Radial microtubule organization by histone H1 on nuclei of cultured tobacco BY-2 cells

In acentriolar higher plant cells, the surface of the nucleus acts as a microtubule-organizing center, substituting for the centrosome. However, the protein factors responsible for this microtubule organization are unknown. The nuclear surfaces of cultured tobacco BY-2 cells possess particles that generate microtubules. We attempted to isolate the proteins in these particles to determine their role in microtubule organization. When incubated with plant or mammalian tubulin, some, but not all, of the isolated nuclei generated abundant microtubules radially from their surfaces. The substance to induce the formation of radial microtubules was confirmed by SDS-PAGE to be a protein with apparent molecular mass of 38 kDa. Partial analysis of the amino acid sequences of the peptide fragments suggested it was a histone H1-related protein. Cloning and cDNA sequence analysis confirmed this and revealed that when the recombinant protein was incubated with tubulin, it could organize microtubules as well as the 38-kDa protein. Histone H1 and tubulin formed complexes immediately, even on ice, and then clusters of these structures were formed. These clusters generated radial microtubules. This microtubule-organizing property was confined to histone H1; all other core histones failed to act as organizers. On immunoblot analysis, rabbit antibodies raised against the 38-kDa protein cross-reacted with histone H1 proteins from tobacco BY-2 cells. These antibodies virtually abolished the ability of the nucleus to organize radial microtubules. Indirect immunofluorescence showed that the antigen was distributed at the nuclear plasm and particularly at nuclear periphery independently from DNA.     

Flow cytometric cell division tracking using nuclei

BACKGROUND: Labeling cells with 5-(and-6) carboxyfluorescein diacetate succinimidyl ester (CFSE) allows their subsequent division history to be determined by flow cytometry. Whether nuclei isolated from CFSE-labeled cells retain any or sufficient dye to reveal the same division history was unknown. If division tracking in nuclei were possible, it would enable the development of new methods for monitoring quantitative changes in nuclei components and how these might vary with successive divisions. METHODS: Nuclei from CFSE-labeled B cells were prepared by lysing whole cells with nonionic detergent Nonidet P-40 (NP-40). The purified nuclei were subsequently fixed with paraformaldehyde and permeabilized with Tween 20 in order to perform intranuclear staining. RESULTS: Purified nuclei displayed the equivalent asynchronous cell division profile as intact cells. Furthermore, the possibility of simultaneously monitoring division history with intranuclear staining was established by labeling bromodeoxyuridine (BrdU) incorporated into DNA during a brief pulse prior to harvesting cells. This result was verified with the staining of proliferating cell nuclear antigen (PCNA). In addition, aminoactinomycin D (7-AAD) staining established that cell cycle stage and cell division history could be simultaneously determined. CONCLUSIONS: Our results demonstrate that cell division history is retained in purified cell nuclei after CFSE labeling and can be used in combination with intranuclear immunofluorescent labeling and DNA staining to provide a comprehensive analysis of nuclei by flow cytometry. This method should prove useful for assessing differential nuclear translocation and accumulation of molecular components during consecutive division rounds and during different stages of the cell cycle.     

Dimethyl Sulfoxide Is Feasible for Plant Tubulin Assembly In vitro: A Comprehensive Analysis

It is much more difficult for tubulin from plant sources to polymerize in vitro than tubulin from animal sources. Taxol, a most widely used reagent in microtubule studies, enhances plant microtubule assembly, but hinders microtubule dynamics. Dimethyl sulfoxide (DMSO), a widely used reagent in animal microtubule studies, is a good candidate for the investigation of plant microtubule assembly in vitro.However, proper investigation is lacking about the effects of DMSO on plant microtubule assembly in vitro.In the present study, DMSO was used to establish optimal conditions for the polymerization of plant tubulin. Tubulin, purified from lily pollen, polymerizes into microtubules at a critical concentration of 1.2mg/mL in the presence of 10% DMSO. The polymers appear to have a normal microtubule structure, as revealed by electron microscopy. In the presence of 10% DMSO, microtubule polymerization decreases when the pH of the medium is increased from 6.5 to 7.4. Both the polymerization rate and the mass of the polymers increase as temperature increases from 25 to 40 ℃. Tubulin polymerizes and depolymerizes along with cycling of temperature, from 37 to 4 ℃, or following the addition to or the removal of Ca2 from the medium. When incubated with nuclei isolated from tobacco BY-2 suspension cells, tubulin assembles onto the nuclear surface in the presence of 10% DMSO. Labeling lily pollen tubulin with 5- (and 6-)carboxytetramethyl-rhodamine succinimidyl ester (NHS-rhodamine) was performed successfully in the presence of 10% DMSO. Labeled tubulin assembles into a radial structure on the surface of BY-2 nuclei. The polymerization of lily pollen tubulin is also enhanced by microtubule-associated proteins from animal sources in the presence of 10% DMSO. All the experimental results indicate that plant tubulin functions normally in the presence of DMSO. Therefore, DMSO is an appropriate reagent for plant tubulin polymerization and investigation of plant microtubules in vitro.     

