems where phase separation is driven by heterotypic interactions. We speculate that such vesicular assemblies play crucial roles in the formation of dynamic multi-layered subcellular membrane-less organelles and can be utilized to fabricate novel stimuli-responsive microscale systems.

scholarly journals Distinct regions of the intrinsically disordered protein MUT-16 mediate assembly of a small RNA amplification complex and promote phase separation of Mutator foci

PLoS Genetics ◽  
2018 ◽  
Vol 14 (7) ◽  
pp. e1007542 ◽  
Author(s):  
Celja J. Uebel ◽  
Dorian C. Anderson ◽  
Lisa M. Mandarino ◽  
Kevin I. Manage ◽  
Stephan Aynaszyan ◽  
...  
Keyword(s):  
Phase Separation ◽  
Small Rna ◽  
Intrinsically Disordered Protein ◽  
Rna Amplification ◽  
Intrinsically Disordered ◽  
Disordered Protein

scholarly journals Phase transition of RNA−protein complexes into ordered hollow condensates

2020 ◽  
Vol 117 (27) ◽  
pp. 15650-15658 ◽  
Author(s):  
Ibraheem Alshareedah ◽  
Mahdi Muhammad Moosa ◽  
Muralikrishna Raju ◽  
Davit A. Potoyan ◽  
Priya R. Banerjee
Keyword(s):  
Phase Separation ◽  
Protein Complexes ◽  
Intrinsically Disordered Protein ◽  
Liquid Droplets ◽  
Isotropic Liquid ◽  
Size Dependent ◽  
Intrinsically Disordered ◽  
Condensate Phase ◽  
Rna Complexes ◽  
Rna Protein Complexes

Liquid−liquid phase separation of multivalent intrinsically disordered protein−RNA complexes is ubiquitous in both natural and biomimetic systems. So far, isotropic liquid droplets are the most commonly observed topology of RNA−protein condensates in experiments and simulations. Here, by systematically studying the phase behavior of RNA−protein complexes across varied mixture compositions, we report a hollow vesicle-like condensate phase of nucleoprotein assemblies that is distinct from RNA−protein droplets. We show that these vesicular condensates are stable at specific mixture compositions and concentration regimes within the phase diagram and are formed through the phase separation of anisotropic protein−RNA complexes. Similar to membranes composed of amphiphilic lipids, these nucleoprotein−RNA vesicular membranes exhibit local ordering, size-dependent permeability, and selective encapsulation capacity without sacrificing their dynamic formation and dissolution in response to physicochemical stimuli. Our findings suggest that protein−RNA complexes can robustly create lipid-free vesicle-like enclosures by phase separation.

Phospho-dependent phase separation of FMRP and CAPRIN1 recapitulates regulation of translation and deadenylation

Science ◽  
2019 ◽  
Vol 365 (6455) ◽  
pp. 825-829 ◽  
Author(s):  
Tae Hun Kim ◽  
Brian Tsang ◽  
Robert M. Vernon ◽  
Nahum Sonenberg ◽  
Lewis E. Kay ◽  
...  
Keyword(s):  
Phase Separation ◽  
Rna Processing ◽  
Intrinsically Disordered Protein ◽  
Resonance Spectroscopy ◽  
Specific Interactions ◽  
Intrinsically Disordered ◽  
Functional Consequences ◽  
Translational Regulators

Membraneless organelles involved in RNA processing are biomolecular condensates assembled by phase separation. Despite the important role of intrinsically disordered protein regions (IDRs), the specific interactions underlying IDR phase separation and its functional consequences remain elusive. To address these questions, we used minimal condensates formed from the C-terminal disordered regions of two interacting translational regulators, FMRP and CAPRIN1. Nuclear magnetic resonance spectroscopy of FMRP-CAPRIN1 condensates revealed interactions involving arginine-rich and aromatic-rich regions. We found that different FMRP serine/threonine and CAPRIN1 tyrosine phosphorylation patterns control phase separation propensity with RNA, including subcompartmentalization, and tune deadenylation and translation rates in vitro. The resulting evidence for residue-specific interactions underlying co–phase separation, phosphorylation-modulated condensate architecture, and enzymatic activity within condensates has implications for how the integration of signaling pathways controls RNA processing and translation.

scholarly journals A modular platform for engineering function of natural and synthetic biomolecular condensates

2021 ◽  
Author(s):  
Keren Lasker ◽  
Steven Boeynaems ◽  
Vinson Lam ◽  
Emma Stainton ◽  
Maarten Jacquemyn ◽  
...  
Keyword(s):  
Phase Separation ◽  
Material Properties ◽  
Asymmetric Cell Division ◽  
Caulobacter Crescentus ◽  
Intrinsically Disordered Protein ◽  
Emergent Properties ◽  
Physiological Importance ◽  
Intrinsically Disordered ◽  
Disordered Protein ◽  
Modular Platform

