Nuclear architecture in the light of gene expression and cell differentiation studies

It is evident that primary DNA sequences, that define genomes, are responsible for genome functions. However, the functional properties of chromatin are additionally regulated by heritable modifications known as epigenetic factors and, therefore, genomes should be also considered with respect to their 'epigenomes'. Nucleosome remodelling, DNA methylation and histone modifications are the most prominent epigenetic changes that play fundamental roles in the chromatin-mediated control of gene expression. Another important nuclear feature with functional relevance is the organization of mammalian chromatin into distinct chromosome territories which are surrounded by the interchromatin compartment that is necessary for transport of regulatory molecules to the targeted DNA. The inner structure of the chromosome territories, as well as the arrangement of the chromosomes within the interphase nuclei, has been found to be non-randomly organized. Therefore, a specific nuclear arrangement can be observed in many cellular processes, such as differentiation and tumour cell transformation.     

Changes of nuclear structure induced by increasing temperatures

Despite the recent improvement in understanding the higher-order structure of chromatin fibers, the organization of interphase chromosomes in specific nuclear domains emerged only recently and it is still controversial. This study took advantage of an integrated approach using complementary techniques in order to investigate the structure and organization of chromatin in interphase nucleus. Native CHO-K1 cells were progressively heated from 310 K to 410 K and the effects of increasing temperatures on nuclear chromatin were analyzed in situ by means of cytometric and calorimetric techniques. Distribution and organization of chromatin domains were analyzed by Fluorescence microscopy, while the mean condensation of nuclear chromatin was measured by Differential scanning calorimetry. The results show as changes of nuclear structures (envelope and matrix, namely) affect significantly organization and condensation of in situ chromatin. Moreover when volume is modified by an external force (the temperature gradient in our case) we observe significant alterations of chromatin structure. These data are in accordance with the hypothesis of an inverse relationship between nuclear volume and chromatin condensation.     

Immunoelectron microscopy of PCNA as an efficient marker for studying replication times and sites during pollen development

Here we report for the first time the ultrastructural localization of DNA replication sites in the nucleus of plant cells and the timing of replication through the pollen developmental programme by proliferating cell nuclear antigen (PCNA) immunogold labelling. Replication sites were identified by labelling with anti-PCNA antibodies in fibrils of the interchromatin region close to the condensed chromatin, defining a perichromatin subdomain in the interchromatin space where DNA replication takes place. The same nuclear structures are decorated by anti-BrdU (5-bromo-2'-deoxyuridine) immunogold after short pulses of BrdU labelling. Double immunogold labelling for PCNA and DNA show colocalization on these perichromatin structures. PCNA immunoelectron microscopy also allows correlation of replicative activity with the dynamics of chromatin condensation. DNA replication was also monitored at different phases during pollen development by PCNA immunoelectron microscopy, revealing two peaks of DNA synthesis, at the beginning (early tetrad), and the end (late vacuolate), of microspore interphase. High-resolution autoradiography after [3H]thymidine incorporation also showed high replicative activity at the same two periods of microspore interphase. In the bicellular pollen grain, PCNA immunogold labelling revealed that DNA replication in the generative cell starts at an intermediate stage of pollen maturation, whereas the vegetative nucleus does not replicate and is arrested in G1. The use of anti-PCNA antibodies at the ultrastructural level is an easier, faster and more feasible method than the detection of in vivo-incorporated nucleotides, especially in plant systems with long cell cycles. PCNA immunogold labelling is, therefore, proposed as an efficient marker for mapping the sites and timing of replication at the electron microscopy level.     

