Nascent Pre-rRNA Sorting via Phase Separation Drives the Assembly of Dense Fibrillar Components in the Human Nucleolus.

Fibrillar centers (FCs) and dense fibrillar components (DFCs) are essential morphologically distinct sub-regions of mammalian cell nucleoli for rDNA transcription and pre-rRNA processing. Here, we report that a human nucleolus consists of several dozen FC/DFC units, each containing 2-3 transcriptionally active rDNAs at the FC/DFC border. Pre-rRNA processing factors, such as fibrillarin (FBL), form 18-24 clusters that further assemble into the DFC surrounding the FC. Mechanistically, the 5' end of nascent 47S pre-rRNA binds co-transcriptionally to the RNA-binding domain of FBL. FBL diffuses to the DFC, where local self-association via its glycine- and arginine-rich (GAR) domain forms phase-separated clusters to immobilize FBL-interacting pre-rRNA, thus promoting directional traffic of nascent pre-rRNA while facilitating pre-rRNA processing and DFC formation. These results unveil FC/DFC ultrastructures in nucleoli and suggest a conceptual framework for considering nascent RNA sorting using multivalent interactions of their binding proteins.

The glycine arginine-rich domain of the RNA-binding protein nucleolin regulates its subcellular localization.

Nucleolin is a multifunctional RNA Binding Protein (RBP) with diverse subcellular localizations, including the nucleolus in all eukaryotic cells, the plasma membrane in tumor cells, and the axon in neurons. Here we show that the glycine arginine rich (GAR) domain of nucleolin drives subcellular localization via protein-protein interactions with a kinesin light chain. In addition, GAR sequences mediate plasma membrane interactions of nucleolin. Both these modalities are in addition to the already reported involvement of the GAR domain in liquid-liquid phase separation in the nucleolus. Nucleolin transport to axons requires the GAR domain, and heterozygous GAR deletion mice reveal reduced axonal localization of nucleolin cargo mRNAs and enhanced sensory neuron growth. Thus, the GAR domain governs axonal transport of a growth controlling RNA-RBP complex in neurons, and is a versatile localization determinant for different subcellular compartments. Localization determination by GAR domains may explain why GAR mutants in diverse RBPs are associated with neurodegenerative disease.

3D-CLEM Reveals that a Major Portion of Mitotic Chromosomes Is Not Chromatin.

Recent studies have revealed the importance of Ki-67 and the chromosome periphery in chromosome structure and segregation, but little is known about this elusive chromosome compartment. Here we used correlative light and serial block-face scanning electron microscopy, which we term 3D-CLEM, to model the entire mitotic chromosome complement at ultra-structural resolution. Prophase chromosomes exhibit a highly irregular surface appearance with a volume smaller than metaphase chromosomes. This may be because of the absence of the periphery, which associates with chromosomes only after nucleolar disassembly later in prophase. Indeed, the nucleolar volume almost entirely accounts for the extra volume found in metaphase chromosomes. Analysis of wild-type and Ki-67-depleted chromosomes reveals that the periphery comprises 30%-47% of the entire chromosome volume and more than 33% of the protein mass of isolated mitotic chromosomes determined by quantitative proteomics. Thus, chromatin makes up a surprisingly small percentage of the total mass of metaphase chromosomes.

Acetylation-mediated remodeling of the nucleolus regulates cellular acetyl-CoA responses.

The metabolite acetyl-coenzyme A (acetyl-CoA) serves as an essential element for a wide range of cellular functions including adenosine triphosphate (ATP) production, lipid synthesis, and protein acetylation. Intracellular acetyl-CoA concentrations are associated with nutrient availability, but the mechanisms by which a cell responds to fluctuations in acetyl-CoA levels remain elusive. Here, we generate a cell system to selectively manipulate the nucleo-cytoplasmic levels of acetyl-CoA using clustered regularly interspaced short palindromic repeat (CRISPR)-mediated gene editing and acetate supplementation of the culture media. Using this system and quantitative omics analyses, we demonstrate that acetyl-CoA depletion alters the integrity of the nucleolus, impairing ribosomal RNA synthesis and evoking the ribosomal protein-dependent activation of p53. This nucleolar remodeling appears to be mediated through the class IIa histone deacetylases (HDACs). Our findings highlight acetylation-mediated control of the nucleolus as an important hub linking acetyl-CoA fluctuations to cellular stress responses.

