Biomolecular condensates are found throughout eukaryotic cells, including in the nucleus, in the cytoplasm and on membranes. They are also implicated in a wide range of cellular functions, organizing molecules that act in processes ranging from RNA metabolism to signalling to gene regulation. Early work in the field focused on identifying condensates and understanding how their physical properties and regulation arise from molecular constituents. Recent years have brought a focus on understanding condensate functions. Studies have revealed functions that span different length scales: from molecular (modulating the rates of chemical reactions) to mesoscale (organizing large structures within cells) to cellular (facilitating localization of cellular materials and homeostatic responses). In this Roadmap, we discuss representative examples of biochemical and cellular functions of biomolecular condensates from the recent literature and organize these functions into a series of non-exclusive classes across the different length scales. We conclude with a discussion of areas of current interest and challenges in the field, and thoughts about how progress may be made to further our understanding of the widespread roles of condensates in cell biology.

A framework for understanding the functions of biomolecular condensates across scales.

Coacervates are condensed liquid-like droplets, usually formed with oppositely charged polymeric molecules. They have been studied extensively in colloid and interface science for their remarkable material properties. The liquid–liquid phase separation underlying coacervate formation also plays an important role in the formation of various membraneless organelles (MLOs) that are found in many living cells. Therefore, there is an increasing interest to use well-characterized coacervates as in vitro models that mimic specific aspects of MLOs. Here, we review five aspects – physical and chemical properties, hierarchical organization, uptake selectivity, formation dynamics, and maturation – that are of particular interest and discuss how useful coacervates are to better understand these aspects of MLOs.

Coacervates as models of membraneless organelles

Biomolecular condensates concentrate macromolecules into discrete cellular foci without an encapsulating membrane. Condensates are often presumed to increase enzymatic reaction rates through increased concentrations of enzymes and substrates (mass action), although this idea has not been widely tested and other mechanisms of modulation are possible. Here we describe a synthetic system where the SUMOylation enzyme cascade is recruited into engineered condensates generated by liquid–liquid phase separation of multidomain scaffolding proteins. SUMOylation rates can be increased up to 36-fold in these droplets compared to the surrounding bulk, depending on substrate KM. This dependency produces substantial specificity among different substrates. Analyses of reactions above and below the phase-separation threshold lead to a quantitative model in which reactions in condensates are accelerated by mass action and changes in substrate KM, probaby due to scaffold-induced molecular organization. Thus, condensates can modulate reaction rates both by concentrating molecules and physically organizing them. A chemically induced dimerization strategy was used to recruit SUMOylation enzymes into condensates, enabling quantification of the effect of phase separation on the activity of a SUMOylation enzyme cascade reaction.

Mechanistic dissection of increased enzymatic rate in a phase-separated compartment

Water scarcity is rapidly spreading across the planet, threatening the population across the five continents and calling for global sustainable solutions Water reclamation is the most ecological approach for supplying clean drinking water However, current water purification technologies are seldom sustainable, due to high-energy consumption and negative environmental footprint Here, we review the cutting-edge technologies based on protein nanofibrils as water purification agents and we highlight the benefits of this green, efficient and affordable solution to alleviate the global water crisis We discuss the different protein nanofibrils agents available and analyze them in terms of performance, range of applicability and sustainability We underline the unique opportunity of designing protein nanofibrils for efficient water purification starting from food waste, as well as cattle, agricultural or dairy industry byproducts, allowing simultaneous environmental, economic and social benefits and we present a case analysis, including a detailed life cycle assessment, to establish their sustainable footprint against other common natural-based adsorbents, anticipating a bright future for this water purification approach

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Protein nanofibrils for next generation sustainable water purification.

Living cells have long been a source of inspiration for chemists. Their capacity of performing complex tasks relies on the spatiotemporal coordination of matter and energy fluxes. Recent years have witnessed growing interest in the bottom-up construction of cell-like models capable of reproducing aspects of such dynamic organisation. Liquid-liquid phase-separation (LLPS) processes in water are increasingly recognised as representing a viable compartmentalisation strategy through which to produce dynamic synthetic cells. Herein, we highlight examples of the dynamic properties of LLPS used to assemble synthetic cells, including their biocatalytic activity, reversible condensation and dissolution, growth and division, and recent directions towards the design of higher-order structures and behaviour.

