Chromosome territories--a functional nuclear landscape

Understanding nuclear architecture is indispensable for understanding the cell-type-dependent orchestration of active and silent genes and other nuclear functions, such as RNA splicing, DNA replication and repair. Yet, while it is now generally agreed that chromosomes in the cell nucleus are organized as chromosome territories, present models of chromosome territory architecture differ widely with respect to the possible functional implications of dynamic changes of this architecture during the cell cycle and terminal cell differentiation.     

Hypophosphorylation of the architectural chromatin protein DEK in death-receptor-induced apoptosis revealed by the isotope coded protein label proteomic platform

During apoptosis nuclear morphology changes dramatically due to alterations of chromatin architecture and cleavage of structural nuclear proteins. To characterize early events in apoptotic nuclear dismantling we have performed a proteomic study of apoptotic nuclei. To this end we have combined a cell-free apoptosis system with a proteomic platform based on the differential isotopic labeling of primary amines with N-nicotinoyloxy-succinimide. We exploited the ability of this system to produce nuclei arrested at different stages of apoptosis to analyze proteome alterations which occur prior to or at a low level of caspase activation. We show that the majority of proteins affected at the onset of apoptosis are involved in chromatin architecture and RNA metabolism. Among them is DEK, an architectural chromatin protein which is linked to autoimmune disorders. The proteomic analysis points to the occurrence of multiple PTMs in early apoptotic nuclei. This is confirmed by showing that the level of phosphorylation of DEK is decreased following apoptosis induction. These results suggest the unexpected existence of an early crosstalk between cytoplasm and nucleus during apoptosis. They further establish a previously unrecognized link between DEK and cell death, which will prove useful in the elucidation of the physiological function of this protein.     

The Role of Chromosome–Nuclear Envelope Attachments in 3D Genome Organization

Chromosomes are intricately folded and packaged in the cell nucleus and interact with the nuclear envelope. This complex nuclear architecture has a profound effect on how the genome works and how the cells function. The main goal of review is to highlight recent studies on the effect of chromosome–nuclear envelope interactions on chromatin folding and function in the nucleus. The data obtained suggest that chromosome–nuclear envelope attachments are important for the organization of nuclear architecture in various organisms. A combination of experimental cell biology methods with computational modeling offers a unique opportunity to explore the fundamental relationships between different aspects of 3D genome organization in greater details. This powerful interdisciplinary approach could reveal how the organization and function of the genome in the nuclear space is affected by the chromosome–nuclear envelope attachments and will enable the development of novel approaches to regulate gene expression.     

Advances in the computational and molecular understanding of the prostate cancer cell nucleus

Nuclear alterations are a hallmark of many types of cancers, including prostate cancer (PCa). Recent evidence shows that subvisual changes, ones that may not be visually perceptible to a pathologist, to the nucleus and its ultrastructural components can precede visual histopathological recognition of cancer. Alterations to nuclear features, such as nuclear size and shape, texture, and spatial architecture, reflect the complex molecular‐level changes that occur during oncogenesis. Quantitative nuclear morphometry, a field that uses computational approaches to identify and quantify malignancy‐induced nuclear changes, can enable a detailed and objective analysis of the PCa cell nucleus. Recent advances in machine learning–based approaches can now automatically mine data related to these changes to aid in the diagnosis, decision making, and prediction of PCa prognoses. In this review, we use PCa as a case study to connect the molecular‐level mechanisms that underlie these nuclear changes to the machine learning computational approaches, bridging the gap between the clinical and computational understanding of PCa. First, we will discuss recent developments to our understanding of the molecular events that drive nuclear alterations in the context of PCa: the role of the nuclear matrix and lamina in size and shape changes, the role of 3‐dimensional chromatin organization and epigenetic modifications in textural changes, and the role of the tumor microenvironment in altering nuclear spatial topology. We will then discuss the advances in the applications of machine learning algorithms to automatically segment nuclei in prostate histopathological images, extract nuclear features to aid in diagnostic decision making, and predict potential outcomes, such as biochemical recurrence and survival. Finally, we will discuss the challenges and opportunities associated with translation of the quantitative nuclear morphometry methodology into the clinical space. Ultimately, accurate identification and quantification of nuclear alterations can contribute to the field of nucleomics and has applications for computationally driven precision oncologic patient care.     

