Membraneless Assemblies Research Articles

Phase Separation and Aggregation of a Globular Folded Protein Small Ubiquitin-like Modifier 1 (SUMO1).

Liquid-liquid phase separation (LLPS) plays a crucial role in cellular organization, primarily driven by intrinsically disordered proteins (IDPs) leading to the formation of biomolecular condensates. A folded protein SUMO that post-translationally modifies cellular proteins has recently emerged as a regulator of LLPS. Given its compact structure and limited flexibility, the precise role of SUMO in condensate formation remains to be investigated. Here, we show the rapid phase separation of SUMO1 into micrometer-sized liquid-like condensates in inert crowders under physiological conditions. Subsequent time-dependent conformational changes and aggregation are probed by label-free methods (tryptophan fluorescence and Raman spectroscopy). Remarkably, experiments on a SUMO1 variant lacking the N-terminal disordered region further corroborate the role of its structured part in phase transitions. Our findings highlight the potential of folded proteins to engage in LLPS and emphasize further investigation into the influence of the SUMO tag on IDPs associated with membrane-less assemblies in cells.

Poly(A)-binding protein is an ataxin-2 chaperone that regulates biomolecular condensates

Biomolecular condensation underlies the biogenesis of an expanding array of membraneless assemblies, including stress granules (SGs), which form under a variety of cellular stresses. Advances have been made in understanding the molecular grammar of a few scaffold proteins that make up these phases, but how the partitioning of hundreds of SG proteins is regulated remains largely unresolved. While investigating the rules that govern the condensation of ataxin-2, an SG protein implicated in neurodegenerative disease, we unexpectedly identified a short 14 aa sequence that acts as a condensation switch and is conserved across the eukaryote lineage. We identify poly(A)-binding proteins as unconventional RNA-dependent chaperones that control this regulatory switch. Our results uncover a hierarchy of cis and trans interactions that fine-tune ataxin-2 condensation and reveal an unexpected molecular function for ancient poly(A)-binding proteins as regulators of biomolecular condensate proteins. These findings may inspire approaches to therapeutically target aberrant phases in disease.

Membraneless Compartmentalization of Nuclear Assembly Sites during Murine Cytomegalovirus Infection.

Extensive reorganization of infected cells and the formation of large structures known as the nuclear replication compartment (RC) and cytoplasmic assembly compartment (AC) is a hallmark of beta-herpesvirus infection. These restructurings rely on extensive compartmentalization of the processes that make up the virus manufacturing chain. Compartmentalization of the nuclear processes during murine cytomegalovirus (MCMV) infection is not well described. In this study, we visualized five viral proteins (pIE1, pE1, pM25, pm48.2, and pM57) and replicated viral DNA to reveal the nuclear events during MCMV infection. As expected, these events can be matched with those described for other beta and alpha herpesviruses and contribute to the overall picture of herpesvirus assembly. Imaging showed that four viral proteins (pE1, pM25, pm48.2, and pM57) and replicated viral DNA condense in the nucleus into membraneless assemblies (MLAs) that undergo a maturation sequence to form the RC. One of these proteins (pM25), which is also expressed in a cytoplasmic form (pM25l), showed similar MLAs in the AC. Bioinformatics tools for predicting biomolecular condensates showed that four of the five proteins had a high propensity for liquid-liquid phase separation (LLPS), suggesting that LLPS may be a mechanism for compartmentalization within RC and AC. Examination of the physical properties of MLAs formed during the early phase of infection by 1,6-hexanediol treatment in vivo revealed liquid-like properties of pE1 MLAs and more solid-like properties of pM25 MLAs, indicating heterogeneity of mechanisms in the formation of virus-induced MLAs. Analysis of the five viral proteins and replicated viral DNA shows that the maturation sequence of RC and AC is not completed in many cells, suggesting that virus production and release is carried out by a rather limited number of cells. This study thus lays the groundwork for further investigation of the replication cycle of beta-herpesviruses, and the results should be incorporated into plans for high-throughput and single-cell analytic approaches.