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Dynamic Synthetic Cells Based on Liquid–Liquid Phase Separation

Recent findings indicate that a class of disordered amino acid sequences promotes functional phase transition of biomolecules in nature Such sequences consist of low complexity domains (LCDs) that are rich in specific amino acids In this work, we exploit these sequences by conjugating them to soluble globular domains to develop molecular adhesives that enable sensitive, controlled self-assembly of these proteins into supramolecular architectures In particular, we used the enzyme adenylate kinase and the green fluorescent protein as soluble domains, and we show that the addition of low complexity regions induces the formation of protein particles via a multistep process This multistep pathway involves an initial liquid-liquid phase transition, which creates protein-rich droplets that mature into protein aggregates over time These protein aggregates consist of permeable structures that maintain activity and release active soluble proteins We show that the LCDs dictate specific noncovalent intermolecular interactions and phase properties that are largely independent of the given globular domain We further demonstrate that this feature, together with the dynamic state of the initial dense liquid phase, allows one to directly assemble different globular domains within the same architecture, thereby enabling the generation of both static multifunctional biomaterials and dynamic microscale bioreactors

Multifunctional Protein Materials and Microreactors using Low Complexity Domains as Molecular Adhesives.

https://onlinelibrary.wiley.com/doi/pdf/10.1002/syst.202000001

Acceleration of an Enzymatic Reaction in Liquid Phase Separated Compartments Based on Intrinsically Disordered Protein Domains

Biomolecular condensates are emerging as an efficient strategy developed by cells to control biochemical reactions in space and time by locally modifying composition and environment. Yet, local increase in protein concentration within these compartments could promote aberrant aggregation events, including the nucleation and growth of amyloid fibrils. Understanding protein stability within the crowded and heterogeneous environment of biological condensates is therefore crucial, not only when the aggregation-prone protein is the scaffold element of the condensates but also when proteins are recruited as client molecules within the compartments. Here, we investigate the partitioning and aggregation kinetics of the amyloidogenic peptide Abeta42 (Aβ-42), the peptide strongly associated with Alzheimer's disease, recruited into condensates based on low complexity domains (LCDs) derived from the DEAD-box proteins Laf1, Dbp1 and Ddx4, which are associated with biological membraneless organelles. We show that interactions between Aβ-42 and the scaffold proteins promote sequestration and local increase of the peptide concentration within the condensates. Yet, heterotypic interactions within the condensates inhibit the formation of amyloid fibrils. These results demonstrate that biomolecular condensates could sequester aggregation-prone proteins and prevent aberrant aggregation events, despite the local increase in their concentration. Biomolecular condensates could therefore work not only as hot-spots of protein aggregation but also as protective reservoirs, since the heterogenous composition of the condensates could prevent the formation of ordered fibrillar aggregates.

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Sequestration within biomolecular condensates inhibits Aβ-42 amyloid formation

A hydrophobic low-complexity region regulates aggregation of the yeast pyruvate kinase Cdc19 into amyloid-like aggregates in vitro.

The assembly of protein and inorganic nanoparticles represents an attractive approach to generate composite materials with multiple functions. Herein, we functionalize inorganic nanoparticles with intrinsically disordered protein domains associated with the formation of membraneless compartments in cells. These protein sequences, defined as low complexity domains (LCDs), encode intermolecular interactions that drive highly controlled, dynamic self-assembly in response to environmental changes. We show that the properties of the LCDs can be transferred to inorganic nanoparticles, inducing controlled phase separation that is dynamic and responsive to ionic strength and pH. Specifically, we hybridize magnetic nanoparticles with multi-domain proteins consisting of LCD domains and a globular enzyme, generating dynamic protein-composite compartments that locally confine hybrid chemoenzymatic reactions and respond to external magnetic fields and changes in solution conditions.

https://onlinelibrary.wiley.com/doi/pdf/10.1002/anie.202005761

Lenka Faltova

Papers

Multifunctional Protein Materials and Microreactors using Low Complexity Domains as Molecular Adhesives.

Acceleration of an Enzymatic Reaction in Liquid Phase Separated Compartments Based on Intrinsically Disordered Protein Domains

Sequestration within biomolecular condensates inhibits Aβ-42 amyloid formation

A hydrophobic low-complexity region regulates aggregation of the yeast pyruvate kinase Cdc19 into amyloid-like aggregates in vitro.

Adaptive Chemoenzymatic Microreactors Composed of Inorganic Nanoparticles and Bioinspired Intrinsically Disordered Proteins.