Nuclear movement in filamentous fungi

One of the most striking features of eukaryotic cells is the organization of specific functions into organelles such as nuclei, mitochondria, chloroplasts, the endoplasmic reticulum, vacuoles, peroxisomes or the Golgi apparatus. These membrane-surrounded compartments are not synthesized de novo but are bequeathed to daughter cells during cell division. The successful transmittance of organelles to daughter cells requires the growth, division and separation of these compartments and involves a complex machinery consisting of cytoskeletal components, mechanochemical motor proteins and regulatory factors. Organelles such as nuclei, which are present in most cells in a single copy, must be precisely positioned prior to cytokinesis. In many eukaryotic cells the cleavage plane for cell division is defined by the location of the nucleus prior to mitosis. Nuclear positioning is thus absolutely crucial in the unequal cell divisions that occur during development and embryogenesis. Yeast and filamentous fungi are excellent organisms for the molecular analysis of nuclear migration because of their amenability to a broad variety of powerful analytical methods unavailable in higher eukaryotes. Filamentous fungi are especially attractive models because the longitudinally elongated cells grow by apical tip extension and the organelles are often required to migrate long distances. This review describes nuclear migration in filamentous fungi, the approaches used for and the results of its molecular analysis and the projection of the results to other organisms.     

The ultrastructure of nuclear division in a suctorian,Tokophrya infusionum

Summary Ultrastructural changes in the micro- and macronucleus throughout division were followed in synchronized cultures of the suctorian, Tokophrya infusionum. After an initial swelling, the micronucleus elongates enormously; microtubules within the micronucleus proliferate and lengthen as the micronucleus elongates. Changes in the macronucleus become visible only after micronuclear division is well underway. The chromatin bodies fuse into long chromatin strands, and the large bundles of microtubules present in the resting macronucleus break up into small groups which parallel the chromatin strands. Colchicine, which prevents reproduction in Tokophrya, seems to block division at a very early stage. The macronucleus appears the same as the resting nucleus of untreated organisms, with numerous microtubules and distinct chromatin bodies. The chromatin in the micronucleus aggregates into large clumps, however, and proliferation of microtubules does not occur.Supported by a Graduate Fellowship at The Rockefeller University.Supported by Grant A1-01407-12 USPHS and Grant A1-08989-01 USPHS.     

Status of nuclear division in arbuscular mycorrhizal fungi during in vitro development

Summary The number of nuclei in spores and along hyphae of an arbuscular mycorrhizal fungiGigaspora margarita was measured in digital images of fluorescence arising from mithramycin stained cultures. Typical dormant spores (250 m diameter) contained 2000 nuclei. Eight hundred nuclei were mobilized during the first 3 days of germination. The number of nuclei in the spores nearly returned to the initial number after 22 days of hyphal growth. The average relative DNA content in the nuclei of dormant spores and in the nuclei of spores incubated for 22 days was comparable, as judged from fluorescence intensity. Hyphal elongation occurred with 460 nuclei per cm under a special set of in vitro conditions that promote extensive hyphal growth of arbuscular mycorrhizal fungi. We found an average total of 26000 hyphal nuclei per germinating spore after 22 days. The specific DNA polymerase inhibitor aphidicolin did not inhibit spore germination but it rapidly reduced the rate of hyphal growth and arrested growth after 4 days. No nuclei were produced de novo during this time. These results demonstrate thatG. margarita replicates nuclear DNA and undergoes nuclear division when grown in vitro even in the absence of a plant host.     

Selection of the germinal micronucleus in Paramecium caudatum: nuclear division and nuclear death

Each cell of Paramecium caudatum has a germinal micronucleus. When a bi-micronucleate state was created artificially by micronuclear transplantation, both micronuclei divided for at least 2 cell cycles after nuclear transplantation. However, this bi-micronucleate state was unstable and reduced to a uni-micronucleate state after several fissions. Although the number of micronuclei was usually 1 during the vegetative phase, 4 presumptive micronuclei differentiated after conjugation. At the first post-conjugational fission, only 1 of the 4 micronuclei divided, indicating that there is tight regulation of micronuclear number in exconjugants. Micronuclei that did not divide at the first post-conjugational fission may persist through the first and second post-conjugational cell cycles. The decision to divide appears to be separate from the decision to degenerate, as evidenced by division of a remaining micronucleus upon removal of the dividing micronucleus at the first division. Degeneration of micronuclei in exconjugants differs from that of haploid nuclei after meiosis. Nutritional state affected micronuclear degeneration. Under well-fed conditions, the micronuclei destined to degenerate lost the ability to divide earlier than after starvation treatment, suggesting that micronuclear degeneration is an "apoptotic" phenomenon, probably under the control of the new macronuclei (macronuclear anlagen).     

