Localization of nuclear matrix core filament proteins at interphase and mitosis.

The gentle removal of chromatin uncovers a nuclear matrix consisting of two parts: a nuclear lamina connected to the intermediate filaments of the cytoskeleton and an internal matrix of thick, polymorphic fibers connecting the lamina to masses in the nuclear interior. This internal nuclear matrix can be further fractionated to uncover a highly branched network of 9 nm and 13 nm core filaments retaining some enmeshed bodies. The core filament network retains most of the nuclear RNA, as well as the fA12RNP antigen, and may be the most basic or core element of internal nuclear structure. One high molecular weight protein component of the core filament network, the H1B2 antigen, is normally masked in the interphase nucleus and is uncovered as the chromatin condenses at mitosis. This protein is associated with a fibrogranular network surrounding and connected to the chromosomes. The core filament-associated fA12 antigen also becomes associated with this perichromosomal network. We propose that the core filament nuclear matrix structure may not completely disassemble at mitosis but, rather, that parts remain as a structural network connected to chromosomes and other mitotic structures. These mitotic networks may, in turn, serve as the core structures on which the nuclear matrices of daughter cells are built.     

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.     

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.     

A differential staining technique for simultaneous visualization of mitotic spindle and chromosomes in mammalian cells

A new technique has been devised for staining the mitotic spindle in mammalian cells while preserving spindle structure and chromosome number. The cells are trypsinized and fixed with a 3:1 methanol:acetic acid solution containing 4 mM MgCl2 and 1.5 mM CaCl2 at room temperature. The cells are then placed on slides and treated with 5% perchloric acid before staining with a 10% acetic acid solution containing safranin O and brilliant blue R. The preserved spindles appear dark blue against a light cytoplasmic background with chromosomes stained bright red. Individual chromosomes and chromatids are clearly visible. Positioning of the chromosomes relative to the spindle apparatus is readily ascertained allowing easy study of mitotic spindle and chromosome behavior.     

A Differential Staining Technique for Simultaneous Visualization of Mitotic Spindle and Chromosomes in Mammalian Cells

A new technique has been devised for staining the mitotic spindle in mammalian cells while preserving spindle structure and chromosome number. The cells are trypsinized and fixed with a 3:1 methanobacetic acid solution containing 4 mM MgCl₂ and 1.5 mM CaCl₂ at room temperature. The cells are then placed on slides and treated with 5% perchloric acid before staining with a 10% acetic acid solution containing safranin O and brilliant blue R. The preserved spindles appear dark blue against a light cytoplasmic background with chromosomes stained bright red. Individual chromosomes and chromatids are clearly visible. Positioning of the chromosomes relative to the spindle apparatus is readily ascertained allowing easy study of mitotic spindle and chromosome behavior.     

Spoke: a 120-kD protein associated with a novel filamentous structure on or near kinetochore microtubules in the mitotic spindle

We have characterized an antiserum that recognizes a single 120-kD protein in CHO cells which is soluble and cytoplasmically localized in interphase, but which is associated with a novel filamentous structure localized on or near kinetochore microtubules in mid-mitosis. These filaments, one per sister chromatid, run from near the mitotic spindle pole to within approximately 0.3 microns of each kinetochore. In metaphase, the staining pattern shows considerable substructure at light microscopy resolution, appearing as bright nodes or striations, often with a kinked or helical appearance. This overall localization pattern is retained throughout anaphase, with the filaments shortening as the chromosomes move toward the mitotic spindle poles. Also in anaphase, a separate ring-like structure lacking a tubulin-staining component appears near the spindle poles. As cells exit mitosis, the amount of this antigen in the cell decreases seven- to tenfold. The unusual staining pattern and the specific localization of this antigen on or near kinetochore microtubules in mid-mitosis indicate that the 120-kD protein defines or is associated with an important and previously unrecognized structural element of the mitotic spindle.     

