Dynamics of cytoskeleton microtubules in higher plant meiosis. II. Perinuclear band formation

Analyses of correspondent meiotic abnormalities is a good tool for studying cytoskeletal rearrangements during plant cell division. The paper reports on the wheat x wheatgrass F1 hybrids, showing various abnormalities during organization of the prophase perinuclear band of microtubules (PNB) in male meiosis. Based on these data, it may be concluded that the perinuclear system of microtubules (MT) in higher plant meiosis is formed from fibrils of the radial system as a result of their translocation in the cell cytoplasm space. According to our data, at this stage the radial MT arrays pass through the following consequence of events: separating from the nuclear envelope, 2) approaching, 3) tangential orientation to the nuclear surface, 4) bending, 5) co-orientation, lateral interaction. As a result, a flat ring of well organized concentric bent MT bundles encircling the nucleus meridionally is organized.     

Dynamics of cytoskeleton microtubules in higher plant meiosis. III. Early prometaphase stage

The early prometaphase and initial stages of meiotic spindle formation in higher plant PMCs were studied by means of a new approach worked out by the authors: a morphological dissection that consists in the analysis of various abnormalities of the process under study. Wide cereal hybrids F1 were used as a source of such abnormalities: phenotypes with C-, S-shaped and combined spindle, with spindles surrounded by microtubule (MT) ring and phenotypes with chaotic circular MT system in M1. Three stages of early prometaphase not described before (disintegration of perinuclear MT band, straightening of its bundles, and their translocation throughout the cytoplasm) were revealed.     

The distribution and arrangement of microtubules in mammalian skeletal muscle fibers

The distribution and arrangement of microtubules (MTs) in skeletal muscle fibers of the rat and mouse diaphragm were examined by thin-section electron microscopy. In the central portion of muscle fibers, most MTs ran longitudinally between myofibrils and beneath the sarcolemma, and some MTs ran transversely predominantly at the level of the I band, especially of the A-I junction, thus forming a lattice-like arrangement. At the fiber periphery, MTs were aggregated in the perinuclear region, from which they radiated to take a longitudinal course beneath the sarcolemma and to run in a transverse direction at the I-band level. In the end portion of muscle fibers, MTs were abundant and ran longitudinally into sarcoplasmic processes. MTs were often found to be spatially associated with membranous organelles. Quantitative analyses indicated that the longitudinally running MTs were remarkably more numerous in the peripheral zone of muscle fibers than in the deeper zones. The density of MTs in the central portion was almost the same in both red and white muscle fibers. The density was significantly higher at the fiber ends, though it varied considerably among different fibers. These results are discussed with special reference to the possible involvement of MTs in intracellular transport as well as structural support.     

Caenorhabditis elegans Aurora A kinase is required for the formation of spindle microtubules in female meiosis

In many animals, female meiotic spindles are assembled in the absence of centrosomes, the major microtubule (MT)-organizing centers. How MTs are formed and organized into meiotic spindles is poorly understood. Here we report that, in Caenorhabditis elegans, Aurora A kinase/AIR-1 is required for the formation of spindle microtubules during female meiosis. When AIR-1 was depleted or its kinase activity was inhibited in C. elegans oocytes, although MTs were formed around chromosomes at germinal vesicle breakdown (GVBD), they were decreased during meiotic prometaphase and failed to form a bipolar spindle, and chromosomes were not separated into two masses. Whereas AIR-1 protein was detected on and around meiotic spindles, its kinase-active form was concentrated on chromosomes at prometaphase and on interchromosomal MTs during late anaphase and telophase. We also found that AIR-1 is involved in the assembly of short, dynamic MTs in the meiotic cytoplasm, and these short MTs were actively incorporated into meiotic spindles. Collectively our results suggest that, after GVBD, the kinase activity of AIR-1 is continuously required for the assembly and/or stabilization of female meiotic spindle MTs.     