Macronuclear division and cytokinesis in Tetrahymena

Tetrahymena contains a micronucleus and a macronucleus. The micronucleus divides with typical mitosis, while the macronucleus divides amitotically. Although the mechanism responsible for macronuclear division was previously unknown, we clarified the organization of microtubules during macronuclear division. The macronuclear microtubules dynamically changed their distribution in an organized way throughout the macronuclear division. The macronuclear microtubules and the cytoplasmic microtubules cooperatively carried out the macronuclear division. When the micronuclear division was finished, p85 appeared at the presumptive division plane prior to the cytokinesis. The p85 directly interacted with calmodulin in a Ca(2+)-dependent manner, and p85 and CaM colocalized to the division furrow during cytokinesis. Moreover, the Ca(2+)/CaM inhibitor, W7, inhibited the direct interaction between p85 and CaM, the localization of both proteins to the division plane, and the formation of the division furrow. Thus, Ca(2+)/CaM and p85 have important roles in initiation and progression of cytokinesis in Tetrahymena.     

Vanadate affects nuclear division and induces aberrantly-shaped cells during subsequent cytokinesis in Tetrahymena

Sodium orthovanadate at 0.1-5.0 mM affected cell proliferation of Tetrahymena in a dose-dependent manner. At 1 h the cell increment was 76-12% of the control (100%), but after lag periods in 1-5 mM the growth rate remained at 76% of control in 0.1 mM vanadate and at 64-61% of control in 0.2-5.0 mM vanadate. Endocytosis was affected in both a time- and dose-dependent manner; an increasing number of cells did not form vacuoles. Cell motility increased initially in 0.1 mM vanadate but decreased later as it did in 0.5-2.0 mM vanadate where the proportion of immobile cells increased with time. Cell divisions occurred at all concentrations but macronuclear elongation was disturbed and subsequent cytokinesis resulted in daughter cells containing the entire G2 macronucleus, a large or small portion of it, or no nucleus at all. Moreover, odd cell shapes appeared with time. The size of the cell and nucleus increased but there was great variation with disturbed cytoplasm/nucleus ratios. Treated cells had dilated rough endoplasmic reticulum that included dense material, presumed to be vanadate, which was not seen in control cells. Scant amounts of dense material were found in dense granules, small vacuoles, and abundantly in contractile vacuoles. It is argued that interference with proper microtubular function is the main effect of vanadate.     

Nuclear migration and positioning in filamentous fungi

Genetic analyses of nuclear distribution mutants have indicated that functions of the microtubule motor, cytoplasmic dynein, and its regulators are important for nuclear positioning in filamentous fungi. Here we review these studies and also present the need to further dissect how dynein and its associated microtubule cytoskeleton are involved mechanistically in nuclear positioning in the multinucleated hyphae.     

Appressorium formation and nuclear division in Colletotrichum truncatum

Conidia of the soybean anthracnose fungus, Colletotrichum truncatum differentiate to form appressoria required for host invasion when the germ tube touches a hard surface. This thigmotrophic stimulus appears to be translated by the fungus during the second round of nuclear division. Inhibiting the second round of DNA synthesis by fluorodeoxyuridine or hydroxyurea blocked appearance of appressoria but not emergence of the germ tube. DNA synthesis and mitosis resumed upon removal of FUdR but only mycelia formed, and infection structures did not appear. In addition, actinomycin D reversibly blocked development of appressoria and synthesis of polyadenylate, but nuclear division was not affected. The data suggest that anthracnose conidia produce appressoria in response to germ tube contact by altering the messenger program of its germ tube nucleus. This study has also shown that mitochondrial DNA had an unusual bimodal distribution in CsCl at 1.690 and 1.719 g/cm³, respectively.Non-Standard Abbreviations FUdR 5-fluorodeoxyuridine - polyA polyadenylic acid     

