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Journal ArticleDOI: 10.1021/ACS.JPCB.0C11606

Biomolecular Condensates: Sequence Determinants of Phase Separation, Microstructural Organization, Enzymatic Activity, and Material Properties

04 Mar 2021-Journal of Physical Chemistry B (American Chemical Society (ACS))-Vol. 125, Iss: 14, pp 3441-3451
Abstract: This perspective article highlights recent progress and emerging challenges in understanding the formation and function of membraneless organelles (MLOs). A long-term goal in the MLO field is to identify the sequence-encoded rules that dictate the formation of compositionally controlled biomolecular condensates, which cells utilize to perform a wide variety of functions. The molecular organization of the different components within a condensate can vary significantly, ranging from a homogeneous mixture to core-shell droplet structures. We provide many examples to highlight the richness of the observed behavior and potential research directions for improving our mechanistic understanding. The tunable environment within condensates can, in principle, alter enzymatic activity significantly. We examine recent examples where this was demonstrated, including applications in synthetic biology. An important question about MLOs is the role of liquid-like material properties in biological function. We discuss the need for improved quantitative characterization tools and the development of sequence-structure-dynamics relationships.

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Journal ArticleDOI: 10.1016/J.JMB.2021.167151
Wei Li1, Hao Jiang1Institutions (1)
Abstract: Our understanding of the spatiotemporal regulation of eukaryotic gene expression has recently been greatly stimulated by the findings that many of the regulators of chromatin, transcription, and RNA processing form biomolecular condensates often assembled through liquid-liquid phase separation. Increasing number of reports suggest that these condensates functionally regulate gene expression, largely by concentrating the relevant biomolecules in the liquid-like micro-compartments. However, it remains poorly understood how the physicochemical properties, especially the material properties, of the condensates regulate gene expression activity. In this review, we discuss current data on various nuclear condensates and their biophysical properties with the underlying molecular interactions, and how they may functionally impact gene expression at the level of chromatin organization and activities, transcription, and RNA processing.

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Topics: Regulation of gene expression (59%), Chromatin (57%), Gene expression (54%) ... show more

2 Citations


Open accessPosted ContentDOI: 10.1101/2021.05.28.446223
29 May 2021-bioRxiv
Abstract: Cells contain membraneless compartments that assemble due to liquid-liquid phase separation, including biomolecular condensates with complex morphologies. For instance, certain condensates are surrounded by a film of distinct composition, such as Ape1 condensates coated by a layer of Atg19, required for selective autophagy in yeast. Other condensates are multiphasic, with nested liquid phases of distinct compositions and functions, such as in the case of ribosome biogenesis in the nucleolus. The size and structure of such condensates must be regulated for proper biological function. We leveraged a bio-inspired approach to discover how amphiphilic, surfactant-like proteins may contribute to the structure and size regulation of biomolecular condensates. We designed and examined families of amphiphilic proteins comprising one phase-separating domain and one non-phase separating domain. In particular, these proteins contain the soluble structured domain glutathione S-transferase (GST) or maltose binding protein (MBP), fused to the intrinsically disordered RGG domain from P granule protein LAF-1. When one amphiphilic protein is mixed in vitro with RGG-RGG, the proteins assemble into enveloped condensates, with RGG-RGG at the core, and the amphiphilic protein forming the surface film layer. Importantly, we found that MBP-based amphiphiles are surfactants and control droplet size, with increasing surfactant concentration resulting in smaller droplet radii. In contrast, GST-based amphiphiles at increased concentrations co-assemble with RGG-RGG into multiphasic structures. We propose a mechanism for these experimental observations, supported by molecular simulations of a minimalist model. We speculate that surfactant proteins may play a significant role in regulating the structure and function of biomolecular condensates.

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2 Citations


Open accessJournal ArticleDOI: 10.1016/J.BPR.2021.100011
08 Sep 2021-
Abstract: The material properties of biomolecular condensates have been suggested to play important biological and pathological roles. Despite the rapid increase in the number of biomolecules identified that undergo liquid-liquid phase separation, quantitative studies and direct measurements of the material properties of the resulting condensates have been severely lagging behind. Here, we develop a micropipette-based technique that uniquely, to our knowledge, allows quantifications of both the surface tension and viscosity of biomolecular condensates, independent of labeling and surface-wetting effects. We demonstrate the accuracy and versatility of this technique by measuring condensates of LAF-1 RGG domains and a polymer-based aqueous two-phase system. We further confirm our measurements using established condensate fusion and fluorescence recovery after photobleaching assays. We anticipate the micropipette-based technique will be widely applicable to biomolecular condensates and will resolve several limitations regarding current approaches.

