Rapid and reversible dissolution of biomolecular condensates using light-controlled recruitment of a solubility tag.

Biomolecular condensates are broadly implicated in both normal cellular regulation and disease. Consequently, several chemical biology and optogenetic approaches have been developed to induce phase separation of a protein of interest. However, few tools are available to perform the converse function - dissolving a condensate of interest on demand. Such a tool would aid in testing whether the condensate plays specific functional roles. Here we show that light-gated recruitment of a solubilizing domain, maltose-binding protein (MBP), results in rapid and controlled dissolution of condensates formed from proteins of interest. Our optogenetic MBP-based dissolution strategy (OptoMBP) is rapid, reversible, and can be spatially controlled with subcellular precision. We also provide a proof-of-principle application of OptoMBP by disrupting condensation of the oncogenic fusion protein FUS-CHOP and reverting FUS-CHOP driven transcriptional changes. We envision that the OptoMBP system could be broadly useful for disrupting constitutive protein condensates to probe their biological functions.

Membrane prewetting by condensates promotes tight-junction belt formation.

Biomolecular condensates enable cell compartmentalization by acting as membraneless organelles1. How cells control the interactions of condensates with other cellular structures such as membranes to drive morphological transitions remains poorly understood. We discovered that formation of a tight-junction belt, which is essential for sealing epithelial tissues, is driven by a wetting phenomenon that promotes the growth of a condensed ZO-1 layer2 around the apical membrane interface. Using temporal proximity proteomics in combination with imaging and thermodynamic theory, we found that the polarity protein PATJ mediates a transition of ZO-1 into a condensed surface layer that elongates around the apical interface. In line with the experimental observations, our theory of condensate growth shows that the speed of elongation depends on the binding affinity of ZO-1 to the apical interface and is constant. Here, using PATJ mutations, we show that ZO-1 interface binding is necessary and sufficient for tight-junction belt formation. Our results demonstrate how cells exploit the collective biophysical properties of protein condensates at membrane interfaces to shape mesoscale structures.

Phase separation of polyubiquitinated proteins in UBQLN2 condensates controls substrate fate.

Ubiquitination is one of the most common posttranslational modifications in eukaryotic cells. Depending on the architecture of polyubiquitin chains, substrate proteins can meet different cellular fates, but our understanding of how chain linkage controls protein fate remains limited. UBL-UBA shuttle proteins, such as UBQLN2, bind to ubiquitinated proteins and to the proteasome or other protein quality control machinery elements and play a role in substrate fate determination. Under physiological conditions, UBQLN2 forms biomolecular condensates through phase separation, a physicochemical phenomenon in which multivalent interactions drive the formation of a macromolecule-rich dense phase. Ubiquitin and polyubiquitin chains modulate UBQLN2's phase separation in a linkage-dependent manner, suggesting a possible link to substrate fate determination, but polyubiquitinated substrates have not been examined directly. Using sedimentation assays and microscopy we show that polyubiquitinated substrates induce UBQLN2 phase separation and incorporate into the resulting condensates. This substrate effect is strongest with K63-linked substrates, intermediate with mixed-linkage substrates, and weakest with K48-linked substrates. Proteasomes can be recruited to these condensates, but proteasome activity toward K63-linked and mixed linkage substrates is inhibited in condensates. Substrates are also protected from deubiquitinases by UBQLN2-induced phase separation. Our results suggest that phase separation could regulate the fate of ubiquitinated substrates in a chain-linkage-dependent manner, thus serving as an interpreter of the ubiquitin code.

Initiation of hnRNPA1 Low-Complexity Domain Condensation Monitored by Dynamic Light Scattering.

