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Meiotin-1, a meiosis-enriched protein present in normal leptotene chromosomes and lacking in precociously condensed leptotene chromosomes.

During mitotic prophase, chromosomes progressively compact to their metaphase length. In contrast, meiotic chromosomes condense moderately until late in prophase I, then they condense more dramatically (coil) to their fully condensed state. Meiotin-1 is a meiosis-enriched, chromosomal protein. We propose that it delays coiling until after reciprocal genetic exchange. We have used immunoblotting and immunocytochemistry with normal lily cells undergoing meiosis to demonstrate that meiotin-1 is present during the early portions of prophase I, but diminishes at the time when meiotic chromosomes begin to coil. Additionally, we have examined lily meiotic nuclei undergoing the reversible phenomenon of precocious leptotene chromosome condensation (precocious coiling). The leptotene chromosomes that are precociously condensed lack meiotin-1 immunostaining. Furthermore nuclei returning to the normal state of moderate prophase I condensation acquire meiotin-1.

Antibodies, Monoclonal↗

Meiosis in primary trisomics of rye: considerations for models of chromosome pairing.

Meiotic chromosome pairing of primary trisomics of rye was analysed by electron microscopy in surface-spread prophase I nuclei and compared with light-microscopic observations of metaphase I cells. Despite the large-sized chromosomes of rye, prophase I trivalent frequencies were close to the two thirds expected on a simple model with two terminal independent pairing initiation sites per trisome (set of three homologous chromosomes). Direct observations mostly reveal one pairing partner switch (PPS) in prophase I trivalents, which confirms this supposition. There were no significant differences between the number of trivalent and bivalent plus univalent configurations observed at prophase and metaphase I; therefore, synapsed segments form chiasmata. In all of the trisomics, the three homologues showed variations not only in the number of telomeric C-bands but also in the amount of heterochromatin of these bands, which allowed identification of chromosomes or chromosome arms associated in most metaphase I configurations. In trisomics for chromosomes 2, 3 and 5, some metaphase I chromosome configuration frequencies did not fit those expected under the assumption of random chromosome association among all partners, suggesting the existence of preferences for pairing between two given chromosome arms of the trisome. No preferential associations either at metaphase I or pachytene were observed in the trisomics for chromosome 6. The fit between theoretical pairing models and the experimental data is also discussed.

Cell Nucleus↗

SUMO modifications control assembly of synaptonemal complex and polycomplex in meiosis of Saccharomyces cerevisiae.

The synaptonemal complex (SC) is a proteinaceous complex that apparently mediates synapsis between homologous chromosomes during meiotic prophase. In Saccharomyces cerevisiae, the Zip1 protein is the integral component of the SC. In the absence of a DNA double-strand break or the SC initiation protein Zip3, Zip1 proteins aggregate to form a polycomplex (PC). In addition, Zip1 is also responsible for DSB-independent nonhomologous centromere coupling at early meiotic prophase. We report here that Zip3 is a SUMO (small ubiquitin-related modifier) E3 ligase and that Zip1 is a binding protein for SUMO-conjugated products. Our results also suggest that at early meiotic prophase, Zip1 interacts with Zip3-independent Smt3 conjugates (e.g., Top2) to promote nonhomologous centromere coupling. At and after mid-prophase, the Zip1 protein begins to associate with Zip3-dependent Smt3 conjugates (e.g., Red1) along meiotic chromosomes in the wild-type cell to form SCs and with Smt3 polymeric chains in the zip3 mutant to form PCs.

Amino Acid Sequence↗

Long duration of mitosis and consequences for the cell cycle concept, as seen in the isthmal cells of the mouse pyloric antrum. I. Identification of early and late steps of mitosis.

In this series of two articles, the duration of mitosis and that of the cell cycle were examined in a group of proliferating cells located in the mouse pyloric antrum and known as isthmal cells. However, before measuring the duration of mitosis, as described in the second article, it is necessary to identify the early and late steps of the mitotic process. This is attempted in the present article, in which the four phases of mitosis and the interphase are described in semithin (0.5 micron thick) Epon serial sections stained with hemalun. The frequency of these phases is then estimated. The beginning of prophase is indicated by the appearance in the nucleus of numerous 0.2-0.3 micron thick basophilic threads. The threads gradually increase in thickness to become the typical chromosomes (about 0.7-micron thick) observed at the end of prophase. Metaphase and anaphase show no remarkable features. At telophase, chromosomes separate from one another, gradually acquire pale segments along their length eventually to look like rows of alternating dark and light patches, and finally vanish. When prophases and telophases are defined in this manner, the enumeration of isthmal cells yields a high proportion of prophases (28%) and telophases (31%), but a low proportion of metaphases (1%) and anaphases (0.3%). Forty per cent of the cells are in interphase.

