Chromosomes In Mitotic Cells Research Articles

P2P-R protein localizes to the nucleolus of interphase cells and the periphery of chromosomes in mitotic cells which show maximum P2P-R immunoreactivity.

P2P-R is a nuclear protein that can bind both p53 and Rb1. Its functions include roles in the control of RNA metabolism, apoptosis, and p53-dependent transcription. The expression of P2P-R also is repressed in G1 arrested terminally differentiated cells. The current studies therefore evaluated if P2P-R undergoes cell cycle-associated changes in its abundance and/or localization. Western blots show that relative to G0 quiescent cells, P2P-R protein levels are higher in populations of G2/M cells prepared by the physiological parasynchronization technique of serum deprivation followed by serum stimulation. More striking is the > 10-fold enrichment of P2P-R protein in specimens of highly purified mitotic cells prepared by the mitotic shake-select technique, or by synchrony with the mitotic spindle disruption agents nocodazole or vinblastine. These changes in P2P-R protein occur without a concomitant change in P2P-R mRNA expression suggesting that P2P-R immunoreactivity increases during mitosis. Confocal microscopy next established the localization of P2P-R to nucleoli in interphase cells and at the periphery of chromosomes in mitotic cells that lack nucleoli. The high levels of P2P-R localized to the periphery of chromosomes in mitotic cells suggest that P2P-R shares characteristics with other nucleolar proteins that associate with the periphery of chromosomes during mitosis. These include: nucleolin, B23, Ki67, and fibrillarin.

Novel complex integrating mitochondria and the microtubular cytoskeleton with chromosome remodeling and tumor suppressor RASSF1 deduced by in silico homology analysis, interaction cloning in yeast, and colocalization in cultured cells.

Availability of the complete sequence of the human genome and sequence homology analysis has accelerated new protein discovery and clues to protein function. Protein-protein interaction cloning suggests multisubunit complexes and pathways. Here, we combine these molecular approaches with cultured cell colocalization analysis to suggest a novel complex and a pathway that integrate the mitochondrial location and the microtubular cytoskeleton with chromosome remodeling, apoptosis, and tumor suppression based on a novel leucine-rich pentatricopeptide repeat-motif-containing protein (LRPPRC) that copurified with the fibroblast growth factor receptor complex. One round of interaction cloning and sequence homology analysis defined a primary LRPPRC complex with novel subunits cat eye syndrome chromosome region candidate 2 (CECR2), ubiquitously expressed transcript (UXT), and chromosome 19 open reading frames 5 (C19ORF5) but still of unknown function. Immuno, deoxyribonucleic acid (DNA), and green fluorescent protein (GFP) tag colocalization analyses revealed that LRPPRC appears in both cytosol and nuclei of cultured cells, colocalizes with mitochondria and beta-tubulin rather than with alpha-actin in the cytosol of interphase cells, and exhibits phase-dependent organization around separating chromosomes in mitotic cells. GFP-tagged CECR2B was strictly nuclear and colocalized with condensed DNA in apoptotic cells. GFP-tagged UXT and GFP-tagged C19ORF5 appeared in both cytosol and nuclei and colocalized with LRPPRC and beta-tubulin. Cells exhibiting nuclear C19ORF5 were apoptotic. Screening for interactive substrates with the primary LRPPRC substrates in the human liver complementary DNA library revealed that CECR2B interacted with chromatin-associated TFIID-associated protein TAFII30 and ribonucleic acid splicing factor SRP40, UXT bridged to CBP/p300-binding factor CITED2 and kinetochore-associated factor BUB3, and C19ORF5 complexed with mitochondria-associated NADH dehydrogenase I and cytochrome c oxidase I. C19ORF5 also interacted with RASSF1, providing a bridge to apoptosis and tumor suppression.