Biomolecular condensates (BMCs) exhibit physiological and pathological relevance in biological systems. Both liquid and solid condensates play significant roles in the spatiotemporal regulation and organization of macromolecules and their biological activities. Some pathological solid condensates, such as Lewy Bodies and other fibrillar aggregates, have been hypothesized to originate from liquid condensates. With the prevalence of BMCs having functional and dysfunctional roles, it is imperative to understand the mechanism of biomolecular condensate formation and initiation. Using the low-complexity domain (LCD) of heterogenous ribonuclear protein A1 (hnRNPA1) as our model, we monitored initial assembly events using dynamic light scattering (DLS) while modulating pH and salt conditions to perturb macromolecule and condensate properties. We observed the formation of nanometer-sized BMCs (nano-condensates) distinct from protein monomers and micron-sized condensates. We also observed that conditions that solubilize micron-sized protein condensates do not solubilize nano-condensates, indicating that the balance of forces that stabilize nano-condensates and micron-sized condensates are distinct. These findings provide insight into the forces that drive protein phase separation and potential nucleation structures of macromolecular condensation.

Fluorescence imaging of lamellipodin-mediated biomolecular condensates on solid supported lipid bilayer membranes.

Biomolecular condensates play a major role in numerous cellular processes, including several that occur on the surface of lipid bilayer membranes. There is increasing evidence that cellular membrane trafficking phenomena, including the internalization of the plasma membrane through endocytosis, are mediated by multivalent protein-protein interactions that can lead to phase separation. We have recently found that proteins involved in the clathrin-independent endocytic pathway named Fast Endophilin Mediated Endocytosis can undergo liquid-liquid phase separation (LLPS) in solution and on lipid bilayer membranes. Here, the protein solution concentrations required for phase separation to be observed are significantly smaller compared to those required for phase separation in solution. LLPS is challenging to systematically characterize in cellular systems in general, and on biological membranes in particular. Model membrane approaches are more suitable for this purpose as they allow for precise control over the nature and amount of the components present in a mixture. Here we describe a method that enables the imaging of LLPS domain formation on solid supported lipid bilayers. These allow for facile imaging, provide long-term stability, and avoid clustering of vesicles and vesicle-attached features (such as buds and tethers) in the presence of multi-valent membrane interacting proteins.

Measuring Partition Coefficients of In Vitro Biomolecular Condensates Using Fluorescence Correlation Spectroscopy.

Liquid-liquid phase separation is a widespread organizing principle of live cells, including, for example, the spatiotemporal organization of bacterial chromatin. The biophysics of phase-separating systems is often studied in vitro to avoid the complexity of the live-cell environment and facilitate application of advanced biophysical methods. One attractive method for measuring, e.g., partition coefficients, in such systems is fluorescence correlation spectroscopy (FCS). FCS circumvents some of the limitations of the widespread confocal laser scanning microscopy image-based measurements. Here, we describe how to perform partition coefficient measurements in biological phase-separating systems. Our protocol details typical workflows for the preparation of in vitro reconstituted condensates, FCS data acquisition, and subsequent data analysis, including corrections of some common artifacts. Our recommendations should help avoid many pitfalls of partition coefficient determination in these challenging systems.

Squishy to crusty: Biophysics reveal the molecular details of FUS droplet maturation.

In a recent issue of Nature Chemical Biology, Emmanouilidis et al. (2024) investigate the maturation of biomolecular condensates of FUS1-267 and probe the molecular details of droplet aging. They observe that the liquid-to-solid transition of the droplet is mediated at the surface by FUS1-267 molecules that have adopted ß-strand conformations.

Modular RNA motifs for orthogonal phase separated compartments.

Recent discoveries in biology have highlighted the importance of protein and RNA-based condensates as an alternative to classical membrane-bound organelles. Here, we demonstrate the design of pure RNA condensates from nanostructured, star-shaped RNA motifs. We generate condensates using two different RNA nanostar architectures: multi-stranded nanostars whose binding interactions are programmed via linear overhangs, and single-stranded nanostars whose interactions are programmed via kissing loops. Through systematic sequence design, we demonstrate that both architectures can produce orthogonal (distinct and immiscible) condensates, which can be individually tracked via fluorogenic aptamers. We also show that aptamers make it possible to recruit peptides and proteins to the condensates with high specificity. Successful co-transcriptional formation of condensates from single-stranded nanostars suggests that they may be genetically encoded and produced in living cells. We provide a library of orthogonal RNA condensates that can be modularly customized and offer a route toward creating systems of functional artificial organelles for the task of compartmentalizing molecules and biochemical reactions.