AbstractPhase separation is emerging as a universal principle for how cells use dynamic subcompartmentalization to organize biochemical reactions in time and space1,2. Yet, whether the emergent physical properties of these biomolecular condensates are important for their biological function remains unclear. The intrinsically disordered protein PopZ forms membraneless condensates at the poles of the bacterium Caulobacter crescentus and selectively sequesters kinase-signaling cascades to regulate asymmetric cell division3–5. By dissecting the molecular grammar underlying PopZ phase separation, we find that unlike many eukaryotic examples, where unstructured regions drive condensation6,7, a structured domain of PopZ drives condensation, while conserved repulsive features of the disordered region modulate material properties. By generating rationally designed PopZ mutants, we find that the exact material properties of PopZ condensates directly determine cellular fitness, providing direct evidence for the physiological importance of the emergent properties of biomolecular condensates. Our work codifies a clear set of design principles illuminating how sequence variation in a disordered domain alters the function of a widely conserved bacterial condensate. We used these insights to repurpose PopZ as a modular platform for generating synthetic condensates of tunable function in human cells.

scholarly journals The disordered P granule protein LAF-1 drives phase separation into droplets with tunable viscosity and dynamics

2015 ◽  
Vol 112 (23) ◽  
pp. 7189-7194 ◽  
Author(s):  
Shana Elbaum-Garfinkle ◽  
Younghoon Kim ◽  
Krzysztof Szczepaniak ◽  
Carlos Chih-Hsiung Chen ◽  
Christian R. Eckmann ◽  
...  
Keyword(s):  
Phase Separation ◽  
Phase Boundary ◽  
Single Molecule ◽  
Protein Interactions ◽  
Driving Forces ◽  
Early Embryo ◽  
P Granules ◽  
Intrinsically Disordered

P granules and other RNA/protein bodies are membrane-less organelles that may assemble by intracellular phase separation, similar to the condensation of water vapor into droplets. However, the molecular driving forces and the nature of the condensed phases remain poorly understood. Here, we show that the Caenorhabditis elegans protein LAF-1, a DDX3 RNA helicase found in P granules, phase separates into P granule-like droplets in vitro. We adapt a microrheology technique to precisely measure the viscoelasticity of micrometer-sized LAF-1 droplets, revealing purely viscous properties highly tunable by salt and RNA concentration. RNA decreases viscosity and increases molecular dynamics within the droplet. Single molecule FRET assays suggest that this RNA fluidization results from highly dynamic RNA–protein interactions that emerge close to the droplet phase boundary. We demonstrate than an N-terminal, arginine/glycine rich, intrinsically disordered protein (IDP) domain of LAF-1 is necessary and sufficient for both phase separation and RNA–protein interactions. In vivo, RNAi knockdown of LAF-1 results in the dissolution of P granules in the early embryo, with an apparent submicromolar phase boundary comparable to that measured in vitro. Together, these findings demonstrate that LAF-1 is important for promoting P granule assembly and provide insight into the mechanism by which IDP-driven molecular interactions give rise to liquid phase organelles with tunable properties.

scholarly journals Viroplasms: Assembly and Functions of Rotavirus Replication Factories

Viruses ◽  
2021 ◽  
Vol 13 (7) ◽  
pp. 1349
Author(s):  
Guido Papa ◽  
Alexander Borodavka ◽  
Ulrich Desselberger
Keyword(s):  
Phase Separation ◽  
Liquid Phase ◽  
Reverse Genetics ◽  
Intrinsically Disordered Protein ◽  
Structural Proteins ◽  
Virion Assembly ◽  
Intrinsically Disordered ◽  
Disordered Protein ◽  
Infected Cells ◽  
Liquid Liquid Phase Separation

Viroplasms are cytoplasmic, membraneless structures assembled in rotavirus (RV)-infected cells, which are intricately involved in viral replication. Two virus-encoded, non-structural proteins, NSP2 and NSP5, are the main drivers of viroplasm formation. The structures (as far as is known) and functions of these proteins are described. Recent studies using plasmid-only-based reverse genetics have significantly contributed to elucidation of the crucial roles of these proteins in RV replication. Thus, it has been recognized that viroplasms resemble liquid-like protein–RNA condensates that may be formed via liquid–liquid phase separation (LLPS) of NSP2 and NSP5 at the early stages of infection. Interactions between the RNA chaperone NSP2 and the multivalent, intrinsically disordered protein NSP5 result in their condensation (protein droplet formation), which plays a central role in viroplasm assembly. These droplets may provide a unique molecular environment for the establishment of inter-molecular contacts between the RV (+)ssRNA transcripts, followed by their assortment and equimolar packaging. Future efforts to improve our understanding of RV replication and genome assortment in viroplasms should focus on their complex molecular composition, which changes dynamically throughout the RV replication cycle, to support distinct stages of virion assembly.