Anaphase↗

Meiosis readiness in Lilium.

It was observed in five cultivars and two hybrids of Lilium that premeiotic prophase is retarded in anthers approaching meiosis. The occurrence of premeiotic despiralization was related to the degree of retardation of premeiotic prophase. It is proposed that meiosis is initiated by stimuli arising outside the premeiotic cells. It is suggested that an accumulation of meiosis-inducing substances in the cytoplasm of the premeiotic cells causes prophase to slow down; when a critical level ("meiosis readiness") is reached, mitotic division is no longer possible and cells in premeiotic prophase despiralize to interphase.

Meiosis↗

Kinetics of meiosis in azoospermic males: a joint histological and cytological approach.

We have developed a protocol for the identification of aberrant chromosome behavior during human male meiosis up to metaphase of the secondary spermatocyte. Histological evaluation by the Johnsen score of a testicular biopsy was combined with immunofluorescence of first meiotic prophase spermatocytes, using antibodies against synaptonemal complex protein 3 (SYCP3) and the product of the ataxia telangiectasia and rad3-related gene (ATR). This combination enables accurate meiotic prophase substaging and the identification of pachytene spermatocytes with asynapsis. Furthermore, we also investigated the competence of late pachytene primary spermatocytes to complete the first meiotic division up to metaphase and of secondary spermatocytes to transform into metaphase by an in vitro challenge with okadaic acid (OA). We tested this protocol on five males with normal Johnsen scores that presented with obstructive azoospermia, five males with low Johnsen scores and non-obstructive azoospermia and six vasectomized control males of proven fertility and normal Johnsen scores. In all azoospermics, the profiling of meiotic prophase stages by immunofluorescence increases the resolving power of the Johnsen score. In both obstructive and non-obstructive azoospermic patients, relatively more leptotene meiotic prophase stages were counted compared to the controls. In non-obstructive azoospermics, a marked heterogeneity in spermatogenesis was found, after combining the results of all three approaches, pointing at functional mosaicism of the germinal epithelium. Asynaptic pachytene spermatocytes were rarely encountered. Also, when first meiotic metaphase could be induced by OA, chiasma counts were normal. In none of the non-obstructive azoospermic males did the pattern of spermatogenesis resemble that of knock-out mouse azoospermics. We conclude that this combined histological and cytological approach enables a detailed phenotypic classification of infertile males, at a level comparable to that applied for male-sterile knock-out mice with a meiotic defect. This may facilitate the identification of candidate genes for human male infertility.

Animals↗

Loss of synaptonemal complex protein-1, a synaptonemal complex protein, contributes to the initiation of follicular assembly in the developing rat ovary.

In the rat ovary, germ and somatic cells become organized into primordial follicles 48-72 h after birth. Although several genes have been implicated in the control of early follicular growth, less is known about the factors involved in the formation of primordial follicles. Using the method of differential display of mRNAs, we found several genes differentially expressed at the time of follicular assembly. One of them encodes synaptonemal complex protein-1 (SCP1), a core component of the protein complex that maintains recombining chromosomes together during prophase I of the first meiotic division in germ cells. This association, evident during the pachytene stage, ends when chromosomal desynapsis begins in the diplotene stage at the end of prophase I. Oocytes become arrested in the diplotene/dictate stage before becoming enclosed into primordial follicles, suggesting that oocytes must complete meiotic prophase I before becoming competent to direct follicle assembly. We now show that attainment of the diplotene stage results in follicular formation. In developing rat ovaries, SCP1 mRNA expression is confined to oocytes and decreases precipitously within 24 h after birth, preceding the organization of primordial follicles. The premature loss of SCP1, achieved via treatment with an antisense oligodeoxynucleotide targeting SCP1 mRNA, resulted in more oocytes reaching the diplotene stage, as evidenced by a decrease in the number of oocytes containing germ cell nuclear antigen-1 (a nuclear protein whose expression ceases in diplotene) and an increase in the number of oocytes expressing MSY2 (a cytoplasmic Y box protein expressed in oocytes that have become arrested in diplotene). SCP1-deficient ovaries exhibited an increased number of newly formed follicles, suggesting that completion of meiotic prophase I endows oocytes with the ability to orchestrate follicular assembly.

