Condensation Aggregation Research Articles

Protein Condensate Formation via Controlled Multimerization of Intrinsically Disordered Sequences.

Many proteins harboring low complexity or intrinsically disordered sequences (IDRs) are capable of undergoing liquid-liquid phase separation to form mesoscale condensates that function as biochemical niches with the ability to concentrate or sequester macromolecules and regulate cellular activity. Engineered disordered proteins have been used to generate programmable synthetic membraneless organelles in cells. Phase separation is governed by the strength of interactions among polypeptides with multivalency enhancing phase separation at lower concentrations. Previously, we and others demonstrated enzymatic control of IDR valency from multivalent precursors to dissolve condensed phases. Here, we develop noncovalent strategies to multimerize an individual IDR, the RGG domain of LAF-1, using protein interaction domains to regulate condensate formation in vitro and in living cells. First, we characterize modular dimerization of RGG domains at either terminus using cognate high-affinity coiled-coil pairs to form stable condensates in vitro. Second, we demonstrate temporal control over phase separation of RGG domains fused to FRB and FKBP in the presence of dimerizer. Further, using a photocaged dimerizer, we achieve optically induced condensation both in cell-sized emulsions and within live cells. Collectively, these modular tools allow multiple strategies to promote phase separation of a common core IDR for tunable control of condensate assembly.

Long-distance association of topological boundaries through nuclear condensates

The eukaryotic genome is partitioned into distinct topological domains separated by boundary elements. Emerging data support the concept that several well-established nuclear compartments are ribonucleoprotein condensates assembled through the physical process of phase separation. Here, based on our demonstration that chemical disruption of nuclear condensate assembly weakens the insulation properties of a specific subset (∼20%) of topologically associated domain (TAD) boundaries, we report that the disrupted boundaries are characterized by a high level of transcription and striking spatial clustering. These topological boundary regions tend to be spatially associated, even interchromosomally, segregate with nuclear speckles, and harbor a specific subset of "housekeeping" genes widely expressed in diverse cell types. These observations reveal a previously unappreciated mode of genome organization mediated by conserved boundary elements harboring highly and widely expressed transcription units and associated transcriptional condensates.

Stochastic particle unbinding modulates growth dynamics and size of transcription factor condensates in living cells

Liquid-liquid phase separation (LLPS) is emerging as a key physical principle for biological organization inside living cells, forming condensates that play important regulatory roles. Inside living nuclei, transcription factor (TF) condensates regulate transcriptional initiation and amplify the transcriptional output of expressed genes. However, the biophysical parameters controlling TF condensation are still poorly understood. Here we applied a battery of single-molecule imaging, theory, and simulations to investigate the physical properties of TF condensates of the progesterone receptor (PR) in living cells. Analysis of individual PR trajectories at different ligand concentrations showed marked signatures of a ligand-tunable LLPS process. Using a machine learning architecture, we found that receptor diffusion within condensates follows fractional Brownian motion resulting from viscoelastic interactions with chromatin. Interestingly, condensate growth dynamics at shorter times is dominated by Brownian motion coalescence (BMC), followed by a growth plateau at longer timescales that result in nanoscale condensate sizes. To rationalize these observations, we extended on the BMC model by including the stochastic unbinding of particles within condensates. Our model reproduced the BMC behavior together with finite condensate sizes at the steady state, fully recapitulating our experimental data. Overall, our results are consistent with condensate growth dynamics being regulated by the escaping probability of PR molecules from condensates. The interplay between condensation assembly and molecular escaping maintains an optimum physical condensate size. Such phenomena must have implications for the biophysical regulation of other nuclear condensates and could also operate in multiple biological scenarios.

A Feedback Regulatory Loop Involving dTrbd/dTak1 in Controlling IMD Signaling in Drosophila Melanogaster.

