Ultrastructural changes associated with the induction of premature chromosome condensation in Vicia faba root meristem cells

Key message

PCC induction is regulated by several signaling pathways, and all observed effects associated with PCC induction are strongly dependent on the mechanism of action of each PCC inducer used.

Abstract

Electron microscopic observations of cells with symptoms of premature chromosome condensation (PCC) showed that the interphase chromatin and mitotic chromosomes differed with respect to a chemical compound inducing PCC. Induction of this process under the influence of hydroxyurea and caffeine as well as hydroxyurea and sodium metavanadate led to a slight decrease in interphase chromatin condensation and the formation of chromosomes with a considerably loosened structure in comparison with the control. Incubation in the mixture of hydroxyurea and 2-aminopurine brought about clear chromatin dispersion in interphase and very strong mitotic chromosome condensation. Electron microscopic examinations also revealed the characteristic features of the structural organization of cytoplasm of Vicia faba root meristems, which seemed to be dependent on the type of the PCC inducer used. The presence of the following was observed: (i) large plastids filled with starch grains (caffeine), (ii) mitochondria and plastids of electron dense matrix with dilated invaginations of their internal membranes (2-aminopurine), and (iii) large mitochondria of electron clear matrix and plastids containing protein crystals in their interior (sodium metavanadate). Moreover, since caffeine causes either the most effective loosening of chromatin fibrils (within the prematurely condensed chromosomes) or induction of starch formation (in the plastids surrounding the nuclei), this may be a proof that demonstrates the existence of a link between physical accessibility to chromatin and the effectiveness of cellular signaling (e.g., phosphothreonine-connected).     

Differential chromatin condensation of female and male pronuclei in mouse zygotes

Mouse zygotes or halves of zygotes, containing either a female or a male pronucleus, were fused with ovulated metaphase II oocytes. In 59.7% of the resulting hybrid cells, the pronuclei underwent premature chromosome condensation (PCC). In some of these heterokaryons the 2 pronuclei differed in the dynamics of condensation. Detectability of differential PCC of pronuclei (dPCC) depended on the type of preparation. In hybrids with PCC, produced by fusion of intact zygotes with metaphase II oocytes and processed for whole-mount preparations, one pronucleus was more advanced in the condensation process in 47% of cases. In air-dried preparations dPCC was detected in as many as 94% of hybrids. Experiments with the fusion of halves of zygotes with metaphase II oocytes have shown that the differential reaction of pronuclei to condensation factor depended on their parental origin. Maternal chromatin responded faster to the condensation factor and attained more advanced stages of PCC than paternal chromatin. Different responses of the maternal and paternal pronucleus to the condensation factor suggests that the 2 pronuclei are not identical with regard to the organization of chromatin and/or the lamin composition of the nuclear envelope. © 1993 Wiley-Liss, Inc.     

The human leulocyte test system. VII. Further investigations concerning micronucleus-derived premature chromosome condensation.

Premature chromosome condensation (PCC) from X-ray induced micronuclei shows a dose-effect relationship in human leukocytes in vitro. Preparations at different culture times without colcemide treatment reveal complex variations of the frequencies of micronuclei and PCC correlated with the fixation time. The positions of PCC patches in the metaphase plate and the frequencies of different PCC types (S and G2) ar independent on the X-ray dose. The latter indicates that the slowing down of the micronuclei in the cell cycle, which is the reason for the formation of PCC, may be an outcome rather of a regulatory phenomenon than of an unspecific physiological damage of the chromatin included in the micronuclei. This is especially evident from labeling experiments with tritiated thymidine, showing that the extent of asynchrony between main nuclei and micronuclei is independent on the X-ray dose. Labeling experiments with tritiated uridine reveal a X-ray dose dependent suppression of RNA synthesis in cells with main nuclei and micronuclei. THE S-phase nature of "pulverized" PCC patches could be verified by incorporation of tritiated thymidine in aound 50%. Staining of centromeric heterochromatin in micronuclei reveal a frequency of micronuclei with centromeric heterochromatin resembling the frequency of G2-phase PCC found in mitoses.     

A physical model for the condensation and decondensation of eukaryotic chromosomes

During the eukaryotic cell cycle, chromatin undergoes several conformational changes, which are believed to play key roles in gene expression regulation during interphase, and in genome replication and division during mitosis. In this paper, we propose a scenario for chromatin structural reorganization during mitosis, which bridges all the different scales involved in chromatin architecture, from nucleosomes to chromatin loops. We build a model for chromatin, based on available data, taking into account both physical and topological constraints DNA has to deal with. Our results suggest that the mitotic chromosome condensation/decondensation process is induced by a structural change at the level of the nucleosome itself.     

