TDP-43 nuclear condensation and neurodegenerative proteinopathies.

RNA-binding proteins (RBPs) can undergo phase separation and form condensates, processes that, in turn, can be critical for their functionality. In a recent study, Huang, Ellis, and colleagues show that cellular stress can trigger transient alterations in nuclear TAR DNA-binding protein 43 (TDP-43), leading to changes crucial for proper neuronal function. These findings have implications for understanding neurological TDP-43 proteinopathies.

MLOsMetaDB, a meta-database to centralize the information on liquid-liquid phase separation proteins and membraneless organelles.

Over the past few years, there has been a focus on proteins that create separate liquid phases in the intracellular liquid environment, known as membraneless organelles (MLOs). These organelles allow for the spatiotemporal associations of macromolecules that dynamically exchange within the cellular milieu. They provide a form of compartmentalization crucial for organizing key functions in many cells. Metabolic processes and signaling pathways in both the cytoplasm and nucleus are among the functions performed by MLOs, which are facilitated by diverse combinations of proteins and nucleic acids. However, disruptions in these liquid-liquid phase separation processes (LLPS) may lead to several diseases, such as neurodegenerative disorders and cancer, among others. To foster the study of this process and MLO function, we present MLOsMetaDB (http://mlos.leloir.org.ar), a comprehensive resource of information on MLO- and LLPS-related proteins. Our database integrates and centralizes available information for every protein involved in MLOs, which is otherwise disseminated across a plethora of different databases. Our manuscript outlines the development and features of MLOsMetaDB, which provides an interactive and user-friendly environment with modern biological visualizations and easy and quick access to proteins based on LLPS role, MLO location, and organisms. In addition, it offers an advanced search for making complex queries to generate customized information. Furthermore, MLOsMetaDB provides evolutionary information by collecting the orthologs of every protein in the same database. Overall, MLOsMetaDB is a valuable resource as a starting point for researchers studying the many processes driven by LLPS proteins and membraneless organelles.

Protein nanocondensates: the next frontier.

Over the past decade, myriads of studies have highlighted the central role of protein condensation in subcellular compartmentalization and spatiotemporal organization of biological processes. Conceptually, protein condensation stands at the highest level in protein structure hierarchy, accounting for the assembly of bodies ranging from thousands to billions of molecules and for densities ranging from dense liquids to solid materials. In size, protein condensates range from nanocondensates of hundreds of nanometers (mesoscopic clusters) to phase-separated micron-sized condensates. In this review, we focus on protein nanocondensation, a process that can occur in subsaturated solutions and can nucleate dense liquid phases, crystals, amorphous aggregates, and fibers. We discuss the nanocondensation of proteins in the light of general physical principles and examine the biophysical properties of several outstanding examples of nanocondensation. We conclude that protein nanocondensation cannot be fully explained by the conceptual framework of micron-scale biomolecular condensation. The evolution of nanocondensates through changes in density and order is currently under intense investigation, and this should lead to the development of a general theoretical framework, capable of encompassing the full range of sizes and densities found in protein condensates.

Integrative Analysis of the Ethanol Tolerance of Saccharomyces cerevisiae.

Ethanol (EtOH) alters many cellular processes in yeast. An integrated view of different EtOH-tolerant phenotypes and their long noncoding RNAs (lncRNAs) is not yet available. Here, large-scale data integration showed the core EtOH-responsive pathways, lncRNAs, and triggers of higher (HT) and lower (LT) EtOH-tolerant phenotypes. LncRNAs act in a strain-specific manner in the EtOH stress response. Network and omics analyses revealed that cells prepare for stress relief by favoring activation of life-essential systems. Therefore, longevity, peroxisomal, energy, lipid, and RNA/protein metabolisms are the core processes that drive EtOH tolerance. By integrating omics, network analysis, and several other experiments, we showed how the HT and LT phenotypes may arise: (1) the divergence occurs after cell signaling reaches the longevity and peroxisomal pathways, with CTA1 and ROS playing key roles; (2) signals reaching essential ribosomal and RNA pathways via SUI2 enhance the divergence; (3) specific lipid metabolism pathways also act on phenotype-specific profiles; (4) HTs take greater advantage of degradation and membraneless structures to cope with EtOH stress; and (5) our EtOH stress-buffering model suggests that diauxic shift drives EtOH buffering through an energy burst, mainly in HTs. Finally, critical genes, pathways, and the first models including lncRNAs to describe nuances of EtOH tolerance are reported here.

