Dynamics of structural and functional association of nucleolar chromosomes in cells of the hexaploid wheat Triticum aestivum L. at different stages of the cell cycle and during genome polyploidization.

Quantitative analysis of interphase association of the nucleolar chromosomes at different stages of the cell cycle and during genome polyploidization was carried out. Cells of various tissues of hexaploid wheat Triticum aestivum L. (Moskovskaya-35) were used, including diploid root meristematic cells, endopolyploid root cells, triploid endosperm cells and antipodal cells with polytene chromosomes. Interphase nucleoli impregnated with silver or stained with autoimmune antibodies to 53 kDa nucleolar protein served as markers of the nucleolar chromosome association. The following data were obtained: (1) silver-staining revealed two pairs of homologous chromosomes 1B and 6B with active nucleolus-organizing regions in the root meristematic cells; (2) maximal number of nucleoli in diploid meristematic cells reaches four, which corresponds to the number of chromosomes with active organizers; (3) analysis of cells at different stages of the cell cycle has shown that the tendency to the nucleoli association is observed as soon as cells pass individual stages of the cycle; (4) after DNA and chromosome reduplication, the nucleolus-organizing regions in sister chromatids function as a common structure-functional complex; (5) in endopolyploid root cells and antipodal cells with polytene chromosomes, the number of nucleoli does not correlate with ploidy level, and an additional nucleolus revealed in some cells is the result of activation of the latent organizer in one of the nucleolar chromosomes; (6) in the triploid endosperm nucleologenesis, the stage of prenucleolar bodies is missing. Our data suggest that "fusion" of nucleoli and reduction of their number due to the "satellite" association of the nucleolar chromosomes are two independent processes regulated by different mechanisms.

Nuclear matrix proteins are carried within peripheral material of mitotic chromosomes.

Immunofluorescent analysis has shown that autoimmune sera M-222 and M-260 are bound to interphase nuclei and mitotic chromosomes of the pig embryo kidney cell culture. The fluorescent stain is diffuse in nuclei and forms a thin fluorescent area around each nucleolus, whereas the nucleolar cores are unstained. The periphery of each mitotic chromosome is stained distinctly. After removal of histones and DNA by the cell treatment with 2 M NaCl and DNase I, the Hoechst 33258 staining of nuclei and chromosomes disappears completely, whereas the pattern of staining with antibodies is not changed as compared with normal cells. Electron microscopy revealed in interphase nuclei after such treatment only lamina, residual nucleoli, and the intranuclear matrix network, and antibodies are bound just to these elements. Molecular mass of proteins bound to these antibodies was determined by immunoblotting. Serum M-260 contained antibodies to a single 65 kDa polypeptide, whereas antibodies to two polypeptides of 47 and 65 kDa were found in M-222. After chromatin removal and revealing nuclear protein matrix, M-222 binds only to 65 kDa polypeptides. Thus, peripheral chromosomal material is involved in transfer of the nuclear matrix polypeptide to daughter nuclei during mitosis.

Exclusively juvenile centrioles in Xenopus laevis oocytes injected with preparations of mature centrioles.

Activated oocytes of Xenopus laevis were injected with centriole preparations isolated either from spermatozoa of loach fish Misgurnus fossilis or from rat liver. These injections induced the development of cytasters in the ooplasm and egg cleavage. Electron microscopic study of cytasters was made at the stage that corresponded to interphase between first and second cleavage divisions. This study revealed in cytasters singleton centrioles surrounded by pericentriolar material and numerous microtubules. Surprisingly, the ultrastructure of centrioles in cytasters corresponded to that of juvenile, newly formed vertebrate centrioles, whereas the injected preparations contained only adult mature centrioles. We suggested that xenogenic centrioles injected to Xenopus laevis oocytes could dissolve after formation of centrioles made from molecules of oocyte origin. A special mechanism that eliminates male centrioles after egg fertilization is speculated.

