Chaotization of division spindle,phragmoplast, and telophase chromosome groups in wheat wheatgrass F1 hybrids meiosis

The phenomenon of the disorientation of completely formed systemic cytoskeleton structures, i.e., the division spindle and phragmoplast, into the constituent elements and their transformation into a network of disoriented fibers in the course of cell division is described. The phenomenon of the disintegration and dispersion in the cytoplasm of completely formed telophase chromosome groups, which is not associated with the chaotization of the cytoskeleton structures, is also described. These abnormalities are revealed in the meiosis of pollen mother cells of the first generation of wheat-wheatgrass hybrids. The chaotization of cytoskeleton structures is a only normal phenomenon in plant-cell division in late prophase-early prometaphase, whereas, at stages of metaphase and telophase, it can indicate a disturbance in the time regulation of the cytoskeleton cycle in the course of meiotic division. The disintegration of the chromosome telophase groups and their movement backwards to the spindle equator can indicate the untimely involvement of processes of prometaphase, specifically the activation of chromokinesins. The significance of the process of cytoskeleton chaotization in the biology of a plant cell is discussed.     

Dynamics of microtubular cytoskeleton in higher plant meiosis. VII. Mechanisms of the simultaneous cytokinesis

Rearrangements of microtubular cytoskeleton during telophase in pollen mother cells of some dicotyledon plants with the simultaneous cytokinesis during normal and abnormal meiosis were studied. At telophase I, a potentially functional phragmoplast forms between daughter nuclei, but no cell plate is present. During interkinesis, the phragmoplast plays the role of an interphase cytoskeleton array. Dynamics of microtubule reorganization in polar regions of the telophase spindle is discussed in addition to the role played by microtubule convergence centers in cytoskeleton rearrangements during meiosis.     

洋葱小孢子母细胞减数分裂过程中微管分布变化

应用间接免疫荧光标记技术和激光共聚焦扫描显微镜成像技术观察洋葱小孢子母细胞减数分裂过程中微管分布变化。减数分裂之前,小孢子母细胞中的微管较短,呈辐射状,由细胞核表面向四周扩散。减数分裂开始后,细胞质中的一部分微管蛋白聚集成纺锤体微管,控制染色体的分布。进入减数分裂I后期,纺锤体微管变为牵引染色体移向两极的着丝粒微管和连接纺锤体两极的极丝微管。之后,所有微管集中在两个核之间,构成成膜体。然后,微管解聚成微管蛋白弥散在细胞质中。减数分裂I完成后,二分体2个子细胞中的微管蛋白又聚集成2个纺锤体微管,开始减数分裂II过程。经过减数分裂II中期,2个二分体细胞中的微管再次集中在2个细胞核之间形成成膜体,隔离2个细胞核。此后,微管蛋白解聚,弥散分布在小孢子细胞质中。     

Microtubule organization during successive microsporogenesis in Allium cepa and simultaneous cytokinesis in Nicotiana tabacum

Microtubule cytoskeleton organization during microspore mother cell (MMC) meiosis in Allium cepa L. and microsporogenesis in Nicotiana tabacum L. was examined. The MMC microtubules (MTs) were short and well dispersed in the cytoplasm of both taxa. As the MMCs of both species entered metaphase of meiosis I, the MTs constructed a spindle that facilitated the chromosomes to orient in the meridian plane. At anaphase of meiosis I, the spindle MTs differentiated into two types: one MT type became short, pulled the chromosomes toward the two poles, and was designated as centromere MTs; the second type of MT connected the two poles, and was designated as pole MTs. In A. cepa, where successive cytokinesis was observed, pole MTs assumed a tubbish shape. Some new short MTs aggregated in the meridian plane and constricted to form a phragmoplast, which developed into a cell plate, divided the cytoplasm into two parts and produced a dyad. However, in tobacco, a phragmoplast was not generated in anaphase of meiosis I and II and cytokinesis did not occur. The spindle MTs depolymerized and reorganized the radial arrangement of MTs from the nucleate surface to the periplasm during anaphase. Following telophase of meiosis II, the cytoplasm produced centripetal furrows, which met in the center of the cell and divided it into four parts, serving as a form of cytokinesis. In this process, MTs appeared to bear no relationship to cytokinesis.     

