Sister Nuclei Research Articles

Evidence from automixis with inverted meiosis for the maintenance of sex by loss of complementation.

The adaptive value of sexual reproduction is still debated. A short-term disadvantage of asexual reproduction is loss of heterozygosity, which leads to the unmasking of recessive deleterious mutations. The cost of this loss of complementation is predicted to be higher than the twofold cost of meiosis for most types of asexual reproduction. Automixis with terminal fusion of sister nuclei is especially vulnerable to the effect of loss of complementation. It is found, however, in some taxa including oribatid mites, the most prominent group of ancient asexuals. Here, I show that automixis with terminal fusion is stable if it is associated with inverted meiosis and that this appears to be the case in nature, notably in oribatid mites. The existence of automixis with terminal fusion, and its co-occurrence with inverted meiosis, therefore, is consistent with the hypothesis that loss of complementation is important in the evolution of sexual reproduction.

Defects in microtubule cytoskeleton during microsporogenesis relate to pollen abortion in male-sterile pepper (Capsicum annuum L.)

Microtubule (MT) cytoskeleton of pepper male-sterile microsporocytes during meiosis was examined to characterise the defects during microsporogenesis by comparison with that of fertile ones. In sterile microsporocytes, after prophase I, abnormal arrangement of MTs occurred until the end of meiosis. At metaphase I, about 10% of microsporocytes initially formed abnormal spindles, including monopolar spindle, non-polar spindle with short kinetochore MTs, and atypical bipolar spindles with two half arrays of MTs not on the same plane. Then, the homologous chromosomes could not separate, or they could move towards the two poles with lagged chromosomes at anaphase I. At telophase I, anisotropic short MTs were formed around the two sister nuclei, or the interzonal MTs depolymerised and nuclei agglutinated. About 10% of microsporocytes degraded in advance from metaphase I to telophase I. About 90% of microsporocytes proceeded to meiosis II. At metaphase II, MTs constructed two spindles at a shallow angle with upper and lower assignment, or four spindles with different size. During anaphase II and telophase II, four nuclei were arranged in a non-tetrahedral distribution with unevenly interzonal MTs between two nuclei; or the cell formed more than four nuclei with different size. The cytokinesis formed the aberrant microspores with different size, irregular shape, and even different-sized nuclei. Therefore, the defects of MTs during microsporogenesis in the male-sterile pepper affected the process of meiosis and cytokinesis, leading to some microsporocytes degraded in advance and the microspores at the end of meiosis completely decayed.

Auxosporulation in Chaetoceros acadianus sp. nov. (Bacillariophyceae), a new member of the Section Compressa

ABSTRACTWe document the fine structure of auxospores in a Chaetoceros species isolated from the Acadian coast of New Brunswick, Canada. Auxospore development in this species occurs in a terminal rather than lateral position, a characteristic never before observed in this genus. Our observations suggest that auxosporulation was uniparental, probably an extreme form of autogamy with sister nuclei fusing following meiosis II. Mature auxospores were adze-shaped to sub-globular and contained both scales and transverse perizonia in their walls. The transverse perizonial band structure was similar to longitudinal perizonial bands found in other species of Chaetoceros and differed from the pinnate bands of pennate transverse perizonia, which consisted of a central rib and bilateral fimbria. Instead, the band structure in C. acadianus was more similar to unilateral fimbriate bands in cymatosiroids. We also propose that our diatom represents a species new to science and is a member of the Chaetoceros Section Compressa. We provide its morphological, molecular and reproductive characterization.

Abnormal meiosis in an intersectional allotriploid of Populus L. and segregation of ploidy levels in 2x × 3x progeny.

