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.

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.

The Heat Shock Response as a Condensate Cascade.

The heat shock response (HSR) is a gene regulatory program controlling expression of molecular chaperones implicated in aging, cancer, and neurodegenerative disease. Long presumed to be activated by toxic protein aggregates, recent work suggests a new functional paradigm for the HSR in yeast. Rather than toxic aggregates, adaptive biomolecular condensates comprised of orphan ribosomal proteins (oRP) and stress granule components have been shown to be physiological chaperone clients. By titrating away the chaperones Sis1 and Hsp70 from the transcription factor Hsf1, these condensates activate the HSR. Upon release from Hsp70, Hsf1 forms spatially distinct transcriptional condensates that drive high expression of HSR genes. In this manner, the negative feedback loop controlling HSR activity - in which Hsf1 induces Hsp70 expression and Hsp70 represses Hsf1 activity - is embedded in the biophysics of the system. By analogy to phosphorylation cascades that transmit information via the dynamic activity of kinases, we propose that the HSR is organized as a condensate cascade that transmits information via the localized activity of molecular chaperones.

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.

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.

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.

Peptide diffusion in biomolecular condensates.

Diffusion determines the turnover of biomolecules in liquid-liquid phase-separated condensates. We considered the mean square displacement and thus the diffusion constant for simple model systems of peptides GGGGG, GGQGG, and GGVGG in aqueous solutions after phase separation by simulating atomic-level models. These solutions readily separate into aqueous and peptide-rich droplet phases. We noted the effect of the peptides being in a solvated, surface, or droplet state on the peptide's diffusion coefficients. Both sequence and peptide conformational distribution were found to influence diffusion and condensate turnover in these systems, with sequence dominating the magnitude of the differences. We found that the most compact structures for each sequence diffused the fastest in the peptide-rich condensate phase. This model result may have implications for turnover dynamics in signaling systems.

A closer look at mammalian antiviral condensates.

Several biomolecular condensates assemble in mammalian cells in response to viral infection. The most studied of these are stress granules (SGs), which have been proposed to promote antiviral innate immune signaling pathways, including the RLR-MAVS, the protein kinase R (PKR), and the OAS-RNase L pathways. However, recent studies have demonstrated that SGs either negatively regulate or do not impact antiviral signaling. Instead, the SG-nucleating protein, G3BP1, may function to perturb viral RNA biology by condensing viral RNA into viral-aggregated RNA condensates, thus explaining why viruses often antagonize G3BP1 or hijack its RNA condensing function. However, a recently identified condensate, termed double-stranded RNA-induced foci, promotes the activation of the PKR and OAS-RNase L antiviral pathways. In addition, SG-like condensates known as an RNase L-induced bodies (RLBs) have been observed during many viral infections, including SARS-CoV-2 and several flaviviruses. RLBs may function in promoting decay of cellular and viral RNA, as well as promoting ribosome-associated signaling pathways. Herein, we review these recent advances in the field of antiviral biomolecular condensates, and we provide perspective on the role of canonical SGs and G3BP1 during the antiviral response.

Fluorescent protein tags affect the condensation properties of a phase-separating viral protein.

Fluorescent protein (FP) tags are extensively used to visualize and characterize the properties of biomolecular condensates despite a lack of investigation into the effects of these tags on phase separation. Here, we characterized the dynamic properties of µNS, a viral protein hypothesized to undergo phase separation and the main component of mammalian orthoreovirus viral factories. Our interest in the sequence determinants and nucleation process of µNS phase separation led us to compare the size and density of condensates formed by FP::µNS to the untagged protein. We found an FP-dependent increase in droplet size and density, which suggests that FP tags can promote µNS condensation. To further assess the effect of FP tags on µNS droplet formation, we fused FP tags to µNS mutants to show that the tags could variably induce phase separation of otherwise noncondensing proteins. By comparing fluorescent constructs with untagged µNS, we identified mNeonGreen as the least artifactual FP tag that minimally perturbed µNS condensation. These results show that FP tags can promote phase separation and that some tags are more suitable for visualizing and characterizing biomolecular condensates with minimal experimental artifacts.

Advanced surface passivation for high-sensitivity studies of biomolecular condensates.