Ribosomal DNA and the Nucleolus as Keystones of Nuclear Architecture, Organization, and Function.

The multicopy ribosomal DNA (rDNA) array gives origin to the nucleolus, a large nonmembrane-bound organelle that occupies a substantial volume within the cell nucleus. The rDNA/nucleolus has emerged as a coordinating hub in which seemingly disparate cellular functions converge, and from which a variety of cellular and organismal phenotypes emerge. However, the role of the nucleolus as a determinant and organizer of nuclear architecture and other epigenetic states of the genome is not well understood. We discuss the role of rDNA and the nucleolus in nuclear organization and function - from nucleolus-associated domains (NADs) to the regulation of imprinted loci and X chromosome inactivation, as well as rDNA contact maps that anchor and position the rDNA relative to the rest of the genome. The influence of the nucleolus on nuclear organization undoubtedly modulates diverse biological processes from metabolism to cell proliferation, genome-wide gene expression, maintenance of epigenetic states, and aging.

Two contrasting classes of nucleolus-associated domains in mouse fibroblast heterochromatin.

In interphase eukaryotic cells, almost all heterochromatin is located adjacent to the nucleolus or to the nuclear lamina, thus defining nucleolus-associated domains (NADs) and lamina-associated domains (LADs), respectively. Here, we determined the first genome-scale map of murine NADs in mouse embryonic fibroblasts (MEFs) via deep sequencing of chromatin associated with purified nucleoli. We developed a Bioconductor package called NADfinder and demonstrated that it identifies NADs more accurately than other peak-calling tools, owing to its critical feature of chromosome-level local baseline correction. We detected two distinct classes of NADs. Type I NADs associate frequently with both the nucleolar periphery and the nuclear lamina, and generally display characteristics of constitutive heterochromatin, including late DNA replication, enrichment of H3K9me3, and little gene expression. In contrast, Type II NADs associate with nucleoli but do not overlap with LADs. Type II NADs tend to replicate earlier, display greater gene expression, and are more often enriched in H3K27me3 than Type I NADs. The nucleolar associations of both classes of NADs were confirmed via DNA-FISH, which also detected Type I but not Type II probes enriched at the nuclear lamina. Type II NADs are enriched in distinct gene classes, including factors important for differentiation and development. In keeping with this, we observed that a Type II NAD is developmentally regulated, and present in MEFs but not in undifferentiated embryonic stem (ES) cells.

Single-shot quantitative phase imaging as an extension of differential interference contrast microscopy.

Various studies have been conducted to obtain quantitative phase information based on differential interference contrast (DIC) microscopy. As one such attempt, we propose in this study a single-shot quantitative phase imaging (QPI) method by combining two developments. First, an add-on optical system to a commercialized DIC microscope was developed to perform quantitative phase gradient imaging (QPGI) with single image acquisition using a polarization camera. Second, an algorithm was formulated to reconstitute QPI from the obtained QPGI by reducing linear artifacts, which arise in simply integrated QPGI images. To demonstrate the applicability of the developed system in cell biology, the system was used to measure various cell lines and compared with fluorescence microscopy images of the same field of view. Consistent with previous studies, nucleoli and lipid droplets can be imaged by the system with greater optical path lengths (OPL). The results also implied that combining fluorescence microscopy and the developed system might be more informative for cell biology research than using these methods individually. Exploiting the single-shot performance of the developed system, time-lapse imaging was also conducted to visualize the dynamics of intracellular granules in monocyte-/macrophage-like cells. Our proposed approach may accelerate the implementation of QPI in standard biomedical laboratories.

