Ultrastructure of meiosis in the hollyhock rust fungus,Puccinia malvacearum

Summary Changes in the spindle pole body (SPB) and meiotic nuclei from interphase I through interphase II in the hollyhock rustPuccinia malvacearum are analyzed ultrastructurally by three-dimensional reconstructions from serial sections. Interphase I nuclei undergo a coordinated migration and rotation during which the SPBs approach the convex face of the lateral promycelial wall. During the transition from interphase I to prometaphase II, the collateral disc (co-disc) apparently enlarges and fuses with the main disc of the SPB. The resulting single SPB nucleates two confluent half spindles and about 225 astral microtubules (MTs). Co-discs and middle pieces (MPs) are absent during division II. SPBs separate and form metaphase II intranuclear spindles oriented in a predictable manner. Tubular cisternae are present within the spindle at early metaphase II. The architecture of the spindle at division II is essentially identical to that reported for division I except that the spindle is about half as long. Anaphase-telophase II nuclear envelope constriction, separation of the sibling nuclei, and externalization of the SPBs is identical to that reported for division I. Genesis of the duplicated interphase II SPB apparently occurs rapidly and involves formation of the MP followed by the three-layered SPB discs. General aspects of the division II spindle are discussed. A model for the meiotic SPB cycle in a rust is presented and its phylogenetic and functional significance in relation to other basidiomycetes and ascomycetes is discussed.     

Spindle Assembly and Mitosis without Centrosomes in Parthenogenetic Sciara Embryos

In Sciara, unfertilized embryos initiate parthenogenetic development without centrosomes. By comparing these embryos with normal fertilized embryos, spindle assembly and other microtubule-based events can be examined in the presence and absence of centrosomes. In both cases, functional mitotic spindles are formed that successfully proceed through anaphase and telophase, forming two daughter nuclei separated by a midbody. The spindles assembled without centrosomes are anastral, and it is likely that their microtubules are nucleated at or near the chromosomes. These spindles undergo anaphase B and successfully segregate sister chromosomes. However, without centrosomes the distance between the daughter nuclei in the next interphase is greatly reduced. This suggests that centrosomes are required to maintain nuclear spacing during the telophase to interphase transition. As in Drosophila, the initial embryonic divisions of Sciara are synchronous and syncytial. The nuclei in fertilized centrosome-bearing embryos maintain an even distribution as they divide and migrate to the cortex. In contrast, as division proceeds in embryos lacking centrosomes, nuclei collide and form large irregularly shaped nuclear clusters. These nuclei are not evenly distributed and never successfully migrate to the cortex. This phenotype is probably a direct result of a failure to form astral microtubules in parthenogenetic embryos lacking centrosomes. These results indicate that the primary function of centrosomes is to provide astral microtubules for proper nuclear spacing and migration during the syncytial divisions. Fertilized Sciara embryos produce a large population of centrosomes not associated with nuclei. These free centrosomes do not form spindles or migrate to the cortex and replicate at a significantly reduced rate. This suggests that the centrosome must maintain a proper association with the nucleus for migration and normal replication to occur.     

The DNA-based structure of human chromosome 5 in interphase

In contrast to those of metaphase chromosomes, the shape, length, and architecture of human interphase chromosomes are not well understood. This is mainly due to technical problems in the visualization of interphase chromosomes in total and of their substructures. We analyzed the structure of chromosomes in interphase nuclei through use of high-resolution multicolor banding (MCB), which paints the total shape of chromosomes and creates a DNA-mediated, chromosome-region-specific, pseudocolored banding pattern at high resolution. A microdissection-derived human chromosome 5-specific MCB probe mixture was hybridized to human lymphocyte interphase nuclei harvested for routine chromosome analysis, as well as to interphase nuclei from HeLa cells arrested at different phases of the cell cycle. The length of the axis of interphase chromosome 5 was determined, and the shape and MCB pattern were compared with those of metaphase chromosomes. We show that, in lymphocytes, the length of the axis of interphase chromosome 5 is comparable to that of a metaphase chromosome at 600-band resolution. Consequently, the concept of chromosome condensation during mitosis has to be reassessed. In addition, chromosome 5 in interphase is not as straight as metaphase chromosomes, being bent and/or folded. The shape and banding pattern of interphase chromosome 5 of lymphocytes and HeLa cells are similar to those of the corresponding metaphase chromosomes at all stages of the cell cycle. The MCB pattern also allows the detection and characterization of chromosome aberrations. This may be of fundamental importance in establishing chromosome analyses in nondividing cells.     

