Biomolecular condensates, often formed through liquid-liquid phase separation, are dynamic cellular compartments. Here, we demonstrate that a wide range of fluorescently tagged proteins undergo inadvertent, condensate-mediated crosslinking, resulting in rapid solidification of condensates under common fluorescence imaging conditions. The process is driven by excitation-induced, short-lived reactive oxygen species (ROS), whose otherwise limited crosslinking potential becomes uniquely enabled in the dense phase. In live cells, excitation-induced ROS potently trigger stress granule formation, while the ROS-driven solidification of condensates is modulated by compartment-dependent antioxidant buffering. Our findings demonstrate that condensates create a distinct environment that enables ROS chemistry unlikely to occur in the bulk cytosol. Furthermore, the cellular redox level can be a general regulator of condensate rheology. Beyond biological insights, our findings underscore the need for scrutiny when examining fluorophore-labeled condensates.

Abstract Alpha-synuclein (αSyn) inclusions are a defining neuropathological feature of Parkinson’s disease, but the cellular events that initiate their formation and promote neurotoxicity remain incompletely understood. Aberrant liquid–liquid phase separation has emerged as a potential early step in αSyn dysregulation, yet the physiological triggers and functional consequences of this process are unclear. Here, we show that lipid droplets promote the spontaneous phase separation of wild-type and E46K mutant αSyn into condensates. These condensates sequester lipid droplets and impair their turnover, indicating disruption of cellular lipid homeostasis. Mitochondria in close proximity to αSyn condensates exhibit reduced membrane potential and increased mitophagy. Correlative light and electron microscopy further reveals αSyn oligomers associated with mitochondrial membranes displaying structural abnormalities. Together, these findings identify lipid droplets as drivers of aberrant αSyn phase separation and suggest that lipid droplet-rich condensates contribute to mitochondrial dysfunction and impaired energy homeostasis. Given the enrichment of lipid droplets within neuromelanin-containing dopaminergic neurons of the substantia nigra, this mechanism may be relevant to the selective neuronal vulnerability observed in Parkinson’s disease.

Fused in sarcoma (FUS) undergoes liquid-liquid phase separation (LLPS) to support essential cellular functions, but aberrant phase transitions promote toxic aggregation in neurodegenerative disease. Short RNA oligonucleotides can reverse this behavior, yet the structural determinants that govern RNA activity remain poorly defined. Here, we identify RNA G-quadruplexes (rG4s) as tunable structural motifs that potently modulate FUS LLPS. rG4 activity depends on its concentration and is modulated by rG4 length and stability: increasing repeat number switches rG4s from inhibitor to nucleator of FUS assembly, whereas chemical modifications that stabilize rG4 enhance inhibitory function and render these activities resilient to ionic perturbation. Although short rG4s interact with both soluble and condensed FUS, they preferentially engage the soluble pool, likely shifting the equilibrium toward dispersion. Leveraging these mechanistic insights, we developed a bioinformatic pipeline that uncovered more rG4 inhibitors that robustly reverse FUS LLPS and aggregation. Our findings establish rG4s as chemically programmable regulators of protein phase behavior and provide a blueprint for engineering RNA-based therapeutics that dissolve pathogenic FUS assemblies. More broadly, this work directly links RNA secondary structure to distinct functional outcomes in phase behavior, establishing a structure-function paradigm for RNA control of condensates, demonstrating implications in both fundamental biology and therapeutic development.

Cells use post-translational modifications (PTMs) to reconfigure biomolecular condensates across length scales, space, and time.1,2 While charged PTMs are well-known electrostatic switches,3,4 how ubiquitous neutral PTMs shape condensate plasticity and hierarchy remains unclear. Here, we establish a set of design principles for using site-specific lipidation, a class of neutral hydrophobic PTMs, to rationally control properties and interactions of engineered biomolecular condensates. Through systematic analysis of over 80 lipidated synthetic intrinsically disordered proteins (IDPs), we uncovered two distinct axes of control. First, the interplay between the lipid and the local three-residue sequence of its attachment site acts as a programmable switch for cohesion-the homotypic interactions that define the material state of the condensed phase-directing assemblies toward dynamic liquids, arrested gels, or ordered fibrillar solids. Second, the lipid, together with the global properties of the IDP scaffold, tunes adhesion-the heterotypic interactions that govern condensate miscibility and hierarchical organization. We harnessed these principles to rationally engineer complex, multi-phase architectures and create hybrid hydrogels with programmed microstructure and material properties that guide the morphogenesis of functional intestinal organoids. These findings establish a new framework for lipoengineering advanced biomaterials and provide a blueprint for dissecting structure-property relationships across diverse classes of PTMs.

Tau and α-Synuclein (αSyn) frequently co-aggregate in various neurodegenerative disorders. Recently, Tau has been shown to form dynamic, liquid-like condensates that can recruit αSyn, and potentially serve as a precursor to pathological aggregation. However, the quantitative impact of αSyn on the material properties of these condensates remains elusive. Here, we measure the viscosity and interfacial tension of Tau condensates and determine how these properties are modulated by αSyn monomers and fibril seeds. We find that while both forms of αSyn partition efficiently into Tau condensates, they exert vastly different effects on the condensate's material state. The viscosity of Tau condensates remains unchanged in the presence of αSyn monomers at concentrations up to 200 μM, accompanied by a moderate reduction in the condensates' interfacial tension. In contrast, the addition of only 5 μM αSyn fibril seeds triggers rapid solidification of Tau condensates, manifested by a nearly 100-fold increase in condensate viscosity within one hour. These findings provide quantitative insights into condensate mechanics, highlighting the unique capacity of αSyn seeds to drive the liquid-to-solid transition of Tau condensates that may underlie the formation of pathological aggregates.

