Higher levels of organization in the interphase nucleus of cycling and differentiated cells.

The review examines the structured organization of interphase nuclei using a range of examples from the plants, animals, and fungi. Nuclear organization is shown to be an important phenomenon in cell differentiation and development. The review commences by examining nuclei in dividing cells and shows that the organization patterns can be dynamic within the time frame of the cell cycle. When cells stop dividing, derived differentiated cells often show quite different nuclear organizations. The developmental fate of nuclei is divided into three categories. (i) The first includes nuclei that undergo one of several forms of polyploidy and can themselves change in structure during the course of development. Possible function roles of polyploidy is given. (ii) The second is nuclear reorganization without polyploidy, where nuclei reorganize their structure to form novel arrangements of proteins and chromosomes. (iii) The third is nuclear disintegration linked to programmed cell death. The role of the nucleus in this process is described. The review demonstrates that recent methods to probe nuclei for nucleic acids and proteins, as well as to examine their intranuclear distribution in vivo, has revealed much about nuclear structure. It is clear that nuclear organization can influence or be influenced by cell activity and development. However, the full functional role of many of the observed phenomena has still to be fully realized.     

Relationship between Endopolyploidy and Cell Size in Epidermal Tissue of Arabidopsis

Relative quantities of DNA in individual nuclei of stem and leaf epidermal cells of Arabidopsis were measured microspectrofluorometrically using epidermal peels. The relative ploidy level in each nucleus was assessed by comparison to root tip mitotic nuclei. A clear pattern of regular endopolyploidy is evident in epidermal cells. Guard cell nuclei contain levels of DNA comparable to dividing root cells, the 2C level (i.e., one unreplicated copy of the nuclear DNA). Leaf trichome nuclei had elevated ploidy levels of 4C, 8C, 16C, 32C, and 64C, and their cytology suggested that the polyploidy represents a form of polyteny. The nuclei of epidermal pavement cells were 2C, 4C, and 8C in stem epidermis, and 2C, 4C, 8C, and 16C in leaf epidermis. Morphometry of epidermal pavement cells revealed a direct proportionality between nuclear DNA level and cell size. A consideration of the development process suggests that the cells of highest ploidy level are developmentally oldest; consequently, the developmental pattern of epidermal tissues can be read from the ploidy pattern of the cells. This observation is relevant to theories of stomate spacing and offers opportunities for genetic analysis of the endopolyploidy/polyteny phenomenon.     

Ultrastructure of meristematic cells of dormant and released buds in Tradescantia paludosa

The lateral bud meristems of Tradescantia paludosa show a characteristic cytohistological zonation during dormancy. The cells comprising this so called ‘zone of inhibition’, which is located at the extreme tip of the bud apex, rarely synthesize nuclear DNA or undergo mitotic division. These nuclei are as large as prophase nuclei, yet contain only telophase (2C) amounts of DNA and significantly lower amounts of histone as compared to the 2C nuclei of the actively dividing cells.Ultrastructural observations of the nuclei in the ‘zone of inhibition’ show that a large proportion of the chromatin is organized as less condensed, diffuse, euchromatin fibrils; however, the chromatin of the actively dividing nuclei of the cells outside the ‘zone of inhibition’ or in the released bud meristems is organized to a greater extent as condensed clumps of heterochromatin. When the dormancy is released, the nuclei in the ‘zone of inhibition’ synthesize DNA and histone and undergo cell division in approx. 4 days. Striking changes in the organization of chromatin fibrils take place during this transition period. The diffuse chromatin fibrils of the nuclei in the ‘zone of inhibition’ progressively become more and more condensed as the cell prepares to undergo the first mitotic division after the release of dormancy. This change which is coupled with the synthesis of histones in the nuclei of the ‘zone of inhibition’ suggests a prominent structural role of these basic proteins in the organization of the chromatin. The large volume of 2C nuclei of the ‘zone of inhibition’ seems, therefore, to result not from a great nuclear mass, but probably from a relatively small degree of condensation of chromatin.     

