scholarly journals Suppression of liquid-liquid phase separation by 1,6-hexanediol partially compromises the 3D genome organization in living cells

2020 ◽  
Author(s):  
Sergey V. Ulianov ◽  
Artem K. Velichko ◽  
Mikhail D. Magnitov ◽  
Artem V. Luzhin ◽  
Arkadiy K. Golov ◽  
...  
Keyword(s):  
Phase Separation ◽  
Liquid Phase ◽  
Spatial Organization ◽  
Living Cells ◽  
Liquid Phase Separation ◽  
Nuclear Space ◽  
3D Genome ◽  
Chromatin Folding ◽  
Optical Reconstruction ◽  
Liquid Liquid Phase Separation

AbstractLiquid-liquid phase separation (LLPS) contributes to the spatial and functional segregation of molecular processes. However, the role played by LLPS in chromatin folding in living cells remains unclear. Here, using stochastic optical reconstruction microscopy (STORM) and Hi-C techniques, we studied the effects of 1,6-hexanediol (1,6-HD)-mediated LLPS modulation on higher-order chromatin organization in living cells. We found that 1,6-HD treatment caused the enlargement of nucleosome nanodomains and their more uniform distribution in the nuclear space. At a megabase-scale, chromatin underwent moderate but irreversible perturbations that resulted in the partial mixing of A and B compartments. The removal of 1,6-HD from the culture medium did not allow chromatin to acquire initial configurations, but increased further mixing of the chromatin compartments and resulted in more compact repressed chromatin than in untreated cells. 1,6-HD treatment also weakened enhancer-promoter interactions but did not considerably affect CTCF-dependent loops. Our results suggest that 1,6-HD-sensitive LLPS plays a limited role in chromatin spatial organization by constraining its folding patterns and facilitating compartmentalization at different levels.

scholarly journals Suppression of liquid–liquid phase separation by 1,6-hexanediol partially compromises the 3D genome organization in living cells

2021 ◽  
Author(s):  
Sergey V Ulianov ◽  
Artem K Velichko ◽  
Mikhail D Magnitov ◽  
Artem V Luzhin ◽  
Arkadiy K Golov ◽  
...  
Keyword(s):  
Phase Separation ◽  
Liquid Phase ◽  
Spatial Organization ◽  
Living Cells ◽  
Liquid Phase Separation ◽  
Nuclear Space ◽  
3D Genome ◽  
Chromatin Folding ◽  
Optical Reconstruction ◽  
Liquid Liquid Phase Separation

Abstract Liquid–liquid phase separation (LLPS) contributes to the spatial and functional segregation of molecular processes within the cell nucleus. However, the role played by LLPS in chromatin folding in living cells remains unclear. Here, using stochastic optical reconstruction microscopy (STORM) and Hi-C techniques, we studied the effects of 1,6-hexanediol (1,6-HD)-mediated LLPS disruption/modulation on higher-order chromatin organization in living cells. We found that 1,6-HD treatment caused the enlargement of nucleosome clutches and their more uniform distribution in the nuclear space. At a megabase-scale, chromatin underwent moderate but irreversible perturbations that resulted in the partial mixing of A and B compartments. The removal of 1,6-HD from the culture medium did not allow chromatin to acquire initial configurations, and resulted in more compact repressed chromatin than in untreated cells. 1,6-HD treatment also weakened enhancer-promoter interactions and TAD insulation but did not considerably affect CTCF-dependent loops. Our results suggest that 1,6-HD-sensitive LLPS plays a limited role in chromatin spatial organization by constraining its folding patterns and facilitating compartmentalization at different levels.

scholarly journals The proline-rich domain promotes Tau liquid–liquid phase separation in cells

2020 ◽  
Vol 219 (11) ◽  
Author(s):  
Xuemei Zhang ◽  
Michael Vigers ◽  
James McCarty ◽  
Jennifer N. Rauch ◽  
Glenn H. Fredrickson ◽  
...  
Keyword(s):  
Phase Separation ◽  
Liquid Phase ◽  
Dynamic Instability ◽  
Living Cells ◽  
Liquid Phase Separation ◽  
State Transitions ◽  
Specific Domain ◽  
Rich Domain ◽  
Liquid Liquid Phase Separation

