Project description:Intracellular aggregation of repeat expanded RNA has been implicated in many neurological disorders. Here, we study the role of biomolecular condensates on irreversible RNA clustering. We find that physiologically relevant and disease-associated repeat RNAs spontaneously undergo an age-dependent percolation transition inside multi-component protein-nucleic acid condensates to form nanoscale clusters. Homotypic RNA clusters drive the emergence of multiphasic condensate structures with an RNA-rich solid core surrounded by an RNA-depleted fluid shell. The timescale of the RNA clustering, which drives a liquid-to-solid transition of biomolecular condensates, is determined by the sequence features, stability of RNA secondary structure, and repeat length. Importantly, G3BP1, the core scaffold of stress granules, introduces heterotypic buffering to homotypic RNA-RNA interactions and impedes intra-condensate RNA clustering in an ATP-independent manner. Our work suggests that biomolecular condensates can act as sites for RNA aggregation. It also highlights the functional role of RNA-binding proteins in suppressing aberrant RNA phase transitions.
Project description:The phase separation of biomolecules into biomolecular condensates has emerged as a ubiquitous cellular process. Understanding how intrinsically disordered protein sequence controls condensate formation and material properties has provided fundamental biological insights and led to the development of functional synthetic condensates. While these studies provide a valuable framework to understand subcellular organization via phase separation they have largely ignored the presence of folded domains and their impact on condensate properties. We set out to determine how the distribution of sticker interactions across a globular protein contributes to rheological properties of condensates and to what extent globular protein-containing condensates differ from those formed from two disordered components. We designed three variants of green fluorescent protein with different charge patterning and used dynamic light scattering microrheology to measure the viscoelastic spectrum of coacervates formed with poly-lysine over a timescale of 10-6 to 10 seconds, elucidating the response of protein condensates in this range for the first time. We further showed that the phase behavior and rheological characteristics of the condensates varied as a function of both protein charge distribution and polymer/protein ratio, behavior that was distinct to condensates formed with folded domains. Together, this work enhances our fundamental understanding of dynamic condensed biomaterials across biologically relevant length- and time-scales.
Project description:RNA localization and biomolecular condensate formation are key biological strategies for organizing the cytoplasm and generating cellular polarity. In Xenopus oocytes, RNAs required for germ layer patterning localize in biomolecular condensates, termed Localization bodies (L-bodies). Here, we have used an L-body RNA-binding protein, PTBP3, to test the role of RNA-protein interactions in regulating the biophysical characteristics of L-bodies in vivo and PTBP3-RNA condensates in vitro. Our results reveal that RNA-protein interactions drive recruitment of PTBP3 and localized RNA to L-bodies and that multivalent interactions tune the dynamics of the PTBP3 after localization. In a concentration-dependent manner, RNA becomes non-dynamic and interactions with the RNA determine PTBP3 dynamics within these biomolecular condensates in vivo and in vitro. Importantly, RNA, and not protein, is required for maintenance of the PTBP3-RNA condensates in vitro, pointing to a model where RNA serves as a non-dynamic substructure in these condensates.
Project description:Several RNA binding proteins involved in membraneless organelles can form pathological amyloids associated with neurodegenerative diseases, but the mechanisms of how this aggregation is modulated remain elusive. Here we investigate how heterotypic protein-RNA interactions modulate the condensation and the liquid to amyloid transition of hnRNPA1A, a protein involved in amyothropic lateral sclerosis. In the absence of RNA, formation of condensates promotes hnRNPA1A aggregation and fibrils are localized at the interface of the condensates. Addition of RNA modulates the soluble to amyloid transition of hnRNPA1A according to different pathways depending on RNA/protein stoichiometry. At low RNA concentrations, RNA promotes both condensation and amyloid formation, and the catalytic effect of RNA adds to the role of the interface between the dense and dilute phases. At higher RNA concentrations, condensation is suppressed according to re-entrant phase behaviour but formation of hnRNPA1A amyloids is observed over longer incubation times. Our findings show how heterotypic nucleic acid-protein interactions affect the kinetics and molecular pathways of amyloid formation.
Project description:Biomolecular condensates are cellular compartments that can form by phase separation in the absence of limiting membranes. Studying the P granules of Caenorhabditis elegans, we find that condensate dynamics are regulated by protein clusters that adsorb to the condensate interface. Using in vitro reconstitution, live observations, and theory, we demonstrate that localized assembly of P granules is controlled by MEG-3, an intrinsically disordered protein that forms low dynamic assemblies on P granules. Following classic Pickering emulsion theory, MEG-3 clusters lower surface tension and slow down coarsening. During zygote polarization, MEG-3 recruits the DYRK family kinase MBK-2 to accelerate spatially regulated growth of the P granule emulsion. By tuning condensate-cytoplasm exchange, interfacial clusters regulate the structural integrity of biomolecular condensates, reminiscent of the role of lipid bilayers in membrane-bound organelles.