Localization of actin filaments on mitotic apparatus in tobacco BY-2 cells

Actin filaments are among the major components of the cytoskeleton, and participate in various cellular dynamic processes. However, conflicting results had been obtained on the localization of actin filaments on the mitotic apparatus and their participation in the process of chromosome segregation. We demonstrated by using rhodamine-phalloidin staining, the localization of actin filaments on the mitotic spindles of tobacco BY-2 cells when the cells were treated with cytochalasin D. At prophase, several clear spots were observed at or near the kinetochores of the chromosomes. At anaphase, the actin filaments that appeared to be pulling chromosomes toward the division poles were demonstrated. However, as there was a slight possibility that these results might have been the artifacts of cytochalasin D treatment or the phalloidin staining, we analyzed the localization of actin filaments at the mitotic apparatus immunologically. We cloned a novel BY-2 -type actin cDNA and prepared a BY-2 actin antibody. The fluorescence of the anti-BY-2 actin antibody was clearly observed at the mitotic apparatus in both non-treated and cytochalasin D-treated BY-2 cells during mitosis. The facts that similar results were obtained in both actin staining with rhodamine-phalloidin and immunostaining with actin antibody strongly indicate the participation of actin in the organization of the spindle body or in the process of chromosome segregation. Furthermore, both filamentous actin and spindle bodies disappeared in the cells treated with propyzamide, which depolymerizes microtubules, supporting the notion that actin filaments are associated with microtubules organizing the spindle body.Hiroshi Yasuda and Katsuhiro Kanda contributed equally.     

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.     

Distribution of c-myc, c-myb, and Ki-67 antigens in interphase and mitotic human cells evidenced by immunofluorescence staining technique

Using specific antibodies and the immunofluorescence staining technique we found a similar subcellular distribution pattern of the cellular proto-oncogene proteins c-myc and c-myb in interphase and mitotic HL60 and Molt4 cells. Antibodies against c-myc as well as those against c-myb protein gave rise to a nuclear staining excluding the nucleoli. In mitotic cells both proteins are apparently not associated with the chromatin of the condensed chromosomes, but appear diffusely distributed throughout the cytoplasm. In contrast, immunostaining using the proliferation marker antibody Ki-67 yielded in both cell lines several prominent specks in the nucleus and a weak finely dispersed staining throughout the nucleoplasm. No fluorescence was detectable in the cytoplasm. In dividing cells Ki-67 immunofluorescence was found to be associated with the surface of the chromosomes. The functional significance of the different localizations of the proteins is discussed in light of what is currently known about nuclear antigens.     

Distribution of a matrix component of the midbody during the cell cycle in Chinese hamster ovary cells

Monoclonal antibodies were raised against isolated spindles of CHO (Chinese hamster ovary) cells to probe for molecular components specific to the mitotic apparatus. One of the antibodies, CHO1, recognized an antigen localized to the midbody during mitosis. Immunofluorescence staining of metaphase cells showed that although the total spindle area was labeled faintly, the antigen corresponding to CHO1 was preferentially localized in the equatorial region of the spindle. With the progression of mitosis, the antigen was further organized into discrete short lines along the spindle axis, and eventually condensed into a bright fluorescent dot at the midzone of the intercellular bridge between two daughter cells. Parallel immunostaining of tubulin showed that the CHO1-stained area corresponded to the dark region where microtubules are entrapped by the amorphous dense matrix components and possibly blocked from binding to tubulin antibody. Immunoblot analysis indicated that CHO1 recognized two polypeptides of mol wt 95,000 and 105,000. The immunoreaction was always stronger in preparations of isolated midbodies than in mitotic spindle fractions. The protein doublet was retained in the particulate matrix fraction after Sarkosyl extraction (Mullins, J. M., and J. R. McIntosh. 1982. J. Cell Biol. 94:654-661), suggesting that CHO1 antigen is indeed a component of the dense matrix. In addition to the equatorial region of spindles and midbodies, CHO1 also stained interphase centrosomes, and nuclei in a speckled pattern that was cell cycle-dependent. Thus, the midbody appears to share either common molecular component(s) or a similar epitope with interphase centrosomes and nuclei.     