Abnormalities of spindle and cytokine behavior leading to the formation of meiotic restitution nuclei in intergeneric cereal hybrids

Mobile stages of meiosis have been analysed by visualizing the spindle in fertile cereal F1 hybrids. We describe four different mechanisms of the formation of restitution nuclei in meiotic division: (1) centripetal migration of telophase chromosome groups from the poles of a curved spindle at early telophase; (2) centripetal migration of the chromosome groups at late telophase when cell plate formation has failed; (3) preferable migration of univalents to one of the poles although spindle appearance is morphologically normal; and (4) in the absence of chromosome segregation where kinetochore fibers have failed to form.     

The dynamics of the actin cytoskeleton during sporogenesis in Psilotum nudum L.

The actin cytoskeleton (microfilaments, MFs) accompanies the tubulin cytoskeleton (microtubules) during the meiotic division of the cell, but knowledge about the scope of their physiological competence and cooperation is insufficient. To cast more light on this issue, we analysed the F-actin distribution during the meiotic division of the Psilotum nudum sporocytes. Unfixed sporangia of P. nudum were stained with rhodamine-phalloidin and 4′,6-diamidino-2-phenylindole dihydrochloride, and we monitored the changes in the actin cytoskeleton and nuclear chromatin throughout sporogenesis. We observed that the actin cytoskeleton in meiotically dividing cells is not only part of the kariokinetic spindle and phragmoplast but it also forms a well-developed network in the cytoplasm present in all phases of meiosis. Moreover, in telophase I F-actin filaments formed short-lived phragmoplast, which was adjacent to the plasma membrane, exactly at the site of future cell wall formation. Additionally, the meiocytes were pre-treated with cytochalasin-B at a concentration that causes damage to the MFs. This facilitated observation of the effect of selective MFs damage on the course of meiosis and sporogenesis of P. nudum. Changes were observed that occurred in the cytochalasin-treated cells: the daughter nuclei were located abnormally close to each other, there was no formation of the equatorial plate of organelles and, consequently, meiosis did not occur normally. It seems possible that, if the actin cytoskeleton only is damaged, regular cytokinesis will not occur and, hence, no viable spores will be produced.     

The cytoplast concept in dividing plant cells: cytoplasmic domains and the evolution of spatially organized cell

The unique cytokinetic apparatus of higher plant cells comprises two cytoskeletal systems: a predictive preprophase band of microtubules (MTs), which defines the future division site, and the phragmoplast, which mediates crosswall formation after mitosis. We review features of plant cell division in an evolutionary context and from the viewpoint that the cell is a domain of cytoplasm (cytoplast) organized around the nucleus by a cytoskeleton consisting of a single "tensegral" unit. The term "tensegrity" is a contraction of "tensional integrity" and the concept proposes that the whole cell is organized by an integrated cytoskeleton of tension elements (e.g., actin fibers) extended over compression-resistant elements (e.g., MTs).During cell division, a primary role of the spindle is seen as generating two cytoplasts from one with separation of chromosomes a later, derived function. The telophase spindle separates the newly forming cytoplasts and the overlap between half spindles (the shared edge of two new domains) dictates the position at which cytokinesis occurs. Wall MTs of higher plant cells, like the MT cytoskeleton in animal and protistan cells, spatially define the interphase cytoplast. Redeployment of actin and MTs into the preprophase band (PPB) is the overt signal that the boundary between two nascent cytoplasts has been delineated. The "actin-depleted zone" that marks the site of the PPB throughout mitosis may be a more persistent manifestation of this delineation of two domains of cortical actin. The growth of the phragmoplast is controlled by these domains, not just by the spindle. These domains play a major role in controlling the path of phragmoplast expansion. Primitive land plants show different morphological changes that reveal that the plane of division, with or without the PPB, has been determined well in advance of mitosis.The green alga Spirogyra suggests how the phragmoplast system might have evolved: cytokinesis starts with cleavage and then actin-related determinants stimulate and positionally control cell-plate formation in a phragmoplast arising from interzonal MTs from the spindle. Actin in the PPB of higher plants may be assembling into a potential furrow, imprinting a cleavage site whose persistent determinants (perhaps actin) align the outgrowing edge of the phragmoplast, as in Spirogyra. Cytochalasin spatially disrupts polarized mitosis and positioning of the phragmoplast. Thus, the tensegral interaction of actin with MTs (at the spindle pole and in the phragmoplast) is critical to morphogenesis, just as they seem to be during division of animal cells. In advanced green plants, intercalary expansion driven by turgor is controlled by MTs, which in conjunction with actin, may act as stress detectors, thereby affecting the plane of division (a response clearly evident after wounding of tissue). The PPB might be one manifestation of this strain detection apparatus.     

