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Prion-like low-complexity domains (PLCDs) have distinctive sequence grammars that determine their driving forces for phase separation. Here we uncover the physicochemical underpinnings of how evolutionarily conserved compositional biases influence the phase behaviour of PLCDs. We interpret our results in the context of the stickers-and-spacers model for the phase separation of associative polymers. We find that tyrosine is a stronger sticker than phenylalanine, whereas arginine is a context-dependent auxiliary sticker. In contrast, lysine weakens sticker–sticker interactions. Increasing the net charge per residue destabilizes phase separation while also weakening the strong coupling between single-chain contraction in dilute phases and multichain interactions that give rise to phase separation. Finally, glycine and serine residues act as non-equivalent spacers, and thus make the glycine versus serine contents an important determinant of the driving forces for phase separation. The totality of our results leads to a set of rules that enable comparative estimates of composition-specific driving forces for PLCD phase separation. The complex link between protein sequence and phase behaviour for a family of prion-like low-complexity domains (PLCDs) has now been revealed. The results have uncovered a set of rules—which are interpreted using a stickers-and-spacers model—that govern the sequence-encoded phase behaviour of such PLCDs and enable physicochemical rationalizations that are connected to the underlying sequence composition.

Intracellular organelles are either membrane-bound vesicles or membrane-less compartments that are made up of proteins and RNA. These organelles play key biological roles, by compartmentalizing the cell to enable spatiotemporal control of biological reactions. Recent studies suggest that membrane-less intracellular compartments are multicomponent viscous liquid droplets that form via phase separation. Proteins that have an intrinsic tendency for being conformationally heterogeneous seem to be the main drivers of liquid–liquid phase separation in the cell. These findings highlight the relevance of classical concepts from the physics of polymeric phase transitions for understanding the assembly of intracellular membrane-less compartments. However, applying these concepts is challenging, given the heteropolymeric nature of protein sequences, the complex intracellular environment, and non-equilibrium features intrinsic to cells. This provides new opportunities for adapting established theories and for the emergence of new physics. The internal structure of cells is organized into compartments, many of which lack a confining membrane and instead resemble viscous liquid droplets. Evidence is mounting that these compartments form via spontaneous phase transitions. The internal structure of cells is organized into compartments, many of which lack a confining membrane and instead resemble viscous liquid droplets. Evidence is mounting that these compartments form via spontaneous phase transitions.

Liquid phase separation into two or more coexisting phases has emerged as a new paradigm for understanding subcellular organization, prebiotic life, and the origins of disease. The design principles underlying biomolecular phase separation have the potential to drive the development of novel liquid-based organelles and therapeutics, however, an understanding of how individual molecules contribute to emergent material properties, and approaches to directly manipulate phase dynamics are lacking. Here, using microrheology, we demonstrate that droplets of poly-arginine coassembled with mono/polynucleotides have approximately 100 fold greater viscosity than comparable lysine droplets, both of which can be finer tuned by polymer length. We find that these amino acid-level differences can drive the formation of coexisting immiscible phases with tunable formation kinetics and can be further exploited to trigger the controlled release of droplet components. Together, this work provides a novel mechanism for leveraging sequence-level components in order to regulate droplet dynamics and multiphase coexistence. The design principles underlying biomolecular phase separation of membrane-less organelles remain poorly understood. Using model homopolymers, Fisher et al. show that the formation kinetics of coexisting liquid phases can be tuned by exploiting differences between arginine and lysine residues.

All structures within living cells must form at the right time and place. This includes condensates such as the nucleolus, Cajal bodies and stress granules, which form via liquid–liquid phase separation of biomolecules, particularly proteins enriched in intrinsically disordered regions (IDRs) 1 , 2 . In non-living systems, the initial stages of nucleated phase separation arise when thermal fluctuations overcome an energy barrier due to surface tension. This phenomenon can be modelled by classical nucleation theory (CNT), which describes how the rate of droplet nucleation depends on the degree of supersaturation, whereas the location at which droplets appear is controlled by interfacial heterogeneities 3 , 4 . However, it remains unknown whether this framework applies in living cells, owing to the multicomponent and highly complex nature of the intracellular environment, including the presence of diverse IDRs, whose specificity of biomolecular interactions is unclear 5 – 8 . Here we show that despite this complexity, nucleation in living cells occurs through a physical process similar to that in inanimate materials, but the efficacy of nucleation sites can be tuned by their biomolecular features. By quantitatively characterizing the nucleation kinetics of endogenous and biomimetic condensates in living cells, we find that key features of condensate nucleation can be quantitatively understood through a CNT-like theoretical framework. Nucleation rates can be substantially enhanced by compatible biomolecular (IDR) seeds, and the kinetics of cellular processes can impact condensate nucleation rates and specificity of location. This quantitative framework sheds light on the intracellular nucleation landscape, and paves the way for engineering synthetic condensates precisely positioned in space and time. Experiments using endogenous and biomimetic condensates in cells show that nucleation in cells resembles the physical process in inanimate materials, but is tuned by biomolecular features.