Biomolecular condensates: Formation mechanisms, biological functions, and therapeutic targets.

Biomolecular condensates are cellular structures composed of membraneless assemblies comprising proteins or nucleic acids. The formation of these condensates requires components to change from a state of solubility separation from the surrounding environment by undergoing phase transition and condensation. Over the past decade, it has become widely appreciated that biomolecular condensates are ubiquitous in eukaryotic cells and play a vital role in physiological and pathological processes. These condensates may provide promising targets for the clinic research. Recently, a series of pathological and physiological processes have been found associated with the dysfunction of condensates, and a range of targets and methods have been demonstrated to modulate the formation of these condensates. A more extensive description of biomolecular condensates is urgently needed for the development of novel therapies. In this review, we summarized the current understanding of biomolecular condensates and the molecular mechanisms of their formation. Moreover, we reviewed the functions of condensates and therapeutic targets for diseases. We further highlighted the available regulatory targets and methods, discussed the significance and challenges of targeting these condensates. Reviewing the latest developments in biomolecular condensate research could be essential in translating our current knowledge on the use of condensates for clinical therapeutic strategies.

New insights into the centrosome-associated spliceosome components as regulators of ciliogenesis and tissue identity.

Biomolecular condensates are membrane-less assemblies of proteins and nucleic acids. Centrosomes are biomolecular condensates that play a crucial role in nuclear division, cytoskeletal remodeling, and cilia formation in animal cells. Spatial omics technology is providing new insights into the dynamic exchange of spliceosome components between the nucleus and the centrosome/cilium. Intriguingly, centrosomes are emerging as cytoplasmic sites for information storage, enriched with RNA molecules and RNA-processing proteins. Furthermore, growing evidence supports the view that nuclear spliceosome components assembled at the centrosome function as potential coordinators of splicing subprograms, pluripotency, and cell differentiation. In this article, we first discuss the current understanding of the centrosome/cilium complex, which controls both stem cell differentiation and pluripotency. We next explore the molecular mechanisms that govern splicing factor assembly and disassembly around the centrosome and examine how RNA processing pathways contribute to ciliogenesis. Finally, we discuss numerous unresolved compelling questions regarding the centrosome-associated spliceosome components and transcript variants within the cytoplasm as sources of RNA-based secondary messages in the regulation of cell identity and cell fate determination. This article is categorized under: RNA-Based Catalysis > RNA Catalysis in Splicing and Translation RNA Interactions with Proteins and Other Molecules > RNA-Protein Complexes RNA Processing > Splicing Regulation/Alternative Splicing RNA Processing > RNA Processing.

An in vitro Assay to Probe the Formation of Biomolecular Condensates.

Biomolecular condensates are membrane-less assemblies of proteins and nucleic acids formed through liquid-liquid phase separation (LLPS). These assemblies are known to temporally and spatially regulate numerous biological activities and cellular processes in plants and animals. In vitro phase separation assay using recombinant proteins represents one of the standard ways to examine the properties of proteins undergoing LLPS. Here, we present a detailed protocol to investigate in vitro LLPS using in vitro expressed and purified recombinant proteins.

Lipid Surfaces and Glutamate Anions Enhance Formation of Dynamic Biomolecular Condensates Containing Bacterial Cell Division Protein FtsZ and Its DNA-Bound Regulator SlmA.