MeCP2 Levels Regulate the 3D Structure of Heterochromatic Foci in Mouse Neurons.

Methyl-CpG binding protein 2 (MeCP2) is a nuclear protein critical for normal brain function, and both depletion and overexpression of MeCP2 lead to severe neurodevelopmental disease, Rett syndrome (RTT) and MECP2 multiplication disorder, respectively. However, the molecular mechanism by which abnormal MeCP2 dosage causes neuronal dysfunction remains unclear. As MeCP2 expression is nearly equivalent to that of core histones and because it binds DNA throughout the genome, one possible function of MeCP2 is to regulate the 3D structure of chromatin. Here, to examine whether and how MeCP2 levels impact chromatin structure, we used high-resolution confocal and electron microscopy and examined heterochromatic foci of neurons in mice. Using models of RTT and MECP2 triplication syndrome, we found that the heterochromatin structure was significantly affected by the alteration in MeCP2 levels. Analysis of mice expressing either MeCP2-R270X or MeCP2-G273X, which have nonsense mutations in the upstream and downstream regions of the AT-hook 2 domain, respectively, showed that the magnitude of heterochromatin changes was tightly correlated with the phenotypic severity. Postnatal alteration in MeCP2 levels also induced significant changes in the heterochromatin structure, which underscored importance of correct MeCP2 dosage in mature neurons. Finally, functional analysis of MeCP2-overexpressing mice showed that the behavioral and transcriptomic alterations in these mice correlated significantly with the MeCP2 levels and occurred in parallel with the heterochromatin changes. Taken together, our findings demonstrate the essential role of MeCP2 in regulating the 3D structure of neuronal chromatin, which may serve as a potential mechanism that drives pathogenesis of MeCP2-related disorders.SIGNIFICANCE STATEMENT Neuronal function is critically dependent on methyl-CpG binding protein 2 (MeCP2), a nuclear protein abundantly expressed in neurons. The importance of MeCP2 is underscored by the severe childhood neurologic disorders, Rett syndrome (RTT) and MECP2 multiplication disorders, which are caused by depletion and overabundance of MeCP2, respectively. To clarify the molecular function of MeCP2 and to understand the pathogenesis of MECP2-related disorders, we performed detailed structural analyses of neuronal nuclei by using mouse models and high-resolution microscopy. We show that the level of MeCP2 critically regulates 3D structure of heterochromatic foci, and this is mediated in part by the AT-hook 2 domain of MeCP2. Our results demonstrate that one primary function of MeCP2 is to regulate chromatin structure.

G-patch domain-containing protein 4 localizes to both the nucleoli and Cajal bodies and regulates cell growth and nucleolar structure.

Ribosome formation occurs in the nucleolus through interaction with various trans-acting factors. Therefore, hundreds of nucleolar proteins have a function in ribosome formation, although the precise function of each nucleolar protein in ribosome formation is largely unclear. We have previously identified an uncharacterized protein, G-patch domain-containing protein 4 (GPATCH4 or G4), as a component of the pre-ribosomes purified with either nucleolin (NCL) or NPM1. In this present study, we sought to clarify the localization and function of G4. We identified that G4 localizes to both the nucleolus and the Cajal body. Although knockdown of G4 did not have a significant effect on pre-ribosomal RNA processing, cell growth did decrease. Interestingly, G4 knockdown also decreased the number of fibrillar center and dense fibrillar component regions inside the nucleolus. This data has identified G4 as a novel nucleolar protein involved in the regulation of cell growth and nucleolar structure.

RNA helicase, DDX27 regulates skeletal muscle growth and regeneration by modulation of translational processes.

