Nucleolar Material Research Articles

Multiscale biophysical analysis of nucleolus disassembly during mitosis

During cell division, precise and regulated distribution of cellular material between daughter cells is a critical step and is governed by complex biochemical and biophysical mechanisms. To achieve this, membraneless organelles and condensates often require complete disassembly during mitosis. The biophysical principles governing the disassembly of condensates remain poorly understood. Here, we used a physical biology approach to study how physical and material properties of the nucleolus, a prominent nuclear membraneless organelle in eukaryotic cells, change during mitosis and across different scales. We found that nucleolus disassembly proceeds continuously through two distinct phases with a slow and reversible preparatory phase followed by a rapid irreversible phase that was concurrent with the nuclear envelope breakdown. We measured microscopic properties of nucleolar material including effective diffusion rates and binding affinities as well as key macroscopic properties of surface tension and bending rigidity. By incorporating these measurements into the framework of critical phenomena, we found evidence that near mitosis surface tension displays a power-law behavior as a function of biochemically modulated interaction strength. This two-step disassembly mechanism maintains structural and functional stability of nucleolus while enabling its rapid and efficient disassembly in response to cell cycle cues.

Two new species of the genus Leptomyxa (Goodey, 1915) – Leptomyxa botanica n. sp. and Leptomyxa monrepos n. sp. from the soil habitats of Northwestern Russia

Amoebae of the genus Leptomyxa have variable morphologies and can only be reliably identified using molecular data. However, species distinction based on the 18S rRNA gene sequence is difficult due to the very low level of sequence divergence among morphologically different species. The database for other genes is much smaller, and genomic data on Leptomyxa is almost absent. In this study, we describe two new terrestrial species of the genus Leptomyxa isolated from Northwestern Russia, Leptomyxa botanica n. sp. and Leptomyxa monrepos n. sp. Both species easily adopt an expanded fan-shaped form and have a complex structure of the nucleolar material. Phylogenetic analyses show a derived status of these two species. They form a clade with Leptomyxa valladaresi. Our tree confirms that the 18S rRNA gene sequences of Leptomyxa species are split into two large clades. The morphological synapomorphies of these clades are not obvious. This analysis is complicated by the lack of reliable morphological data on many sequenced strains and probable misidentification of some isolates.

Viscoelasticity and advective flow of RNA underlies nucleolar form and function

The nucleolus is the largest biomolecular condensate and facilitates transcription, processing, and assembly of ribosomal RNA (rRNA). Although nucleolar function is thought to require multiphase liquid-like properties, nucleolar fluidity and its connection to the highly coordinated transport and biogenesis of ribosomal subunits are poorly understood. Here, we use quantitative imaging, mathematical modeling, and pulse-chase nucleotide labeling to examine nucleolar material properties and rRNA dynamics. The mobility of rRNA is several orders of magnitude slower than that of nucleolar proteins, with rRNA steadily moving away from the transcriptional sites in a slow (∼1Å/s), radially directed fashion. This constrained but directional mobility, together with polymer physics-based calculations, suggests that nascent rRNA forms an entangled gel, whose constant production drives outward flow. We propose a model in which progressive maturation of nascent rRNA reduces its initial entanglement, fluidizing the nucleolar periphery to facilitate the release of assembled pre-ribosomal particles.

Trypanosoma cruzi PARP is enriched in the nucleolus and is present in a thread connecting nuclei during mitosis.

Poly (ADP-ribose) polymerase (PARP) is responsible for the synthesis of ADP-ribose polymers, which are involved in a wide range of cellular processes such as preservation of genome integrity, DNA damage signaling and repair, molecular switches between distinct cell death pathways, and cell cycle progression. Previously, we demonstrated that the only PARP present in T. cruzi migrates to the nucleus upon genotoxic stimulus. In this work, we identify the N-terminal domain as being sufficient for TcPARP nuclear localization and describe for the first time that TcPARP is enriched in the parasite's nucleolus. We also describe that TcPARP is present in a thread-like structure that connects two dividing nuclei and co-localizes with nucleolar material and microtubules. Furthermore, ADP-ribose polymers could also be detected in this thread during mitosis. These findings represent a first approach to new potential TcPARP functions inside the nucleus and will help understand its role well beyond the largely described DNA damage response protein in trypanosomatids.