Triploid plants are usually highly aborted owing to unbalanced meiotic chromosome segregation, but limited viable gametes can participate in the transition to different ploidy levels. In this study, numerous meiotic abnormalities were found with high frequency in an intersectional allotriploid poplar (Populus alba × P. berolinensis ‘Yinzhong’), including univalents, precocious chromosome migration, lagging chromosomes, chromosome bridges, micronuclei, and precocious cytokinesis, indicating high genetic imbalance in this allotriploid. Some micronuclei trigger mini-spindle formation in metaphase II and participate in cytokinesis to form polyads with microcytes. Unbalanced chromosome segregation and chromosome elimination resulted in the formation of microspores with aneuploid chromosome sets. Fusion of sister nuclei occurs in microsporocytes with precocious cytokinesis, which could form second meiotic division restitution (SDR)-type gametes. However, SDR-type gametes likely contain incomplete chromosome sets due to unbalanced segregation of homologous chromosomes during the first meiotic division in triploids. Misorientation of spindles during the second meiotic division, such as fused and tripolar spindles with low frequency, could result in the formation of first meiotic division restitution (FDR)-type unreduced gametes, which most likely contain three complete chromosome sets. Although ‘Yinzhong’ yields 88.7% stainable pollen grains with wide diameter variation from 23.9 to 61.3 μm, the pollen viability is poor (2.78% ± 0.38). A cross of ‘Yinzhong’ pollen with a diploid female clone produced progeny with extensive segregation of ploidy levels, including 29 diploids, 18 triploids, 4 tetraploids, and 48 aneuploids, suggesting the formation of viable aneuploidy and unreduced pollen in ‘Yinzhong’. Individuals with different chromosome compositions are potential to analyze chromosomal function and to integrate the chromosomal dosage variation into breeding programs of Populus.

High frame-rate resolution of cell division during Candida albicans filamentation

The commensal yeast, Candida albicans, is an opportunistic pathogen in humans and forms filaments called hyphae and pseudohyphae, in which cell division requires precise temporal and spatial control to produce mononuclear cell compartments. High-frame-rate live-cell imaging (1frame/min) revealed that nuclear division did not occur across the septal plane. We detected the presence of nucleolar fragments that may be extrachromosomal molecules carrying the ribosomal RNA genes. Cells occasionally maintained multiple nucleoli, suggesting either polyploidy, multiple nuclei and/or aneuploidy of ChrR., while the migration pattern of sister nuclei differed between unbranched and branched hyphae. The presented movie challenges and extends previous concepts of C. albicans cell division.

Spore behaviors reveal a category of mating-competent infertile heterokaryons in the offspring of the medicinal fungus Agaricus subrufescens.

Strain breeding is much less advanced in the edible and medicinal species Agaricus subrufescens than in Agaricus bisporus, the button mushroom. Both species have a unifactorial system of sexual incompatibility, a mating type locus tightly linked to a centromere, and basidia producing both homokaryotic (n) and heterokaryotic (n + n) spores. In A. bisporus, breeding is mainly based on direct selection among the heterokaryotic offspring and on hybridization between homokaryotic offspring. The parental heterozygosity is highly maintained in the heterokaryotic offspring due to suppression of recombination and preferential pairing in the spores of nuclei, each one per second meiotic divisions; such "non-sister nuclei" heterokaryons are fertile. In A. subrufescens, recent studies revealed that recombination is not suppressed and that nuclei from the same second meiotic division can also be paired in a spore that give rise to a "sister nuclei" heterokaryon in which the nuclei bear the same mating type allele. The objective of the present work was to investigate the potential function of the different categories of spores in A. subrufescens and their possible use in a genetic breeding program. Using eight co-dominant molecular markers, we found that half of the offspring of the A. subrufescens strain WC837 were heterokaryotic, one quarter of them being sister nuclei heterokaryons. These heterokaryons were infertile and behaved like homokaryons, being even able to cross between each other. In contrast, non-sister nuclei heterokaryons could fruit but inconsistently due to inbreeding depression. Potential roles of these two categories of heterokaryons in nature and consequences for strain breeding are discussed.

Metakaryotic stem cell nuclei use pangenomic dsRNA/DNA intermediates in genome replication and segregation

Bell shaped nuclei of metakaryotic cells double their DNA content during and after symmetric and asymmetric amitotic fissions rather than in the separate, pre-mitotic S-phase of eukaryotic cells. A parsimonious hypothesis was tested that the two anti-parallel strands of each chromatid DNA helix were first segregated as ssDNA-containing complexes into sister nuclei then copied to recreate a dsDNA genome. Metakaryotic nuclei that were treated during amitosis with RNase A and stained with acridine orange or fluorescent antibody to ssDNA revealed large amounts of ssDNA. Without RNase treatment metakaryotic nuclei in amitosis stained strongly with an antibody complex specific to dsRNA/DNA. Images of amitotic figures co-stained with dsRNA/DNA antibody and DAPI indicated that the entire interphase dsDNA genome (B-form helices) was transformed into two dsRNA/DNA genomes (A-form helices) that were segregated in the daughter cell nuclei then retransformed into dsDNA. As this process segregates DNA strands of opposite polarity in sister cells it hypothetically offers a sequential switching mechanism within the diverging stem cell lineages of development.