Cytological changes and alterations in polyamine contents induced by cadmium in tobacco BY-2 cells.

Changes in cell viability, proliferation, cell and nuclear morphology including nuclear and DNA fragmentation induced by 0.05 and 1 mM CdSO4 (Cd2+) in tobacco BY-2 cell line (Nicotiana tabacum L.) were studied in the course of 7 days. Simultaneously changes in endogenous contents of both free and conjugated forms of polyamines (PAs) were investigated for 3 days. The application of 0.05 mM Cd2+ evoked decline of cell viability to approximately 60% during the first 24 h of treatment. Later on degradation of cytoplasmic strands, formation of the stress granules and vesicles, modifications in size and shape of the nuclei, including their fragmentation, were observed in the surviving cells. Their proliferation was blocked and cells elongated. Beginning the first day of treatment TUNEL-positive nuclei were detected in cells cultivated in medium containing 0.05 mM Cd2+. Treatment with highly toxic 1 mM Cd2+ induced fast decrease of cell viability (no viable cells remained after 6-h treatment) and cell death occurred before DNA cleavage might be initiated. The exposure of tobacco BY-2 cells to 0.05 mM Cd2+ resulted in a marked accumulation of total PAs (represented by the sum of free PAs and their perchloric acid (PCA)-soluble and PCA-insoluble conjugates) during 3-day treatment. The increase in total PA contents was primarily caused by the increase in putrescine (Put) concentration. The accumulation of free spermidine (Spd) and spermine (Spm) at 12 and 24 h in 0.05 mM Cd2+ treated BY-2 cells and high contents of Spd and especially Spm determined in dead cells after I mM Cd2+ application was observed. The participation of PA conjugation with hydroxycinnamic acids and PA oxidative deamination in maintaining of free PA levels in BY-2 cells under Cd2+-induced oxidative stress is discussed.     

Different subcellular localization and glycosylation for a functional antibody expressed in Nicotiana tabacum plants and suspension cells

Genes encoding the heavy and light chains of LO-BM2, a therapeutic IgG antibody, were assembled in the tandem or inverted convergent orientation and expressed in Nicotiana tabacum plants and BY-2 suspension cells. The tandem construct allowed higher expression in both expression systems. A similar degradation pattern was observed for the secreted antibody recovered from the leaf intercellular fluid and BY-2 culture medium. Degradation increased with leaf age or culture time. Antibodies purified from leaf tissues and BY-2 cells were both functional. However, MS analysis of the N-glycosylation showed complex plant-type glycans to be the major type in the antibody purified from plants, whereas, oligomannosidic was the major glycosylation type in that purified from BY-2 cells. LO-BM2 was observed mainly in the endoplasmic reticulum of BY-2 cells while, in leaf cells, it was localized mostly to vesicles resembling prevacuolar compartments. These results and those from endoglycosidase H studies suggest that LO-BM2 is secreted from BY-2 cells more readily than from leaf cells where it accumulates in a post-Golgi compartment. Electronic supplementary material  Supplementary material is available in the online version of this article at doi: and is accessible for authorized users.     

Spindle formation in Aspergillus is coupled to tubulin movement into the nucleus

In many important organisms, including many algae and most fungi, the nuclear envelope does not disassemble during mitosis. This fact raises the possibility that mitotic onset and/or exit might be regulated, in part, by movement of important mitotic proteins into and out of the nucleoplasm. We have used two methods to determine whether tubulin levels in the nucleoplasm are regulated in the fungus Aspergillus nidulans. First, we have used benomyl to disassemble microtubules and create a pool of free tubulin that can be readily observed by immunofluorescence. We find that tubulin is substantially excluded from interphase nuclei, but is present in mitotic nuclei. Second, we have observed a green fluorescent protein/alpha-tubulin fusion in living cells by time-lapse spinning-disk confocal microscopy. We find that tubulin is excluded from interphase nuclei, enters the nucleus seconds before the mitotic spindle begins to form, and is removed from the nucleoplasm during the M-to-G1 transition. Our data indicate that regulation of intranuclear tubulin levels plays an important, perhaps essential, role in the control of mitotic spindle formation in A. nidulans. They suggest that regulation of protein movement into the nucleoplasm may be important for regulating mitotic onset in organisms with intranuclear mitosis.     

