Early Metaphase Research Articles

Mechanism of the formation of contractile ring in dividing cultured animal cells. II. Cortical movement of microinjected actin filaments.

The contractile ring in dividing animal cells is formed primarily through the reorganization of existing actin filaments (Cao, L.-G., and Y.-L. Wang. 1990. J. Cell Biol. 110:1089-1096), but it is not clear whether the process involves a random recruitment of diffusible actin filaments from the cytoplasm, or a directional movement of cortically associated filaments toward the equator. We have studied this question by observing the distribution of actin filaments that have been labeled with fluorescent phalloidin and microinjected into dividing normal rat kidney (NRK) cells. The labeled filaments are present primarily in the cytoplasm during prometaphase and early metaphase, but become associated extensively with the cell cortex 10-15 min before the onset of anaphase. This process is manifested both as an increase in cortical fluorescence intensity and as movements of discrete aggregates of actin filaments toward the cortex. The concentration of actin fluorescence in the equatorial region, accompanied by a decrease of fluorescence in polar regions, is detected 2-3 min after the onset of anaphase. By directly tracing the distribution of aggregates of labeled actin filaments, we are able to detect, during anaphase and telophase, movements of cortical actin filaments toward the equator at an average rate of 1.0 micron/min. Our results, combined with previous observations, suggest that the organization of actin filaments during cytokinesis probably involves an association of cytoplasmic filaments with the cortex, a movement of cortical filaments toward the cleavage furrow, and a dissociation of filaments from the equatorial cortex.

Regulation of mitosis onset and thymidine kinase activity during the cell cycle of Physarum polycephalum plasmodia: Effect of fluorodeoxyuridine

The effects of fluorodeoxyuridine were investigated during three events of the cell cycle: S-phase, mitosis, and the cyclic synthesis of thymidine kinase in the synchronous plasmodium of the myxomycete Physarum. DNA synthesis was inhibited, and there was limited action on other macromolecular syntheses. When DNA synthesis was slowed down, onset of the following increase of thymidine kinase synthesis occurred at approximately the same time as in the control, but mitosis was blocked in a very early prophase stage and metaphase was never observed. These effects were suppressed when the action of fluorodeoxyuridine was prevented by the addition of thymidine to the medium. In agreement with the action of aphidicolin and hydroxyurea, these observations show that: 1) perturbation of the S-phase does not prevent the nuclei from entering a very early prophase stage, but it does prevent them from proceeding through metaphase; 2) blockage of DNA synthesis does not perturb the normal timing of the triggering of thymidine kinase synthesis; and 3) the signal that triggers the arrest of thymidine kinase synthesis is postmitotic and does not require extensive DNA synthesis. In contrast with hydroxyurea and aphidicolin, in the presence of fluorodeoxyuridine metaphase was not observed. Thus, the triggering of thymidine kinase synthesis is unambiguously dissociated from metaphase and postmitotic events. Because synthesis of thymidine kinase remains under the control of temperature shifts from 22 to 32 degrees C, a simple model of the cell cycle involving two regulatory pathways could account for the triggering of thymidine kinase synthesis, early prophase stage, and metaphase.

The ultrastructure and timing of events in the nuclear cycle of the fungus Entomophaga aulicae

ABSTRACT The ultrastructure of the mitotic nuclear division cycle of the fungus Entomophaga aulicae was studied from serial sections of hyphal tips and protoplasts. The extranuclear bar-shaped nucleus-associated organelle (NAO) remained associated with the persistent nuclear envelope throughout. Prior to spindle formation, a patch of intranuclear NAO-associated chromatin detached from the nuclear envelope to yield a chromatinfree zone containing fine filaments and a linear array of presumptive kinetochores. Early metaphase spindles less than 1 pm in length were characterized by a ‘fused’ metaphase plate consisting of kinetochore-associated chromatin and a full complement of at least 15 kinetochore microtubules per half-spindle, while most of the chromatin was remote from the intranuclear spindle. Analysis of the distribution of antiparallel spindle microtubules indicated that polar separation and concomitant spindle elongation through metaphase were not accompanied by intermicrotubule sliding. Anaphase exhibited extensive decondensation of the large patches of condensed chromatin characteristic of all other stages. In a logarithmically growing protoplast population all nuclei contained spindle microtubules, with metaphase occupying approximately 66% of the nuclear cycle time. The calculated genome size of 4-3 pg, and average DNA content per chromosome of 0-3 pg, are extremely high for fungi.