Cellular aspects of hypertrophic cardiomyopathy pathogenesis: the role of cardiomyocyte polyploidy and activation of the proliferating cell nuclear antigen in the myocardium

Mechanisms of hypertrophy development in hypertrophic obstructive cardiomyopathy (HOCM) have not been enough investigated. In our study, there have been examined patients with severe HOCM at different ages, including children, and patients with essential arterial hypertension (EAH). There was found, that HOCM in children compared to adults was characterized by considerable interventricular septum (IVS) hypertrophy and it was accompanied by the acceleration of cardiomyocyte polyploidy. The average ploidy level of cardiomyocytes in children with HOCM was higher than analogous indices in adults. The average ploidy level of nuclei, the part of PCNA-positive nuclei and polyploidic nuclei of cardiomyocytes in aduls with HOCM were authentically higher than in patients with EAH. Activation of the nuclear antigen in stromal cells was detected only in patients with HOCM. Our findings provide evidence of an important role of cardiomyocyte polyploidy and activation of the proliferating cell nuclear antigen in development of the myocardial hypertrophy in patients with HOCM.     

Nuclear envelope and nuclear pore complex structure and organization in tobacco BY-2 cells

The nuclear envelope (NE) is a fundamental structure of eukaryotic cells with a dual role: it separates two distinct compartments, and enables communication between them via nuclear pore complexes (NPCs). Little is known about NPCs and NE structural organization in plants. We investigated the structure of NPCs from both sides of the NE in tobacco BY-2 cells. We detected structural differences between the NPCs of dividing and quiescent nuclei. Importantly, we also traced the organizational pattern of the NPCs, and observed non-random NPC distribution over the nuclear surface. Lastly, we observed an organized filamentous protein structure that underlies the inner nuclear membrane, and interconnects NPCs. The results are discussed within the context of the current understanding of NE structure and function in higher eukaryotes.     

Nuclear pore biogenesis into an intact nuclear envelope

Nuclear pore complexes (NPCs) serve as transport channels across the nuclear membrane, a double lipid bilayer that physically separates the nucleoplasm and cytoplasm of eukaryotic cells. New evidence suggests that the multiprotein nuclear pores also play a role in chromatin organization and gene expression. Given the importance of NPC function, it is not surprising that a growing list of human diseases and developmental defects have been linked to its malfunction. In order to fully understand the functional repertoire of NPCs and their essential role for nuclear organization, it is critical to determine the sequence of events that lead to the formation of nuclear pores. This is particularly relevant since NPC number, and possibly composition, are tightly linked to metabolic activity. Most of our knowledge is derived from NPC formation that occurs in dividing cells at the end of mitosis when the nuclear envelope (NE) and NPCs reform from disassembled precursors. However, NPC assembly also takes place during interphase into an intact NE. Importantly, this process is not restricted to dividing cells but also occurs during cell differentiation. Here, we will review aspects unique to this process, namely the regulation of nuclear expansion and the mechanisms of fusion between the outer and inner nuclear membranes. We will then discuss conserved and diverging mechanisms between post-mitotic and interphase assembly of the proteinaceous structure in light of recently published data.     

Cellular aspects of pathogenesis of hypertrophic cardiomyopathy: Role of cardiomyocyte polyploidy and activation of nuclear antigen of the proliferating cell in myocardium

For a comparative analysis of cytomorphological characteristics of hypertrophied interventricular septum (IVS), both patients different ages with severe courses of obstructive hypertrophic cardiomyopathy (OHCMP) were examined, including children, and patients with essential arterial hypertension (EAH). The course of OHCMP in children as compared with adults was found to be characterized by considerable IVS hypertrophy that was accompanied by an acceleration of cardiomyocyte polyploidization. The mean ploidy level of cardiomyocytes in children with OHCMP was higher than in adult patients. The mean ploidy level of nuclei, the number of prolipherative cell nuclear antigen (PCNA)-positive nuclei, and the number of polyploid cardiomyocyte nuclei in adult patients with OHCMP were significantly higher statistically than in patients with EAH. The PCNA-positive labels in stromal cells were revealed only in patients with OHCMP. The obtained data indicate an important role of cardiomyocyte polyploidy and of activation of the proliferating cell nuclear antigen in development of myocardial hypertrophy in patients with OHCMP.     

Suggestive Structural Evidence for Macronuclear "Subnuclei" in Tetrahymena pyriformis GL