Negative regulators of the inflammatory responses are essential for the maintenance of immune homeostasis and organismal fitness. In Drosophila, the deubiquitinase (Dub) dTrbd selectively restricts the K63-linked ubiquitination modification of dTak1, a pivotal kinase of the IMD signaling pathway, to regulate the IMD innate immune response. However, which domain and how it functions to enable dTrbd’s activity remain unexplored. Here, we provide compelling evidence showing that the NZF domain of dTrbd is essential for its association with dTak1. Meanwhile, the Linker region of dTrbd is involved in modulating its condensation, a functional state representing the Dub enzymatical activity of dTrbd. Of interest, the activated IMD signals following bacterial stimuli enhance the dTrbd/dTak1 interaction, as well as the condensate assembly and Dub enzymatical activity of dTrbd. Collectively, our studies shed light on the dual mechanisms by which the IMD signaling-mediated feedback loop of dTrbd/dTak1 precisely regulates the innate immune response in Drosophila.

Integration of light and temperature sensing by liquid-liquid phase separation of phytochrome B

Light and temperature in plants are perceived by a common receptor, phytochrome B (phyB). How phyB distinguishes these signals remains elusive. Here, we report that phyB spontaneously undergoes phase separation to assemble liquid-like droplets. This capacity is driven by its C terminus through self-association, whereas the intrinsically disordered N-terminal extension (NTE) functions as a biophysical modulator of phase separation. Light exposure triggers a conformational change to subsequently alter phyB condensate assembly, while temperature sensation is directly mediated by the NTE to modulate the phase behavior of phyB droplets. Multiple signaling components are selectively incorporated into phyB droplets to form concentrated microreactors, allowing switch-like control of phyB signaling activity through phase transitions. Therefore, light and temperature cues are separately read out by phyB via allosteric changes and spontaneous phase separation, respectively. We provide a conceptual framework showing how the distinct but highly correlated physical signals are interpreted and sorted by one receptor.

DNA condensation and formation of ultrathin nanosheets via DNA assisted self-assembly of an amphiphilic fullerene derivative.

DNA nanotechnology propose various assembly strategies to develop novel functional nanostructures utilizing unique interactions of DNA with small molecules, nanoparticles, polymers, and other biomolecules. Although, well defined nanostructures of DNA and amphiphilic small molecules were achieved through hybridization of covalently modified DNA, attaining precise organization of functional moieties through non-covalent interactions remain as a challenging task. Herein, we report mutually assisted assembly of an amphiphilic fullerene derivative and various DNA structures through non-covalent interactions, which leads to initial DNA condensation and subsequent assembly yielding ordered fullerene-DNA nanosheets. The molecular design of the cationic, amphiphilic fullerene derivative (FPy) ensures molecular solubility in the 10% DMSO-PBS buffer system and facile interactions with DNA through groove binding and electrostatic interactions of fullerene moiety and positively charged pyridinium moiety, respectively. The formation of FPy/DNA nanostructures were thoroughly investigated in the presence of λ-DNA, pBR322 plasmid DNA, and single and double stranded 20-mer oligonucleotides using UV-visible spectroscopy, AFM and TEM analysis. λ-DNA and pBR322 plasmid DNA readily condense in presence of FPy leading to micrometer sized few layer nanosheets with significant crystallinity due to ordered arrangement of fullerenes. Similarly, single and double stranded 20-mer oligonucleotides also interact efficiently with FPy and form highly crystalline nanosheets, signifying the role of electrostatic interaction and subsequent charge neutralization in the condensation triggered assembly. However, there is significant differences in the crystallinity and ordered arrangements of fullerenes between these two cases, where longer DNA form condensed structures and less ordered nanosheets while short oligonucleotides lead to more ordered and highly crystalline nanosheets, which could be attributed to the differential DNA condensation. Finally, we have demonstrated the addressability of the assembly using a cyanine modified single strand DNA, which also forms highly crystalline nanosheets and exhibit efficient quenching of the cyanine fluorescence upon self-assembly. These results open up new prospects in the development of functional DNA nanostructures through non-covalent interactions and hence have potential applications in the context of DNA nanotechnology.