Nuclear Repulsion Enables Division Autonomy in a Single Cytoplasm

Current models of cell-cycle control, based on classic studies of fused cells, predict that nuclei in a shared cytoplasm respond to the same CDK activities to undergo synchronous cycling. However, synchrony is rarely observed in naturally occurring syncytia, such as the multinucleate fungus Ashbya gossypii. In this system, nuclei divide asynchronously, raising the question of how nuclear timing differences are maintained despite sharing a common milieu. We observe that neighboring nuclei are highly variable in division-cycle duration and that neighbors repel one another to space apart and demarcate their own cytoplasmic territories. The size of these territories increases as a nucleus approaches mitosis and can influence cycling rates. This nonrandom nuclear spacing is regulated by microtubules and is required for nuclear asynchrony, as nuclei that transiently come in very close proximity will partially synchronize. Sister nuclei born of the same mitosis are generally not persistent neighbors over their lifetimes yet remarkably retain similar division cycle times. This indicates that nuclei carry a memory of their birth state that influences their division timing and supports that nuclei subdivide a common cytosol into functionally distinct yet mobile compartments. These findings support that nuclei use cytoplasmic microtubules to establish "cells within cells." Individual compartments appear to push against one another to compete for cytoplasmic territory and insulate the division cycle. This provides a mechanism by which syncytial nuclei can spatially organize cell-cycle signaling and suggests size control can act in a system without physical boundaries.

Theoretical approaches for detection of sequence-specific Sister-Chromatid Exchanges in interphase cells

Two procedures for detection of Sister-Chromatid Exchanges (SCEs) in interphase cells within specific DNA sequence areas are delineated. Proliferating cells cultured with 5-bromodeoxyuridine (BrdU) for one cell cycle are subjected to cytokinesis-block to produce binucleated cells. Then, the BrdU-substituted DNA strands are removed as in the chromosome orientation-FISH (CO-FISH) procedure. In one case, the intact unsubstituted DNA strands may be hybridized with differentially labelled direct and reverse single-stranded DNA (ssDNA) probes from the target area. After formation of a SCE during the first cycle, specifically within the target area, three signals should be visualized in each nucleus of the binucleated cell. Two of them, of different color, must appear colocalized or adjacent simultaneously in both sister nuclei, corresponding to the SCE site, being accompanied by a signal from the other homologous locus showing a complementary color pattern in both sister nuclei. Other possibility is hybridizing two separated direct ssDNA probes flanking the target sequence area labelled with the same color and the two reverse complementary ssDNA probes labelled with other color. The presence of three signals with similar color and one of different color in each nucleus from the binucleated cell, following a complementary color pattern between both nuclei, would indicate the presence of a SCE within the DNA sequence area flanked by the DNA probes. The availability of complementary ssDNA probes specific for single locus should make the Interphase SCE-FISH a suitable procedure.

KIF4 Regulates Midzone Length during Cytokinesis

Midzones, also called central spindles, are an array of antiparallel microtubules that form during cytokinesis between the separated chromosomes. Midzones can be considered to be platforms that recruit specific proteins and orchestrate cytokinetic events, such as sister nuclei being kept apart, furrow ingression, and abscission. Despite this important role, many aspects of midzone biology remain unknown, including the dynamic organization of midzone microtubules. Investigating midzone microtubule dynamics has been difficult in part because their plus ends are interdigitated and buried in a dense matrix, making them difficult to observe. We employed monopolar cytokinesis to reveal that midzone plus ends appear to be nondynamic. We identified the chromokinesin KIF4 as a negative regulator of midzone plus-end dynamics whose activity controls midzone length but not stability. KIF4 is required to terminate midzone elongation in late anaphase. In the absence of KIF4, midzones elongate abnormally, and their overlap regions are unfocused. Electron-dense material and midbodies are both absent from the elongated midzones, and actin filaments from the furrow cortex are not disassembled after ingression. KIF4-mediated midzone length regulation appears to occur by terminating midzone elongation at a specific time during cytokinesis, making midzones and mitotic spindles differ in their dynamics and length-regulating mechanisms.

