scholarly journals A role for liquid-liquid phase separation in ESCRT-mediated nuclear envelope reformation

2019 ◽  
Author(s):  
Alexander von Appen ◽  
Dollie LaJoie ◽  
Isabel E. Johnson ◽  
Mike Trnka ◽  
Sarah M. Pick ◽  
...  
Keyword(s):  
Phase Separation ◽  
Liquid Phase ◽  
Nuclear Envelope ◽  
Genome Stability ◽  
Transmembrane Protein ◽  
Low Complexity ◽  
Liquid Phase Separation ◽  
Endosomal Sorting ◽  
Spindle Disassembly ◽  
Liquid Liquid Phase Separation

At mitotic exit, microtubule arrays are dismantled in concert with the reformation of the nuclear envelope. We show how the inner nuclear membrane protein, LEM2, exploits liquid-liquid phase separation to direct microtubule remodeling and nuclear envelope sealing via the Endosomal Sorting Complexes Required for Transport (ESCRT) pathway. LEM2 tethers membrane to chromatin disks through direct binding between its LEM motif and the chromatin-associated barrier-to-autointegration factor (BAF). Concurrently, a low-complexity domain within LEM2 undergoes liquid-liquid phase separation to coat spindle microtubule bundles. Spatially restricted, LEM2’s winged helix (WH) domain activates the ESCRT-II/ESCRT-III hybrid protein, CHMP7. Together LEM2 and CHMP7 copolymerize around microtubule bundles to form a molecular “O-ring” that promotes nuclear compartmentalization and initiates downstream ESCRT factor recruitment. These results demonstrate how multivalent interactions of a transmembrane protein, including those that mediate phase separation, coordinate localized ESCRT polymerization, mitotic spindle disassembly, and membrane fusion. Defects in this pathway compromise spindle disassembly, nuclear integrity, and genome stability.

scholarly journals Liquid–Liquid Phase Separation Enhances TDP-43 LCD Aggregation but Delays Seeded Aggregation

Biomolecules ◽  
2021 ◽  
Vol 11 (4) ◽  
pp. 548
Author(s):  
Donya Pakravan ◽  
Emiel Michiels ◽  
Anna Bratek-Skicki ◽  
Mathias De Decker ◽  
Joris Van Lindt ◽  
...  
Keyword(s):  
Phase Separation ◽  
Liquid Phase ◽  
Hydrophobic Interactions ◽  
Low Complexity ◽  
Liquid Phase Separation ◽  
End Stage ◽  
Positive Effect ◽  
Self Aggregation ◽  
Lateral Sclerosis ◽  
Liquid Liquid Phase Separation

Aggregates of TAR DNA-binding protein (TDP-43) are a hallmark of several neurodegenerative disorders, including amyotrophic lateral sclerosis (ALS). Although TDP-43 aggregates are an undisputed pathological species at the end stage of these diseases, the molecular changes underlying the initiation of aggregation are not fully understood. The aim of this study was to investigate how phase separation affects self-aggregation and aggregation seeded by pre-formed aggregates of either the low-complexity domain (LCD) or its short aggregation-promoting regions (APRs). By systematically varying the physicochemical conditions, we observed that liquid–liquid phase separation (LLPS) promotes spontaneous aggregation. However, we noticed less efficient seeded aggregation in phase separating conditions. By analyzing a broad range of conditions using the Hofmeister series of buffers, we confirmed that stabilizing hydrophobic interactions prevail over destabilizing electrostatic forces. RNA affected the cooperativity between LLPS and aggregation in a “reentrant” fashion, having the strongest positive effect at intermediate concentrations. Altogether, we conclude that conditions which favor LLPS enhance the subsequent aggregation of the TDP-43 LCD with complex dependence, but also negatively affect seeding kinetics.

