Synchronous Nuclear Division Research Articles

Kinesin Motors in the Filamentous Basidiomycetes in Light of the Schizophyllum commune Genome.

Kinesins are essential motor molecules of the microtubule cytoskeleton. All eukaryotic organisms have several genes encoding kinesin proteins, which are necessary for various cell biological functions. During the vegetative growth of filamentous basidiomycetes, the apical cells of long leading hyphae have microtubules extending toward the tip. The reciprocal exchange and migration of nuclei between haploid hyphae at mating is also dependent on cytoskeletal structures, including the microtubules and their motor molecules. In dikaryotic hyphae, resulting from a compatible mating, the nuclear location, synchronous nuclear division, and extensive nuclear separation at telophase are microtubule-dependent processes that involve unidentified molecular motors. The genome of Schizophyllum commune is analyzed as an example of a species belonging to the Basidiomycota subclass, Agaricomycetes. In this subclass, the investigation of cell biology is restricted to a few species. Instead, the whole genome sequences of several species are now available. The analyses of the mating type genes and the genes necessary for fruiting body formation or wood degrading enzymes in several genomes of Agaricomycetes have shown that they are controlled by comparable systems. This supports the idea that the genes regulating the cell biological process in a model fungus, such as the genes encoding kinesin motor molecules, are also functional in other filamentous Agaricomycetes.

Genetic transformation of Spizellomyces punctatus, a resource for studying chytrid biology and evolutionary cell biology.

Chytrids are early-diverging fungi that share features with animals that have been lost in most other fungi. They hold promise as a system to study fungal and animal evolution, but we lack genetic tools for hypothesis testing. Here, we generated transgenic lines of the chytrid Spizellomyces punctatus, and used fluorescence microscopy to explore chytrid cell biology and development during its life cycle. We show that the chytrid undergoes multiple rounds of synchronous nuclear division, followed by cellularization, to create and release many daughter 'zoospores'. The zoospores, akin to animal cells, crawl using actin-mediated cell migration. After forming a cell wall, polymerized actin reorganizes into fungal-like cortical patches and cables that extend into hyphal-like structures. Actin perinuclear shells form each cell cycle and polygonal territories emerge during cellularization. This work makes Spizellomyces a genetically tractable model for comparative cell biology and understanding the evolution of fungi and early eukaryotes.

Rhinosporidium seeberi Nuclear Cycle Activities Using Confocal Microscopy.

Rhinosporidium seeberi is an uncultivated Ichthyosporean infecting animals, including humans. Recent studies suggested R. seeberi undergoes synchronized nuclear division without cytokinesis. We used confocal microscopy to investigate R. seeberi nuclear division cycles in formalin-fixed tissues stained with DAPI and phalloidin. We report that R. seeberi nuclei in juvenile and intermediary sporangia synchronously divided without cytokinesis. Intermediary sporangia display numerous 3-4 μm nuclei at different mitotic stages as well as a thick inner layer with strong affinity for phalloidin. Mature sporangia showed numerous 5-12 μm cell-walled endospores, each containing a 2-4 μm in diameter nucleus. Phalloidin did not bind to the inner layers of mature sporangia or endospores. The development of a "germinative zone" in the inner layer of mature sporangia containing hundreds of nuclei was also confirmed. This study establishes that during the R. seeberi life cycle synchronous nuclear divisions without cytokinesis takes place, resulting in the formation of thousands of nuclei. Cytokinesis, on the other hand, is a 1-time event and occurs in the latest stages of intermediate sporangia, after the formation of thousands of nuclei and just before mature sporangia development.

Crumbs and Xpd in mitosis.

Crumbs and Xpd in mitosis.

Maize early endosperm growth and development: from fertilization through cell type differentiation.