SYNOPSIS Structural changes in the Feulgen-positive material of the Tetrahymena pyriformis GL macronucleus have been observed during the cell cycle. From the finely granulated appearance in the interphase cell it appears as small rods, often arranged in pairs (probably the endomitotic stage) during early morphogenesis and as larger (and fewer) aggregates of granules during the nuclear division. These latter aggregates are also visible in dividing nuclei in the electron microscope where groups of chromation granules are separated by fairly empty nucleoplasm. It is suggested that these Feulgen positive aggregates in dividing nuclei are macronuciear segregation units or "subnuclei." The number per dividing macronucleus may vary from one experiment to another, but the variation seems to be related to cell volume. The distribution of the aggregates among the daughter nuclei is almost equal. The total number per dividing macronucleus is about 80 which is close to the estimated number of "subnuclei" in the T. pyriformis macronucleus (Allen and Nanney, 1958).
Some calculations are made on the polyploidy of the T. pyriformis GL macronucleus. Using published electron micrographs of micronuclei of known age to calculate the total number of chromatin granules per haploid nucleus, the polyploidy of the strain GL macronucleus is about 40. This figure is half of that expected from Allen and Nanney's estimation, since they assumed that the "subnuclei" were diploid; however, it is in agreement with the reported haploid nature of the "subnuclei" as found by Woodard, Gorovsky & Kaneshiro, 1968. Further calculations suggest that each macronuclear "chromosome" is composed of about 40 chromatin granules; an indication of such a chain arrangement of the chromatin granules has been observed in the phase contrast and electron microscope during the earliest macronuclear events, i.e., at the macronuclear "prophase."     

Occurrence of amitotic division of trophoblast cell nuclei in blastocysts of the western spotted skunk (Spilogale putorius latifrons)

A cytogenetic examination of spreaded cells of diapausing and early activated blastocysts obtained from 7 female western spotted skunks was performed. Mitosis was not observed in 1626 cells obtained from 9 diapausing blastocysts; however, 12 (1.5%) figures of diploid mitosis were seen in 851 cells from 5 early activated embryos. Diameter of the cell nuclei varied from 4 to 29 microm during diapause, and from 5 to 40 microm in activated blastocyst, and the heterogeneity in nuclear size was significantly different between diapausing and activated embryos (P<0.01). About 80% of nuclei from diapausing blastocysts measured 9 to 16 microm, whereas a similar percentage of nuclei from activated blastocysts ranged from 15 to 27 microm. Many enlarged nuclei exhibited morphological features characteristic of mammalian polytene (i.e. endopolyploid with polytenic organization of chromosomes) trophoblast cells. The number of silver stained nucleoli in all the nuclei did not exceed 2, which corresponds to the number of nucleolus organizers in the diploid karyotype in this species of skunk and suggests the polytene organization of chromosomes in enlarged nuclei. About 10% of large interphase nuclei were observed to undergo amitosis, i.e. direct division by constriction. The resulting nuclear fragments in diapausing blastocysts usually had normal morphology and active nucleoli. In activated embryos, nearly 15% of amitotically divided nuclei appeared to be dividing into fragments of unequal size, one of which had normal cell nuclear morphology and extremely large silver positive nucleoli, and the other fragment exhibited signs of cell death. We interpret these data as indicating that 1) amitotic division of trophoblast endopolyploid cell nuclei in the skunk blastocysts may generate new trophoblast cells which contribute to increased cell number during both diapause and activation stages, and 2) activation of blastocysts after diapause is related to the production of trophoblast cells with enhanced synthetic capabilities.     

Review: the dynamics of the nuclear lamins during the cell cycle-- relationship between structure and function

The nuclear lamins are members of the intermediate filament (IF) family of proteins. The lamins have an essential role in maintaining nuclear integrity, as do the other IF family members in the cytoplasm. Also like cytoplasmic IFs, the organization of lamins is dynamic. The lamins are found not only at the nuclear periphery but also in the interior of the nucleus, as distinct nucleoplasmic foci and possibly as a network throughout the nucleus. Nuclear processes such as DNA replication may be organized around these structures. In this review, we discuss changes in the structure and organization of the nuclear lamins during the cell cycle and during cell differentiation. These changes are correlated with changes in nuclear structure and function. For example, the interactions of lamins with chromatin and nuclear envelope components occur very early during nuclear assembly following mitosis. During S-phase, the lamins colocalize with markers of DNA replication, and proper lamin organization must be maintained for replication to proceed. When cells differentiate, the expression pattern of lamin isotypes changes. In addition, changes in lamin organization and expression patterns accompany the nuclear alterations observed in transformed cells. These lamin structures may modulate nuclear function in each of these processes.     