Reassembly of functional nucleoli following in situ unraveling by low-ionic-strength treatment of cultured mammalian cells.

In order to determine the most persistent components of the nucleolus that might serve as "core" nucleolar elements, we studied the reactivity of nucleoli in living mammalian cells subjected to hypotonic buffer saline followed by the incubation of the cells in an isotonic medium. To document as precisely as possible the fine structural changes which occurred, the cells were examined by video-enhanced optical microscopy, fluorescence confocal laser scanning microscopy, and electron microscopy combined with cytochemistry. Light microscopic autoradiography was used to demonstrate the transcriptional characteristics of the reassembled nucleoli. It was shown that all the major compartments of the intact nucleolus could be substantially affected by reduction of the osmolarity of the environmental media. The dynamic events of the nucleolar unraveling in low-salt buffers occurred in the following order: dispersion of the nucleolar pars granulosa, disassociation of the fibrillar complexes into discrete fibrillar centers (FCs) and the dense fibrillar component (DFC), and the almost complete unraveling of the DFC and FCs. At the terminal stages of nucleolar dispersion, the nuclear interior was mainly composed of a loose filamentous meshwork, and none of the typically discerned nucleolar constituents was recognized. Nevertheless, when hypotonically treated cells were returned to isotonic conditions, the nucleolar bodies rapidly began to reassemble. Within 1-2 h of cell incubation under isotonicity, the nucleoli not only became clearly visible, but also reconstituted to their initial size, shape, and position within the nucleus. The ultrastructure and functional activity of the reassembled nucleoli were also found to be fully comparable to those of the untreated controls. These data indicate that the architectural composition of the interphase nucleolus is strictly controlled by the cell. As far as could be determined, none of the usual substructures of the intact nucleolus that could be substituted by complete reassembly of the nucleolar bodies in normotonic conditions, including FCs and the DFC, remained clearly preserved in the terminal stage of nucleolar unraveling. We concluded that the integrity of the nucleolus was mainly preserved by the nuclear or nucleolar matrix system rather than by any other nucleolar structural domains.

The silver-stained NOR and argentophilic nuclear proteins in early mouse embryogenesis: a cytological study.

Silver staining (Howell and Black, 1980) was used in light and electron microscopic studies for detecting the localization of argentophilic nuclear proteins in fertilized ova and cleaving mouse embryos. No silver-stained nucleolus organizing regions (NORs) (Ag-NORs) were visualized in the metaphase chromosomes of the first cleavage mitosis. From the 2-cell stage on, metaphase chromosomes contained Ag-NORs. Argentophilic proteins were detected in the pronuclei of the 1-cell embryos, i.e. before transcription of the ribosomal genes started. After fertilization these proteins accumulated on the decondensing sperm chromatin and telophase maternal chromosomes, then migrated into the pronuclei to be stored in pronucleoli, and, during mitosis, were transferred into the cytoplasm. In the metaphase chromosomes of the cleaving embryos Ag-NORs adequately reflected the transcriptional activity of the ribosomal genes, whereas in pronuclei of the 1-cell stage embryos argentophilic proteins were not involved in this process, but are likely to play a part in the formation and maturation of pronucleoli, and in the cell cycle regulation.

Differential decondensation of mitotic chromosomes during hypotonic treatment of living cells as a possible cause of G-banding: an ultrastructural study.

The chromosomal ultrastructure of Chinese hamster cells treated with 0.075 M KCl - a solution ordinarily used for making preparations of spread chromosomes - was studied. The hypotonic treatment was shown to result in differential decondensation of chromosomes which consists in the uneven distribution of deoxyribonucleoprotein (DNP) fibrils along chromatids. Fixation of cells with methanol acetic acid causes an abrupt restructuring of chromosomes. However, the DNP preserves its uneven distribution along chromatids. As seen on ultra-thin sections of marker nucleolus organizer chromosomes, the densely packed regions may correspond to G-bands detected in the selfsame chromosomes by standard methods of differential staining. The results suggest that the capacity of chromosomes for differential staining is based on the different resistance of G- and R-bands to the decondensing action of hypotonic solutions on living cells.

