Postmetaphase nuclear formation: loss of a chromosomal epitope coincident with apparent chromatid coalescence.

Previously, we have conceptualized mitotic nuclear formation following metaphase as a morphogenic process and have suggested that sets of chromatids, after separation from a metaphase plate, can be thought of as prenuclei. Such structures can be grouped temporally as either early or late prenuclei based on morphologic, morphometric and density characteristics. Sequential ordering of early prenuclei is of particular interest because it reveals that condensed chromatids coalesce with the resulting formation of a unique chambered structure. In this paper we describe data obtained with a newly raised monoclonal antibody (mAb-2) that initially recognizes an epitope(s) on metaphase chromosomes. Light and confocal fluorescent microscopy of early prenuclei reveal that the chromosomal epitope can no longer be detected about chromatids after their apparent coalescence. Immunoblot analysis of dispersed polypeptides of metaphase plates and early prenuclei indicates that the major protein antigens recognized by mAb-2 have apparent molecular masses of approximately 106000 and 80500 and that each is likely composed of multiple charge isomers. A dual fluorescent analysis using mAb-2 and high-titer anti-lamin B serum provides additional evidence that chromatid coalescence is a separate, early event that precedes nuclear lamina formation.

Computer graphics of SEM images facilitate recognition of chromosome position in isolated human metaphase plates.

There is general agreement that at the time of mitosis chromosomes occupy precise positions and that these positions likely affect subsequent nuclear function in interphase. However, before such ideas can be investigated in human cells, it is necessary to determine first the precise position of each chromosome with regard to its neighbors. It has occurred to us that stereo images, produced by scanning electron microscopy, of isolated metaphase plates could form the basis whereby these positions could be ascertained. In this paper we describe a computer graphic technique that permits us to keep track of individual chromosomes in a metaphase plate and to compare chromosome positions in different metaphase plates. Moreover, the computer graphics provide permanent, easily manipulated, rapid recall of stored chromosome profiles. These advantages are demonstrated by a comparison of the relative position of group A-specific and groups D- and G-specific chromosomes to the full complement of chromosomes in metaphase plates isolated from a nearly triploid human-derived cell (HeLa S3) to a hypo-diploid human fetal lung cell.

Intermediate structures in nuclear morphogenesis following metaphase from HeLaS3 cells can be isolated and temporally grouped.

Previously nuclear reformation following metaphase in HeLaS3 cells was conceptualized in terms of a stepwise process which was continuous throughout anaphase and telophase. This concept was based on a three-dimensional visualization by scanning electron microscopy (SEM) of individual, organically prepared chromatid structures (prenuclei) which could be sequentially arranged. Morphologic analysis revealed unique topographies and morphometric properties which suggested that it should be possible to isolate populations of prenuclei aqueously. Such an isolation using detergents and density centrifugation is presented which yields metaphase plates and two populations of prenuclei with distinctive morphology. Essentially, prenuclei are freed from late mitotic cells in suspension cultures of synchronized HeLaS3 cells by treatment with 0.1% Nonidet-P40 followed by treatment with a mixture of Tween 40-desoxycholate (0.5%). Critical for the isolation is the presence of a divalent cation (5 mM Mg(+)+) and an acid pH (approximately 5.8). After density centrifugation, 2N decondensing structures (late intermediates) are recovered from 42% Percoll, and a mixture of 2N predecondensing (early intermediates) and 4N metaphase plates are recovered from 52% Percoll. The latter intermediates can be further separated into highly enriched populations (greater than 94% pure) by fluorescence-activated sorting. Predecondensing structures are of the same overall morphology as prenuclei isolated previously by organic means, can also be ordered sequentially to demonstrate nuclear morphogenesis, and retain centromere/kinetochore loci. These chromosomal loci based on immunostaining of individual structures appear to be positioned centrally during chromatid reassociation and then appear to be dispersed prior to structural rearrangements leading to formation of a disc-like prenucleus.(ABSTRACT TRUNCATED AT 250 WORDS)

Chromatid behavior in late mitosis: a scanning electron microscopy analysis of mammalian cell lines with various chromosome numbers.