Gold-nanoparticle-assisted laser perturbation of chromatin assembly reveals unusual aspects of nuclear architecture within living cells

Chromatin organization within the nucleus is a vital regulator of genome function, yet its mechanical coupling to the nuclear architecture has remained elusive. To directly investigate this coupling, we locally modulated chromatin structure in living cells using nanoparticle-based laser perturbation. Unusual differences in the response of the cell nucleus were observed depending on the nuclear region that was perturbed--the heterochromatin, the euchromatin, and the nuclear envelope. This response varied under different conditions of cellular perturbations such as ATP depletion, apoptosis, and inhibition of histone deacetylases. Our studies implicate heterochromatin organization in imparting mechanical stability to the cell nucleus and suggest that nuclear size and shape are the result of interplay between nuclear and cytoplasmic anchors.     

Genome-wide nuclear morphology screen identifies novel genes involved in nuclear architecture and gene-silencing in Saccharomyces cerevisiae

Organisation of the cell nucleus is crucial for the regulation of gene expression but little is known about how nuclei are structured. To address this issue, we designed a genomic screen to identify factors involved in nuclear architecture in Saccharomyces cerevisiae. This screen is based on microscopic monitoring of nuclear pore complexes and nucleolar proteins fused with the green fluorescent protein in a collection of approximately 400 individual deletion mutants. Among the 12 genes identified by this screen, most affect both the nuclear envelope and the nucleolar morphology. Corresponding gene products are localised preferentially to the nucleus or close to the nuclear periphery. Interestingly, these nuclear morphology alterations were associated with chromatin-silencing defects. These genes provide a molecular context to explore the functional link between nuclear architecture and gene silencing.     

Three-dimensional localization and dynamics of centromeres in mouse oocytes during folliculogenesis

Very little is known about oocyte nuclear architecture during folliculogenesis. Using antibodies to reveal centromeres, Hoechst-staining to detect the AT-rich pericentromeric heterochromatin (chromocenters), combined with confocal microscopy for the three-dimensional analysis of the nucleus, we demonstrate that during mouse folliculogenesis the oocyte nuclear architecture undergoes dynamic changes. In oocytes isolated from primordial and primary follicles, centromeres and chromocenters were preferentially located at the periphery of the nucleus. During oocyte growth, centromeres and chromocenters were initially found spread within the nucleus and then progressively clustered around the periphery of the nucleolus. Our results indicate that the oocyte nuclear achitecture is developmentally regulated and they contribute to a further understanding of the role of nuclear organization in the regulation of genome functioning during differentiation and development.     

Morphometry of nuclear and nucleolar structures in a CaCo-2 cell line

The number of the nucleoli in a CaCo-2 cell nucleus does not generally depend on the quantity of DNA in the nucleus, but nucleolar DNA content is directly proportional to total nuclear DNA. However, in multinucleolar cells (three or more nucleoli), the nucleolar DNA content increases after 96 h incubation in culture without concomitant quantitative changes in nuclear DNA. The percentage of multinucleolar cells and the average number of nucleoli per nucleus increase with increasing incubation time. After 72 and 96 h in culture, multinucleolar cells show distinctive morphologies. The ratio of the sum of nucleolar perimeters to the nuclear perimeter increases linearly when the number of nucleoli in a nucleus increases, but there is no concomitant increase in total nucleolar area or DNA content, except in the 72 and 96 h populations. When the number of nucleoli in CaCo-2 cells increases after 48 and 60 h in culture, the amount of DNA per nucleolus decreases.     

Importin β2 Mediates the Spatio-temporal Regulation of Anillin through a Noncanonical Nuclear Localization Signal

The compartmentalization of cell cycle regulators is a common mechanism to ensure the precise temporal control of key cell cycle events. For instance, many mitotic spindle assembly factors are known to be sequestered in the nucleus prior to mitotic onset. Similarly, the essential cytokinetic factor anillin, which functions at the cell membrane to promote the physical separation of daughter cells at the end of mitosis, is sequestered in the nucleus during interphase. To address the mechanism and role of anillin targeting to the nucleus in interphase, we identified the nuclear targeting motif. Here, we show that anillin is targeted to the nucleus by importin β2 in a Ran-dependent manner through an atypical basic patch PY nuclear localization signal motif. We show that although importin β2 binding does not regulate anillin''s function in mitosis, it is required to prevent the cytosolic accumulation of anillin, which disrupts cellular architecture during interphase. The nuclear sequestration of anillin during interphase serves to restrict anillin''s function at the cell membrane to mitosis and allows anillin to be rapidly available when the nuclear envelope breaks down to remodel the cellular architecture necessary for successful cell division.     