Reversible chromosome condensation induced in Drosophila embryos by anoxia: visualization of interphase nuclear organization

We have studied the morphology of nuclei in Drosophila embryos during the syncytial blastoderm stages. Nuclei in living embryos were viewed with differential interference-contrast optics; in addition, both isolated nuclei and fixed preparations of whole embryos were examined after staining with a DNA-specific fluorescent dye. We find that: (a) The nuclear volumes increase dramatically during interphase and then decrease during prophase of each nuclear cycle, with the magnitude of the nuclear volume increase being greatest for those cycles with the shortest interphase. (b) Oxygen deprivation of embryos produces a rapid developmental arrest that is reversible upon reaeration. During this arrest, interphase chromosomes condense against the nuclear envelope and the nuclear volumes increase dramatically. In these nuclei, individual chromosomes are clearly visible, and each condensed chromosome can be seen to adhere along its entire length to the inner surface of the swollen nuclear envelope, leaving the lumen of the nucleus devoid of DNA. (c) In each interphase nucleus the chromosomes are oriented in the "telophase configuration," with all centromeres and all telomeres at opposite poles of the nucleus; all nuclei at the embryo periphery (with the exception of the pole cell nuclei) are oriented with their centromeric poles pointing to the embryo exterior.     

Relationship of the DNA content in the nuclei of Amoeba proteus to the food regimen]

The relative DNA content of isolated Amoeba proteus nuclei has been measured by cytofluorometry. With the amoeba strain studied, the generation time is roughly equal to 48 hours at 25 degrees C, and with the presence of food in the medium. After the synchronous divisions, amoebae were maintained in the medium either with or without food organisms (Tetrahymena pyriformis). DNA contents in the nuclei of both the amoebae groups were measured within 4 and 48 hours after division. Before 16 hours, the nuclear DNA contents did not differ in either group. Starting from 20 hours, the DNA amount in fed amoebae exceeded that in starved animals. On the whole, the differences in DNA quantity increased by a 48th hour after division, when the nuclei of the former contained 145% DNA of the latter. The results obtained suggest that the DNA synthesis in amoeba nuclei may proceed during the whole interphase, and that during the second half of interphase the content of DNA may depend on the feeding intensity in amoebae. After refeeding the starved animals, DNA contents in their nuclei increased to reach the same level as in the constantly fed amoebae seen in the end of interphase.     

A proposed model of chromosomal organization in nuclei at fertilization

Evidence is presented to support the proposition that the position of chromosomes within nuclei is determined by the following factors: (1) the location of centromeres on one side of the nucleus and telomeres(ends) on the other (reflecting the telophase orientation brought about by their poleward anaphase migration); (2) attachment of telomeres to the nuclear membrane (site of attachment in relation to the poles and equator being dependent on the length of the individual arms and point 1 above); (3) telomere-to-telomere attachment of nonhomologues in a specific sequence; (4) telomere-to-telomere attachment of certain homologous chromosomes.It is proposed that a specific arrangement of nonhomologues occurs within gametic nuclei following meiosis, while initial homologous alignment takes place during karyogamy (fusion of gametic nuclei). The method of homologous association of telomeres is dependent on whether or not karyogamy within the species is between interphase pronuclei or occurs during the first cleavage division. A model of chromosome behavior for both these type of karyogamy is presented.     

Microtubules in Candida albicans hyphae drive nuclear dynamics and connect cell cycle progression to morphogenesis

Candida albicans is an opportunistic fungal pathogen whose virulence is related to its ability to switch between yeast, pseudohyphal, and true-hyphal morphologies. To ask how long-distance nuclear migration occurs in C. albicans hyphae, we identified the fundamental properties of nuclear movements and microtubule dynamics using time-lapse microscopy. In hyphae, nuclei migrate to, and divide across, the presumptive site of septation, which forms 10 to 15 microm distal to the basal cell. The mother nucleus returns to the basal cell, while the daughter nucleus reiterates the process. We used time-lapse microscopy to identify the mechanisms by which C. albicans nuclei move over long distances and are coordinated with hyphal morphology. We followed nuclear migration and spindle dynamics, as well as the time and position of septum specification, defined it as the presumptum, and established a chronology of nuclear, spindle, and morphological events. Analysis of microtubule dynamics revealed that premitotic forward nuclear migration is due to the repetitive sliding of astral microtubules along the cell cortex but that postmitotic forward and reverse nuclear migrations are due primarily to spindle elongation. Free microtubules exhibit cell cycle regulation; they are present during interphase and disappear at the time of spindle assembly. Finally, a growth defect in strains expressing Tub2-green fluorescent protein revealed a connection between hyphal elongation and the nuclear cell cycle that is coordinated by hyphal length and/or volume.     