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 Exploiting mammalian low-complexity domains for liquid-liquid phase separation–driven underwater adhesive coatings

2019 ◽  
Vol 5 (8) ◽  
pp. eaax3155 ◽  
Author(s):  
Mengkui Cui ◽  
Xinyu Wang ◽  
Bolin An ◽  
Chen Zhang ◽  
Xinrui Gui ◽  
...  
Keyword(s):  
Phase Separation ◽  
Liquid Phase ◽  
Soft Materials ◽  
Low Complexity ◽  
Liquid Phase Separation ◽  
Biologically Inspired ◽  
Underwater Adhesion ◽  
Assembly Mechanism ◽  
Adhesion Performance ◽  
Liquid Liquid Phase Separation

Many biological materials form via liquid-liquid phase separation (LLPS), followed by maturation into a solid-like state. Here, using a biologically inspired assembly mechanism designed to recapitulate these sequential assemblies, we develop ultrastrong underwater adhesives made from engineered proteins containing mammalian low-complexity (LC) domains. We show that LC domain–mediated LLPS and maturation substantially promotes the wetting, adsorption, priming, and formation of dense, uniform amyloid nanofiber coatings on diverse surfaces (e.g., Teflon), and even penetrating difficult-to-access locations such as the interiors of microfluidic devices. Notably, these coatings can be deposited on substrates over a broad range of pH values (3 to 11) and salt concentrations (up to 1 M NaCl) and exhibit strong underwater adhesion performance. Beyond demonstrating the utility of mammalian LC domains for driving LLPS in soft materials applications, our study illustrates a powerful example of how combining LLPS with subsequent maturation steps can be harnessed for engineering protein-based materials.

scholarly journals Poly-glutamine-dependent self-association as a potential mechanism for regulation of androgen receptor activity

2021 ◽  
Author(s):  
Josep Rizo ◽  
Carlos M. Roggero ◽  
Victoria Esser ◽  
Lingling Duan ◽  
Allyson M. Rice ◽  
...  
Keyword(s):  
Prostate Cancer ◽  
Phase Separation ◽  
Androgen Receptor ◽  
Liquid Phase ◽  
Transcriptional Activity ◽  
Molecular Mechanisms ◽  
Catalytic Domain ◽  
Low Complexity ◽  
Liquid Phase Separation ◽  
Liquid Liquid Phase Separation

The androgen receptor (AR) plays a central role in prostate cancer. Development of castration resistant prostate cancer (CRPC) requires androgen-independent activation of AR, which involves its large N-terminal domain (NTD) and entails dramatic epigenetic changes depending in part on histone lysine demethylases (KDMs) that interact with AR. The AR-NTD is rich in low-complexity sequences, including a polyQ repeat. Longer polyQ sequences were reported to decrease transcriptional activity and to protect against prostate cancer. However, the molecular mechanisms underlying these observations are unclear. Using NMR spectroscopy, here we identify weak interactions between the AR-NTD and the KDM4A catalytic domain, and between the AR ligand-binding domain and a central KDM4A region that also contains low-complexity sequences. We also show that the AR-NTD can undergo liquid-liquid phase separation in vitro, with longer polyQ sequences phase separating more readily. Moreover, longer polyQ sequences hinder nuclear localization in the absence of hormone and increase the propensity for formation of AR-containing puncta in the nucleus of cells treated with dihydrotestosterone. These results lead us to hypothesize that polyQ-dependent liquid-liquid phase separation may provide a mechanism to decrease the transcriptional activity of AR, potentially opening new opportunities to design effective therapies against CRPC.

scholarly journals The Role of Methionine Residues in the Regulation of Liquid-Liquid Phase Separation

Biomolecules ◽  
2021 ◽  
Vol 11 (8) ◽  
pp. 1248
Author(s):  
Juan Carlos Aledo
Keyword(s):  
Phase Separation ◽  
Liquid Phase ◽  
Low Complexity ◽  
Liquid Phase Separation ◽  
Modular Architecture ◽  
Residue Interaction ◽  
Methionine Residues ◽  
Molecular Forces ◽  
Liquid Liquid Phase Separation