Intracellular bodies such as nucleoli, Cajal bodies and various signalling assemblies represent membraneless organelles, or condensates, that form via liquid–liquid phase separation (LLPS) 1 , 2 . Biomolecular interactions—particularly homotypic interactions mediated by self-associating intrinsically disordered protein regions—are thought to underlie the thermodynamic driving forces for LLPS, forming condensates that can facilitate the assembly and processing of biochemically active complexes, such as ribosomal subunits within the nucleolus. Simplified model systems 3 – 6 have led to the concept that a single fixed saturation concentration is a defining feature of endogenous LLPS 7 – 9 , and has been suggested as a mechanism for intracellular concentration buffering 2 , 7 , 8 , 10 . However, the assumption of a fixed saturation concentration remains largely untested within living cells, in which the richly multicomponent nature of condensates could complicate this simple picture. Here we show that heterotypic multicomponent interactions dominate endogenous LLPS, and give rise to nucleoli and other condensates that do not exhibit a fixed saturation concentration. As the concentration of individual components is varied, their partition coefficients change in a manner that can be used to determine the thermodynamic free energies that underlie LLPS. We find that heterotypic interactions among protein and RNA components stabilize various archetypal intracellular condensates—including the nucleolus, Cajal bodies, stress granules and P-bodies—implying that the composition of condensates is finely tuned by the thermodynamics of the underlying biomolecular interaction network. In the context of RNA-processing condensates such as the nucleolus, this manifests in the selective exclusion of fully assembled ribonucleoprotein complexes, providing a thermodynamic basis for vectorial ribosomal RNA flux out of the nucleolus. This methodology is conceptually straightforward and readily implemented, and can be broadly used to extract thermodynamic parameters from microscopy images. These approaches pave the way for a deeper understanding of the thermodynamics of multicomponent intracellular phase behaviour and its interplay with the nonequilibrium activity that is characteristic of endogenous condensates. Heterotypic multicomponent interactions are shown to dominate the liquid–liquid phase separation that enables the formation of intracellular condensates.

Expansions of short nucleotide repeats produce several neurological and neuromuscular disorders including Huntington disease, muscular dystrophy, and amyotrophic lateral sclerosis. A common pathological feature of these diseases is the accumulation of the repeat-containing transcripts into aberrant foci in the nucleus. RNA foci, as well as the disease symptoms, only manifest above a critical number of nucleotide repeats, but the molecular mechanism governing foci formation above this characteristic threshold remains unresolved. Here we show that repeat expansions create templates for multivalent base-pairing, which causes purified RNA to undergo a sol–gel transition in vitro at a similar critical repeat number as observed in the diseases. In human cells, RNA foci form by phase separation of the repeat-containing RNA and can be dissolved by agents that disrupt RNA gelation in vitro . Analogous to protein aggregation disorders, our results suggest that the sequence-specific gelation of RNAs could be a contributing factor to neurological disease. Nucleotide repeat expansions create templates for multivalent base-pairing, which causes RNA to undergo a sol–gel phase transition and may explain the formation of nuclear RNA foci that are commonly observed in several neurological and neuromuscular diseases. Phase transitions make lengthened RNAs toxic Expansion in the length of certain trinucleotide sequences underlies several neurological diseases. The lengthened repeats are thought to make the RNA transcripts 'toxic', and at disease-associated lengths these RNAs form foci. Ankur Jain and Ron Vale find that the reason foci formation requires a particular length repeat is because at the disease length, the RNA undergoes a phase separation. This suggests that, similar to neurological diseases caused by protein aggregation, the gelation of RNAs may contribute to the pathology of trinucleotide-repeat diseases.

