Physical limits to acceleration of enzymatic reactions inside phase-separated compartments.

We present a theoretical analysis of phase-separated compartments to facilitate enzymatic chemical reactions. While phase separation can facilitate reactions by increasing local concentration, it can also hinder the mobility of reactants. In particular, we find that the attractive interactions that concentrate reactants within the dense phase can inhibit reactions by lowering the mobility of the reactants. This mobility loss severely limits the potential to enhance reaction rates. Phase separation provides greater benefit in situations where multiple sequential reactions occur and/or high order reactions, provided the enzymes are unsaturated, transport to the condensate is not limiting, and the reactants are mobile. We show that mobility can be maintained if recruitment to the condensed phase is driven by multiple attractive moieties that can bind and release independently. However, the spacers necessary to ensure independence between stickers are prone to entangle with the dense phase scaffold. We find an optimal sticker affinity that balances the need for rapid binding/unbinding kinetics and minimal entanglement. Reaction rates can be accelerated by shrinking the size of the dense phase with a corresponding increase in the number of stickers. Our results showcase the potential capabilities of phase-separated compartments to act as biochemical reaction crucibles within living cells.

Interfacial Water Is Separated from a Hydrophobic Silica Surface by a Gap of 1.2 nm.

The interaction of liquid water with hydrophobic surfaces is ubiquitous in life and technology. Yet, the molecular structure of interfacial liquid water on these surfaces is not known. By using a 3D atomic force microscope, we characterize with angstrom resolution the structure of interfacial liquid water on hydrophobic and hydrophilic silica surfaces. The combination of 3D AFM images and molecular dynamics simulations reveals that next to a hydrophobic silica surface, there is a 1.2 nm region characterized by a very low density of water. In contrast, the 3D AFM images obtained of a hydrophilic silica surface reveal the presence of hydration layers next to the surface. The gap observed on hydrophobic silica surfaces is filled with two-to-three layers of straight-chain alkanes. We developed a 2D Ising model that explains the formation of a continuous hydrocarbon layer on hydrophobic silica surfaces.

Crossing boundaries of light microscopy resolution discerns novel assemblies in the nucleolus.

The nucleolus is the largest membraneless organelle and nuclear body in mammalian cells. It is primarily involved in the biogenesis of ribosomes, essential macromolecular machines responsible for synthesizing all proteins required by the cell. The assembly of ribosomes is evolutionarily conserved and accounts for the most energy-consuming cellular process needed for cell growth, proliferation, and homeostasis. Despite the significance of this process, the substructural mechanistic principles of the nucleolar function in preribosome biogenesis have only recently begun to emerge. Here, we provide a new perspective using advanced super-resolution microscopy and single-molecule MINFLUX nanoscopy on the mechanistic principles governing ribosomal RNA-seeded nucleolar formation and the resulting tripartite suborganization of the nucleolus driven, in part, by liquid-liquid phase separation. With recent advances in the cryogenic electron microscopy (cryoEM) structural analysis of ribosome biogenesis intermediates, we highlight the current understanding of the step-wise assembly of preribosomal subunits in the nucleolus. Finally, we address how novel anticancer drug candidates target early steps in ribosome biogenesis to exploit these essential dependencies for growth arrest and tumor control.

Prediction of Antibody Viscosity from Dilute Solution Measurements.

The high antibody doses required to achieve a therapeutic effect often necessitate high-concentration products that can lead to challenging viscosity issues in production and delivery. Predicting antibody viscosity in early development can play a pivotal role in reducing late-stage development costs. In recent years, numerous efforts have been made to predict antibody viscosity through dilute solution measurements. A key finding is that the entanglement of long, flexible complexes contributes to the sharp rise in antibody viscosity at the required dosing. This entanglement model establishes a connection between the two-body binding affinity and the many-body viscosity. Exploiting this insight, this study connects dilute solution measurements of self-association to high-concentration viscosity profiles to quantify the relationship between these regimes. The resulting model has exhibited success in predicting viscosity at high concentrations (around 150 mg/mL) from dilute solution measurements, with only a few outliers remaining. Our physics-based approach provides an understanding of fundamental physics, interpretable connections to experimental data, the potential to extrapolate beyond training conditions, and the capacity to effectively explain the physical mechanics behind these outliers. Conducting hypothesis-driven experiments that specifically target the viscosity and relaxation mechanisms of outlier molecules may allow us to unravel the intricacies of their behavior and, in turn, enhance the performance of our model.

Reduction of oligomer size modulates the competition between cluster formation and phase separation of the tumor suppressor SPOP.