• Given the worldwide economic importance of maize endosperm, it is surprising that its development is not the most comprehensively studied of the cereals. We present detailed morphometric and cytological descriptions of endosperm development in the maize inbred line B73, for which the genome has been sequenced, and compare its growth with four diverse Nested Association Mapping (NAM) founder lines.• The first 12 d of B73 endosperm development were described using semithin sections of plastic-embedded kernels and confocal microscopy. Longitudinal sections were used to compare endosperm length, thickness, and area.• Morphometric comparison between Arizona- and Michigan-grown B73 showed a common pattern. Early endosperm development was divided into four stages: coenocytic, cellularization through alveolation, cellularization through partitioning, and differentiation. We observed tightly synchronous nuclear divisions in the coenocyte, elucidated that the onset of cellularization was coincident with endosperm size, and identified a previously undefined cell type (basal intermediate zone, BIZ). NAM founders with small mature kernels had larger endosperms (0-6 d after pollination) than lines with large mature kernels.• Our B73-specific model of early endosperm growth links developmental events to relative endosperm size, while accounting for diverse growing conditions. Maize endosperm cellularizes through alveolation, then random partitioning of the central vacuole. This unique cellularization feature of maize contrasts with the smaller endosperms of Arabidopsis, barley, and rice that strictly cellularize through repeated alveolation. NAM analysis revealed differences in endosperm size during early development, which potentially relates to differences in timing of cellularization across diverse lines of maize.

MYB64 and MYB119 Are Required for Cellularization and Differentiation during Female Gametogenesis in Arabidopsis thaliana

In angiosperms, the egg cell forms within the multicellular, haploid female gametophyte. Female gametophyte and egg cell development occurs through a unique process in which a haploid spore initially undergoes several rounds of synchronous nuclear divisions without cytokinesis, resulting in a single cell containing multiple nuclei. The developing gametophyte then forms cell walls (cellularization) and the resulting cells differentiate to generate the egg cell and several accessory cells. The switch between free nuclear divisions and cellularization-differentiation occurs during developmental stage FG5 in Arabidopsis, and we refer to it as the FG5 transition. The molecular regulators that initiate the FG5 transition during female gametophyte development are unknown. In this study, we show using mutant analysis that two closely related MYB transcription factors, MYB64 and MYB119, act redundantly to promote this transition. MYB64 and MYB119 are expressed during the FG5 transition, and most myb64 myb119 double mutant gametophytes fail to initiate the FG5 transition, resulting in uncellularized gametophytes with supernumerary nuclei. Analysis of cell-specific markers in myb64 myb119 gametophytes that do cellularize suggests that gametophytic polarity and differentiation are also affected. We also show using multiple-mutant analysis that MYB119 expression is regulated by the histidine kinase CKI1, the primary activator of two-component signaling (TCS) during female gametophyte development. Our data establish a molecular pathway regulating the FG5 transition and implicates CKI1-dependent TCS in the promotion of cellularization, differentiation, and gamete specification during female gametogenesis.

Number of Nuclear Divisions in the Drosophila Blastoderm Controlled by Onset of Zygotic Transcription

The cell number of the early Drosophila embryo is determined by exactly 13 rounds of synchronous nuclear divisions, allowing cellularization and formation of the embryonic epithelium. The pause in G2 in cycle 14 is controlled by multiple pathways, such as activation of DNA repair checkpoint, progression through S phase, and inhibitory phosphorylation of Cdk1, involving the genes grapes, mei41, and wee1. In addition, degradation of maternal RNAs and zygotic gene expression are involved. The zinc finger Vielfältig (Vfl) controls expression of many early zygotic genes, including the mitotic inhibitor frühstart. The functional relationship of these pathways and the mechanism for triggering the cell-cycle pause have remained unclear. Here, we show that a novel single-nucleotide mutation in the 3' UTR of the RNPII215 gene leads to a reduced number of nuclear divisions that is accompanied by premature transcription of early zygotic genes and cellularization. The reduced number of nuclear divisions in mutant embryos depends on the transcription factor Vfl and on zygotic gene expression, but not on grapes, the mitotic inhibitor Frühstart, and the nucleocytoplasmic ratio. We propose that activation of zygotic gene expression is the trigger that determines the timely and concerted cell-cycle pause and cellularization.