Gene expression in a tissue-specific context depends on the combined efforts of epigenetic, transcriptional and post-transcriptional processes that lead to the production of specific proteins that are important determinants of cellular identity. Ribosomes are a central component of the protein biosynthesis machinery in cells; however, their regulatory roles in the translational control of gene expression in skeletal muscle remain to be defined. In a genetic screen to identify critical regulators of myogenesis, we identified a DEAD-Box RNA helicase, DDX27, that is required for skeletal muscle growth and regeneration. We demonstrate that DDX27 regulates ribosomal RNA (rRNA) maturation, and thereby the ribosome biogenesis and the translation of specific transcripts during myogenesis. These findings provide insight into the translational regulation of gene expression in myogenesis and suggest novel functions for ribosomes in regulating gene expression in skeletal muscles.

Migration speed of nucleolus precursor bodies in human male pronuclei: a novel parameter for predicting live birth.

PURPOSE: To study the relationship between the migration speed of nucleolus precursor bodies (NPBs) in male and female pronuclei (mPN; fPN) and human embryo development during assisted reproduction. METHODS: The migration speed of 263 NPBs from 47 zygotes was quantitated, and embryonic development was observed until the blastocyst stage. The central coordinates of mPN, fPN, and NPBs were noted at multiple timepoints. Then, the distance traveled by the NPBs between two sequential images was measured, and migration speed was calculated. Additionally, we investigated the relationship between NPB migration speed and ploidy status (N = 33) or live birth/ongoing pregnancy (LB/OP) (N = 60) after assisted reproduction. RESULTS: The NPB migration speed in both mPN and fPN was significantly faster in the zygotes that developed into blastocysts (N = 25) than that in the zygotes that arrested (N = 22). The timing of blastulation was negatively correlated with NPB migration speed in the mPN. Faster NPB migration was significantly correlated with LB/OP. In multivariate logistic analysis, NPB migration speed in the mPN was the only morphokinetic parameter associated with LB/OP. In a receiver-operating characteristic curve analysis of LB/OP by the NPB migration speed in the mPN, the cut-off value was 4.56 µm/h. When this cut-off value was applied to blastocysts with preimplantation genetic testing for aneuploidy, 100% of the blastocysts faster than or equal to the cut-off value were euploid. CONCLUSION: The NPBs migrated faster in zygotes having the potential to develop into a blastocyst, and eventually into a baby. This predictor could be an attractive marker for non-invasive embryo selection.

Visualization of Chromatin in the Yeast Nucleus and Nucleolus Using Hyperosmotic Shock.

Unlike in most eukaryotic cells, the genetic information of budding yeast in the exponential growth phase is only present in the form of decondensed chromatin, a configuration that does not allow its visualization in cell nuclei conventionally prepared for transmission electron microscopy. In this work, we studied the distribution of chromatin and its relationships to the nucleolus using different cytochemical and immunocytological approaches applied to yeast cells subjected to hyperosmotic shock. Our results show that osmotic shock induces the formation of heterochromatin patches in the nucleoplasm and intranucleolar regions of the yeast nucleus. In the nucleolus, we further revealed the presence of osmotic shock-resistant DNA in the fibrillar cords which, in places, take on a pinnate appearance reminiscent of ribosomal genes in active transcription as observed after molecular spreading ("Christmas trees"). We also identified chromatin-associated granules whose size, composition and behaviour after osmotic shock are reminiscent of that of mammalian perichromatin granules. Altogether, these data reveal that it is possible to visualize heterochromatin in yeast and suggest that the yeast nucleus displays a less-effective compartmentalized organization than that of mammals.

Histone H4 variant, H4G, drives ribosomal RNA transcription and breast cancer cell proliferation by loosening nucleolar chromatin structure.

The hominidae-specific histone variant H4G is expressed in breast cancer patients in a stage-dependent manner. H4G localizes primarily in the nucleoli via its interaction with nucleophosmin (NPM1). H4G is involved in rDNA transcription and ribosome biogenesis, which facilitates breast cancer cell proliferation. However, the molecular mechanism underlying this process remains unknown. Here, we show that H4G is not stably incorporated into nucleolar chromatin, even with the chaperoning assistance of NPM1. H4G likely form transient nucleosome-like-structure that undergoes rapid dissociation. In addition, the nucleolar chromatin in H4GKO cells is more compact than WT cells. Altogether, our results suggest that H4G relaxes the nucleolar chromatin and enhances rRNA transcription by forming destabilized nucleosome in breast cancer cells.