Tau protein in vitro can undergo liquid–liquid phase separation (LLPS); however, observations of this phase transition in living cells are limited. To investigate protein state transitions in living cells, we attached Cry2 to Tau and studied the contribution of each domain that drives the Tau cluster in living cells. Surprisingly, the proline-rich domain (PRD), not the microtubule binding domain (MTBD), drives LLPS and does so under the control of its phosphorylation state. Readily observable, PRD-derived cytoplasmic condensates underwent fusion and fluorescence recovery after photobleaching consistent with the PRD LLPS in vitro. Simulations demonstrated that the charge properties of the PRD predicted phase separation. Tau PRD formed heterotypic condensates with EB1, a regulator of plus-end microtubule dynamic instability. The specific domain properties of the MTBD and PRD serve distinct but mutually complementary roles that use LLPS in a cellular context to implement emergent functionalities that scale their relationship from binding α-beta tubulin heterodimers to the larger proportions of microtubules.

A Short Peptide Synthon for Liquid-Liquid Phase Separation

2020 ◽  
Author(s):  
Manzar Abbas ◽  
Wojciech P. Lipiński ◽  
Karina K. Nakashima ◽  
Wilhelm T.S. Huck ◽  
Evan Spruijt
Keyword(s):  
Phase Separation ◽  
Liquid Phase ◽  
Living Cells ◽  
Liquid Phase Separation ◽  
Disordered Proteins ◽  
Liquid Droplets ◽  
Short Peptide ◽  
Peptide Analogues ◽  
Single Peptide ◽  
Liquid Liquid Phase Separation

Liquid-liquid phase separation of disordered proteins has emerged as a ubiquitous route to membraneless compartments in living cells, and similar coacervates may have played a role when the first cells formed. However, existing coacervates are typically made of multiple macromolecular components, and designing short peptide analogues capable of self-coacervation has proven difficult. Here, we present a short peptide synthon for phase separation, made of only two dipeptide stickers linked via a flexible, hydrophilic spacer. These small-molecule compounds self-coacervate into micrometre-sized liquid droplets at sub-mM concentrations, which retain up to 75 weight-% water. The design is general and we derive guidelines for the required sticker hydrophobicity and spacer polarity. To illustrate their potential as protocells, we create a disulphide-linked derivative that undergoes reversible compartmentalisation controlled by redox chemistry. The resulting coacervates sequester and melt nucleic acids, and act as microreactors that catalyse two different anabolic reactions yielding molecules of increasing complexity. This provides a stepping stone for new protocells made of single peptide species.<br>

A Short Peptide Synthon for Liquid-Liquid Phase Separation

2020 ◽  
Author(s):  
Manzar Abbas ◽  
Wojciech P. Lipiński ◽  
Karina K. Nakashima ◽  
Wilhelm T.S. Huck ◽  
Evan Spruijt
Keyword(s):  
Phase Separation ◽  
Liquid Phase ◽  
Living Cells ◽  
Liquid Phase Separation ◽  
Disordered Proteins ◽  
Liquid Droplets ◽  
Short Peptide ◽  
Peptide Analogues ◽  
Single Peptide ◽  
Liquid Liquid Phase Separation

Liquid-liquid phase separation of disordered proteins has emerged as a ubiquitous route to membraneless compartments in living cells, and similar coacervates may have played a role when the first cells formed. However, existing coacervates are typically made of multiple macromolecular components, and designing short peptide analogues capable of self-coacervation has proven difficult. Here, we present a short peptide synthon for phase separation, made of only two dipeptide stickers linked via a flexible, hydrophilic spacer. These small-molecule compounds self-coacervate into micrometre-sized liquid droplets at sub-mM concentrations, which retain up to 75 weight-% water. The design is general and we derive guidelines for the required sticker hydrophobicity and spacer polarity. To illustrate their potential as protocells, we create a disulphide-linked derivative that undergoes reversible compartmentalisation controlled by redox chemistry. The resulting coacervates sequester and melt nucleic acids, and act as microreactors that catalyse two different anabolic reactions yielding molecules of increasing complexity. This provides a stepping stone for new protocells made of single peptide species.<br>

scholarly journals Physical observables to determine the nature of membrane-less cellular sub-compartments

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Mathias L Heltberg ◽  
Judith Miné-Hattab ◽  
Angela Taddei ◽  
Aleksandra M Walczak ◽  
Thierry Mora
Keyword(s):  
Phase Separation ◽  
Liquid Phase ◽  
Binding Site ◽  
Binding Sites ◽  
Spatial Organization ◽  
Liquid Droplet ◽  
Liquid Phase Separation ◽  
Site Model ◽  
Multiple Binding ◽  
Liquid Liquid Phase Separation