Driving force of biomolecular liquid-liquid phase separation probed by nuclear magnetic resonance spectroscopy.

The assembly of biomolecular condensates is driven by liquid-liquid phase separation. To understand the structure and functions of these condensates, it is essential to characterize the underlying driving forces, e.g., protein-protein and protein-RNA interactions. As both structured and low-complexity domains are involved in the phase separation process, NMR is probably the only technique that can be used to depict the binding topology and interaction modes for the structured and nonstructured domains simultaneously. Atomic-resolution analysis for the intramolecular and intermolecular interactions between any pair of components sheds light on the mechanism for phase separation and biomolecular condensate assembly and disassembly. Herein, we describe the procedures used for the most extensively employed NMR techniques to characterize key interactions for biomolecular phase separation.

Axonal TDP-43 condensates drive neuromuscular junction disruption through inhibition of local synthesis of nuclear encoded mitochondrial proteins

Mislocalization of the predominantly nuclear RNA/DNA binding protein, TDP-43, occurs in motor neurons of ~95% of amyotrophic lateral sclerosis (ALS) patients, but the contribution of axonal TDP-43 to this neurodegenerative disease is unclear. Here, we show TDP-43 accumulation in intra-muscular nerves from ALS patients and in axons of human iPSC-derived motor neurons of ALS patient, as well as in motor neurons and neuromuscular junctions (NMJs) of a TDP-43 mislocalization mouse model. In axons, TDP-43 is hyper-phosphorylated and promotes G3BP1-positive ribonucleoprotein (RNP) condensate assembly, consequently inhibiting local protein synthesis in distal axons and NMJs. Specifically, the axonal and synaptic levels of nuclear-encoded mitochondrial proteins are reduced. Clearance of axonal TDP-43 or dissociation of G3BP1 condensates restored local translation and resolved TDP-43-derived toxicity in both axons and NMJs. These findings support an axonal gain of function of TDP-43 in ALS, which can be targeted for therapeutic development.

‘RNA modulation of transport properties and stability in phase-separated condensates

One of the key mechanisms employed by cells to control their spatiotemporal organization is the formation and dissolution of phase-separated condensates. The balance between condensate assembly and disassembly can be critically regulated by the presence of RNA. In this work, we use a chemically-accurate sequence-dependent coarse-grained model for proteins and RNA to unravel the impact of RNA in modulating the transport properties and stability of biomolecular condensates. We explore the phase behavior of several RNA-binding proteins such as FUS, hnRNPA1, and TDP-43 proteins along with that of their corresponding prion-like domains and RNA recognition motifs from absence to moderately high RNA concentration. By characterizing the phase diagram, key molecular interactions, surface tension, and transport properties of the condensates, we report a dual RNA-induced behavior: on the one hand, RNA enhances phase separation at low concentration as long as the RNA radius of gyration is comparable to that of the proteins, whereas at high concentration, it inhibits the ability of proteins to self-assemble independently of its length. On the other hand, along with the stability modulation, the viscosity of the condensates can be considerably reduced at high RNA concentration as long as the length of the RNA chains is shorter than that of the proteins. Conversely, long RNA strands increase viscosity even at high concentration, but barely modify protein self-diffusion which mainly depends on RNA concentration and on the effect RNA has on droplet density. On the whole, our work rationalizes the different routes by which RNA can regulate phase separation and condensate dynamics, as well as the subsequent aberrant rigidification implicated in the emergence of various neuropathologies and age-related diseases.

A CENH3 mutation promotes meiotic exit and restores fertility in SMG7-deficient Arabidopsis.