Ultrastructural changes in nucleoli and fibrillar centers under the effect of local ultraviolet microbeam irradiation of interphase culture cells.

As shown previously, ultraviolet (uv) microbeam irradiation of one of the two mature nucleoli within an interphase cell nucleus causes significant diminution and inactivation of the irradiated nucleolus and compensatory growth and activation of the nonirradiated one. In the present work we describe the results of an ultrastructural study of this phenomenon. The changes in the nucleoli were examined by means of complete series of ultrathin sections obtained from seven irradiated pig kidney cells. The compensatory hypertrophy of the nonirradiated nucleoli is shown to be accompanied by a nearly twofold increase in the number of fibrillar centers (FCs) and by a decrease in their linear dimensions compared with the control cells of the same ploidy. In the degraded nucleoli the number of FCs decreases, but their dimensions increase. Ultraviolet microbeam irradiation causes dramatic diminution of the dense fibrillar component within the irradiated nucleoli as well. The nucleolar capacity for compensatory hypertrophy indicates that in addition to active ribosomal genes, mature nucleoli also contain "silent" genes capable of being activated under extreme conditions to sustain the required level of rRNA synthesis. It is assumed that activation of latent ribosomal genes is accompanied by FC "fragmentation" without a considerable increase in their total volume per cell.

Changes in the number and volume of fibrillar centres with the inactivation of nucleoli at erythropoiesis.

The number and volume of fibrillar centres, the structural components of interphase cell nucleoli on the surface of which rRNA is synthesized, have been studied in differentiating erythroblasts of mouse embryo liver. Complete series of ultrathin sections of erythroblast nuclei have been used at the main stages of differentiation: proerythroblast, basophilic erythroblast, polychromatophilic erythroblast and normoblast. It has been shown that in the active nucleoli of proerythroblasts the number of fibrillar centres is correlated with cell ploidy and exceeds by several-fold the number of nucleolus-organizing regions of chromosomes. The total volumes of fibrillar centres in 2C (0.369 micron 3) and 4C (0.749 micron 3) proerythroblasts are proportional to number of nucleolus-organizing regions. With the maturation of erythroblasts the total number of fibrillar centres declines and in normoblasts it is 3- to 10-fold less than that of the nucleolus-organizing regions. The total volume of fibrillar centres in normoblasts (0.102 micron 3) is threefold smaller than that in proerythroblasts (0.369 micron 3), even though the mean volumes of individual fibrillar centres are significantly higher (0.0042 micron 3 in proerythroblasts and 0.039 in normoblasts). The optical density of fibrillar centres in normoblasts can be higher compared with that of proerythroblasts. It has been suggested that the inactivation of nucleoli at erythropoiesis is accompanied by the fusion of individual fibrillar centres and, possibly, by the compaction of their material.

Coupling membranes as energy-transmitting cables. I. Filamentous mitochondria in fibroblasts and mitochondrial clusters in cardiomyocytes.

