The centriolar cycle in polykaryons containing prematurely condensed chromosomes or telophase-like nuclei]

In fused interphase-mitotic cells, either interphase nuclei are induced to premature chromosome condensation (PCC) or mitotic chromosomes are induced to telophase-like nuclei (TLN) formation. This study concerns structural and functional changes in centrioles of fused cells in which PCC or TLN are induced. Embryonic pig kidney cells were fused using a modified PEG-DMSO-serum method. Cell cycle period of the nuclei was determined before cell fusion using double-labeling autoradiography. Polykaryons containing desirable type of PCC or interphase nuclear combination in TLN were selected on the basis of isotope labeling after being embedded in epon. Selected cells were cut into serial sections and studied under electron microscope. The data obtained showed that centrioles at every interphase period undergo mitotic activation when their nuclei are induced to PCC. They acquire fibrillar halo and form half-spindles. Daughter centrioles at G1, S and G2 periods are also capable of mitotic activation when separated from their mother centriole. Inert centrioles were found in some cells with G1-PCC. When mitotic nuclei are induced to TLN formation, their centrioles also become inactivated. They lose fibrillar halo and mitotic spindles break down. Some mitotic centrioles develop features characteristic of interphase period such as satellites and vacuoles. Induced nuclear and centriolar changes are simultaneous and may be controlled by the same factor. Mitotic factor of mitotic cell partner which induces PCC may also induce interphase centrioles to mitotic activation. Degradation of the mitotic factor leading to TLN formation may also cause the loss of the mitotic activity of centrioles and disorganization of mitotic spindles.     

The identification of mammalian centrosomal antigens using human autoimmune anticentrosome antisera.

Human autoimmune sera were screened for the presence of anticentrosome autoantibodies. Two high titer sera were identified that reacted with HeLa, CHO, and PtK2 centrosomes by immunofluorescence, although the fluorescent patterns that were obtained using the two antisera were separate and distinct. Serum obtained from patient IJ contained antibodies that reacted with epitopes present only in mitotic centrosomes; staining of interphase centrosomes was never detected uing IJ antiserum. Immunoblot analysis demonstrated that antibodies present in IJ antiserum reacted with a 190 kD spindle pole antigen. Immunofluorescent staining of cultured mammalian cells demonstrated that antibodies present in serum obtained from patient SPJ reacted with both interphase and mitotic centrosomes. Characterization of SPJ antiserum by immunoblotting demonstrated that antibodies present in the SPJ serum recognized proteins of Mrs of 39, 185, and 220 kD, although the possibility that the 185 kD polypeptide was a proteolytic breakdown product of the 220 kD protein has not been eliminated. Neither antiserum was able to inhibit microtubule nucleation from centrosomes in a lysed cell system in which pure 6S tubulin was added to permeabilized cells following pretreatment of the cells with either SPJ or IJ antiserum. These antisera should be useful probes for studying the biochemistry of the mammalian centrosome.     

Nuclear lamins during prophasing and telophasing in heterophasic HeLa and Chinese hamster homokaryons

We have perturbed the dynamics of the nuclear lamins by means of cell fusion between mitotic and interphase cells and have studied redistribution of lamins in fused cells as a function of extracellular pH levels. We show here that in heterophasic M-1 HeLa homokaryons disassembly of interphase lamins predominates at low pH levels between 7.0 to 7.3, whereas deposition of cytoplasmic lamins around condensed metaphase chromosomes was observed at pH 8.0. In HeLa homokaryons lamina disassembly and lamina deposition around chromosomes are mutually exclusive. Using heterophasic M-1 homokaryons of the Chinese hamster cell line DON we observed that disassembly of interphase lamins and deposition of lamins around condensed chromosomes coexisted in the same homokaryon kept at pH 7.0. Disassembly of lamins developed synchronously with premature chromosome condensation (PCC) whereas lamina deposition around the condensed M-chromosomes was followed by telophasing. In fusions kept at pH 8.0 cytoplasmic lamins were exclusively deposited around mitotic chromosomes. The results are interpreted as showing that pH regulates the lamina dynamics in homokaryons of mitotic and interphase cells.     