A conserved G1 regulatory circuit promotes asynchronous behavior of nuclei sharing a common cytoplasm

Synthesis and accumulation of conserved cell cycle regulators such as cyclins are thought to promote G1/S and G2/M transitions in most eukaryotes. 1 When cells at different stages of the cell cycle are fused to form heterokaryons, the shared complement of regulators in the cytoplasm induces the nuclei to become synchronized.2 However, multinucleate fungi often display asynchronous nuclear division cycles, even though the nuclei inhabit a shared cytoplasm. 3 Similarly, checkpoints can induce nuclear asynchrony in multinucleate cells by arresting only the nucleus that receives damage. 4-6 The cell biological basis for nuclear autonomy in a common cytoplasm is not known. Here we show that in the filamentous fungus Ashbya gossypii, sister nuclei born from one mitosis immediately lose synchrony in the subsequent G1 interval. A conserved G1 transcriptional regulatory circuit involving the Rb-analogue Whi5p promotes the asynchronous behavior yetWhi5 protein is uniformly distributed among nuclei throughout the cell cycle. The homologous Whi5p circuit in S. cerevisiae employs positive feedback to promote robust and coherent entry into the cell cycle. We propose that positive feedback in this same circuit generates timing variability in a multinucleate cell. These unexpected findings indicate that a regulatory program whose products (mRNA transcripts) are translated in a common cytoplasm can nevertheless promote variability in the individual behavior of sister nuclei.

XRCC1 deficiency influences the cytotoxicity and the genomic instability induced by Me-lex, a specific inducer of N3-methyladenine

Me-lex is a sequence-specific alkylating agent synthesized to preferentially (>90%) generate N3-methyladenine (3-mA) in the minor groove of double-strand DNA, in A-T rich regions. In this paper we investigated the effect of XRCC1 deficiency in the processing of 3-mA adducts generated by Me-lex, through the molecular analysis of the Hprt mutations and the evaluation of cytogenetic end points such as sister chromatid exchanges (SCEs), micronuclei (MN) and nucleus fragmentation. EM-C11 cells, deficient in XRCC1 activity, showed a 2.5-fold higher sensitivity to the toxicity of Me-lex compared to the DNA repair proficient parental CHO-9 cells, but were not hyper mutable. The spontaneous mutation spectrum at the Hprt locus generated in EM-C11 cells revealed a high percentage of genomic deletions. After Me-lex treatment, the percentage of genomic deletions did not increase, but a class of mutations which appeared to target regulatory regions of the gene significantly increased ( p = 0.0277), suggesting that non-coding Hprt genomic sequences represent a strong target for the rare mutations induced by Me-lex. The number of SCEs per chromosome increased 3-fold above background in 50 μМ Me-lex treated CHO-9 cells, while at higher Me-lex concentrations a sharp increase in the percentage of MN and fragmented nuclei was observed. In EM-C11 cells the background level of SCEs (0.939 ± 0.182) was approximately 10-fold higher than in CHO-9 (0.129 ± 0.027) and higher levels of multinucleated cells and MN were also found. In EM-C11, even low doses of Me-lex (25 μM) led to a significant increase in genomic damage. These results indicate that XRCC1 deficiency can lead to genomic instability even in the absence of an exogenous genotoxic insult and low levels of Me-lex-induced lesions, i.e., 3-mA and/or a BER intermediate, can exacerbate this instability.

Distribution of Microtubular Cytoskeletons and Organelle Nucleoids During Microsporogenesis in a 2n Pollen Producer of Hybrid Populus

AbstractThe distribution of microtubular cytoskeletons and organelle nucleoids during microsporogenesis in Populus simonii Carr. x P. nigra L. ‘Tongliao’ was studied by indirect immunofluorescence and 4’,6-diamidino-2- phenylindole (DAPI) staining. Our purpose was to reveal the relationship between changes in microtubule and chromosome behavior and cytokinesis and to explain the mechanism of 2n pollen production. We observed stable frequencies of 2n pollen production, ranging from 1.56% to 2.29%, between 2006 and 2008. Moreover, conjoined pollen grains were also observed. Meiotic abnormalities, including univalents, lagging chromosomes and micronuclei, were observed during microsporogenesis. Triads and dyads were also detected in meiotic products. Parallel spindles in metaphase II were unable to fuse, owing to the existence of an organelle band. Regularly, in telophase II, primary phragmoplasts were organized between sister nuclei, and secondary phragmoplasts formed between non-sister nuclei. Cell plates were initiated by centrifugal expansion of phragmoplasts and cytoplasmic infurrowing started at the junctions between the microsporocyte wall and the expanded phragmoplasts. However, a secondary phragmoplast was absent in some microsporocytes. These observations suggest that the occurrence of 2n pollen may result from the partial failure of cytokinesis caused by the absence of secondary phragmoplasts in this hybrid.