Characterization of a novel nucleolar protein that transiently associates with the condensed chromosomes in mitotic cells

We report the isolation and characterization of a murine gene encoding a conserved mammalian nucleolar protein. The protein, called Tsg118, has a predicted molecular mass of 59.4 kDa and a high content of basic amino acids. A homologous human gene was localized to chromosome 16p12.3. The Tsg118 protein is predominantly expressed in proliferating somatic cells and in male germ cells. Indirect immunofluorescence microscopy analysis using an affinity-purified anti-Tsg118 serum shows colocalization of Tsg118 and a known nucleolar protein, fibrillarin, to the dense fibrillar component of the nucleolus. The nucleolar localization of the Tsg118 protein appears to be temporally restricted to the interphase stages of the somatic cell cycle and to the meiotic phase of spermatogenesis. We find that the Tsg118 protein localizes to the nucleolus in both proliferating and serum-starved cells. Interestingly, as the nucleolar signal disappears in mitotic cells, the Tsg118 protein instead becomes associated with the surface of the condensed chromosomes.

Pictures in cell biology: A new marker for mitosis

Specific posttranslational modifications of histones, particularly acetylation and phosphorylation, have long been correlated with chromatin dynamics. Phosphorylation of histone H3, for example, correlates closely with mitosis[ 1 Gurley L.R. et al. Eur. J. Biochem. 1978; 84: 1-15 Crossref PubMed Scopus (404) Google Scholar , 2 Paulson J.R. Taylor S.S. J. Biol. Chem. 1982; 257: 6064-6072 Abstract Full Text PDF PubMed Google Scholar , 3 Allis C.D. Gorovsky M.A. Biochemistry. 1981; 20: 3828-3833 Crossref PubMed Scopus (65) Google Scholar ]. Recently, using antiserum that is highly selective for the phosphorylated (serine 10) form of H3, we have shown that this modification correlates closely with mitotic chromosome condensation[ 4 Hendzel M.J. et al. Chromosoma. 1997; 106: 348-360 Crossref PubMed Scopus (1511) Google Scholar , 5 Wei, Y. et al. Proc. Natl. Acad. Sci. U. S. A. (in press) Google Scholar ]. Fig. 1 shows HeLa cells doubly stained for phosphorylated H3 (red) and tubulin (green). The majority of cells show no detectable phosphorylated H3 signal, but metaphase chromosomes in mitotic cells are stained brightly. Similar specificity is observed with Caenorhabditis elegans (Fig. 2) or Drosophila melanogaster (Fig. 3) embryos. A tight correlation between H3 phosphorylation and mitosis has also been observed in Aspergillus nidulans (G. S. May, pers. commun.) and maize (E. Kaszas and Z. Cande, pers. commun.). Thus, mitotic H3 phosphorylation is a highly conserved event, making this antiserum a powerful in situ marker for mitotic cells. A rapid and transient phosphorylation of H3 also correlates with an immediate-early response to mitogens in mammalian cells[ 8 Mahadevan L.C. Willis A.C. Barratt M.J. Cell. 1991; 65: 775-783 Abstract Full Text PDF PubMed Scopus (374) Google Scholar ]. This antibody also recognizes mitogen-induced H3 phosphorylation (C. D. Allis, unpublished) and can therefore also be a useful reagent for studying this H3 phosphorylation outside mitosis. Fig. 2Confocal images of a Caenorhabditis elegans embryo stained with the antibody against phosphorylated histone H3 (a) and the DNA-specific dye propidium iodide (b). The two channels are merged in (c). The arrow denotes a cell in metaphase of mitosis, and the arrowhead marks a cell exhibiting a DNA morphology indicative of an early stage of pre-mitotic chromosome condensation (also see Ref. [6]). Images were kindly provided by Jason Lieb and Barbara Meyer of the Howard Hughes Medical Institute and the University of California, Berkeley, USA. View Large Image Figure Viewer Fig. 3Precellular blastoderm stage Drosophila embryos (synchronous divisions) were stained with the antibody against phosphorylated histone H3 (a, c) or DAPI for DNA (b, d). Note that the chromosomes of mitotic nuclei (a, b) stain with the antibody, whereas interphase nuclei (c, d) do not. Postgastrulation-stage embryos (asynchronous divisions) were stained with the antibody marking mitotic domains[7]at 200 (e) and 220 (f) min after egg deposition. Images were kindly provided by Bruce Edgar of the Fred Hutchinson Cancer Research Center, Seattle, WA, USA. View Large Image Figure Viewer

Two-step delivery of retroviruses to postmitotic, terminally differentiated cells.