Membraneless organelles are non-stoichiometric supramolecular structures in the micron scale. These structures can be quickly assembled/disassembled in a regulated fashion in response to specific stimuli. Membraneless organelles contribute to the spatiotemporal compartmentalization of the cell, and they are involved in diverse cellular processes often, but not exclusively, related to RNA metabolism. Liquid-liquid phase separation, a reversible event involving demixing into two distinct liquid phases, provides a physical framework to gain insights concerning the molecular forces underlying the process and how they can be tuned according to the cellular needs. Proteins able to undergo phase separation usually present a modular architecture, which favors a multivalency-driven demixing. We discuss the role of low complexity regions in establishing networks of intra- and intermolecular interactions that collectively control the phase regime. Post-translational modifications of the residues present in these domains provide a convenient strategy to reshape the residue–residue interaction networks that determine the dynamics of phase separation. Focus will be placed on those proteins with low complexity domains exhibiting a biased composition towards the amino acid methionine and the prominent role that reversible methionine sulfoxidation plays in the assembly/disassembly of biomolecular condensates.

scholarly journals Pericentromeric heterochromatin is hierarchically organized and spatially contacts H3K9me2 islands in euchromatin

2019 ◽  
Author(s):  
Yuh Chwen G. Lee ◽  
Yuki Ogiyama ◽  
Nuno M. C. Martins ◽  
Brian J. Beliveau ◽  
David Acevedo ◽  
...  
Keyword(s):  
Phase Separation ◽  
Liquid Phase ◽  
Genome Stability ◽  
Repetitive Sequences ◽  
Liquid Phase Separation ◽  
Hierarchical Organization ◽  
Pericentromeric Heterochromatin ◽  
Chromosome Dynamics ◽  
Technical Challenges ◽  
Liquid Liquid Phase Separation

AbstractMembraneless pericentromeric heterochromatin (PCH) domains play vital roles in chromosome dynamics and genome stability. However, our current understanding of 3D genome organization does not include PCH domains because of technical challenges associated with repetitive sequences enriched in PCH genomic regions. We investigated the 3D architecture of Drosophila melanogaster PCH domains and their spatial associations with euchromatic genome by developing a novel analysis method that incorporates genome-wide Hi-C reads originating from PCH DNA. Combined with cytogenetic analysis, we reveal a hierarchical organization of the PCH domains into distinct “territories.” Strikingly, H3K9me2/3-enriched regions embedded in the euchromatic genome show prevalent 3D interactions with the PCH domain. These spatial contacts require H3K9me2/3 enrichment, are likely mediated by liquid-liquid phase separation, and may influence organismal fitness. Our findings have important implications for how PCH architecture influences the function and evolution of both repetitive heterochromatin and the gene-rich euchromatin.Author summaryThe three dimensional (3D) organization of genomes in cell nuclei can influence a wide variety of genome functions. However, most of our understanding of this critical architecture has been limited to the gene-rich euchromatin, and largely ignores the gene-poor and repeat-rich pericentromeric heterochromatin, or PCH. PCH comprises large part of most eukaryotic genomes, forms 3D PCH domains in nuclei, and plays vital role in chromosome dynamics and genome stability. In this study, we developed a new method that overcomes the technical challenges imposed by the highly repetitive PCH DNA, and generated a comprehensive picture of its 3D organization. Combined with image analyses, we revealed a hierarchical organization of the PCH domains. Surprisingly, we showed that distant euchromatic regions enriched for repressive epigenetic marks also dynamically interact with the main PCH domains. These 3D interactions are mediated by liquid-liquid phase separation mechanisms, similar to how oil and vinegar separate in salad dressing, and can influence the fitness of individuals. Our discoveries have strong implications for how seemingly “junk” DNA could impact functions in the gene-rich euchromatin.

Tau Conformers in FTLD-MAPT  Undergo Liquid-liquid Phase Separation and Perturb the Nuclear Envelope

2020 ◽  
Author(s):  
Sang-Gyun Kang ◽  
Zhuang Zhuang Han ◽  
Nathalie Daude ◽  
Emily McNamara ◽  
Serene Wohlgemuth ◽  
...  
Keyword(s):  
Cell Death ◽  
Phase Separation ◽  
Liquid Phase ◽  
Nuclear Envelope ◽  
Liquid State ◽  
Critical Mass ◽  
Liquid Phase Separation ◽  
Single Mutation ◽  
Cytoplasmic Transport ◽  
Liquid Liquid Phase Separation

