Microtubules as platforms for probing liquid–liquid phase separation in cells – application to RNA-binding proteins

AbstractTightly packed complexes of nucleocapsid protein and genomic RNA form the core of viruses and may assemble within viral factories, dynamic compartments formed within the host cells. Here, we examine the possibility that the multivalent RNA-binding nucleocapsid protein (N) from the severe acute respiratory syndrome coronavirus (SARS-CoV-2) compacts RNA via protein-RNA liquid-liquid phase separation (LLPS) and that N interactions with host RNA-binding proteins are mediated by phase separation. To this end, we created a construct expressing recombinant N fused to a N-terminal maltose binding protein tag which helps keep the oligomeric N soluble for purification. Using in vitro phase separation assays, we find that N is assembly-prone and phase separates avidly. Phase separation is modulated by addition of RNA and changes in pH and is disfavored at high concentrations of salt. Furthermore, N enters into in vitro phase separated condensates of full-length human hnRNPs (TDP-43, FUS, and hnRNPA2) and their low complexity domains (LCs). However, N partitioning into the LC of FUS, but not TDP-43 or hnRNPA2, requires cleavage of the solubilizing MBP fusion. Hence, LLPS may be an essential mechanism used for SARS-CoV-2 and other RNA viral genome packing and host protein co-opting, functions necessary for viral replication and hence infectivity.

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FUS-dependent liquid-liquid phase separation is an early event in double-strand break repair

10.1101/798884 ◽

2019 ◽

Cited By ~ 2

Author(s):

Brunno R. Levone ◽

Silvia C. Lenzken ◽

Marco Antonaci ◽

Andreas Maiser ◽

Alexander Rapp ◽

...

Keyword(s):

Dna Damage ◽

Phase Separation ◽

Liquid Phase ◽

Rna Binding ◽

Rna Binding Proteins ◽

Liquid Phase Separation ◽

Early Event ◽

Structured Illumination Microscopy ◽

Liquid Liquid Phase Separation

AbstractRNA-binding proteins (RBPs) are emerging as important effectors of the cellular DNA damage response (DDR). The RBP FUS is implicated in RNA metabolism and DNA repair, and it undergoes reversible liquid-liquid phase separation (LLPS) in vitro. Here, we demonstrate that FUS-dependent LLPS is necessary for the initiation of the DDR. Using laser microirradiation in FUS-knockout cells, we show that FUS is required for the recruitment to DNA damage sites of the DDR factors KU80, NBS1, 53BP1, and of SFPQ, another RBP implicated in the DDR. The relocation of KU80, NBS1, and SFPQ is similarly impaired by LLPS inhibitors, or LLPS-deficient FUS variants. We also show that LLPS is necessary for efficient γH2AX foci formation. Finally, using super-resolution structured illumination microscopy, we demonstrate that the absence of FUS impairs the proper arrangement of γH2AX nano-foci into higher-order clusters. These findings demonstrate the early requirement for FUS-dependent LLPS in the activation of the DDR and the proper assembly of DSBs repair complexes.

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FUS-dependent liquid–liquid phase separation is important for DNA repair initiation

The Journal of Cell Biology ◽

10.1083/jcb.202008030 ◽

2021 ◽

Vol 220 (5) ◽

Cited By ~ 1

Author(s):

Brunno R. Levone ◽

Silvia C. Lenzken ◽

Marco Antonaci ◽

Andreas Maiser ◽

Alexander Rapp ◽

...

Keyword(s):