Dynamic chromosomal interactions and control of heterochromatin positioning by Ki‐67

Ki‐67 is a chromatin‐associated protein with a dynamic distribution pattern throughout the cell cycle and is thought to be involved in chromatin organization. The lack of genomic interaction maps has hampered a detailed understanding of its roles, particularly during interphase. By pA‐DamID mapping in human cell lines, we find that Ki‐67 associates with large genomic domains that overlap mostly with late‐replicating regions. Early in interphase, when Ki‐67 is present in pre‐nucleolar bodies, it interacts with these domains on all chromosomes. However, later in interphase, when Ki‐67 is confined to nucleoli, it shows a striking shift toward small chromosomes. Nucleolar perturbations indicate that these cell cycle dynamics correspond to nucleolar maturation during interphase, and suggest that nucleolar sequestration of Ki‐67 limits its interactions with larger chromosomes. Furthermore, we demonstrate that Ki‐67 does not detectably control chromatin‐chromatin interactions during interphase, but it competes with the nuclear lamina for interaction with late‐replicating DNA, and it controls replication timing of (peri)centromeric regions. Together, these results reveal a highly dynamic choreography of genome interactions and roles for Ki‐67 in heterochromatin organization.

Physiological functions and roles in cancer of the proliferation marker Ki-67.

What do we know about Ki-67, apart from its usefulness as a cell proliferation biomarker in histopathology? Discovered in 1983, the protein and its regulation of expression and localisation throughout the cell cycle have been well characterised. However, its function and molecular mechanisms have received little attention and few answers. Although Ki-67 has long been thought to be required for cell proliferation, recent genetic studies have conclusively demonstrated that this is not the case, as loss of Ki-67 has little or no impact on cell proliferation. In contrast, Ki-67 is important for localising nucleolar material to the mitotic chromosome periphery and for structuring perinucleolar heterochromatin, and emerging data indicate that it also has critical roles in cancer development. However, its mechanisms of action have not yet been fully identified. Here, we review recent findings and propose the hypothesis that Ki-67 is involved in structuring cellular sub-compartments that assemble by liquid-liquid phase separation. At the heterochromatin boundary, this may control access of chromatin regulators, with knock-on effects on gene expression programmes. These changes allow adaptation of the cell to its environment, which, for cancer cells, is a hostile one. We discuss unresolved questions and possible avenues for future exploration.

Nucleolar-based Dux repression is essential for embryonic two-cell stage exit.

Upon fertilization, the mammalian embryo must switch from dependence on maternal transcripts to transcribing its own genome, and in mice this involves the transient up-regulation of MERVL transposons and MERVL-driven genes at the two-cell stage. The mechanisms and requirement for MERVL and two-cell (2C) gene up-regulation are poorly understood. Moreover, this MERVL-driven transcriptional program must be rapidly shut off to allow two-cell exit and developmental progression. Here, we report that robust ribosomal RNA (rRNA) synthesis and nucleolar maturation are essential for exit from the 2C state. 2C-like cells and two-cell embryos show similar immature nucleoli with altered structure and reduced rRNA output. We reveal that nucleolar disruption via blocking RNA polymerase I activity or preventing nucleolar phase separation enhances conversion to a 2C-like state in embryonic stem cells (ESCs) by detachment of the MERVL activator Dux from the nucleolar surface. In embryos, nucleolar disruption prevents proper nucleolar maturation and Dux silencing and leads to two- to four-cell arrest. Our findings reveal an intriguing link between rRNA synthesis, nucleolar maturation, and gene repression during early development.

Glomerulosomes: morphologically distinct nuclear organelles of unknown nature.

