Changes in distribution of nuclear matrix antigens during the mitotic cell cycle

We examined the distribution of nonlamin nuclear matrix antigens during the mitotic cell cycle in mouse 3T3 fibroblasts. Four monoclonal antibodies produced against isolated nuclear matrices were used to characterize antigens by the immunoblotting of isolated nuclear matrix preparations, and were used to localize the antigens by indirect immunofluorescence. For comparison, lamins and histones were localized using human autoimmune antibodies. At interphase, the monoclonal antibodies recognized non-nucleolar and nonheterochromatin nuclear components. Antibody P1 stained the nuclear periphery homogeneously, with some small invaginations toward the interior of the nucleus. Antibody I1 detected an antigen distributed as fine granules throughout the nuclear interior. Monoclonals PI1 and PI2 stained both the nuclear periphery and interior, with some characteristic differences. During mitosis, P1 and I1 were chromosome-associated, whereas PI1 and PI2 dispersed in the cytoplasm. Antibody P1 heavily stained the periphery of the chromosome mass, and we suggest that the antigen may play a role in maintaining interphase and mitotic chromosome order. With antibody I1, bright granules were distributed along the chromosomes and there was also some diffuse internal staining. The antigen to I1 may be involved in chromatin/chromosome higher-order organization throughout the cell cycle. Antibodies PI1 and PI2 were redistributed independently during prophase, and dispersed into the cytoplasm during prometaphase. Antibody PI2 also detected antigen associated with the spindle poles.     

Dynamics of the spatial organization of the chromosome set in cells of <Emphasis Type="Italic">Drosophila melanogaster</Emphasis> imaginal disks normally and under the action of the tumor-inducing mutation <Emphasis Type="Italic">Merlin</Emphasis>

Fluorescence of H3-p histone and DAPI was studi ed at different stages of interphase and mitosis in cells of imaginal disks of third-instar Drosophila melanogaster larvae. Three stages differing in the spatial organization of the chromosome set in mitosis were revealed. At the first stage (prophase, prometaphase), the histone 3 phosphorylation level rises, and the volume occupied by the chromosome set in the nucleus increases. The distinctive features of the second stage (metaphase) are a gradual decrease in the histone 3 phosphorylation (the density of phosphorylation remaining constant) and a reduction of the volume occupied by the chromosome set. At the third stage (anaphase, telophase), the intensity and density of the signal from H3-p histone decrease, and the volume occupied by the chromosome set reduces. At this stage, in Mer ⁴ larvae, in contrast to the control strain, the cells prematurely pass from anaphase into telophase. In addition, a subpopulation of cells with an abnormally large volume of nuclear DNA during the G₁ period was revealed in Mer ⁴ larvae. The cells of this subpopulation do not enter into the DNA synthesis and quit the cycle.     

The chromosome periphery during mitosis

A complex structure, visible by electron microscopy, surrounds each chromosome during mitosis. The organization of this structure is distinct from that of the chromosomes and the cytoplasm. It forms a perichromosomal layer that can be isolated together with the chromosomes. This layer covers the chromosomes except in centromeric regions. The perichromosomal layer includes nuclear and nucleolar proteins as well as ribonucleoproteins (RNPs). The list of proteins and RNAs identified includes nuclear matrix proteins (perichromin, peripherin), nucleolar proteins (perichro-monucleolin, Ki-67 antigen, B23 protein, fibrillarin, p103, p52), ribosomal proteins (S1) and snRNAs (U3 RNAs). Only limited information is available about how and when the perichromosomal layer is formed. During early prophase, the proteins extend from the nucleoli towards the periphery of the nucleus. Thin cordon-like structures reach the nuclear envelope delimiting areas in which chromosomes condense. At telophase, the proteins are associated with the part of the chromosomes remaining condensed and accumulate in newly formed nucleoli in regions where chromatin is already decondensed. The perichromosomal layer contains several different classes of proteins and RNPs and it has been attributed various roles: (1) in chromosome organization, (2) as a barrier around the chromosomes, (3) involvement in compartmentation of the cells in prophase and telophase and (4) a binding site for chromosomal passenger proteins necessary to the early process of nuclear assembly.     