Biomolecular condensates are cellular compartments that concentrate biomolecules without an encapsulating membrane. In recent years, significant advances have been made in the understanding of condensates through biochemical reconstitution and microscopic detection of these structures. Quantitative visualization and biochemical assays of biomolecular condensates rely on surface passivation to minimize background and artifacts due to condensate adhesion. However, the challenge of undesired interactions between condensates and glass surfaces, which can alter material properties and impair observational accuracy, remains a critical hurdle. Here, we introduce an efficient, broadly applicable, and simple passivation method employing self-assembly of the surfactant Pluronic F127 (PF127). The method greatly reduces nonspecific binding across a range of condensates systems for both phase-separated droplets and biomolecules in dilute phase. Additionally, by integrating PF127 passivation with the Biotin-NeutrAvidin system, we achieve controlled multipoint attachment of condensates to surfaces. This not only preserves condensate properties but also facilitates long-time fluorescence recovery after photobleaching imaging and high-precision single-molecule analyses. Using this method, we have explored the dynamics of polySIM molecules within polySUMO/polySIM condensates at the single-molecule level. Our observations suggest a potential heterogeneity in the distribution of available polySIM-binding sites within the condensates.

Demixing is a default process for biological condensates formed via phase separation.

Excitatory and inhibitory synapses do not overlap even when formed on one submicron-sized dendritic protrusion. How excitatory and inhibitory postsynaptic cytomatrices or densities (e/iPSDs) are segregated is not understood. Broadly, why membraneless organelles are naturally segregated in cellular subcompartments is unclear. Using biochemical reconstitutions in vitro and in cells, we demonstrate that ePSDs and iPSDs spontaneously segregate into distinct condensed molecular assemblies through phase separation. Tagging iPSD scaffold gephyrin with a PSD-95 intrabody (dissociation constant ~4 nM) leads to mistargeting of gephyrin to ePSD condensates. Unexpectedly, formation of iPSD condensates forces the intrabody-tagged gephyrin out of ePSD condensates. Thus, instead of diffusion-governed spontaneous mixing, demixing is a default process for biomolecules in condensates. Phase separation can generate biomolecular compartmentalization specificities that cannot occur in dilute solutions.

Transient crosslinking controls the condensate formation pathway within chromatin networks.

The network structure of densely packed chromatin within the nucleus of eukaryotic cells acts in concert with nonequilibrium processes. Using statistical physics simulations, we explore the control provided by transient crosslinking of the chromatin network by structural-maintenance-of-chromosome (SMC) proteins over (i) the physical properties of the chromatin network and (ii) condensate formation of embedded molecular species. We find that the density and lifetime of transient SMC crosslinks regulate structural relaxation modes and tune the sol-vs-gel state of the chromatin network, which imparts control over the kinetic pathway to condensate formation. Specifically, lower density, shorter-lived crosslinks induce sollike networks and a droplet-fusion pathway, whereas higher density, longer-lived crosslinks induce gellike networks and an Ostwald-ripening pathway.

Ionic Effect on the Microenvironment of Biomolecular Condensates.

Biomolecules such as proteins and RNA could organize to form condensates with distinct microenvironments through liquid-liquid phase separation (LLPS). Recent works have demonstrated that the microenvironment of biomolecular condensates plays a crucial role in mediating biological activities, such as the partition of biomolecules, and the subphase organization of the multiphasic condensates. Ions could influence the phase transition point of LLPS, following the Hofmeister series. However, the ion-specific effect on the microenvironment of biomolecular condensates remains unknown. In this study, we utilized fluorescence lifetime imaging microscopy (FLIM), fluorescence recovery after photobleaching (FRAP), and microrheology techniques to investigate the ion effect on the microenvironment of condensates. We found that ions significantly affect the microenvironment of biomolecular condensates: salting-in ions increase micropolarity and reduce the microviscosity of the condensate, while salting-out ions induce opposing effects. Furthermore, we manipulate the miscibility and multilayering behavior of condensates through ion-specific effects. In summary, our work provides the first quantitative survey of the microenvironment of protein condensates in the presence of ions from the Hofmeister series, demonstrating how ions impact micropolarity, microviscosity, and viscoelasticity of condensates. Our results bear implications on how membrane-less organelles would exhibit varying microenvironments in the presence of continuously changing cellular conditions.

Protein condensates in the the secretory pathway: Unraveling biophysical interactions and function.

The emergence of phase separation phenomena among macromolecules has identified biomolecular condensates as fundamental cellular organizers. These condensates concentrate specific components and accelerate biochemical reactions without relying on membrane boundaries. Although extensive studies have revealed a large variety of nuclear and cytosolic membraneless organelles, we are witnessing a surge in the exploration of protein condensates associated with the membranes of the secretory pathway, such as the endoplasmic reticulum and the Golgi apparatus. This review focuses on protein condensates in the secretory pathway and discusses their impact on the organization and functions of this cellular process. Moreover, we explore the modes of condensate-membrane association and the biophysical and cellular consequences of protein condensate interactions with secretory pathway membranes.