Protein conformation and biomolecular condensates.

Protein conformation and cell compartmentalization are fundamental concepts and subjects of vast scientific endeavors. In the last two decades, we have witnessed exciting advances that unveiled the conjunction of these concepts. An avalanche of studies highlighted the central role of biomolecular condensates in membraneless subcellular compartmentalization that permits the spatiotemporal organization and regulation of myriads of simultaneous biochemical reactions and macromolecular interactions. These studies have also shown that biomolecular condensation, driven by multivalent intermolecular interactions, is mediated by order-disorder transitions of protein conformation and by protein domain architecture. Conceptually, protein condensation is a distinct level in protein conformational landscape in which collective folding of large collections of molecules takes place. Biomolecular condensates arise by the physical process of phase separation and comprise a variety of bodies ranging from membraneless organelles to liquid condensates to solid-like conglomerates, spanning lengths from mesoscopic clusters (nanometers) to micrometer-sized objects. In this review, we summarize and discuss recent work on the assembly, composition, conformation, material properties, thermodynamics, regulation, and functions of these bodies. We also review the conceptual framework for future studies on the conformational dynamics of condensed proteins in the regulation of cellular processes.

The perinuclear region concentrates disordered proteins with predicted phase separation distributed in a 3D network of cytoskeletal filaments and organelles.

Membraneless organelles have emerged during the evolution of eukaryotic cells as intracellular domains in which multiple proteins organize into complex structures to perform specialized functions without the need of a lipid bilayer compartment. Here we describe the perinuclear space of eukaryotic cells as a highly organized network of cytoskeletal filaments that facilitates assembly of biomolecular condensates. Using bioinformatic analyses, we show that the perinuclear proteome is enriched in intrinsic disorder with several proteins predicted to undergo liquid-liquid phase separation. We also analyze immunofluorescence and transmission electron microscopy images showing the association between the nucleus and other organelles, such as mitochondria and lysosomes, or the labeling of specific proteins within the perinuclear region of cells. Altogether our data support the existence of a perinuclear dense sub-micron region formed by a well-organized three-dimensional network of structural and signaling proteins, including several proteins containing intrinsically disordered regions with phase behavior. This network of filamentous cytoskeletal proteins extends a few micrometers from the nucleus, contributes to local crowding, and organizes the movement of molecular complexes within the perinuclear space. Our findings take a key step towards understanding how membraneless regions within eukaryotic cells can serve as hubs for biomolecular condensates assembly, in particular the perinuclear space. Finally, evaluation of the disease context of the perinuclear proteins revealed that alterations in their expression can lead to several pathological conditions, and neurological disorders and cancer are among the most frequent.

Insight into membraneless organelles and their associated proteins: Drivers, Clients and Regulators.

In recent years, attention has been devoted to proteins forming immiscible liquid phases within the liquid intracellular medium, commonly referred to as membraneless organelles (MLO). These organelles enable the spatiotemporal associations of cellular components that exchange dynamically with the cellular milieu. The dysregulation of these liquid-liquid phase separation processes (LLPS) may cause various diseases including neurodegenerative pathologies and cancer, among others. Until very recently, databases containing information on proteins forming MLOs, as well as tools and resources facilitating their analysis, were missing. This has recently changed with the publication of 4 databases that focus on different types of experiments, sets of proteins, inclusion criteria, and levels of annotation or curation. In this study we integrate and analyze the information across these databases, complement their records, and produce a consolidated set of proteins that enables the investigation of the LLPS phenomenon. To gain insight into the features that characterize different types of MLOs and the roles of their associated proteins, they were grouped into categories: High Confidence MLO associated (including Drivers and reviewed proteins), Potential Clients and Regulators, according to their annotated functions. We show that none of the databases taken alone covers the data sufficiently to enable meaningful analysis, validating our integration effort as essential for gaining better understanding of phase separation and laying the foundations for the discovery of new proteins potentially involved in this important cellular process. Lastly, we developed a server, enabling customized selections of different sets of proteins based on MLO location, database, disorder content, among other attributes (https://forti.shinyapps.io/mlos/).