An hypothesis considering mitochondria as intracellular power-transmitting protonic cables was tested in human fibroblasts where mitochondria are thin and long and in rat cardiomyocytes where they show cluster organization. Mitochondria in the cell were specifically stained with fluorescent-penetrating cation ethylrhodamine, which electrophoretically accumulates in the mitochondrial matrix. A 40-micron-long mitochondrial filament of fibroblast was illuminated by a very narrow (less than or equal to 0.5 micron) laser beam to induce local damage of the mitochondrial membranes. Such a treatment was found to induce quenching of the ethylrhodamine fluorescence in the entire filament. According to the electron microscope examination, the laser-treated filament retained its continuity after the laser illumination. Other mitochondrial filaments (some of which were localized at a distance less than 10 micron from the laser-treated one) remained fluorescent. In a cell where mitochondrial filaments seemed to be united in a network, laser illumination of one filament resulted in fluorescence quenching in the whole network, whereas fluorescence of small mitochondria not connected with the network was unaffected. The illumination of cardiomyocyte was found to result in the fluorescence quenching not only in a laser-illuminated mitochondrion but also in a large cluster of organelles composed of many mitochondria. Electron microscopy showed that all the mitochondria in the cluster change from the orthodox to the condensed state. It was also found that mitochondria in the cluster are connected to one another with specific junctions. If a mitochondrion did not form junctions with a quenched cluster, its fluorescence was not decreased even when this mitochondrion was localized close to an illuminated one. The size of the mitochondrial cluster may be as long as 50 micron. The cluster is formed by branched chains of contacting mitochondria, which may be defined as Streptio mitochondriale. In the cardiomyocyte there are several mitochondrial clusters or, alternatively, the quenched cluster is a result of decomposition of a supercluster uniting all the mitochondria of the cell. Cluster organization of mitochondria could also be revealed when a single mitochondrion was punctured in situ with a microcapillary. The obtained data are in agreement with the idea that mitochondrial junctions are H+ permeable so that, within the cluster, delta psi may be transmitted from one mitochondrion to another. The above results are consistent with the assumption that mitochondrial filaments or networks represent a united electrical system.(ABSTRACT TRUNCATED AT 400 WORDS)

The centriolar rim. The structure that maintains the configuration of centrioles and basal bodies in the absence of their microtubules.

Preparations of centrioles from bovine spleen were incubated in solutions of NaCl, MgCl2, HCl, NaOH, EDTA and heparin. Their effects on the centrioles were studied by electron microscopy of ultrathin sections. It was found that the microtubules of centriolar cylinders gradually disintegrate at a higher than physiological ionic strength and at a pH value lower than 3.5 and higher than 8.5. After microtubule extraction, a closely apposed rim or sheath of dense centriolar matrix remains which has the same dimensions of length and width as the original centriole. Some other centriolar structures, including the pericentriolar satellites and certain structures in the cylinders (hub) are also preserved. The basal bodies of fish spermatozoa revealed similar structures, including the centriolar rim and hub, after microtubule extraction. Thus, the microtubule triplets are not involved in maintaining the structure of the centriolar cylinder; this role is rather carried out by amorphous material--the matrix, surrounding the microtubules.

The dynamics of reconstitution of microtubules around the cell center after cooling.

In interphase PE cells, after cooling (2 h at 0 degree C), some microtubules are retained in the cytoplasm. After the transfer of the cells to a thermostat (37 degrees C), the reconstitution of the microtubule network begins near the cell center. At this time in most of the cells around the center one can see the electron-dense foci of convergence of microtubules which then disappear. The number of microtubules diverging radially from the mother centriole reaches a maximum after 15 to 16 min, that of microtubules growing from the daughter centriole 12 min after the cells are placed at 37 degrees C. 45 min after the heating started the number of radially diverging microtubules somewhat exceeds the control level. These data show that microtubules are associated with the centers only during their growth. The mature microtubule is separated from the center and may be replaced by a new one. Thus, most, of not all, microtubules originate from the cell center, but at any moment only some of the microtubules are associated with it.

Ontogenesis of mitochondrial reticulum in rat diaphragm muscle.

