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Biological Liquid-Liquid Phase Separation, Biomolecular Condensates, and Membraneless Organelles

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A special issue of International Journal of Molecular Sciences (ISSN 1422-0067). This special issue belongs to the section "Molecular Biophysics".

Deadline for manuscript submissions: closed (28 February 2023) | Viewed by 27498

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Prof. Dr. Vladimir N. Uversky

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Department of Molecular Medicine, USF Health Byrd Alzheimer’s Research Institute, Morsani College of Medicine, University of South Florida, 12901 Bruce B. Downs Blvd, MDC07, Tampa, FL 33612, USA
Interests: intrinsically disordered proteins; protein folding; protein misfolding; partially folded proteins; protein aggregation; protein structure; protein function; protein stability; protein biophysics; protein bioinformatics; conformational diseases; protein–ligand interactions; protein–protein interactions; liquid-liquid phase transitions
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Dear Colleagues,

Currently, there is immense interest among the scientific community in intracellular liquid–liquid phase separation (LLPS) and resulting biomolecular condensates (BMCs) and membrane-less organelles (MLOs). Obviously, such BMCs and MLOs, which do not have enclosing membranes, are mysterious subjects, whose components can directly contact, and exchange with, the exterior environment and whose biogenesis and structural integrity rely exclusively on protein–protein and/or protein–nucleic acid interactions. BMCs/MLOs, are large, highly dynamic, macromolecular ensembles visible under the light microscope as spherical micron-sized droplets. They demonstrate liquid-like behavior, being able to drip, formation of spherical structures upon fusion, and wetting. Therefore, MLOs are condensed liquid droplets formed as a result of reversible and highly controlled LLPS. MLOs are different in size, shape, and composition, and have important and diverse biological functions. Typically, MLOs are formed in response to specific cellular activities or stress. Alterations of their biogenesis might have pathological consequences, and many MLOs are associated with the pathogenesis of various diseases. This Special Issue includes research papers and reviews dedicated to the different aspects of LLPS, BMCs, and MLOs.

Dr. Vladimir N. Uversky
Guest Editor

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Published Papers (11 papers)

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Editorial

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6 pages, 375 KiB

Open AccessEditorial

Biological Liquid–Liquid Phase Separation, Biomolecular Condensates, and Membraneless Organelles: Now You See Me, Now You Don’t

by Vladimir N. Uversky

Int. J. Mol. Sci. 2023, 24(17), 13150; https://doi.org/10.3390/ijms241713150 - 24 Aug 2023

Cited by 1 | Viewed by 1143

Abstract

Liquid–liquid phase separation (LLPS, also known as biomolecular condensation) and the related biogenesis of various membraneless organelles (MLOs) and biomolecular condensates (BMCs) are now considered fundamental molecular mechanisms governing the spatiotemporal organization of the intracellular space [...] Full article

(This article belongs to the Special Issue Biological Liquid-Liquid Phase Separation, Biomolecular Condensates, and Membraneless Organelles)

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Research

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24 pages, 7735 KiB

Open AccessArticle

Molecular Determinants of Fibrillation in a Viral Amyloidogenic Domain from Combined Biochemical and Biophysical Studies

by Juliet F. Nilsson, Hakima Baroudi, Frank Gondelaud, Giulia Pesce, Christophe Bignon, Denis Ptchelkine, Joseph Chamieh, Hervé Cottet, Andrey V. Kajava and Sonia Longhi

Int. J. Mol. Sci. 2023, 24(1), 399; https://doi.org/10.3390/ijms24010399 - 26 Dec 2022

