Membraneless Organelles Research Articles

Enhanced liquid-liquid phase separation of stress granules in a reconstructed model and their cytoplasmic targeting using a DNA nanodevice.

Biomolecular condensates (BCs) are crucial membraneless organelles formed through the process of liquid-liquid phase separation (LLPS) involving proteins and nucleic acids. These LLPS processes are tightly linked with essential cellular activities. Stress granules (SGs), functioning as cytoplasmic BCs, play indispensable roles in maintaining cellular homeostasis and are implicated in diseases like cancers and neurodegenerative disorders. However, devices that can regulate SG LLPS are lacking. Herein, a triangular prism-shaped DNA nanostructure containing polythymidine (ΔDNA(polyT)) is presented as a nanodevice to investigate the LLPS process of in vitro reconstructed SGs (rSGs), a mixture of marker protein G3BP1 and total RNAs. Our observations reveal that the concentration threshold required for rSG LLPS decreases upon addition of ΔDNA(polyT), suggesting an enhancement in SG LLPS efficiency. It is speculated that ΔDNA(polyT) can concentrate mRNAs onto its surface via polyT hybridization with poly-adenosine sequences (polyA) in mRNAs. This alteration in the spatial distribution of mRNAs subsequently affects the multivalency interactions between G3BP1 and mRNAs. Furthermore, ΔDNA(polyT) exhibits excellent colocalization with cytoplasmic SGs under stressed conditions. This DNA-based nanodevice presents a new artificial approach for the targeted regulation of BC LLPS and holds promise for future studies focusing on BCs.

Feedback-induced phase separation of hollow condensates to create biomimetic membraneless compartments.

Intracellular macromolecules have the ability to form membraneless compartments, such as vacuoles and hollow condensates, through liquid-liquid phase separation (LLPS) in order to adapt to changes in their environment. The development of artificial non-homogeneous compartments, such as multiphase hollow or multicavity condensates, has gained significant attention due to their potential to uncover the mechanisms underlying the formation of artificial condensates and biomolecular condensates. However, the complexity of design and construction has hindered progress, particularly in creating dynamic non-homogeneous compartments. In this study, we present a dynamic membraneless compartment using peptide-oligonucleotide conjugates derived from short elastin-like polypeptides (sELP-ONs), which undergo pH-mediated phase transition. Below pH 8.8, the microcompartment exists as microdroplets that transform into non-homogeneous hollow condensates above pH 8.8. Notably, these hollow condensates retain liquid properties and high molecular ordering, and effectively sequester guest molecules with a hollow condensed layer. Furthermore, our sELP-ON microcompartments exhibit a feedback-induced phase transition in response to environmental pH fluctuations generated by complex enzymatic reactions mimicking cellular metabolism, providing a novel dynamic model for creating biomimetic membraneless compartments.

Bioinspired programmable coacervate droplets and self-assembled fibers through pH regulation of monomers.

Phase separation and phase transitions pervade the biological domain, where proteins and RNA engage in liquid-liquid phase separation (LLPS), forming liquid-like membraneless organelles. The misregulation or dysfunction of these proteins culminates in the formation of solid aggregates via a liquid-to-solid transition, leading to pathogenic conditions. To decipher the underlying mechanisms, synthetic LLPS has been examined through complex coacervate formation from charged polymers. Nonetheless, temporal control over phase transitions from prebiotically relevant small organic synthons remains largely unexplored. Herein, we propose utilizing pH modulation to regulate the charge of small molecular building blocks, thereby controlling the LLPS process. Through a bio-inspired, enzyme-mediated pH-regulated reaction, we introduce temporal control over both LLPS and the transition from coacervates to supramolecular polymers. Additionally, by incorporating antagonistic pH modulators, we achieve transient LLPS and further temporal regulation of supramolecular polymer disassembly. Our investigation into pH-regulated LLPS provides a new avenue for exploring the stimuli-responsive, dynamic, and transient nature of LLPS.

Assessing the Effect of Chromatin-Binding Enzymes on Condensed Chromatin with Optical Tweezers.