The reduction of chromosome number in meiosis is determined by properties built into the chromosomes

In meiosis I, two chromatids move to each spindle pole. Then, in meiosis II, the two are distributed, one to each future gamete. This requires that meiosis I chromosomes attach to the spindle differently than meiosis II chromosomes and that they regulate chromosome cohesion differently. We investigated whether the information that dictates the division type of the chromosome comes from the whole cell, the spindle, or the chromosome itself. Also, we determined when chromosomes can switch from meiosis I behavior to meiosis II behavior. We used a micromanipulation needle to fuse grasshopper spermatocytes in meiosis I to spermatocytes in meiosis II, and to move chromosomes from one spindle to the other. Chromosomes placed on spindles of a different meiotic division always behaved as they would have on their native spindle; e.g., a meiosis I chromosome attached to a meiosis II spindle in its normal fashion and sister chromatids moved together to the same spindle pole. We also showed that meiosis I chromosomes become competent meiosis II chromosomes in anaphase of meiosis I, but not before. The patterns for attachment to the spindle and regulation of cohesion are built into the chromosome itself. These results suggest that regulation of chromosome cohesion may be linked to differences in the arrangement of kinetochores in the two meiotic divisions.     

Dynamics of microtubular cytoskeleton in higher plant meiosis. VI. Mechanisms of the successive cytokinesis

Intracellular morphological processes of successive cytokinesis in cereal pollen mother cells during normal and abnormal meiosis were studied. It was shown that the central spindle fiber system transforms into a phragmoplast at telophase. A model of centrifugal movement of the phragmoplast as a modification of B-anaphase has been proposed.     

{gamma}-Tubulin and microtubule organization during meiosis in the liverwort Ricciocarpus natans (Ricciaceae)

Extant liverworts are "living fossils" considered sister to all other plants and as such provide clues to the evolution of the microtubule organizing center (MTOC) in anastral cells. This report is the first on microtubule arrays and their γ-tubulin-nucleating sites during meiosis in a member of the Ricciales, a specialized, species-rich group of complex thalloid (marchantioid) liverworts. In meiotic prophase, γ-tubulin becomes concentrated at several sites adjacent to the nuclear envelope. Microtubules organized at these foci give rise to a multipolar prometaphase spindle. By metaphase I, the spindle has matured into a bipolar structure with truncated poles. In both first and second meiosis, γ-tubulin forms box-like caps at the spindle poles. γ-Tubulin moves from spindle poles to the proximal surfaces of telophase chromosomes where interzonal microtubules are nucleated. Although a phragmoplast is organized, no cell plate is deposited, and second division occurs simultaneously in the undivided sporocyte. γ-Tubulin surrounds each of the tetrad nuclei, and phragmoplasts initiated between both sister and nonsister nuclei direct simultaneous cytokinesis. The overall pattern of meiosis (unlobed polyplastidic sporocytes, nuclear envelope MTOC, multipolar spindle origin, spindles with box-like poles, and simultaneous cytokinesis) more closely resembles that of Conocephalum than other marchantiod liverworts.     