Project description:Temperature can impact every reaction and molecular interaction essential to a cell. For organisms that cannot regulate their own temperature, a major challenge is how to adapt to temperatures that fluctuate unpredictability and on variable timescales. Biomolecular condensation offers a possible mechanism for encoding temperature-responsiveness and robustness into cell biochemistry and organization. To explore this idea, we examined temperature adaptation in a filamentous-growing fungus called Ashbya gossypii that engages biomolecular condensates containing the RNA-binding protein Whi3 to regulate mitosis and morphogenesis. We collected wild isolates of Ashbya that originate in different climates and found that mitotic asynchrony and polarized growth, which are known to be controlled by the condensation of Whi3, are temperature sensitive. Sequence analysis in the wild strains revealed changes to specific domains within Whi3 known to be important in condensate formation. Using an in vitro condensate reconstitution assay we found that temperature impacts the relative abundance of protein to RNA within condensates and that this directly impacts the material properties of the droplets. Finally, we found that exchanging Whi3 genes between warm and cold isolates was sufficient to rescue some, but not all, condensate-related phenotypes. Together these data demonstrate that material properties of Whi3 condensates are temperature sensitive, that these properties are important for function, and that sequence optimizes properties for a given climate.
Project description:Biomolecular condensates form by phase separation of biological polymers and have important functions in the cell - functions that are inherently connected to their physical properties. A remarkable aspect of such condensates is that their viscoelastic properties can vary by orders of magnitude, but it has remained unclear how these pronounced differences are rooted in the nanoscale dynamics at the molecular level. Here we investigate a series of condensates formed by complex coacervation that span about two orders of magnitude in molecular dynamics, diffusivity, and viscosity. We find that the nanoscale chain dynamics on the nano- to microsecond timescale can be accurately related to both translational diffusion and mesoscale condensate viscosity by analytical relations from polymer physics. Atomistic simulations reveal that the observed differences in friction - a key quantity underlying these relations - are caused by differences in inter-residue contact lifetimes, leading to the vastly different dynamics among the condensates. The rapid exchange of inter-residue contacts we observe may be a general mechanism for preventing dynamic arrest in compartments densely packed with polyelectrolytes, such as the cell nucleus.
Project description:While eukaryotic cells have a myriad of membrane-bound organelles enabling the isolation of different chemical environments, prokaryotic cells lack these defined reaction vessels. Biomolecular condensates-organelles that lack a membrane-provide a strategy for cellular organization without a physical barrier while allowing for the dynamic, responsive organization of the cell. It is well established that intrinsically disordered protein domains drive condensate formation via liquid-liquid phase separation; however, the role of globular protein domains on intracellular phase separation remains poorly understood. We hypothesized that the overall charge of globular proteins would dictate the formation and concentration of condensates and systematically probed this hypothesis with supercharged proteins and nucleic acids in E. coli. Within this study, we demonstrated that condensates form via electrostatic interactions between engineered proteins and RNA and that these condensates are dynamic and only enrich specific nucleic acid and protein components. Herein, we propose a simple model for the phase separation based on protein charge that can be used to predict intracellular condensate formation. With these guidelines, we have paved the way to designer functional synthetic membraneless organelles with tunable control over globular protein function.
Project description:Proteins containing both intrinsically disordered regions (IDRs) and RNA binding domains (RBDs) can phase separate in vitro, forming bodies similar to cellular biomolecular condensates. However, how IDR and RBD domains contribute to in vivo recruitment of proteins to biomolecular condensates remains poorly understood. Here, we analyzed the roles of IDRs and RBDs in L-bodies, biomolecular condensates present in Xenopus oocytes. We show that a cytoplasmic isoform of hnRNPAB, which contains two RBDs and an IDR, is highly enriched in L-bodies. While both of these domains contribute to hnRNPAB self-association and phase separation in vitro and mediate enrichment into L-bodies in oocytes, neither the RBDs nor the IDR replicate the localization of full-length hnRNPAB. Our results suggest a model where the combined effects of the IDR and RBDs regulate hnRNPAB partitioning into L-bodies. This model likely has widespread applications as proteins containing RBD and IDR domains are common biomolecular condensate residents.
Project description:Evidence accumulated over the past decade provides support for liquid-liquid phase separation as the mechanism underlying the formation of biomolecular condensates, which include not only 'membraneless' organelles such as nucleoli and RNA granules, but additional assemblies involved in transcription, translation and signaling. Understanding the molecular mechanisms of condensate function requires knowledge of the structures of their constituents. Current knowledge suggests that structures formed via multivalent domain-motif interactions remain largely unchanged within condensates. Two different viewpoints exist regarding structures of disordered low-complexity domains within condensates; one argues that low-complexity domains remain largely disordered in condensates and their multivalency is encoded in short motifs called 'stickers', while the other argues that the sequences form cross-β structures resembling amyloid fibrils. We review these viewpoints and highlight outstanding questions that will inform structure-function relationships for biomolecular condensates.