Animals↗

Relocation of nucleolar proteins around chromosomes at mitosis. A study by confocal laser scanning microscopy.

The behaviour of nucleolar antigens known to associate with chromosomes at mitosis was investigated in mammalian cells (HeLa, HEp-2, PtK1, CHO) by immunofluorescence and confocal laser scanning microscopy. Serial optical sections through mitotic cells, from prophase to telophase, were used to generate three-dimensional images of the antigen distribution. Our results indicate that, at the onset of mitosis, these antigens leave the nucleoli in a highly ordered manner to form a network extending from the nucleoli towards the nuclear envelope. The migration begins at very early prophase, when the condensation of the chromosomes is not yet visible. After completion of the migration at late prophase, the labelling is found at the chromosome periphery. The antigens remain distributed as a sheath surrounding the chromosomes from prophase to telophase. Therefore, the proteins involved in the formation of this perichromosomal layer have different behaviour than those of the prenucleolar bodies. The antigens appear to interact strongly with chromosomes, since they are not lost during chromosome isolation in hypotonic buffer. Each chromosome is entirely covered from one telomere to the other, except in the centromeric region. Thus the relocation of these nucleolar proteins does not appear to be the result of a passive accumulation at the chromosome periphery, but seems rather to be due to an active targeting to specific sites. Consequently, these proteins may have a determining function in the progression of the cells through mitosis, possibly by participating in the protection and stabilization of the chromosomes.

Animals↗

Cytoplasmic accumulation of cdc25B phosphatase in mitosis triggers centrosomal microtubule nucleation in HeLa cells.

The formation of the mitotic spindle is an essential prerequisite for successful mitosis. The dramatic changes in the level of microtubule (Mt) nucleation at the centrosomes and Mt dynamics that occur in prophase are presumed to be initiated through the activity of cdc2/cyclin B. Here we present data that the cdc25B isoform functions to activate the cytoplasmic pool of cdc2/cyclin B responsible for these events. In contrast to cdc25C, cdc25B is present at low levels in HeLa cells during interphase, but sharply increases in prophase, when cdc25B accumulation in the cytoplasm correlates with prophase spindle formation. Overexpression of wild type and dominant negative mutants of cdc25B and cdc25C shows that prophase Mt nucleation is a consequence of cytoplasmic cdc25B activity, and that cdc25C regulates nuclear G2/M events. Our data also suggest that the functional status of the centrosome can regulate nuclear mitotic events.

Antibodies, Monoclonal↗

Variations in the distribution and migration of centriole duplexes in mitotic PtK2 cells studied by immunofluorescence microscopy.

The localization and migration of centriole duplexes have been studied in PtK2 cells by indirect immunofluorescence microscopy using specific tubulin antibodies. The study demonstrated the usefulness of the immunofluorescence technique to quantitate studies of centriole migration and concomitant events such as cytoplasmic microtubule breakdown in large populations of cells. Centriole duplex locations in normal and Colcemid-treated interphase populations have been compared with duplex locations in prophase cells. A higher percentage of duplexes were found close to the nucleus in prophase than in interphase cells, but approximately 5% of the duplexes remained in the cytoplasm far removed from the nucleus in prophase and throughout the course of duplex separation. Duplex separation occurred along a wide variety of paths and duplexes did not have to be closely juxtaposed to the nuclear envelope for separation to occur. Some duplexes separated in the cytoplasm with no detectable nuclear attachment, with spindles forming far to the side of the condensing chromosomes. The timing of duplex separation did not always coincide either with chromosome condensation or with nuclear membrane breakdown, and in a small percentage of the cells separation occurred as late as prometaphase. These data suggest that normal spindle formation can occur despite the large variability in initial and final centriole duplex location, their migration patterns, and the timing of the different events. Breakdown of cytoplasmic microtubules began in prophase and progressed until prometaphase; the last cytoplasmic microtubules disappeared soon after the loss of the nuclear membrane.

Cell Line↗

The mechanism of secondary nondisjunction in Drosophila melanogaster females.