The formation of biomolecular condensates in vitro and in vivo has become an increasingly important subject of studies. One particular area of interest is the phase separation of chromatin in the nucleus. However, the interplay of condensed chromatin and chromatin-binding enzymes has barely been studied as of now. Here, we show an optical tweezer-based assay that uses controlled fusion of two condensates to probe the effect of enzymes, such as chromatin remodeling motor proteins, on the fluidity of condensates. The assay provides a powerful tool that enables the study of if and how chromatin-enzyme interactions alter biophysical properties in these dense chromatin condensates.

Chlorophyll-Based Optogenetics to Control Membraneless Organelles.

Membraneless organelles (MLOs) formed via protein phase separation have garnered significant attention recently due to their relevance to cellular physiology and pathology. However, there is a lack of tools available to study their behavior and control their bioactivity in complex biological systems. This chapter describes a new optogenetic tool based on water-soluble chlorophyll protein (WSCP), a redlight-induced singlet oxygen-generating protein, to control syntheticMLOs. Upon exposure to red light, WSCP generates singlet oxygen, which triggers the crosslinking of the proteins in theMLOs, resulting in their liquid-to-solid phase transition. The effective delivery of chlorophylls enables the successful reconstitution of WSCP in living cells, thusoffering a potential approach to biological regulation at the subcellular level.

Directed stochasticity: Building biomolecular condensates in the right place

Controlling biomolecular condensate formation within the nucleus is critical for genome function. In this issue, Xu et al. (https://doi.org/10.1083/jcb.202401036) report that KPNA3 promotes histone locus body formation and expression of replication-dependent histone genes by both importing NPAT into the nucleus and preventing NPAT condensation from improperly occurring in the cytoplasm.

Imaging-Based Quantitative Assessment of Biomolecular Condensates in vitro and in Cells.

The formation of biomolecular condensates contributes to intracellular compartmentalization, and plays an important role in many cellular processes. The characterization of condensates is however challenging, requiring advanced biophysical or biochemical methods that are often less suitable for in vivo studies. A particular need for easily accessible yet thorough methods that enable the characterization of condensates across different experimental systems thus remains. To address this, we present PhaseMetrics, a semi-automated FIJI-based image analysis pipeline tailored for quantifying particle properties from microscopy data. Tested using the FG-domain of yeast nucleoporin Nup100, PhaseMetrics accurately assesses particle properties across diverse experimental setups, including particles formed in vitro in chemically defined buffers or in Xenopus egg extracts, and in cellular systems. Comparing the results with biochemical assays, we conclude that PhaseMetrics reliably detects changes induced by various conditions, including the presence of polyethylene glycol, 1,6-hexanediol, or a salt gradient, as well as the activity of the molecular chaperone DNAJB6b and the protein disaggregase Hsp104. Given the flexibility in its analysis parameters, the pipeline should also be applicable to other condensate-forming systems and we show it application for detecting TDP-43 particles. By enabling the accurate representation of the variability within the population and the detection of subtle changes at the single-condensate level, the method complements conventional biochemical assays. Combined, PhaseMetrics is an easily accessible, customizable pipeline that enables imaging-based quantitative assessment of biomolecular condensates in vitro and in cells, providing a valuable addition to the current toolbox.

Liquid–liquid phase separation in hepatocellular carcinoma

Liquid-liquid phase separation (LLPS) drives the formation of membraneless intracellular compartments within both cytoplasm and nucleus. These compartments can form distinct physicochemical environments, and in particular display different concentrations of proteins, RNA, and macromolecules compared to the surrounding cytosol. Recent studies have highlighted the significant role of aberrant LLPS in cancer development and progression, impacting many core processes such as oncogenic signalling pathways, transcriptional dysregulation, and genome instability. In hepatocellular carcinoma (HCC), aberrant formation of biomolecular condensates has been observed in a number of preclinical models, highlighting their significance as an emerging factor in understanding cancer biology and its molecular underpinnings. In this review, we summarize emerging evidence and recent advances in understanding the role of LLPS in HCC, with a particular focus on the regulation and dysregulation of cytoplasmic and nuclear condensates in cancer cells. We finally discuss how an emerging understanding of phase separation processes in HCC opens up new potential treatment avenues.