scholarly journals Negative regulation of a ribonucleoprotein condensate driven by dilute phase oligomerization

2021 ◽  
Author(s):  
Ian Seim ◽  
Ammon E. Posey ◽  
Wilton T. Snead ◽  
Benjamin M. Stormo ◽  
Daphne Klotsa ◽  
...  
Keyword(s):  
Phase Separation ◽  
Phase Behavior ◽  
Material Properties ◽  
Driving Forces ◽  
Negative Regulation ◽  
Regulatory Control ◽  
Sequence Motif ◽  
Rich Region ◽  
Intrinsically Disordered ◽  
Intrinsically Disordered Regions

AbstractRibonucleoprotein bodies are exemplars of membraneless biomolecular condensates that can form via spontaneous or driven phase transitions. The fungal protein Whi3 forms compositionally distinct ribonucleoprotein condensates that are implicated in key processes such as cell-cycle control and cell polarity. Whi3 has a modular architecture that includes a Q-rich intrinsically disordered region and a tandem RNA recognition module. Here, we uncover localized order-to-disorder transitions within a 21-residue stretch of the Q-rich region. This region, which can form alpha-helical conformations, is shown to modulate protein density within Whi3-RNA condensates by driving dilute phase oligomerization. Specifically, enhancing helicity within this region enhances oligomerization in the dilute phase. This weakens the associations among disordered Q-rich regions thereby diluting the concentration of Whi3 in condensates. The opposite behavior is observed when helicity within the 21-residue stretch of the Q-rich region is abrogated. Thus, dilute phase oligomers, driven by a specific sequence motif, lead to negative regulation of the stoichiometry of protein versus RNA in the dense phase. Our findings stand in contrast to other systems where oligomerization is known to enhance the drive for phase separation. Our results highlight distinctive regulatory effects over phase behavior due to local order-to-disorder transitions within intrinsically disordered regions. This provides a way to leverage molecular scale conformational preferences and coupled intermolecular associations to regulate mesoscale phase behavior and material properties of condensates.SignificanceA large sub-class of biomolecular condensates are linked to RNA regulation and known as ribonucleoprotein (RNP) bodies. While extensive work has identified driving forces of biomolecular condensates, relatively little is known about negative regulation of assembly. Here, using a fungal RNP component, Whi3, we show that its intrinsically-disordered, Q-rich region exerts regulatory control over condensate formation through a cryptic helical region that enables the formation 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 equilibria can enable control over micron-scale phase equilibria.

scholarly journals Interplay Between Short-range Attraction and Long-range Repulsion Controls Reentrant Liquid Condensation of Ribonucleoprotein-RNA Complexes

2019 ◽  
Author(s):  
Ibraheem Alshareedah ◽  
Taranpreet Kaur ◽  
Jason Ngo ◽  
Hannah Seppala ◽  
Liz-Audrey Djomnang Kounatse ◽  
...  
Keyword(s):  
Phase Separation ◽  
Long Range ◽  
Short Range ◽  
Rna Binding ◽  
Driving Forces ◽  
Low Complexity ◽  
Model Systems ◽  
Supramolecular Organization ◽  
Cluster Phase ◽  
Rna Complexes

AbstractIn eukaryotic cells, ribonucleoproteins (RNPs) form mesoscale condensates by liquid-liquid phase separation that play essential roles in subcellular dynamic compartmentalization. The formation and dissolution of many RNP condensates are finely dependent on the RNA-to-RNP ratio, giving rise to a window-like phase separation behavior. This is commonly referred to as reentrant liquid condensation (RLC). Here, using RNP-inspired polypeptides with low-complexity RNA-binding sequences as well as the C-terminal disordered domain of the ribonucleoprotein FUS as model systems, we investigate the molecular driving forces underlying this non-monotonous phase transition. We show that an interplay between short-range cation-π attractions and long-range electrostatic forces governs the heterotypic RLC of RNP-RNA complexes. Short-range attractions, which can be encoded by both polypeptide chain primary sequence and nucleic acid base sequence, are activated by RNP-RNA condensate formation. After activation, the short-range forces regulate material properties of polypeptide-RNA condensates and subsequently oppose their reentrant dissolution. In the presence of excess RNA, a competition between short-range attraction and long-range electrostatic repulsion drives the formation of a colloid-like cluster phase. With increasing short-range attraction, the fluid dynamics of the cluster phase is arrested, leading to the formation of a colloidal gel. Our results reveal that phase behavior, supramolecular organization, and material states of RNP-RNA assemblies are controlled by a dynamic interplay between molecular interactions at different length scales.

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