Expression of calcium-binding proteins in cerebellar- and inferior olivary-projecting neurons in the nucleus lentiformis mesencephali of pigeons

In the avian brain, the optokinetic response is controlled by two retinal-recipient nuclei: the nucleus of the basal optic root (nBOR) of the accessory optic system and the pretectal nucleus lentiformis mesencephali (LM). Although considered sister nuclei because of their similar response properties and function, there are both similarities and differences with respect to efferent projections and neurochemistry. Both nBOR and LM project to the cerebellum (Cb) directly as mossy fibers but also indirectly via the inferior olive (IO). In a previous report, we showed that the cerebellar- and inferior olivary-projecting neurons in nBOR of pigeons differentially express the calcium-binding proteins calretinin (CR) and parvalbumin (PV). Both CR and PV are expressed in the somata of LM neurons, although the latter is not as prevalent, and whether expression of CR and PV reflects cerebellar and IO projections is not known. In this report, by combining retrograde neuronal tracing from the Cb and IO with fluorescent immunohistochemistry, we examined the expression of these calcium-binding proteins in the pigeon LM. Half (52%) of the cerebellar-projecting neurons were CR+ve, but only 15% were PV+ve. Almost all (>95%) these PV+ve cells also expressed CR. In contrast, few of the IO-projecting neurons expressed CR or PV (<or=5%). This is strikingly similar to what we observed in nBOR and reveals that calcium-binding protein expression is concordant with projection patterns in two nuclei that share similar functions.

γ‐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.

Interphase chromosome arrangement in Arabidopsis thaliana is similar in differentiated and meristematic tissues and shows a transient mirror symmetry after nuclear division.

Whole-mount fluorescence in situ hybridization (FISH) was applied to Arabidopsis thaliana seedlings to determine the three-dimensional (3D) interphase chromosome territory (CT) arrangement and heterochromatin location within the positional context of entire tissues or in particular cell types of morphologically well-preserved seedlings. The interphase chromosome arrangement was found to be similar between all inspected meristematic and differentiated root and shoot cells, indicating a lack of a gross reorganization during differentiation. The predominantly random CT arrangement (except for a more frequent association of the homologous chromosomes bearing a nucleolus organizer) and the peripheric location of centromeric heterochromatin were as previously observed for flow-sorted nuclei, but centromeres tend to fuse more often in nonendoreduplicating cells and NORs in differentiated cells. After mitosis, sister nuclei revealed a symmetric arrangement of homologous CTs waning with the progress of the cell cycle or in the course of differentiation. Thus, the interphase chromosome arrangement in A. thaliana nuclei seems to be constrained mainly by morphological features such as nuclear shape, presence or absence of a nucleolus organizer on chromosomes, nucleolar volume, and/or endopolyploidy level.

Mitotic Microtubule Development and Histone H3 Phosphorylation in the Holocentric Chromosomes of Rhynchospora Tenuis (Cyperaceae)

In the present work we report the phosphorylation pattern of histone H3 and the development of microtubular structures using immunostaining techniques, in mitosis of Rhynchospora tenuis (2n = 4), a Cyperaceae with holocentric chromosomes. The main features of the holocentric chromosomes of R. tenuis coincide with those of other species namely: the absence of primary constriction in prometaphase and metaphase, and the parallel separation of sister chromatids at anaphase. Additionaly, we observed a highly conserved chromosome positioning at anaphase and early telophase sister nuclei. Four microtubule arrangements were distinguished during the root tip cell cycle. Interphase cells showed a cortical microtubule arrangement that progressively forms the characteristic pre-prophase band. At prometaphase the microtubules were homogeneously distributed around the nuclear envelope. Metaphase cells displayed the spindle arrangement with kinetochore microtubules attached throughout the entire chromosome extension. At anaphase kinetochoric microtubules become progressively shorter, whereas bundles of interzonal microtubules became increasingly broader and denser. At late telophase the microtubules were observed equatorially extended beyond the sister nuclei and reaching the cell wall. Immunolabelling with an antibody against phosphorylated histone H3 revealed the four chromosomes labelled throughout their entire extension at metaphase and anaphase. Apparently, the holocentric chromosomes of R. tenuis function as an extended centromeric region both in terms of cohesion and H3 phosphorylation.