Phase separation compartmentalizes many cellular pathways. Given that the same interactions that drive phase separation mediate the formation of soluble complexes below the saturation concentration, the contribution of condensates versus complexes to function is sometimes unclear. Here, we characterized several new cancer-associated mutations of the tumor suppressor speckle-type POZ protein (SPOP), a substrate recognition subunit of the Cullin3-RING ubiquitin ligase. This pointed to a strategy for generating separation-of-function mutations. SPOP self-associates into linear oligomers and interacts with multivalent substrates, and this mediates the formation of condensates. These condensates bear the hallmarks of enzymatic ubiquitination activity. We characterized the effect of mutations in the dimerization domains of SPOP on its linear oligomerization, binding to its substrate DAXX, and phase separation with DAXX. We showed that the mutations reduce SPOP oligomerization and shift the size distribution of SPOP oligomers to smaller sizes. The mutations therefore reduce the binding affinity to DAXX but unexpectedly enhance the poly-ubiquitination activity of SPOP toward DAXX. Enhanced activity may be explained by enhanced phase separation of DAXX with the SPOP mutants. Our results provide a comparative assessment of the functional role of complexes versus condensates and support a model in which phase separation is an important factor in SPOP function. Our findings also suggest that tuning of linear SPOP self-association could be used by the cell to modulate activity and provide insights into the mechanisms underlying hypermorphic SPOP mutations. The characteristics of cancer-associated SPOP mutations suggest a route for designing separation-of-function mutations in other phase-separating systems.

Polyubiquitin ligand-induced phase transitions are optimized by spacing between ubiquitin units.

Biomolecular condensates form via multivalent interactions among key macromolecules and are regulated through ligand binding and/or posttranslational modifications. One such modification is ubiquitination, the covalent addition of ubiquitin (Ub) or polyubiquitin chains to target macromolecules. Specific interactions between polyubiquitin chains and partner proteins, including hHR23B, NEMO, and UBQLN2, regulate condensate assembly or disassembly. Here, we used a library of designed polyubiquitin hubs and UBQLN2 as model systems for determining the driving forces of ligand-mediated phase transitions. Perturbations to either the UBQLN2-binding surface of Ub or the spacing between Ub units reduce the ability of hubs to modulate UBQLN2 phase behavior. By developing an analytical model based on polyphasic linkage principles that accurately described the effects of different hubs on UBQLN2 phase separation, we determined that introduction of Ub to UBQLN2 condensates incurs a significant inclusion energetic penalty. This penalty antagonizes the ability of polyUb hubs to scaffold multiple UBQLN2 molecules and cooperatively amplify phase separation. The extent to which polyubiquitin hubs promote UBQLN2 phase separation is encoded in the spacings between Ub units. This spacing is modulated by chains of different linkages and designed chains of different architectures, thus illustrating how the ubiquitin code regulates functionality via the emergent properties of the condensate. The spacing in naturally occurring linear polyubiquitin chains is already optimized to promote phase separation with UBQLN2. We expect our findings to extend to other condensates, emphasizing the importance of ligand properties, including concentration, valency, affinity, and spacing between binding sites in studies and designs of condensates.

Architectural basis for cylindrical self-assembly governing Plk4-mediated centriole duplication in human cells.

Proper organization of intracellular assemblies is fundamental for efficient promotion of biochemical processes and optimal assembly functionality. Although advances in imaging technologies have shed light on how the centrosome is organized, how its constituent proteins are coherently architected to elicit downstream events remains poorly understood. Using multidisciplinary approaches, we showed that two long coiled-coil proteins, Cep63 and Cep152, form a heterotetrameric building block that undergoes a stepwise formation into higher molecular weight complexes, ultimately generating a cylindrical architecture around a centriole. Mutants defective in Cep63•Cep152 heterotetramer formation displayed crippled pericentriolar Cep152 organization, polo-like kinase 4 (Plk4) relocalization to the procentriole assembly site, and Plk4-mediated centriole duplication. Given that the organization of pericentriolar materials (PCM) is evolutionarily conserved, this work could serve as a model for investigating the structure and function of PCM in other species, while offering a new direction in probing the organizational defects of PCM-related human diseases.

Distinct growth regimes of α-synuclein amyloid elongation.

Addition of amyloid seeds to aggregation-prone monomers allows for amyloid fiber growth (elongation) omitting slow nucleation. We here combine Thioflavin T fluorescence (probing formation of amyloids) and solution-state NMR spectroscopy (probing disappearance of monomers) to assess elongation kinetics of the amyloidogenic protein, α-synuclein, for which aggregation is linked to Parkinson's disease. We found that both spectroscopic detection methods give similar kinetic results, which can be fitted by applying double exponential decay functions. When the origin of the two-phase behavior was analyzed by mathematical modeling, parallel paths as well as stop-and-go behavior were excluded as possible explanations. Instead, supported by previous theory, the experimental elongation data reveal distinct kinetic regimes that depend on instantaneous monomer concentration. At low monomer concentrations (toward end of experiments), amyloid growth is limited by conformational changes resulting in ß-strand alignments. At the higher monomer concentrations (initial time points of experiments), growth occurs rapidly by incorporating monomers that have not successfully completed the conformational search. The presence of a fast disordered elongation regime at high monomer concentrations agrees with coarse-grained simulations and theory but has not been detected experimentally before. Our results may be related to the wide range of amyloid folds observed.