Meiosis in angiosperm plants is followed by mitotic divisions to form multicellular haploid gametophytes. Termination of meiosis and transition to gametophytic development is, in Arabidopsis, governed by a dedicated mechanism that involves SMG7 and TDM1 proteins. Mutants carrying the smg7-6 allele are semi-fertile due to reduced pollen production. We found that instead of forming tetrads, smg7-6 pollen mother cells undergo multiple rounds of chromosome condensation and spindle assembly at the end of meiosis, resembling aberrant attempts to undergo additional meiotic divisions. A suppressor screen uncovered a mutation in centromeric histone H3 (CENH3) that increased fertility and promoted meiotic exit in smg7-6 plants. The mutation led to inefficient splicing of the CENH3 mRNA and a substantial decrease of CENH3, resulting in smaller centromeres. The reduced level of CENH3 delayed formation of the mitotic spindle but did not have an apparent effect on plant growth and development. We suggest that impaired spindle re-assembly at the end of meiosis limits aberrant divisions in smg7-6 plants and promotes formation of tetrads and viable pollen. Furthermore, the mutant with reduced level of CENH3 was very inefficient haploid inducer indicating that differences in centromere size is not the key determinant of centromere-mediated genome elimination.

ALS-linked mutations impair UBQLN2 stress-induced biomolecular condensate assembly in cells.

Mutations in ubiquilin-2 (UBQLN2), a ubiquitin-binding shuttle protein involved in several protein quality control processes, can lead to amyotrophic lateral sclerosis (ALS). We previously found that wild-type UBQLN2 forms dynamic, membraneless biomolecular condensates upon cellular stress, and undergoes liquid-liquid phase separation in vitro. However, the impact of ALS-linked mutations on UBQLN2 condensate formation in cells remains unknown. Here, we overexpress mCherry-fused UBQLN2 with five patient-derived ALS-linked mutations and employ live-cell imaging and photokinetic analysis to investigate how each of these mutations impact stress-induced UBQLN2 condensate assembly and condensate material properties. Unlike endogenous UBQLN2, exogenously introduced UBQLN2 forms condensates distinct from stress granules. Both wild-type and mutant UBQLN2 condensates are generally cytoplasmic and liquid-like. However, mutant UBQLN2 forms fewer stress-induced UBQLN2 condensates than wild-type UBQLN2. Exogenously expressed P506T UBQLN2 forms the lowest number of stress-induced condensates of all UBQLN2 mutants, and these condensates are significantly smaller than those of wild-type UBQLN2. Fluorescence recovery after photobleaching (FRAP) analysis of UBQLN2 condensates revealed higher immobile fractions for UBQLN2 mutants, especially P506T. P497S and P497H mutations differentially impact condensate properties, demonstrating that the effects of ALS-linked mutations are both position- and amino acid-dependent. Collectively, our data show that disease mutations hinder assembly and alter viscoelastic properties of stress-induced UBQLN2 condensates, potentially leading to aggregates commonly observed in ALS.

O-Linked-N-Acetylglucosaminylation of the RNA-Binding Protein EWS N-Terminal Low Complexity Region Reduces Phase Separation and Enhances Condensate Dynamics.

Many membraneless organelles are thought to be biomolecular condensates formed by phase separation of proteins and other biopolymers. Post-translational modifications (PTMs) can impact protein phase separation behavior, although for many PTMs this aspect of their function is unknown. O-linked β-D-N-acetylglucosaminylation (O-GlcNAcylation) is an abundant form of intracellular glycosylation whose roles in regulating biomolecular condensate assembly and dynamics have not been delineated. Using an in vitro approach, we found that O-GlcNAcylation reduces the phase separation propensity of the EWS N-terminal low complexity region (LCRN) under different conditions, including in the presence of the arginine- and glycine-rich RNA-binding domains (RBD). O-GlcNAcylation enhances fluorescence recovery after photobleaching (FRAP) within EWS LCRN condensates and causes the droplets to exhibit more liquid-like relaxation following fusion. Following extended incubation times, EWS LCRN+RBD condensates exhibit diminished FRAP, indicating a loss of fluidity, while condensates containing the O-GlcNAcylated LCRN do not. In HeLa cells, EWS is less O-GlcNAcylated following OGT knockdown, which correlates with its increased accumulation in a filter retardation assay. Relative to the human proteome, O-GlcNAcylated proteins are enriched with regions that are predicted to phase separate, suggesting a general role of O-GlcNAcylation in regulation of biomolecular condensates.