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2 Citations



Open accessJournal ArticleDOI: 10.1002/ADMA.202104837
19 Oct 2021-Advanced Materials
Abstract: Increasing evidence indicates that cells can regulate biochemical functions in time and space by generating membraneless compartments with well-defined mesoscopic properties. One important mechanism underlying this control is simple coacervation driven by associative disordered proteins that encode multivalent interactions. Inspired by these observations, we developed programmable droplets based on simple coacervation of responsive synthetic polymers that mimic the "stickers-and-spacers" architecture of biological disordered proteins. We designed zwitterionic polymers that undergo an enthalpy-driven liquid-liquid phase separation process and form liquid droplets that remarkably exclude most molecules. Starting from this reference material, we progressively added different functional groups in the zwitterionic polymer to encode an increasing number of different intermolecular interactions. This strategy allowed us to independently control multiple emerging properties of the droplets, such as stimulus-responsiveness, polarity, selective uptake of client molecules, fusion times, and miscibility. By exploiting this high programmability, we reproduced a model of cellular compartmentalization and generated droplets capable of confining different molecules in space without physical barriers. Moreover, we demonstrated that these biomolecular sorters can localize, separate and enable the detection of target molecules even within complex mixtures, opening attractive applications in bioseparation and diagnostics. This article is protected by copyright. All rights reserved.

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113 results found


Open accessJournal ArticleDOI: 10.1038/NRM.2017.7
Abstract: In addition to membrane-bound organelles, eukaryotic cells feature various membraneless compartments, including the centrosome, the nucleolus and various granules. Many of these compartments form through liquid–liquid phase separation, and the principles, mechanisms and regulation of their assembly as well as their cellular functions are now beginning to emerge. Biomolecular condensates are micron-scale compartments in eukaryotic cells that lack surrounding membranes but function to concentrate proteins and nucleic acids. These condensates are involved in diverse processes, including RNA metabolism, ribosome biogenesis, the DNA damage response and signal transduction. Recent studies have shown that liquid–liquid phase separation driven by multivalent macromolecular interactions is an important organizing principle for biomolecular condensates. With this physical framework, it is now possible to explain how the assembly, composition, physical properties and biochemical and cellular functions of these important structures are regulated.

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Topics: Biological phase (52%), Ribosome biogenesis (51%)

1,988 Citations


Journal ArticleDOI: 10.1038/35051719
Alexander M. Klibanov1Institutions (1)
11 Jan 2001-Nature
Abstract: The technological utility of enzymes can be enhanced greatly by using them in organic solvents rather than their natural aqueous reaction media. Studies over the past 15 years have revealed not only that this change in solvent is feasible, but also that in such seemingly hostile environments enzymes can catalyse reactions impossible in water, become more stable, and exhibit new behaviour such as 'molecular memory'. Of particular importance has been the discovery that enzymatic selectivity, including substrate, stereo-, regio- and chemoselectivity, can be markedly affected, and sometimes even inverted, by the solvent. Enzyme-catalysed reactions in organic solvents, and even in supercritical fluids and the gas phase, have found numerous potential applications, some of which are already commercialized.

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Topics: Solvent effects (52%)

1,820 Citations


Journal ArticleDOI: 10.1126/SCIENCE.1172046
26 Jun 2009-Science
Abstract: In sexually reproducing organisms, embryos specify germ cells, which ultimately generate sperm and eggs In Caenorhabditis elegans, the first germ cell is established when RNA and protein-rich P granules localize to the posterior of the one-cell embryo Localization of P granules and their physical nature remain poorly understood Here we show that P granules exhibit liquid-like behaviors, including fusion, dripping, and wetting, which we used to estimate their viscosity and surface tension As with other liquids, P granules rapidly dissolved and condensed Localization occurred by a biased increase in P granule condensation at the posterior This process reflects a classic phase transition, in which polarity proteins vary the condensation point across the cell Such phase transitions may represent a fundamental physicochemical mechanism for structuring the cytoplasm