RNA-dependent RNA polymerase of predominant human norovirus forms liquid-liquid phase condensates as viral replication factories.

Many viral proteins form biomolecular condensates via liquid-liquid phase separation (LLPS) to support viral replication and evade host antiviral responses, and thus, they are potential targets for designing antivirals. In the case of nonenveloped positive-sense RNA viruses, forming such condensates for viral replication is unclear and less understood. Human noroviruses (HuNoVs) are positive-sense RNA viruses that cause epidemic and sporadic gastroenteritis worldwide. Here, we show that the RNA-dependent RNA polymerase (RdRp) of pandemic GII.4 HuNoV forms distinct condensates that exhibit all the signature properties of LLPS with sustained polymerase activity and the capability of recruiting components essential for viral replication. We show that such condensates are formed in HuNoV-infected human intestinal enteroid cultures and are the sites for genome replication. Our studies demonstrate the formation of phase-separated condensates as replication factories in a positive-sense RNA virus, which plausibly is an effective mechanism to dynamically isolate RdRp replicating the genomic RNA from interfering with the ribosomal translation of the same RNA.

Nucleo-cytoplasmic environment modulates spatiotemporal p53 phase separation.

Liquid-liquid phase separation of various transcription factors into biomolecular condensates plays an essential role in gene regulation. Here, using cellular models and in vitro studies, we show the spatiotemporal formation and material properties of p53 condensates that might dictate its function. In particular, p53 forms liquid-like condensates in the nucleus of cells, which can bind to DNA and perform transcriptional activity. However, cancer-associated mutations promote misfolding and partially rigidify the p53 condensates with impaired DNA binding ability. Irrespective of wild-type and mutant forms, the partitioning of p53 into cytoplasm leads to the condensate formation, which subsequently undergoes rapid solidification. In vitro studies show that abundant nuclear components such as RNA and nonspecific DNA promote multicomponent phase separation of the p53 core domain and maintain their liquid-like property, whereas specific DNA promotes its dissolution into tetrameric functional p53. This work provides mechanistic insights into how the life cycle and DNA binding properties of p53 might be regulated by phase separation.

RNA encodes physical information.

Most amino acids are encoded by multiple codons, making the genetic code degenerate. Synonymous mutations affect protein translation and folding, but their impact on RNA itself is often neglected. We developed a genetic algorithm that introduces synonymous mutations to control the diversity of structures sampled by an mRNA. The behavior of the designed mRNAs reveals a physical code layered in the genetic code. We find that mRNA conformational heterogeneity directs physical properties and functional outputs of RNA-protein complexes and biomolecular condensates. The role of structure and disorder of proteins in biomolecular condensates is well appreciated, but we find that RNA conformational heterogeneity is equally important. This feature of RNA enables both evolution and engineers to build cellular structures with specific material and responsive properties.

Delivery of Peptide Coacervates to Form Stable Interaction Hubs in Cells.

Cells contain membrane-bound and membraneless organelles that operate as spatially distinct biochemical niches. However, these subcellular reaction centers lose fidelity with aging and as a result of disease. A grand challenge for biomedicine is restoring or augmenting cellular functionalities. Although commonly tackled by gene replacement therapy, an excited new strategy is the delivery of protein-based materials that can directly interact with and alter biological networks inside a cell. In this study we sought to develop long-lasting materials capable of cellular uptake and incorporation, akin to an artificial organelle or intracellular interaction hub. Drawing inspiration from protein-based membranelles organelles, we developed a new delivery method to transplant micron size peptide-based compartments into living cells. We determined conditions to form large stable coacervates that are efficiently taken up by a variety of useful cell types and demonstrate their intracellular stability over time. We developed tools to enhance the extent and spatial organization of cargo loading into these coacervates, including co-assembly of nanobodies that selectively bind to targets of interest. Combining them together, we demonstrate successful targeting of GFP protein inside cells. These results represent an important first step toward the development of deliverable synthetic organelles that can be fabricated in vitro and taken up by cells for applications in cell engineering and regenerative medicine.

Discrimination between Purine and Pyrimidine-Rich RNA in Liquid-Liquid Phase-Separated Condensates with Cationic Peptides and the Effect of Artificial Crowding Agents.