A multi-step nucleation process determines the kinetics of prion-like domain phase separation

Compartmentalization by liquid-liquid phase separation (LLPS) has emerged as a ubiquitous mechanism underlying the organization of biomolecules in space and time. Here, we combine rapid-mixing time-resolved small-angle X-ray scattering (SAXS) approaches to characterize the assembly kinetics of a prototypical prion-like domain with equilibrium techniques that characterize its phase boundaries and the size distribution of clusters prior to phase separation. We find two kinetic regimes on the micro- to millisecond timescale that are distinguished by the size distribution of clusters. At the nanoscale, small complexes are formed with low affinity. After initial unfavorable complex assembly, additional monomers are added with higher affinity. At the mesoscale, assembly resembles classical homogeneous nucleation. Careful multi-pronged characterization is required for the understanding of condensate assembly mechanisms and will promote understanding of how the kinetics of biological phase separation is encoded in biomolecules.

Observing a Zeolite Nucleus (Subcrystal) with a Uniform Framework Structure and Its Oriented Attachment without Single-Molecule Addition.

Multiple and complex crystallization process of zeolite including complementary single-molecule condensation and particle assembly, and alternately dominant nucleation and growth behavior, plays the critical role in zeolite crystallization but meanwhile makes us hard to study the respective effects. Herein, we strip nuclei from the synthetic solution and find that high-ordered nucleus (subcrystal) is the premise to ignite high-speed growth of zeolite crystal. The high-ordered subcrystals with the size of only 6-10 nm possess regular aperture structure and microporous area similar to zeolite nanocrystal. Interestingly, a unitary oriented aggregation process of the subcrystals towards nanosheets is well observed and characterized where single-molecule addition process is greatly repressed. If a wider range of zeotype nuclei can be expanded, a new synthetic strategy of zeotype materials with heterogeneous framework and active sites may be expected, which may novelize zeolite catalytic properties.

Observing a Zeolite Nucleus (Subcrystal) with a Uniform Framework Structure and Its Oriented Attachment without Single‐Molecule Addition

AbstractMultiple and complex crystallization process of zeolite including complementary single‐molecule condensation and particle assembly, and alternately dominant nucleation and growth behavior, plays the critical role in zeolite crystallization but meanwhile makes us hard to study the respective effects. Herein, we strip nuclei from the synthetic solution and find that high‐ordered nucleus (subcrystal) is the premise to ignite high‐speed growth of zeolite crystal. The high‐ordered subcrystals with the size of only 6–10 nm possess regular aperture structure and microporous area similar to zeolite nanocrystal. Interestingly, a unitary oriented aggregation process of the subcrystals towards nanosheets is well observed and characterized where single‐molecule addition process is greatly repressed. If a wider range of zeotype nuclei can be expanded, a new synthetic strategy of zeotype materials with heterogeneous framework and active sites may be expected, which may novelize zeolite catalytic properties.