Dna Damage ◽

Dna Repair ◽

Phase Separation ◽

Liquid Phase ◽

Rna Binding ◽

Rna Binding Proteins ◽

Liquid Phase Separation ◽

Structured Illumination Microscopy ◽

Liquid Liquid Phase Separation

RNA-binding proteins (RBPs) are emerging as important effectors of the cellular DNA damage response (DDR). The RBP FUS is implicated in RNA metabolism and DNA repair, and it undergoes reversible liquid–liquid phase separation (LLPS) in vitro. Here, we demonstrate that FUS-dependent LLPS is necessary for the initiation of the DDR. Using laser microirradiation in FUS-knockout cells, we show that FUS is required for the recruitment to DNA damage sites of the DDR factors KU80, NBS1, and 53BP1 and of SFPQ, another RBP implicated in the DDR. The relocation of KU80, NBS1, and SFPQ is similarly impaired by LLPS inhibitors, or LLPS-deficient FUS variants. We also show that LLPS is necessary for efficient γH2AX foci formation. Finally, using superresolution structured illumination microscopy, we demonstrate that the absence of FUS impairs the proper arrangement of γH2AX nanofoci into higher-order clusters. These findings demonstrate the early requirement for FUS-dependent LLPS in the activation of the DDR and the proper assembly of DSB repair complexes.

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Taurine suppresses liquid-liquid phase separation of lysozyme protein

10.1101/2021.01.26.428332 ◽

2021 ◽

Author(s):

Kanae Tsubotani ◽

Sayuri Maeyama ◽

Shigeru Murakami ◽

Stephen W Schaffer ◽

Takashi Ito

Keyword(s):

Phase Separation ◽

Polyethylene Glycol ◽

Critical Temperature ◽

Liquid Phase ◽

Liquid Phase Separation ◽

Compatible Osmolyte ◽

Hen Egg ◽

Liquid Liquid Phase Separation ◽

In Cells

AbstractTaurine is a compatible osmolyte that infers stability to proteins. Recent studies have revealed that liquid-liquid phase separation (LLPS) of proteins underlie the formation of membraneless organelles in cells. In the present study, we evaluated the role of taurine on LLPS of hen egg lysozyme. We demonstrated that taurine decreases the turbidity of the polyethylene glycol-induced crowding solution of lysozyme. We also demonstrated that taurine attenuates LLPS-dependent cloudiness of lysozyme solution with 0.5 or 1M NaCl at a critical temperature. Moreover, we observed that taurine inhibits LLPS formation of a heteroprotein mix solution of lysozyme and ovalbumin. These data indicate that taurine can modulate the formation of LLPS of proteins.

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Liquid–Liquid Phase Separation in Crowded Environments

International Journal of Molecular Sciences ◽

10.3390/ijms21165908 ◽

2020 ◽

Vol 21 (16) ◽

pp. 5908 ◽

Cited By ~ 4

Author(s):

Alain A. M. André ◽

Evan Spruijt

Keyword(s):

Phase Separation ◽

Liquid Phase ◽

Macromolecular Crowding ◽

Liquid Phase Separation ◽

The Impact ◽

Crowded Environments ◽

Liquid Liquid Phase Separation ◽

In Cells

Biomolecular condensates play a key role in organizing cellular fluids such as the cytoplasm and nucleoplasm. Most of these non-membranous organelles show liquid-like properties both in cells and when studied in vitro through liquid–liquid phase separation (LLPS) of purified proteins. In general, LLPS of proteins is known to be sensitive to variations in pH, temperature and ionic strength, but the role of crowding remains underappreciated. Several decades of research have shown that macromolecular crowding can have profound effects on protein interactions, folding and aggregation, and it must, by extension, also impact LLPS. However, the precise role of crowding in LLPS is far from trivial, as most condensate components have a disordered nature and exhibit multiple weak attractive interactions. Here, we discuss which factors determine the scope of LLPS in crowded environments, and we review the evidence for the impact of macromolecular crowding on phase boundaries, partitioning behavior and condensate properties. Based on a comparison of both in vivo and in vitro LLPS studies, we propose that phase separation in cells does not solely rely on attractive interactions, but shows important similarities to segregative phase separation.

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Organization and Regulation of Chromatin by Liquid-Liquid Phase Separation

10.1101/523662 ◽

2019 ◽

Cited By ~ 13

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.

