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Membraneless organelles (MLOs) are detected in cells as dots of mesoscopic size. By undergoing phase separation into a liquid-like or gel-like phase, MLOs contribute to intracellular compartmentalization of specific biological functions. In eukaryotes, dozens of MLOs have been identified, including the nucleolus, Cajal bodies, nuclear speckles, paraspeckles, promyelocytic leukaemia protein (PML) nuclear bodies, nuclear stress bodies, processing bodies (P bodies) and stress granules. MLOs contain specific proteins, of which many possess intrinsically disordered regions (IDRs), and nucleic acids, mainly RNA. Many MLOs contribute to gene regulation by different mechanisms. Through sequestration of specific factors, MLOs promote biochemical reactions by simultaneously concentrating substrates and enzymes, and/or suppressing the activity of the sequestered factors elsewhere in the cell. Other MLOs construct inter-chromosomal hubs by associating with multiple loci, thereby contributing to the biogenesis of macromolecular machineries essential for gene expression, such as ribosomes and spliceosomes. The organization of many MLOs includes layers, which might have different biophysical properties and functions. MLOs are functionally interconnected and are involved in various diseases, prompting the emergence of therapeutics targeting them. In this Review, we introduce MLOs that are relevant to gene regulation and discuss their assembly, internal structure, gene-regulatory roles in transcription, RNA processing and translation, particularly in stress conditions, and their disease relevance.Membraneless organelles (MLOs) contribute to intracellular compartmentalization and to various cellular processes. This Review provides a guide to MLOs involved in gene regulation in eukaryotes, discussing their assembly, structure, roles in transcription, RNA processing and translation — particularly in stress conditions — and their disease relevance.

Key Points In addition to canonical membrane-bound organelles, eukaryotic cells contain numerous membraneless compartments, or biomolecular condensates, that concentrate specific collections of proteins and nucleic acids. Biomolecular condensates behave as phase-separated liquids and are enriched in multivalent molecules. Theoretical concepts from polymer and physical chemistry regarding the behaviour of multivalent molecules provide a mechanistic framework that can explain a wide range of cellular behaviours exhibited by biomolecular condensates, including plausible mechanisms by which their assembly, composition, and biochemical and cellular functions can be regulated. In addition to membrane-bound organelles, eukaryotic cells feature various membraneless compartments, including the centrosome, the nucleolus and various granules. Many of these compartments form through liquid–liquid phase separation, and the principles, mechanisms and regulation of their assembly as well as their cellular functions are now beginning to emerge. Biomolecular condensates are micron-scale compartments in eukaryotic cells that lack surrounding membranes but function to concentrate proteins and nucleic acids. These condensates are involved in diverse processes, including RNA metabolism, ribosome biogenesis, the DNA damage response and signal transduction. Recent studies have shown that liquid–liquid phase separation driven by multivalent macromolecular interactions is an important organizing principle for biomolecular condensates. With this physical framework, it is now possible to explain how the assembly, composition, physical properties and biochemical and cellular functions of these important structures are regulated.

The distinct spatial architecture of the apical actin cables (or actin cap) facilitates rapid biophysical signaling between extracellular mechanical stimuli and intracellular responses, including nuclear shaping, cytoskeletal remodeling, and the mechanotransduction of external forces into biochemical signals. These functions are abrogated in lamin A/C-deficient mouse embryonic fibroblasts that recapitulate the defective nuclear organization of laminopathies, featuring disruption of the actin cap. However, how nuclear lamin A/C mediates the ability of the actin cap to regulate nuclear morphology remains unclear. Here, we show that lamin A/C expressing cells can form an actin cap to resist nuclear deformation in response to physiological mechanical stresses. This study reveals how the nuclear lamin A/C-mediated formation of the perinuclear apical actin cables protects the nuclear structural integrity from extracellular physical disturbances. Our findings highlight the role of the physical interactions between the cytoskeletal network and the nucleus in cellular mechanical homeostasis. An actin cap protects the morphology of the nucleus during cellular mechanical stress. Here, the authors show that the nuclear lamina protein lamin A/C mediates the formation of the actin cap in response to stress, and model the distribution of forces in the presence and absence of the actin cap.

