Prophase bands of microtubules occur in protoplast cultures of Vicia hajastana Grossh

Circumnuclear bands of microtubules (MT) have been found in the prophase of mitoses in cultured protoplasts of Vicia hajastana. The timing of the appearance and disappearance of the prophase band of MT (PB) relative to the stage of mitosis was studied using simultaneous staining of MT by immunofluorescence and DNA by Hoechst 33258. These protoplasts regenerate into unorganized tissue. Pre-prophase bands of MT have previously been found only in highly organized tissues of higher plants. The role of PB in cell division is discussed.Abbreviations MT microtubule(s) - PB prophase band(s) - FPB pre-prophase band(s) - PNF perinuclear fluorescence     

Mitosis and microtubule organizational changes in rice root-tip cells

The pattern of change of the microtubule cytoskeleton of the root-tip cells of rice during mitosis was studied using immunofluorescence technic and confocal laser scanning microscopy. All the major stages of ceil division including preprophase, prophase, metaphase, anaphase and telophase were observed. The most significant finding was that in the preprophase cells microtubules radiating from the nuclear surface to the cortex were frequently seen. During development these microtubules became closely associated with the preprophase band and prophase spindie indicating that the microtubules radiating from the nuclear surface, the preprophase band and the prophazc spindle were structurally and functionally closely related to each other. Granule-like anchorage sites for the radiating microtubules at the muclear surface were often seen and the possibility that these gramle-like anchorage sites might represent the microtubule organizing centres was discussed.     

DNA replication and the development of preprophase bands in soybean protoplast cultures

Nuclear DNA replication and the development of preprophase bands (PPBs) are two chronologically close processes during the higher plant cell cycle. However, it is not clear whether occurrence of PPBs is coupled with DNA replication. A soybean protoplast culture with a high frequency of PPBs was used to study the relationship between the two processes when treated with aphidicolin, a potent and specific inhibitor of eukaryotic DNA polymerase-α. When DNA replication was partially inhibited by 10 mg l^-1 aphidicolin, both the percentage of cells with PPBs and the mitotic index (MI) decreased in absolute terms, but there were proportionately more PPBs than mitoses. Since PPBs change in appearance as they develop, they were divided into categories of early (interphase associated) and late (prophase associated). The increased PPB/MI ratio was associated with an increased proportion of early stage PPBs relative to late stage PPBs. When DNA replication was completely blocked by 50 mg l^-1 aphidicolin, both MI and the percentage of cells with PPBs were close to zero. These results suggest that development of PPBs was to a large extent coupled DNA replication. We propose that the increased PPB/MI ratio at 10 mg l^-1 aphidicolin was due to a linkage between the duration of interphase and the time period in which early stage PPBs are visible. The increased duration of early PPBs partially compensates for the reduced number of nuclei reaching the stage of PPB initiation. Furthermore, in cultures containing aphidicolin, the percentage of PPBs with simultaneous perinuclear fluorescence (PNF, accumulation of microtubules on nuclear envelope) was reduced and whenever PNF was prominent and dense on the nuclear envelope the nucleus showed chromatin condensation. These observations indicated that the transition from PPB to PNF and then to the prophase spindle is closely related to the progress of the nuclear cycle.     

The microtubular cytoskeleton during megasporogenesis in the Nun orchid, Phaius tankervilliae

This study examines the microtubular cytoskeleton during megasporogenesis in the Nun orchid, Phaius tankervilliae . The subepidermal cell located at the terminal end of the nucellar filament differentiates first into an archesporial cell and then enlarges to become the megasporocyte. The megasporocyte undergoes the first meiotic division, giving rise to two dyad cells of unequal size. Immunostaining reveals that microtubules become more abundant as the megasporocyte increases in size. Microtubules congregate around the nucleus forming a distinct perinuclear array and many microtubules radiate directly from the nuclear envelope. In the megasporocyte, prominent microtubules are readily detected at the chalazal end of the cell cytoplasm. After meiosis I, the chalazal dyad cell expands in size at the expense of the micropylar dyad cell. At this stage, new microtubule organizing centres can be found at the corners of the cells. The appearance of these structures is stage-specific and they are not found at any other stages of megasporogenesis. The functional dyad cell undergoes the second meiotic division, resulting in the formation of two megaspores of unequal size. The chalazal megaspore enlarges and eventually gives rise to the embryo sac. As the functional megaspore expands, the microtubules again form a distinct perinuclear array with many microtubules radiating from the nuclear envelope. A defined cortical array of microtubules has not been found in P. tankervilliae during the course of megasporogenesis.     