Chromatin arrangements in intact interphase nuclei examined by laser confocal microscopy

Using a laser confocal microscope, chromatin arrangements in intact interphase nuclei were investigated in four plant species. Chromosomes in these plants have specific segments that can be stained with the fluorescent dye chromomycin A₃ (CMA). We stained centromeres inHordeum vulgare, sub-telomeric regions inSecale cereale, satellites inChrysanthemum multicore, and the satellites and the short arms of chromosomes with satellites inHemerocallis middendorfii. The following points were shown: (1) In mitotic interphase nuclei, the centromere and the telomeres of both arms touched the nuclear membrane and had evident polarity. Some CMA-bodies in sub-telomeric regions do not contact the nuclear membrane. (2) Differentiated nuclei had a non-random construction. Polarity of chromosomes is maintained, however, the chromosomes are far apart from the nuclear membrane. (3) Associations in sub-telomeric regions in the interphase nuclei ofSecale cereale were probably due to the association of heterochromatic regions with identical repeated sequences rather than telomere associlations. (4) In interphase nuclei ofChrysanthemum multicore, satellites fused during interphase.     

Onset of the DNA replication checkpoint in the early Drosophila embryo

The Drosophila embryo is a promising model for isolating gene products that coordinate S phase and mitosis. We have reported before that increasing maternal Cyclin B dosage to up to six copies (six cycB) increases Cdk1-Cyclin B (CycB) levels and activity in the embryo, delays nuclear migration at cycle 10, and produces abnormal nuclei at cycle 14. Here we show that the level of CycB in the embryo inversely correlates with the ability to lengthen interphase as the embryo transits from preblastoderm to blastoderm stages and defines the onset of a checkpoint that regulates mitosis when DNA replication is blocked with aphidicolin. A screen for modifiers of the six cycB phenotypes identified 10 new suppressor deficiencies. In addition, heterozygote dRPA2 (a DNA replication gene) mutants suppressed only the abnormal nuclear phenotype at cycle 14. Reduction of dRPA2 also restored interphase duration and checkpoint efficacy to control levels. We propose that lowered dRPA2 levels activate Grp/Chk1 to counteract excess Cdk1-CycB activity and restore interphase duration and the ability to block mitosis in response to aphidicolin. Our results suggest an antagonistic interaction between DNA replication checkpoint activation and Cdk1-CycB activity during the transition from preblastoderm to blastoderm cycles.     

Condensation of chromatin into chromosomes preserves an open configuration but alters the DNase I hypersensitive cleavage sites of the transcribed gene

DNase I was used to probe the molecular organization of the chicken ovalbumin (OV) gene and glyceraldehyde 3-phosphate dehydrogenase (GPD) gene in interphase nuclei and in metaphase chromosomes of cultured chicken lymphoblastoid cells (MSB-1 line). The OV gene was not transcribed in this cell line, whereas the GPD gene was constitutively expressed. The GPD gene was more sensitive to DNase I digestion than the OV gene in both interphase nuclei and metaphase chromosomes, as determined by Southern blotting and liquid hybridization techniques. In addition, we observed DNase I hypersensitive sites around the 5' region of the GPD gene. These hypersensitive sites were not always at the same locations between the interphase nuclei and metaphase chromosomes. Our results suggest that chromatin condensation and decondensation during cell cycle alters nuclease hypersensitive cleavage sites.     

Microtubule nucleating sites in higher plant cells identified by an auto-antibody against pericentriolar material

Human scleroderma serum 5051, which is known to recognize the amorphous pericentriolar microtubule organizing center material of a variety of vertebrate cells, was found to immunostain spindle poles of meristematic higher plants from pre-prophase to late anaphase. Subsequently, during cytokinesis, staining was redistributed around the reforming telophase nuclei, but was not evident in the cytokinetic phragmoplast. At the transition between telophase and interphase, before the typical cortical interphase microtubule array was established, short microtubules radiated from the nucleus and in such cells the material recognized by 5051 was located around the daughter nuclei and not the cortex. These observations have led us to propose that the perinuclear region, or the nuclear surface, may function as a nucleation center for both spindle and interphase microtubules in higher plant cells.     

Yeast importin-β is required for nuclear import of the Mig2 repressor

Background

In interphase nuclei of a wide range of species chromosomes are organised into their own specific locations termed territories. These chromosome territories are non-randomly positioned in nuclei which is believed to be related to a spatial aspect of regulatory control over gene expression. In this study we have adopted the pig as a model in which to study interphase chromosome positioning and follows on from other studies from our group of using pig cells and tissues to study interphase genome re-positioning during differentiation. The pig is an important model organism both economically and as a closely related species to study human disease models. This is why great efforts have been made to accomplish the full genome sequence in the last decade.

Results

This study has positioned most of the porcine chromosomes in in vitro cultured adult and embryonic fibroblasts, early passage stromal derived mesenchymal stem cells and lymphocytes. The study is further expanded to position four chromosomes in ex vivo tissue derived from pig kidney, lung and brain.