Recombinant replication-defective retroviral vectors are currently the most commonly used vectors for introducing foreign genes into human cells in gene therapy protocols. Their genomes stably incorporate in the host chromosomes of mitotic cells, thus ensuring stable expression. However, the applications of retroviruses to gene therapy are limited by their inability to infect postmitotic cells such as muscle fibers. In an attempt to overcome such limitations, we have developed a novel two-step transduction protocol that allows integration and expression of retroviral genes in differentiated cells. We induced DNA synthesis in terminally differentiated cultured mouse myotubes derived from both established myogenic cell lines and from primary myoblasts. We infected the postmitotic cells with a recombinant replication-defective adenoviral vector encoding the SV40 large T antigen as a mitogen. Subsequently we transduced the adenovirus-infected cells with a Moloney retroviral vector bearing the LacZ gene. Histochemical analysis revealed the coincident expression of LacZ gene in those myotubes that had been induced to synthesize DNA.

Presence of tau in isolated nuclei from human brain

Previous studies have demonstrated that the microtubule-associated protein (MAP) tau is present in the axonal and somatodendritic compartment of neurons. In cultured primate cell lines, tau has been found localized to the NOR regions of the acrocentric chromosomes in mitotic cells and the dense fibrillar regions of nucleoli in interphase cells. We report here the presence of nuclear tau in nuclei isolated from fresh, frozen human frontal cortex. Using several monoclonal antibodies against tau, Tau-1, Tau 46.1, and 5132, we have established by both indirect immunofluorescence and Western blotting that tau is an integral component of nuclei isolated from Alzheimer's disease (AD) and pathologically normal control brains. Brain nuclear tau, like nuclear tau in primate cells, is insoluble in SDS and must first be extracted with formic acid prior to analysis by Western blot. Immunoblot analysis of isolated brain nuclei displays the characteristic ladder of tau proteins and demonstrates that all isoforms of tau are present. It is unclear whether levels of nuclear tau can be correlated to pathologic events in AD, but its insoluble nature along with reports of intranuclear PHFs warrant further studies of nuclear tau as a molecular candidate in the genesis of AD.

Differential expression of a phosphoepitope at the kinetochores of moving chromosomes.

A phosphorylated epitope is differentially expressed at the kinetochores of chromosomes in mitotic cells and may be involved in regulating chromosome movement and cell cycle progression. During prophase and early prometaphase, the phosphoepitope is expressed equally among all the kinetochores. In mid-prometaphase, some chromosomes show strong labeling on both kinetochores; others exhibit weak or no labeling; while in other chromosomes, one kinetochore is intensely labeled while its sister kinetochore is unlabeled. Chromosomes moving toward the metaphase plate express the phosphoepitope strongly on the leading kinetochore but weakly on the trailing kinetochore. This is the first demonstration of a biochemical difference between the two kinetochores of a single chromosome. During metaphase and anaphase, the kinetochores are unlabeled. At metaphase, a single misaligned chromosome can inhibit further progression into anaphase. Misaligned chromosomes express the phosphoepitope strongly on both kinetochores, even when all the other chromosomes of a cell are assembled at the metaphase plate and lack expression. This phosphoepitope may be involved in regulating chromosome movement to the metaphase plate during prometaphase and may be part of a cell cycle checkpoint by which the onset of anaphase is inhibited until complete metaphase alignment is achieved.