MPF Induction of Microtubule Assembly at Interphase Kinetochore of CHO Cells

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A novel function of plant histone H1: microtubule nucleation and continuous plus end association

In higher plant cells, various microtubular arrays can be seen despite of their lack of structurally defined microtubule-organizing centers (MTOCs) like centrosomes in animal cells. Little is known about the molecular properties of the microtubule-organizing centers in higher plant cells. The nuclear surface contains one of these microtubule-organizing centers and generates microtubules radially toward the cell periphery (radial microtubules). Previously, we reported that histone H1 possessed the microtubule-organizing activity, and it was suggested that histone H1 localized on the nuclear surfaces in Tobacco BY-2 cells (Nakayama, T., Ishii, T., Hotta, T., and Mizuno, K. J. Biol. Chem. (submitted)). Here we show that histone H1 forms ring-shaped complexes with tubulin, and these complexes nucleated and elongated the radial microtubules continuously (processively) associating with their proximal ends where the incorporation of tubulin occurred. Furthermore, the polarity of radial microtubules was determined to be proximal end plus. Immunofluorescence microscopy of the isolated nuclei revealed that histone H1 localized on the nuclear surfaces, distinct from that in the chromatin. These results indicate that radial microtubules are organized by a novel MTOC that is totally different from MTOCs previously found in either plant or animal cells.     

Putative involvement of a 49 kDa protein in microtubule assembly in vitro

In higher plant cells, thus far only a few molecules have been inferred to be involved in microtubule organizing centers (MTOCs). Examination of a 49 kDa tobacco protein, homologous to a 51 kDa protein involved in sea urchin MTOCs, showed that it also accumulated at the putative MTOC sites in tobacco BY-2 cells. In this report, we show that the 49 kDa protein is likely to play a significant role in microtubule organization in vitro. We have established a system prepared from BY-2 cells, capable of organizing microtubules in vitro. The fraction, which was partially purified from homogenized miniprotoplasts (evacuolated protoplasts) by salt extraction and subsequent ion exchange chromatography, contained many particles of diameters about 1 micron after desalting by dialysis. When this fraction was incubated with purified porcine brain tubulin, microtubules were elongated radially from the particles and organized into structures similar to the asters observed in animal cells, and therefore also termed "asters" here. Since we could hardly detect BY-2 tubulin molecules in this fraction, the microtubules in "asters" seemed to be solely composed of the added porcine tubulin. Tubulin molecules were newly polymerized at the ends of the microtubules distal to the particles, and the elongation rate of microtubules was more similar to the reported rate of the plus-ends than that of the minus-ends in vitro. By fluorescence microscopy, the 49 kDa protein was shown to be located at the particles. Thus, its location at the centers of the "asters" suggests that the protein plays a role in microtubule organization in vitro.     

pH Change in Culture Medium Induces Reorganization of Intranuclear Actin in Two-Cell Mouse Embryos

Actin is a permanent component of the cell nucleus involved in many nuclear processes. However, some nuclear functions of actin remain insufficiently explored. The role played by various extracellular stimuli in regulation of nuclear actin still remains enigmatic. Deviation of basic parameters of culture medium from optimal values is a member of the group of extracellular stimuli that are very important for mammalian embryos cultured in vitro. Change in culture medium pH from the level optimal for embryo homeostasis is one such signals. The purpose of this study was to investigate the intranuclear actin distribution in nuclei of two-cell mouse embryos under stress conditions induced by changes in extracellular pH. The pattern of actin localization has been tracked after short-term culturing of the embryos at optimal (pH 7.2), increased (pH 7.8), or decreased (pH 6.5) pH conditions. Analysis was carried out with confocal microscopy using methods of direct fluorescent and indirect immunofluorescent identification of actin. It has been shown that the change of culture medium pH from the optimum value is the signal that alters intranuclear actin distribution in nuclei of the embryonic cells. Culture of two-cell mouse embryos in suboptimal pH conditions (pH 6.5 and pH 7.8) induced alterations in the intranuclear actin localization, which, in particular, were expressed in accumulation of monomeric actin and the appearance of phalloidin-stainable actin in the nuclei. These changes, in our opinion, show some signs of similarity with stress-induced changes in nuclear-actin distribution, which, as has been reported earlier by a number of researchers, have been observed in the nuclei of somatic cells.     