Review: The Dynamics of the Nuclear Lamins during the Cell Cycle— Relationship between Structure and Function

The nuclear lamins are members of the intermediate filament (IF) family of proteins. The lamins have an essential role in maintaining nuclear integrity, as do the other IF family members in the cytoplasm. Also like cytoplasmic IFs, the organization of lamins is dynamic. The lamins are found not only at the nuclear periphery but also in the interior of the nucleus, as distinct nucleoplasmic foci and possibly as a network throughout the nucleus. Nuclear processes such as DNA replication may be organized around these structures. In this review, we discuss changes in the structure and organization of the nuclear lamins during the cell cycle and during cell differentiation. These changes are correlated with changes in nuclear structure and function. For example, the interactions of lamins with chromatin and nuclear envelope components occur very early during nuclear assembly following mitosis. During S-phase, the lamins colocalize with markers of DNA replication, and proper lamin organization must be maintained for replication to proceed. When cells differentiate, the expression pattern of lamin isotypes changes. In addition, changes in lamin organization and expression patterns accompany the nuclear alterations observed in transformed cells. These lamin structures may modulate nuclear function in each of these processes.     

The radial positioning of chromatin is not inherited through mitosis but is established de novo in early G1

The organization of chromatin in the nucleus is nonrandom. Different genomic regions tend to reside in preferred nuclear locations, relative to radial position and nuclear compartments. Several lines of evidence support a role for chromatin localization in the regulation of gene expression. Therefore, a key problem is how the organization of chromatin is established and maintained in dividing cell populations. There is controversy about the extent to which chromatin organization is inherited from mother to daughter nucleus. We have used time-lapse microscopy to track specific human loci after exit from mitosis. In comparison to later stages of interphase, we detect increased chromatin mobility during the first 2 hr of G1, and during this period association of loci with nuclear compartments is both gained and lost. Although chromatin in daughter nuclei has a rough symmetry in its spatial distribution, we show, for the first time, that the association of loci with nuclear compartments displays significant asymmetry between daughter nuclei and therefore cannot be inherited from the mother nucleus. We conclude that the organization of chromatin in the nucleus is not passed down precisely from one cell to its descendents but is more plastic and becomes refined during early G1.     

Functional role of newly formed pore complexes in postmitotic nuclear reorganization

Many nuclear proteins are released into the cytoplasm at prometaphase and are transported back into the daughter nuclei at the end of mitosis. To determine the role of this reentry in nuclear remodelling during early interphase, we experimentally manipulated nuclear protein uptake in dividing cells. Recently we and others have shown that signal-dependent, pore complex-mediated uptake of nuclear protein is blocked in living cells on microinjection of the lectin wheat germ agglutinin (WGA), or of antibodies such as PI1 that are directed against WGA-binding pore complex glycoproteins. In the present study, we microinjected mitotic PtK₂ cells with WGA or antibody PI1 and followed nuclear reorganization of the daughter cells by immunofluorescence and electron microscopy. The inhibitory effect on nuclear protein uptake was monitored by co-injection of the karyophilic protein nucleoplasmin. When injected by itself early in mitosis, nucleoplasmin became sequestered into the daughter nuclei as they entered telophase. In contrast, nucleoplasmin was excluded from the daughter nuclei in the presence of WGA or antibody PI1. Although PtK₂ cells with blocked nuclear protein uptake completed cytokinesis, their nuclei showed a telophaselike organization characterized by highly condensed chromatin surrounded by a nuclear envelope containing a few pore complexes. These findings suggest that pore complexes become functional as early as telophase, in close coincidence with nuclear envelope reformation. They further indicate that the extensive structural rearrangement of the nucleus during the telophase-G1 transition is dependent on the influx of karyophilic proteins from the cytoplasm through the pore complexes, and is not due solely to chromosome-associated components.Abbreviations WGA wheat germ agglutinin - GlcNAc N-acetylglucosamine     

Study of the structural and structure-functional subdomains in the interphase nucleus by photostabilization

The role of the nuclear matrix components in the organization of structural and functional domains of interphase nuclei was studied using irradiation with blue light in the presence of a photosensibilized agent (Ethidium bromide). Nuclear domain resistance to extractive solution (2 M NaCl) treatment served as a criterion of irradiation-induced stabilization of different nuclear domains. The following results have been obtained: 1) the structural organization of the complexes of chromatin and clusters of replication does not depend on the state of the nuclear matrix in isolated nuclei; 2) chemical stabilization of the nuclear matrix by Cu(2+)-ions is not sufficient for the organization of chromatin domains; 3) irradiation in the presence of Ethidium bromide stabilizes domains of the nuclei, but does not lead to stabilization of the nuclear matrix internal network. Hence, the irradiation prevented extraction from the nuclear domains of nonhistone proteins which were not standard matrix proteins. Based on the results obtained, a hypothesis was proposed about a coexistence of two groups of nonhistone proteins in the cell nucleus. The first group includes proteins of the nuclear matrix involved in immobilization of scafford attachment regions and active genes. The second group includes some hypothetical structural proteins participating only in compaction of DNA of condensed chromatin.     