Agarose embedding affects cell wall regeneration and microtubule organization in sunflower hypocotyl protoplasts

Sunflower hypocotyl protoplasts ( Helianthus annuus L. cv. Emil) divide symmetrically to form loosely associated microcolonies when cultured in liquid medium, whereas when embedded in agarose beads they divide asymmetrically to give rise to embryo-like structures. To understand the relationship between protoplast embedding and cell division patterns, we studied the deposition of β-linked glucan and the dynamics of microtubules during early phases of culture. After one day in culture, under both culture conditions, a small proportion of the protoplasts had already begun to rebuild a β-glucan cell wall and the process reached completion in all protoplasts after 10 days. Callose deposition was faster in agarose than in liquid medium but it concerned only 30–40% of the protoplasts and was not related to either division type. No marked differences were observed in cortical arrays of microtubules. However, in embedded protoplasts perinuclear microtubules formed a well-defined basket around the nucleus; these microtubules were never observed in liquid-cultured protoplasts. A narrow preprophase band was present only in dividing protoplasts cultured in liquid medium. The results suggest that asymmetric division could be related to the lack of a narrow preprophase band and that protoplast embedding enhances nucleation or stabilization of microtubules.     

Aberrant microtubule organization can result in genetic abnormalities in protoplast cultures of Vicia hajastana Grossh

Protoplast cultures of Vicia hajastana have a high division frequency. However, 20–40% of the microcolonies fail to develop beyond the 20-30-cell stage. Aneuploids and polyploids were found in early divisions and persisted in older cultures. The resulting protoplast-derived suspension culture differed karyologically from the original culture. Karyokinesis and cytokinesis were studied using simultaneous staining of microtubules (MT) by immunofluorescence, DNA by Hoechst 33258 (2-[2-(4-hydroxyphenyl)-6-benzimidazoyl]-6-[1-methyl-4-piperazyl]benzimidazole) and cell walls by Calcofluor. Freshly prepared protoplasts showed mitoses and high frequencies of binucleate cells, which probably resulted mainly from failure of cytokinesis. In early divisions, many mitoses showed metaphase chromosomes with kinetochore MT but lacking polar MT. These aberrant mitoses probably accounted for an increase in hyperploid cells observed in protoplast cultures. Multipolar spindles, which gave rise to hypoploid cells, were also seen in the early divisions. Telophase abnormalities included dislocated phragmoplasts and incomplete formation of cross walls. Many divisions resulted in daughter nuclei of unequal size. Unequal segregation of chromosomes was detected by cytofluorimetric measurements of telophase nuclei stained with Hoechst. After 5 d of culture, 91% of the divisions with incomplete cross walls also contained different-size nuclei; conversely, 78% of the divisions with fully formed cross walls contained nuclei of equal size. The malfunctioning of spindles and phragmoplasts in the same cells indicates a functional interdependence of the different MT configurations in mitosis. During the first 24 h of culture, a high frequency of abnormalities was found in spindles, cross-wall formation and chromosome segregation; this was reduced substantially in the cells undergoing first division by 48 h. The data indicate that it may be possible to manipulate the frequency of abnormalities by controlling the onset of the first division in protoplast cultures.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - MT microtubule(s) - PB prophase band(s) - PNF perinuclear fluorescence - PPB pre-prophase band     

Disruption of organization of mitotic microtubules in root meristem cells of Allium cepa induced by chloral hydrate