The spatial organization of complex biochemical reactions is essential for the regulation of cellular processes. Membrane-less structures called foci containing high concentrations of specific proteins have been reported in a variety of contexts, but the mechanism of their formation is not fully understood. Several competing mechanisms exist that are difficult to distinguish empirically, including liquid-liquid phase separation, and the trapping of molecules by multiple binding sites. Here we propose a theoretical framework and outline observables to differentiate between these scenarios from single molecule tracking experiments. In the binding site model, we derive relations between the distribution of proteins, their diffusion properties, and their radial displacement. We predict that protein search times can be reduced for targets inside a liquid droplet, but not in an aggregate of slowly moving binding sites. We use our results to reject the multiple binding site model for Rad52 foci, and find a picture consistent with a liquid-liquid phase separation. These results are applicable to future experiments and suggest different biological roles for liquid droplet and binding site foci.

scholarly journals Characterization of Liquid-liquid phase Separation in 3D Genome Organization

2020 ◽  
Author(s):  
Tingting Li ◽  
Jiaqing Xing ◽  
Tao Li ◽  
Teng Li ◽  
Weihua Li
Keyword(s):  
Phase Separation ◽  
Liquid Phase ◽  
Genome Organization ◽  
Enrichment Analysis ◽  
Liquid Phase Separation ◽  
Rna Recognition Motif ◽  
3D Genome ◽  
Shiny App ◽  
Modular Domains ◽  
Liquid Liquid Phase Separation

Abstract Many proteins have been demonstrated to participate in 3D genome organization through liquid-liquid phase separation (LLPS) such as RNPII, HP1a. However, systematic investigation of relationships between LLPS and 3D genome organization remains lacking. Here, we predicted the intrinsic disordered regions (IDRs) and modular domains of all human proteins and performed GSEA analysis according to their proportions of IDRs. Our results showed that main biological processes involved in 3D genome organization are highly enriched with IDRs, including chromatin organization, RNA splicing and histone modification, demonstrating the key role of LLPS in regulating nuclear structure. Of the 3885 IDR-rich proteins, 1427 proteins are involved in 3D genome organization. IDR regions of these proteins have strong preference of Ser, Leu, Pro, Ala, Gly, Glu and Lys, and lack of hydrophobic amino acids such as Trp, Tyr, Phe and Met, suggesting dipolar interactions rather than aromatic-involved interactions involved. Further motif enrichment analysis suggests that RNA recognition motif and zinc finger motif are the two most abundant repeatedly-occurred modular domains within IDR-containing proteins. Finally, we developed a Shiny APP named phasepro that interactively analyze and visualize a protein’s potential of LLPS, including IDRs, motifs, amino acid preferences and electric charges.

scholarly journals Biomolecular condensates as arbiters of biochemical reactions inside the nucleus

2020 ◽  
Vol 3 (1) ◽  
Author(s):  
Guillaume Laflamme ◽  
Karim Mekhail
Keyword(s):  
Phase Separation ◽  
Liquid Phase ◽  
Environmental Conditions ◽  
Genome Organization ◽  
Liquid Phase Separation ◽  
Nuclear Space ◽  
Biological Processes ◽  
Biochemical Reactions ◽  
Dynamic Structures ◽  
Liquid Liquid Phase Separation

AbstractLiquid-liquid phase separation (LLPS) has emerged as a central player in the assembly of membraneless compartments termed biomolecular condensates. These compartments are dynamic structures that can condense or dissolve under specific conditions to regulate molecular functions. Such properties allow biomolecular condensates to rapidly respond to changing endogenous or environmental conditions. Here, we review emerging roles for LLPS within the nuclear space, with a specific emphasis on genome organization, expression and repair. Our review highlights the emerging notion that biomolecular condensates regulate the sequential engagement of molecules in multistep biological processes.

scholarly journals Hyperactivation of L-lactate oxidase by liquid-liquid phase separation

2020 ◽  
Author(s):  
Tomoto Ura ◽  
Ako Kagawa ◽  
Hiromasa Yagi ◽  
Naoya Tochio ◽  
Takanori Kigawa ◽  
...  
Keyword(s):  
Phase Separation ◽  
Liquid Phase ◽  
Liquid Droplet ◽  
Living Cells ◽  
Enzyme Activation ◽  
Liquid Phase Separation ◽  
Enzymatic Reactions ◽  
Liquid Droplets ◽  
Essential Information ◽  
Liquid Liquid Phase Separation

