scholarly journals Hyperosmotic phase separation: Condensates beyond inclusions, granules and organelles

2020 ◽  
pp. jbc.REV120.010899
Author(s):  
Ameya P Jalihal ◽  
Andreas Schmidt ◽  
Guoming Gao ◽  
Saffron Little ◽  
Sethuramasundaram Pitchiaya ◽  
...  
Keyword(s):  
Phase Separation ◽  
Cellular Response ◽  
In Vivo Studies ◽  
Cloud Formation ◽  
Live Cells ◽  
Rapid Phase ◽  
Biological Liquid ◽  
External Fluctuations ◽  
Liquid Liquid Phase Separation

Biological liquid-liquid phase separation has gained considerable attention in recent years as a driving force for the assembly of subcellular compartments termed membraneless organelles. The field has made great strides in elucidating the molecular basis of biomolecular phase separation in various disease, stress-response and developmental contexts. Many important biological consequences of such “condensation” are now emerging from in vivo studies. Here we review recent work from our group and others showing that many proteins undergo rapid, reversible condensation in the cellular response to ubiquitous environmental fluctuations such as osmotic changes. We discuss molecular crowding as an important driver of condensation in these responses and suggest that a significant fraction of the proteome is poised to undergo phase separation under physiological conditions. In addition, we review methods currently emerging to visualize, quantify and modulate the dynamics of intracellular condensates in live cells. Finally, we propose a metaphor for rapid phase separation based on cloud formation, reasoning that our familiar experiences with the readily reversible condensation of water droplets help understand the principle of phase separation. Overall, we provide an account of how biological phase separation supports the highly intertwined relationship between the composition and dynamic internal organization of cells, thus facilitating extremely rapid reorganization in response to internal and external fluctuations.

scholarly journals Integration of Data from Liquid–Liquid Phase Separation Databases Highlights Concentration and Dosage Sensitivity of LLPS Drivers

2021 ◽  
Vol 22 (6) ◽  
pp. 3017
Author(s):  
Nazanin Farahi ◽  
Tamas Lazar ◽  
Shoshana J. Wodak ◽  
Peter Tompa ◽  
Rita Pancsa
Keyword(s):  
Phase Separation ◽  
Liquid Phase ◽  
Systematic Difference ◽  
Liquid Phase Separation ◽  
In Vivo Studies ◽  
Supporting Evidence ◽  
Condensate Formation ◽  
Liquid Liquid Phase Separation

Liquid–liquid phase separation (LLPS) is a molecular process that leads to the formation of membraneless organelles, representing functionally specialized liquid-like cellular condensates formed by proteins and nucleic acids. Integrating the data on LLPS-associated proteins from dedicated databases revealed only modest agreement between them and yielded a high-confidence dataset of 89 human LLPS drivers. Analysis of the supporting evidence for our dataset uncovered a systematic and potentially concerning difference between protein concentrations used in a good fraction of the in vitro LLPS experiments, a key parameter that governs the phase behavior, and the proteomics-derived cellular abundance levels of the corresponding proteins. Closer scrutiny of the underlying experimental data enabled us to offer a sound rationale for this systematic difference, which draws on our current understanding of the cellular organization of the proteome and the LLPS process. In support of this rationale, we find that genes coding for our human LLPS drivers tend to be dosage-sensitive, suggesting that their cellular availability is tightly regulated to preserve their functional role in direct or indirect relation to condensate formation. Our analysis offers guideposts for increasing agreement between in vitro and in vivo studies, probing the roles of proteins in LLPS.

scholarly journals Phase separation enables heterochromatin domains to do mechanical work

2020 ◽  
Author(s):  
Jessica F. Williams ◽  
Ivan V. Surovtsev ◽  
Sarah M. Schreiner ◽  
Hang Nguyen ◽  
Yan Hu ◽  
...  
Keyword(s):  
Mechanical Properties ◽  
Phase Separation ◽  
Mechanical Work ◽  
Live Cells ◽  
Fluctuation Analysis ◽  
Cellular Organization ◽  
Heterochromatin Protein ◽  
Liquid Liquid Phase Separation