Circuit Topology Approach for the Comparative Analysis of Intrinsically Disordered Proteins.

Intrinsically disordered proteins (IDPs) lack a stable native conformation, making it challenging to characterize their structure and dynamics. Key topological motifs with fundamental biological relevance are often hidden in the conformational noise, eluding detection. Here, we develop a circuit topology toolbox to extract conformational patterns, critical contacts, and timescales from simulated dynamics of intrinsically disordered proteins. We follow the dynamics of IDPs by providing a smart low-dimensionality representation of their three-dimensional (3D) configuration in the topology space. Such an approach allows us to quantify topological similarity in dynamic systems, therefore providing a pipeline for structural comparison of IDPs.

Reduction of oligomer size modulates the competition between cluster formation and phase separation of the tumor suppressor SPOP.

Phase separation is a ubiquitous process that compartmentalizes many cellular pathways. Given that the same interactions that drive phase separation mediate the formation of complexes below the saturation concentration, the contribution of condensates vs complexes to function is not always clear. Here, we characterized several new cancer-associated mutations of the tumor suppressor Speckle-type POZ protein (SPOP), a substrate recognition subunit of the Cullin3-RING ubiquitin ligase (CRL3), which pointed to a strategy for generating separation-of-function mutations. SPOP self-associates into linear oligomers and interacts with multivalent substrates, and this mediates the formation of condensates. These condensates bear the hallmarks of enzymatic ubiquitination activity. We characterized the effect of mutations in the dimerization domains of SPOP on its linear oligomerization, binding to the substrate DAXX, and phase separation with DAXX. We showed that the mutations reduce SPOP oligomerization and shift the size distribution of SPOP oligomers to smaller sizes. The mutations therefore reduce the binding affinity to DAXX, but enhance the poly-ubiquitination activity of SPOP towards DAXX. This unexpectedly enhanced activity may be explained by enhanced phase separation of DAXX with the SPOP mutants. Our results provide a comparative assessment of the functional role of clusters versus condensates and support a model in which phase separation is an important factor in SPOP function. Our findings also suggest that tuning of linear SPOP self-association could be used by the cell to modulate its activity, and provide insights into the mechanisms underlying hypermorphic SPOP mutations. The characteristics of these cancer-associated SPOP mutations suggest a route for designing separation-of-function mutations in other phase-separating systems.

Decoding optimal ligand design for multicomponent condensates.

Biomolecular condensates form via multivalent interactions among key macromolecules and are regulated through ligand binding and/or post-translational modifications. One such modification is ubiquitination, the covalent addition of ubiquitin (Ub) or polyubiquitin chains to target macromolecules for various cellular processes. Specific interactions between polyubiquitin chains and partner proteins, including hHR23B, NEMO, and UBQLN2, regulate condensate assembly or disassembly. Here, we used a library of designed polyubiquitin hubs and UBQLN2 as model systems for determining the driving forces of ligand-mediated phase transitions. Perturbations to the UBQLN2-binding surface of Ub or deviations from the optimal spacing between Ub units reduce the ability of hubs to modulate UBQLN2 phase behavior. By developing an analytical model that accurately described the effects of different hubs on UBQLN2 phase diagrams, we determined that introduction of Ub to UBQLN2 condensates incurs a significant inclusion energetic penalty. This penalty antagonizes the ability of polyUb hubs to scaffold multiple UBQLN2 molecules and cooperatively amplify phase separation. Importantly, the extent to which polyubiquitin hubs can promote UBQLN2 phase separation are encoded in the spacings between Ub units as found for naturally-occurring chains of different linkages and designed chains of different architectures, thus illustrating how the ubiquitin code regulates functionality via the emergent properties of the condensate. We expect our findings to extend to other condensates necessitating the consideration of ligand properties, including concentration, valency, affinity, and spacing between binding sites in studies and designs of condensates.

Spatially non-uniform condensates emerge from dynamically arrested phase separation.