Presence of the betaII isotype of tubulin in the nuclei of cultured mesangial cells from rat kidney

Tubulin has generally been considered to be a cytosolic protein whose only function is to form microtubules. This assumption is supported by a great deal of evidence derived from immunohistochemical studies using antibodies directed against whole tubulin or its component polypeptides alpha- and beta-tubulin. We have re-examined the intracellular distribution of tubulin using monoclonal antibodies specific for the betaI, betaII, betaIII, and betaIV isotypes of beta-tubulin. Our test system is the cultured rat kidney mesangial cell. We have found that betaIII is absent from these cells and that beta1 and betaIV are present in microtubules throughout the cytosol. In contrast, betaII is present largely in the nuclei. Immunoblotting of purified nuclear extracts shows that the betaII-reactive antigen co-migrates with beta-tubulin. Extraction of the cytosol and chromatin suggests that betaII is concentrated in the nucleoli and also in a reticulated network in the rest of the nucleoplasm. An antibody to tyrosinated alpha-tubulin shows that alpha is also present in the nucleoli. Treatment of the cells with fluorescent colchicine shows an accumulation of colchicine in the nucleoli. Finally, fluorescently labeled alphabetaII-tubulin dimers, when microinjected into the cells, enter the nuclei and are concentrated in the nucleoli. These results suggest that the betaII isotype of tubulin is present as an alphabetaII dimer in the nuclei of cultured mesangial cells and suggest the possibility that different tubulin isotypes may have specific functions within the cell.     

Synthesis of histone mRNA sequences in isolated nuclei of cleavage stage sea urchin embryos

A qualitative assay for detection of histone mRNA sequences in nuclear RNA was developed using actinomycin D-CsCl gradients to separate histone DNA from bulk DNA by differences in buoyant density. A significant amount of RNA synthesized in vitro in isolated nuclei from early blastula stage sea urchin embryos hybridized coincident with the histone DNA satellite, and this hybridization was competed out by unlabeled “9S” polysomal RNA purified from embryos at the same stage of development. The biogenesis of these histone mRNA sequences appeared similar as observed during in vivo and in vitro synthesis. Nuclear RNA from embryos pulse labeled in vivo was found to lack histone sequences, suggesting a rapid exit time for these sequences from the nucleus. Attempts to study the exit of histone sequences from isolated nuclei labeled in vitro also suggested a rapid exit time for histone sequences. The histone sequences were synthesized to a much lesser extent in isolated nuclei from late blastula stage embryos, as anticipated from the much reduced amount of histone mRNA labeled on polysomes at this stage.     

Microtubule-nucleation sites on nuclei of higher plant cells

Summary The nucleation and the elongation of microtubules from isolated nuclei of higher plant cells were investigated. Isolated intact nuclei failed to nucleate microtubules at their surface when they were incubated with purified tubulin from plant or animal sources. However, frozen and thawed nuclei or nuclear particles obtained by gentle nuclei homogenization nucleated microtubules and nucleated microtubules elongated radially from the surface of nuclei or from the nuclear particles. Microtubules radiating from the nuclear particles were very much shorter than those radiating from frozen and thawed nuclei. The washing of the nuclear particles diminished the ability of the particles to nucleate microtubules. The ability of the washed nuclear particles to nucleate microtubules was restored by the addition of the soluble fraction of a nuclear homogenate. The complexes of radiating microtubules could easily be observed under a phasecontrast microscope. Electron microscopy demonstrated that microtubules in the complexes formed bundles. The staining with a monoclonal antibody specific for plant tubulin of the complexes of radiating microtubules, prepared by successive polymerization of animal tubulin and plant tubulin, revealed that microtubules in the complex incorporated tubulin at their proximal ends. This result indicates that the mode of incorporation of tubulin onto frozen and thawed nuclei or onto the nuclear particles is different from that in pericentriolar bodies in animal cells. Mg²⁺ seems to participate in the regulatory mechanism that determines the length of microtubules on the complexes.Abbreviations MTOC microtubule-organizing center - MES 2-(N-morpholino) ethane-sulfonic acid - PIPES piperazine-N,N-bis(2-ethanesulfonic acid) - PMSF phenylmethyl sulfonyl fluoride - EDTA ethylenediaminetetraacetic acid - EGTA ethylene glycol bis (-aminoethylether)-N,N,N,N-tetraacetic acid - GTP guanosine triphosphate - NP-40 Nonidet P-40 - DMSO dimethylsulfoxide - EPC ethyl N-phenylcarbamate - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis - DAPI 4,6-diamidiho-2-phenylindole     

Effect of cycloheximide on lipid metabolism in cells, nuclei and subcellular fractions in the rat liver

Well coordinated stages of inhibition, restoration and stimulation of protein, DNA and RNA synthesis were observed after administration of cycloheximide (3 mg/kg). The changes in lipid synthesis and composition in the nuclei and intranuclear structures were studied at different stages of cycloheximide action. The accumulation and stimulation of lipid synthesis in the nuclei during the inhibition and restoration of protein and DNA syntheses were followed by electron microscopy and labeled precursors methods. Dramatic changes were observed in the phospholipid composition of chromatin and nuclear matrix. The accumulation of minor phospholipid fractions in intranuclear structures was observed during DNA synthesis. The sphingomyelin concentration was predominant and commensurable with those of phosphatidylcholine and phosphatidylethanolamine.     