Bridges (1916) observed that X chromosome nondisjunction was much more frequent in XXY females than it was in genetically normal XX females. In addition, virtually all cases of X nondisjunction in XXY females were due to XX <--> Y segregational events in oocytes in which the two X chromosomes had failed to undergo crossing over. He referred to these XX <--> Y segregation events as "secondary nondisjunction." Cooper (1948) proposed that secondary nondisjunction results from the formation of an X-Y-X trivalent, such that the Y chromosome directs the segregation of two achiasmate X chromosomes to opposite poles on the first meiotic spindle. Using in situ hybridization to X and YL chromosomal satellite sequences, we demonstrate that XX <--> Y segregations are indeed presaged by physical associations of the X and Y chromosomal heterochromatin. The physical colocalization of the three sex chromosomes is observed in virtually all oocytes in early prophase and maintained at high frequency until midprophase in all genotypes examined. Although these XXY associations are usually dissolved by late prophase in oocytes that undergo X chromosomal crossing over, they are maintained throughout prophase in oocytes with nonexchange X chromosomes. The persistence of such XXY associations in the absence of exchange presumably facilitates the segregation of the two X chromosomes and the Y chromosome to opposite poles on the developing meiotic spindle. Moreover, the observation that XXY pairings are dissolved at the end of pachytene in oocytes that do undergo X chromosomal crossing over demonstrates that exchanges can alter heterochromatic (and thus presumably centromeric) associations during meiotic prophase.

Animals↗

Taxol-induced mitotic block triggers rapid onset of a p53-independent apoptotic pathway.

BACKGROUND: At therapeutic concentrations, the antineoplastic agent taxol selectively perturbs mitotic spindle microtubules. Taxol has recently been shown to induce apoptosis, similar to the mechanism of cell death induced by other antineoplastic agents. However, taxol has shown efficacy against drug-refractory cancers, raising the possibility that this pharmacological agent may trigger an alternative apoptotic pathway. MATERIALS AND METHODS: The kinetics and IC50 of mitotic (M) block, aberrant mitosis, and cytotoxicity following taxol treatment were analyzed in human cell lines as well as normal mouse embryo fibroblasts (MEFs) and MEFs derived from p53-null mice. Apoptosis was followed by DNA gel electrophoresis and by in situ DNA end-labeling (TUNEL). RESULTS: Taxol induced two forms of cell cycle arrest: either directly in early M at prophase or, for those cells progressing through aberrant mitosis, arrest in G1 as multimininucleated cells. TUNEL labeling revealed that DNA nicking occurred within 30 min of the arrest in prophase. In contrast, G1-arrested, multimininucleated cells became TUNEL positive only after several days. In the subset of cells that became blocked directly in prophase, both wt p53-expressing and p53-null MEFs responded similarly to taxol, showing rapid onset of DNA nicking and apoptosis. However, p53-null MEFs progressing through aberrant mitosis failed to arrest in the subsequent G1 phase or to become TUNEL positive, and remained viable. CONCLUSIONS: Taxol induces two forms of cell cycle arrest, which in turn induce two independent apoptotic pathways. Arrest in prophase induces rapid onset of a p53-independent pathway, whereas G1-block and the resulting slow (3-5 days) apoptotic pathway are p53 dependent.

Animals↗

A genetic strategy for differential screening of meiotic germ-cell cDNA libraries.

The goals of this work were to create germ-cell-stage-specific cDNA libraries from mouse spermatogenic cells and to employ a novel two-step genetic screen to identify gene sequences present during the critical meiotic stage of spermatogenesis. Highly enriched germ-cell fractions were prepared from adult and juvenile mouse testes, and purity of these fractions was extensively analyzed by light and electron microscopy. Standard techniques were used to prepare cDNA libraries from populations of mixed leptotene and zygotene (L/Z) spermatocytes, pachytene (P) spermatocytes, and round spermatids. These libraries were analyzed with respect to representation of sequences from ubiquitously expressed genes, and from genes expressed at specific germ-cell stages as well as from genes expressed in testicular somatic cells. For the first step of the screening procedure, testicular cDNA was prepared from mutant mice carrying the T(X;11)38H chromosomal translocation that causes spermatogenic arrest at early meiotic prophase. This mixed cDNA probe was used to screen the libraries from L/Z and P spermatocytes to detect sequences failed to hybridize. The clones identified were characterized for ability to hybridize to various germ-cell-specific cDNAs to verify that they represented sequences present in normal spermatogenic meiotic cells. These clones were then subjected to a second screening with another mutant probe; this time the cDNA probe was from testes of sterile mice bearing the T(X;16)16H chromosomal translocation that causes spermatogenic arrest at late meiotic prophase. This screen identified 27 clones that were not represented in testicular cDNA from T38-bearing mice or from T16-bearing mice. These clones may represent sequences essential for normal completion of the genetic events of meiosis during spermatogenesis. Likewise, the secondary screen identified 19 clones that were not represented in testicular cDNA from T38-bearing mice but were represented in testicular cDNA of T16-bearing mice. These clones are thus gene sequences present in spermatogenic cells during the time from early meiotic prophase to mid-to-late prophase. This strategy represents the first use of genetic aberrations in differential screening to identify genes expressed at specific times during mammalian spermatogenesis.