Premature chromosome condensation : Conformational changes of chromatin associated with phytohemagglutinin stimulation of peripheral lymphocytes

Phytohemagglutinin (PHA)-stimulated human peripheral blood lymphocytes were shown to be distinguishable from unstimulated lymphocytes by the technique of premature chromosome condensation (PCC). Greater than 70% of the PCC from lymphocytes stimulated by incubating with PHA for 18–22 h showed greatly extended PCC as compared with only 30% in the unstimulated cultures. This decondensation pattern of the PCC paralleled with the previously reported increase in the template activity of chromatin. The PCC method can be useful in determining the proliferative potential of bone marrows of leukemic patients during and after chemotherapy.     

Premature chromosme condensation in the bone marrow of Chinese hamster after application of bleomycin in vivo.

The Chinese hamster bone marrow was used as a test system in vivo to analyse the chromosome-danaging effect of bleomycin. Both chromosome and chromatid aberrations were found. Mitoses with aberrations (Ma) show a linear dose-effect relationship after a recovery time of 24 h, the same hold true for cells with micronuclei (Cm) and for mitoses with premature chromosome condensation (PCC). The dose-effect relationships for Ma, Cm and PCC run parallel to each other with Ma at the highest and PCC at the lowest level (Ma greater than Cm greater than PCC). The time-effect relationships for Ma, Cm and PCC show that after 12 h recovery time there are no PCCs but the highest frequencies of Ma and Cm indicating that most cells are in their first post-treatment mitoses or Gi-phases at this fixation time. In addition to the frequency determinations autoradiographic analysis were performed to clarigy the nature of the PCCs. The results are interpreted as follows: bleomycin induces chromosomal aberrations that in turn give rise to micronuclei by means of lagging chromatin, main and micronuclei eventually become asynchronous in their cell cycles and mitosing main nuclei induce PCC in the micronuclei.     

Optimization of calyculin A-induced premature chromosome condensation assay for chromosome aberration studies

Calyculin A-induced premature chromosome condensation (PCC) assay is a simple and useful method to assess structural and numerical chromosome aberrations in cells. Our hypothesis in this study is that suboptimum calyculin A induction of PCC resulting in fuzzy compactness and/or shortened length chromosomes would decrease the detection sensitivity of numerical and structural chromosome aberrations such as small PCC rings and small excess fragments. In this study, an optimization of calyculin A exposure on chromosome morphology and PCC induction frequency was investigated using a human peripheral blood lymphocyte (PBL) ex vivo irradiation ((60) Co-γ rays; ～0.6 Gy/min; 0-30 Gy) model. Treatment with calyculin A (50 nM) for 15 and 30 min resulted in 11.3 ± 2.7 and 9.9 ± 1.6-fold increases in the frequency of G(2) /M-PCC cells with extended length chromosomes compared with the 60-min treated group over a broad dose range (0 to 20 Gy), respectively. The G(2) /M-PCC scoring index per PCC in 15- and 30-min treated groups was increased by 1.9 ± 0.2 (P = 0.001) and 1.8 ± 0.2 (P = 0.001) compared with the 60-min treated group over 0-20 Gy, respectively. The G(2) /M-PCC efficiency of 30-min treated group was highest in the three conditions (i.e., 15-, 30-, and 60-min treatment) of calyculin A exposure. Calyculin A (50 nM) treatment for 30 min before the 48-h harvest of mitogen-stimulated human PBL is optimum for the formation of suitable chromosome morphology necessary to assess structural chromosome aberrations induced by exposure to radiation using the chemical induced-PCC assay. Published 2011 Wiley Periodicals, Inc.     

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.     

X-ray crystal structure of MENT: evidence for functional loop-sheet polymers in chromatin condensation

Most serpins are associated with protease inhibition, and their ability to form loop-sheet polymers is linked to conformational disease and the human serpinopathies. Here we describe the structural and functional dissection of how a unique serpin, the non-histone architectural protein, MENT (Myeloid and Erythroid Nuclear Termination stage-specific protein), participates in DNA and chromatin condensation. Our data suggest that MENT contains at least two distinct DNA-binding sites, consistent with its simultaneous binding to the two closely juxtaposed linker DNA segments on a nucleosome. Remarkably, our studies suggest that the reactive centre loop, a region of the MENT molecule essential for chromatin bridging in vivo and in vitro, is able to mediate formation of a loop-sheet oligomer. These data provide mechanistic insight into chromatin compaction by a non-histone architectural protein and suggest how the structural plasticity of serpins has adapted to mediate physiological, rather than pathogenic, loop-sheet linkages.     