DFMRP and Caprin, translational regulators of synaptic plasticity, control the cell cycle at the Drosophila mid-blastula transition

The molecular mechanisms driving the conserved metazoan developmental shift referred to as the mid-blastula transition (MBT) remain mysterious. Typically, cleavage divisions give way to longer asynchronous cell cycles with the acquisition of a gap phase. In Drosophila, rapid synchronous nuclear divisions must pause at the MBT to allow the formation of a cellular blastoderm through a special form of cytokinesis termed cellularization. Drosophila Fragile X mental retardation protein (dFMRP; FMR1), a transcript-specific translational regulator, is required for cellularization. The role of FMRP has been most extensively studied in the nervous system because the loss of FMRP activity in neurons causes the misexpression of specific mRNAs required for synaptic plasticity, resulting in mental retardation and autism in humans. Here, we show that in the early embryo dFMRP associates specifically with Caprin, another transcript-specific translational regulator implicated in synaptic plasticity, and with eIF4G, a key regulator of translational initiation. dFMRP and Caprin collaborate to control the cell cycle at the MBT by directly mediating the normal repression of maternal Cyclin B mRNA and the activation of zygotic frühstart mRNA. These findings identify two new targets of dFMRP regulation and implicate conserved translational regulatory mechanisms in processes as diverse as learning, memory and early embryonic development.

Nuclear behaviour of pea leaf protoplasts

Detailed cytological studies of protoplasts isolated from mesophyll tissue of pea leaves were made. The diploid nuclei of mononucleate protoplasts showed normal mitotic division. Binucleate protoplasts showed synchronous nuclear division of two nuclei leading to multinucleate formation. Some of the nuclei of binucleate protoplasts fused prior to nuclear division. The fused nuclei had chromatin connections. Abnormalities in nuclear division were noted in some cases. Protoplasts isolated from older leaves showed a lower frequency of mitotic division than those from younger leaves.

Molecular and ultrastructural characterization of Andreanna caspii n. gen., n. sp. (Microsporida: Amblyosporidae), a parasite of Ochlerotatus caspius (Diptera: Culicidae)

A new genus and species of microsporidia, Andreanna caspii n. gen., n. sp. is described from the mosquito, Ochlerotatus caspius (Pallas) based on ultrastructural morphology, developmental characteristics, and comparative sequence analyses of the small subunit (SSU) ribosomal DNA (rDNA). Parasite development is confined to fat body tissue and infected larvae appear swollen with dull white masses within the thorax and abdomen. Meronts have diplokaryotic nuclei and are delineated by a simple plasmalemma contiguous with the host cell cytoplasm. Merogony occurs by synchronous binary division followed by cytokinesis. Diplokaryotic sporonts undergo meiosis and synchronous nuclear division forming sporogonial plasmodia with two, four and eight nuclei enclosed within a persistent sporophorous vesicle. Cytokinesis of sporogonial plasmodia results in the formation of eight uninucleate spores. The episporontal space of early sporonts is filled with a homogeneous accumulation of electron dense granular inclusions and ovoid vesicles of various dimensions, transforming into an interwoven matrix during the initial phase of sporogenesis. Spores are oval, uninucleate and measure 4.8 ± 0.3 × 3.1 ± 0.4 μm (fixed). The spore wall is 260 μm thick with an irregular exospore consisting of two layers (150–170 μm) and a thinner endospore (90–100 μm). The anchoring disk is well developed and is contiguous with a lamellar polaroplast that occupies the anterior third of the spore and possess more narrow lamellae on the posterior end. The polar filament is gradually tapered and arranged in a single row consisting of six coils ranging from 180 to 150 μm in diameter. The posterior vacuole (posterosome) is moderately sized and filled with a matrix of moderate electron density. Phylogenetic analysis of SSU rDNA from A. caspii and 30 other species of microsporidia including 11 genera parasitic in mosquitoes using maximum parsimony, neighbor joining and maximum likelihood methods showed A. caspii to be a sister group to the clade containing all of the Amblyospora species, including Culicospora, Edhazardia and Intrapredatorus, as well as Culicosporella and Hyalinocysta thus providing strong support for establishment of Andreanna as a separate genus.