Animals↗

Dense bodies in silver-stained spermatocytes of the Chinese hamster: behavior and cytochemical nature.

From the silver staining behavior of various organelles in the nucleus we have divided meiotic prophase (leptotene to the diffuse stage) of the male Chinese hamster into five stages. Components within the nucleus, such as synaptonemal complex (SC), sex bivalent (SB), nucleolus organizer regions (NORs), chromatin and the dense bodies, showed a characteristic feature in each stage of meiotic prophase. The lampbrush chromosome stage was found to be followed by the diffuse stage. The chromatin around SC began to be organized at early pachytene and formed a brush-like structure at late pachytene. During early prophase stages a dramatic change in SB morphology occurred. Three types of morphology of SB were recognized: (1) the XY pair with long synapsis and fusiform or diffuse thickening of the unpaired portions (late zygotene and early pachytene), (2) desynapsed, branched, and anastomosed axes seen at late pachytene. Two types of the dense body were found during meiotic prophase; the double body in early stage (leptotene to early pachytene) and the single body in later stages (mid pachytene to diffuse stage). The small precursors of the double body existed at early leptotene but they increased in size and also changed the silver stainability during zygotene, becoming the characteristic double body consisted of one light body (L-body) and one dark body (D-body). These two bodies can also be recognized after Giemsa or acridine orange (AO) staining. The L-body fluoresced reddish orange after AO staining. The single body, which is probably formed by amalgamation of the D- and the L-bodies, showed a staining reaction similar to that of the D-body. Data from pancreatic lipase and protease treatments suggest that the D-body contained a lipoprotein.

Animals↗

Cytogenetics of two species of Euceraphis (Homoptera, Aphididae).

Somatic cell divisions, spermatogenesis, and the prophase stages of primary oocytes, are described for two species of birch aphid, Euceraphis betulae (Koch) and E. punctipennis (Zettersted). Females of E. betulae have two autosome pairs, two pairs of X-chromosomes of different lengths, and two B-chromosomes. Females of E. punctipennis have the same number of X-chromosomes. The sex determination system is X1X2O. E. punctipennis males sometimes have only one B-chromosome. In the spermatogenesis of E. Betulae, pairing of homologous autosomes occurs in early prophase I, but no evidence was found of chiasmata or end-to-end alignment of homologues. Instead, homologues remain closely aligned in parallel as they condense into metaphase, and anaphase I separates the products of pairing in a strictly reductional manner. The two unpaired X-chromosomes and both B-chromosomes are stretched on the anaphase I spindle and all four pass into the larger secondary spermatocyte. The second division is equational. The B-chromosomes thus show accumulation in spermatogenesis, which must be compensated in some way by an elimination mechanism in oogenesis. Meiosis of E. punctipennis is highly anomalous. The two autosomes pair but separate again in early prophase I, then one homologue becomes heterchromatic and is apparently rejected from the late prophase nucleus. A single, equational maturation division follows. In female neiosis I, both species show highly characteristic diplotene figures with multiple chiasmata, the B-chromosomes remaining unpaired. These results are discussed in relation to previous work on aphid cytogenetics.

Animals↗

Synchronous nuclear-envelope breakdown and anaphase onset in plant multinucleate cells.