Cited by 5 | Viewed by 1629

Abstract

The Nipah and Hendra viruses (NiV and HeV) are biosafety level 4 human pathogens classified within the Henipavirus genus of the Paramyxoviridae family. In both NiV and HeV, the gene encoding the Phosphoprotein (P protein), an essential polymerase cofactor, also encodes the V and W proteins. These three proteins, which share an intrinsically disordered N-terminal domain (NTD) and have unique C-terminal domains (CTD), are all known to counteract the host innate immune response, with V and W acting by either counteracting or inhibiting Interferon (IFN) signaling. Recently, the ability of a short region within the shared NTD (i.e., PNT3) to form amyloid-like structures was reported. Here, we evaluated the relevance of each of three contiguous tyrosine residues located in a previously identified amyloidogenic motif (EYYY) within HeV PNT3 to the fibrillation process. Our results indicate that removal of a single tyrosine in this motif significantly decreases the ability to form fibrils independently of position, mainly affecting the elongation phase. In addition, we show that the C-terminal half of PNT3 has an inhibitory effect on fibril formation that may act as a molecular shield and could thus be a key domain in the regulation of PNT3 fibrillation. Finally, the kinetics of fibril formation for the two PNT3 variants with the highest and the lowest fibrillation propensity were studied by Taylor Dispersion Analysis (TDA). The results herein presented shed light onto the molecular mechanisms involved in fibril formation. Full article

(This article belongs to the Special Issue Biological Liquid-Liquid Phase Separation, Biomolecular Condensates, and Membraneless Organelles)

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17 pages, 11694 KiB

Open AccessArticle

Cytoophidia Maintain the Integrity of Drosophila Follicle Epithelium

by Qiao-Qi Wang, Dong-Dong You and Ji-Long Liu

Int. J. Mol. Sci. 2022, 23(23), 15282; https://doi.org/10.3390/ijms232315282 - 04 Dec 2022

Cited by 4 | Viewed by 2271

Abstract

CTP synthase (CTPS) forms a filamentous structure termed the cytoophidium in all three domains of life. The female reproductive system of Drosophila is an excellent model for studying the physiological function of cytoophidia. Here, we use CTPS^H355A, a point mutation that destroys the cytoophidium-forming ability of CTPS, to explore the in vivo function of cytoophidia. In CTPS^H355A egg chambers, we observe the ingression and increased heterogeneity of follicle cells. In addition, we find that the cytoophidium-forming ability of CTPS, rather than the protein level, is the cause of the defects observed in CTPS^H355A mutants. To sum up, our data indicate that cytoophidia play an important role in maintaining the integrity of follicle epithelium. Full article

(This article belongs to the Special Issue Biological Liquid-Liquid Phase Separation, Biomolecular Condensates, and Membraneless Organelles)

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14 pages, 2318 KiB

Open AccessArticle

Super-Resolution Imaging Reveals Dynamic Reticular Cytoophidia

by Yi-Fan Fang, Yi-Lan Li, Xiao-Ming Li and Ji-Long Liu

Int. J. Mol. Sci. 2022, 23(19), 11698; https://doi.org/10.3390/ijms231911698 - 02 Oct 2022

Cited by 2 | Viewed by 3254

Abstract

CTP synthase (CTPS) can form filamentous structures termed cytoophidia in cells in all three domains of life. In order to study the mesoscale structure of cytoophidia, we perform fluorescence recovery after photobleaching (FRAP) and stimulated emission depletion (STED) microscopy in human cells. By using an EGFP dimeric tag as a tool to explore the physical properties of cytoophidia, we find that cytoophidia are dynamic and reticular. The reticular structure of CTPS cytoophidia may provide space for other components, such as IMPDH. In addition, we observe CTPS granules with tentacles. Full article

(This article belongs to the Special Issue Biological Liquid-Liquid Phase Separation, Biomolecular Condensates, and Membraneless Organelles)

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13 pages, 4138 KiB

Open AccessArticle

The Patterning and Proportion of Charged Residues in the Arginine-Rich Mixed-Charge Domain Determine the Membrane-Less Organelle Targeted by the Protein

by Tamami Miyagi, Rio Yamazaki, Koji Ueda, Satoshi Narumi, Yuhei Hayamizu, Hiroshi Uji-i, Masahiko Kuroda and Kohsuke Kanekura