Male or Female? The Answer Depends on When You Ask

Male or Female? The Answer Depends on When You Ask

Root-knot nematodes manipulate plant cell functions during a compatible interaction

Sedentary endoparasitic nematodes are root parasites that interact with their hosts in a remarkable way. These obligate biotrophic pathogens establish an intimate relationship with their host plants, inducing the redifferentiation of root cells into specialized feeding cells. The successful establishment of feeding cells is essential for nematode development. Root-knot nematodes, of the genus Meloidogyne, have evolved strategies enabling them to induce feeding cell formation in thousands of plant species, probably by manipulating fundamental elements of plant cell development. Feeding cells enlarge and are converted into multinucleate giant cells through synchronous nuclear divisions without cell division. Fully differentiated giant cells may contain more than a hundred polyploid nuclei that may have undergone extensive endoreduplication. Hyperplasia and hypertrophy of the surrounding cells lead to the formation of the typical root gall. Giant cell formation requires extensive changes to gene expression. The induction of feeding cells remains poorly understood, but it is thought that effectors secreted by the nematode play a key role in parasitism, with potential direct effects on recipient host cells. In this review, we focus on the most recent investigations of the molecular basis of the plant-root-knot nematode interaction. Recently, microarray technology has been used to study the plant response to Meloidogyne spp. infection. Such a genome-wide expression profiling provides a global view of transcriptional changes, especially for genes involved in cell wall, transport processes and plant defense responses during giant cell formation. The identification of nematode-responsive plant genes constitutes a major step toward understanding how root-knot nematodes dramatically alter root development to induce and maintain giant cells. The characterization of nematode secretions as parasitism effectors and the development of RNAi technology should improve our understanding of the molecular events and regulatory mechanisms involved in plant parasitism. Finally, Meloidogyne genome sequences should provide further insight into plant-root-knot nematode interactions.

Dividing to their own beat

Multiple fungal nuclei within a common cytoplasm divide independently, based on findings on page 347. Gladfelter et al. suggest that the Ashbya gossypii filamentous fungus evolved a new means to control cyclin activity that might make asynchronous nuclear division possible. Figure 1 Cyclin (red) is present at every nucleus (blue) regardless of cell cycle stage in A. gossypii. Cyclin activity drives the mitotic cycle in eukaryotes. To prevent untimely mitosis, most cells degrade mitotic cyclins at telophase and show an oscillating pattern of cyclin protein levels. As cyclins can enter and exit nuclei and diffuse throughout the cytoplasm, multinucleated cells normally have synchronous nuclear divisions. But the new report shows that A. gossypii nuclei divide independently of their neighbors. Nuclei at various stages of the cell cycle were found within single cells. Artificially synchronized nuclei did not remain in synch for long, suggesting that each nucleus divides on its own time frame. Such autonomy might allow the cell to grow in some spots (perhaps where nutrients are richest) without burdening the other nuclei. This unusual nuclear independence might be possible, according to the authors, because A. gossypii does not rely on oscillations in cyclin levels to control mitosis. G1 and mitotic cyclins were present at every nucleus independent of its division status. And the loss of proteosome-mediated cyclin degradation did not interfere with cell cycle progression, as it does in other cells. The group has not proven the mechanistic basis of asynchrony, but they propose that cyclin activity, rather than abundance, drives the A. gossypii cell cycle. Cyclin activity might be controlled by Sic1, which in budding yeast is a CDK–cyclin inhibitor. Budding yeast cells normally degrade Sic1 to escape G1, but the filamentous fungus instead seems to regulate its localization. Before division, Sic1 was found throughout the nucleoplasm. At times when cyclin activity should be highest, Sic1 was concentrated at spindle pole bodies, perhaps thereby turning it off. Most cells have intricate and redundant mechanisms that assure precise cell cycle progression. If indeed cyclin oscillation has been lost as one such means, A. gossypii might have traded nuclear accuracy for autonomy. Its abundant nuclei, however, may protect the fungus from any resulting sloppiness.