Characterization of a family of nuclear and chromosomal proteins identified by a monoclonal antibody

A monoclonal antibody (3C5) isolated from a mouse immunized with human chromatin stained the nuclei of all cultured cell types tested by indirect immunofluorescence. Experiments with HeLa and PtK1 cells demonstrated striking cell-cycle-related changes in the staining properties of the target antigen. A rapid increase in nuclear fluorescence was seen in prophase, with antigen located between the condensing chromosomes. In metaphase and anaphase cells antigen was present throughout the cytoplasm with the chromosomes apparently unstained. However, isolated metaphase chromosomes showed intense, peripheral staining. In telophase cells immunofluorescent staining was most intense among the decondensing chromosomes and by early G1 staining was predominantly nuclear. Nuclear fluorescence faded as cells progressed through interphase. By protein blotting and immunostaining, 3C5 recognized protein bands with subunit molecular weights of 130, 73, 50, 38, 32 and 22 to 25 kDa. These bands were present in all human and rodent cultured cell types tested. All bands were extracted by 6 M urea or 1% sodium dodecyl sulfate (SDS) but not by Triton X-100. Our results provide evidence against the involvement of a common carbohydrate moiety, in vitro proteolysis or non-specific cross reaction in this multi-banded pattern. The same family of proteins was detected in mitotic and interphase cells, suggesting that the changes in immunofluorescent staining through mitosis are due to changes in antigen accessibility. Subcellular fractionation experiments showed that all major bands were present in the nuclear fraction. Only two (50 and 32 kDa) were detected also in the post-nuclear membrane fraction and none were present in the soluble cytoplasmic fraction.(ABSTRACT TRUNCATED AT 250 WORDS)     

Inhibition of aurora B kinase blocks chromosome segregation,overrides the spindle checkpoint,and perturbs microtubule dynamics in mitosis

How kinetochores correct improper microtubule attachments and regulate the spindle checkpoint signal is unclear. In budding yeast, kinetochores harboring mutations in the mitotic kinase Ipl1 fail to bind chromosomes in a bipolar fashion. In C. elegans and Drosophila, inhibition of the Ipl1 homolog, Aurora B kinase, induces aberrant anaphase and cytokinesis. To study Aurora B kinase in vertebrates, we microinjected mitotic XTC cells with inhibitory antibody and found several related effects. After injection of the antibody, some chromosomes failed to congress to the metaphase plate, consistent with a conserved role for Aurora B in bipolar attachment of chromosomes. Injected cells exited mitosis with no evidence of anaphase or cytokinesis. Injection of anti-Xaurora B antibody also altered the microtubule network in mitotic cells with an extension of the astral microtubules and a reduction of kinetochore microtubules. Finally, inhibition of Aurora B in cultured cells and in cycling Xenopus egg extracts caused escape from the spindle checkpoint arrest induced by microtubule drugs. Our findings implicate Aurora B as a critical coordinator relating changes in microtubule dynamics in mitosis, chromosome movement in prometaphase and anaphase, signaling of the spindle checkpoint, and cytokinesis.     

Isolation of monoclonal antibodies to chromatin and preliminary characterization of target antigens

This paper describes the isolation of monoclonal antibodies to chromatin-associated protein antigens and their use in the characterization of such proteins by indirect immunofluorescence. Hybridomas were derived by fusion of the mouse myeloma Ag8653 with spleen cells from mice immunized with chromatin from human liver, rat liver or a human lymphoblastoid cell line. Hybrids were screened by solid-phase radioimmunoassay. The proportion of positive hybrids varied with the immunizing chromatin as follows: human liver 55/83, human lymphoblast 8/183 and rat liver 2/82. Fifteen antibodies derived from these fusions (7, 7 and 1 respectively) were subjected to further analysis. Most of these (11/13) were IgM and recognized both human and rat chromatin (12/15). Most of the target antigens were protease sensitive (8/13) and nuclease resistant. In fact the binding of five antibodies to lymphoblast chromatin was more than doubled by preincubation with DNAase I. The subcellular location of target antigens was examined by indirect immunofluorescence. Seven antibodies stained at least one of several cultured cell lines tested. Three gave staining patterns consistent with the in vivo association of the target antigen with chromatin recognizing, respectively, the interphase nucleus and metaphase chromosomes, the nuclear periphery and the mitotic spindle and other microtubule-containing structures. The remaining four all recognized antigens associated with the intermediate filament network.     