Int. J. Mol. Sci. 2022, 23(14), 7658; https://doi.org/10.3390/ijms23147658 - 11 Jul 2022

Cited by 4 | Viewed by 2380

Abstract

Membrane-less organelles (MLOs) are formed by biomolecular liquid–liquid phase separation (LLPS). Proteins with charged low-complexity domains (LCDs) are prone to phase separation and localize to MLOs, but the mechanism underlying the distributions of such proteins to specific MLOs remains poorly understood. Recently, proteins with Arg-enriched mixed-charge domains (R-MCDs), primarily composed of R and Asp (D), were found to accumulate in nuclear speckles via LLPS. However, the process by which R-MCDs selectively incorporate into nuclear speckles is unknown. Here, we demonstrate that the patterning of charged amino acids and net charge determines the targeting of specific MLOs, including nuclear speckles and the nucleolus, by proteins. The redistribution of R and D residues from an alternately sequenced pattern to uneven blocky sequences caused a shift in R-MCD distribution from nuclear speckles to the nucleolus. In addition, the incorporation of basic residues in the R-MCDs promoted their localization to the MLOs and their apparent accumulation in the nucleolus. The R-MCD peptide with alternating amino acids did not undergo LLPS, whereas the blocky R-MCD peptide underwent LLPS with affinity to RNA, acidic poly-Glu, and the acidic nucleolar protein nucleophosmin, suggesting that the clustering of R residues helps avoid their neutralization by D residues and eventually induces R-MCD migration to the nucleolus. Therefore, the distribution of proteins to nuclear speckles requires the proximal positioning of D and R for the mutual neutralization of their charges. Full article

(This article belongs to the Special Issue Biological Liquid-Liquid Phase Separation, Biomolecular Condensates, and Membraneless Organelles)

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23 pages, 2959 KiB

Open AccessArticle

The Multivalent Polyampholyte Domain of Nst1, a P-Body-Associated Saccharomyces cerevisiae Protein, Provides a Platform for Interacting with P-Body Components

by Yoon-Jeong Choi, Yujin Lee, Yuxi Lin, Yunseok Heo, Young-Ho Lee and Kiwon Song

Int. J. Mol. Sci. 2022, 23(13), 7380; https://doi.org/10.3390/ijms23137380 - 02 Jul 2022

Cited by 2 | Viewed by 1777

Abstract

The condensation of nuclear promyelocytic leukemia bodies, cytoplasmic P-granules, P-bodies (PBs), and stress granules is reversible and dynamic via liquid–liquid phase separation. Although each condensate comprises hundreds of proteins with promiscuous interactions, a few key scaffold proteins are required. Essential scaffold domain sequence elements, such as poly-Q, low-complexity regions, oligomerizing domains, and RNA-binding domains, have been evaluated to understand their roles in biomolecular condensation processes. However, the underlying mechanisms remain unclear. We analyzed Nst1, a PB-associated protein that can intrinsically induce PB component condensations when overexpressed. Various Nst1 domain deletion mutants with unique sequence distributions, including intrinsically disordered regions (IDRs) and aggregation-prone regions, were constructed based on structural predictions. The overexpression of Nst1 deletion mutants lacking the aggregation-prone domain (APD) significantly inhibited self-condensation, implicating APD as an oligomerizing domain promoting self-condensation. Remarkably, cells overexpressing the Nst1 deletion mutant of the polyampholyte domain (PD) in the IDR region (Nst1_∆PD) rarely accumulate endogenous enhanced green fluorescent protein (EGFP)-tagged Dcp2. However, Nst1_∆PD formed self-condensates, suggesting that Nst1 requires PD to interact with Dcp2, regardless of its self-condensation. In Nst1_∆PD-overexpressing cells treated with cycloheximide (CHX), Dcp2, Xrn1, Dhh1, and Edc3 had significantly diminished condensation compared to those in CHX-treated Nst1-overexpressing cells. These observations suggest that the PD of the IDR in Nst1 functions as a hub domain interacting with other PB components. Full article