Cytocortical organization during natural and prolonged mitosis of mouse 8-cell blastomeres.

Late 8-cell blastomeres were harvested within the first 45 min after entering mitosis. Some mitotic cells were analysed within the ensuing 2 h for the organization of their surface in relation to their progress through mitosis. Whereas in most late interphase cells microvilli were restricted to a discrete polar region, in mitotic cells at all stages from early metaphase to immediately postcytokinesis microvilli were found to be present over more of the cell surface. Other mitotic cells were placed in nocodazole to arrest them in M-phase for up to 10 h. They were found to show an even more extensive distribution of microvilli over the whole surface, the longer periods of incubation yielding more extended coverage such that many cells no longer appeared to have any residual surface polarity. Removal from nocodazole at all time points from 1 to 10 h resulted in most cells completing mitosis to yield pairs of cells which, in most cases, resembled pairs derived from nonarrested blastomeres and in which a defined polar area of microvilli was restored. However, the percentage of differentiative divisions decreased after 6 h arrest. If, instead of removing cells from nocodazole, they were placed in both nocodazole and cytochalasin D (CCD) for periods of up to 3 h, most microvilli retracted to reveal a tight polar zone of CCD-resistant microvilli. This result suggests that a heterogeneity of cytocortical organization may still exist within the arrested mitotic cell. We propose a model to explain the origin of this heterogeneity of organization and its relationship to the generation of cell diversity.

The human inactivated X chromosome folds in early metaphase, prometaphase, and prophase.

The inactivated X chromosome has a site of unusually frequent folding in region Xq1, whereas a fold in Xq1 is uncommon on the active X. We investigated the pattern of X chromosome folding in high-resolution GTG- and RBG-stained preparations from four women. In early metaphase cells, slightly more than 50% of late-replicating Xs folded at Xq1----Xq21, compared with about 6% of early replicating Xs. The late-replicating X folded in about 80% of prometaphase cells; the early, in only about 14% of these cells. and the late-replicating X folded in 19 of 20 prophase cells. Occasionally, one X had an omega-shaped loop or apparent physical connection between Xq13 and Xq21.1. It is possible that a segment of Xq1 never completely uncoils and may help to provide continuity for the Barr body from one interphase to the next.

Idiograms of horse chromosomes at prometaphase, early metaphase, and midmetaphase after R-banding by BrdU incorporation followed by the fluorochrome-photolysis-Giemsa technique.

We present three idiograms of equine chromosomes, R-banded after BrdU incorporation and stained by the fluorochrome-photolysis-Giemsa technique. The haploid set of prometaphasic chromosomes shows 591 bands (range 7-38 per chromosome), the early metaphasic set 404 (range 5-26), and the midmetaphasic set 272 (range 3-18). Following cell synchronization with thymidine, more than twice as many R-bands were revealed on the resulting prometaphasic chromosomes, making possible the establishment of a very accurate and characteristic representation of this banding pattern in the domestic horse.

Sequential G- to R-banding for high resolution chromosome analysis

A sequential staining protocol for revealing first G- and then R-bands on early metaphase chromosomes is presented. Lymphocyte cultures are synchronized with methotrexate, and released from the S-phase block with bromodeoxyuridine. The resulting early metaphase cells are initially G-banded with Wright's stain and then R-banded by the fluorescence plus Giemsa (FPG) technique.

Metaphase chromosome folds and X-inactivation.

To evaluate the suggested nonrandom folding of Xq13-q21 (center of Barr body condensation) of the inactivated X chromosomes, metaphases from nine subjects with or without X chromosome abnormalities (eight females and one male) were investigated with RBG-staining. A significant increase (p less than .05) in the number of arm folds (Xq13-q21) of the late-replicating X chromosome, particularly in early to mid-metaphase, was observed in four of eight females. Therefore, the stage of chromosome contraction was an important factor with more folds observed at the centromeres and in longer chromosomes in early metaphase compared with mid- to late metaphase. X chromosome folds were present in cells of subjects treated with or without bromodeoxyuridine. While our study agrees with the relationship of Xq13-q21 fold with the X-inactivation center, the correlation of extended chromosomes and folding limits this method as a sole indicator of X-inactivation in routine mid-metaphase, but is useful in the analysis of early metaphase chromosomes.