The nucleus of some representatives of the genus Pelomyxa (Amoebozoa, Archamoebae, Pelobiontida) contains specific bodies (membrane-less organelles). They may be either embedded in the nucleolar mass or detached from the nucleolus. We termed these nuclear bodies the glomerulosomes for their characteristic ultrastructural appearance. The glomerulosomes are distinct nuclear bodies, about 1μm in diameter. The morphological and diagnostic unit of a glomerulosome is an electron-dense thread/string, about 30-40nm in thickness. These threads are not direct continuation of the nucleolar material. The threads create the unique geometric appearance of the glomerulosome by being organized into precisely parallel rows/cords. Each cord of the threads can curve at different angles within the glomerulosome body, but the threads themselves are not coiled. Nowadays, the glomerulosomes have been discovered in P. palustris, P.stagnalis, P.paradoxa, and Pelomyxa sp. Despite the unique appearance of glomerulosomes, their existence may be a more common phenomenon in eukaryotic cells than just a specific feature of the nucleus of elected pelomyxes.

Nucleolar maturation of the human small subunit processome.

The human small subunit processome mediates early maturation of the small ribosomal subunit by coupling RNA folding to subsequent RNA cleavage and processing steps. We report the high-resolution cryo–electron microscopy structures of maturing human small subunit (SSU) processomes at resolutions of 2.7 to 3.9 angstroms. These structures reveal the molecular mechanisms that enable crucial progressions during SSU processome maturation. RNA folding states within these particles are communicated to and coordinated with key enzymes that drive irreversible steps such as targeted exosome-mediated RNA degradation, protein-guided site-specific endonucleolytic RNA cleavage, and tightly controlled RNA unwinding. These conserved mechanisms highlight the SSU processome’s impressive structural plasticity, which endows this 4.5-megadalton nucleolar assembly with the distinctive ability to mature the small ribosomal subunit from within.

Assessing the effect of interspecies oocyte nucleolar material dosage on embryonic development

Sequence differences are considered to be the basic cause of developmental failure in interspecies embryos when more distant species are combined. However, other phenomena, such as insufficient or excessive quantity of specific cellular factors, might also influence the outcome. These effects are usually not considered. One of the organelles shown to contain different amount of proteins is the oocyte nucleolus-like body. Here we show that upon interspecies transfer, a single porcine nucleolus-like body is unable to support the development of a mouse parthenogenetic embryo derived from an enucleolated oocyte. However, when the amount of the porcine nucleolar material is increased to equalize the amount of mouse nucleolar material by transferring two nucleolus-like bodies, mouse embryos are able to pass the developmental block elicited by enucleolation. These embryos progress to the blastocyst stage at rates comparable to controls. Thus, using the model of an interspecies nucleolus-like body transplantation between mouse and pig oocytes, we show that an inadequate amount of nucleolar factors, rather than the species origin, affects the development. In a wider context of interspecies nuclear transfer schemes, the observed incompatibility between more distant species might not stem simply from sequence differences but also from improper dosage of key cellular factors.

The nucleolar protein NOP2 is required for nucleolar maturation and ribosome biogenesis during preimplantation development in mammals.

The maternal nucleolus plays an indispensable role in zygotic genome activation (ZGA) and early embryonic development in mice. During oocyte-to-embryo transition, the nucleolus is subject to substantial transformation. Despite the primary role of the nucleolus is ribosome biogenesis, accumulating evidence has uncovered its functions in various other cell processes. However, the regulation of nucleolar maturation and ribosome biogenesis and the molecules involved remain unclear during early embryonic development. In this study, we observed that nucleolar protein 2 (NOP2) is restrictedly localized within the nucleolus, first detected in the late two-cell embryos, and increases to a peak level at the eight-cell stage in mice. RNAi-mediated NOP2 depletion leads to a developmental arrest during the morula-to-blastocyst transition. RNA-seq analyses reveal that 208 genes are differentially expressed, including multiple lineage-specific genes and several genes encoding ribosome proteins. Indeed, we observe a failure of the first lineage specification with reduced TEA domain transcription factor 4(TEAD4) (trophectoderm-specific), tir na nog (NANOG), and kruppel-like factor 4 (KLF4) (inner cell mass-specific). Importantly, by TransmissionElectronMicroscopy (TEM), we noted a decrease in the ratio of the nucleolus size and an increase in the ratio of the size of the nucleolus precursor body, suggesting the nucleolar maturation is disrupted. Moreover, both qPCR and FluorescenceInSituHybridization (FISH) data showcase a significant decrease in the abundance of ribosome RNAs. Similarly, NOP2 depletion causes reduced developmental potential and decreased rRNA level in bovine early embryos, suggesting a functional conservation of NOP2 in mammals. Taken together, these results suggest that NOP2 is required for mammalian preimplantation development, presumably by regulating nucleolar maturation and ribosome biogenesis.