Biomolecular condensates play a key role in organizing cellular reactions by concentrating a specific set of biomolecules. However, whether condensate formation is accompanied by an increase in the total mass concentration within condensates or by the demixing of already highly crowded intracellular components remains elusive. Here, using refractive index imaging, we quantify the mass density of several condensates, including nucleoli, heterochromatin, nuclear speckles, and stress granules. Surprisingly, the latter two condensates exhibit low densities with a total mass concentration similar to the surrounding cyto- or nucleoplasm. Low-density condensates display higher permeability to cellular protein probes. We find that RNA tunes the biomolecular density of condensates. Moreover, intracellular structures such as mitochondria heavily influence the way phase separation proceeds, impacting the localization, morphology, and growth of condensates. These findings favor a model where segregative phase separation driven by non-associative or repulsive molecular interactions together with RNA-mediated selective association of specific components can give rise to low-density condensates in the crowded cellular environment. The cell interior is organized by diverse membrane-less condensates. Here, the authors reveal that the densities of certain condensates are surprisingly low, similar to the surrounding protoplasm and driven by cellular RNA as well as the crowded milieu.

As the home of cellular genetic information, the nucleus has a critical role in determining cell fate and function in response to various signals and stimuli. In addition to biochemical inputs, the nucleus is constantly exposed to intrinsic and extrinsic mechanical forces that trigger dynamic changes in nuclear structure and morphology. Emerging data suggest that the physical deformation of the nucleus modulates many cellular and nuclear functions. These functions have long been considered to be downstream of cytoplasmic signalling pathways and dictated by gene expression. In this Review, we discuss an emerging perspective on the mechanoregulation of the nucleus that considers the physical connections from chromatin to nuclear lamina and cytoskeletal filaments as a single mechanical unit. We describe key mechanisms of nuclear deformations in time and space and provide a critical review of the structural and functional adaptive responses of the nucleus to deformations. We then consider the contribution of nuclear deformations to the regulation of important cellular functions, including muscle contraction, cell migration and human disease pathogenesis. Collectively, these emerging insights shed new light on the dynamics of nuclear deformations and their roles in cellular mechanobiology.Nuclei are subject to various deformations, being pulled, pushed, squeezed and stretched by a plethora of intracellular and extracellular forces. Recent work is unravelling how nuclei sense and respond to these deformations, including with changes in genome organization and function, cell signalling, and cell mechanics.

The nucleolus is the most prominent nuclear body and serves a fundamentally important biological role as a site of ribonucleoprotein particle assembly, primarily dedicated to ribosome biogenesis. Despite being one of the first intracellular structures visualized historically, the biophysical rules governing its assembly and function are only starting to become clear. Recent studies have provided increasing support for the concept that the nucleolus represents a multilayered biomolecular condensate, whose formation by liquid–liquid phase separation (LLPS) facilitates the initial steps of ribosome biogenesis and other functions. Here, we review these biophysical insights in the context of the molecular and cell biology of the nucleolus. We discuss how nucleolar function is linked to its organization as a multiphase condensate and how dysregulation of this organization could provide insights into still poorly understood aspects of nucleolus-associated diseases, including cancer, ribosomopathies and neurodegeneration as well as ageing. We suggest that the LLPS model provides the starting point for a unifying quantitative framework for the assembly, structural maintenance and function of the nucleolus, with implications for gene regulation and ribonucleoprotein particle assembly throughout the nucleus. The LLPS concept is also likely useful in designing new therapeutic strategies to target nucleolar dysfunction.The nucleolus is a membraneless organelle involved in ribonucleoprotein assembly, including ribosome biogenesis. Recent evidence indicates that the nucleolus is a biomolecular condensate that forms via liquid–liquid phase separation (LLPS), and insights from studies within the LLPS framework are increasing our understanding of the relationship between nucleolar structure and function.

Damaged DNA shows increased mobility, which can promote interactions with repair-conducive nuclear pore complexes (NPCs). This apparently random mobility is paradoxically abrogated upon disruption of microtubules or kinesins, factors that typically cooperate to mediate the directional movement of macromolecules. Here, we resolve this paradox by uncovering DNA damage-inducible intranuclear microtubule filaments (DIMs) that mobilize damaged DNA and promote repair. Upon DNA damage, relief of centromeric constraint induces DIMs that cooperate with the Rad9 DNA damage response mediator and Kar3 kinesin motor to capture DNA lesions, which then linearly move along dynamic DIMs. Decreasing and hyper-inducing DIMs respectively abrogates and hyper-activates repair. Accounting for DIM dynamics across cell populations by measuring directional changes of damaged DNA reveals that it exhibits increased non-linear directional behavior in nuclear space. Abrogation of DIM-dependent processes or repair-promoting factors decreases directional behavior. Thus, inducible and dynamic nuclear microtubule filaments directionally mobilize damaged DNA and promote repair. Following DNA damage, different processes come to action to aid repair. The authors here find that microtubule filaments within the cell nucleus capture and non-randomly mobilize damaged chromatin to mediate DNA repair.