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1,513 Citations


Journal ArticleDOI: 10.1126/SCIENCE.AAF4382
Yongdae Shin1, Clifford P. Brangwynne1Institutions (1)
22 Sep 2017-Science
Abstract: BACKGROUND Living cells contain distinct subcompartments to facilitate spatiotemporal regulation of biological reactions. In addition to canonical membrane-bound organelles such as secretory vesicles and endoplasmic reticulum, there are many organelles that do not have an enclosing membrane yet remain coherent structures that can compartmentalize and concentrate specific sets of molecules. Examples include assemblies in the nucleus such as the nucleolus, Cajal bodies, and nuclear speckles and also cytoplasmic structures such as stress granules, P-bodies, and germ granules. These structures play diverse roles in various biological processes and are also increasingly implicated in protein aggregation diseases. ADVANCES A number of studies have shown that membrane-less assemblies exhibit remarkable liquid-like features. As with conventional liquids, they typically adopt round morphologies and coalesce into a single droplet upon contact with one another and also wet intracellular surfaces such as the nuclear envelope. Moreover, component molecules exhibit dynamic exchange with the surrounding nucleoplasm and cytoplasm. These findings together suggest that these structures represent liquid-phase condensates, which form via a biologically regulated (liquid-liquid) phase separation process. Liquid phase condensation increasingly appears to be a fundamental mechanism for organizing intracellular space. Consistent with this concept, several membrane-less organelles have been shown to exhibit a concentration threshold for assembly, a hallmark of phase separation. At the molecular level, weak, transient interactions between molecules with multivalent domains or intrinsically disordered regions (IDRs) are a driving force for phase separation. In cells, condensation of liquid-phase assemblies can be regulated by active processes, including transcription and various posttranslational modifications. The simplest physical picture of a homogeneous liquid phase is often not enough to capture the full complexity of intracellular condensates, which frequently exhibit heterogeneous multilayered structures with partially solid-like characters. However, recent studies have shown that multiple distinct liquid phases can coexist and give rise to richly structured droplet architectures determined by the relative liquid surface tensions. Moreover, solid-like phases can emerge from metastable liquid condensates via multiple routes of potentially both kinetic and thermodynamic origins, which has important implications for the role of intracellular liquids in protein aggregation pathologies. OUTLOOK The list of intracellular assemblies driven by liquid phase condensation is growing rapidly, but our understanding of their sequence-encoded biological function and dysfunction lags behind. Moreover, unlike equilibrium phases of nonliving matter, living cells are far from equilibrium, with intracellular condensates subject to various posttranslational regulation and other adenosine triphosphate–dependent biological activity. Efforts using in vitro reconstitution, combined with traditional cell biology approaches and quantitative biophysical tools, are required to elucidate how such nonequilibrium features of living cells control intracellular phase behavior. The functional consequences of forming liquid condensates are likely multifaceted and may include facilitated reaction, sequestration of specific factors, and organization of associated intracellular structures. Liquid phase condensation is particularly interesting in the nucleus, given the growing interest in the impact of nuclear phase behavior on the flow of genetic information; nuclear condensates range from micrometer-sized bodies such as the nucleolus to submicrometer structures such as transcriptional assemblies, all of which directly interact with and regulate the genome. Deepening our understanding of these intracellular states of matter not only will shed light on the basic biology of cellular organization but also may enable therapeutic intervention in protein aggregation disease by targeting intracellular phase behavior.

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Topics: Biological phase (58%)

1,512 Citations


Open accessJournal ArticleDOI: 10.1016/J.CELL.2015.07.047
Avinash Patel1, Hyun O. Lee1, Louise Jawerth1, Shovamayee Maharana1  +16 moreInstitutions (2)
27 Aug 2015-Cell
Abstract: Many proteins contain disordered regions of low-sequence complexity, which cause aging-associated diseases because they are prone to aggregate. Here, we study FUS, a prion-like protein containing intrinsically disordered domains associated with the neurodegenerative disease ALS. We show that, in cells, FUS forms liquid compartments at sites of DNA damage and in the cytoplasm upon stress. We confirm this by reconstituting liquid FUS compartments in vitro. Using an in vitro "aging" experiment, we demonstrate that liquid droplets of FUS protein convert with time from a liquid to an aggregated state, and this conversion is accelerated by patient-derived mutations. We conclude that the physiological role of FUS requires forming dynamic liquid-like compartments. We propose that liquid-like compartments carry the trade-off between functionality and risk of aggregation and that aberrant phase transitions within liquid-like compartments lie at the heart of ALS and, presumably, other age-related diseases.

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Topics: Protein aggregation (51%)

1,395 Citations


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