Induced synchrony in Cryptococcus neoformans after release from G2-arrest.

Cryptococcus neoformans was grown first to OD 4 under moderate aeration, then diluted 2.5 times with fresh medium, and grown under limited aeration for 5 h. Oxygen concentration decreased from 5-6 mg l(-1) to 1.5 mg l(-1) 1 h after the shift to limited aeration, and remained at a similar level thereafter. In all the eleven strains examined the shift caused unbudded G(2)-arrest in more than half of the cells. In three strains more than 80% of the cells were arrested in unbudded G(2), and, therefore they were selected for synchrony experiments. After being shifted to extensive aeration again, the cells resumed growth by synchronous budding, followed by synchronous nuclear division. This method has turned out to be a good tool to prepare synchronized culture in C. neoformans, especially when a large amount of synchronized cells is needed. This is worthy of attention, since synchronous cultures after release from G(2)-arrest have not been reported yet in any yeast species.

Studying nuclear organization in embryos using antibody tools.

One of the major foci in cell biology is to understand the process of nuclear division. In each cell cycle, the chromosomes must be faithfully replicated and the complex nuclear structure has to be duplicated and reorganized (, , , ). Our understanding of the cell cycle and mitosis has increased dramatically in the last several years, as a result of cross-disciplinary approaches combining molecular, cell biological, and genetic techniques (reviewed in refs. , , , , ). An organism offering a particularly advantageous model system for such studies of mitosis is the early embryo of Drosophila melanogaster. The cytoskeleton and mitotic spindle are large and easily visualized, thus facilitating structural analysis. The embryo undergoes 13 rapid and nearly synchronous nuclear divisions giving rise to about 5000 nuclei before cell boundaries form after 3 h of development (, , ). This syncytial organization of nuclei affords excellent accessibility for experimental perturbations (e.g., using antibodies [, , , , ] or pharmacological tools [,, , ]).

The High Mobility Group I/Y Protein Is Hypophosphorylated in Endoreduplicating Maize Endosperm Cells and Is Involved in Alleviating Histone H1-mediated Transcriptional Repression

The High Mobility Group I/Y Protein Is Hypophosphorylated in Endoreduplicating Maize Endosperm Cells and Is Involved in Alleviating Histone H1-mediated Transcriptional Repression

The centrosome in early drosophila embryogenesis

Publisher Summary The early Drosophila embryo is a useful system for functional studies of the centrosome. This organelle plays essential and diverse roles in guiding nuclear movement and cytoskeletal reorganization during the initial 3 hours of development. Drosophila embryogenesis begins with 13 synchronous nuclear divisions that occur without accompanying cytokinesis. The divisions immediately following fertilization proceed as an expanding ball in the interior of the embryo. The centrosome-based activities coordinate nuclear and cytoskeletal dynamics during these divisions. Because Drosophila is amenable to molecular genetic, biochemical, and a variety of cellular approaches, the mechanisms underlying many activities of the centrosome in the early embryo are addressed. This chapter discusses the mechanisms that control centrosome movement and migration, the role of the centrosome in coordinating nuclear and cytoskeletal events of the cell cycle, and how the centrosome directs the dramatic microfilament and plasma membrane rearrangements at the cortex. The mechanism of centrosome duplication and its regulation and the issues of ultrastructure, composition, duplication, separation, fertilization, mitotic, and precortial division are described and provide insights into centrosome function.

Changes in Cell Number and Cell Division Activity during Endosperm Development in Allohexaploid Wheat, Triticum aestivum L.