SummaryLiquid-liquid phase separation (LLPS) has emerged as a major driver of cellular organization. However, it remains unexplored whether the mechanical properties of LLPS domains are functionally important. The heterochromatin protein HP1-α (and the orthologous Swi6 in S. pombe) is capable of LLPS in vitro and promotes formation of LLPS heterochromatin domains in vivo. Here, we demonstrate that LLPS of Swi6 contributes to the emergent mechanical properties of nuclei. Using nuclear fluctuation analysis in live cells and force spectroscopy of isolated nuclei, we find that disrupting histone H3K9 methylation or depleting Swi6 compromises nuclear stiffness, while heterochromatin spreading through loss of the H3K9 demethylase, Epe1, increases nuclear stiffness. Leveraging a separation-of-function allele, we demonstrate that phase separation of Swi6—but not its histone binding or dimerization—is essential for Swi6’s mechanical role. These findings demonstrate that altering chromatin state has mechanical consequences and highlights that phase-separated domains can do mechanical work.

scholarly journals Targeting NSD2-mediated SRC-3 liquid–liquid phase separation sensitizes bortezomib treatment in multiple myeloma

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Jing Liu ◽  
Ying Xie ◽  
Jing Guo ◽  
Xin Li ◽  
Jingjing Wang ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Drug Resistance ◽  
Phase Separation ◽  
Liquid Phase ◽  
Liquid Phase Separation ◽  
Acquired Drug Resistance ◽  
Bortezomib Treatment ◽  
Liquid Liquid Phase Separation

AbstractDevelopment of chemoresistance is the main reason for failure of clinical management of multiple myeloma (MM), but the genetic and epigenetic aberrations that interact to confer such chemoresistance remains unknown. In the present study, we find that high steroid receptor coactivator-3 (SRC-3) expression is correlated with relapse/refractory and poor outcomes in MM patients treated with bortezomib (BTZ)-based regimens. Furthermore, in immortalized cell lines, high SRC-3 enhances resistance to proteasome inhibitor (PI)-induced apoptosis. Overexpressed histone methyltransferase NSD2 in patients bearing a t(4;14) translocation or in BTZ-resistant MM cells coordinates elevated SRC-3 by enhancing its liquid–liquid phase separation to supranormally modify histone H3 lysine 36 dimethylation (H3K36me2) modifications on promoters of anti-apoptotic genes. Targeting SRC-3 or interference of its interactions with NSD2 using a newly developed inhibitor, SI-2, sensitizes BTZ treatment and overcomes drug resistance both in vitro and in vivo. Taken together, our findings elucidate a previously unrecognized orchestration of SRC-3 and NSD2 in acquired drug resistance of MM and suggest that SI-2 may be efficacious for overcoming drug resistance in MM patients.

scholarly journals Emerging Silk Material Trends: Repurposing, Phase Separation and Solution-Based Designs

Materials ◽  
2021 ◽  
Vol 14 (5) ◽  
pp. 1160
Author(s):  
F. Philipp Seib
Keyword(s):  
Phase Separation ◽  
Recent Progress ◽  
Fiber Formation ◽  
Live Cells ◽  
Specific Reference ◽  
New Developments ◽  
Silk Fabrics ◽  
Induced Crystallization ◽  
Liquid Liquid Phase Separation

Silk continues to amaze. This review unravels the most recent progress in silk science, spanning from fundamental insights to medical silks. Key advances in silk flow are examined, with specific reference to the role of metal ions in switching silk from a storage to a spinning state. Orthogonal thermoplastic silk molding is described, as is the transfer of silk flow principles for the triggering of flow-induced crystallization in other non-silk polymers. Other exciting new developments include silk-inspired liquid–liquid phase separation for non-canonical fiber formation and the creation of “silk organelles” in live cells. This review closes by examining the role of silk fabrics in fashioning facemasks in response to the SARS-CoV-2 pandemic.