Mitosis in Tilia americana endosperm

The endosperm cells of the American basswood Tilia americana are favorable experimental material for investigating the birefringence of living plant spindles and anaphase movement of chromosomes. The behavior of the chromosomes in anaphase and the formation of the phragmoplast are unique. The numerous (3 n equals 123), small chromosomes move in precise, parallel rows until midanaphase when they bow away from the poles. Such a pattern of anaphase chromosome distribution has been described once before, but was ascribed to fusion of the chromosomes. The bowing of chromosome rows in Tilia is explainable quantitatively by the constant poleward velocity of the chromosomes during anaphase. Peripheral chromosomes are moving both relative to the spindle axis and laterally closer to the axis, whereas chromosomes lying on the spindle axis possess no lateral component in their motion, and thus at uniform velocity progress more rapidly than peripheral chromosomes relative to the spindle axis. The chromosomes are moved poleward initially by pole-to-pole elongation of the spindle, then moved farther apart by shortening of the kinetochore fibers. In contrast to other plant cells where the phragmoplast forms in telophase, the phragmoplast in Tilia endosperm is formed before midanaphase and the cell during midanaphase, while the chromosomes are still in poleward transit.     

Meiosis spindle formation and chromosome behavior in diploid potatoes with 'fused spindles' mutation

Chromosomal behaviour and spindle morphology were studied in microsporogenesis of two kinds of diploid potato clones: with normal meiosis, and with "fused spindles" (fs) occurring during the second meiotic division from prometaphase II (proMII) to telophase II (TII). For the first time, morphological effect of fs was found at the late proMII stage to be expressed as two interrelated processes: 1) abnormal chromosome movement, which resulted in joining two groups of chromosomes in the central zone of meiocytes, and 2) abnormal formation of two spindles in the direction to two division poles instead of four poles that actually led to the formation of a united bipolar spindle. Thus, it is not the fusion of two parallel spindles but the formation of united bipolar spindle that constitutes fs abnormality, while the parallel co-orientation of two spatially separated meiotic spindles is a norm in diploid potato. These primary abnormalities detected at proMII resulted in abnormalities at its subsequent meiotic stages: formation of fused spindle and united metaphase plate at MII, bipolar chromosome segration at anaphase II, formation of two telophase nuclei at TII and dyads at the tetrad stage. The results obtained evidence the polar division disturbance in diploid potato clones with fs abnormality.     

MITOSIS IN THE ALGA VACUOLARIA VIRESCENS

Details of mitosis in the chloromonadophycean alga Vacuolaria virescens Cienk. have been studied with the light microscope. The chromosomes are relatively large (up to μ in length at metaphase) and so mitotic stages are readily distinguishable. Chromosomes can be recognized in interphase nuclei as fine strands of chromatin. Contraction of these chromosomes marks the beginning of mitosis and continues progressively until the transition from metaphase to anaphase. Disintegration of nucleoli is complete by late prophase and nucleolar reformation begins in telophase. Some chromosomes exhibit less densely stained regions; centromeres are also present as indicated by their differential staining and by the behavior of chromosomes at metaphase and anaphase. At anaphase progeny chromosomes move apart parallel to the division axis of the nucleus. As anaphase progresses the chromosomes fuse at the polar surface of the progeny chromosome groups. This process continues in telophase and the chromosome groups become more spherical. By the end of telophase nucleolar reformation has begun and the chromosomes have relaxed to their interphase condition.     