Location of nuclear antigen(s) recognized by DSB389 MAb, a monoclonal antibody against desmin, observed by confocal laser scanning fluorescence microscopy

The location of antigen(s) of DSB389 MAb, a monoclonal antibody which was raised against an intermediate filament protein, desmin, was investigated in HeLa S3 cells by indirect immuno-electron microscopy and by confocal laser scanning fluorescence microscopy. At interphase, the antigen(s) locates mainly in the intra-nuclear space adjacent to chromatin and sometimes near the nuclear periphery. At mitosis, they locate at the periphery of the chromosomes -first at each chromosome, then around the mass of chromosomes. The antigen(s) are thought to be a component of one type of nuclear matrix which is present outside both chromatin and chromosomes and which has some structural role(s) in organizing them in the nucleus or in the nuclear region.     

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.     

Kid-mediated chromosome compaction ensures proper nuclear envelope formation

Toward the end of mitosis, neighboring chromosomes gather closely to form a compact cluster. This is important for reassembling the nuclear envelope around the entire chromosome mass but not individual chromosomes. By analyzing mice and cultured cells lacking the expression of chromokinesin Kid/kinesin-10, we show that Kid localizes to the boundaries of anaphase and telophase chromosomes and contributes to the shortening of the anaphase chromosome mass along the spindle axis. Loss of Kid-mediated anaphase chromosome compaction often causes the formation of multinucleated cells, specifically at oocyte meiosis II and the first couple of mitoses leading to embryonic death. In contrast, neither male meiosis nor somatic mitosis after the morula-stage is affected by Kid deficiency. These data suggest that Kid-mediated anaphase/telophase chromosome compaction prevents formation of multinucleated cells. This protection is especially important during the very early stages of development, when the embryonic cells are rich in ooplasm.     

A class of nonribosomal nucleolar components is located in chromosome periphery and in nucleolus-derived foci during anaphase and telophase

The subcellular location of several nonribosomal nucleolar proteins was examined at various stages of mitosis in synchronized mammalian cell lines including HeLa, 3T3, COS-7 and HIV-1 Rev-expressing CMT3 cells. Nucleolar proteins B23, fibrillarin, nucleolin and p52 as well as U3 snoRNA were located partially in the peripheral regions of chromosomes from prometaphase to early telophase. However, these proteins were also found in large cytoplasmic particles, 1–2 μm in diameter, termed nucleolus-derived foci (NDF). The NDF reached maximum numbers (as many as 100 per cell) during mid- to late anaphase, after which their number declined to a few or none during late telophase. The decline in the number of NDF approximately coincided with the appearance of prenucleolar bodies and reforming nucleoli. The HIV-1 Rev protein and a mutant Rev protein defective in its nuclear export signal were also found in the NDF. The mutant Rev protein precisely followed the pattern of localization of the above nucleolar proteins, whereas the wild-type Rev did not enter nuclei until G1 phase. The nucleolar shuttling phosphoprotein Nopp 140 did not follow the above pattern of localization during mitosis: it dispersed in the cytoplasm from prometaphase through early telophase and was not found in the NDF. Although the NDF and mitotic coiled bodies disappeared from the cytoplasm at approximately the same time during mitosis, protein B23 was not found in mitotic coiled bodies, nor was p80 coilin present in the NDF. These results suggest that a class of proteins involved in preribosomal RNA processing associate with chromosome periphery and with NDF as part of a system to conserve and deliver preexisting components to reforming nucleoli during mitosis. Edited by: S. A. Gerbi     

The ultrastructure of mitosis during sporangiogenesis inWoronina pythii (Plasmodiophoromycetes)