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Polymer Stiffness Regulates Multivalent Binding and Liquid-Liquid Phase Separation

10.1101/2020.05.13.093245 ◽

2020 ◽

Cited By ~ 1

Author(s):

E. Zumbro ◽

A. Alexander-Katz

Keyword(s):

Phase Separation ◽

Liquid Phase ◽

Rational Design ◽

Rna Binding ◽

Liquid Phase Separation ◽

Random Coil ◽

Small Target ◽

Toxic Protein ◽

Multivalent Binding ◽

Liquid Liquid Phase Separation

AbstractMultivalent binding is essential to many biological processes because it builds high affinity bonds by using several weak binding interactions simultaneously. Multivalent polymers have shown promise as inhibitors of toxins and other pathogens, and they are important components in the formation of biocondensates. Explaining how structural features of these polymers change their binding and subsequent control of phase separation is critical to designing better pathogen inhibitors and also to understanding diseases associated with membraneless organelles. In this work, we will examine the binding of a multivalent polymer to a small target. This scenario could represent a polymeric inhibitor binding to a toxic protein or RNA binding to an RNA-binding protein in the case of liquid-liquid phase separation. We use simulation and theory to show that flexible random-coil polymers bind more strongly than stiff rod-like polymers and that flexible polymers nucleate condensed phases at lower energies than their rigid analogues. We hope these results will provide insight into the rational design of polymeric inhibitors and improve understanding of membraneless organelles.Statement of SignificanceMultivalent polymers are essential for many biological systems, including targeting pathogens and controlling the formation of liquid-liquid phase separated biocondensates. Here, we explain how increasing polymer stiffness can reduce multivalent binding affinity to a small target such as a toxic protein and how modulating polymer stiffness can change the phase boundary for liquid-liquid phase separation. These results have implications for designing stronger pathogen inhibitors and provide insights on neurodegenerative diseases associated with abnormal biocondensate formation.

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Pressure-Jump Kinetics of Liquid–Liquid Phase Separation: Comparison of Two Different Condensed Phases of the RNA-Binding Protein, Fused in Sarcoma

Journal of the American Chemical Society ◽

10.1021/jacs.1c07571 ◽

2021 ◽

Author(s):

Ryo Kitahara ◽

Ryota Yamazaki ◽

Fumika Ide ◽

Shujie Li ◽

Yutaro Shiramasa ◽

...

Keyword(s):

Phase Separation ◽

Liquid Phase ◽

Rna Binding ◽

Rna Binding Protein ◽

Liquid Phase Separation ◽

Pressure Jump ◽

Condensed Phases ◽

Fused In Sarcoma ◽

Kinetics Of ◽

Liquid Liquid Phase Separation

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

10.1101/2021.02.19.430946 ◽

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.

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Liquid-Liquid Phase Separation of Patchy Particles Illuminates Diverse Effects of Regulatory Components on Protein Droplet Formation

10.1101/294058 ◽

2018 ◽

Author(s):

Valery Nguemaha ◽

Huan-Xiang Zhou

Keyword(s):

Phase Separation ◽

Liquid Phase ◽

Rna Binding ◽

Liquid Phase Separation ◽

Steric Repulsion ◽

Cellular Functions ◽

Binding Domains ◽

Patchy Particles ◽

Droplet Phase ◽

Liquid Liquid Phase Separation

AbstractRecently many cellular functions have been associated with membraneless organelles, or protein droplets, formed by liquid-liquid phase separation (LLPS). Proteins in these droplets often contain RNA-binding domains, but the effects of RNA on LLPS have been controversial. To gain better understanding on the roles of RNA, here we used Gibbs-ensemble simulations to determine phase diagrams of two-component patchy particles, as models for mixtures of proteins with RNA or other regulatory components. Protein-like particles have four patches, with attraction strength εPP; regulatory particles experience mutual steric repulsion but have two attractive patches toward proteins, with the strength εPR tunable. At low εPR, the regulator, due to steric repulsion, preferentially partitions in the dispersed phase, thereby displacing the protein into the droplet phase and promoting LLPS. At moderate εPR, the regulator starts to partition and displace the protein in the droplet phase, but only to weaken bonding networks and thereby suppress LLPS. At εPR > εPP, the enhanced bonding ability of the regulator initially promotes LLPS, but at higher amounts, the resulting displacement of the protein suppresses LLPS. These results illustrate how RNA can have disparate effects on LLPS, thus able to perform diverse functions in different organelles.

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