Abstract Background: Germline mutations in the MAPT gene cause some forms of frontotemporal lobar degeneration (FTLD). Recent studies show that a single mutation in MAPT can promote alternative tau misfolding pathways engendering divergent tau conforms and representing clinical heterogeneity, and that under conditions of cell-free molecular crowding the repertoire of tau forms can include liquid-liquid phase separation (LLPS). Methods: Neuronal nuclear morphologies in FTLD patients and TgTauP301L transgenic mice were analyzed by immunohistochemistry of nuclear lamina. Tau conformers associated with a common behavioral variant of frontotemporal dementia were cloned by endpoint dilution; the cells were assayed for viability and biochemical markers of cell death and were also assessed by video microscopy and photobleaching to determine dynamic aspects of aggregate formation.Results: Analysis of post-mortem tissues from aged neurologically normal controls and other neurodegenerative syndromes indicated that microtubule-associated nuclear clefts were associated with chronological aging and disruptions of the nuclear envelope with FTLD-MAPT. Tau conformers present in FTLD cases and transduced into reporter cells had a high propensity to condense on the nuclear envelope and to disrupt nuclear-cytoplasmic transport. Nuclear envelope fluorescent tau signals and small fluorescent inclusions in a stable clonal line behaved as a demixed liquid state under live cell conditions; indicative of LLPS effects, these droplets exhibited spherical morphology, fusion events and recovery from photobleaching. While pathogenic mutations in some proteins can interfere with physiological functions of membrane-less organelles, a disease-causing MAPT mutation perturbed nuclear-cytoplasmic transport by gain-of-function formation of LLPS on the nuclear envelope, this acting as a molecular cue to trigger regulated cell death. Thioflavin S-positive intracellular aggregates were prevalent in tau-derived inclusions with a size bigger than 3 µm2, inferring that a threshold of critical mass in the liquid state condensation may drive liquid-solid phase transitions. Conclusions: Our findings indicate that within a spectrum of alternative conformers, tau undergoing LLPS is a notably toxic species; demixed droplets on the nuclear envelope hindering nuclear-cytoplasmic transport can serve to trigger cytotoxic pathways and may act as nurseries for the abundant fibrillar structures present at end-stage disease.

scholarly journals The C-terminal low-complexity domain involved in liquid–liquid phase separation is required for BRD4 function in vivo

2019 ◽  
Vol 11 (9) ◽  
pp. 807-809
Author(s):  
Chenlu Wang ◽  
Erhao Zhang ◽  
Fan Wu ◽  
Yufeng Sun ◽  
Yingcheng Wu ◽  
...  
Keyword(s):  
Phase Separation ◽  
Liquid Phase ◽  
Low Complexity ◽  
Liquid Phase Separation ◽  
Liquid Liquid Phase Separation

scholarly journals Inhibition of amyloid formation of the Nucleoprotein of SARS-CoV-2

2021 ◽  
Author(s):  
Einav Tayeb-Fligelman ◽  
Xinyi Cheng ◽  
Christen Tai ◽  
Jeannette T. Bowler ◽  
Sarah Griner ◽  
...  
Keyword(s):  
Phase Separation ◽  
Liquid Phase ◽  
Low Complexity ◽  
Fibril Formation ◽  
Liquid Phase Separation ◽  
Amyloid Formation ◽  
Rna Packaging ◽  
Atomic Structures ◽  
Amyloid Fibril Formation ◽  
Liquid Liquid Phase Separation

The SARS-CoV-2 Nucleoprotein (NCAP) functions in RNA packaging during viral replication and assembly. Computational analysis of its amino acid sequence reveals a central low-complexity domain (LCD) having sequence features akin to LCDs in other proteins known to function in liquid-liquid phase separation. Here we show that in the presence of viral RNA, NCAP, and also its LCD segment alone, form amyloid-like fibrils when undergoing liquid-liquid phase separation. Within the LCD we identified three 6-residue segments that drive amyloid fibril formation. We determined atomic structures for fibrils formed by each of the three identified segments. These structures informed our design of peptide inhibitors of NCAP fibril formation and liquid-liquid phase separation, suggesting a therapeutic route for Covid-19.

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