Autologous bone (AB) is the gold standard for bone-replacement surgeries 1 , despite its limited availability and the need for an extra surgical site. Traditionally, competitive biomaterials for bone repair have focused on mimicking the mineral aspect of bone, as evidenced by the widespread clinical use of bioactive ceramics 2 . However, AB also exhibits hierarchical organic structures that might substantially affect bone regeneration. Here, using a range of cell-free biomimetic-collagen-based materials in murine and ovine bone-defect models, we demonstrate that a hierarchical hybrid microstructure—specifically, the twisted plywood pattern of collagen and its association with poorly crystallized bioapatite—favourably influences bone regeneration. Our study shows that the most structurally biomimetic material has the potential to stimulate bone growth, highlighting the pivotal role of physicochemical properties in supporting bone formation and offering promising prospects as a competitive bone-graft material. By examining several cell-free biomimetic-collagen-based materials in murine and ovine bone-defect models, the twisted plywood pattern of collagen-based materials is shown to favourably influence bone regeneration and contributes to bone autograft performance.

Cells harbour numerous mesoscale membraneless compartments that house specific biochemical processes and perform distinct cellular functions. These protein- and RNA-rich bodies are thought to form through multivalent interactions among proteins and nucleic acids, resulting in demixing via liquid–liquid phase separation. Proteins harbouring intrinsically disordered regions (IDRs) predominate in membraneless organelles. However, it is not known whether IDR sequence alone can dictate the formation of distinct condensed phases. We identified a pair of IDRs capable of forming spatially distinct condensates when expressed in cells. When reconstituted in vitro, these model proteins do not co-partition, suggesting condensation specificity is encoded directly in the polypeptide sequences. Through computational modelling and mutagenesis, we identified the amino acids and chain properties governing homotypic and heterotypic interactions that direct selective condensation. These results form the basis of physicochemical principles that may direct subcellular organization of IDRs into specific condensates and reveal an IDR code that can guide construction of orthogonal membraneless compartments. Cells spatially organize biochemical reactions within membrane-bound and membraneless compartments. The extent to which intrinsically disordered proteins themselves can form discrete compartments or condensed phases is poorly understood. Now a pair of model IDRs that display orthogonality in condensation and the chain features governing selective assembly have been identified.

The endothelial cell (EC) outgrowth in both vasculogenesis and angiogenesis starts with remodeling surrounding matrix and proceeds with the crosstalk between cells for the multicellular vasculature formation. The mechanical plasticity of matrix, defined as the ability to permanently deform by external traction, is pivotal in modulating cell behaviors. Nevertheless, the implications of matrix plasticity on cell-to-cell interactions during EC outgrowth, along with the molecular pathways involved, remain elusive. Here we develop a collagen-hyaluronic acid based hydrogel platform with tunable plasticity by using compositing strategy of dynamic and covalent networks. We show that although the increasing plasticity of the hydrogel facilitates the matrix remodeling by ECs, the largest tubular lumens and the longest invading distance unexpectedly appear in hydrogels with medium plasticity instead of the highest ones. We unravel that the high plasticity of the hydrogels promotes stable integrin cluster of ECs and recruitment of focal adhesion kinase with an overenhanced contractility which downregulates the vascular endothelial cadherin expression and destabilizes the adherens junctions between individual ECs. Our results, further validated with mathematical simulations and in vivo angiogenic tests, demonstrate that a balance of matrix plasticity facilitates both cell-matrix binding and cell-to-cell adherens, for promoting vascular assembly and invasion. It is vital to unveil the effects of extracellular matrix cues on endothelial cell (EC) outgrowth for desirably governing vasculature formation, but the role of matrix plasticity on EC outgrowth is elusive. Here, the authors develop hydrogels with tunable mechanical plasticity independent of stiffness, and elucidate the plasticity-mediated responses of ECs during vasculogenesis and angiogenesis.

Cells compartmentalize their intracellular environment to orchestrate countless simultaneous biochemical processes. Many intracellular tasks rely on membrane-less organelles, multicomponent condensates that assemble by liquid–liquid phase separation. A decade of intensive research has provided a basic understanding of the biomolecular driving forces underlying the form and function of such organelles. Here we review the technologies enabling these developments, along with approaches to designing spatiotemporally actuated organelles based on multivalent low-affinity interactions. With these recent advances, it is now becoming possible both to modulate the properties of native condensates and to engineer entirely new structures, with the potential for widespread biomedical and biotechnological applications. Engineering and manipulating phase-separated liquid organelles is the latest frontier in the quest to mimic and interrogate living systems at the molecular level.