(This article belongs to the Special Issue Biological Liquid-Liquid Phase Separation, Biomolecular Condensates, and Membraneless Organelles)

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18 pages, 3643 KiB

Open AccessArticle

BIAPSS: A Comprehensive Physicochemical Analyzer of Proteins Undergoing Liquid–Liquid Phase Separation

by Aleksandra E. Badaczewska-Dawid, Vladimir N. Uversky and Davit A. Potoyan

Int. J. Mol. Sci. 2022, 23(11), 6204; https://doi.org/10.3390/ijms23116204 - 31 May 2022

Cited by 7 | Viewed by 2632

Abstract

The liquid–liquid phase separation (LLPS) of biomolecules is a phenomenon which is nowadays recognized as the driving force for the biogenesis of numerous functional membraneless organelles and cellular bodies. The interplay between the protein primary sequence and phase separation remains poorly understood, despite intensive research. To uncover the sequence-encoded signals of protein capable of undergoing LLPS, we developed a novel web platform named BIAPSS (Bioinformatics Analysis of LLPS Sequences). This web server provides on-the-fly analysis, visualization, and interpretation of the physicochemical and structural features for the superset of curated LLPS proteins. Full article

(This article belongs to the Special Issue Biological Liquid-Liquid Phase Separation, Biomolecular Condensates, and Membraneless Organelles)

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13 pages, 4601 KiB

Open AccessArticle

Free Cholesterol Accelerates Aβ Self-Assembly on Membranes at Physiological Concentration

by Mohtadin Hashemi, Siddhartha Banerjee and Yuri L. Lyubchenko

Int. J. Mol. Sci. 2022, 23(5), 2803; https://doi.org/10.3390/ijms23052803 - 03 Mar 2022

Cited by 10 | Viewed by 2038

Abstract

The effects of membranes on the early-stage aggregation of amyloid β (Aβ) have come to light as potential mechanisms by which neurotoxic species are formed in Alzheimer’s disease. We have shown that direct Aβ-membrane interactions dramatically enhance the Aβ aggregation, allowing for oligomer assembly at physiologically low concentrations of the monomer. Membrane composition is also a crucial factor in this process. Our results showed that apart from phospholipids composition, cholesterol in membranes significantly enhances the aggregation kinetics. It has been reported that free cholesterol is present in plaques. Here we report that free cholesterol, along with its presence inside the membrane, further accelerate the aggregation process by producing aggregates more rapidly and of significantly larger sizes. These aggregates, which are formed on the lipid bilayer, are able to dissociate from the surface and accumulate in the bulk solution; the presence of free cholesterol accelerates this dissociation as well. All-atom molecular dynamics simulations show that cholesterol binds Aβ monomers and significantly changes the conformational sampling of Aβ monomer; more than doubling the fraction of low-energy conformations compared to those in the absence of cholesterol, which can contribute to the aggregation process. The results indicate that Aβ-lipid interaction is an important factor in the disease prone amyloid assembly process. Full article

(This article belongs to the Special Issue Biological Liquid-Liquid Phase Separation, Biomolecular Condensates, and Membraneless Organelles)

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Review

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13 pages, 3890 KiB

Open AccessReview

The Role of Liquid–Liquid Phase Separation in Actin Polymerization

by Olga I. Povarova, Iuliia A. Antifeeva, Alexander V. Fonin, Konstantin K. Turoverov and Irina M. Kuznetsova

Int. J. Mol. Sci. 2023, 24(4), 3281; https://doi.org/10.3390/ijms24043281 - 07 Feb 2023

