Tomas Sneideris

Biomolecular condensates are membraneless compartments, crucial for organising and regulating diverse cellular processes. Current approaches to study condensate biology either use simplified recombinant protein systems with limited physiological relevance, or complex live-cell models with restricted experimental control and scalability. Here, we present ExVivo PhaseScan, a droplet microfluidics platform that couples mammalian lysate-based reconstitution with scalable analysis to generate high-resolution phase diagrams of compositionally complex protein condensates. We apply this approach to study two multicomponent condensate systems, stress granules and nucleoli, and dissect the physicochemical interactions that influence their stability. We further developed a machine learning pipeline to analyse condensate morphology which we use to reveal how mutations in the amyotrophic lateral sclerosis (ALS)-linked protein Fused in Sarcoma (FUS) remodels condensate properties. We identify liquid-to-solid transitions of mutant FUS within stress granules and nucleoli, and show that these transitions can be reversed by RNA aptamer-based interventions. Together, these findings establish ExVivo PhaseScan as a versatile tool for dissecting the physicochemical and pathological regulation of condensates, with potential to inform therapeutic strategies for diseases driven by aberrant phase transitions.

Characterizing the mechanical properties of single colloids is a central problem in soft matter physics. It also plays a key role in cell biology through biopolymer condensates, which function as membraneless compartments. Such systems can also malfunction, leading to the onset of a number of diseases, including many neurodegenerative diseases; the functional and pathological condensates are commonly differentiated by their mechanical signature. Comprehensive understanding of mechanical properties of biopolymer condensates has, however, remained challenging. In this study, we demonstrate that acoustic trapping can be used to probe the bulk modulus of biopolymer condensates in a contactless manner. We find that acoustic fields exert an acoustic radiation force on condensates, leading to their migration to a trapping point where acoustic potential energy is minimized. Furthermore, our results show that the positional fluctuation of a condensate undergoing Brownian motion in an acoustic potential well can be used as a probe for its bulk modulus. Using this framework, we infer changes in the bulk modulus of polyadenylic acid condensates in response to variations in ionic strength. Our results show that acoustic trapping opens up a path to characterize the bulk modulus of soft colloids in a noninvasive manner with applications in biology, materials science, and beyond.

Respiratory syncytial virus (RSV) causes severe respiratory infections, with viral replication occurring in cytoplasmic membraneless viral factories formed by liquid-liquid phase separation (LLPS). The interactions among the RSV nucleoprotein N, phosphoprotein P, transcription factor M2-1, and RNA drive these condensates. Here, we used a microfluidic PhaseScan platform, together with biochemical and cellular assays, to systematically characterize LLPS involving RSV proteins and RNA. We identified optimal concentrations of oligomeric N and P tetramers for condensate formation in vitro without crowding agents, demonstrated that monomeric N inhibits LLPS and revealed that M2-1 enhances condensate formation by increasing multivalency. Notably, we found that M2-1 preferentially binds 5′ capped RNA, distinguishing it from N, which binds uncapped RNA. These findings elucidate molecular determinants of RSV viral factory assembly and subcompartmentalization, providing insights into viral replication mechanisms and informing potential antiviral strategies targeting LLPS processes.

Neurofibrillary tangles (NFTs) formed from the protein tau disrupt neuronal function in Alzheimer’s disease and are strongly associated with cognitive decline. Early events in tau aggregation are increasingly linked to the formation of biomolecular condensates, which lower the energetic barriers to pathological aggregation by acting as intermediates that transition into insoluble assemblies, a mechanism also implicated in other neurodegenerative diseases, such as amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Despite growing evidence for this pathway, the molecular basis by which reversible condensates evolve into irreversible, pathogenic aggregates has remained unclear. Here, we map the phase behavior, structural transitions, and thermodynamic reversibility of tau during condensate aging. Our results reveal that the two hallmark features of the pathological end state, β-sheet enrichment and irreversible aggregation, emerge at different rates and occupy distinct regions of the phase space, indicating that these properties are mechanistically uncoupled. Notably, we identify tau condensate phases that are β-sheet rich yet thermodynamically reversible, as well as irreversible intermediates that lack β-sheet structure. These findings expand the landscape of tau aggregate species beyond a simple linear progression toward fibrils and highlight a diverse array of intermediates with distinct structural and thermodynamic properties. This decoupling of structure and irreversibility has important implications for understanding tau aggregation mechanisms and may offer targets for therapeutic intervention.