ABSTRACTLiquid droplets formed by liquid-liquid phase separation are attracting attention as functional states of proteins in living cells. Liquid droplets are thought to activate enzymatic reactions by assembling the required molecules. Thus, liquid droplets usually increase the affinity of an enzyme to its substrates, leading to decreased KM values. In this study, we demonstrate a new mechanism of enzyme activation in the droplets using Llactate oxidase (LOX). In the presence of poly-L-lysine (PLL), LOX formed droplets with diameters of hundreds of nanometers to tens of micrometers, stabilized by electro-static interaction. The enzyme activity of LOX in the droplets was significantly enhanced by a fourfold decrease in KM and a tenfold increase in kcat. To our knowledge, this represents the first report for increasing kcat by the formation of the liquid droplet. Interestingly, the conformation of LOX changed in the liquid droplet, probably leading to increased kcat value. Understanding enzyme activation in the droplets provides essential information about enzyme function in living cells in addition to biotechnology applications.

scholarly journals The Proline-rich Domain Promotes Tau Liquid Liquid Phase Separation in Cells

2020 ◽  
Author(s):  
Xuemei Zhang ◽  
Michael Vigers ◽  
James McCarty ◽  
Jennifer N. Rauch ◽  
Glenn H. Fredrickson ◽  
...  
Keyword(s):  
Phase Separation ◽  
Liquid Phase ◽  
Dynamic Instability ◽  
Living Cells ◽  
Liquid Phase Separation ◽  
State Transitions ◽  
Specific Domain ◽  
Rich Domain ◽  
Liquid Liquid Phase Separation

AbstractTau protein in vitro can undergo liquid liquid phase separation (LLPS); however, observations of this phase transition in living cells are limited. To investigate protein state transitions in living cells we found that Cry2 can optogentically increase the association of full lengh tau with microtubules. To probe this mechanism, we identified tau domains that drive tau clustering on microtubules in living cells. The polyproline rich domain (PRD) drives LLPS and does so under the control of phosphorylation. These readily observable cytoplasmic condensates underwent fusion and fluorescence recovery after photobleaching consistent with the ability of the PRD to undergo LLPS in vitro. In absence of the MTBD, the tau PRD co-condensed with EB1, a regulator of plus-end microtubule dynamic instability. The specific domain properties of the MTBD and PRD serve distinct but mutually complementary roles that utilize LLPS in a cellular context to implement emergent functionalities that scale their relationship from binding alpha-beta tubulin heterodimers to the larger proportions of microtubules.

scholarly journals Consistent Force Field Captures Homolog Resolved HP1 Phase Separation

2021 ◽  
Author(s):  
Andrew P. Latham ◽  
Bin Zhang
Keyword(s):  
Phase Separation ◽  
Liquid Phase ◽  
Force Field ◽  
Large Scale ◽  
Spatial Organization ◽  
Structural Difference ◽  
Liquid Phase Separation ◽  
Coarse Grained ◽  
Disordered Proteins ◽  
Liquid Liquid Phase Separation

Many proteins have been shown to function via liquid-liquid phase separation. Computational modeling could offer much needed structural details of protein condensates and reveal the set of molecular interactions that dictate their stability. However, the presence of both ordered and disordered domains in these proteins places a high demand on the model accuracy. Here, we present an algorithm to derive a coarse-grained force field, MOFF, that can model both ordered and disordered proteins with consistent accuracy. It combines maximum entropy biasing, least-squares fitting, and basic principles of energy landscape theory to ensure that MOFF recreates experimental radii of gyration while predicting the folded structures for globular proteins with lower energy. The theta temperature determined from MOFF separates ordered and disordered proteins at 300 K and exhibits a strikingly linear relationship with amino acid sequence composition. We further applied MOFF to study the phase behavior of HP1, an essential protein for posttranslational modification and spatial organization of chromatin. The force field successfully resolved the structural difference of two HP1 homologs, despite their high sequence similarity. We carried out large scale simulations with hundreds of proteins to determine the critical temperature of phase separation and uncover multivalent interactions that stabilize higher-order assemblies. In all, our work makes significant methodological strides to connect theories of ordered and disordered proteins and provides a powerful tool for studying liquid-liquid phase separation with near-atomistic details.

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