Nuclear matrix proteins are carried within peripheral material of mitotic chromosomes

Immunofluorescent analysis has shown that autoimmune sera M-222 and M-260 are bound to interphase nuclei and mitotic chromosomes of the pig embryo kidney cell culture. The fluorescent stain is diffuse in nuclei and forms a thin fluorescent area around each nucleolus, whereas the nucleolar cores are unstained. The periphery of each mitotic chromosome is stained distinctly. After removal of histones and DNA by the cell treatment with 2 M NaCl and DNase I, the Hoechst 33258 staining of nuclei and chromosomes disappears completely, whereas the pattern of staining with antibodies is not changed as compared with normal cells. Electron microscopy revealed in interphase nuclei after such treatment only lamina, residual nucleoli, and the intranuclear matrix network, and antibodies are bound just to these elements. Molecular mass of proteins bound to these antibodies was determined by immunoblotting. Serum M-260 contained antibodies to a single 65 kDa polypeptide, whereas antibodies to two polypeptides of 47 and 65 kDa were found in M-222. After chromatin removal and revealing nuclear protein matrix, M-222 binds only to 65 kDa polypeptides. Thus, peripheral chromosomal material is involved in transfer of the nuclear matrix polypeptide to daughter nuclei during mitosis.     

Specific attachment of nuclear-mitotic apparatus protein to metaphase chromosomes and mitotic spindle poles: possible function in nuclear reassembly

NuMA protein is the largest, abundant, primate-specific chromosomal protein. The protein was purified from HeLa cells and monospecific monoclonal antibodies were prepared that react exclusively with NuMA protein in immunoblot analysis. These antibodies were used to define the intracellular location and properties of NuMA protein. Using indirect immunofluorescence, NuMA protein was detected only in the nucleus of interphase cells and on the chromosomes in mitotic cells. One class of monoclonal antibody called the 2E4-type antibody, caused NuMA protein (or a complex of proteins including NuMA) to be released from its binding site on metaphase or anaphase chromosomes. The separation of NuMA protein from chromosomes was observed either with the immunofluorescence assay or in electrophoretic analyses of proteins released from isolated metaphase chromosomes after reaction with 2E4 antibody. The immunofluorescence studies also showed that after release of the NuMA protein from chromosomes of metaphase or anaphase cells, the protein bound specifically to the polar region of the mitotic spindle. It was shown that exogenously added NuMA antigen/antibody complex bound only to the mitotic spindle poles of permeabilized primate cells and not to the spindle poles of other mammalian cells, thus demonstrating the specificity of the spindle-pole interaction. The antibody mediated transfer of NuMA from chromosomes to poles was blocked when the chromosomes were treated with cross-linking fixatives. Results suggest that the NuMA protein has specific attachment sites on both metaphase chromosomes and mitotic spindle poles (the site where post-mitotic nuclear assembly occurs). A model is proposed suggesting that a protein having such dual binding sites could function during nuclear reassembly to link mitotic chromosomes into the reforming nucleus.     

Cell-cycle modulation of MPM-2-specific spindle pole body phosphorylation in Aspergillus nidulans

MPM-2 is a monoclonal antibody that interacts with mitosis-specific phosphorylated proteins in many different organisms. Immunocytochemistry of tissue culture cells has shown that MPM-2 stains centrosomes, chromosomes, kinetochores, and spindles. In this paper, we demonstrate that MPM-2 staining colocalizes with the spindle pole body (SPB) of Aspergillus nidulans and that SPB staining varies during the mitotic cycle. In an unsynchronized population, about one-fourth to one-third of the cells stain with MPM-2 at the spindle plaques or SPBs. Nuclei in mitosis have two SPBs localized at the ends of the spindle, both of which stain with MPM-2. To determine when MPM-2 staining appears, we have examined the effects of temperature-sensitive cell-cycle mutations that block nuclear division in S or G2. Only a very small fraction of cells blocked in S-phase stain with MPM-2. In contrast, a large fraction of cells blocked in G2 stain brightly at the SPB. These data suggest that MPM-2 reactivity of SPBs appears in G2. Moreover, the fact that cells blocked in G2 showed MPM-2 staining but no spindles suggests that reactivity of SPBs occurs prior to mitosis but is not sufficient to trigger spindle formation. When G2-blocked cells were downshifted to permissive temperature, they generated a mitotic spindle with an SPB at each end. Both SPBs stained with MPM-2 in all of the mitotic cells.     