SnapShot: Condensates in plant biology.

Plant cells share a number of biological condensates with cells from other eukaryotes. There are, however, a growing number of plant-specific condensates that support different cellular functions. Condensates operating in different plant tissues contribute to aspects of development and stress responses. To view this SnapShot, open or download the PDF.

Biomolecular Condensates Decipher Molecular Codes of Cell Fate: From Biophysical Fundamentals to Therapeutic Practices.

Cell fate is precisely modulated by complex but well-tuned molecular signaling networks, whose spatial and temporal dysregulation commonly leads to hazardous diseases. Biomolecular condensates (BCs), as a newly emerging type of biophysical assemblies, decipher the molecular codes bridging molecular behaviors, signaling axes, and clinical prognosis. Particularly, physical traits of BCs play an important role; however, a panoramic view from this perspective toward clinical practices remains lacking. In this review, we describe the most typical five physical traits of BCs, and comprehensively summarize their roles in molecular signaling axes and corresponding major determinants. Moreover, establishing the recent observed contribution of condensate physics on clinical therapeutics, we illustrate next-generation medical strategies by targeting condensate physics. Finally, the challenges and opportunities for future medical development along with the rapid scientific and technological advances are highlighted.

Biomolecular Condensates: Structure, Functions, Methods of Research.

The term "biomolecular condensates" is used to describe membraneless compartments in eukaryotic cells, accumulating proteins and nucleic acids. Biomolecular condensates are formed as a result of liquid-liquid phase separation (LLPS). Often, they demonstrate properties of liquid-like droplets or gel-like aggregates; however, some of them may appear to have a more complex structure and high-order organization. Membraneless microcompartments are involved in diverse processes both in cytoplasm and in nucleus, among them ribosome biogenesis, regulation of gene expression, cell signaling, and stress response. Condensates properties and structure could be highly dynamic and are affected by various internal and external factors, e.g., concentration and interactions of components, solution temperature, pH, osmolarity, etc. In this review, we discuss variety of biomolecular condensates and their functions in live cells, describe their structure variants, highlight domain and primary sequence organization of the constituent proteins and nucleic acids. Finally, we describe current advances in methods that characterize structure, properties, morphology, and dynamics of biomolecular condensates in vitro and in vivo.

Photoswitchable Endocytosis of Biomolecular Condensates in Giant Vesicles.

Interactions between membranes and biomolecular condensates can give rise to complex phenomena such as wetting transitions, mutual remodeling, and endocytosis. In this study, light-triggered manipulation of condensate engulfment is demonstrated using giant vesicles containing photoswitchable lipids. UV irradiation increases the membrane area, which can be stored in nanotubes. When in contact with a condensate droplet, the UV light triggers rapid condensate endocytosis, which can be reverted by blue light. The affinity of the protein-rich condensates to the membrane and the reversibility of the engulfment processes is quantified from confocal microscopy images. The degree of photo-induced engulfment, whether partial or complete, depends on the vesicle excess area and the relative sizes of vesicles and condensates. Theoretical estimates suggest that utilizing the light-induced excess area to increase the vesicle-condensate adhesion interface is energetically more favorable than the energy gain from folding the membrane into invaginations and tubes. The overall findings demonstrate that membrane-condensate interactions can be easily and quickly modulated via light, providing a versatile system for building platforms to control cellular events and design intelligent drug delivery systems for cell repair.

Proteomic composition of eukaryotic and bacterial RNA decay condensates suggests convergent evolution.

Bacterial cells have a unique challenge to organize their cytoplasm without the use of membrane-bound organelles. Biomolecular condensates (henceforth BMCs) are a class of nonmembrane-bound organelles, which, through the physical process of phase separation, can form liquid-like droplets with proteins/nucleic acids. BMCs have been broadly characterized in eukaryotic cells, and BMCs have been recently identified in bacteria, with the first and best studied example being bacterial ribonucleoprotein bodies (BR-bodies). BR-bodies contain the RNA decay machinery and show functional parallels to eukaryotic P-bodies (PBs) and stress granules (SGs). Due to the finding that mRNA decay machinery is compartmentalized in BR-bodies and in eukaryotic PBs/SGs, we will explore the functional similarities in the proteins, which are known to enrich in these structures based on recent proteomic studies. Interestingly, despite the use of different mRNA decay and post-transcriptional regulatory machinery, this analysis has revealed evolutionary convergence in the classes of enriched enzymes in these structures.