Nuclear or cell number, and the mitotic index, were recorded in endosperms of Triticum aestivum cv. Mardler to test if a particular stage of endosperm development was critical in determining the final grain weight. The basal four florets of emasculated spikelets (controls), and the third and fourth florets of spikelets where the two basal ovaries were removed (ovary removed), were sampled at various times up to 360 h after hand-pollination. The coenocytic phase in endosperms ended about 84 h after pollination regardless of both grain position and the treatment. The onset of the cellular stage was characterized by the final large fluctuations in the mitotic index reflecting the culmination of the synchronous nuclear division of the coenocytic stage. Thereafter, the mitotic index fluctuated with smaller amplitudes and, by 216 h after pollination, was <1%. Neither floret position in the spikelet nor the treatment affected the pattern of alteration to the mitotic index. However, ovary removal from first and second florets resulted in significantly heavier grains and higher endosperm cell number in the 3rd and 4th florets compared with the controls. In all florets, mean endosperm cell number peaked at 280 h but decreased by 360 h after pollination. At this time, the mean cell numbers in endosperms of the 3rd and 4th florets of ovary-removed spikelets were significantly higher than in the corresponding endosperms in the controls. Thus, a key contributory factor in determining the final endosperm cell number may be the number of cells which are lost during the late period of the cellular stage of endosperm development.

Retarded nuclear migration in Drosophila embryos with aberrant F-actin reorganization caused by maternal mutations and by cytochalasin treatment

Three X-linked mutations of Drosophila melanogaster, gs(1)N26, gs(1)N441 and paralog, had a common maternal-effect phenotype. Mutant embryos show reduced egg contraction that normally occurs at an early cleavage stage in wild-type embryos. In addition, the mutants exhibited retarded nuclear migration while synchronous nuclear divisions were unaffected. The retarded migration causes nuclei to remain in the anterior part of the embryo retaining their spherical distribution even in a late cleavage stage. This consequently results in an extreme delay in nuclear arrival in the posterior periplasm. A mutant phenocopy was induced in wild-type embryos that were treated with cytochalasin B or D at a very early cleavage stage. Remarkable differences were noticed in the organization of cortical F-actin between the mutants and the wild type throughout the cleavage stage: obvious F-actin aggregates were dispersed in the cortex of mutant embryos, in contrast to the wild type where the cortical F-actin layer was smooth and underlying F-actin aggregates were smaller than those in the mutants; the transition of the distribution pattern of F-actin in the yolk mass, from the centralized to the fragmented type, occurred later in the mutants than in wild type. The results suggest that these mutations affect the mechanism underlying establishment and transition of F-actin organization required for normal egg contraction and nuclear migration in the cleavage embryos.

Formation of multinucleated cells in a Hodgkin's-disease-derived cell line.

The proliferative potential of multinucleated Reed-Sternberg-like cells and the process of multinuclear formation were studied on the Hodgkin's-disease-derived cell line HDLM-2. No difference in surface antigen expression was found between mono- and multinucleated cells as determined by immunolabelling with characteristic markers. After sorting and reculture of purified mononucleated cells, polykaryons emerged subsequently in these cultures, indicating that mononucleated cells give rise to multinucleated variants. The morphological observation of mitotic figures and immunostaining with the cell cycle indicators Ki-67 and BrdU provided evidence of DNA synthesis and nuclear division in multinucleated cells. The presence of mitotic figures demonstrated that multinucleated cells are able to undergo synchronous nuclear division. However, while polykaryons were clearly mitotically active and capable of DNA synthesis, the absence of telophases and the failure of active replication suggest a disturbed cytokinesis. Co-cultivation of BrdU-labelled and unlabelled populations did not lead to hybrid polykaryons with negative and positive nuclei. Therefore, multinucleated giant cell formation of HDLM-2 cells appears to involve nuclear endomitosis without cell division rather than cell fusion.