Summary Mitotic divisions during sporangiogenous plasmodial cleavage inWoronina pythii were studied with transmission electron microscopy. We conclude that these nuclear divisions (e.g., transitional nuclear division, and sporangial mitoses) share basic similarities with the cruciform nuclear divisions inW. pythii and other plasmo-diophoraceous taxa. The major distinction appeared to be the absence of nucleoli during sporangial mitosis and the presence of nucleoli during cruciform nuclear division. The similarities were especially evident with regard to nuclear envelope breakdown and reformation. The mitotic divisions during formation of sporangia were centric, and closed with polar fenestrae, and characterized by the formation of intranuclear membranous vesicles. During metaphase, anaphase, and telophase, these vesicles appeard to bleb from the inner membrane of the original nuclear envelope and appeared to coalesce on the surface of the separating chromatin masses. By late telophase, the formation of new daughter nuclear envelopes was complete, and original nuclear envelope was fragmented. New observation pertinent to the mechanisms of mitosis in thePlasmodiophoromycetes include a evidence for the incorporation of membrane fragments of the original nuclear envelope into new daughter nuclear envelopes, and b the change in orientation of paired centrioles during sporangial mitosis.     

Immunocytochemical localization of the major polypeptides of the nuclear pore complex-lamina fraction. Interphase and mitotic distribution

This laboratory has previously isolated a fraction from rat liver nuclei consisting of nuclear pore complexes associated with the proteinaceous lamina which underlies the inner nuclear membrane. Using protein eluted from sodium dodecyl sulfate (SDS) gels, we have prepared antibodies in chickens to each of the three predominant pore complex- lamina bands. Ouchterlony double diffusion analysis shows that each of these individual bands cross-reacts strongly with all three antisera. In immunofluorescence localization performed on tissue culture cells with these antibodies, we obtain a pattern of intense staining at the periphery of the interphase nucleus, with little or no cytoplasmic reaction. Electron microscope immunoperoxidase staining of rat liver nuclei with these antibodies labels exclusively the nuclear periphery. Furthermore, reaction occurs in areas which contain the lamina, but not at the pore complexes. While our isolation procedure extracts the internal contents of nuclei completely, semiquantitative Ouchterlony analysis shows that it releases negligible amounts of these lamina antigens. Considered together, our results indicate that these three bands represent major components of a peripheral nuclear lamina, and are not structural elements of an internal "nuclear protein matrix." Fluorescence microscopy shows that the perinuclear interphase localization of these lamina proteins undergoes dramatic changes during mitosis. Concomitant with nuclear envelope disassembly in prophase, these antigens assume a diffuse localization throughout the cell. This distribution persists until telophase, when the antigens become progressively and completely localized at the surface of the daughter chromosome masses. We propose that the lamina is a biological polymer which can undergo reversible disassembly during mitosis.     

Autoradiographic and cytophotometric study of DNA synthesis in the oocytes of the domestic hen at the preleptotene prophase stage of meiosis

3H-thymidine incorporation into the fowl oocytes was established radioautographically at the middle preleptotene, when chromosomes are condensed and associated in the complex chromocenters. According to the cytophotometry of Feulgen stained oocyte nuclei, their DNA value increases during preleptotene from 2 to 4c. So, the DNA synthesis observed is characteristic of chromosome reduplication, rather than of nuclear organizer amplification. The preleptotene should be considered as the initial stage of meiotic prophase because it involves spiralization and individualization of chromosomal threads. Both the analysis of literary data and of our own results enable us to conclude that in different species the meiotic chromosome reduplication may proceed in different periods between telophase of last gonial mitosis and the beginning of homologous chromosome conjugation.     

Entamoeba histolytica: microtubule movement during mitosis

The movement of microtubules (MTs) during nuclear division of Entamoeba histolytica was ultrastructurally studied. Regarding this MT movement, five stages of mitosis could be defined: prophase, metaphase, anaphase A, anaphase B, and telophase. In early stages of mitosis, chromatinic material appeared condensed, and MTs were detected in the center of the nucleus. Later, MTs seemed to grow from an electron-dense body located in the center of the nucleus. This body might be the microtubule organizing center, which organized the MTs, first in a lateral way, and later to form the mitotic spindle, which was made of a bundle of MTs joined by their ends. This junction of MTs to themselves could also be observed in cross-sections. The last stage of mitosis was the nuclear separation. Two different morphological types of intranuclear vesicles were also observed, which seemed to have different types of membrane. Both intranuclear vesicles were present during nuclear division, generally in clusters, and located close to the nuclear periphery.     