Reproductive Aspects of Chagas Disease Vectors: Evidence of Transcriptional Activity during the Nucleolar Persistence Phenomenon in the Spermatogenesis of Triatomines.

The subfamily Triatominae currently consists of 154 species, most of them being of great importance for public health because they are considered potential vectors of the protozoan Trypanosoma cruzi, the etiologic agent of Chagas disease. In addition to their epidemiological importance, these insects are considered important biological models for cell studies because they have peculiar characteristics in their cells, for example, persistence of the nucleolus during spermatogenesis. This phenomenon is characterized by the presence of the nucleolus or nucleolar corpuscles during all phases of meiosis. To date, all knowledge is restricted to the study of the presence/absence of the nucleolus during the triatomine meiosis, so the present work aimed to analyze if this persistent nucleolar material has transcriptional activity. Analysis of the meiotic metaphases of Rhodnius montenegrensis and Panstrongylus megistus by using fluorochrome acridine orange made it possible to characterize the presence of RNA in the nucleolar material. Thus, it was demonstrated, for the first time, that the persistent nucleolar material during triatomine meiosis is transcriptionally active, supporting the hypothesis of the relationship between nucleolar persistence during meiosis of these insects and the formation of the chromoid body, an organelle responsible for the support of all transcriptional activities during spermiogenesis.

Meiotic cellular rejuvenation is coupled to nuclear remodeling in budding yeast.

Production of healthy gametes in meiosis relies on the quality control and proper distribution of both nuclear and cytoplasmic contents. Meiotic differentiation naturally eliminates age-induced cellular damage by an unknown mechanism. Using time-lapse fluorescence microscopy in budding yeast, we found that nuclear senescence factors - including protein aggregates, extrachromosomal ribosomal DNA circles, and abnormal nucleolar material - are sequestered away from chromosomes during meiosis II and subsequently eliminated. A similar sequestration and elimination process occurs for the core subunits of the nuclear pore complex in both young and aged cells. Nuclear envelope remodeling drives the formation of a membranous compartment containing the sequestered material. Importantly, de novo generation of plasma membrane is required for the sequestration event, preventing the inheritance of long-lived nucleoporins and senescence factors into the newly formed gametes. Our study uncovers a new mechanism of nuclear quality control and provides insight into its function in meiotic cellular rejuvenation.

Controlling the material properties and rRNA processing function of the nucleolus using light

The nucleolus is a prominent nuclear condensate that plays a central role in ribosome biogenesis by facilitating the transcription and processing of nascent ribosomal RNA (rRNA). A number of studies have highlighted the active viscoelastic nature of the nucleolus, whose material properties and phase behavior are a consequence of underlying molecular interactions. However, the ways in which the material properties of the nucleolus impact its function in rRNA biogenesis are not understood. Here we utilize the Cry2olig optogenetic system to modulate the viscoelastic properties of the nucleolus. We show that above a threshold concentration of Cry2olig protein, the nucleolus can be gelled into a tightly linked, low mobility meshwork. Gelled nucleoli no longer coalesce and relax into spheres but nonetheless permit continued internal molecular mobility of small proteins. These changes in nucleolar material properties manifest in specific alterations in rRNA processing steps, including a buildup of larger rRNA precursors and a depletion of smaller rRNA precursors. We propose that the flux of processed rRNA may be actively tuned by the cell through modulating nucleolar material properties, which suggests the potential of materials-based approaches for therapeutic intervention in ribosomopathies.

Nucleoli in embryos: a central structural platform for embryonic chromatin remodeling?