Multinucleate plant cells with genetically balanced nuclei can be generated by inhibiting cytokinesis in sequential telophases. These cells can be used to relate the effect of changes in the distribution of nuclei in the cytoplasm to the control of the timing of cell cycle transitions. Which mitotic cell cycle events are sensitive to differences in the amount of cytoplasm surrounding each chromosomal complement has not been determined. To address this, we maximized the cell size by transiently inhibiting replication, while cell growth was not affected. The nuclei of 93% of the elongated cells reached prophase asynchronously compared to 46% of normal-sized multinucleate cells. The asynchronous prophases of normal-sized cells became synchronous at the time of nuclear-envelope breakdown, and the ensuing metaphase plate formation and anaphase onset and progression occurred synchronously. The elongated multinucleate cells were also very efficient in synchronizing the prophases at nuclear-envelope breakdown, in the prophase-to-prometaphase transition. However, 2.4% of these cells broke down the nuclear envelope asynchronously, though they became synchronous at the metaphase-to-anaphase transition. The kinetochore-microtubular cycle, responsible for coordinating the metaphase-to-anaphase transition and for the rate of sister segregation to opposite spindle poles during anaphase, remained strictly controlled and synchronous in the different mitoses of a single cell, independently of differences in the amount of cytoplasm surrounding each mitosis or its ploidy. Moreover, the degree of chromosome condensation varied considerably within the different mitotic spindles, being higher in the mitoses with the largest surrounding cytoplasm.

Allium↗

Meiotic events at the centromeric heterochromatin: histone H3 phosphorylation, topoisomerase II alpha localization and chromosome condensation.

Mechanisms of chromosome condensation and segregation during the first meiotic division are not well understood. Resolution of recombination events to form chiasmata is important, for it is chiasmata that hold homologous chromosomes together for their oppositional orientation on the meiotic metaphase spindle, thus ensuring their accurate segregation during anaphase I. Events at the centromere are also important in bringing about proper attachment to the spindle apparatus. This study was designed to correlate the presence and activity of two proteins at the centromeric heterochromatin, topoisomerase II alpha (TOP2A) and histone H3, with the processes of chromosome condensation and individualization of chiasmate bivalents in murine spermatocytes. We tested the hypothesis that phosphorylation of histone H3 is a key event instigating localization of TOP2A to the centromeric heterochromatin and condensation of chromosomes as spermatocytes exit prophase and progress to metaphase. Activity of topoisomerase II is required for condensation of chromatin at the end of meiotic prophase. Histone H3 becomes phosphorylated at the end of prophase, beginning with its phosphorylation at the centromeric heterochromatin in the diplotene stage. However, it cannot be involved in localization of TOP2A, since TOP2A is localized to the centromeric heterochromatin throughout most of meiotic prophase. This observation suggests a meiotic function for TOP2A in addition to its role in chromatin condensation. The use of kinase inhibitors demonstrates that phosphorylation of histone H3 can be uncoupled from meiotic chromosome condensation; therefore other proteins, such as those constituting metaphase-promoting factor, must be involved. These results define the timing of important meiotic events at the centromeric heterochromatin and provide insight into mechanisms of chromosome condensation for meiotic metaphase.

Animals↗

Polar organizers and girdling bands of microtubules are associated with gamma-tubulin and act in establishment of meiotic quadripolarity in the hepatic Aneura pinguis (Bryophyta).

Meiosis in Aneura pinguis is preceded by extensive cytoplasmic preparation for quadripartitioning of the diploid sporocyte into a tetrad of haploid spores. In early prophase the four future spore domains are defined by lobing of the cytoplasm and development of a quadripolar prophase spindle focused at polar organizers (POs) centered in the lobes. Cells entering the reproductive phase become isolated and, instead of hooplike cortical microtubules, have endoplasmic microtubule systems centered on POs. These archesporial cells proliferate by mitosis before entering meiosis. In prophase of each mitosis, POs containing a distinct concentration of gamma-tubulin appear de novo at tips of nuclei and initiate the bipolar spindle. Cells entering meiosis become transformed into quadrilobed sporocytes with four POs, one in each lobe. This transition is a complex process encompassing assembly of two opposite POs which subsequently disperse into intersecting bands of microtubules that form around the central nucleus. The girdling bands define the future planes of cytokinesis and the cytoplasm protrudes through the restrictive bands becoming quadrilobed. Two large POs reappear in opposite cleavage furrows. Each divides and the resulting POs migrate into the tetrahedral lobes of cytoplasm. Cones of microtubules emanating from the four POs interact to form a quadripolar microtubule system (QMS) that surrounds the nucleus in meiotic prophase. The QMS is subsequently transformed into a functionally bipolar metaphase spindle by migration of poles in pairs to opposite cleavage furrows. These findings contribute to knowledge of microtubule organization and the role of microtubules in spatial regulation of cytokinesis in plants.

Bryophyta↗