T antigen banding on chromosomes of simian virus 40 infected muntjac cells.

Chromosomes were prepared from mitotic munjac cells 48 to 72 h after infection with SV40 virus. When stained for SV40 T antigen by indirect immunofluorescence, all chromosomes within an infected cell were fluorescent, indicating the presence of T antigen. Furthermore, the chromosomes were not uniformly stained but appeared to have regions of high and low fluorescence intensity. A variety of controls showed that the banding patterns are specific and highly reproducible and may indeed reflect the binding sites of T antigen. The bright, fluorescent bands T antigen were found to correspond to bands visualized by trypsin-Giesma staining (G-bands) and also by quinacrine staining (Q-bands). Current knowledge of chromosome banding indicates that Q-bands reflect the distribution of AT-rich regions along the chromosome. From the DNA sequence of SV40, it is known that one of the T antigen binding sites contains AT-rich sequences; thus, T antigen banding might be due to the base-specific binding of T antigen to chromatin. In addition, these bands have been implicated as centers for chromosome condensation and units in control of DNA replication. While the functional significance of T antigen binding has yet to be determined, the SV40-muntjac system provides an unusual opportunity to study the interaction of a known regulatory protein with mammalian chromosomes.     

Bcl-2 is an integral component of mitotic chromosomes

We previously demonstrated that phospho-Thr56 Bcl-2 colocalizes with Ki-67 and nucleolin in nuclear structures in prophase cells and is detected on mitotic chromosomes in later mitotic phases. To gain insight into the fine localization of Bcl-2 on mitotic chromosomes, we further investigated Bcl-2 localization by immunostaining of Bcl-2 with known components of metaphase chromosomes and electron microscopic immunocytochemistry. Immunofluorescence analysis on HeLa mitotic cells together with chromatin immunoprecipitation assays showed that Bcl-2 is associated with the condensed chromatin. Co-immunostaining experiments performed on mitotic chromosome spreads demonstrated that Bcl-2 is not localized on the longitudinal axis of chromatids with the condensin complex, but partially colocalizes with histone H3 on some regions of the mitotic chromosome. Finally, most of the Bcl-2 staining overlaps with Ki-67 staining at the chromosome periphery. Bcl-2 localization at the periphery and over the mitotic chromosome was confirmed by immunoelectron microscopy on mitotic cells.Our results indicate that Bcl-2 is an integral component of the mitotic chromosome.     

A microtubule-associated protein antigen unique to mitotic spindle microtubules in PtK1 cells

Microtubule-associated proteins (MAPs) that copurify with tubulin through multiple cycles of in vitro assembly have been implicated as regulatory factors and effectors in the in vivo activity of microtubules. As an approach to the analysis of the functions of these molecules, a collection of lymphocyte hybridoma monoclonal antibodies has been generated using MAPs from HeLa cell microtubule protein as antigen. Two of the hybridoma clones secrete IgGs that bind to distinct sites on what appears to be a 200,000-dalton polypeptide. Both immunoglobulin preparations stain interphase and mitotic apparatus microtubules in cultured human cells. One of the clones (N-3B4.3.10) secretes antibody that reacts only with cells of human origin, while antibody from the other hybridoma (N-2B5.11.2) cross-reacts with BSC and PtK1 cells, but not with 3T3 cells. In PtK1 cells the N-2B5 antigen is associated with the microtubules of the mitotic apparatus, but there is no staining of the interphase microtubule array; rather, the antibody stains an ill-defined juxtanuclear structure. Further, neither antibody stains vinblastine crystals in either human or marsupial cells at any stage of the cell cycle. N-2B5 antibody microinjected into living PtK1 cells binds to the mitotic spindle, but does not cause a rapid dissolution of either mitotic or interphase microtubule structures. When injected before the onset of anaphase, however, the N-2B5 antibody inhibits proper chromosome partition in mitotic PtK1 cells. N-2B5 antibody injected into interphase cells causes a redistribution of MAP antigen onto the microtubule network.