MULTIPOLAR MEIOSIS IN DIPLOID CRESTED WHEATGRASS,AGROPYRON CRISTATUM

A new model of spindle organizers is proposed: The spindle organizer in a higher plant is similar to the centriole of animal cells. It is a unit cell organelle which follows regular cell division cycles and is genome specific. Each genome carries its own spindle organizer. During fertilization, a male spindle organizer enters the egg cell. It may fuse with the female spindle organizer, or either one may degenerate. In a hybrid, both male and female spindle organizers may exist, and multipolar divisions separate different genomes into different groups. The same mechanism can be used to explain the formation of a polyhaploid from a polyploid. The chromosome behavior of an individual is believed to be an interaction of chromosome homology and the homology between chromosomes and their spindle organizers. This model is based on observations of multipolar meiosis which occurred in two cultures of diploid crested wheatgrass, Agropyron cristatum (L.) Gaertn. In these two cultures multipolar meiosis occurred at every stage after late diakinesis. The seven bivalents were separated into groups at late diakinesis. More than one equatorial plate was formed at metaphase I. Each micrometaphase plate behaved as an independent unit and had its own anaphase movement. Usually the chromosome complement separated into two groups with (4–3), (5–2), and (6–1) separations observed in about an equal number of cells. Cells with chromosomes divided into three or four groups were found less frequently. Multipolar meiosis may take place at either first or second division. Cell plates were formed across each spindle apparatus, cleaving each group of chromosomes into smaller micro-cells. At the “quartet” stage, 4- to 12-celled “quartets” were observed. Pollen stainability was measured at above 75% in both cultures. Stained pollen grains could be classified into two distinct size classes. Darkly stained, small pollen grains represented the result of multipolar meiosis and may have been viable. Multipolar cell divisions provided a mechanism which polyploids might reduce their ploidy level.     

Chromosomal behavior in starfish (Asterina pectinifera) zygotes under the effect of aphidicolin, an inhibitor of DNA polymerase

When calf thymus histones were labeled fluorescently and microinjected into oocytes of the starfish, Asterina pectinifera, the labeled histones visualized chromosomes during maturation division and cleavage. In doing so, we confirmed the previously reported phenomenon that chromosomes became incompetent at the first cleavage in the aphidicolin-treated egg, although cleavage itself took place. Moreover, we found that chromosomes were aligned at the equator of the metaphase spindle of the first cleavage and that they did not separate into two groups at all, but made a lump in the middle of the spindle. Chromosomes finally entered one blastomere, although they did not participate in the following karyokinesis. DNA and microtubules were examined by cytochemistry and immunofluorescence in order to investigate the relation between chromosome movement and the microtubular cytoskeleton. The mitotic apparatus developed and grew in the aphidicolin-treated cells in the same manner as those in normal cells without normal chromatin condensation or chromosome movement during the first cleavage. However, the mitotic apparatus consisted of two asters without the spindle formed at subsequent cleavages. Electron microscopic study revealed that chromosomes did not condense normally and kinetochores were not detected during the first cleavage. These results indicate that the dynamic changes in microtubular structures during mitosis have poor relation with the chromosome behavior such as prophase chromosome condensation and anaphase chromosome movement.     

“Bouquet arrest”, monopolar chromosomes segregation, and correction of the abnormal spindle

According to our data, the arrest of univalents in bouquet arrangement is a widespread meiotic feature in cereal haploids and allohaploids (wide hybrids F₁). We have analyzed 83 different genotypes of cereal haploids and allohaploids with visualization of the cytoskeleton and found a bouquet arrest in 45 of them (in 30% to 100% pollen mother cells (PMCs)). The meiotic plant cell division in 26 various genotypes with a zygotene bouquet arrest was analyzed in detail. In three of them in PMCs, a very specific monopolar conic-shaped figure at early prometaphase is formed. This monopolar figure consists of mono-oriented univalents and their kinetochore fibers converging in pointed pole. Such figures are never observed at wild-type prometaphase or in asynaptic meiosis in the variants without a bouquet arrest. Later at prometaphase, the bipolar central spindle fibers join in this monopolar figure, and a bipolar spindle with all univalents connected to one pole is formed. As a result of monopolar chromosome segregation at anaphase and normal cytokinesis at telophase, a dyad with one member carrying a restitution nucleus and the other enucleated is formed. However, such phenotype has only three genotypes among 26 analyzed with a bouquet arrest. In the remaining 23 haploids and allohaploids, the course of prometaphase was altered after the conic monopolar figure formation. In these variants, the completely formed conic monopolar figure was disintegrated into a chaotic network of spindle fibers and univalents acquired a random orientation. This arrangement looks like a mid-prometaphase in the wild-type meiosis. At late prometaphase, a bipolar spindle is formed with the univalents distributed more or less equally between two poles, similar to the phenotypes without a bouquet arrest. The product of cell division is a dyad with aneuploid members. Thus, the spindle abnormality—monopolar chromosome orientation—is corrected. In some cells the correction of the prometaphase monopolus occurs by means of its splitting into two half-spindles and their rotation along the future division axis.     