Chromatid activity during the process of nuclear reformation following metaphase is a period of mitosis where little precise information is available. Nuclear reformation requires that chromosomes, at metaphase and chromatids during anaphase and telophase align, position and associate in a clearly defined sequence to insure the specific design of each nucleus. Four cell lines with chromosome numbers ranging from seven to almost seventy were chosen to determine whether the process of nuclear assembly is the same throughout. Chromosomal alignment at metaphase is found to be radial in all four cell lines. Chromosome positioning is essentially the same in all four, where the smaller chromosomes are located centrally and longer ones are positioned peripherally in a radial alignment. Chromosomal association is directly related to chromosome number. The more chromosomes in a one dimensional plane occupying a given area, the closer the association. In comparing the HeLaS3 and muntjac chromatids, the former has the closer association at metaphase. Since association is the most important aspect of chromatid behavior in nuclear reformation, chromatid positioning becomes a vital process during anaphase movement. Chromatid positions established during anaphase determines later positioning in the interphase nucleus because of the subsequent interconnection of adjacent chromatids by the formation of a fibrous meshwork. This fibrous meshwork, formed in anaphase and early telophase, functions to stabilize chromatids following their positioning and it also serves as a substrate or matrix for the assembly of nuclear envelope.

Nuclear reformation following metaphase in HeLa S3 cells: three-dimensional visualization of chromatid rearrangements.

Nuclear reformation from chromatids following metaphase was visualized three-dimensionally for the first time in mammalian cells (HeLa S3) by scanning electron microscopy (SEM). Anaphase and telophase configurations free of mitotic apparatus, cytoskeletal elements and nuclear envelope were prepared using a slightly modified standard cytological procedure which permitted visualization of chromatid position and orientation. Mid-anaphase alignments were observed to be more complex than previously revealed by light and transmission electron microscopy (TEM). One pole consisted of chromatids joined along their lateral length, the other pole consisted of telomeres, apparently of the longest chromatids, aligned in a double concentric layer. As anaphase progressed, re-association of these chromatids appeared to occur progressively along their lateral length toward their telomeres. Morphological evidence is presented suggesting that this lateral re-association may involve interchromatid fibers. After complete joining, structures resembling a hollow half sphere had formed. Based on different preparative procedures for SEM and published TEM analysis, it is this shell-like configuration upon which the nuclear envelope is reestablished in early telophase. As telophase progressed there was loss in depth of the internal chamber resulting in a disc configuration. Following loss of chromatid outline from the surface of this structure, interphase nuclear shape was assumed. Morphometric determinations revealed relative dimensions of chromatid configurations and supported the conclusion that nuclear reformations proceeded by discrete steps. The complexity of such a process, as revealed by SEM analysis, is discussed.

A scanning electron microscopic technique for three-dimensional visualization of the spatial arrangement of metaphase, anaphase and telophase chromatids.

Chromosome and chromatid alignment in mitotic configurations remains a topic of interest because there is little precise information. For example, reconstruction of mitotic configurations from serial sections collected with transmission electron microscopy has proven to be neither practical nor a sensitive method for conceptualizing these arrangements. Similarly light microscopy has been even more unsatisfactory because of its limited resolution and lack of three-dimensional capabilities. These limitations conceivably could be overcome by visualization of mitotic configurations by scanning electron microscopy (SEM). However, SEM has its limitations, of which the most obvious with regard to visualization of mitotic configurations, is that such structures in dividing cells are obscured from the beam by membranes, cellular organelles, and the mitotic apparatus. These "contaminants," we have found, can be removed by the appropriate procedure such that a direct three-dimensional visualization of intact life-like mitotic configurations of chromatids from mammalian cells is possible. We also demonstrate that these configurations, although some artifacts may exist, retain the same basic shape and chromatid arrangements throughout metaphase, anaphase, and telophase when compared to configurations isolated with a non-ionic detergent and neutral buffers.

Chromosome stabilizing structures in mitotic Indian muntjac (Muntiacus muntjak) cells.

A new technique which removes all membranes, cytoskeletal elements, organelles, but preserves intact metaphase, anaphase and telophase configurations is combined with scanning electron microscopy (SEM) as an approach for direct visualization of chromosomal behavior in late mitosis. With this approach we are able to confirm the presence of a centromeric ring which stabilizes the centromeres during the cell cycle and present evidence for a lattice-like sheet of interchromatidic fibers in late mitosis.