Characterization of the absence of an unique DNA-binding protein in senescent but not in their young growing and nongrowing counterparts provides the means to mark the final stage of the cellular aging process

In senescent fibroblasts, the incapability of cell replication is permanent, and may involve the irreversible loss of gene expressions potentiating the engagement in DNA replication. The initial attempt is to identify gene products that are permanently lost in irreversibly growth-arrested cells. In this article, we report the success in identifying a DNA-associated S6 antigen found in the nuclei of growing and growth-arrested young cells, but not in the nuclei of their senescent counterparts. The presence of the S6 antigen is uniform throughout the nucleoplasm, except in the regions of the nucleoli, and found to be associated with condensed chromosomes in mitotic cells. Treatment with RNase does not abolish the antibody staining activity in the nuclei, while treatment with DNase does remove the activity. Equal intensity of S6 antibody staining is observed in transformed, growing, and contact-inhibited young fibroblasts. Significant reduced level of S6 antibody staining activity is found in the nuclei of senescent fibroblasts, indicating the loss of the expression of this protein during cellular aging process. Immunoblotting assay shows that the S6 antigen is of 50 kDa and with a possibility of a 100 kDa as a dimeric precursor. Our results suggests that a permanent turning off of unique gene expression is associated with the onset of senescence or terminal differentiation, and this hypothesis is supported by the characterization of S6 antigen's absence in in vitro-aged fibroblasts.

The 180-kDa isoform of topoisomerase II is localized in the nucleolus and belongs to the structural elements of the nucleolar remnant

Monoclonal antibodies raised against two isoforms (170 and 150 180 kDa) of DNA topoisomerase II showed distinct fluorescence patterns in HeLa cells in different moments of the cell cycle ( C. Negri et al., 1992 , Exp. Cell Res. 200, 452–459). The ultrastructural distribution of the 150 180 -kDa isoform, which in immunofluorescence showed a localization into the nucleolar region, has been analyzed by electron microscopy with a gold-conjugated secondary antibody in HeLa and K562 cells. The results indicate that this isoform of the enzyme is exclusively localized in the nucleolus, mainly in the dense fibrillar component, while the nucleoplasm of interphase cells and the chromosomes of mitotic cells are completely negative. The antibody also reacts with the nucleolus of isolated nuclei and with the nucleolar remnant of purified nuclear matrices. A quantitative evaluation of the label distribution indicates that the percentage of label in the nucleolar remnant of isolated matrix is almost identical to that of the nucleolus in whole cells. The interaction with the insoluble proteins of the isolated nuclear matrix is also demonstrated by quantitative immunoblotting in which the MoAb specifically stains a unique band corresponding to the 150 180 -kDa isoform of topoisomerase II. The localization of the 150 180 -kDa isoform of topoisomerase II in the nucleolar remnant strongly suggests that it represents a structural element for the spatial organization and for the regulation of transcription of the ribosomal genes.

Chromosomal complements of two forms of Neanthes japonica (Polychaeta, Nereididae) with evidence of male-heterogametic sex chromosomes

Karyotypes of the brackish-water polychaeteNeanthes japonica (Izuka) collected from five rivers in Japan from 1984 to 1988 were examined with air-drying and flame-drying methods using materials consisting of regenerating tails, clumps of spermatogonia and youngN. japonica specimens (embryos, larvae or juveniles). A diploid number of 28 was determined in well-spread metaphase chromosomes of mitotic cells. The presence of an XX-XY (male heterogametic) sex chromosome system was established for the first time in polychaetes. The Y chromosome was larger than the X chromosome. Slight differences in karyotype were found between two forms (the small- and large-egg forms), which are very similar in adult morphology but can be distinguished by reproductive and developmental characteristics.

Association of ubiquitin-activating enzyme with HeLa cell chromosomes during mitosis.

Ubiquitin-activating enzyme (E1) is the first enzyme in the pathway leading to formation of ubiquitin-protein conjugates. Antibodies raised against E1 were affinity purified and used for immunostaining HeLa cells. Condensed chromosomes in mitotic cells were found to be strongly immunoreactive. Chromosomes from metaphase-arrested HeLa cells were isolated and chromosome-associated proteins were analyzed by Western blotting. E1 was detected in fractions containing isolated chromosomes. These results suggest that E1 is associated with condensed chromosomes during mitosis.