Ultrastructure of cell fusion and premature chromosome condensation (PCC) of thymocyte nuclei in metaphase II mouse oocytes

Following PEG (polyethylene glycol) treatment of ovulated metaphase II mouse oocytes aggregated with thymocytes, fusion of cell membranes occurs. Prerequisites for cell fusion are: close apposition of lectin-agglutinated (phytohemagglutinin-treated) membranes of both cells, formation of firm punctual adhesion sites, and expansion of adhesion sites over a certain area. Establishment of the firm cell-cell contact is associated with development of actin-like filaments along both of the adhering plasma membranes. Membrane fusion occurs at single or multiple sites, and is followed by internalization of thymocyte-oocyte membrane complexes decorated with actin filaments into the hybrid cell cytoplasm. A filamentous actin layer forms also along the inner surface of newly formed hybrid oocyte-thymocyte plasma membrane. Thymocyte nuclei incorporated into oocyte cytoplasm undergo nuclear envelope breakdown and premature chromosome condensation (PCC) leading, eventually, to formation of single chromatids complete with kinetochores. Concomitantly with chromatin condensation an extensive polymerization of microtubules starts in the center of the chromatin mass which leads to the formation of an apparently non-functional spindle-like structure.     

Thermodynamics of condensation of nuclear chromatin. A differential scanning calorimetry study of the salt-dependent structural transitions

We present a detailed thermodynamic investigation of the conformational transitions of chromatin in calf thymus nuclei. Differential scanning calorimetry was used as the leading method, in combination with infrared spectroscopy, electron microscopy, and techniques for the molecular characterization of chromatin components. The conformational transitions were induced by changes in the counterion concentration. In this way, it was possible to discriminate between the interactions responsible for the folding of the higher order structure and for the coiling of nucleosomal DNA. Our experiments confirm that the denaturation of nuclear chromatin at physiological ionic strength occurs at the level of discrete structural domains, the linker and the core particle, and we were able to rule out that the actual denaturation pattern might be determined by dissociation of the nucleohistone complex and successive migration of free histones toward native regions, as recently suggested. The sequence of the denaturation events is (1) the conformational change of the histone complement at 66 degrees C, (2) the unstacking of the linker DNA at 74 degrees C, and (3) the unstacking of the core particle DNA, that can be observed either at 90 or at 107 degrees C, depending on the degree of condensation of chromatin. Nuclear chromatin unfolds in low-salt buffers, and can be refolded by increasing the ionic strength, in accordance with the well-known behavior of short fragments. The process is athermal, therefore showing that the stability of the higher order structure depends on electrostatic interactions. The transition between the folded conformation and the unfolded one proceeds through an intermediate condensation state, revealed by an endotherm at 101 degrees C. The analysis of the thermodynamic parameters of denaturation of the polynucleosomal chain demonstrates that the wrapping of the DNA around the histone octamer involves a large energy change. The most striking observation concerns the linker segment, which melts a few degrees below the peak temperature of naked DNA. This finding is in line with previous thermal denaturation investigations on isolated chromatin at low ionic strength, and suggests that a progressive destabilization of the linker occurs in the course of the salt-induced coiling of DNA in the nucleosome.     

Fluorimetric measurements and chromatin condensation patterns of nuclei from 3T3 cells throughout G1

Using two cytological methods based on nuclear morphology, quinacrine dihydrochloride (QDH) staining and premature chromosome condensation (PCC), it has been possible to identify cell cyle positions within G1 of growing and arrested 3T3 cells. The fluorescent intensity of QDH-stained interphase cells appears to decrease as the cells pass from mitosis to S phase. Likewise, the length and thickness of prematurely condensed chromatids can be related to the cells' position within the G1 period. Data are presented that deal with three interrelated topics: (1) We determined by fluorometric measurements of nuclei from 3T3 cells that the visual observation of the decrease in QDH fluorescence during G1 reflects an actual decrease in total fluorescence and not a dispersion of the fluorescent chromatin in a larger nuclear area. (2) We correlated the results obtained by QDH staining with those of PCC on the same cell samples blocked in G1 by different conditions. Serum-starved and contact-inhibited cell nuclei had the highest intensity, hydroxyurea-treated ones had the lowest intensity, while that of isoleucine-deprived cells was in between. The same relative order of G1 positions was obtained based on PCC morphology. Thus, both methods monitor the state of chromatin condensation and can be used to identify cell cycle position within G1.(3) We showed with both methods that the states of chromatin resulting from the various G1 blocking conditions differ from each other.     