Asymmetric nuclear division in neural stem cells generates sibling nuclei that differ in size,envelope composition,and chromatin organization

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New observations of the nuclear division in Entamoeba histolytica. The chromosomes

The morphologic organization of the nucleus and DNA during the nuclear division of Entamoeba histolytica was examined. The DNA of dividing amebic trophozoites was visualized with the fluorescent probe, Hoechst 33258 for light microscopy, and a DNA-specific antibody and phosphotungstic acid for electron microscopy. These techniques demonstrated features of the dividing amebic nuclei and the presence of spherical DNA-containing bodies corresponding to the condensed chromosomes. Based on light microscopy observations the number of chromosomes in E histolytica is five. Microtubules (MT) radiating from the microtubule organizing center (MTOC) were observed attached to the putative chromosomes.     

Stimulation of septation in mitochondria by diamide,a thiol oxidising agent

Summary Diamide (10^–4M), a thiol oxidizing agent, rapidly promotes septation in isolated frog liver mitochondria and also in situ in liver slices. The effect is partially inhibited by dithioerythritol. DNP does not have this effect, and it is concluded that diamide does not promote septation via an uncoupling action. The septate mitochondria have a different appearance from typical dividing mitochondria previously described; it is suggested that (1) diamide may act by favouring the fusion of the internal membranes, and (2) -SH oxidation is of importance in mitochondria in ageing and in various pathological conditions. The ways in which septa may develop in mitochondria in the orthodox and condensed configuration are discussed.     

Nuclear division of the vegetative cells, conchosporangial cells and conchospores of Porphyra yezoensis (Bangiales, Rhodophyta)

To confirm the position and timing of meiosis in Porphyra yezoensis Ueda, the nuclear division of vegetative cells, conchosporangial cells and conchospores was observed. An improved staining method using modified carbol fuchsin was introduced to stain the chromosomes of Porphyra. Pit‐connections between conchosporangial cells also stained well with this method. Leptotene, zygotene, pachytene, diplotene, diakinesis, metaphase, anaphase and telophase were observed in the conchosporangial cells. During the germination of conchospores, no characteristics of meiosis I were found. No difference between the nuclear division of vegetative cells and that of conchospores was observed, and 2–3 days were needed for the first cell division both in vegetative cells and conchospores. Therefore, the cell division that occurs during conchospore germination is not meiosis I. Our results indicate that the prophase of meiosis I begins during the formation of conchosporangial branches, and metaphase I, anaphase I and telophase I take place during the maturation of conchosporangial branches. Then the three‐bivalent nucleate sporangia complete cell division to form two individual conchospores, each with one three‐univalent nucleus. The conchospores released from the sporangia are at meiotic interphase. Meiosis II occurs at the first nuclear division during conchospore germination, which is a possible explanation for the observation of mosaic thalli in mutant germlings of P. yezoensis. The mosaic thalli might also arise from gene conversion/post meiotic segregation events, comparable to those in Sordaria fimicola (Roberge ex Desm.) Ces. & De Not. and Neurospora crassa Shear & B.O. Dodge.     

Centromeres in cell division, evolution, nuclear organization and disease

As the spindle fiber attachment region of the chromosome, the centromere has been investigated in a variety of contexts. Here, we will review current knowledge about this unique chromosomal region and its relevance for proper cell division, speciation, and disease. Understanding the three-dimensional organization of centromeres in normal and tumor cells is just beginning to emerge. Multidisciplinary research will allow for new insights into its normal and aberrant nuclear organization and may allow for new therapeutic interventions that target events linked to centromere function and cell division.     

Nuclear transport and nuclear pores in yeast

The central features of nuclear import have been conserved during evolution. In yeast the nuclear accumulation of proteins follows the same selective and active transport mechanisms known from higher eukaryotes. Yeast nuclear proteins contain nuclear localization sequences (NLS) which are presumably recognized by receptors in the cytoplasm and the nuclear envelope. Subsequent to this recognition step, nuclear proteins are translocated into the nucleus via the nuclear pore complexes. The structure of the yeast nuclear pore complex resembles that of higher eukaryotes. Recently, the first putative components of the yeast nuclear import machinery have been cloned and sequenced. The genetically amenable yeast system allows for an efficient structural and functional analysis of these components. Due to the evolutionary conservation potential insights into the nuclear import mechanisms in yeast can be transferred to higher eukaryotes. Thus, yeast can be considered as a eukaryotic model system to study nuclear transport.     