Conclusions

It was concluded that porcine chromosomes are also non-randomly positioned within interphase nuclei with few major differences in chromosome position in interphase nuclei between different cell and tissue types. There were also no differences between preferred nuclear location of chromosomes in in vitro cultured cells as compared to cells in tissue sections. Using a number of analyses to ascertain by what criteria porcine chromosomes were positioned in interphase nuclei; we found a correlation with DNA content.     

Proteins recognized by antibodies against isolated cytological heterochromatin from rat liver cells change their localization between cell species and between stages of mitosis (interphase vs metaphase)

Heterochromatin in the cell nucleus seems to concentrate various proteins, such as Drosophila heterochromatin protein 1, which maintain the repressed state of gene expression. However, it still remains obscure how protein composition related to chromatin structure is different between heterochromatin and euchromatin in interphase nuclei. We isolated cytological heterochromatin from sonicated interphase nuclei obtained from rat liver cells and prepared antisera against it. The dense heterochromatic bodies seen in the preparation of intact nuclei were duplicated in a relatively pure form during the preparation of heterochromatin. In the sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis, differences between the fractions of heterochromatin and euchromatin were noted by their protein composition. Isolated heterochromatin was then digested by DNase after partial digestion with trypsin and its dense structure changed to become highly sensitive to DNase. The prepared antibodies reacted with the heterochromatin region of rat liver cell nuclei and isolated cytological heterochromatin; however, they did not react with euchromatin. Using immunohistochemistry, the antibodies bound to each cell nucleus in all tissues observed; some cell types were distinguished by their differential stainability (e.g. staining in the cytoplasm). Staining of the mitotic cells showed that the proteins recognized by the antibodies were localized in the cytoplasm and, in part, on the chromosomes. Based on the results of molecular cloning from rat liver cDNA library using the antibodies as a probe, it seemed that the antibodies mainly recognized two proteins similar to arginase and general vesicular transport factor p115, respectively. The results obtained from these experiments reveal that some proteins located in the heterochromatin of interphase liver cell nuclei seem to play important roles in condensing a portion of the chromatin structure during interphase and suggest that proteins composing heterochromatin might be changed according to cell types or the stage of the cell cycle.     

The ultrastructure of human mitotic chromosomes and interphase nuclei treated by Giemsa banding techniques

Total preparations of mitotic chromosomes and interphase nuclei prepared as for Giemsa banding techniques were investigated by standard transmission electron microscopy and by a method of a three dimensional representation. Chromosomes as well as interphase nuclei appear to be composed of irregularely folded fibrils of at least 300 Å thickness. In the G-band regions the chromosomes are thicker containing more foldings of fibrils. Also the fibrils are darker stained in the G-band regions. Loops of fibrils stick out from chromosomes as well as from interphase nuclei. When chromosomes or interphase nuclei come to lie close enough, such loops may stick together and form fibrillar bridges between them. These as well as interchromatid bridges are considered to be artefacts. The fibrils seem to be built up either of one or of several finer fibrils. No further conclusions regarding the fine structure of the fibrils can be drawn.     

Topological origins of chromosomal territories

Using freely jointed polymer model we compare equilibrium properties of crowded polymer chains whose segments are either permeable or not permeable for other segments to pass through. In particular, we addressed the question whether non-permeability of long chain molecules, in the absence of excluded volume effect, is sufficient to compartmentalize highly crowded polymer chains, similarly to what happens during formation of chromosomal territories in interphase nuclei. Our results indicate that even polymers without excluded volume compartmentalize and show strongly reduced intermingling when they are mutually non-permeable. Judging from the known fact that chromatin fibres originating from different chromosomes show very limited intermingling in interphase nuclei, we propose that regular chromatin fibres during chromosome decondensation can hardly serve as a substrate of cellular type II DNA topoisomerases.     

THE CYTONUCLEOPROTEINS OF AMEBAE : II. Some Aspects of Cytonucleoprotein Behavior and Synthesis

Radioactivity, apparently in cytonucleoproteins, from an amino acid-labeled nucleus implanted into a non-radioactive cell appeared in the host nucleus within 10 minutes, and the typical equilibrium ratio 70:30 donor nucleus radioactivity:host nucleus radioactivity was reached in 4 to 5 hours at 25°C. If such binucleates grew and divided, no localization of radioactivity was observable in cells fixed during mitosis, but the protein label remained concentrated in the daughter interphase nuclei for at least 4 generations. Continued migration of cytonucleoproteins was observed if these daughter nuclei were transplanted to other unlabeled cells. The Q₁₀ (19° to 29°C) of the migration rate of radioactive cytonucleoproteins was ca. 1.3, suggesting that passage through the cytoplasm occurred by diffusion. Both non-migratory nuclear proteins and cytonucleoproteins appear to be synthesized in the cytoplasm.