Nucleoli are the site of ribosomal RNA production and subunit assembly. In contrast to active nucleoli in somatic cells, where three basic sub-compartments can be observed, mammalian oocytes and early embryos contain atypical nucleoli termed "nucleolus-like bodies" or "nucleolus precursor bodies", respectively. Unlike their somatic counterparts, these structures are composed of dense homogenous fibrillar material and exhibit no polymerase activity. Irrespective of these unusual properties, they have been shown to be absolutely essential for embryonic development, as their microsurgical removal results in developmental arrest. Historically, nucleolus-like and nucleolus precursor bodies have been perceived as passive storage sites of nucleolar material, which is gradually utilized by embryos to construct fully functional nucleoli once they have activated their genome and have started to produce ribosomes. For decades, researchers have been trying to elucidate the composition of these organelles and provide the evidence for their repository role. However, only recently has it become clear that the function of these atypical nucleoli is altogether different, and rather than being involved in ribosome biogenesis, they participate in parental chromatin remodeling, and strikingly, the artificial introduction of a single NPB component is sufficient to rescue the developmental arrest elicited by the NPB removal. In this review, we will describe and summarize the experiments that led to the change in our understanding of these unique structures.

Nucleolar Division in the Promastigote Stage of Leishmania major Parasite: A Nop56 Point of View.

Nucleogenesis is the cellular event responsible for the formation of the new nucleoli at the end of mitosis. This process depends on the synthesis and processing of ribosomal RNA (rRNA) and, in some eukaryotes, the transfer of nucleolar material contained in prenucleolar bodies (PNBs) to active transcription sites. The lack of a comprehensive description of the nucleolus throughout the cell cycle of the human pathogen Leishmania major prompted us to analyze the distribution of nucleolar protein 56 (Nop56) during interphase and mitosis in the promastigote stage of the parasite. By in silico analysis we show that the orthologue of Nop56 in L. major (LmNop56) contains the three characteristic Nop56 domains and that its predicted three-dimensional structure is also conserved. Fluorescence microscopy observations indicate that the nucleolar localization of LmNop56 is similar, but not identical, to that of the nucleolar protein Elp3b. Notably, unlike other nucleolar proteins, LmNop56 remains associated with the nucleolus in nonproliferative cells. Moreover, epifluorescent images indicate the preservation of the nucleolar structure throughout the closed nuclear division. Experiments performed with the related parasite Trypanosoma brucei show that nucleolar division is carried out by an analogous mechanism.

Synaptic configuration of quadrivalents and their association with the XY bivalent in spermatocytes of Robertsonian heterozygotes of Mus domesticus

BackgroundThe nuclear architecture of meiotic prophase spermatocytes is based on higher-order patterns of spatial associations among chromosomal domains and consequently is prone to modification by chromosomal rearrangements. We have shown that nuclear architecture is modified in spermatocytes of Robertsonian (Rb) homozygotes of Mus domesticus. In this study we analyse the synaptic configuration of the quadrivalents formed in the meiotic prophase of spermatocytes of mice double heterozygotes for the dependent Rb chromosomes: Rbs 11.16 and 16.17.ResultsElectron microscope spreads of 60 pachytene spermatocytes from four animals of Mus domesticus 2n = 38 were studied and their respective quadrivalents analysed in detail. Normal synaptonemal complex was found between arms 16 of the Rb metacentric chromosomes, telocentrics 11 and 17 and homologous arms of the Rb metacentric chromosomes. About 43% of the quadrivalents formed a synaptonemal complex between the heterologous short arms of chromosomes 11 and 17. This synaptonemal complex is bound to the nuclear envelope through a fourth synapsed telomere, thus dragging the entire quadrivalent to the nuclear envelope. About 57% of quadrivalents showed unsynapsed single axes in the short arms of the telocentric chromosomes. About 90% of these unsynapsed quadrivalents also showed a telomere-to-telomere association between one of the single axes of the telocentric chromosome 11 or 17 and the X chromosome single axis, which was otherwise normally paired with the Y chromosome. Nucleolar material was associated with two bivalents and with the quadrivalent.ConclusionsThe spermatocytes of heterozygotes for dependent Rb chromosomes formed a quadrivalent where four chromosomes are synapsed together and bound to the nuclear envelope through four telomeres. The nuclear configuration is determined by the fourth shortest telomere, which drags the centromere regions and heterochromatin of all the chromosomes towards the nuclear envelope, favouring the reiterated encounter and eventual rearrangement between the heterologous chromosomes. The unsynapsed regions of quadrivalents are frequently bound to the single axis of the X chromosome, possibly perturbing chromatin condensation and gene expression.