The use of field emission in-lens scanning electron microscopy to study the steps of assembly of the nuclear envelope in vitro.

At mitosis the nuclear envelope (NE) is disassembled to allow chromosome separation. In telophase it is reassembled as the chromosomes decondense. Cell-free extracts of Xenopus eggs have been used extensively to study assembly of the NE and the nuclear pore complexes (NPCs), providing several models for the steps involved. The NE is a surface structure which in cell-free extracts is easily exposed. It is appropriate, therefore, to use a surface imaging technique to study NE dynamics. Field emission in-lens scanning electron microscopy (FEISEM) provides the opportunity to image surfaces, directly, and to visualise details of structures such as the NPC. Here we show the feasibility and value of FEISEM to study the steps of NE formation. Nuclei have been assembled in vitro and fixed at different time points during assembly, followed by conductive staining, platinum coating, and visualisation by FEISEM. Changes on the nuclear surface with time are shown. Details of the surface of chromatin and the cytoplasmic face of NPC structure are demonstrated without the need to isolate the structures from the nucleus.     

The proliferating cell marker monoclonal antibody Ki-67 recognizes specific antigens associated with the nuclear envelope of the early Drosophila embryo

Summary— Immunofluorescence and immunoelectron microscopy indicated that the antibody raised against the nuclear antigen Ki-67 of mammalian cells recognized antigenic determinants of early Drosophila embryos, localized on the outside of the nuclear envelope. Hence, the nuclear envelope of Drosophila appears to share a similar epitope with the chromosome scaffold of mitotic mammalian cells. With the progression of mitosis the antigen persisted around the mitotic spindle region and was also found in the pole regions at metaphase and anaphase. The antibody also stained the equatorial regions of the spindles from anaphase to late telophase. The antibody may therefore be used as a biochemical marker of the nuclear envelope for studying nuclear membrane biogenesis and behavior during the mitotic divisions of the Drosophila embryo.     

Ultrastructure of Chilomonas paramecium and the phylogeny of the cryptoprotists

The ultrastructure of the cryptoprotist Chilomonas paramecium is reviewed and compared to earlier accounts. Distinctive features include a complex cytoskeleton which defines the cell organization and interconnects cell components; trichocysts which resemble those in other cryptoprotists; and two non-photosynthetic plastids. During mitosis there is partial dispersal of the nuclear envelope early in prophase but some remains at the nuclear periphery throughout mitosis. At metaphase chromosomes are arranged on the longitudinal axis of an elongated elliptical nucleus. During telophase the chromosomes decondense and the nuclear envelope reforms. Cell structure is compared with that in other cryptoprotists, and origin of this taxon of algae is discussed.     

The use of field emission in-lens scanning electron microscopy to study the steps of assembly of the nuclear envelope in vitro

At mitosis the nuclear envelope (NE) is disassembled to allow chromosome separation. In telophase it is reassembled as the chromosomes decondense. Cell-free extracts of Xenopus eggs have been used extensively to study assembly of the NE and the nuclear pore complexes (NPCs), providing several models for the steps involved. The NE is a surface structure which in cell-free extracts is easily exposed. It is appropriate, therefore, to use a surface imaging technique to study NE dynamics. Field emission in-lens scanning electron microscopy (FEISEM) provides the opportunity to image surfaces, directly, and to visualise details of structures such as the NPC. Here we show the feasibility and value of FEISEM to study the steps of NE formation. Nuclei have been assembled in vitro and fixed at different time points during assembly, followed by conductive staining, platinum coating, and visualisation by FEISEM. Changes on the nuclear surface with time are shown. Details of the surface of chromatin and the cytoplasmic face of NPC structure are demonstrated without the need to isolate the structures from the nucleus.     