Unregulated production of virus and/or sperm specific anti-idiotypic antibodies as a cause of AIDS.

A network of idiotypic and anti-idiotypic antibodies is often suggested as the basis for cellular interactions that maintain a steady-state immunological equilibrium. This hypothesis proposes that repeated exposure to certain external antigens--ie, both viral and sperm--stimulates an unregulated production of a uniquely potent immunomodulating idiotypic antibody(ies). In a genetically predisposed individual, this particular antibody(ies), which is also an autoantibody(ies), results in a cellular immune deficiency. This disruption in the immune system permits opportunistic infection and thus the acquired immune deficiency syndrome. This hypothesis, which is readily testable and which does not involve a primary pathogen, can explain both the active induction of this disease in, as well as its passive transfer to, all at-risk populations.

The endoplasmic reticulum in the cortex of developing guard cells: coordinate studies with chlorotetracycline and osmium ferricyanide.

The distribution of endoplasmic reticulum (ER) was investigated in young guard cells of Vicia faba and Allium cepa in order to gain more information on the control of guard cell development. Young, living guard cells of V. faba fluoresce when exposed to 25-100 microM chlorotetracycline (CTC). Intense fluorescence is restricted to the cytoplasm between the nucleus and adjacent regions of the ventral and paradermal walls. Much of the fluorescence is fibrillar in appearance and seems to arise from endomembranes, but not from particulate organelles such as mitochondria and plastids. A similar fluorescence pattern is produced by the membrane probes oxytetracycline and N-phenyl-1-napthylamine. Procaine and dibucaine render the fluorescence highly prone to photobleaching. Fluorescence appears near the ventral wall during early stages of cell development but declines when the guard cells mature. Epidermal tissue of V. faba and A. cepa was examined in the electron microscope with the aid of osmium ferricyanide staining. ER appears to be concentrated in regions of the guard cell that exhibit intense CTC fluorescence, while no other organelles (e.g., mitochondria) are similarly distributed. Much of the ER consists of a tubular network in close proximity to the plasmalemma. Our results indicate that the ER becomes asymmetrically distributed in young guard cells adjacent to those regions of the cell wall that undergo extensive thickening during cell differentiation. Furthermore, these membranes appear to sequester divalent cations such as Ca2+.

Evidence for a unique profile of phosphatidylcholine synthesis in late mitotic cells.

Evidence is presented that the structural rearrangements in late mitosis are accompanied by an alteration in membrane lipid synthesis. This evidence was derived from analyzing phospholipid classes after rapid-labeling, as well as from determining the intracellular site of incorporation of choline by HeLa S3 cells as they progressed from metaphase into early interphase (G1). Compared with postmitotic cell data, the recent mitotic cell data indicate a specific two- to threefold increase in the net synthesis of phosphatidylcholine (PC) species, which appeared to contain the more saturated fatty acids. Since this was observed with glycerol, choline, and orthophosphate labelings, and not with methyl labeling, it appears that the CDP-choline plus diacylglycerol pathway rather than the phosphatidylethanolamine to PC pathway was augmented. Electron microscope autoradiography of anaphase, telophase, and early G1 cells demonstrated that the reformed nuclear envelope was the incorporation site of a significant proportion of the newly synthesized PC. This incorporation occurred by early telophase prior to chromosome decondensation. The potential significance of PC metabolism with regard to membrane rearrangements, such as nuclear envelope reformation, is discussed.

Nuclear matrix: a cell-cycle-dependent site of increased intranuclear protein phosphorylation.

Evidence is presented that the nuclear matrix is a cell-cycle-dependent site of increased intranuclear protein phosphorylation. The incorporation of radioactive phosphate (32P) is highest during the premitotic (G2) phase and 40-50% less in the postmitotic phase (G1). This is observed for both total matrix protein and for several individual polypeptides ranging in molecular mass from greater than 200 kDa to 19 kDa. The phenomenon can be demonstrated when the matrix is isolated from orthophosphate-labeled intact cells, as well as when the matrix is isolated and then incubated in vitro in a protein kinase reaction mixture. The ability of the isolated matrix to mimic the events in vivo indicates the presence of endogenous protein phosphokinase activity and physiological substrates in this isolated nuclear fraction. Further evidence for such mimicry was obtained when amino acid phosphorylation sites were determined. Phosphoserine is the most abundant phosphoamino acid in the matrix labelled both in vitro and in vivo, although phosphothreonine and phosphotyrosine are also present. On the basis of several pieces of data, the endogenous matrix activity appears to be due to multiple protein phosphokinases. Since the maximum phosphorylation coincides with premitosis, the phosphoproteins may play a role in mitotic events. These observations extend and expand the application of this fraction to the study of nuclear structure/function relationships, particularly at the time of mitosis.