Direct Visualization of a Protein Nuclear Architecture

Whether the cell nucleus is organized by an underlying architecture analagous to the cytoskeleton has been a highly contentious issue since the original isolation of a nuclease and salt-resistant nuclear matrix. Despite electron microscopy studies that show that a nuclear architecture can be visualized after fractionation, the necessity to elute chromatin to visualize this structure has hindered general acceptance of a karyoskeleton. Using an analytical electron microscopy method capable of quantitative elemental analysis, electron spectroscopic imaging, we show that the majority of the fine structure within interchromatin regions of the cell nucleus in fixed whole cells is not nucleoprotein. Rather, this fine structure is compositionally similar to known protein-based cellular structures of the cytoplasm. This study is the first demonstration of a protein network in unfractionated and uninfected cells and provides a method for the ultrastructural characterization of the interaction of this protein architecture with chromatin and ribonucleoprotein elements of the cell nucleus.     

Power-law rheology of isolated nuclei with deformation mapping of nuclear substructures

Force-induced changes in genome expression as well as remodeling of nuclear architecture in development and disease motivate a deeper understanding of nuclear mechanics. Chromatin and green fluorescent protein-lamin B dynamics were visualized in a micropipette aspiration of isolated nuclei, and both were shown to contribute to viscoelastic properties of the somatic cell nucleus. Reversible swelling by almost 200% in volume, with changes in salt, demonstrates the resilience and large dilational capacity of the nuclear envelope, nucleoli, and chromatin. Swelling also proves an effective way to separate the mechanical contributions of nuclear elements. In unswollen nuclei, chromatin is a primary force-bearing element, whereas swollen nuclei are an order of magnitude softer, with the lamina sustaining much of the load. In both cases, nuclear deformability increases with time, scaling as a power law-thus lacking any characteristic timescale-when nuclei are either aspirated or indented by atomic force microscopy. The nucleus is stiff and resists distortion at short times, but it softens and deforms more readily at longer times. Such results indicate an essentially infinite spectrum of timescales for structural reorganization, with implications for regulating genome expression kinetics.     

Well-defined genome architecture in the human sperm nucleus

Using fluorescence in situ hybridization, conventional epifluorescence microscopy, and laser scanning confocal microscopy followed by three-dimensional reconstruction we describe a well-defined higher order packaging of the human genome in the sperm cell nucleus. This was determined by the spatial localization of centromere and telomere regions of all chromosomes and supported by localization of subtelomere sequences of chromosome 3 and the entire chromosome 2. The nuclear architecture in the human sperm is characterized by the clustering of the 23 centromeres into a compact chromocenter positioned well inside the nucleus. The ends of the chromosomes are exposed to the nuclear periphery where both the subtelomere and the telomere sequences of the chromosome arms are joined into dimers. Thus chromosomes in the human sperm nucleus are looped into a hairpin-like configuration. The biological implications of this nuclear architecture in spermatogenesis and male pronuclear formation following fertilization are discussed.     

Estimation of the mechanical connection between apical stress fibers and the nucleus in vascular smooth muscle cells cultured on a substrate

Actin stress fibers (SFs) generate intercellular tension and play important roles in cellular mechanotransduction processes and the regulation of various cellular functions. We recently found, in vascular smooth muscle cells (SMCs) cultured on a substrate, that the apical SFs running across the top surface of the nucleus have a mechanical connection with the cell nucleus and that their internal tension is transmitted directly to the nucleus. However, the effects of the connecting conditions and binding forces between SFs and the nucleus on force transmission processes are unclear at this stage. Here, we estimated the mechanical connection between apical SFs and the nucleus in SMCs, taking into account differences in the contractility of individual SFs, using experimental and numerical approaches. First, we classified apical SFs in SMCs according to their morphological characteristics: one subset appeared pressed onto the apical surface of the nucleus (pressed SFs), and the other appeared to be smoothly attached to the nuclear surface (attached SFs). We then dissected these SFs by laser irradiation to release the pretension, observed the dynamic behavior of the dissected SFs and the nucleus, and estimated the pretension of the SFs and the connection strength between the SFs and the nucleus by using a simple viscoelastic model. We found that pressed SFs generated greater contractile force and were more firmly connected to the nuclear surface than were attached SFs. We also observed line-like concentration of the nuclear membrane protein nesprin 1 and perinuclear DNA that was significantly located along the pressed SFs. These results indicate that the internal tension of pressed SFs is transmitted to the nucleus more efficiently than that of attached SFs, and that pressed SFs have significant roles in the regulation of the nuclear morphology and rearrangement of intranuclear DNA.     

The nuclear lamins and the nuclear envelope

The cell nucleus is separated from the rest of the cell by the nuclear envelope. The nuclear envelope, nuclear envelope proteins and nuclear lamina organise the structure of the entire nucleus and the chromatin via a myriad of interactions. These interactions are dynamic, change with the change (progress) of the cell cycle, with cell differentiation and with changes in cell physiology.