Protein Condensate Atlas from predictive models of heteromolecular condensate composition.

Biomolecular condensates help cells organise their content in space and time. Cells harbour a variety of condensate types with diverse composition and many are likely yet to be discovered. Here, we develop a methodology to predict the composition of biomolecular condensates. We first analyse available proteomics data of cellular condensates and find that the biophysical features that determine protein localisation into condensates differ from known drivers of homotypic phase separation processes, with charge mediated protein-RNA and hydrophobicity mediated protein-protein interactions playing a key role in the former process. We then develop a machine learning model that links protein sequence to its propensity to localise into heteromolecular condensates. We apply the model across the proteome and find many of the top-ranked targets outside the original training data to localise into condensates as confirmed by orthogonal immunohistochemical staining imaging. Finally, we segment the condensation-prone proteome into condensate types based on an overlap with biomolecular interaction profiles to generate a Protein Condensate Atlas. Several condensate clusters within the Atlas closely match the composition of experimentally characterised condensates or regions within them, suggesting that the Atlas can be valuable for identifying additional components within known condensate systems and discovering previously uncharacterised condensates.

Measurement of solubility product reveals the interplay of oligomerization and self-association for defining condensate formation.

Cellular condensates often consist of 10s to 100s of distinct interacting molecular species. Because of the complexity of these interactions, predicting the point at which they will undergo phase separation is daunting. Using experiments and computation, we therefore studied a simple model system consisting of polySH3 and polyPRM designed for pentavalent heterotypic binding. We tested whether the peak solubility product, or the product of the dilute phase concentration of each component, is a predictive parameter for the onset of phase separation. Titrating up equal total concentrations of each component showed that the maximum solubility product does approximately coincide with the threshold for phase separation in both experiments and models. However, we found that measurements of dilute phase concentration include small oligomers and monomers; therefore, a quantitative comparison of the experiments and models required inclusion of small oligomers in the model analysis. Even with the inclusion of small polyPRM and polySH3 oligomers, models did not predict experimental results. This led us to perform dynamic light scattering experiments, which revealed homotypic binding of polyPRM. Addition of this interaction to our model recapitulated the experimentally observed asymmetry. Thus, comparing experiments with simulation reveals that the solubility product can be predictive of the interactions underlying phase separation, even if small oligomers and low affinity homotypic interactions complicate the analysis.

Hydrogen-Bonded Network of Water in Phase-Separated Biomolecular Condensates.

Biomolecular condensates formed via phase separation of intrinsically disordered proteins/regions (IDPs/IDRs) and nucleic acids are associated with cell physiology and disease. Water makes up for ∼60-70% of the condensate volume and is thought to influence the complex interplay of chain-chain and chain-solvent interactions, modulating the mesoscale properties of condensates. The behavior of water in condensates and the key roles of protein hydration water in driving the phase separation remain elusive. Here, we employ single-droplet vibrational Raman spectroscopy to illuminate the structural redistribution within protein hydration water during the phase separation of neuronal IDPs. Our Raman measurements reveal the changes in the water hydrogen bonding network during homotypic and heterotypic phase separation governed by various molecular drivers. Such single-droplet water Raman measurements offer a potent generic tool to unmask the intriguing interplay of sequence-encoded chain-chain and chain-solvent interactions governing macromolecular phase separation into membraneless organelles, synthetic condensates, and protocells.

Oxidative stress elicits the remodeling of vimentin filaments into biomolecular condensates.