Localization and in vitro binding studies suggest that the cytoplasmic/nuclear tobacco lectin can interact in situ with high-mannose and complex N-glycans

The possible in vivo interaction of the Nicotiana tabacum agglutinin (Nictaba) with endogenous glycoproteins was corroborated using a combination of confocal/electron microscopy of an EGFP-Nictaba fusion protein expressed in tobacco Bright Yellow-2 (BY-2) cells and biochemical analyses. In vitro binding studies demonstrated that the expressed EGFP-Nictaba possesses carbohydrate-binding activity. Microscopic analyses confirmed the previously reported cytoplasmic/nuclear location of Nictaba in jasmonate-treated tobacco leaves and provided evidence for the involvement of a nuclear localization signal-dependent transport mechanism. In addition, it became evident that the lectin is not uniformly distributed over the nucleus and the cytoplasm of BY-2 cells. Far Western blot analysis of extracts from whole BY-2 cells and purified nuclei revealed that Nictaba interacts in a glycan inhibitable way with numerous proteins including many nuclear proteins. Enzymatic deglycosylation with PNGase F indicated that the observed interaction depends on the presence of N-glycans. Glycan array screening, which showed that Nictaba exhibits a strong affinity for high-mannose and complex N-glycans, provided a reasonable explanation for this observation. The cytoplasmic/nuclear localization of a plant lectin that has a high affinity for high-mannose and complex N-glycans and specifically interacts with conspecific glycoproteins suggests that N-glycosylated proteins might be more important in the cytoplasm and nucleus than is currently believed.     

A selective block of nuclear actin export stabilizes the giant nuclei of Xenopus oocytes

Actin is a major cytoskeletal element and is normally kept cytoplasmic by exportin 6 (Exp6)-driven nuclear export. Here, we show that Exp6 recognizes actin features that are conserved from yeast to human. Surprisingly however, microinjected actin was not exported from Xenopus laevis oocyte nuclei, unless Exp6 was co-injected, indicating that the pathway is inactive in this cell type. Indeed, Exp6 is undetectable in oocytes, but is synthesized from meiotic maturation onwards, which explains how actin export resumes later in embryogenesis. Exp6 thus represents the first example of a strictly developmentally regulated nuclear transport pathway. We asked why Xenopus oocytes lack Exp6 and observed that ectopic application of Exp6 renders the giant oocyte nuclei extremely fragile. This effect correlates with the selective disappearance of a sponge-like intranuclear scaffold of F-actin. These nuclei have a normal G2-phase DNA content in a volume 100,000 times larger than nuclei of somatic cells. Apparently, their mechanical integrity cannot be maintained by chromatin and the associated nuclear matrix, but instead requires an intranuclear actin-scaffold.     

A plant in vitro system for the nuclear import of proteins

This paper reports the development of an in vitro system that allows the direct assay of protein import into plant nuclei. In this assay the import of fluorescently labelled karyophilic protein substrates into nuclei isolated from evacuolated tobacco BY-2 suspension cells is monitored. It is demonstrated that import of the fluorescently labelled peptide conjugates is rapid, saturable and nuclear localization signal (NLS)-dependent. Exclusion of high molecular weight (70 kDa) dextran and substrates carrying mutated NLS sequences further underline the specificity of this system. Nuclear translocation of karyophilic import substrates in tobacco, similar to mammalian systems, is inhibited by the non-hydrolysable GTP analogue GTP-γ-S. In contrast, protein uptake is not blocked by wheat germ agglutinin, N-ethyl-maleinimide and iodoacetic acid. Furthermore, it is shown that nuclear import of proteins is only partially inhibited by low temperature (0–4°C). The in vitro nuclear import assay does not depend on exogenously added ATP or cytosolic factors. However, a block of nuclear import with GTP-γ-S could be overcome by the addition of cytosolic extract, suggesting the dependence on cytosolic factors or proteins. These data indicate that the characteristics of nuclear protein import in plant and mammalian cells are similar, but may be, at least in some respects, also different from each other.