Characterization of nuclear compartments identified by ectopic markers in mammalian cells with distinctly different karyotype

The functional organization of chromatin in cell nuclei is a fundamental question in modern cell biology. Individual chromosomes occupy distinct chromosome territories in interphase nuclei. Nuclear bodies localize outside the territories and colocalize with ectopically expressed proteins in a nuclear subcompartment, the interchromosomal domain compartment. In order to investigate the structure of this compartment in mammalian cells with distinctly different karyotypes, we analyzed human HeLa cells (3n+=71 chromosomes) and cells of two closely related muntjac species, the Chinese muntjac (2n=46 chromosomes) and the Indian muntjac (2n=6/7 chromosomes). The distribution of ectopically expressed intermediate filament proteins (vimentin and cytokeratins) engineered to contain a nuclear localization sequence (NLS) and a nuclear particle forming protein (murine Mx1) fused to a yellow fluorescent protein (YFP) was compared. The proteins were predominantly localized in regions with poor DAPI staining independent of the cells karyotype. In contrast to NLS-vimentin, the NLS-modified cytokeratins were also found close to the nuclear periphery. In Indian muntjac cells, NLS-vimentin colocalized with Mx1-YFP as well as the NLS-cytokeratins. Since the distribution of the ectopically expressed protein markers is similar in cells with distinctly different chromosome numbers, the property of the delineated, limited compartment might indeed depend on chromatin organization.     

Ultrastructural studies of H-1 parvovirus replication. II. Induced changes in the deoxyribonucleoprotein and ribonucleoprotein components of human NB cell nuclei.

Ultrastructural changes in the nuclear DNP and RNP components of human NB cells induced by synchronous infection with H-1 parvovirus were studied using Bernhard's EDTA method of staining. Early events (12 h after infection) occurred in the nucleolus. Chromatin within the nucleolar fibrous centers condensed thereby converting the centers to vacuoles. DNP associated with the granular nucleolonema also contracted markedly, causing a disruption of this skein-like structure; it then migrated peripherally forming a heterochromatic cortex surrounding the granular nucleolar vestige. Subsequently (24–36 h after inoculation), condensation of extranucleolar chromatin took place concurrently with the accumulation of extensive amounts of interchromatin granules in the nucleus and cytoplasm. Conglomerates of perichromatin fibrils and interchromatin granules were frequently juxtapposed to the condensing chromatin. Large clumps of interchromatin granules were also closely associated with fragmenting nucleoli, and the apparent transformation of nucleolar granules into interchromatin granules was observed. Accumulation of H-1 protein on chromatin evidently fostered its condensation resulting in the pathology described.     

Involvement of Cenp-F in interphase chromatin organization possibly through association with DNA-dependent protein kinase

Cenp-F (also named mitosin) is a 350-kDa human kinetochore protein important for the mitotic progression. It is also a nuclear matrix protein in interphase cells. Here, we showed that overexpression of N-terminal deletion mutants of Cenp-F containing the C-terminal 112 residues induced chromatin condensation into numerous aggregates of varying sizes in interphase nucleus, colocalizing with the exogenous proteins. In situ hybridization using whole chromosome painting probes indicated that the chromatin aggregates were not prematurely condensed individual chromosomes. Neither were they due to apoptosis. We provided evidence showing association of Cenp-F with certain regions of interphase chromatin fibers. Cenp-F associated with the DNA-dependent protein kinase (DNA-PK), a trimeric protein complex critical for genome homeostasis. Moreover, the DNA-PK association activity of Cenp-F mutants correlated with their ability to induce chromatin aggregation. These results imply a role of Cenp-F in organization of interphase chromatin through association and possibly regulation of DNA-PK.     

Cryoelectron microscopy of vitrified sections: a new challenge for the analysis of functional nuclear architecture

Cryoelectron microscopy of vitrified sections has become a powerful tool for investigating the fine structural features of cellular compartments. In the present study, this approach has been applied in order to explore the ultrastructural morphology of the interphase nucleus in different mammalian cultured cells. Rat hepatoma, Chinese hamster ovary and Potorus kidney cells were cryofixed by high-pressure freezing and the cryosections were examined at low temperature by transmission electron microscopy. Our results show that while the contrast of nuclear structural domains is remarkably homogeneous in hydrated sections, some of them can be recognised due to their characteristic texture. Thus, condensed chromatin appears finely granular and the perichromatin region contains rather abundant fibro-granular elements suggesting the presence of dispersed chromatin fibres and of perichromatin fibrils and granules. The interchromatin space looks homogeneous and interchromatin granules have not been identified under these preparative conditions. In the nucleolus, the most striking feature is the granular component, while the other parts of the nucleolar body, which appear less contrasted, are difficult to resolve. The nuclear envelope is easily recognisable with its regular perinuclear space and nuclear pore complexes. Our observations are discussed in the context of results obtained by other, more conventional electron microscopic methods.     