Chromosome numbers, meiotic behavior, and pollen viability of species of Vriesea and Aechmea genera (Bromeliaceae) native to Rio Grande do Sul, Brazil

Chromosome number, meiotic behavior, and pollen viability were analyzed in 15 species of two genera, Vriesea and Aechmea, native to Rio Grande do Sul, Brazil. This study is the first cytogenetic analysis of these taxa. The chromosome numbers are all n = 25, consistent with the proposed base number of x = 25 for Bromeliaceae. All examined taxa displayed regular bivalent pairing and chromosome segregation at meiosis. Observed meiotic abnormalities include univalents in metaphase I; missing or extra chromosomes and precocious division of centromeres in metaphase II; laggards in telophase I and anaphase II/telophase II. The high pollen viability (>88%) reflects a regular meiosis.     

Chromosomal behavior in starfish (Asterina pectinifera) zygotes under the effect of aphidicolin, an inhibitor of DNA polymerase

When calf thymus histones were labeled fluorescently and microinjected into oocytes of the starfish, Asterina pectinifera, the labeled histones visualized chromosomes during maturation division and cleavage. In doing so, we confirmed the previously reported phenomenon that chromosomes became incompetent at the first cleavage in the aphidicolin-treated egg, although cleavage itself took place. Moreover, we found that chromosomes were aligned at the equator of the metaphase spindle of the first cleavage and that they did not separate into two groups at all, but made a lump in the middle of the spindle. Chromosomes finally entered one blastomere, although they did not participate in the following karyokinesis. DNA and microtubules were examined by cytochemistry and immunofluorescence in order to investigate the relation between chromosome movement and the microtubular cytoskeleton. The mitotic apparatus developed and grew in the aphidicolin-treated cells in the same manner as those in normal cells without normal chromatin condensation or chromosome movement during the first cleavage. However, the mitotic apparatus consisted of two asters without the spindle formed at subsequent cleavages. Electron microscopic study revealed that chromosomes did not condense normally and kinetochores were not detected during the first cleavage. These results indicate that the dynamic changes in microtubular structures during mitosis have poor relation with the chromosome behavior such as prophase chromosome condensation and anaphase chromosome movement.     

Kid-mediated chromosome compaction ensures proper nuclear envelope formation

Toward the end of mitosis, neighboring chromosomes gather closely to form a compact cluster. This is important for reassembling the nuclear envelope around the entire chromosome mass but not individual chromosomes. By analyzing mice and cultured cells lacking the expression of chromokinesin Kid/kinesin-10, we show that Kid localizes to the boundaries of anaphase and telophase chromosomes and contributes to the shortening of the anaphase chromosome mass along the spindle axis. Loss of Kid-mediated anaphase chromosome compaction often causes the formation of multinucleated cells, specifically at oocyte meiosis II and the first couple of mitoses leading to embryonic death. In contrast, neither male meiosis nor somatic mitosis after the morula-stage is affected by Kid deficiency. These data suggest that Kid-mediated anaphase/telophase chromosome compaction prevents formation of multinucleated cells. This protection is especially important during the very early stages of development, when the embryonic cells are rich in ooplasm.