High-resolution measurement of breaks in prematurely condensed chromosomes by differential staining

A relatively simple method has been developed to improve the resolution for measuring breaks produced in interphase chromosomes by X rays or other agents following the induction of premature chromosome condensation (PCC). Mitotic HeLa cells, which induce PCC when fused with interphase cells, were obtained from cultures grown for several generations in 5-bromodeoxyuridine (BrdU). These were fused to cells from low-passage confluent cultures of normal human fibroblasts and subsequently stained by a modified fluorescence-plus-Giemsa (FPG) technique. Following this protocol the prematurely condensed chromosomes stain intensely, whereas the mitotic chromosomes of the inducer cell(s), which are intermingled with them, stain very lightly. With this technique the interphase chromosomes and their fragments can be identified unequivocally, making scoring much easier and more accurate. The frequency of breaks produced in G1 phase AG1522 human fibroblasts immediately following X-ray doses of 58 and 117 rad was 3.68 and 7.38 per cell, respectively. Use of this technique should allow the detection of damage from ionizing radiation at doses lower than 10 rad.     

Monoclonal antibody with specificity to mitotic chromosomes of primates

Fusion of a cell in mitosis with a cell in interphase results in the condensation of chromatin in the interphase nucleus into chromosomes. Premature chromosome condensation is caused by certain proteins, called mitotic factors, that are present in the mitotic cell and are localized on chromosomes. Extracts from mitotic cells were used to immunize mice to produce monoclonal antibodies specific for cells in mitosis. Among the antibodies obtained, the MPM-4 antibody defines a 125-kD polypeptide antigen located on mitotic chromosomes by indirect immunofluorescence. Although the polypeptide antigen is present in approximately equal concentrations in extracts of interphase cells and mitotic cells, as revealed by immunoblots, it cannot be detected cytologically in the former. Cell fractionation experiments showed that the 125-kD antigen is found in the cytoplasm of interphase cells and metaphase cells, but is concentrated in fractions containing metaphase chromosomes, although not detectable in interphase nuclei. Even though the antigen is apparently primate-specific, it binds to mitotic chromosomes and prematurely condensed chromosomes in human-rodent cell hybrids without regard to the species of origin of the mitotic inducer. The presence of the antigen in the cytoplasm of interphase cells and the chromosomes of mitotic cells suggests a relationship between the presence of the antigen on chromosomes and the process of chromosome condensation and decondensation.     

Premature chromosome condensation and cell cycle analysis.

The application of the phenomenon of premature chromosome condensation for cell cycle analysis in HeLa and CHO cells has been examined. Random populations of HeLa and CHO cells pulse labelled with H3-TdR were separately fused with mitotic HeLa cells using U.V. inactivated Sendai virus. The resulting prematurely condensed chromosomes (PCC) were scored and classified into G1, S and G2-PCC on the basis of both morphological and autoradiographic data, The results of this study indicated that the G1, S and G2 phase cells are equally susceptible to virus-induced fusion with mitotic cells and subsequent induction into PCC. Hence the PCC method for cell cycle analysis is both practical and accurate. This study also revealed that the process of chromosome decondensation initiated during the telophase of mitosis continues throughout the G1 period reaching an ultimate state of decondensation by the end of G1, at which point the fusion of such cells with those in mitosis yield PCC with the most diffused morphology instead of the discrete single stranded structures characteristic of early G1-PCC. Thus, the decondensation of chromatin during G1 appears to be a prerequisite for the subsequent initiation of DNA synthesis.     

Events associated with the initiation of mitosis in fused multinucleate HeLa cells

Large multinucleate (LMN) HeLa cells with more than 10–50 nuclei were produced by random fusion with polyethylene glycol. The number of nuclei in a particular stage of the cell cycle at the time of fusion was proportionate to the duration of the phase relative to the total cell cycle. The fused cells did not gain generation time. Interaction of various nuclei in these cells has been observed. The nuclei initially belonging to the G1-or S-phase required a much longer time to complete DNA synthesis than in mononucleate cells. Some of the cells reached mitosis 15 h after fusion, whereas others required 24 h. The cells dividing early, contained a larger number of initially early G1-phase nuclei than those cells dividing late. The former very often showed prematurely condensed chromosome (PCC) groups. In cells with a large number of advanced nuclei the few less advanced nuclei could enter mitosis prematurely. On the other hand, the cells having a large number of nuclei belonging initially to late S-or G2-phase took longer to reach mitosis. These nuclei have been taken out of the normal sequence and therefore failed to synthesize the mitotic factors and depended on others to supply them. Therefore the cells as a whole required a longer period to enter mitosis. Although the nuclei became synchronized at metaphase, the cells revealed a gradation in prophase progression in the different nuclei. At the ultrastructural level the effect of advanced nuclei on the less advanced ones was evident with respect to chromosome condensation and nuclear envelope breakdown. Less advanced nuclei trapped among advanced nuclei showed PCC and nuclear envelope breakdown prematurely, whereas mitotic nuclei near interphase or early prophase nuclei retained their nuclear envelopes for a much longer time. PCC is closely related to premature breakdown of the nuclear envelope. Our observations clearly indicate that chromosome condensation and nuclear envelope breakdown are two distinct events. Kinetochores with attached microtubules could be observed on prematurely condensed chromosomes. Kinetochores of fully condensed chromosomes often failed to become connected to spindle elements. This indicates that the formation of a functional spindle is distinct from the other events and may depend on different factors.     