Mitosis in a Green AlgaTriploceras GracileBailey with Particular Reference to the Behaviour of Nuclear Envelope

SUMMARYThe process of mitosis in the desmid Triploceras gracile was studied with an electron microscope paying a particular attention to the behaviour of the nuclear envelope. At prophase, the nucleolus disintegrates, and at early metaphase the nuclear envelope breaks down. The nuclear envelope after dispersion surrounded the individual chromosomes at early metaphase and remained persistent around the chromosomes until late anaphase. At metaphase, the doubled chromosomes formed a precisely aligned metaphase plate. The chromosomes possessed distinct kinetochores. The nuclear envelope of the individual chromosomes fused with one another at telophase to give rise to daughter nuclear envelopes around the reforming nuclei. Many small nucleoli appeared among the chromosomes at early telophase, which later fused to give rise to a large conspicuous nucleolus during telophase.

Reproductive performance and mutagenic response of the wasp Bracon hebetor following treatment with the antibiotic bleomycin

Bleomycin (BM) induced dominant-lethal genetic lesions in the gametes of both sexes of adult Bracon hebetor wasps following ingestion. This effect was demonstrated by decreased fertility in the unfertilized eggs of treated females as well as in eggs fertilized by mature sperm derived from treated males. Death in the unhatched eggs occurred prior to blastoderm formation indicating BM caused chromosome breakage. Affected ovarian cells were limited to those exposed to BM between the developmental stages of vitellogenesis and early metaphase I. Sperm-associated fertility reductions were temporary in duration with substantial reductions being observed starting on the second day following the initiation of oviposition. BM lowered general egg-formation capacity in treated females but, unlike the fertility effects, decreased fecundity was caused by somatic debility. Although the BM-induced genetic damage observed in B. hebetor was radiomimetic in nature, the time of appearance and duration of the effects did not correspond with previous patterns found when B. hebetor adults were exposed to X-rays. While the differences could be due to the BM detoxification or the repair of BM-induced chromosome damage, further investigation would be required to demonstrate the presence of such systems in this hymemopteran.

Cytological identification of acrotrisomy in sugar beets

The analysis of sugar beet cells at three different somatic stages—prophase, early metaphase, and mid-metaphase—permits the identification and physical measurement of individual chromosomes and determines the break-point of some structural changes. This technique is successfully applied in the identification of an acrocentric chromosome 9S9L in the sugar beet.

868 A PRACTICAL METAPHASE MARKER OF THE INACTIVE X CHROMOSOME

The ability to identify the inactivated X chromosome with routine G- or Q-banding would have broad clinical and research applicability. We recently reported that the inactivated X frequently bends or folds in region Xql3Xq21 (Flejter et al, Am J Hum Genet 36:218, 1984). The fold occurs in about 88% of prometaphase, 50% of early metaphase, 30% of midmetaphase, and 10% of late metaphase inactive X's. In prometaphase, the site of folding includes Xq11.2 and Xq13.3, infrequently extending to Xq21.1. An omega-shaped loop is frequently formed between sub-bands Xq11.2 and Xq13.3. It is paradoxical that the inactive X is the only chromosome identifiable in interphase, yet in metaphase it cannot be distinguished from its active homolog. The specific inactivation-associated fold at region Xq1 resolves that paradox and is a useful marker of the inactive X. 1. The KOP translocation, t(X; 14)(q13; q32), has nearly all of Xq translocated to 14q (Allerdice et al, Am J Med Genet 2:223, 1978). Cell line GM0074 has one normal 14, one Xq-, two der (14) chromosomes, and a Y. One der(14) folded in 10/18 cells scored, confirming translocation of the inactivation center adjacent to 14q distal. 2. Metaphase cells from other primates had a specific fold in the same region as in the human X: at Xq13-Xq21 in 2 gorillas, 1 chimp, 2 pygmy chimps, 1 orang, 1 baboon, 1 rhesus and 1 stump-tail monkey. This is further evidence for evolutionary conservation of the X chromosome. One chimpanzee exhibited the fold at Xq24; we suspect a pericentric inv in this individual. We have not seen a frequent fold in the X of a bat (Tadarida brasiliensis), Chinese hamster (Cricetulus griseus), or rat kangaroo (Potorous tridactylis). 3. Regarding the relationship of X inactivation and intelligence in fra(X)(q28) carriers, we observe that in cells of normal carriers the fold was mostly on the fragile X-positive chromosome, whereas in cells of an affected carrier the fold was mostly on the fragile X-negative chromosome. This is evidence that inactivation of the fragile X chromosome is positively correlated with intelligence in carriers.