Multicomponent phase-separating molecular systems play a key role as membraneless organelles in living cells. Phase diagrams are indispensable for probing the concentration-dependent behavior of these condensates, yet their interpretation has remained largely qualitative due to challenges of modeling complex, multicomponent interactions. Here, we present a generic framework for quantitative analysis and interpretation of phase diagrams. We derive an analytical expression for the phase boundary normal vector, which reveals two distinct scaling regimes: one for associative phase separation, characterized by a power-law scaling analogous to mass-action kinetics that allows for direct extraction of dense phase solute molar ratios, and another for condensate dissolution, where an exponential scaling quantifies three-body repulsion effects triggered by the addition of a new component. We analyze a wide range of experimentally measured phase diagrams, covering proteins NSP5, NSP2, FUS, Whi3, G3BP1, and antimicrobial peptides, and we demonstrate that the additional quantitative insights can aid the mechanistic interpretation of their phase behavior.

Electrochemical gradients are essential to the functioning of cells and form across membranes using active transporters. Here we show in contrast that condensed biomolecular systems—often termed condensates—sustain pH gradients without any external energy input. By studying individual condensates on the micrometre scale using a microdroplet platform, we reveal dense-phase pH shifts towards conditions of minimal electrostatic repulsion. We demonstrate that protein condensates can drive substantial alkaline and acidic gradients, which are compositionally tunable and can extend to complex architectures sustaining multiple unique pH conditions simultaneously. Through in silico characterization of human proteomic condensate networks, we further highlight potential wide-ranging electrochemical properties emerging from condensation in nature, while correlating intracellular condensate pH gradients with complex biomolecular composition. Together, the emergent nature of condensation shapes distinct pH microenvironments, thereby creating a regulatory mechanism to modulate biochemical activity in living and artificial systems.

Biomolecular condensates are membraneless compartments, crucial for organising and regulating diverse cellular processes. Current approaches to study condensate biology either use simplified recombinant protein systems with limited physiological relevance, or complex live-cell models with restricted experimental control and scalability. Here, we present ExVivo PhaseScan, a droplet microfluidics platform that couples mammalian lysate-based reconstitution with scalable analysis to generate high-resolution phase diagrams of physiological protein condensates. We apply this approach to study two complex multicomponent condensate systems, stress granules and nucleoli, and dissect the physicochemical interactions that influence their stability. We further developed a machine learning pipeline to analyse condensate morphology which we use to reveal how mutations in the ALS-linked protein FUS remodels condensate phase landscapes. We identify liquid-to-solid transitions of mutant FUS within stress granules and nucleoli, and show that these transitions can be reversed by RNA aptamer-based interventions. Together, these findings establish ExVivo PhaseScan as a versatile tool for dissecting the physicochemical and pathological regulation of condensates, with potential to inform therapeutic strategies for diseases driven by aberrant phase transitions.

Respiratory syncytial virus (RSV) causes severe respiratory infections, with viral replication occurring in cytoplasmic membraneless viral factories formed by liquid-liquid phase separation (LLPS). The interactions between the RSV nucleoprotein N, phosphoprotein P, transcription factor M2-1, and RNA drive these condensates. Here we employed a microfluidic Phase Scan platform, together with biochemical and cellular assays to systematically characterise LLPS involving RSV proteins and RNA. We identified optimal stoichiometric ratios of N oligomers and P tetramers for condensate formation, demonstrated that monomeric N inhibits LLPS, and revealed that M2-1 enhances condensate formation by increasing multivalency. Notably, we discovered that M2-1 preferentially binds 5’ capped RNA, distinguishing it from N, which binds uncapped RNA. These findings elucidate molecular determinants of RSV viral factory assembly and subcompartmentalisation, providing insights into viral replication mechanisms and informing potential antiviral strategies targeting LLPS processes.

Biomolecular phase separation is an emerging theme for protein assembly and cellular organisation. The collective forces driving such condensation, however, remain challenging to characterise. Here we show that tracking the dilute phase concentration of only one component suffices to quantify composition and energetics of multicomponent condensates. Applying this assay to several disease- and stress-related proteins, we find that monovalent ions can either deplete from or enrich within the dense phase in a context-dependent manner. By analysing the effect of the widely used modulator 1,6-hexanediol, we find that the compound inhibits phase separation by acting as a solvation agent that expands polypeptide chains. Extending the strategy to in cellulo data, we even quantify the relative energetic contributions of individual proteins within complex condensates. Together, our approach provides a generic and broadly applicable tool for dissecting the forces governing biomolecular condensation and guiding the rational modulation of condensate behaviour.

Integrating in vitro and in silico experiments allows CTPR biomolecular condensates to be rationally designed in a facile and versatile way, modulating protein–protein interactions in the dense phase through specific recruitment of target proteins.