Protein synthesis requirements for nuclear division, cytokinesis, and cell separation in Saccharomyces cerevisiae.

Protein synthesis inhibitors have often been used to identify regulatory steps in cell division. We used cell division cycle mutants of the yeast Saccharomyces cerevisiae and two chemical inhibitors of translation to investigate the requirements for protein synthesis for completing landmark events after the G1 phase of the cell cycle. We show, using cdc2, cdc6, cdc7, cdc8, cdc17 (38 degrees C), and cdc21 (also named tmp1) mutants, that cells arrested in S phase complete DNA synthesis but cannot complete nuclear division if protein synthesis is inhibited. In contrast, we show, using cdc16, cdc17 (36 degrees C), cdc20, cdc23, and nocodazole treatment, that cells that arrest in the G2 stage complete nuclear division in the absence of protein synthesis. Protein synthesis is required late in the cell cycle to complete cytokinesis and cell separation. These studies show that there are requirements for protein synthesis in the cell cycle, after G1, that are restricted to two discrete intervals.     

A beta-tubulin mutation selectively uncouples nuclear division and cytokinesis in Tetrahymena thermophila

The ciliated protozoan Tetrahymena thermophila contains two distinct nuclei within a single cell-the mitotic micronucleus and the amitotic macronucleus. Although microtubules are required for proper division of both nuclei, macronuclear chromosomes lack centromeres and the role of microtubules in macronuclear division has not been established. Here we describe nuclear division defects in cells expressing a mutant beta-tubulin allele that confers hypersensitivity to the microtubule-stabilizing drug paclitaxel. Macronuclear division is profoundly affected by the btu1-1 (K350M) mutation, producing cells with widely variable DNA contents, including cells that lack macronuclei entirely. Protein expressed by the btu1-1 allele is dominant over wild-type protein expressed by the BTU2 locus. Normal macronuclear division is restored when the btu1-1 allele is inactivated by targeted disruption or expressed as a truncated protein. Immunofluorescence studies reveal elongated microtubular structures that surround macronuclei that fail to migrate to the cleavage furrows. In contrast, other cytoplasmic microtubule-dependent processes, such as cytokinesis, cortical patterning, and oral apparatus assembly, appear to be unaffected in the mutant. Micronuclear division is also perturbed in the K350M mutant, producing nuclei with elongated early-anaphase spindle configurations that persist well after the initiation of cytokinesis. The K350M mutation affects tubulin dynamics, as the macronuclear division defect is exacerbated by three treatments that promote microtubule polymerization: (i) elevated temperatures, (ii) sublethal concentrations of paclitaxel, and (iii) high concentrations of dimethyl sulfoxide. Inhibition of phosphatidylinositol 3-kinase (PI 3-kinase) with 3-methyladenine or wortmannin also induces amacronucleate cell formation in a btu1-1-dependent manner. Conversely, the myosin light chain kinase inhibitor ML-7 has no effect on nuclear division in the btu1-1 mutant strain. These findings provide new insights into microtubule dynamics and link the evolutionarily conserved PI 3-kinase signaling pathway to nuclear migration and/or division in Tetrahymena.     

The nuclear envelope in the plant cell cycle: structure, function and regulation

Background

Higher plants are, like animals, organisms in which successful completion of the cell cycle requires the breakdown and reformation of the nuclear envelope in a highly controlled manner. Interestingly, however, while the structures and processes appear similar, there are remarkable differences in protein composition and function between plants and animals.

Scope

Recent characterization of integral and associated components of the plant nuclear envelope has been instrumental in understanding its functions and behaviour. It is clear that protein interactions at the nuclear envelope are central to many processes in interphase and dividing cells and that the nuclear envelope has a key role in structural and regulatory events.

Conclusion

Dissecting the mechanisms of nuclear envelope breakdown and reformation in plants is necessary before a better understanding of the functions of nuclear envelope components during the cell cycle can be gained.