The integrity of the plasma membrane is vital for nearly all aspects of cell functioning1. Mechanical forces can cause plasma membrane damage2, but it is unclear whether there are large molecules that regulate the integrity of the plasma membrane under mechanical strain. Here we constructed a 384-well cellular-stretch system that delivers precise, reproducible strain to cultured cells. Using the system, we screened 10,843 small interfering RNAs (siRNAs) targeting 2,726 multipass transmembrane proteins for strain-induced membrane permeability changes. The screen identified NINJ1-a protein that was recently proposed to regulate pyroptosis and other lytic cell death3,4-as the top hit. We demonstrate that NINJ1 is a critical regulator of mechanical-strain-induced plasma membrane rupture (PMR), without the need for stimulating any cell death programs. NINJ1 levels on the plasma membrane are inversely correlated with the amount of force required to rupture the membrane. In the pyroptosis context, NINJ1 on its own is not sufficient to fully rupture the membrane, and additional mechanical force is required for full PMR. Our study establishes that NINJ1 functions as a bona fide determinant of membrane biomechanical properties. Our study also suggests that PMR across tissues of distinct mechanical microenvironments is subjected to fine-tuning by differences in NINJ1 expression and external forces.

The nucleolus is a multiphasic biomolecular condensate that facilitates ribosome biogenesis, a complex process involving hundreds of proteins and RNAs. The proper execution of ribosome biogenesis likely depends on the material properties of the nucleolus. However, these material properties remain poorly understood due to the challenges of in vivo measurements. Here, we use micropipette aspiration (MPA) to directly characterize the viscoelasticity and interfacial tensions of nucleoli within transcriptionally active Xenopus laevis oocytes. We examine the major nucleolar subphases, the outer granular component (GC) and the inner dense fibrillar component (DFC), which itself contains a third small phase known as the fibrillar center (FC). We show that the behavior of the GC is more liquid-like, while the behavior of the DFC/FC is consistent with that of a partially viscoelastic solid. To determine the role of ribosomal RNA in nucleolar material properties, we degrade RNA using RNase A, which causes the DFC/FC to become more fluid-like and alters interfacial tension. Together, our findings suggest that RNA underlies the partially solid-like properties of the DFC/FC and provide insights into how material properties of nucleoli in a near-native environment are related to their RNA-dependent function.

Biomolecular condensates are protein-dense regions in cells that often arise from liquid-liquid phase separation. Interfacial tension is a key determinant of biomolecular condensate behavior, influencing condensate size and interactions with intracellular structures. Certain proteins and RNAs are known to selectively localize to the interface of condensates, where they can regulate condensate function in cells. Previously, we designed amphiphilic proteins that preferentially adsorb to the surface of condensates. These proteins contain one condensate-philic domain (RGG) and one condensate-phobic domain (MBP or GST). Here, we demonstrate through direct quantification that these amphiphilic proteins act as surfactants, reducing the interfacial tension of RGG-RGG condensates from ∼260 to ∼100 μN/m in a concentration-dependent manner. Notably, the GST-based surfactant protein exhibits a 10-fold greater efficacy in lowering interfacial tension compared with the MBP-based surfactant. We show that this increased efficacy is due to its higher surface density, driven by GST's ability to oligomerize. We also show that these surfactant proteins slow droplet fusion and reduce the average droplet size, as would be expected of a typical surfactant. Our findings quantitatively show how surfactant proteins can play a critical role in regulating the behavior of biomolecular condensates by modulating their interfacial tension.

Tension-adhesion interplay is a crucial mechanism in multicellular organisms that determines the tension differential among internal and external interfaces, which in turn, mediates tissue surface tension and cell sorting, morphogenesis and remodeling, and cancer progression. Cadherins are widely believed to be involved, yet key aspects of the process are neither well characterized nor quantified. This study demonstrates the critical role of N-cadherin in driving tension polarization throughout the actin cortical network. N-cadherin regulates both tension increase at the cell-medium (external) interface and decrease at the cell-cell (internal) interface, and their quantitative magnitudes, both absolute and relative, strongly depend on the surface density of N-cadherin. Furthermore, the strength of tension polarization also increases with respect to the number of cell-cell interfaces for cells within a multicellular cluster. The cadherin-actin contractility linkage is mediated by Rac1, which serves as a molecular switch to trigger cortex remodeling and contraction via myosin II. Inhibition of Rac1 activity decreases tension polarization and leads to reduced coherence in both small clusters and spheroids. These results provide a pathway to reconcile opposing theories for tissue surface tension generation and perspectives in cancer treatment.

Biomolecular condensates are increasingly recognized as central regulators of numerous cellular processes. The bulk rheology of condensates (e.g., viscoelasticity) balances molecular mobility with structural stability, while the interfacial properties of condensates (e.g., interfacial tension) regulate condensate growth and their interactions with other cellular structures. Here, we review the functional roles of condensate rheology and interfacial properties, as well as diseases associated with their dysregulation. By summarizing emerging methodologies and quantitative measurements of condensate viscoelasticity and interfacial tension in the literature, we highlight key regulators of condensate material properties and discuss their implications in biology.