Ultrastructural study on mitosis and cytokinesis in Scytosiphon lomentaria zygotes (Scytosiphonales,Phaeophyceae) by freeze-substitution

Summary. The ultrastructure of mitosis and cytokinesis in Scytosiphon lomentaria (Lyngbye) Link zygotes was studied by freeze fixation and substitution. During mitosis, the nuclear envelope remained mostly intact. Spindle microtubules (MTs) from the centrosome passed through the gaps of the nuclear envelope and entered the nucleoplasm. In anaphase and telophase, two daughter chromosome masses were partially surrounded with endoplasmic reticulum. After telophase, the nuclear envelope was reconstructed and two daughter nuclei formed. Then, several large vacuoles occupied the space between the daughter nuclei. MTs from the centrosomes extended toward the mid-plane between two daughter nuclei, among the vacuoles. At that time, Golgi bodies near the centrosome actively produced many vesicles. Midway between the daughter nuclei, small globular vesicles and tubular cisternae accumulated. These vesicles derived from Golgi bodies were transported from the centrosome to the future division plane. Cytokinesis then proceeded by fusion of these vesicles, but not by a furrowing of the plasma membrane. After completion of the continuity with the plasma membrane, cell wall material was deposited between the plasma membranes. The tubular cisternae were still observed at the periphery of the newly formed septum. Microfilaments could not be observed by this procedure. We conclude that cytokinesis in the brown algae proceeds by fusion of Golgi vesicles and tubular cisternae, not by a furrowing of the plasma membrane. Received September 12, 2001 Accepted November 12, 2001     

Ultrastructure of mitosis inAmoeba proteus

Summary The fine structure ofAmoeba proteus nuclei has been studied during interphase and mitosis. The interphase nucleus is discoidal, the nuclear envelope is provided with a honeycomb layer on the inside. There are numerous nucleoli at the periphery and many chromatin filaments and nuclear helices in the central part of nucleus.In prophase the nucleus becomes spherical, the numerous chromosomes are condensed, and the number of nucleoli decreases. The mitotic apparatus forms inside the nucleus in form of an acentric spindle. In metaphase the nuclear envelope loses its pore complexes and transforms into a system of rough endoplasmic reticulum cisternae (ERC) which separates the mitotic apparatus from the surrounding cytoplasm; the nucleoli and the honeycomb layer disappear completely. In anaphase the half-spindles become conical, and the system of ERC around the mitotic spindle persists. Electron dense material (possibly microtubule organizing centers—MTOCs) appears at the spindle pole regions during this stage. The spindle includes kinetochore microtubules attached to the chromosomes, and non-kinetochore ones which pierce the anaphase plate. In telophase the spindle disappears, the chromosomes decondense, and the nuclear envelope becomes reconstructed from the ERC. At this stage, nucleoli can already be revealed with the light microscope by silver staining; they are visible in ultrathin sections as numerous electron dense bodies at the periphery of the nucleus.The mitotic chromosomes consist of 10 nm fibers and have threelayered kinetochores. Single nuclear helices still occur at early stages of mitosis in the spindle region.     

Analysis of spindle microtubule organization in untreated and taxol-treated PtK1 cells.

Taxol, a microtubule stabilizing agent, has been used to study changes in spindle microtubule organization during mitosis. PtK₁ cells have been treated with 5 μg/ml taxol for brief periods to determine its effect on spindle architecture. During prophase taxol induces microtubules to aggregate, particularly evident in the region between the nucleus and cell periphery. Taxol induces astral microtubule formation in prometaphase and metaphase cells concomitant with a reduction in spindle length. At anaphase taxol induces an increase in length in astral microtubules and reduces microtubule length in the interzone. Taxol-treated telophase cells show a reduction in the rate of furrowing and astral microtubules lack a discrete focus and are arranged more diffusely on the surface of the nuclear envelope. In summary, taxol treatment of cells prior to anaphase produces an increase in astral microtubules, a reduction in kinetochore microtubules and a decrease in spindle length. Brief taxol treatments during anaphase through early G₁ promotes stabilization of microtubules, an increase in the length of astral microtubules and a delayed rate of cytokinesis.