Factors influencing ADP-ribosylation differences between chromosomal proteins of interphase and metaphase HeLa cells

Fundamental differences were previously discovered in the ADP-ribosylation of proteins from metaphase chromosomes and interphase nuclei of HeLa cells. The number of modified nonhistone species was found to be dramatically reduced for metaphase chromosomes. An investigation has therefore been made of factors which could influence, and therefore be responsible for, this change in ADP-ribosylation during the cell cycle. Modified proteins were detected by autoradiography of sodium dodecyl sulfate-polyacrylamide gels containing mitotic and interphase samples from permeabilized cells that had been incubated with [32P]NAD. Whole cells showed a difference between interphase and metaphase similar to that for isolated nuclei and chromosomes. Chromosome expansion, disruption of chromosomes or nuclei, DNA nicking, and cellular growth activity significantly changed the incorporation of 32P label. Inhibitors of protein, RNA, and DNA synthesis did not, however, greatly affect ADP-ribosylation. The pattern of labeled species was not altered by the presence of nonradioactive NAD, though the extent of labeling declined. The results were not artifactually due to the procedure used to arrest cells in mitosis. Similar results were found with Novikoff rat hepatoma cells, demonstrating that the difference between metaphase and interphase is not confined to HeLa cells.     

Premature chromosome condensation. I. Visualization of x-ray-induced chromosome damage in interphase cells

A new method is described to visualize chromosome damage in interphase cells immediately after exposure to mutagenic agents. This method involves the fusion of treated interphase cells with untreated mitotic cells which results in the induction of premature chromosome condensation (PCC). Chinese hamster ovary (CHO) cells were treated with X-rays and chromosome aberrations were scored in G2-PCC and the mitotic chromosomes. The incidence of aberrations was significantly higher in PCC than that observed in the mitotic chromosomes of the treated cells. Post-irradiation incubation for I h before fusion allowed the repair of some of the chromosome damage. Data are also presented which indicate that the extent of radiation damage visualized in PCC is inversely proportional to the degree of chromosome condensation. These results indicate that the PCC method has a greater senstivity in the detection of induced chromosome damage than the standard method of scoring metaphase chromosomes.     

Chromosome condensation outside of mitosis: mechanisms and new tools

A basic principle of cell physiology is that chromosomes condense during mitosis. However, condensation can be uncoupled from mitotic events under certain circumstances. This phenomenon is known as "premature chromosome condensation (PCC)." PCC provides insights in the mechanisms of chromosome condensation, thus helping clarifying the key molecular events leading to the mitosis. Besides, PCC has proved to be an useful tool for analyzing chromosomes in interphase. For example, using PCC we can visualize genetic damage shortly after the exposure to clastogenic agents. More than 30 years ago, the first report of PCC in interphase cells fused to mitotic cells using Sendai virus was described (virus-mediated PCC). The method paved the way to a great number of fundamental discoveries in cytogenetics, radiation biology, and related fields, but it has been hampered by technical difficulties. The novel drug-induced PCC method was introduced about 10 years ago. While fusion-induced PCC exploits the action of external maturation/mitosis promoting factor (MPF), migrating from the inducer mitotic cell to the interphase recipient, drug-induced PCC exploits protein phosphatase inhibitors, which can activate endogenous intracellular MPF. This method is much simpler than fusion-induced PCC, and has already proven useful in different fields.     

MPM-12: a monoclonal antibody that predominantly stains mitotic cells and recognizes a protein kinase.