Redistribution of nuclear envelope associated antigen during the mitotic cycle.

Murine hybridomas were generated to DNA/tight binding proteins complex isolated from the residual nuclear structure following a procedure analogous to that yielding "empty" shells of nuclear envelope. A monoclonal antibody designated 2A8 was selected because of its differential immunostaining of mitotic cells of a synchronized mouse fibroblast cell culture L-929. The target antigen was rendered insoluble by a sequence of extractions of isolated nuclei of diverse cell types with detergents, urea, DNase I and alkali thus reproducing some solubility properties of proteins constituting an operationally defined residual nuclear matrix. The cognate polypeptide was localized on a subset of proteins of Mr 58-65 kDa, 70 kDa in isolated fibroblast nuclear matrices. The functional implication of the antigen in mitosis-related disassembly-assembly process of the nuclear matrix/envelope was detected. At prophase the antibody decorated the nuclear periphery and nuclear envelope fixed inward filaments. A fibrous network of cytoplasmic localization was stained in metaphase. At anaphase the antigen was dispositioned into peripheral fibrogranular clusters of polar orientation predominantly on one side of the nucleus. Proceeding to telophase a spreading fluorescence was manifested over the entire contour of the nuclear periphery to delineate the reforming nucleus. By immunogold electron microscopy of interphase cells the antigen was identified as evenly distributed in chromatin and interchromatin regions. At initiation of chromosome condensation in mitosis the label was detected predominantly in the chromosomal area.     

Characterization of DIP1, a novel nuclear protein in Drosophila melanogaster

We have recently identified in Drosophila melanogaster a new gene encoding a nuclear protein, DIP1. Here we report the developmental expression and the finding that DIP1 subcellular localization is in the nucleus and at the nuclear periphery during interphase in embryos. Interestingly, in humans, DIP1 antibody identified signals in nuclei from cultured cells and reacted with a rough 30kDa protein in Western blotting experiments, demonstrating evolutionary conservation.     

Nuclear distributions of NUP62 and NUP214 suggest architectural diversity and spatial patterning among nuclear pore complexes

The shape of nuclei in many adherent cultured cells approximates an oblate ellipsoid, with contralateral flattened surfaces facing the culture plate or the medium. Observations of cultured cell nuclei from orthogonal perspectives revealed that nucleoporin p62 (NUP62) and nucleoporin 214 (NUP214) are differentially distributed between nuclear pore complexes on the flattened surfaces and peripheral rim of the nucleus. High resolution stimulated emission depletion (STED) immunofluorescence microscopy resolved individual NPCs, and suggested both heterogeneity and microheterogeneity in NUP62 and NUP214 immunolabeling among in NPC populations. Similar to nuclear domains and interphase chromosome territories, architectural diversity and spatial patterning of NPCs may be an intrinsic property of the nucleus that is linked to the functions and organization of underlying chromatin.     

NuMA interaction with chromatin is vital for proper chromosome decondensation at the mitotic exit

NuMA is an abundant long coiled-coil protein that plays a prominent role in spindle organization during mitosis. In interphase, NuMA is localized to the nucleus and hypothesized to control gene expression and chromatin organization. However, because of the prominent mitotic phenotype upon NuMA loss, its precise function in the interphase nucleus remains elusive. Here, we report that NuMA is associated with chromatin in interphase and prophase but released upon nuclear envelope breakdown (NEBD) by the action of Cdk1. We uncover that NuMA directly interacts with DNA via evolutionarily conserved sequences in its C-terminus. Notably, the expression of the DNA-binding–deficient mutant of NuMA affects chromatin decondensation at the mitotic exit, and nuclear shape in interphase. We show that the nuclear shape defects observed upon mutant NuMA expression are due to its potential to polymerize into higher-order fibrillar structures. Overall, this work establishes the spindle-independent function of NuMA in choreographing proper chromatin decompaction and nuclear shape by directly associating with the DNA.