The prometaphase configuration and chromosome order in early mitosis

We have examined early mitotic stages in HeLa cells, mouse 3T3 fibroblasts and mitogen-activated mouse lymphocytes by immunofluorescence labelling with anti-tubulin and anti-centromere. Chromatin organization was monitored with the DNA-specific fluorochrome Hoechst 33258. This approach has led us to identify a modified Rabl array of chromosomes and spindle microtubules early in mitosis that is distinct from that at metaphase, and which we have called 'the prometaphase configuration'. In the configuration, chromosomes are oriented so that telomeres are clustered at the outer surface, whereas centromeres are clustered inside the configuration, at the surface of a hollow spindle. Observations on cells earlier in mitosis indicate that the configuration is presaged by the spatial relationship between chromosomes and cytoplasmic microtubules in prophase and early prometaphase. We propose a model in which the prometaphase configuration represents an important step linking prophase and metaphase, serving to translate interphase spatial and intragenomic order into order at the metaphase plate

Alterations to nuclear architecture and genome behavior in senescent cells.

The organization of the genome within interphase nuclei, and how it interacts with nuclear structures is important for the regulation of nuclear functions. Many of the studies researching the importance of genome organization and nuclear structure are performed in young, proliferating, and often transformed cells. These studies do not reveal anything about the nucleus or genome in nonproliferating cells, which may be relevant for the regulation of both proliferation and replicative senescence. Here, we provide an overview of what is known about the genome and nuclear structure in senescent cells. We review the evidence that nuclear structures, such as the nuclear lamina, nucleoli, the nuclear matrix, nuclear bodies (such as promyelocytic leukemia bodies), and nuclear morphology all become altered within growth-arrested or senescent cells. Specific alterations to the genome in senescent cells, as compared to young proliferating cells, are described, including aneuploidy, chromatin modifications, chromosome positioning, relocation of heterochromatin, and changes to telomeres

Phosphorothioate antisense oligonucleotides induce the formation of nuclear bodies

Antisense oligonucleotides are powerful tools for the in vivo regulation of gene expression. We have characterized the intracellular distribution of fluorescently tagged phosphorothioate oligodeoxynucleotides (PS-ONs) at high resolution under conditions in which PS-ONs have the potential to display antisense activity. Under these conditions PS-ONs predominantly localized to the cell nucleus where they accumulated in 20-30 bright spherical foci designated phosphorothioate bodies (PS bodies), which were set against a diffuse nucleoplasmic population excluding nucleoli. PS bodies are nuclear structures that formed in cells after PS-ON delivery by transfection agents or microinjection but were observed irrespectively of antisense activity or sequence. Ultrastructurally, PS bodies corresponded to electron-dense structures of 150-300 nm diameter and resembled nuclear bodies that were found with lower frequency in cells lacking PS-ONs. The environment of a living cell was required for the de novo formation of PS bodies, which occurred within minutes after the introduction of PS-ONs. PS bodies were stable entities that underwent noticeable reorganization only during mitosis. Upon exit from mitosis, PS bodies were assembled de novo from diffuse PS-ON pools in the daughter nuclei. In situ fractionation demonstrated an association of PS-ONs with the nuclear matrix. Taken together, our data provide evidence for the formation of a nuclear body in cells after introduction of phosphorothioate oligodeoxynucleotides

Electron microscopy of nuclear matrices prepared in situ under oxidizing conditions

Nuclear matrices (NM) were prepared from mouse 3T3 fibroblasts attached to growth support film in the continuous presence of the disulfide cross-linking reagent, sodium tetrathionate. Intact cells and samples at each stage of NM preparation were fixed, embedded in Epon-Araldite, sectioned and stained conventionally with uranyl-lead. Nuclear size and shape changed little during extraction, but nuclei showed a gradual reduction in internal fibrogranular elements up to 1M NaCl, after which larger spaces were visible in the nucleoplasm. In contrast to similar samples prepared under reducing conditions in other studies, final NMs contained a highly ramified internal network of fibers and granular aggregates

Is DNA topoisomerase IIβ a nucleolar protein?

We have carried out immunofluorescence labelling of two human cell types, HeLa cells and peripheral blood lymphocytes, prepared by several different fixation/permeabilization protocols using a variety of antibodies against DNA Topoisomerase II (Topo II). We have found that the distribution of Topo IIα was overall similar during interphase and mitosis to tha

Centromeres reposition to the nuclear periphery during L6E9 myogenesis in vitro

To test the hypothesis that genome architecture in interphase is related to nuclear function, we have compared the disposition of centromeres in nuclei of undifferentiated rat L6E9 myoblasts with that in nuclei of L6E9 myotubes differentiated in vitro. Immunofluorescence labeling showed that centromeres repositioned to the nuclear periphery during differentiation, and