scholarly journals Phosphorylation triggers presynaptic phase separation of Liprin-α3 to control active zone structure

2020 ◽  
Author(s):  
Javier Emperador-Melero ◽  
Man Yan Wong ◽  
Shan Shan H. Wang ◽  
Giovanni de Nola ◽  
Tom Kirchhausen ◽  
...  
Keyword(s):  
Phase Separation ◽  
Active Zone ◽  
Neurotransmitter Release ◽  
Phosphorylation Site ◽  
Double Knockout ◽  
Zone Structure ◽  
And Function ◽  
Pkc Activation ◽  
Liquid Liquid Phase Separation

AbstractLiquid-liquid phase separation enables the assembly of membrane-less subcellular compartments, but testing its biological functions has been difficult. The presynaptic active zone, protein machinery in nerve terminals that defines sites for neurotransmitter release, may be organized through phase separation. Here, we discover that the active zone protein Liprin-α3 rapidly and reversibly undergoes phase separation upon phosphorylation by PKC at a single site. RIM and Munc13 are co-recruited to membrane-attached condensates, and phospho-specific antibodies establish Liprin-α3 phosphorylation in vivo. At synapses of newly generated Liprin-α2/α3 double knockout mice, RIM, Munc13 and the pool of releasable vesicles were reduced. Re-expression of Liprin-α3 restored these defects, but mutating the Liprin-α3 phosphorylation site to abolish phase condensation prevented rescue. Finally, PKC activation acutely increased RIM, Munc13 and neurotransmitter release, which depended on the presence of phosphorylatable Liprin-α3. We conclude that Liprin-α3 phosphorylation rapidly triggers presynaptic phase separation to modulate active zone structure and function.

scholarly journals Pathologic tau conformer ensembles induce dynamic, liquid-liquid phase separation events at the nuclear envelope

BMC Biology ◽  
2021 ◽  
Vol 19 (1) ◽  
Author(s):  
Sang-Gyun Kang ◽  
Zhuang Zhuang Han ◽  
Nathalie Daude ◽  
Emily McNamara ◽  
Serene Wohlgemuth ◽  
...  
Keyword(s):  
Phase Separation ◽  
Liquid Phase ◽  
Nuclear Envelope ◽  
Guanidine Hydrochloride ◽  
Liquid Phase Separation ◽  
Soluble Form ◽  
Stable Cell Line ◽  
Cross Sectional ◽  
Liquid Liquid Phase Separation

Abstract Background The microtubule-associated protein tau forms aggregates in different neurodegenerative diseases called tauopathies. Prior work has shown that a single P301L mutation in tau gene, MAPT, can promote alternative tau folding pathways that correlate with divergent clinical diagnoses. Using progressive chemical denaturation, some tau preparations from the brain featured complex transitions starting at low concentrations of guanidine hydrochloride (GdnHCl) denaturant, indicating an ensemble of differently folded tau species called conformers. On the other hand, brain samples with abundant, tangle-like pathology had simple GdnHCl unfolding profile resembling the profile of fibrillized recombinant tau and suggesting a unitary conformer composition. In studies here we sought to understand tau conformer progression and potential relationships with condensed liquid states, as well as associated perturbations in cell biological processes. Results As starting material, we used brain samples from P301L transgenic mice containing tau conformer ensembles that unfolded at low GdnHCl concentrations and with signatures resembling brain material from P301L subjects presenting with language or memory problems. We seeded reporter cells expressing a soluble form of 4 microtubule-binding repeat tau fused to GFP or YFP reporter moieties, resulting in redistribution of dispersed fluorescence signals into focal assemblies that could fuse together and move within processes between adjacent cells. Nuclear envelope fluorescent tau signals and small fluorescent inclusions behaved as a demixed liquid phase, indicative of liquid-liquid phase separation (LLPS); these droplets exhibited spherical morphology, fusion events and could recover from photobleaching. Moreover, juxtanuclear tau assemblies were associated with disrupted nuclear transport and reduced cell viability in a stable cell line. Staining for thioflavin S (ThS) became more prevalent as tau-derived inclusions attained cross-sectional area greater than 3 μm2, indicating (i) a bipartite composition, (ii) in vivo progression of tau conformers, and (iii) that a mass threshold applying to demixed condensates may drive liquid-solid transitions. Conclusions Tau conformer ensembles characterized by denaturation at low GdnHCl concentration templated the production of condensed droplets in living cells. These species exhibit dynamic changes and develop in vivo, and the larger ThS-positive assemblies may represent a waystation to arrive at intracellular fibrillar tau inclusions seen in end-stage genetic tauopathies.