Unraveling the molecular interactions involved in phase separation of glucocorticoid receptor.

BACKGROUND: Functional compartmentalization has emerged as an important factor modulating the kinetics and specificity of biochemical reactions in the nucleus, including those involved in transcriptional regulation. The glucocorticoid receptor (GR) is a ligand-activated transcription factor that translocates to the nucleus upon hormone stimulation and distributes between the nucleoplasm and membraneless compartments named nuclear foci. While a liquid-liquid phase separation process has been recently proposed to drive the formation of many nuclear compartments, the mechanisms governing the heterogeneous organization of GR in the nucleus and the functional relevance of foci formation remain elusive. RESULTS: We dissected some of the molecular interactions involved in the formation of GR condensates and analyzed the GR structural determinants relevant to this process. We show that GR foci present properties consistent with those expected for biomolecular condensates formed by a liquid-liquid phase separation process in living human cells. Their formation requires an initial interaction of GR with certain chromatin regions at specific locations within the nucleus. Surprisingly, the intrinsically disordered region of GR is not essential for condensate formation, in contrast to many nuclear proteins that require disordered regions to phase separate, while the ligand-binding domain seems essential for that process. We finally show that GR condensates include Mediator, a protein complex involved in transcription regulation. CONCLUSIONS: We show that GR foci have properties of liquid condensates and propose that active GR molecules interact with chromatin and recruit multivalent cofactors whose interactions with additional molecules lead to the formation of a focus. The biological relevance of the interactions occurring in GR condensates supports their involvement in transcription regulation.

Processing Bodies Oscillate in Neuro 2A Cells.

Circadian rhythms are biological variables that oscillate with periods close to 24 h that are generated internally by biological clocks. Depending on the tissue/cell type, about 5-20% of genes are expressed rhythmically. Unexpectedly, the correlation between the oscillations of messengers and the proteins they encode is low. We hypothesize that these discrepancies could be because in certain phases of the circadian cycle some messengers could be translationally silenced and stored. Processing bodies (PBs) are membraneless organelles formed by ribonucleoprotein aggregates located in the cytoplasm. They contain silenced messengers and factors involved in mRNA processing. A previous work showed that the number of cells containing these mRNA granules varies when comparing two time-points in U2OS cell cultures and that these differences disappear when an essential clock gene is silenced. Here we evaluate whether PBs oscillate in Neuro2A cells. We analyzed in cell cultures synchronized with dexamethasone the variations in the number, the signal intensity of the markers used (GE-1/HEDLS and DDX6), and the area of PBs between 8 and 68 h post-synchronization. All three parameters oscillated with periods compatible with a circadian regulated process. The most robust rhythm was the number of PBs. These rhythms could be generated by oscillations in proteins that have been involved in the nucleation of these foci such as LSM1, TTP, and BRF1. The described phenomenon would allow to explain the differences observed in the temporal profiles of some messengers and their proteins and to understand how circadian clocks can control post-transcriptionally cellular functions.

Strategies for Success. Viral Infections and Membraneless Organelles.

Regulation of RNA homeostasis or "RNAstasis" is a central step in eukaryotic gene expression. From transcription to decay, cellular messenger RNAs (mRNAs) associate with specific proteins in order to regulate their entire cycle, including mRNA localization, translation and degradation, among others. The best characterized of such RNA-protein complexes, today named membraneless organelles, are Stress Granules (SGs) and Processing Bodies (PBs) which are involved in RNA storage and RNA decay/storage, respectively. Given that SGs and PBs are generally associated with repression of gene expression, viruses have evolved different mechanisms to counteract their assembly or to use them in their favor to successfully replicate within the host environment. In this review we summarize the current knowledge about the viral regulation of SGs and PBs, which could be a potential novel target for the development of broad-spectrum antiviral therapies.