Data are presented on the effect of chlorahydrate on microtubule organization in the root meristem of Allium cepa. Our studies show that an incomplete preprophase band commonly appears during G2-prophase transition, yet the major effect is the lack of perinuclear microtubules, leading to inhibition of the prophase spindle formation and transition to C-mitosis. Upon chloralhydrate treatment of metaphase cells, we found cells with chromosomes regularly aligned within the metaphase plate and differently disorganized mitotic spindles. Concurrently, C-metaphase cells with remnants of kinetochore fibers were present. In addition, normal bipolar and abnormal irregular types of chromosome segregation were detected, this representing multipolar and diffuse anaphases. The major difference between them is the presence of polar microtubules during multipolar anaphase, and their lacking during diffuse anaphase. Alternatively, microtubule clusters between segregated groups of chromosomes are typical for cells with diffuse anaphase. During bipolar anaphase, excessive aster-like microtubules emanate from the spindle poles, and in telophase accessory phragmoplasts are observed at the cell periphery. The formation of incomplete phragmoplasts was observed after normal bipolar and abnormal chromosome segregation. We conclude that chloralhydrate may affect the nuclear surface capability to initiate the growth of perinuclear microtubules, thus blocking the prophase spindle formation. It also disturbs the spatial interaction between microtubules, which is crucial for the formation and functioning of various microtubular systems (preprophase band, spindle and phragmoplast).     

THE ULTRASTRUCTURE OF MITOSIS AND CYTOKINESIS IN THE SARCINOID CHLOROKYBUS ATMOPHYTICUS (CHLOROPHYTA,CHAROPHYCEAE) REVEALED BY RAPID FREEZE FIXATION AND FREEZE SUBSTITUTION1

Mitosis and cytokinesis in vegetative cells of the sarcinoid green alga Chlorokybus atmophyticus Geitler were examined with rapid freeze fixation, freeze substitution, and transmission electron microscopy. The taxonomic placement of C. atmophyticus in the class Charophyceae sensu Stewart and Mattox is corroborated by some mitotic and cytokinetic features including development of a microtubular sheath around the prophase nucleus, the almost constant chromosome to pole distance during anaphase, telophase nuclei widely separated by a persistent interzonal spindle, and centripetal plasma membrane invagination. Features, previously unknown in the Charophyceae, include the specific position of the peroxisome lying between the nucleus and adjacent cell wall during interphase and mitosis, the extensive array of microtubules radiating from the centrioles located at the presumptive poles at prophase, involvement of coated vesicles in the furrowing process, and occurrence of transversely aligned cleavage microtubules. Placement of Chlorokybus in the order Klebsormidiales is proposed.     

Preprophase bands in cultured multinucleate soybean protoplasts

Summary Multinucleate cells derived from soybean protoplasts were used to investigate the effect of increased nuclear number on the development and frequency of preprophase bands (PPBs) of microtubules (MTs). The results do not support the assumption that one nucleus establishes one PPB because the majority of multinucleate cells had only one large PPB. However, nuclear number or ploidy level has some influence on PPB development since double PPBs occurred more often in multinucleate than uninucleate cells. Double (divergent) PPBs were present at early and late stages of PPB development, suggesting that they are not a transient stage. PPBs in multinucleate cells developed in a similar fashion to those in uninucleate cells. In multinucleate cells, each dividing nucleus had its own spindle and phragmoplast. Subsequent phragmoplast development was frequently uncoupled from PPB distribution. Most multinucleates contained a single large PPB but at telophase, multiple phragmoplasts oriented in different planes.Abbreviations MT microtubule - MtSB microtubule stabilizing buffer - PBS phosphate buffered saline - PNF perinuclear fluorescence - PPB preprophase band     

High molecular weight protein detected in higher plant cells by antibodies against dynein is associated with vesicular organelles including Golgi apparatus

The cytoplasmic dynein is a multisubunit complex driving organelles along microtubules to their minus-end. We used antibodies against two functional domains (motor and microtubule-binding) of one of principal components of the complex—dynein heavy chain of slime mould Dictyostelium discoideum—to test root meristem cells of wheat Triticum aestivum. The antibodies reacted with a high molecular weight protein (>500 kDa) in the total cell extract and the band recognized by the antibodies in plant extracts had a lower electrophoretic mobility than the high molecular weight band of mammalian dynein. Antibodies coupled to protein A-Sepharose precipitated the high molecular weight protein from the purified cell extracts. Immunocytochemical analysis demonstrated that the antigen recognized by antibodies against dynein heavy chains is associated with the vesicles whose localization depends on the cell cycle stage. The antigen-positive vesicles were localized to the perinuclear region in interphase and early prophase, to the spindle periphery and to spindle pole region during mitosis, and to the interzonal region in the period of fragmoplast and cell plate formation. Some antigen-positive vesicles also reacted with antibodies against Golgi protein markers. The obtained data indicate that higher plant cells contain a high molecular weight protein interacting with antibodies against the motor and microtubules-binding domains of Dictyostelium dynein heavy chain. The revealed antigen was associated with the vesicular structures in the cytoplasm including the Golgi apparatus.     