scholarly journals Rotavirus Replication Factories Are Complex Ribonucleoprotein Condensates

2020 ◽  
Author(s):  
Florian Geiger ◽  
Guido Papa ◽  
William E. Arter ◽  
Julia Acker ◽  
Kadi L. Saar ◽  
...  
Keyword(s):  
Phase Separation ◽  
Unique Feature ◽  
Rna Viruses ◽  
Therapeutic Approaches ◽  
Subcellular Organelles ◽  
Selective Enrichment ◽  
Novel Therapeutic ◽  
Liquid Liquid Phase Separation

AbstractRNA viruses induce formation of subcellular organelles that provide microenvironments conducive to their replication. Here we show that replication factories of rotaviruses represent protein-RNA condensates that are formed via liquid-liquid phase separation. We demonstrate that rotavirus proteins NSP5 and NSP2 undergo phase separation in vitro and form RNA-rich condensates in vivo that can be reversibly dissolved by aliphatic diols. During infection, these RNA-protein condensates became less dynamic and impervious to aliphatic diols, indicating a transition from a liquid to solid state. Some aspects of assembly of rotavirus replication factories mirror the formation of cytoplasmic ribonucleoprotein granules, while the selective enrichment of viral transcripts appears to be a unique feature of these condensates. Such complex RNA-protein condensates that underlie replication of RNA viruses represent an attractive target for developing novel therapeutic approaches.

scholarly journals Organization and Regulation of Chromatin by Liquid-Liquid Phase Separation

2019 ◽  
Author(s):  
B.A. Gibson ◽  
L.K. Doolittle ◽  
L.E. Jensen ◽  
N. Gamarra ◽  
S. Redding ◽  
...  
Keyword(s):  
Phase Separation ◽  
Liquid Phase ◽  
Binding Proteins ◽  
Liquid Phase Separation ◽  
Basic Unit ◽  
Chromatin Binding ◽  
Divalent Salts ◽  
Nuclear Processes ◽  
Liquid Liquid Phase Separation

Genomic DNA is highly compacted in the nucleus of eukaryotic cells as a nucleoprotein assembly called chromatin1. The basic unit of chromatin is the nucleosome, where ∼146 base pair increments of the genome are wrapped and compacted around the core histone proteins2,3. Further genomic organization and compaction occur through higher order assembly of nucleosomes4. This organization regulates many nuclear processes, and is controlled in part by histone post-transtranslational modifications and chromatin-binding proteins. Mechanisms that regulate the assembly and compaction of the genome remain unclear5,6. Here we show that in the presence of physiologic concentrations of mono- and divalent salts, histone tail-driven interactions drive liquid-liquid phase separation (LLPS) of nucleosome arrays, resulting in substantial condensation. Phase separation of nucleosomal arrays is inhibited by histone acetylation, whereas histone H1 promotes phase separation, further compaction, and decreased dynamics within droplets, mirroring the relationship between these modulators and the accessibility of the genome in cells7-10. These results indicate that under physiologically relevant conditions, LLPS is an intrinsic behavior of the chromatin polymer, and suggest a model in which the condensed phase reflects a genomic “ground state” that can produce chromatin organization and compaction in vivo. The dynamic nature of this state could enable known modulators of chromatin structure, such as post-translational modifications and chromatin binding proteins, to act upon it and consequently control nuclear processes such as transcription and DNA repair. Our data suggest an important role for LLPS of chromatin in the organization of the eukaryotic genome.