Assembly of bacterial cell division protein FtsZ into dynamic biomolecular condensates

Biomolecular condensation through phase separation may be a novel mechanism to regulate bacterial processes, including cell division. Previous work revealed that FtsZ, a protein essential for cytokinesis in most bacteria, forms biomolecular condensates with SlmA, a protein that protects the chromosome from damage inflicted by the division machinery in Escherichia coli. The absence of condensates composed solely of FtsZ under the conditions used in that study suggested this mechanism was restricted to nucleoid occlusion by SlmA or to bacteria containing this protein. Here we report that FtsZ alone, under physiologically relevant conditions, can demix into condensates in bulk and when encapsulated in synthetic cell-like systems generated by microfluidics. Condensate assembly depends on FtsZ being in the GDP-bound state and on conditions mimicking the crowded environment of the cytoplasm that promote its oligomerization. Condensates are dynamic and reversibly convert into filaments upon GTP addition. Notably, FtsZ lacking its C-terminal disordered region, a structural element likely to favor biomolecular condensation, also forms condensates, albeit less efficiently. The inherent tendency of FtsZ to form condensates susceptible to modulation by physiological factors, including binding partners, suggests that such mechanisms may play a more general role in bacterial division than initially envisioned.

Theaflavin-regulated Imd condensates control Drosophila intestinal homeostasis and aging

SummaryBlack tea is the most widely consumed tea drink in the world and has consistently been reported to possess anti-aging benefits. However, whether theaflavins, one type of the characteristic phytochemicals in black tea extracts, are involved in regulating aging and lifespan in consumers remains largely unknown. In this study, we show that theaflavins play a beneficial role in preventing age-onset intestinal leakage and dysbiosis, thus delaying aging in Drosophila. Mechanistically, theaflavins regulate the condensate assembly of Imd to negatively govern the overactivation of Imd signals in fruit fly intestines. In addition, theaflavins prevent DSS-induced colitis in mice, suggesting theaflavins play a role in modulating intestinal integrity. Overall, our study reveals a molecular mechanism by which theaflavins regulate gut homeostasis likely through controlling Imd coalescence.

Biomolecular condensates at the nexus of cellular stress, protein aggregation disease and ageing.

Biomolecular condensates are membraneless intracellular assemblies that often form via liquid-liquid phase separation and have the ability to concentrate biopolymers. Research over the past 10 years has revealed that condensates play fundamental roles in cellular organization and physiology, and our understanding of the molecular principles, components and forces underlying their formation has substantially increased. Condensate assembly is tightly regulated in the intracellular environment, and failure to control condensate properties, formation and dissolution can lead to protein misfolding and aggregation, which are often the cause of ageing-associated diseases. In this Review, we describe the mechanisms and regulation of condensate assembly and dissolution, highlight recent advances in understanding the role of biomolecular condensates in ageing and disease, and discuss how cellular stress, ageing-related loss of homeostasis and a decline in protein quality control may contribute to the formation of aberrant, disease-causing condensates. Our improved understanding of condensate pathology provides a promising path for the treatment of protein aggregation diseases.

Driving force of biomolecular liquid–liquid phase separation probed by nuclear magnetic resonance spectroscopy

The assembly of biomolecular condensates is driven by liquid–liquid phase separation. To understand the structure and functions of these condensates, it is essential to characterize the underlying driving forces, <i>e</i>.<i>g</i>., protein–protein and protein–RNA interactions. As both structured and low-complexity domains are involved in the phase separation process, NMR is probably the only technique that can be used to depict the binding topology and interaction modes for the structured and nonstructured domains simultaneously. Atomic-resolution analysis for the intramolecular and intermolecular interactions between any pair of components sheds light on the mechanism for phase separation and biomolecular condensate assembly and disassembly. Herein, we describe the procedures used for the most extensively employed NMR techniques to characterize key interactions for biomolecular phase separation.

Dissecting the complexity of biomolecular condensates.

Biomolecular condensates comprise a diverse and ubiquitous class of membraneless organelles. Condensate assembly is often described by liquid-liquid phase separation. While this process explains many key features, it cannot account for the compositional or architectural complexity that condensates display in cells. Recent work has begun to dissect the rich network of intermolecular interactions that give rise to biomolecular condensates. Here, we review the latest results from theory, simulations and experiments, and discuss what they reveal about the structure-function relationship of condensates.