Stabilizing microtubules with taxol increases microfilament stability during freezing of rye root tips

We have used double fluorescence labelling to investigate the effect of freezing on microtubules and microfilaments in root-tip cells of rye (Secale cereale L. cv Rymin). Freezing to -5°C (which does not kill these cells) caused partial depolymerization of both, but microfilaments were more resistant than microtubules. When microtubules were stabilized against freeze-induced depolymerization by pre-treating seedlings with taxol, microfilaments exhibited enhanced stability as well. Almost all the frozen cells containing taxol-stabilized microtubules also contained microfilaments. When seedlings were treated with the microtubule-destabilizing drug APM prior to freezing, microfilaments became more susceptible to freeze-induced depolymerization than in controls. These data suggest a physical interaction between microtubules and microfilaments in these cells.     

Cytological abnormalities and aberrant microtubule organization during early divisions in mesophyll protoplast cultures of Medicago sativa and Nicotiana tabacum

Karyokinetic and cytokinetic irregularities were examined in cultured mesophyll protoplasts of Medicago sativa L. cv. Regen S and Nicotiana tabacum L. cv. Wisconsin 38 during the early cell divisions. Spindle anomalies, laggine chromalids during anaphase, displaced phragmoplasts during telophase and incomplete cross-walls following the completion of cytokinesis were scored in these cultures. The abnormalities, however, occurred at low frequencies when compared to those found in suspension culture-derived protoplasts of Vicia hajastana . It is suggested that abnormalities are reduced because the mesophyll protoplasts regenerate a substantial cell wall prior to the onset of the first division.
An entirely different class of cytological aberration occurring in 40–50% of the cells was caused by localized protruding of the cytoplasm (budding). The nucleus was often positioned at the site of constriction of the bud. The association of budding with mitosis has led us to postulate a mechanism which may be a cause of budding and to suggest a possible way to prevent it.     

Impact of taxol-mediated stabilization of microtubules on nuclear morphology,ploidy levels and cell growth in maize roots

Direct contact of the radiating perinuclear microtubules (MTs) with the nuclear envelope was visualized with an immunogold technique using specific monoclonal tubulin antibody. The possibility that these perinuclear MT arrays are involved in establishing and maintaining nuclear organization during the interphase of cycling cells in maize root meristems was tested using taxol, a MT-stabilizing agent. Taxol not only stabilized all MTs against the action of the MT-disrupters colchicine and oryzalin but also prevented these agents from their usual induction of nuclear enlargement and decondensation of nuclear chromatin. On the contrary, nuclear size decreased and the chromatin became more compact in mitotically cycling cells of the taxol-treated root apices. Moreover, taxol prevented the stimulation, by colchicine and oryzalin, of the onset of the S phase in cells of the quiescent centre and proximal root meristem. Exposure of maize roots to taxol strongly decreased final cell volumes, suggesting that the more condensed nuclear chromatin is less efficient in genome expression and that this accounts for the restriction of cellular growth. All these findings support the hypothesis that MT arrays, radiating from the nuclear surface, are an essential part of an integrated plant ‘cell body’ consisting of nucleus and the MT cytoskeleton, and that they regulate, perhaps via their impact on chromatin condensation and activity, progress through the plant cell cycle.     