scholarly journals Concentration and dosage sensitivity of proteins driving liquid-liquid phase separation

2021 ◽  
Author(s):  
Nazanin Farahi ◽  
Tamas Lazar ◽  
Shoshana J. Wodak ◽  
Peter Tompa ◽  
Rita Pancsa
Keyword(s):  
Phase Separation ◽  
Liquid Phase ◽  
Liquid Phase Separation ◽  
Condensate Formation ◽  
Associated Proteins ◽  
Liquid Liquid Phase Separation ◽  
In Cells ◽  
Dosage Sensitivity

AbstractLiquid-liquid phase separation (LLPS) is a molecular process that leads to the formation of membraneless organelles (MLOs), i.e. functionally specialized liquid-like cellular condensates formed by proteins and nucleic acids. Integration of data on LLPS-associated proteins from dedicated databases revealed only modest overlap between them and resulted in a confident set of 89 human LLPS driver proteins. Since LLPS is highly concentration-sensitive, the underlying experiments are often criticized for applying higher-than-physiological protein concentrations. To clarify this issue, we performed a naive comparison of in vitro applied and quantitative proteomics-derived protein concentrations and discuss a number of considerations that rationalize the choice of apparently high in vitro concentrations in most LLPS studies. The validity of in vitro LLPS experiments is further supported by in vivo phase-separation experiments and by the observation that the corresponding genes show a strong propensity for dosage sensitivity. This observation implies that the availability of the respective proteins is tightly regulated in cells to avoid erroneous condensate formation. In all, we propose that although local protein concentrations are practically impossible to determine in cells, proteomics-derived cellular concentrations should rather be considered as lower limits of protein concentrations, than strict upper bounds, to be respected by in vitro experiments.

scholarly journals Polycomb Cbx2 Condensates Assemble through Phase Separation

2018 ◽  
Author(s):  
Roubina Tatavosian ◽  
Samantha Kent ◽  
Kyle Brown ◽  
Tingting Yao ◽  
Huy Nguyen Duc ◽  
...  
Keyword(s):  
Phase Separation ◽  
Polycomb Group ◽  
Developmental Defects ◽  
Intrinsically Disordered ◽  
Condensate Formation ◽  
Development And Differentiation ◽  
Polycomb Repressive Complex 1 ◽  
Liquid Liquid Phase Separation

AbstractPolycomb group (PcG) proteins are master regulators of development and differentiation. Mutation and dysregulation of PcG genes cause developmental defects and cancer. PcG proteins form condensates in the nucleus of cells and these condensates are the physical sites of PcG-targeted gene silencing. However, the physiochemical principles underlying the PcG condensate formation remain unknown. Here we show that Polycomb repressive complex 1 (PRC1) protein Cbx2, one member of the Cbx family proteins, contains a long stretch of intrinsically disordered region (IDR). Cbx2 undergoes phase separation to form condensates. Cbx2 condensates exhibit liquid-like properties and can concentrate DNA and nucleosomes. We demonstrate that the conserved residues within the IDR promote the condensate formation in vitro and in vivo. We further indicate that H3K27me3 has minimal effects on the Cbx2 condensate formation while depletion of core PRC1 subunits facilitates the condensate formation. Thus, our results reveal that PcG condensates assemble through liquid-liquid phase separation (LLPS) and suggest that PcG-bound chromatin is in part organized through phase-separated condensates.

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