Uptake and early fate of metaphase chromosomes ingested by the Wi-L2 human lymphoid cell line.

Aspects of the ingestion and early intracellular fate of homologous. [3H]-thymidine-labeled chromosomes (donor) were studied in recipient Wi-L2 cells in the absence of reutilized radioactivity. As much as 67% of the cell-associated radioactivity was resistant to hydrolysis by DNase I after 4 h of incubation. Cell fractionation and electron microscope autoradiography indicated that chromosome uptake was rapid, into both cytoplasmic and nuclear fractions and was facilitator and dose dependent. Sedimentation analysis demonstrated that at 4 h donor DNA of approximate single-strand mol wt of 1--6 X 10(6), as compared to 6--12 X 10(6) for chromosomal DNA, was recoverable in cell fractions. By 6 h, a significant portion of the nucleus-associated donor DNA was converted into material of higher mol wt, although no evidence was found for integration into recipient DNA. Cytoplasmic donor DNA continued to be degraded. An average number of chromosome equivalents of nucleus-associated donor DNA to recipient cell nuclei of 1--4 was obtained and its relationship to the lower frequency of chromosome-mediated gene transfer is discussed.

Nuclear matrix of HeLa S3 cells. Polypeptide composition during adenovirus infection and in phases of the cell cycle.

A subnuclear fraction has been isolated from HeLa S3 nuclei after treatment with high salt buffer, deoxyribonuclease, and dithiothreitol. This fraction retains the approximate size and shape of nuclei and resembles the nuclear matrix recently isolated from rat liver nuclei. Ultrastructural and biochemical analyses indicate that this structure consists of nonmembranous elements as well as some membranous elements. Its chemical composition is 87% protein, 12% phospholipid, 1% DNA, and 0.1% RNA by weight. The protein constituents are resolved in SDS-polyacrylamide slab gels into 30-35 distinguishable bands in the apparent molecular weight range of 14,000 - 200,000 with major peptides at 14,000 - 18,000 and 45,000 - 75,000. Analysis of newly synthesized polypeptides by cylindrical gel electrophoresis reveals another cluster in the 90,000-130,000 molecular weight range. Infection with adenovirus results in an altered polypeptide profile. Additional polypeptides with apparent molecular weights of 21,000, 23,000, and 92,000 become major components by 22 h after infection. Concomitantly, some peptides in the 45,000-75,000 mol wt range become less prominent. In synchronized cells the relative staining capacity of the six bands in the 45,000-75,000 mol wt range changes during the cell cycle. Synthesis of at least some matrix polypeptides occures in all phases of the cell cycle, although there is decreased synthesis in late S/G2. In the absence of protein synthesis after cell division, at least some polypeptides in the 45,000-75,000 mol wt range survive nuclear dispersal and subsequent reformation during mitosis. The possible significance of this subnuclear structure with regard to structure-function relationships within the nucleus during virus replication and during the life cycle of the cell is discussed.

Adenovirus messenger RNA in mammalian cells: failure of polyribosome association in the absence of nuclear cleavage.

The nuclear synthesis of adenovirus-specific RNA late in the infectious cycle in the presence of toyocamycin (an adenosine analogue) has been investigated. There is reduced synthesis of viral RNA with an accumulation of virus-specific RNA in the molecular weight range of at least 4 to 8 x 10(6). No new viral RNA associates with cytoplasmic polyribosomes. In addition, hybridization competition experiments indicate a 70% competition between these large nuclear transcripts and polyribosome-associated viral RNA that was synthesized in the absence of inhibitor. These data are consistent with the following interpretations: complete nuclear processing of viral RNA is necessary for polyribosome association, and precursor viral message(s) contain sequences that are lost normally during post-transcriptional processing.