Study of mitosis in root-tip cells ofTriticum turgidum treated with the DNA-intercalating agent ethidium bromide

Summary This work examines mitosis in root-tip cells ofTriticum turgidum treated with the RNA synthesis inhibitor ethidium bromide, using tubulin immunolabeling and electron microscopy. The following aberrations were observed in ethidium bromideaffected cells: (1) incomplete chromatin condensation and nuclear-envelope breakdown; (2) delay of preprophase microtubule band maturation; (3) preprophase microtubule band assembly in cells displaying an interphase appearance of the nucleus; (4) prevention of the prophase spindle formation, caused by inhibition of perinuclear microtubule (Mt) formation and/or inability of the perinuclear Mts to assume bipolarity; (5) organization of an atypical metaphase spindle which is unable to arrange the chromosomes on the equatorial plane; (6) formation of an atypical perinuclear metaphase spindle in cells in which nuclear-envelope breakdown has been almost completely inhibited; (7) inhibition of the anaphase spindle formation as well as of anaphase chromosome movement; (8) disorganization of the atypical mitotic spindle during transition from mitosis to cytokinesis. The observations favor the following hypotheses. Nucleation of prophase spindle Mts is related to the mechanism that causes nuclear-envelope breakdown. The mitotic poles lack Mtnucleating and -organizing properties, and their function does not account for prophase and metaphase spindle assembly. The organization of the prophase spindle is not a prerequisite for the formation of the metaphase spindle; the metaphase spindle seems to be formed de novo by Mts nucleated on the nuclear envelope and/or in the immediate vicinity of chromosomes.Abbreviations 5-AU 5-aminouracil - EB ethidium bromide - EM electron microscopy - k-Mt kinetochore microtubule - Mt microtubule - MTOC microtubule-organizing center - NE nuclear envelope - NEB nuclear-envelope breakdown - PPB preprophase band of microtubules     

Dynamics of cytoskeleton microtubules in higher plant meiosis. I. The perinuclear band of microtubules and the meiotic spindle formation

A planar meridional perinuclear band of microtubules was observed at the late meiotic prophase I in a range of higher plant species. A distinct high-organized structure and a long time of existence allow to consider it as a new class of MTs dependent on the cell cycle in plant meiosis. MTs of the perinuclear band convert into meiotic spindle through a complex process of spatial rearrangements.     

The effect of taxol onTriticum preprophase root cells: preprophase microtubule band organization seems to depend on new microtubule assembly

Summary To examine whether preprophase microtubule band (PPB) organization occurs by rearrangement of pre-existing, or by assembly of new microtubules (Mts), we treated root cells ofTriticum turgidum with taxol, which stabilizes pre-existing Mts by slowing their depolymerization. With taxol early preprophase cells failed to form a normal PPB and PPB narrowing was prevented in cells that had already formed a wide one. The PPB became persistent in prometaphase cells and the formation of multipolar prophase-prometaphase spindles was induced. These data favour the suggestion that PPB formation and narrowing, as well as prophase spindle development, are dynamic processes depending on continuous Mt assembly at the PPB site and in the perinuclear cytoplasm.Abbreviations Mt microtubule - MTOC microtubule organizing centre - PPB preprophase microtubule band - DMSO dimethyl sulfoxide     

Analysis of spindle microtubule organization in untreated and taxol-treated PtK1 cells.

Taxol, a microtubule stabilizing agent, has been used to study changes in spindle microtubule organization during mitosis. PtK₁ cells have been treated with 5 μg/ml taxol for brief periods to determine its effect on spindle architecture. During prophase taxol induces microtubules to aggregate, particularly evident in the region between the nucleus and cell periphery. Taxol induces astral microtubule formation in prometaphase and metaphase cells concomitant with a reduction in spindle length. At anaphase taxol induces an increase in length in astral microtubules and reduces microtubule length in the interzone. Taxol-treated telophase cells show a reduction in the rate of furrowing and astral microtubules lack a discrete focus and are arranged more diffusely on the surface of the nuclear envelope. In summary, taxol treatment of cells prior to anaphase produces an increase in astral microtubules, a reduction in kinetochore microtubules and a decrease in spindle length. Brief taxol treatments during anaphase through early G₁ promotes stabilization of microtubules, an increase in the length of astral microtubules and a delayed rate of cytokinesis.     