Role of polyamines during chromosome condensation of mammalian cells

The objective of the present study was to investigate the role of polyamines in the process of chromosome condensation. The phenomenon of premature chromosome condensation (PCC) involving fusion between mitotic and interphase cells was used as the assay system. The factors present in the mitotic cells would bring about the breakdown of the nuclear membrane and condensation of the interphase chromatin into chromosomes, similar to that which occurs during the initiation of mitosis. Alpha-difluoromethyl ornithine (DFMO), a specific irreversible inhibitor of ornithine decarboxylase was used to deplete both mitotic and interphase cells of polyamines. The results indicate that the polyamine depleted mitotic cells have a diminished ability to induce PCC. This inhibition could easily be reversed by exogenous addition of polyamines at the time of fusion. Furthermore, exogenously added polyamines hastened the entry of exponentially growing cells into mitosis. These observations suggest an essential role for polyamines during the process of chromosome condensation of mammalian cells.     

Chromosome aberrations induced in human lymphocytes by 3.45 MeV alpha particles analyzed by premature chromosome condensation.

Premature chromosome condensation (PCC) experiments using human lymphocytes with centromere staining have shown that after exposure to 3.45 MeV alpha-particle radiation, the full number of dicentric chromosomes appears when the cell fusion protocol is applied immediately after irradiation. In this case, the time available for repair and misrepair of DNA damage is only about 30 min. The number of dicentrics does not change with a further increase in the time available for chromatin rearrangement. This fast response confirms the expectation based on our previous experiments using PCC with 150 kV X rays in which the alpha component of the yield of dicentrics was found to appear when the cell fusion protocol was applied immediately after irradiation, whereas the beta component was delayed by several hours. The time constant for rejoining of the excess acentric chromosome fragments is found to be donor-specific and not to differ for alpha particles and X rays, but alpha-particle radiation leaves a larger fraction of the excess acentric fragments unrejoined. The RBEs of the 3.45 MeV alpha-particle radiation compared to 150 kV X rays, evaluated for the alpha component for the yield of dicentrics and for the yield of unrepaired acentric fragments, have almost equal values of about 4. This is consistent with data in the literature on chromosome aberrations observed in metaphase that show the equality of the RBE values for production of dicentrics and acentric fragments. Our experimental results concerning the fast kinetics of the alpha component of the yield of exchange-type chromosome aberrations are not consistent with Lea's pairwise lesion interaction model, and they support the proposed alternative mechanism of lesion-nonlesion interaction between chromatin regions carrying clustered DNA damage and intact chromatin regions.     

Cell proliferation in chorionic villi at different gestational ages, as analyzed by premature chromosome condensation

The technique of premature chromosome condensation (PCC) was adapted to human first-trimester chorionic villi cells to analyze the cell-cycle kinetics of interphase chromatin. Uncultured cells of the cytotrophoblast (CT) and the mesenchymal core (MC) were obtained by a two-step digestion. PCC was induced by fusion of the chorionic interphase cells with mitotic Chinese hamster ovary or HeLa cells. Cells showing PCC in G1 (classes 1-6), S, and G2 were found. To analyze further the proliferation stages of chorionic G1 interphases, the proliferation potential index (PPI) of 34 placentae recovered between the 8th and 12th week of gestation was determined. The mean PPI found in the CT and MC cells ranged from 18% to 73%, values similar to those described for intensely proliferating tissues. The highest mean PPI value (73%) was observed in CT cells from placentae recovered at the 9th week of gestation, indicating a high specific proliferative activity of CT cells at this developmental stage.     

Premature chromosome condensation in the bone marrow of chinese hamsters after whole body irradiation with Co-60 gamma rays in vivo.