Cited by 5 | Viewed by 2267

Abstract

To date, it has been shown that the phenomenon of liquid–liquid phase separation (LLPS) underlies many seemingly completely different cellular processes. This provided a new idea of the spatiotemporal organization of the cell. The new paradigm makes it possible to provide answers to many long-standing, but still unresolved questions facing the researcher. In particular, spatiotemporal regulation of the assembly/disassembly of the cytoskeleton, including the formation of actin filaments, becomes clearer. To date, it has been shown that coacervates of actin-binding proteins that arise during the phase separation of the liquid–liquid type can integrate G-actin and thereby increase its concentration to initiate polymerization. It has also been shown that the activity intensification of actin-binding proteins that control actin polymerization, such as N-WASP and Arp2/3, can be caused by their integration into liquid droplet coacervates formed by signaling proteins on the inner side of the cell membrane. Full article

(This article belongs to the Special Issue Biological Liquid-Liquid Phase Separation, Biomolecular Condensates, and Membraneless Organelles)

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28 pages, 3367 KiB

Open AccessReview

Phase Separation-Mediated Chromatin Organization and Dynamics: From Imaging-Based Quantitative Characterizations to Functional Implications

by Woei Shyuan Ng, Hendrik Sielaff and Ziqing Winston Zhao

Int. J. Mol. Sci. 2022, 23(14), 8039; https://doi.org/10.3390/ijms23148039 - 21 Jul 2022

Cited by 4 | Viewed by 3443

Abstract

As an effective and versatile strategy to compartmentalize cellular components without the need for lipid membranes, phase separation has been found to underpin a wide range of intranuclear processes, particularly those involving chromatin. Many of the unique physico-chemical properties of chromatin-based phase condensates are harnessed by the cell to accomplish complex regulatory functions in a spatially and temporally controlled manner. Here, we survey key recent findings on the mechanistic roles of phase separation in regulating the organization and dynamics of chromatin-based molecular processes across length scales, packing states and intranuclear functions, with a particular emphasis on quantitative characterizations of these condensates enabled by advanced imaging-based approaches. By illuminating the complex interplay between chromatin and various chromatin-interacting molecular species mediated by phase separation, this review sheds light on an emerging multi-scale, multi-modal and multi-faceted landscape that hierarchically regulates the genome within the highly crowded and dynamic nuclear space. Moreover, deficiencies in existing studies also highlight the need for mechanism-specific criteria and multi-parametric approaches for the characterization of chromatin-based phase separation using complementary techniques and call for greater efforts to correlate the quantitative features of these condensates with their functional consequences in close-to-native cellular contexts. Full article

(This article belongs to the Special Issue Biological Liquid-Liquid Phase Separation, Biomolecular Condensates, and Membraneless Organelles)

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23 pages, 1703 KiB

Open AccessReview

Phase-Separated Subcellular Compartmentation and Related Human Diseases

by Lin Zhang, Shubo Wang, Wenmeng Wang, Jinming Shi, Daniel B. Stovall, Dangdang Li and Guangchao Sui

Int. J. Mol. Sci. 2022, 23(10), 5491; https://doi.org/10.3390/ijms23105491 - 14 May 2022

Cited by 3 | Viewed by 3292

Abstract

In live cells, proteins and nucleic acids can associate together through multivalent interactions, and form relatively isolated phases that undertake designated biological functions and activities. In the past decade, liquid–liquid phase separation (LLPS) has gradually been recognized as a general mechanism for the intracellular organization of biomolecules. LLPS regulates the assembly and composition of dozens of membraneless organelles and condensates in cells. Due to the altered physiological conditions or genetic mutations, phase-separated condensates may undergo aberrant formation, maturation or gelation that contributes to the onset and progression of various diseases, including neurodegenerative disorders and cancers. In this review, we summarize the properties of different membraneless organelles and condensates, and discuss multiple phase separation-regulated biological processes. Based on the dysregulation and mutations of several key regulatory proteins and signaling pathways, we also exemplify how aberrantly regulated LLPS may contribute to human diseases. Full article

(This article belongs to the Special Issue Biological Liquid-Liquid Phase Separation, Biomolecular Condensates, and Membraneless Organelles)

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