Phosphorylation or DNA binding promotes the physical partitioning of HP1α out of a soluble aqueous phase into droplets, suggesting that the repressive action of heterochromatin may in part be mediated by the phase separation of HP1. HP1α forms reversible droplets The gene-silencing action of heterochromatin is thought to arise from the spread of proteins such as HP1 that compact the underlying chromatin and recruit repressors. Two papers in this issue demonstrate that HP1α has the ability to form phase-separated droplets. Gary Karpen and colleagues show that HP1α can nucleate into foci that display liquid properties during the early stages of heterochromatin domain formation in Drosophila embryos. Geeta Narlikar and colleagues demonstrate that human HP1α protein also forms phase-separated droplets. Phosphorylation or DNA binding promotes the physical partitioning of HP1α out of the soluble aqueous phase into droplets. These related findings suggest that the repressive action of heterochromatin may be in part mediated by the phase separation of HP1, with the droplets being initiated or dissolved by various ligands depending on nuclear context. Gene silencing by heterochromatin is proposed to occur in part as a result of the ability of heterochromatin protein 1 (HP1) proteins to spread across large regions of the genome, compact the underlying chromatin and recruit diverse ligands 1 , 2 , 3 . Here we identify a new property of the human HP1α protein: the ability to form phase-separated droplets. While unmodified HP1α is soluble, either phosphorylation of its N-terminal extension or DNA binding promotes the formation of phase-separated droplets. Phosphorylation-driven phase separation can be promoted or reversed by specific HP1α ligands. Known components of heterochromatin such as nucleosomes and DNA preferentially partition into the HP1α droplets, but molecules such as the transcription factor TFIIB show no preference. Using a single-molecule DNA curtain assay, we find that both unmodified and phosphorylated HP1α induce rapid compaction of DNA strands into puncta, although with different characteristics 4 . We show by direct protein delivery into mammalian cells that an HP1α mutant incapable of phase separation in vitro forms smaller and fewer nuclear puncta than phosphorylated HP1α. These findings suggest that heterochromatin-mediated gene silencing may occur in part through sequestration of compacted chromatin in phase-separated HP1 droplets, which are dissolved or formed by specific ligands on the basis of nuclear context.

Biomolecular condensation partitions cellular contents and has important roles in stress responses, maintaining homeostasis, development and disease. Many nuclear and cytoplasmic condensates are rich in RNA and RNA-binding proteins (RBPs), which undergo liquid–liquid phase separation (LLPS). Whereas the role of RBPs in condensates has been well studied, less attention has been paid to the contribution of RNA to LLPS. In this Review, we discuss the role of RNA in biomolecular condensation and highlight considerations for designing condensate reconstitution experiments. We focus on RNA properties such as composition, length, structure, modifications and expression level. These properties can modulate the biophysical features of native condensates, including their size, shape, viscosity, liquidity, surface tension and composition. We also discuss the role of RNA–protein condensates in development, disease and homeostasis, emphasizing how their properties and function can be determined by RNA. Finally, we discuss the multifaceted cellular functions of biomolecular condensates, including cell compartmentalization through RNA transport and localization, supporting catalytic processes, storage and inheritance of specific molecules, and buffering noise and responding to stress.Recent studies have highlighted the contribution of RNA to cellular liquid–liquid phase separation and condensate formation. RNA features modulate the composition and biophysical properties of RNA–protein condensates, which have various cellular functions, including RNA transport and localization, supporting catalytic processes and responding to stress.

The epigenome involves a complex set of cellular processes governing genomic activity. Dissecting this complexity necessitates the development of tools capable of specifically manipulating these processes. The repurposing of prokaryotic CRISPR systems has allowed for the development of diverse technologies for epigenome engineering. Here, we review the state of currently achievable epigenetic manipulations along with corresponding applications. With future optimization, CRISPR-based epigenomic editing stands as a set of powerful tools for understanding and controlling biological function. Qi and colleagues review CRISPR-based epigenome engineering technologies to modulate histone and DNA modifications and to perturb DNA and RNA regulatory elements and chromatin organization.