γ-Tubulin and microtubule organization during microsporogenesis in Ginkgo biloba

This is the first report on gamma-tubulin and microtubule arrays during microsporogenesis in a gymnosperm. Meiosis in Ginkgo biloba is polyplastidic, as is typical of the spermatophyte clade, and microtubule arrays are organized at various sites during meiosis and cytokinesis. In early prophase, a cluster of gamma-tubulin globules occurs in the central cytoplasm adjacent to the off-center nucleus. These globules diminish in size and spread over the surface of the nucleus. A system of microtubules focused on the gamma-tubulin forms a reticulate pattern in the cytoplasm. As the nucleus migrates to the center of the microsporocyte, gamma-tubulin becomes concentrated at several sites adjacent to the nuclear envelope. Microtubules organized at these foci of gamma-tubulin give rise to a multipolar prophase spindle. By metaphase I, the spindle has matured into a distinctly bipolar structure with pointed poles. In both first and second meiosis, gamma-tubulin becomes distributed throughout the metaphase spindles, but becomes distinctly polar again in anaphase. In telophase I, gamma-tubulin moves from polar regions to the proximal surface of chromosome groups/nuclei where interzonal microtubules are organized. No cell wall is deposited and the interzonal microtubules embrace a plate of organelles between the two nuclear cytoplasmic domains (NCDs) of the dyad. Following second meiosis, phragmoplasts that form between sister and non-sister nuclei fuse to form a complex six-sided structure that directs simultaneous cytokinesis. Gamma-tubulin becomes associated with nuclei after both meiotic divisions and is especially conspicuous in the distal hemisphere of each young microspore where an unusual encircling system of cortical microtubules develops.

?-Tubulin localization changes from discrete polar organizers to anastral spindles and phragmoplasts in mitosis of Marchantia polymorpha L.

Unlike the astral mitotic spindle that is organized at discrete centriolar centrosomes, the spindle of land plants is typically anastral and its origin has remained obscure. Gamma tubulin (gamma-tubulin), an important component of the centrosome, has been demonstrated at microtubule-nucleating sites in plant cells. Mitotic spindles of certain hepatics are initiated at distinct acentriolar polar organizers (POs) that appear de novo at the onset of mitosis. Data on the relationship of gamma-tubulin to POs and to microtubule arrays throughout the cell cycle were collected from rapidly dividing cells of Marchantia polymorpha (Bryophyta) that were triple-stained for gamma-tubulin, microtubules, and nuclei. POs at opposite ends of the elongated nucleus in early prophase stain brightly for gamma-tubulin and astral microtubules emanating from them initiate the spindle. As the spindle develops, however, the gamma-tubulin becomes dispersed from the highly concentrated spherical form of the POs to more diffusely organized cups at tips of the fusiform nucleus. By the end of prophase, all astral microtubules have disappeared and the gamma-tubulin is located in several minipoles along the now broad polar regions of the spindle. At metaphase, gamma-tubulin extends into the spindle itself. By telophase, the gamma-tubulin has migrated from distal to proximal surfaces of the sister nuclei and extends into the phragmoplast. Upon completion of cytokinesis, gamma-tubulin appears diminished and surrounds the nuclear envelopes. These data show that gamma-tubulin is only briefly concentrated in the PO, migrates in a cell-cycle-specific manner, and is consistently present at all putative sites of microtubule nucleation.

Abnormal Nucleolar Cycle in Microsporogenesis of Brachiaria decumbens(Gramineae).

Cytogenetic studies carried out on the tetraploid (2n=4x=36) accession of Brachiaria decumbens (BRA007722) revealed an unusual pattern of the nucleolar cycle during microsporogenesis. Nucleolus behavior was normal during prophase and disappeared at the end of diakinesis. After dissolution, it was fractioned into multiple micronucleoli of different sizes at metaphase which persisted during early anaphase. Some entered in a fusion process in late anaphase: they reduced their number and increased in size. Since fusion process continued throughout the cycle, in late telophase all were rejoined into a unique nucleolus in the sister nuclei. They persisted in this condition until the end of prophase II and then disappeared once more. Such nucleolar behavior was also reported in the second division, while at the end of the meiotic process tetrads had 4 microspores with a normal nucleolus. Pollen viability was not affected by this anomalous nucleolar cycle behavior. A mutation affecting some steps of the nucleolar cycle has been suggested.