The intermediate filament protein vimentin performs an essential role in cytoskeletal interplay and dynamics, mechanosensing and cellular stress responses. In pathology, vimentin is a key player in tumorigenesis, fibrosis and infection. Vimentin filaments undergo distinct and versatile reorganizations, and behave as redox sensors. The vimentin monomer possesses a central α-helical rod domain flanked by N- and C-terminal low complexity domains. Interactions between this type of domains play an important function in the formation of phase-separated biomolecular condensates, which in turn are critical for the organization of cellular components. Here we show that several oxidants, including hydrogen peroxide and diamide, elicit the remodeling of vimentin filaments into small particles. Oxidative stress elicited by diamide induces a fast dissociation of filaments into circular, motile dots, which requires the presence of the single vimentin cysteine residue, C328. This effect is reversible, and filament reassembly can occur within minutes of oxidant removal. Diamide-elicited vimentin droplets recover fluorescence after photobleaching. Moreover, fusion of cells expressing differentially tagged vimentin allows the detection of dots positive for both tags, indicating that vimentin dots merge upon cell fusion. The aliphatic alcohol 1,6-hexanediol, known to alter interactions between low complexity domains, readily dissolves diamide-elicited vimentin dots at low concentrations, in a C328 dependent manner, and hampers reassembly. Taken together, these results indicate that vimentin oxidation promotes a fast and reversible filament remodeling into biomolecular condensate-like structures, and provide primary evidence of its regulated phase separation. Moreover, we hypothesize that filament to droplet transition could play a protective role against irreversible damage of the vimentin network by oxidative stress.

Engineered droplet-forming peptide as photocontrollable phase modulator for fused in sarcoma protein.

The assembly and disassembly of biomolecular condensates are crucial for the subcellular compartmentalization of biomolecules in the control of cellular reactions. Recently, a correlation has been discovered between the phase transition of condensates and their maturation (aggregation) process in diseases. Therefore, modulating the phase of condensates to unravel the roles of condensation has become a matter of interest. Here, we create a peptide-based phase modulator, JSF1, which forms droplets in the dark and transforms into amyloid-like fibrils upon photoinitiation, as evidenced by their distinctive nanomechanical and dynamic properties. JSF1 is found to effectively enhance the condensation of purified fused in sarcoma (FUS) protein and, upon light exposure, induce its fibrilization. We also use JSF1 to modulate the biophysical states of FUS condensates in live cells and elucidate the relationship between FUS phase transition and FUS proteinopathy, thereby shedding light on the effect of protein phase transition on cellular function and malfunction.

Preserving condensate structure and composition by lowering sequence complexity.

Biomolecular condensates play a vital role in organizing cellular chemistry. They selectively partition biomolecules, preventing unwanted cross talk and buffering against chemical noise. Intrinsically disordered proteins (IDPs) serve as primary components of these condensates due to their flexibility and ability to engage in multivalent interactions, leading to spontaneous aggregation. Theoretical advancements are critical at connecting IDP sequences with condensate emergent properties to establish the so-called molecular grammar. We proposed an extension to the stickers and spacers model, incorporating heterogeneous, nonspecific pairwise interactions between spacers alongside specific interactions among stickers. Our investigation revealed that although spacer interactions contribute to phase separation and co-condensation, their nonspecific nature leads to disorganized condensates. Specific sticker-sticker interactions drive the formation of condensates with well-defined networked structures and molecular composition. We discussed how evolutionary pressures might emerge to affect these interactions, leading to the prevalence of low-complexity domains in IDP sequences. These domains suppress spurious interactions and facilitate the formation of biologically meaningful condensates.

Fundamental Aspects of Phase-Separated Biomolecular Condensates.

Biomolecular condensates, formed through phase separation, are upending our understanding in much of molecular, cell, and developmental biology. There is an urgent need to elucidate the physicochemical foundations of the behaviors and properties of biomolecular condensates. Here we aim to fill this need by writing a comprehensive, critical, and accessible review on the fundamental aspects of phase-separated biomolecular condensates. We introduce the relevant theoretical background, present the theoretical basis for the computation and experimental measurement of condensate properties, and give mechanistic interpretations of condensate behaviors and properties in terms of interactions at the molecular and residue levels.