Modifications of microfilaments and microtubules induced by two hepatic tumor promoters,phenobarbital and biliverdin in non-transformed and transformed hepatic cell lines

Microfilaments and microtubules are components of the cytoskeleton which could be implicated in neoplastic transformation. We studied the effect of two hepatic tumor promoters, phenobarbital (PB) and biliverdin (BV), on microfilaments and microtubules of non-transformed (Cl₃) and transformed (FV) hepatic epithelial cells. Cl₃ non-transformed cells cultured in the presence of 1 × 10^–6M BV for 48 h showed a loss of F-actin, fragmentation of actin and the appearance of star-like structures in the cytoplasm, as well as loosening of the peripheral bundle of actin, and some ruffling of cell membranes. In Cl₃ cells exposed to 0.2 × 10^–3M PB a similar disappearance of F-actin staining and a very prominent ruffling of cell membrane were observed. BV and PB also produced in these cells modifications of microtubules characterized by a disappearance of centrosome staining in numerous cells, a condensed ring of tubulin around the nucleus and a depolymerized aspect of the microtubular network. All these modifications of microfilaments and microtubules closely resembled those observed in FV transformed cells in the absence of any treatment (Solvent DMSO only). We did not observe an effect of BV and PB on FV cells.The present data demonstrate that the cytoskeleton of non-transformed epithelial liver cells is sensitive to the action of liver tumor promoters suggesting that it might play a role as to yet be defined in the promotion mechanism.Abbreviations PB phenobarbital - BV biliverdin - TPA 12-0-tetradecanoyl-phorbol 13 acetate - GGT gamma-glutamyl-transpeptidase - DMSO dimethylsulfoxyde     

Nuclear envelope radiating microtubules in plant cells during interphase mitosis transition

Summary The microtubule distribution during the transition from interphase to the mitotic phase was examined at ultrastructural level in large highly vacuolated cells ofNautilocalyx lynchii and in small non-vacuolated cells ofPisum sativum. Both cell types contain, besides preprophase bands and perinuclear microtubules, also microtubules radiating from the nucleus into the transvacuolar cytoplasmic strands and cytoplasm respectively.This microtubule array appears to be nucleated by the cell's nuclear envelope (NE) or NE-surrounding cytoplasm.It is hypothesized that the microtubules radiating from the nucleusinitially play a role in the mobilization of the nucleus whilelater on a stabilized part of this array anchors the nucleus in the plane of cell division, and thus forms a cytoskeletal link between nucleus and division site.Our results are discussed in the light of previous work on cytoplasmic behaviour during interphase-mitosis transition in highly vacuolated plant cells.     

The effect of low temperature on the microtubules in root meristem cells of spring and winter cultivars of wheat Triticum aestivum L

We have studied the response of the interphase and mitotis microtubule arrays in root meristem cells of spring and winter cultivars of wheat Triticum aestivum L. (Moskovskaya 35 and Moskovskaya 39) during cold stress (1 h at 0 degrees C) and acclimation to cold (3-48 h at 0 degrees C). Our data show that interphase microtubules are more resistant to cold than mitotic arrays in both cultivars. During cold stress the density of endoplasmic microtubules increases in interphase cells of winter plants, yet no changes are detected in cells of spring plants. In mitotic cells of both wheat cultivars the density of microtubules within the kinetochore fibers decreases, yet this effect is more evident in the cells of spring plants. During acclimation to cold of both cultivars, we have observed the disorganization of the interphase cortical arrays and the enhanced growth of endoplasmic microtubule arrays, composed of microtubule converging centers. However, the reaction of mitotic microtubule arrays differs in the cells of winter and spring plants. In winter plants, during prophase diffuse tubulin "halo" accumulates first at perinuclear area, followed by the appearance of the microtubule converging centers. In spring plants, we have observed the formation of the prophase spindle, yet later the prophase spindle is not detected. Metaphase cells of both cultivars show similar aberrations of the mitotic spindle, accumulation of abnormal metaphases and the excessive formation of microtubule converging centers. In telophase cells of both cultivars, acclimation induces similar reaction, resulting in the disorganization of the phragmoplast and the formation of multiple microtubule converging centers. The latter are detected in the perinuclear areas of the daughter cells in winter plants and in the cortical cytoplasm of cells in spring plants. Our data point to the common pathways of microtubule response to cold treatment (0 degrees C). The excessive formation of the microtubule converging centers indicates the activation of microtubule assembly during prolonged cold treatment.