Trans-sensing effects from Drosophila to humans

In drosophila and other dipterian insects ,homologous pairing of chromosomes in mitotic cells is a well established phenomenon.Of particular interest here are the significance of homologous pairing for gene expression in drosophila and its wider implications for mammals .

Expression of the acidic nuclear immediate-early protein (IE1) of human cytomegalovirus in stable cell lines and its preferential association with metaphase chromosomes

Stable DNA-transfected Vero cell lines that express the major immediate-early nuclear antigen (IE68) of HCMV-(Towne) have been established. Immunofluorescence staining with monoclonal antibodies revealed that the protein was distributed either in a uniform diffuse nuclear pattern or as punctate nuclear granules in up to 80% of the cells in these cultures. In addition, 1 to 2% of the positive nuclei gave a distinctive staining pattern suggesting an association with the chromosomes of mitotic cells. Colcemid-blocking studies confirmed that most of the IE antigen was localized in the vicinity of condensed chromosomes in all metaphase cells after methanol fixation. In contrast, the SV40 large T-antigen protein was found to be preferentially excluded from metaphase chromosomes in a similar colcemid-treated human cell line. In transient expression assays, 1 to 2% of IE antigen-positive Vero, 293, or Balb/c3T3 cells also displayed a metaphase chromosome association pattern. Mapping studies using deletion and truncation mutants revealed that the monoclonal antibodies recognized epitopes encoded within the small NH2-terminal exons that are common to both the IE1 and IE2 gene products. However, an intact exon-4 (IE1) region, but not the exon-5 (IE2) region of the HCMV IE gene complex, was required for conferring both the normal diffuse nuclear localization pattern and the chromosome-association properties. Furthermore, removal of the glutamic acid-rich COOH-terminal coding portions of exon-4 resulted in aberrant staining patterns with production of large, phase-dense nuclear globules in all positive cells. An association between the IE68 IE1 protein and metaphase chromosomes was also detected after HCMV-(Towne) infection in a small proportion of both nonpermissive Balb/c3T3 cells and permissive HF cells. We conclude that the IE1 acidic nuclear phosphoprotein displays some properties similar to those of the EBNA-1 protein of Epstein-Barr virus and suggest that it may potentially play a role in maintenance of the latent state of HCMV DNA.

Use of a laser-induced optical force trap to study chromosome movement on the mitotic spindle.

A laser-induced optical force trap was used to alter the movement of chromosomes in mitotic cells in vitro. The trap was produced by using a 1.06-microns neodymium YAG (yttrium/aluminum garnet) laser focused through a phase-contrast microscope. The trap was applied to one side of centrophilic chromosomes off the mitotic spindle and to late-moving chromosomes on the mitotic spindle. In both situations, chromosome movement was initiated in the direction opposite to that of the applied force. When the force was applied, chromosomes moved at velocities 10-20 times normal. These studies verify and extend the feasibility of using this new technique to study factors that influence organelle motility.

Natural hybridization and flower color inheritance in Sisyrinchium rosulatum bickn.

Plants showing intermediate features between Sisyrinchium rosulatum BICKN. and a pale-purple flowered race (L-race) occur on the university campus of the authors. Based on morphological and cytological studies it was confirmed that these plants are natural hybrids between S. rosulatum and L-race. S. rosulatum, L-race and their hybrids had 32 chromosomes in mitotic cells, but their hybrids were sterile and showed a multipolar division in PMC and a low pollen fertility. L-race may be a related but separate taxon of S. rosulatum. Genetic investigation of flower color indicated that the white is dominant over the purple by a single Mendelian factor in S. rosulatum. Flower color variation in S. rosulatum may have resulted from the polymorphic occurrence of flower color controlled by major gene (s) and natural hybridization with the related species.

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.

Localization of SV40 T antigen in mitotic cells by an immunoperoxidase method.