The formation of biomolecular condensates through phase separation from proteins and nucleic acids is emerging as a spatial organisational principle used broadly by living cells. Many such biomolecular condensates are not, however, homogeneous fluids, but possess an internal structure consisting of distinct sub-compartments with different compositions. Notably, condensates can contain compartments that are depleted in the biopolymers that make up the condensate. Here, we show that such double-emulsion condensates emerge via dynamically arrested phase transitions. The combination of a change in composition coupled with a slow response to this change can lead to the nucleation of biopolymer-poor droplets within the polymer-rich condensate phase. Our findings demonstrate that condensates with a complex internal architecture can arise from kinetic, rather than purely thermodynamic driving forces, and provide more generally an avenue to understand and control the internal structure of condensates in vitro and in vivo.

Beneficial and detrimental effects of non-specific binding during DNA hybridization.

DNA strands have to sample numerous states to find the alignment that maximizes Watson-Crick-Franklin base pairing. This process depends strongly on sequence, which affects the stability of the native duplex as well as the prevalence of non-native inter- and intramolecular helices. We present a theory that describes DNA hybridization as a three-stage process: diffusion, registry search, and zipping. We find that non-specific binding affects each of these stages in different ways. Mis-registered intermolecular binding in the registry search stage helps DNA strands sample different alignments and accelerates the hybridization rate. Non-native intramolecular structure affects all three stages by rendering portions of the molecule inert to intermolecular association, limiting mis-registered alignments to be sampled, and impeding the zipping process. Once in-register base pairs are formed, the stability of the native structure is important to hold the molecules together long enough for non-native contacts to break.

Conformational entropy limits the transition from nucleation to elongation in amyloid aggregation.

The formation of ß-sheet-rich amyloid fibrils in Alzheimer's disease and other neurodegenerative disorders is limited by a slow nucleation event. To understand the initial formation of ß-sheets from disordered peptides, we used all-atom simulations to parameterize a lattice model that treats each amino acid as a binary variable with ß- and non-ß-sheet states. We show that translational and conformational entropy give the nascent ß-sheet an anisotropic surface tension that can be used to describe the nucleus with 2D classical nucleation theory. Since translational entropy depends on concentration, the aspect ratio of the critical ß-sheet changes with protein concentration. Our model explains the transition from the nucleation phase to elongation as the point where the ß-sheet core becomes large enough to overcome the conformational entropy cost to straighten the terminal molecule. At this point the ß-strands in the nucleus spontaneously elongate, which results in a larger binding surface to capture new molecules. These results suggest that nucleation is relatively insensitive to sequence differences in coaggregation experiments because the nucleus only involves a small portion of the peptide.

Low-dissipation self-assembly protocols of active sticky particles.

We use neuroevolutionary learning to identify time-dependent protocols for low-dissipation self-assembly in a model of generic active particles with interactions. When the time allotted for assembly is sufficiently long, low-dissipation protocols use only interparticle attractions, producing an amount of entropy that scales as the number of particles. When time is too short to allow assembly to proceed via diffusive motion, low-dissipation assembly protocols instead require particle self-propulsion, producing an amount of entropy that scales with the number of particles and the swim length required to cause assembly. Self-propulsion therefore provides an expensive but necessary mechanism for inducing assembly when time is of the essence.

How Hierarchical Interactions Make Membraneless Organelles Tick Like Clockwork.

Biomolecular condensates appear throughout the cell, serving many different biochemical functions. We argue that condensate functionality is optimized when the interactions driving condensation vary widely in affinity. Strong interactions provide structural specificity needed to encode functional properties but carry the risk of kinetic arrest, while weak interactions allow the system to remain dynamic but do not restrict the conformational ensemble enough to sustain specific functional features. To support our opinion, we describe illustrative examples of the interplay of strong and weak interactions that are found in the nucleolus, SPOP/DAXX condensates, polySUMO/polySIM condensates, chromatin, and stress granules. The common feature of these systems is a hierarchical assembly motif in which weak, transient interactions condense structurally defined functional units.

Structure-Function Properties in Disordered Condensates.

Biomolecular condensates appear throughout the cell serving a wide variety of functions. Many condensates appear to form by the assembly of multivalent molecules, which produce phase-separated networks with liquidlike properties. These networks then recruit client molecules, with the total composition providing functionality. Here we use a model system of poly-SUMO and poly-SIM proteins to understand client-network interactions and find that the structure of the network plays a strong role in defining client recruitment and thus functionality. The basic unit of assembly in this system is a zipperlike filament composed of alternating poly-SUMO and poly-SIM molecules. These filaments have defects of unsatisfied bonds that allow for both the formation of a 3D network and the recruitment of clients. The filamentous structure constrains the scaffold stoichiometries and the distribution of client recruitment sites that the network can accommodate. This results in a nonmonotonic client binding response that can be tuned independently by the client valence and binding energy. These results show how the interactions within liquid states can be disordered yet still contain structural features that provide functionality to the condensate.