Nucleolus structural integrity during the first meiotic prophase in rat spermatocytes.

A typical nucleolus structure is shaped by three components. A meshwork of fine fibers forming the fibrillar center (FC) is surrounded by densely packed fibers forming the dense fibrillar component (DFC). Meanwhile, wrapping the FC and DFC is the granular component (GC). During the mitotic prophase, the nucleolus undergoes disassembling of its components. On the contrary, throughout the first meiotic prophase that occurs in the cells of the germ line, small nucleoli are assembled into one nucleolus by the end of the prophase. These nucleoli are transcriptionally active, suggesting that they are fully functional. Electron microscopy analysis has suggested that these nucleoli display their three main components but a typical organization has not been observed. Here, by immunolabeling and electron microscopy, we show that the nucleolus has its three main components. The GC is interlaced with the DFC and is not as well defined as previously thought during leptotene and zygotene stage.

Nucleolar residence of the seckel syndrome protein TRAIP is coupled to ribosomal DNA transcription.

The RING finger protein TRAIP protects genome integrity and its mutation causes Seckel syndrome. TRAIP encodes a nucleolar protein that migrates to UV-induced DNA lesions via a direct interaction with the DNA replication clamp PCNA. Thus far, mechanistically how UV mobilizes TRAIP from the nucleoli remains unknown. We found that PCNA binding is dispensable for the nucleolus-nucleoplasm shuttling of TRAIP following cell exposure to UV irradiation, and that its redistribution did not rely on the master DNA damage kinases ATM and ATR. Interestingly, I-PpoI-induced ribosomal DNA damage led to TRAIP exclusion from the nucleoli, raising the possibility that active ribosomal DNA transcription may underlie TRAIP retention in the nuclear sub-compartments. Accordingly, chemical inhibition of RNA polymerase I activity led to TRAIP diffusion into the nucleoplasm, and was coupled with marked reduction of DNA/RNA hybrids in the nucleoli, suggesting that TRAIP may be sequestered via binding to nucleic acid structures in the nucleoli. Consistently, cell pre-treatment with DNase/RNase effectively released TRAIP from the nucleoli. Taken together, our study defines a bipartite mechanism that drives TRAIP trafficking in response to UV damage, and highlights the nucleolus as a stress sensor that contributes to orchestrating DNA damage responses.

Dominant Nucleolus in the Progenitor Cell Using Human Bone Marrow Erythroid and Granulocytic Cell Lineages as a Model. A Morphological and Cytochemical Note.

Progenitor cells of the human erythroid and granulocytic cell lineages are characterized by the presence of several nucleoli. One of these nucleoli is larger and possesses more fibrillar centres than others. Such nucleolus is apparently dominant in respect of both size and main nucleolar function such as nucleolar-ribosomal RNA transcription. Such nucleolus is also visible in specimens using conventional visualization procedures, in contrast to smaller nucleoli. In the terminal differentiation nucleated stages of the erythroid and granulocytic development, dominant nucleoli apparently disappeared, since these cells mostly contained very small nucleoli of a similar size with one fibrillar centre. Thus, the easily visible dominant nucleoli appear to be useful markers of the progenitor cell state, such as proliferation, and differentiation potential.

Relationships between the structural and functional organization of the turtle cell nucleolus.