Dynamic biomolecular condensates formed by liquid-liquid phase separation can regulate the spatial and temporal organization of proteins, thus modulating their functional activity in cells. Previous studies showed that the cell division protein FtsZ from Escherichia coli formed dynamic phase-separated condensates with nucleoprotein complexes containing the FtsZ spatial regulator SlmA under crowding conditions, with potential implications for condensate-mediated spatiotemporal control of FtsZ activity in cell division. In the present study, we assessed formation of these condensates in the presence of lipid surfaces and glutamate ions to better approximate the E. coli intracellular environment. We found that potassium glutamate substantially promoted the formation of FtsZ-containing condensates when compared to potassium chloride in crowded solutions. These condensates accumulated on supported lipid bilayers and eventually fused, resulting in a time-dependent increase in the droplet size. Moreover, the accumulated condensates were dynamic, capturing protein from the external phase. FtsZ partitioned into the condensates at the lipid surface only in its guanosine diphosphate (GDP) form, regardless of whether it came from FtsZ polymer disassembly upon guanosine triphosphate (GTP) exhaustion. These results provide insights into the behavior of these GTP-responsive condensates in minimal membrane systems, which suggest how these membraneless assemblies may tune critical bacterial division events during the cell cycle.

An Introduction to the Stickers-and-Spacers Framework as Applied to Biomolecular Condensates.

Cellular organization is determined by a combination of membrane-bound and membrane-less biomolecular assemblies that range from clusters of tens of molecules to micrometer-sized cellular bodies. Over the last decade, membrane-less assemblies have come to be referred to as biomolecular condensates, reflecting their ability to condense specific molecules with respect to the remainder of the cell. In many cases, the physics of phase transitions provides a conceptual framework and a mathematical toolkit to describe the assembly, maintenance, and dissolution of biomolecular condensates. Among the various quantitative and qualitative models applied to understand intracellular phase transitions, the stickers-and-spacers framework offers an intuitive yet rigorous means to map biomolecular sequences and structure to the driving forces needed for higher-order assembly. This chapter introduces the fundamental concepts behind the stickers-and-spacers model, considers its application to different biological systems, and discusses limitations and misconceptions around the model.

Modulating biomolecular condensates: a novel approach to drug discovery.

In the past decade, membraneless assemblies known as biomolecular condensates have been reported to play key roles in many cellular functions by compartmentalizing specific proteins and nucleic acids in subcellular environments with distinct properties. Furthermore, growing evidence supports the view that biomolecular condensates often form by phase separation, in which a single-phase system demixes into a two-phase system consisting of a condensed phase and a dilute phase of particular biomolecules. Emerging understanding of condensate function in normal and aberrant cellular states, and of the mechanisms of condensate formation, is providing new insights into human disease and revealing novel therapeutic opportunities. In this Perspective, we propose that such insights could enable a previously unexplored drug discovery approach based on identifying condensate-modifying therapeutics (c-mods), and we discuss the strategies, techniques and challenges involved.

EIF3a Destabilization and TDP-43 Alter Dynamics of Heat-Induced Stress Granules.

Stress granules (SGs) are membrane-less assemblies arising upon various stresses in eukaryotic cells. They sequester mRNAs and proteins from stressful conditions and modulate gene expression to enable cells to resume translation and growth after stress relief. SGs containing the translation initiation factor eIF3a/Rpg1 arise in yeast cells upon robust heat shock (HS) at 46 °C only. We demonstrate that the destabilization of Rpg1 within the PCI domain in the Rpg1-3 variant leads to SGs assembly already at moderate HS at 42 °C. These are bona fide SGs arising upon translation arrest containing mRNAs, which are components of the translation machinery, and associating with P-bodies. HS SGs associate with endoplasmatic reticulum and mitochondria and their contact sites ERMES. Although Rpg1-3-labeled SGs arise at a lower temperature, their disassembly is delayed after HS at 46 °C. Remarkably, the delayed disassembly of HS SGs after the robust HS is reversed by TDP-43, which is a human protein connected with amyotrophic lateral sclerosis. TDP-43 colocalizes with HS SGs in yeast cells and facilitates cell regrowth after the stress relief. Based on our results, we propose yeast HS SGs labeled by Rpg1 and its variants as a novel model system to study functions of TDP-43 in stress granules disassembly.