The immunoperoxidase technique has clearly demonstrated that SV 40 T antigen is dissociated from the chromosomes in mitotic cells, and massive transport of T antigen from the cytoplasm to the nucleus appears to take place during or immediately after the telophase.

Giemsa C-Banding, Somatic Association and Orientation of Interphase Chromosomes inTrigonella Foenum-Graecum(L.)

SUMMARYGiemsa C-bands on somatic chromosomes of Trigonella foenum-graecum are present at telomeric end of short arm of chromosomes except chromosome number 2 where the band is centromeric and the nucleolar chromosomes where the bands are present on terminal end of distal arm and in the region of secondary constriction.Arrangement of somatic chromosomes in mitotic cells is non-random and nucleolar chromosomes lie closely associated. At interphase, chromosomes remain polarized and heterochromatic regions form a heterochromatic ring towards one pole. The ring like configuration of heterochromatic regions at anaphase is maintained till next interphase and even early prophase through telophase. The regularity of non random arrangement of chromosomes throughout the division cycle is indicated.

Mitotic cell studies based on in vivo observations. X. Problems on the objectivity of fixation cytology, of genetic information and of the teleonomy in the structure and function of the karyokinetic spindle.

In this report the author has enumerated the differences in interpretations of the mitosis studied both in fixed preparations and in in vivo observations. The author especially maintains the opinion that any movement of chromosomes in mitotic cells should be reinvestigated thoroughly by in vivo observations in the same way as studies on any movement of intracellular organelles, e.g. plastids, mitochondria, rotation of nucleus, streaming of protoplasm, etc.1. Practically all behavior of mitotic cells described in cytology books has scarcely made reference to the genetic back-ground of the mitosis.2. The breakdown of nuclear membrane before spindle formation in higher plants and animals is an impossibility. The nuclear membrane in eukaryotes is predicted and controlled by genes and genetic information which exist as long as the cell lives.3. The participation of centrioles in the formation of spindle fibers and in the chromosome movement in anaphase is also a misinterpretation due to a disregard of genes and genetic information under which centrioles behave as primordia of organelles for cell movement.4. In in vivo observations, the continuous movement of chromosomes in anaphase can be seen objectively in a three dimensional space of living spindles, whereas the same behavior of chromosomes in fixation cytology is made up by animating a series of chromosome images in sectioned plane mitotic cells in fixed preparations under observers' subjective influences.5. The teleonomy and the cause and effect relationship among the behavior of mitotic cells, viz. reversible transformation of nuclear sap proteins, the change of nuclear contour from sphere to spindle-shape, the development of spindle background fibrils, the movement of chromosomes in anaphase by shortening of kinetochore fibers, formation of daughter nuclei, the accumulation of ATP at spindle poles, etc. are practically disregarded of the descriptions on the mitosis in current cytology books.

Histochemical detection of cyclic nucleotide phosphodiesterase activity in germinating onion seed

A cyclic nucleotide phosphodiesterase(s) capable of hydrolyzing both purine and pyrimidine substrate has been detected histochemically in a plant root meristem. Activity was enhanced in the presence of exogenous nucleotidases. Acrolein was inhibitory but glutaraldehyde or paraformaldehyde were suitable fixatives. The enzyme(s) displayed no detectable inhibition with theophylline or caffeine or activation with imidazole. Low spurious reaction products, which could not be significantly enhanced by gentle treatments with detergents were detectable in small pieces of tissue incubated in vivo or in vitro. Cell surface activity was not enhanced by incubating small pieces of tissue in vivo with insulin. In the growing root the highest activities were found in peripheral cells of the cortex and the root cap. In sections incubated with exogenous nucleotidases, especially in those fixed, virtually all the nuclei display positive activity beyond control staining intensities while a significant proportion possessed a more intense reaction product; a similar heavy staining was observed on some chromosomes of mitotic cells. Activity was especially detectable in cell wall regions of epidermal tissues in ungerminated embryos and it intesified rapidly after germination. Whereas provascular and nuclear tissues demonstrated low levels of activity, significant reaction products were found in the epicotyl.