The nucleolus is a multifunctional structure of the eukaryotic cell nucleus. However, its primary role is ribosome formation. Although the factors and mechanisms involved in ribogenesis are well conserved in eukaryotes, two types of nucleoli have been observed under the electron microscope: a tricompartmentalized nucleolus in amniotes and a bicompartmentalized nucleolus in other species. A recent study has also revealed that turtles, although belonging to amniotes, displayed a nucleolus with bipartite organization, suggesting that this reptile group may have carried out a reversion phenomenon during evolution. In this study, we examine in great detail the functional organization of the turtle nucleolus. In liver and spleen cells cultured in vitro, we confirm that the turtle nucleolus is mainly formed by two components: a fibrillar zone surrounded by a granular zone. We further show that the fibrillar zone includes densely-contrasted strands, which are positive after silver-stained Nucleolar Organizer Region (Ag-NOR) staining and DNA labelling. We also reveal that the dense strands condensed into a very compact mass within the fibrillar zone after a treatment with actinomycin D or 5,6-dichlorobenzimidazole riboside. Finally, by using pulse-chase experiments with BrUTP, three-dimensional image reconstructions of confocal optical sections, and electron microscopy analysis of ultrathin sections, we show that the topological and spatial dynamics of rRNA within the nucleolus extend from upstream binding factor (UBF)-positive sites in the fibrillar zone to the granular zone, without ever releasing the positive sites for the UBF. Together, these results seem to clearly indicate that the compartmentalization of the turtle nucleolus into two main components reflects a less orderly organization of ribosome formation.

Ki-67: more than a proliferation marker.

Ki-67 protein has been widely used as a proliferation marker for human tumor cells for decades. In recent studies, multiple molecular functions of this large protein have become better understood. Ki-67 has roles in both interphase and mitotic cells, and its cellular distribution dramatically changes during cell cycle progression. These localizations correlate with distinct functions. For example, during interphase, Ki-67 is required for normal cellular distribution of heterochromatin antigens and for the nucleolar association of heterochromatin. During mitosis, Ki-67 is essential for formation of the perichromosomal layer (PCL), a ribonucleoprotein sheath coating the condensed chromosomes. In this structure, Ki-67 acts to prevent aggregation of mitotic chromosomes. Here, we present an overview of functional roles of Ki-67 across the cell cycle and also describe recent experiments that clarify its role in regulating cell cycle progression in human cells.

MacroH2A histone variants maintain nuclear organization and heterochromatin architecture.

Genetic loss-of-function studies on development, cancer and somatic cell reprogramming have suggested that the group of macroH2A histone variants might function through stabilizing the differentiated state by a yet unknown mechanism. Here, we present results demonstrating that macroH2A variants have a major function in maintaining nuclear organization and heterochromatin architecture. Specifically, we find that a substantial amount of macroH2A is associated with heterochromatic repeat sequences. We further identify macroH2A on sites of interstitial heterochromatin decorated by histone H3 trimethylated on K9 (H3K9me3). Loss of macroH2A leads to major defects in nuclear organization, including reduced nuclear circularity, disruption of nucleoli and a global loss of dense heterochromatin. Domains formed by DNA repeat sequences are disorganized, expanded and fragmented, and mildly re-expressed when depleted of macroH2A. At the molecular level, we find that macroH2A is required for the interaction of repeat sequences with the nucleostructural protein lamin B1. Taken together, our results argue that a major function of macroH2A histone variants is to link nucleosome composition to higher-order chromatin architecture.

A RNA-Targeted Two-Photon Bioprobe with High Selective Permeability into Nuclear Pore Complexes for Dynamically Tracking the Autophagy Process among Multi-Organelles.

Dynamic tracking of the spatiotemporal coordination among various organelles to in-depth understanding of the mechanism of autophagy have attracted considerable attention. However, the monitor of nucleoli participation in autophagy was somehow neglected. Herein, we report a RNA-targeted bioprobe (ADAP) with high selective permeability into nuclear pore complexes, which induced a two-photon (TP) fluorescence "off-on" response by groove combination with RNA, dynamically monitoring the autophagy process among multiorganelles (nucleoli, mitochondria, and mitochondria-containing lysosomes). This work provides a simple and convenient way to observe the dynamic behavior of multiorganelles during the autophagy process, which benefits the understanding of cellular metabolism.