The Dynamic Composition of an Archetypal Plant Condensate Highlights a Tug-of-War between Condensates and Cell Vertex

Cellular condensates are usually ribonucleoprotein membrane-less assemblies that can shuttle between liquid or less fluid states. These phase transitions could impact a broad range of biological outcomes. Here, by developing proximity-biotinylation approaches, we unveil the composition of the condensate known as processing bodies (PBs) in the model plant Arabidopsis. We show that PBs comprise both conserved and dynamic networks with unexpected connections to membrane subdomains. We study further one of these networks and discover a cell vertex-defining module with membrane remodeling properties. This module functions through a liquid/solid-like phase transition that impacts growth. The underlying mechanism is developmentally regulated by a tug-of-war between PBs and the plasma membrane instructed by a phospho-switch. We thus provide a framework for the elucidation of condensates in plants and describe a growth module based on phase transitions of condensates at a membranous interface.

Poly(A)-Binding Protein Is an Ataxin-2 Chaperone That Emulsifies Biomolecular Condensates

Biomolecular condensation underlies the biogenesis of an array of membraneless assemblies, including stress granules (SGs) which form under a variety of cellular stresses. Advances have been made in understanding the molecular grammar that dictates the behavior of key scaffold proteins that make up these phases but how the partitioning of hundreds of other SG proteins is regulated remains largely unresolved. While investigating the rules that govern the condensation of ataxin-2, a SG protein implicated in neurodegenerative disease, we unexpectedly identified a short 14aa sequence that acts as an ataxin-2 condensation switch and is conserved across eukaryotes. We identify poly(A)-binding proteins as unconventional RNA-dependent chaperones that control this regulatory switch. Our results uncover a hierarchy of cis and trans interactions that fine-tune ataxin-2 condensation and reveal a new molecular function for ancient poly(A)-binding proteins as biomolecular condensate emulsifiers. These findings may inspire novel approaches to therapeutically target aberrant phases in disease.

Replication Compartments of DNA Viruses in the Nucleus: Location, Location, Location.

DNA viruses that replicate in the nucleus encompass a range of ubiquitous and clinically important viruses, from acute pathogens to persistent tumor viruses. These viruses must co-opt nuclear processes for the benefit of the virus, whilst evading host processes that would otherwise attenuate viral replication. Accordingly, DNA viruses induce the formation of membraneless assemblies termed viral replication compartments (VRCs). These compartments facilitate the spatial organization of viral processes and regulate virus–host interactions. Here, we review advances in our understanding of VRCs. We cover their initiation and formation, their function as the sites of viral processes, and aspects of their composition and organization. In doing so, we highlight ongoing and emerging areas of research highly pertinent to our understanding of nuclear-replicating DNA viruses.

Implications of kappa-casein evolutionary diversity for the self-assembly and aggregation of casein micelles.

Milk alpha-, beta- and kappa-casein proteins assemble into casein micelles in breast epithelial cells. The glycomacropeptide (GMP) tails of kappa-casein that extend from the surface of the micelle are key to assembly and aggregation. Aggregation is triggered by stomach pepsin cleavage of GMP from para-kappa-casein (PKC). While one casein micelle model emphasizes the importance of hydrophobic interactions, another focuses on polar residues. We performed an evolutionary analysis of kappa-casein primary sequence and predicted features that potentially impact on protein interactions. We noted more rapid change in the earlier period (166 to 60 Ma). Pepsin and plasmin cleavage sites were avoided in the GMP, which may partly explain its amino acid composition. Short tandem repeats have led to modest expansions of PKC, and to large GMP expansions, suggesting the GMP is less length constrained. Amino acid compositional constraints were assessed across species. Polarity and hydrophobicity properties were insufficient to explain differences between PKC and GMP. Among polar residues, threonine dominates the GMP, compared to serine, probably reflecting its preference for O-glycosylation over phosphorylation. Glutamine, enriched in the bovine PQ-rich region, is not positionally conserved in other species. Among hydrophobic residues, isoleucine is clearly preferred over leucine in the GMP, and patches of hydrophobicity are not markedly positionally conserved. PKC tyrosine and charged residues showed stronger conservation of position, suggesting a role for pi-interactions, seen in other structurally dynamic protein membraneless assemblies. Independent acquisitions of cysteines are consistent with a trend of increasing stabilization of multimers by covalent disulphide bonds, over evolutionary time. In conclusion, kappa-casein compositional and positional constraints appear to be influenced by modification preferences, protease evasion and protein–protein interactions.