Membraneless organelles, often referred to as condensates or coacervates, are liquid-liquid phase-separated systems formed between noncoding RNAs and intrinsically disordered proteins. While the importance of different amino acid residues in short peptide-based condensates has been investigated, the role of the individual nucleobases or the type of heterocyclic structures, the purine vs pyrimidine nucleobases, is less researched. The cell's crowded environment has been mimicked in vitro to demonstrate its ability to induce the formation of condensates, but more research in this area is required, especially with respect to RNA-facilitated phase separation and the properties of the crowding agent, poly(ethylene glycol) (PEG). Herein, we have shown that the nucleotide base sequence of RNA can greatly influence its propensity to undergo phase separation with cationic peptides, with the purine-only RNA decamer (AG)5 readily doing so while the pyrimidine-only (CU)5 does not. Furthermore, we show that the presence and size of a PEG macromolecular crowder affects both the ability to phase separate and the stability of coacervates formed, possibly due to co-condensation of PEG with the RNA and peptides. This work sheds light on the presence of low-complexity long purine- or pyrimidine-rich noncomplementary repeat (AG or CU) sequences in various noncoding RNAs found in biology.

PICNIC accurately predicts condensate-forming proteins regardless of their structural disorder across organisms

Biomolecular condensates are membraneless organelles that can concentrate hundreds of different proteins in cells to operate essential biological functions. However, accurate identification of their components remains challenging and biased towards proteins with high structural disorder content with focus on self-phase separating (driver) proteins. Here, we present a machine learning algorithm, PICNIC (Proteins Involved in CoNdensates In Cells) to classify proteins that localize to biomolecular condensates regardless of their role in condensate formation. PICNIC successfully predicts condensate members by learning amino acid patterns in the protein sequence and structure in addition to the intrinsic disorder. Extensive experimental validation of 24 positive predictions in cellulo shows an overall ~82% accuracy regardless of the structural disorder content of the tested proteins. While increasing disorder content is associated with organismal complexity, our analysis of 26 species reveals no correlation between predicted condensate proteome content and disorder content across organisms. Overall, we present a machine learning classifier to interrogate condensate components at whole-proteome levels across the tree of life.

Recent Progress in Modeling and Simulation of Biomolecular Crowding and Condensation Inside Cells.

Macromolecular crowding in the cellular cytoplasm can potentially impact diffusion rates of proteins, their intrinsic structural stability, binding of proteins to their corresponding partners as well as biomolecular organization and phase separation. While such intracellular crowding can have a large impact on biomolecular structure and function, the molecular mechanisms and driving forces that determine the effect of crowding on dynamics and conformations of macromolecules are so far not well understood. At a molecular level, computational methods can provide a unique lens to investigate the effect of macromolecular crowding on biomolecular behavior, providing us with a resolution that is challenging to reach with experimental techniques alone. In this review, we focus on the various physics-based and data-driven computational methods developed in the past few years to investigate macromolecular crowding and intracellular protein condensation. We review recent progress in modeling and simulation of biomolecular systems of varying sizes, ranging from single protein molecules to the entire cellular cytoplasm. We further discuss the effects of macromolecular crowding on different phenomena, such as diffusion, protein-ligand binding, and mechanical and viscoelastic properties, such as surface tension of condensates. Finally, we discuss some of the outstanding challenges that we anticipate the community addressing in the next few years in order to investigate biological phenomena in model cellular environments by reproducing in vivo conditions as accurately as possible.

Liquid-liquid separation in gut immunity.

Gut immunity is essential for maintaining intestinal health. Recent studies have identified that intracellular liquid-liquid phase separation (LLPS) may play a significant role in regulating gut immunity, however, the underlying mechanisms remain unclear. LLPS refers to droplet condensates formed through intracellular molecular interactions, which are crucial for the formation of membraneless organelles and biomolecules. LLPS can contribute to the formation of tight junctions between intestinal epithelial cells and influence the colonization of probiotics in the intestine, thereby protecting the intestinal immune system by maintaining the integrity of the intestinal barrier and the stability of the microbiota. Additionally, LLPS can affect the microclusters on the plasma membrane of T cells, resulting in increased density and reduced mobility, which in turn influences T cell functionality. The occurrence of intracellular LLPS is intricately associated with the initiation and progression of gut immunity. This review introduces the mechanism of LLPS in gut immunity and analyzes future research directions and potential applications of this phenomenon.