Sexing of mouse eggs at the first cleavage division by the use of C-staining method.

One-celled eggs were recovered from mated, colchicine-primed dd female mice, and chromosome preparations were made by the air-drying and C-staining methods. Their mitotic stages were mostly at late prometaphase with two haploid groups of chromosomes and at early metaphase, a stage of syngamy, and partly at the chromatid stage of metaphase. The Y, a small chromosome with no C-band, was distinguished not only at the chromatid stage of metaphase but also at the other stages above-mentioned. Out of 2, 053 eggs examined 767 eggs could be sexed, sexing rate being 37.4%. Of these, 334 eggs were identified as males and 433 females, with a sex ratio of 43.5% males. The sex ratio was further classified according to the stages above-mentioned. Sex ratio before syngamy was significantly higher than that at syngamy and the chromatid stage of metaphase.

The high-resolution RBA-banding pattern of bovine chromosomes.

The high-resolution RBA-banding pattern of bovine chromosomes (Bos taurus L.), together with a diagrammatic representation, are presented. The haploid set of prometaphase chromosomes shows 521 bands, 183 of which are positive, 257 negative, and 81 variable. Compared to early metaphase chromosomes, the prometaphase chromosomes show 50% more bands, resulting in a more accurate description of the banding pattern of individual chromosomes of this species.

Anther development and the orientation of the interchange quadrivalent in pearl millet

The effects of developmental stage of the anther, ageing of the plant, inbreeding and season on meiotic segregation of an interchange chain quadrivalent were studied in pearl millet, Pennisetum americanum (L.) Leeke. The frequency of adjacent orientation (and segregation) increased with developmental stage of the anther, independent of other factors. Plant age and degree of inbreeding had no demonstrable effects, but there was an indication that high temperature favoured adjacent orientation. Chromosome contraction as measured by change in chromosome length appeared to be negligible during metaphase-anaphase. Therefore, increased adjacent orientation cannot readily be explained by metaphase reorientation resulting from the straightening of chromosomes caused by an increase in their rigidity. It is probable that the unoriented or “mal-oriented” quadrivalents observed regularly at early metaphase I continue to straighten out prior to their delayed orientation. When they finally orientate late in metaphase, their orientation will more likely be adjacent than alternate.

Evidence of nonchiasmate bonds at metaphase I in inbred rye

The loss of bound chromosome arms through early, middle, and late metaphase I has been analyzed in a plant of inbred rye (Secale cereale L.) heterozygous for a terminal heterochromatic C-band of the long arm of chromosome 1R. From the increase in the number of univalent pairs due to bound arm loss, and from the comparison between the frequencies of bound arms at metaphase I and recombinant chromosomes at anaphase I, it is concluded that some of the chromosome bonds appearing at metaphase I are actually nonchiasmate associations that can be considered as remnants of prophase pairing. Conclusions concerning recombination obtained solely from the analysis of chiasma frequency measured as bound arms may be invalid.Key words: inbred rye, C-heterochromatin, meiotic pairing, nonchiasmate bonds.

Action spectrum for changes in spindle fibre birefringence after ultraviolet microbeam irradiations of single chromosomal spindle fibres in crane-fly spermatocytes.