Effects of cadmium on nucleolus in root tips of Hordeum vulgare

Background Hordeum vulgare is an economically important crop that is consumed in large quantities in the world. In a previous investigation, we found that cadmium (Cd) could induce some nucleolar materials containing the argyrophilic proteins scattered in the nuclei and located in the cytoplasm in the root tip cells of H. vulgare . However, what kinds of nucleolar proteins are affected has not been reported. In order to further confirm the cytological effects of Cd on nucleolus and nucleolar proteins, alterations in the cellular localization and expression of two major nucleolar proteins: fibrillarin and nucleolin in the root tips of H. vulgare were studied in the present investigation.
 Methods Cytological effects of Cd on nucleolus were observed by silver-staining method and two nucleolarproteins: nucleolin and fibrillarin were observed and analyzed by means of indirect immunofluorescent microscopy and western blotting.
 Results Results from the present investigation demonstrated that under Cd stress (cadmium chloride at 50 and 100 ?M), Cd could disturb the nucleolar cycle with the extrusion of silver-stained materials containing argyrophilic proteins from the nucleolus into the cytoplasm. Nucleolar material containing nucleolin and fibrillarin and their movement into the cytoplasm following Cd stress was detected with indirect immunofluorescence techniques. Western blotting revealed higher expression of these two major nucleoproteins in Cd-treated roots, which was consistent with the results obtained by indirect immunofluorescence.
 Conclusion The findings of the present study confirm that Cd can have toxic effects on nucleolus and affect the expression of nucleolar proteins, nucleolin and fibrillarin. The data obtained here can provide valuable information for monitoring and forecast early effects of exposure to Cd in polluted environments.

Behavior of nucleolus in the tobacco male meiocytes involved in cytomixis.

Behavior of nucleolus during the nuclear migration between plant cells (cytomixis) is studied for the first time in the tobacco male meiosis. As is shown, the nucleolus is located in a nonrandom manner in the migrating nuclei. In the majority of cases, the nucleolus resides on the nuclear pole strictly opposite to the cytomictic channel. Owing to this localization, the nucleolus extremely rare enters the recipient cell, so that the nucleolar material is in most cases undetectable in the micronuclei formed after cytomixis. When a whole nucleus migrates from a donor cell to recipient, the nucleolus can leave the nucleus and remain in the donor cells either alone or with a small amount of chromatin. The causes underlying a nonrandom location of the nucleolus in cytomictic cells are discussed. It is assumed that the nucleolar material contacts the cytoplasmic cytoskeleton, which prevents migration of the nucleolus into another cell within the nucleus. The potential use of cytomixis as a model for studying the nuclear motion is discussed.

Nucleolar Persistence: Peculiar Characteristic of Spermatogenesis of the Vectors of Chagas Disease (Hemiptera, Triatominae).

All species of triatomines are considered potential vectors of Chagas disease and the reproductive biology of these bugs has been studied by different approaches. In 1999, nucleolar persistence during meiosis was observed in the subfamily for the first time. Recently, it has been observed that all species within the genus Rhodnius exhibit the same phenomenon, suggesting that it may be a synapomorphy of the triatomines. Thus, this article aims to analyze the nucleolar behavior during spermatogenesis of 59 triatomine species. All analyzed species exhibited nucleolar persistence during meiosis. Recently, it has been suggested that nucleolar persistence may be fundamental for the spermatogenesis of these vectors, since it is related to the formation of the chromatoid body. Therefore, we emphasize that this phenomenon is a peculiarity of the Triatominae subfamily and that further studies are required to analyze whether the nucleolar material that persists is active.