Ontogenesis of mitochrondrial reticulum discovered in rat diaphragm has been studied with an electron microscope by the serial section technique. The amount of mitochondrial material in the diaphragm of rat embryos was found to be small. There are rare inclusions of single mitochondria which form columns paralleling the long axis of muscle fiber. After birth, a progressive increase in the amount of mitochondrial material takes place. This process is accompanied by the appearance of new components inherent in the unitary system of mitochondrial reticulum, namely mitochondrial junctions, as well as branches and tubules of mitochondrial material paralleling Z-discs and localized on the levels of the isotropic regions of sarcomeres. The first mitochondrial junctions were found to appear as early as on the second day, and the mitochondrial branches between the second and the ninth days of postnatal development. For a one-month-old rat, a three-dimensional model of a part of mitochondrial reticulum corresponding to one sarcomere was reconstituted. It revealed columns of mitochondrial material crossing the anisotropic region of sarcomere. Such columns connect two networks of mitochondrial profiles localized on the levels of two isotropic regions of sarcomere. Formation of mitochondrial reticulum is completed within two postnatal months.

The ultrastructure of centriole in mammalian tissue culture cells.

Structural polarity of centriole has been shown by analyzing serial sections of centrioles in the tissue culture cells of mouse, man, pig and Chinese hamster. Its major component is nine microtubule triplets. The inclination of the triplets towards the radius at the proximal end of the centriole is smaller than at the distal one. The internal tubule of the triplet has a smaller diameter than the middle and external ones; The triplets are bound by links of various nature all over their length. In the middle part, in the centriole lumen there is an amorphous hub; in the distal part, a thin fibre that is helically wound. In the proximal part, there are bases along the triplets, and handles stretch from the internal tubules. In the middle and distal parts, there are accumulations of an electron dense substance along the middle tubules. At the distal end, the centriole lumen is filled with an amorphous substance, whereas the proximal end is free from it. From outside, appendages are attached to the triplets at the distal end. The centriole structure is identical in all the cell types studied, except for those of Chinese hamster.

On the association of centrioles with the interphase nucleus.

Preparations of nuclei from rat liver and bovine spleen purified by centrifugation through dense sucrose solutions are shown to contain centrioles. These centrioles retain their in situ ultrastructure and are surrounded by a network of filaments adjacent to the nucleus and probably attached to it. The number of centrioles in isolated nuclei depends on the conditions of cell homogenization. Under certain conditions of homogenization, the fraction of purified nuclei contains almost all centrioles of the original tissue. The number of centrioles in isolated nuclei sharply decreases if the nuclei are rehomogenized under conditions that do not cause damage to nuclei. The number of nucleus-associated centrioles does not decrease after solubilization of nuclear membranes by Triton X-100. Nuclei retain the associated centrioles after treatmentwith RNase-free DNase I. It is concluded that in interphase the centrioles are associated with the nucleus and that this association which is probably mediated by filaments involves nuclear structures other than nuclear membranes or whole chromatin.

Partial purification of centrioles from spleen cells.

Isolation of a fraction enriched with centrioles from bovine spleen cells is described. The method involves preparation of a nuclear-centriolar complex and detachment of centrioles from the complex by mechanical treatment in a homogenizer. Purification of centrioles was achieved by centrifugation through a sucrose concentration gradient after treatment by ultrasound, DNase I and Triton X-100. The resulting centrioles retain their in situ ultrastructure.

Electron microscopic studies on the polytene chromosomes of Chironomus thummi salivary glands. I. Ultrastructural mapping.

The method of ultrathin sections of unsquashed salivary gland polytene chromosomes of Ch. thummi was applied to their ultrastrual mapping. There was a good agreement between electron micrographs and Hägele's light microscopic map (1970) with respect to the pattern and number of bands. 94% of bands were identified in larval and prepupal chromosomes. In Ch. thummi, band thickness varied from 0.05-0.5 mum. Most characteristic were 0.2-0.3 mum bands. Morphologically, bands were classified as: continuous (frequently with holes and gaps), discrete, dotted and continuous-discrete, discrete-dotted. Band morphology is related to band size, such that smaller bands, as a rule, were also dotted. Bands beginning to puff likewise became dotted. Interbands in unsquashed chromosome sections were from 0.05-0.15 mum. The smallest interbands contained only fibrils, in the larger interbands few granules could be observed. This makes interbands distinguishable from a typical puff with many such granules.