Endogenous oligomer formation underlies DVL2 condensates and promotes Wnt/β-catenin signaling

Activation of the Wnt/β-catenin pathway crucially depends on the polymerization of dishevelled 2 (DVL2) into biomolecular condensates. However, given the low affinity of known DVL2 self-interaction sites and its low cellular concentration, it is unclear how polymers can form. Here, we detect oligomeric DVL2 complexes at endogenous protein levels in human cell lines, using a biochemical ultracentrifugation assay. We identify a low-complexity region (LCR4) in the C-terminus whose deletion and fusion decreased and increased the complexes, respectively. Notably, LCR4-induced complexes correlated with the formation of microscopically visible multimeric condensates. Adjacent to LCR4, we mapped a conserved domain (CD2) promoting condensates only. Molecularly, LCR4 and CD2 mediated DVL2 self-interaction via aggregating residues and phenylalanine stickers, respectively. Point mutations inactivating these interaction sites impaired Wnt pathway activation by DVL2. Our study discovers DVL2 complexes with functional importance for Wnt/β-catenin signaling. Moreover, we provide evidence that DVL2 condensates form in two steps by pre-oligomerization via high-affinity interaction sites, such as LCR4, and subsequent condensation via low-affinity interaction sites, such as CD2.

Endogenous oligomer formation underlies DVL2 condensates and promotes Wnt/β-catenin signaling.

Activation of the Wnt/β-catenin pathway crucially depends on the polymerization of dishevelled 2 (DVL2) into biomolecular condensates. However, given the low affinity of known DVL2 self-interaction sites and its low cellular concentration, it is unclear how polymers can form. Here, we detect oligomeric DVL2 complexes at endogenous protein levels in human cell lines, using a biochemical ultracentrifugation assay. We identify a low-complexity region (LCR4) in the C-terminus whose deletion and fusion decreased and increased the complexes, respectively. Notably, LCR4-induced complexes correlated with the formation of microscopically visible multimeric condensates. Adjacent to LCR4, we mapped a conserved domain (CD2) promoting condensates only. Molecularly, LCR4 and CD2 mediated DVL2 self-interaction via aggregating residues and phenylalanine stickers, respectively. Point mutations inactivating these interaction sites impaired Wnt pathway activation by DVL2. Our study discovers DVL2 complexes with functional importance for Wnt/β-catenin signaling. Moreover, we provide evidence that DVL2 condensates form in two steps by pre-oligomerization via high-affinity interaction sites, such as LCR4, and subsequent condensation via low-affinity interaction sites, such as CD2.

The role of ER exit sites in maintaining P-body organization and integrity during Drosophila melanogaster oogenesis.

Processing bodies (P-bodies) are cytoplasmic membrane-less organelles which host multiple mRNA processing events. While the fundamental principles of P-body organization are beginning to be elucidated in vitro, a nuanced understanding of how their assembly is regulated in vivo remains elusive. Here, we investigate the potential link between ER exit sites and P-bodies in Drosophila melanogaster egg chambers. Employing a combination of live and super-resolution imaging, we find that P-bodies associated with ER exit sites are larger and less mobile than cytoplasmic P-bodies, indicating that they constitute a distinct class of P-bodies. Moreover, we demonstrate that altering the composition of ER exit sites has differential effects on core P-body proteins (Me31B, Cup, and Trailer Hitch), suggesting a potential role for ER exit sites in P-body organization. Furthermore, we show that in the absence of ER exit sites, P-body integrity is compromised and the stability and translational repression efficiency of the maternal mRNA, oskar, are reduced. Together, our data highlights the crucial role of ER exit sites in governing P-body organization.

Targeting the interplay between biomolecular condensates and regulatory RNAs in cancer

Targeting the interplay between biomolecular condensates and regulatory RNAs in cancer