In the Chinese hamster bone marrow chromosomal aberrations were induced after whole body irradiation with Co-60 gamma rays in vivo. Aberrant mitoses give rise to lagging chromatin that forms micronuclei. Eventually the micronuclei are slowed down in their cell cycles in comparison to the main nuclei. The mitotic chromatin of the main nuclei induces premature chromosome condensation (PCC) in the micronuclei that are still in the interphase state of their cell cycles.     

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.     

A model for co-translational translocation: ribosome-regulated nascent polypeptide translocation at the protein-conducting channel

The protein-conducting channel (PCC) must allow both the translocation of soluble polypeptide regions across, and the lateral partitioning of hydrophobic transmembrane helices (TMHs) into, the membrane. We have analyzed existing structures of ribosomes and ribosome-PCC complexes and observe conformational changes suggesting that the ribosome may sense and orient the nascent polypeptide and also facilitate conformational changes in the PCC, subsequently directing the nascent polypeptide into the appropriate PCC-mediated translocation mode. The PCC is predicted to be able to accommodate one central, consolidated channel or two segregated pores with different lipid accessibilities, which may enable the lipid-mediated partitioning of a TMH from one pore, while the other, aqueous, pore allows translocation of a hydrophilic polypeptide segment. Our hypothesis suggests a plausible mechanism for the transitioning of the PCC between different configurations.     

Premature chromosome condensation is not essential for nuclear reprogramming in bovine somatic cell nuclear transfer

Premature chromosome condensation (PCC) was believed to promote nuclear reprogramming and to facilitate cloning by somatic cell nuclear transfer (NT) in mammalian species. However, it is still uncertain whether PCC is necessary for the successful reprogramming of an introduced donor nucleus in cattle. In the present study, fused NT embryos were subjected to immediate activation (IA, simultaneous fusion and activation), delayed activation (DA, activation applied 4 h postfusion), and IA with aged oocytes (IAA, activation at the same oocyte age as group DA). The morphologic changes, such as nuclear swelling, the occurrence of PCC, and microtubule/aster formation, were analyzed in detail by laser-scanning confocal microscopy. When embryos were subjected to IA in both IA and IAA groups, the introduced nucleus gradually became swollen, and a pronuclear-like structure formed within the oocyte, but PCC was not observed. In contrast, delaying embryo activation resulted in 46.5%-91.2% of NT embryos exhibiting PCC. This PCC was observed beginning at 4 h postcell fusion and was shown as one, two, or multiple chromosomal complexes. Subsequently, a diversity of pronuclear-like structures existed in NT embryos, characterized as single, double, and multiple nuclei. In the oocytes exhibiting PCC, the assembled spindle structure was observed to be an interactive mass, closely associated with condensed chromosomes, but no aster had formed. Regardless of whether they were subjected to IA, IAA, or DA treatments, if the oocytes contained pronuclear-like structures, either one or two asters were observed in proximity to the nuclei. A significantly higher rate of development to blastocysts was achieved in embryos that were immediately activated (IA, 59.1%; IAA, 40.7%) than in those for which activation was delayed (14.2%). The development rate was higher in group IA than in group IAA, but it was not significant (P = 0.089). Following embryo transfer, there was no statistically significant difference in the pregnancy rates (Day 70) between two of the groups (group IA, 11.7%, n = 94 vs. group DA, 12.3%, n = 130; P > 0.05) or live term development (group IA, 4.3% vs. group DA, 4.6%; P > 0.05). Our study has demonstrated that the IA of bovine NT embryos results in embryos with increased competence for preimplantational development. Moreover, PCC was shown to be unnecessary for the reprogramming of a transplanted somatic genome in a cattle oocyte.     

The change in the nucleus pattern during chromatin condensation in Eurygaster integriceps spermatids

When spermiogenesis of Eurygaster integriceps occurs, the pattern of the round nucleus of the spermaiid changes under the influence of a manchette consisting of two large groups of microtubules (up to 130–140 in each). The pericentriolar matter was found to be involved in the changes in the nuclear pattern; it takes part in the formation of the asymmetric intranuclear channel, open at one side. Two zones appear in the nucleus that differ greatly in their rates of chromatin condensation. The two main parts of the future nucleus are thereby established: the apical one with a greater rate of chromatin condensation, which gives origin to the two-walled cylinder, and the outer part, which grows for a long time and extends caudally to the middle piece. At the later stages of nucleus formation, when its larger part is separated from the surface of the middle piece, the vesicles of the endoplasmic reticulum that penetrate between the Nebenkern and the larger part of the nucleus, play a certain role.