The monoclonal antibody MPM-12, raised by using partially purified extract of mitotic HeLa cells as the immunogen, preferentially stains the cytoplasm of mitotic cells by indirect immunofluorescence without exhibiting any species specificity. On immunoblots, MPM-12 recognizes three bands, of 155, 88, and 68 kDa, in mitotic HeLa cell extract but only the 68-kDa band in interphase cell extract. The 68-kDa band seems to be associated with chromatin while the other two are not. All three MPM-12 reactive peptides are phosphorylated, and the phosphorylation seems to be required for MPM-12 reactivity. The MPM-12 immunocomplexes exhibit autophosphorylating and histone H1 kinase activity.     

Perturbation of mammalian cell division. II. Studies on the isolation and characterization of human mini segregant cells.

A method is described for the isolation, according to size, of mini segregants produced by the abnormal cleavage of reversibly arrested mitotic HeLa cells. Many of these mini segregants contain small amounts of DNA, as judged by Feulgen staining and chromosome analysis. After fusion with mitotic HeLa cells, the interphase chromosomes of the mini segregants are seen as either monovalent or bivalent prematurely condensed chromosomes (PCC), some of which are damaged. A proportion of isolated mini segregants synthesize DNA, RNA and protein. Fusion of mini segregants with interphase HeLa cells gives rise to cells with 'hybrid' karyotypes.     

Induction of prematurely condensed chromosomes from testicular cells of the mouse

Mitotic CHO cells and mouse testicular cells were fused with polyethylene glycol. Several types of prematurely condensed chromosomes were observed. From chromosome morphology it was possible to determine that most of the PCC represented mouse cells. Labeling of either the CHO cells in vitro or the testicular cells in vivo with ³H-TdR prior to fusion also demonstrated that the PCC were derived from the mouse cells. In some PCC, 20 chromosomes could be counted, the haploid number for mouse. It is assumed that these PCC were induced in mouse spermatid nuclei.     

Spectrin redistributes to the cytosol and is phosphorylated during mitosis in cultured cells

Dramatic changes in morphology and extensive reorganization of membrane-associated actin filaments take place during mitosis in cultured cells, including rounding up; appearance of numerous actin filament-containing microvilli and filopodia on the cell surface; and disassembly of intercellular and cell-substratum adhesions. We have examined the distribution and solubility of the membrane-associated actin-binding protein, spectrin, during interphase and mitosis in cultured CHO and HeLa cells. Immunofluorescence staining of substrate-attached, well-spread interphase CHO cells reveals that spectrin is predominantly associated with both the dorsal and ventral plasma membranes and is also concentrated at the lateral margins of cells at regions of cell-cell contacts. In mitotic cells, staining for spectrin is predominantly in the cytoplasm with only faint staining at the plasma membrane on the cell body, and no discernible staining on the membranes of the microvilli and filopodia (retraction fibers) which protrude from the cell body. Biochemical analysis of spectrin solubility in Triton X-100 extracts indicates that only 10-15% of the spectrin is soluble in interphase CHO or HeLa cells growing attached to tissue culture plastic. In contrast, 60% of the spectrin is soluble in mitotic CHO and HeLa cells isolated by mechanical "shake-off" from nocodazole-arrested synchronized cultures, which represents a four- to sixfold increase in the proportion of soluble spectrin. This increase in soluble spectrin may be partly due to cell rounding and detachment during mitosis, since the amount of soluble spectrin in CHO or HeLa interphase cells detached from the culture dish by trypsin-EDTA or by growth in spinner culture is 30-38%. Furthermore, mitotic cells isolated from synchronized spinner cultures of HeLa S3 cells have only 2.5 times as much soluble spectrin (60%) as do synchronous interphase cells from these spinner cultures (25%). The beta subunit of spectrin is phosphorylated exclusively on serine residues both in interphase and mitosis. Comparison of steady-state phosphorylation levels of spectrin in mitotic and interphase cells demonstrates that solubilization of spectrin in mitosis is correlated with a modest increase in the level of phosphorylation of the spectrin beta subunit in CHO and HeLa cells (a 40% and 70% increase, respectively). Two-dimensional phosphopeptide mapping of CHO cell spectrin indicates that this is due to mitosis-specific phosphorylation of beta-spectrin at several new sites. This is independent of cell rounding and dissociation from other cells and the substratum, since no changes in spectrin phosphorylation take place when cells are detached from culture dishes with trypsin-EDTA.(ABSTRACT TRUNCATED AT 400 WORDS)     