Phase separation in biology and disease-a symposium report.

Phase separation of multivalent protein and RNA molecules enables cells the formation of reversible nonstoichiometric, membraneless assemblies. These assemblies, referred to as biomolecular condensates, help with the spatial organization and compartmentalization of cellular matter. Each biomolecular condensate is defined by a distinct macromolecular composition. Distinct condensates have distinct preferential locations within cells, and they are associated with distinct biological functions, including DNA replication, RNA metabolism, signal transduction, synaptic transmission, and stress response. Several proteins found in biomolecular condensates have also been implicated in disease, including Huntington's disease, amyotrophic lateral sclerosis, and several types of cancer. Disease-associated mutations in these proteins have been found to affect the material properties of condensates as well as the driving forces for phase separation. Understanding the intrinsic and extrinsic forces driving the formation and dissolution of biomolecular condensates via spontaneous and driven phase separation is an important step in understanding the processes associated with biological regulation in health and disease.

Dynamic fibrils drive protein assembly

Structural Biology A low-complexity domain of the protein FUS plays a role in forming RNA granules. Luo et al. identify and structurally characterize two reversible amyloid cores (RACs) in this domain. In stable amyloid fibrils, β-strands stack to form β-sheets that pack tightly and exclude water. In contrast, RAC1 forms a kinked coil that stacks along the fibril axis; two such layers interact through tyrosine ring stacking. RAC2 forms β-sheets, but with water molecules between mating sheets. It is already established that the LARK (low-complexity amyloid-like kinked segment) may be broadly involved in membraneless assemblies. The RAC2 structure suggests that reversible fibril formation may occur without kinking. Nat. Struct. Mol. Biol. 25 , 341 (2018).

Atomic structures of low-complexity protein segments reveal kinked β sheets that assemble networks.

Subcellular membraneless assemblies are a reinvigorated area of study in biology, with spirited scientific discussions on the forces between the low-complexity protein domains within these assemblies. To illuminate these forces, we determined the atomic structures of five segments from protein low-complexity domains associated with membraneless assemblies. Their common structural feature is the stacking of segments into kinked β sheets that pair into protofilaments. Unlike steric zippers of amyloid fibrils, the kinked sheets interact weakly through polar atoms and aromatic side chains. By computationally threading the human proteome on our kinked structures, we identified hundreds of low-complexity segments potentially capable of forming such interactions. These segments are found in proteins as diverse as RNA binders, nuclear pore proteins, and keratins, which are known to form networks and localize to membraneless assemblies.

Methods for improving the reliability and information content of chromatographic analyses of biologically active compounds

A series of methods was developed for improving the reliability of chromatographic analyses and to augment their information content and reproducibility. In high-performance liquid chromatography (HPLC) and thin-layer chromatography (TLC) the possibility of utilizing azeotropic and isobaric solvent systems was substantiated. To control variations of the composition of the eluent systems during storage and analysis in TLC and HPLC, gas chromatography (GC) was found to be applicable. To improve the reliability of GC analyses, a membraneless assembly for introduction of samples, which allows false peaks in a chromatogram to be removed, is recommended. In order to augment the reliability and information content of chromatographic analyses of several bioactive compounds, complex chromatographic methods based on GC, TLC and HPLC were found to be desirable.