Disruption of Nuclear Lamin Organization Alters the Distribution of Replication Factors and Inhibits DNA Synthesis

The nuclear lamina is a fibrous structure that lies at the interface between the nuclear envelope and the nucleoplasm. The major proteins comprising the lamina, the nuclear lamins, are also found in foci in the nucleoplasm, distinct from the peripheral lamina. The nuclear lamins have been associated with a number of processes in the nucleus, including DNA replication. To further characterize the specific role of lamins in DNA replication, we have used a truncated human lamin as a dominant negative mutant to perturb lamin organization. This protein disrupts the lamin organization of nuclei when microinjected into mammalian cells and also disrupts the lamin organization of in vitro assembled nuclei when added to Xenopus laevis interphase egg extracts. In both cases, the lamina appears to be completely absent, and instead the endogenous lamins and the mutant lamin protein are found in nucleoplasmic aggregates. Coincident with the disruption of lamin organization, there is a dramatic reduction in DNA replication. As a consequence of this disruption, the distributions of PCNA and the large subunit of the RFC complex, proteins required for the elongation phase of DNA replication, are altered such that they are found within the intranucleoplasmic lamin aggregates. In contrast, the distribution of XMCM3, XORC2, and DNA polymerase α, proteins required for the initiation stage of DNA replication, remains unaltered. The data presented demonstrate that the nuclear lamins may be required for the elongation phase of DNA replication.     

In planta quantification of endoreduplication using fluorescent in situ hybridization (FISH)

Endopolyploidy, i.e. amplification of the genome in the absence of mitosis, occurs in many plant species and happens along with organ and cell differentiation. Deciphering the functional roles of endopolyploidy is hampered by the fact that polyploid tissues generally comprise cells with various ploidy levels. In some fleshy fruits (amongst them tomato fruit) the ploidy levels present at the end of development range from 2C to 256C in the same tissue. To investigate the temporal and spatial distribution of endopolyploidy it is necessary to address the DNA content of individual nuclei in situ. Conventional methods such as fluorometry or densitometry can be used for some tissues displaying favorable characteristics, e.g. small cells, small nuclei, organization in a monolayer, but high levels of varying polyploidy are usually associated with large sizes of nuclei and cells, in a complex three dimensional (3-D) organization of the tissues. The conventional methods are inadequate for such tissue, becoming semi-quantitative and imprecise. We report here the development of a new method based on fluorescent in situ bacterial artificial chromosome hybridizations that allows the in situ determination of the DNA ploidy level of individual nuclei. This method relies on the counting of hybridization signals and not on intensity measurements and is expected to provide an alternative method for mapping endopolyploidy patterns in mature, 3-D organized plant tissues as illustrated by the analysis of ploidy level and cell size in pericarp from mature green tomato fruit.     

A NOVEL PATTERN OF APICAL CELL POLYPLOIDY,SEQUENTIAL POLYPLOIDY REDUCTION AND INTERCELLULAR NUCLEAR TRANSFER IN THE RED ALGA POLYSIPHONIA

Nearly a century ago, Rosenvinge published a now-classic paper reporting nuclear transfer between cells of Polysiphonia during secondary pit connection (SPC) formation. While reinvestigating this phenomenon, we discovered that the uninucleate apical cell, which is the progenitor of all cells in the plant, has many times (ca. 64–128 ×) the level of nuclear DNA characteristic of nuclei of gametes or mature pericentral cells. Via a regular sequence of cell divisions, the polyploid apical cell gives rise to tiers of cells, each composed of a number of pericentral cells which surround a single central cell. A large proportion of the nuclear divisions are not accompanied by DNA replication. Thus, as the number of nuclei within elongating pericentral cells increases, the DNA level of nuclei in these cells “cascades” down to the DNA level expected for the particular life history generation (i.e., gametophyte or tetrasporophyte). In mature pericentral cells, the number of nuclei is proportional to the volume of the cell. The pattern of nuclear division, reduction in ploidy level and the timing of intercellular nuclear transfer via SPC formation is regular and characteristic of a species. Nuclei transferred from one cell to an adjacent cell participate in the further nuclear divisions of the recipient cell. The degree of polyploidy in apical cells may determine the number of cells in a “determinant” branch or even the number of cells in “indeterminant” axes. In addition, the highly polyploid state of the germinating spore and its pattern of development may provide for the rapid initial growth so characteristic of this taxon.