Single chromosomal spindle fibres in Nephrotoma suturalis (crane-fly) spermatocytes in metaphase and anaphase were irradiated with monochromatic ultraviolet light focussed to a 2 micrometer spot. In cells in both metaphase and anaphase either the birefringence of the irradiated spindle fibre was altered in the irradiated region, or there was no change, depending on the dose and wavelength of ultraviolet light used for the irradiation. When there was an area of reduced birefringence (ARB), it moved poleward regardless of whether the associated chromosome moved poleward. When cells were irradiated in early metaphase they remained in metaphase until the ARB reached the pole. In some cells irradiated in late metaphase the chromosomes began anaphase before the ARB reached the pole; in many such cells anaphase was abnormal in that all six half-bivalents separated at the start of anaphase but none moved polewards. In all cases the ARB moved poleward at the same speed as subsequent chromosome movement; that is, at about 0.8 micrometer/min. In cells irradiated in anaphase, spindle fibre birefringence was reduced independently of blockage of chromosome movement. Because birefringence and movement were altered independently there were four classes of results: (1) in some cases there was no effect on the movement of the chromosome associated with the irradiated spindle fibre and no effect on the birefringence of the irradiated spindle fibre. (2)In some cases, primarily with 260 nm wavelength light, there was no effect on the movement of the chromosome associated with the irradiated spindle fibre and there was an effect on the birefringence of the irradiated spindle fibre. (3) In some cases, primarily with 290 nm wavelength light, there was an effect on the movement of the chromosome associated with the irradiated spindle fibre and no effect on the birefringence of the irradiated spindle fibre. (4) In some cases, primarily with 270 nm and 280 nm wavelength light, there was an effect on the movement of the chromosomes associated with the irradiated spindle fibre and there was an effect on the birefringence of the irradiated spindle fibre. The action spectrum for reducing spindle fibre birefringence in crane-fly spermatocytes had two peaks, one at 260 nm and the other, less sensitive, at 280 nm. For irradiations at 270 nm, 280 nm and 290 nm, five to fifty times more energy was needed to reduce spindle fibre birefringence than to stop chromosome movement, but for irradiations at 260 nm five times less energy was needed to reduce spindle fibre birefringence than to stop chromosome movement. The action spectrum for reducing spindle fibre birefringence is quite different from that for stopping chromosome movement.

A cell-cycle-dependent protein of Physarum polycephalum revealed by two-dimensional gel electrophoresis.

Silver-stained two-dimensional polyacrylamide gels of Physarum polycephalum extracts have revealed no major changes in protein accumulation during the synchronous nuclear division cycle. However, one protein, or apparent Mr 32 000 and isoelectric point 4.9, shows a reproducible ten-fold increase in total amount between early G2 phase and metaphase. This protein represents about 0.005% of total plasmodial protein.

Microtubule‐containing detergent‐extracted cytoskeletons in sea urchin eggs from fertilization through cell division: Antitubulin immunofluorescence microscopy

AbstractThe microtubule‐containing structures that appear in eggs during fertilization and cell division in the sea urchins Lytechinus variegatus and Arbacia punctulata were detected by antitubulin immunofluorescence microscopy of detergent extracted cytoskeletal preparations. The extraction buffer, which is composed of 0.55 mM MgCl2, 10 mM EGTA, 25 mM MES, 25% glycerol, 1% Nonidet P‐40, and 25 μM PMSF, pH 6.7, allows for dramatically improved fluorescent images compared to those obtained using conventional staining procedures, with residual background staining being reduced to near zero.The immunofluorescent images obtained using this technique provide information on several motile events that occur during the first cell cycle. This technique demonstrates that all of the cytoplasmic microtubules are associated with the incorporated sperm's centrioles during female pronuclear migration. This changes during the centration of the male and female pronuclei at which time a monastral array of microtubules forms in the egg's cytoplasm. A large proportion of the monastral microtubules do not appear to be associated with the centrioles. At prophase and early metaphase, the centrioles are the dominant microtubule organizing centers (MTOCs) consistent with mitotic theories that the kinetochore catches, but does not initiate, microtubules. Observations of intercentriolar distances show that there are three stages of pole separation during the first cell cycle. The initial separation occurs during pronuclear centration, the second during the streak stage, and the final one during the late stages of mitosis. At telophase, polar microtubules appear to extend into the cortex supporting the cell surface at all regions except the presumptive cleavage site.

High resolution G-banding patterns of Syrian hamster chromosomes.

The late prophase, prometaphase, early metaphase, and metaphase chromosomes of Syrian hamster embryo fibroblast cells were G-banded using a technique combining actinomycin D pretreatment and Wright/Giemsa staining. Late prophase chromosomes have approximately twice as many bands as metaphase chromosomes, whereas prometaphase and early metaphase chromosomes have intermediate numbers of bands. An idiogram for metaphase and late prophase chromosomes is presented, and a nomenclatural system for identifying individual bands of Syrian hamster chromosomes is proposed.