Stability and deformation of biomolecular condensates under the action of shear flow.

Biomolecular condensates play a key role in cytoplasmic compartmentalization and cell functioning. Despite extensive research on the physico-chemical, thermodynamic, or crowding aspects of the formation and stabilization of the condensates, one less studied feature is the role of external perturbative fluid flow. In fact, in living cells, shear stress may arise from streaming or active transport processes. Here, we investigate how biomolecular condensates are deformed under different types of shear flows. We first model Couette flow perturbations via two-way coupling between the condensate dynamics and fluid flow by deploying Lattice Boltzmann Molecular Dynamics. We then show that a simplified approach where the shear flow acts as a static perturbation (one-way coupling) reproduces the main features of the condensate deformation and dynamics as a function of the shear rate. With this approach, which can be easily implemented in molecular dynamics simulations, we analyze the behavior of biomolecular condensates described through residue-based coarse-grained models, including intrinsically disordered proteins and protein/RNA mixtures. At lower shear rates, the fluid triggers the deformation of the condensate (spherical to oblated object), while at higher shear rates, it becomes extremely deformed (oblated or elongated object). At very high shear rates, the condensates are fragmented. We also compare how condensates of different sizes and composition respond to shear perturbation, and how their internal structure is altered by external flow. Finally, we consider the Poiseuille flow that realistically models the behavior in microfluidic devices in order to suggest potential experimental designs for investigating fluid perturbations in vitro.

Synthetic Condensates and Cell-Like Architectures from Amphiphilic DNA Nanostructures.

Synthetic droplets and condensates are becoming increasingly common constituents of advanced biomimetic systems and synthetic cells, where they can be used to establish compartmentalization and sustain life-like responses. Synthetic DNA nanostructures have demonstrated significant potential as condensate-forming building blocks owing to their programmable shape, chemical functionalization, and self-assembly behavior. We have recently demonstrated that amphiphilic DNA "nanostars", obtained by labeling DNA junctions with hydrophobic moieties, constitute a particularly robust and versatile solution. The resulting amphiphilic DNA condensates can be programmed to display complex, multi-compartment internal architectures, structurally respond to various external stimuli, synthesize macromolecules, capture and release payloads, undergo morphological transformations, and interact with live cells. Here, we demonstrate protocols for preparing amphiphilic DNA condensates starting from constituent DNA oligonucleotides. We will address (i) single-component systems forming uniform condensates, (ii) two-component systems forming core-shell condensates, and (iii) systems in which the condensates are modified to support in vitro transcription of RNA nanostructures.

Nonspecific Interactions in Transcription Regulation and Organization of Transcriptional Condensates.

Eukaryotic cells are characterized by a high degree of compartmentalization of their internal contents, which ensures precise and controlled regulation of intracellular processes. During many processes, including different stages of transcription, dynamic membraneless compartments termed biomolecular condensates are formed. Transcription condensates contain various transcription factors and RNA polymerase and are formed by high- and low-specificity interactions between the proteins, DNA, and nearby RNA. This review discusses recent data demonstrating important role of nonspecific multivalent protein-protein and RNA-protein interactions in organization and regulation of transcription.

Biomolecular condensates as drivers of membrane trafficking and remodelling.

Membrane remodelling is essential for the trafficking of macromolecules throughout the cell, a process that regulates various aspects of cellular health and pathology. Recent studies implicate the role of biomolecular condensates in regulating multiple steps of the membrane trafficking pathway including but not limited to the organization of the trafficking machinery, dynamic remodeling of membranes, spatial and functional regulation, and response to cellular signals. The implicated proteins contain key structural elements, most notably prion-like domains within intrinsically disordered regions that are necessary for biomolecular condensate formation at fusion sites in processes like endocytic assembly, autophagy, organelle biosynthesis and synaptic vesicle fusion. Experimental and theoretical advances in the field continue to demonstrate that protein condensates can perform mechanical work, the implications of which can be extrapolated to diverse areas of membrane biology.