Conservation of interphase chromatin nonhistone antigens as components of metaphase chromosomes

The degree of conservation of HeLa interphase chromatin nonhistone antigens among the nonhistones of isolated metaphase chromosomes was determined with immunological procedures. Proteins were separated on SDS-polyacrylamide gels and electrophoretically transferred to diazophenylthioether (DPT)-paper, which was then overlaid with antiserum to chromatin from interphase nuclei. The bound antibodies were detected with 125I-labeled protein A. Alternatively, polyacrylamide gels were directly overlaid with antiserum and with 125I-protein A. Densitometry of autoradiograms and stained gels revealed the degree of conservation of nonhistone antigenic determinants from interphase to metaphase to be over 90% for chromatin.     

An autoimmune antibody from scleroderma patients recognizes a component of the plant cell nucleolus

Summary An auto-antibody from human serum of patients with the autoimmune disease scleroderma was used to localize the nucleolus in meristematic cells of onion and soybean roots using indirect immunofluorescence microscopy. Similar lots of antiserum recognized a single 34 kD, nucleolar protein, fibrillarin, in a variety of animal cells (Ochs, et al. 1984, 1985). In both plants, antibody linked fluorescence is associated with the one to several nucleoli present in the interphase nucleus. The fluorescence becomes diffuse around condensing prophase chromosomes and becomes more diffused at metaphase with slightly more intense fluorescence surrounding the chromosomes. At anaphase-telophase the fluorescence is localized in dense areas within the chromosomes, presumably representing prenucleolar bodies which will form the interphase nucleoli of the daughter nuclei. This antiserum provides a new, valuable tool for the study of the nucleolus and the highly conversed nucleolar antigen(s) that it recognizes.     

An 83-kDa embryonic-type nuclear antigen is detected within the germinal vesicles of oocytes of the ascidianHalocynthia roretzi

Summary Monoclonal antibodies were raised against germinal vesicles which were isolated from fully grown oocytes of the ascidianHalocynthia roretzi. Immunoblot analyses revealed that one of the antibodies, designated Hgv-2, recognized a single band with a molecular weight of about 83 kDa. The antibody, visualized by indirect immunohistochemistry, reacted only with the germinal vesicles of oocytes and did not react with test cells, follicle cells, and other somatic cells of the gonad. During embryogenesis the antigenicity was found in interphase nuclei of all embryonic cells. The antibody did not react with chromosomes or the mitotic apparatus. The antigenicity was retained by interphase nuclei of larval cells, but it disappeared from nuclei of juveniles about 7 days after metamorphosis.     

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)     

Generation and characterization of a monoclonal antibody, mH1, raised against mitotic HeLa cells

Hybridoma cell lines were prepared from spleen cells of mouse immunized with mitotic HeLa cells. A monoclonal antibody (mH1), which intensively reacted with cleavage furrows of dividing HeLa cells in immunofluorescence, was obtained. In interphase, this antibody diffusely stained whole HeLa cells. Immunoelectron microscopy showed that mH1 antigens were localized at microvillus projections at the surface of dividing HeLa cells, but definite localization of that antigen was not observed in interphasic cells. Immunoblot analysis showed that mH1 is reactive to 42-kDa and 130-kDa components. Further, the 42-kDa component was identified as a gamma-actin homolog by N-terminal amino acid sequence analysis.     

Fusion between interphase and mitotic plant protoplasts : Induction of premature chromosome condensation

Morphological changes in interphase nuclei were cytologically studied in heterophasic dinucleate cells formed by the fusion of mitotic and interphase plant protoplasts. Mitotic protoplasts were isolated from a partially synchronized suspension culture of wheat (Triticum monococcum). The mitotic cells were accumulated by colchicine after release of hydroxyurea block. Treatment of protoplast populations with polyethylene glycol-dimethyl sulphoxide solution resulted in metaphase-interphase fusion. Three hours after fusion, the appearance of chromosomes with single chromatid as well as of fragmented, pulverized chromatin in heterophasic cells indicated the induction of premature chromosome condensation (PCC) in somatic wheat cells. Condensation in interphase nuclei of mitotically inactive rice protoplasts was also detected after fusion with mitotic wheat protoplasts.