Orientation of microtubules suggests a role in mRNA transportation in fertilized eggs of Chinese pine (Pinus tabulaeformis)

Summary. Polysomes become associated with microtubules (MTs) in egg cells of Chinese pine upon fertilization, providing direct evidence for MT-based intracellular mRNA and polysome localization. We have investigated by immunoelectron microscopy the orientation and spatial distribution of MTs and their association with polysomes in the fertilized egg cells. There is a perinuclear accumulation of MTs and polysomes in the zygote soon after fertilization. At this time, some of the MTs are perpendicular to the nuclear envelope and directly connected to the outer membrane or nuclear-pore complexes (NPC) at one end, and the other ends reach to the outer tier or cortical MTs that are parallel to the long axis of the zygote. The polysomes in the perinuclear region show the same spatial and temporal pattern as the MTs. Immunolocalization of the mRNA-binding protein hnRNP indicates that the mRNAs are loaded onto the nucleus-associated MTs immediately after their export from the nuclear-pore complexes. The polysomes and mRNAs are then transported from these MTs to the outer tier and/or cortical MTs, where they further localize to the polar region of the cell. Correspondence (present address): Fengli Guo, Stowers Institute for Medical Research, 1000 E 50th Street, Kansas City, MO 64110, U.S.A.     

The origin and organization of cortical microtubules during the transition between M and G1 phases of the cell cycle as observed in highly synchronized cells of tobacco BY-2

The organization of microtubules (MTs) during the transition from the M phase to the G₁ phase of the cell cycle was followed in highly synchronized suspension-cultured cells ofNicotiana tabacum L. (tobacco BY-2) by sequential treatment of cells with aphidicolin and propyzamide. Short MTs were first formed in the perinuclear regions at the expense of phragmoplasts, but when these short MTs elongated to reach the cell cortex, they grew parallel to the long axis and towards the distal end of the cells. As soon as, or shortly before the tips of elongated MTs reached the distal end, transverse cortical MTs were formed in the region proximal to the division plane. Thereafter, almost all cells retained cortical MTs which were transversely orientated to the long axis of cells and could be observed in the G₁ phase. Thus, in the organization of cortical MTs, there are two steps that have been overlooked thus far. This novel observation provides a new scheme for the organization of cortical MTs, which could unify two contrasting hypotheses, i.e. organization in the perinuclear regions versus that in the cell cortex. These observations are discussed in relation to the microtubule-organizing center of plant cells.     

Stable, detyrosinated microtubules function to localize vimentin intermediate filaments in fibroblasts

Separate populations of microtubules (MTs) distinguishable by their level of posttranslationally modified tubulin subunits and by their stability in vivo have been described. In polarized 3T3 cells at the edge of an in vitro wound, we have found a striking preferential coalignment of vimentin intermediate filaments (IFs) with detyrosinated MTs (Glu MTs) rather than with the bulk of the MTs, which were tyrosinated MTs (Tyr MTs). Vimentin IFs were not stabilizing the Glu MTs since collapse of the IF network to a perinuclear location, induced by microinjection of monoclonal anti-IF antibody, had no noticeable effect on the array of Glu MTs. To test whether Glu MTs may affect the organization of IFs we regrew MTs in cells that had been treated with nocodazole to depolymerize all the MTs and to collapse IFs; the reextension of IFs into the lamella lagged behind the rapid regrowth of Tyr MTs, but was correlated with the slower reformation of Glu MTs. Similar realignment of IFs with newly formed Glu MTs was observed in serum-starved cells treated with either serum or taxol to induce the formation of Glu MTs. Next, we microinjected affinity purified antibodies specific for Glu tubulin (polyclonal SG and monoclonal 4B8) and specific for Tyr tubulin (polyclonal W2 and monoclonal YL1/2) into 3T3 cells. Both injected SG and 4B8 antibodies labeled the subset of endogenous Glu MTs; W2 and YL1/2 antibodies labeled virtually all of the cytoplasmic MTs. Injection of SG or 4B8 resulted in the collapse of IFs to a perinuclear region. This collapse was comparable to that observed after complete MT depolymerization by nocodazole. Injection of W2, YL1/2, or nonspecific control IgGs did not result in collapse of the IFs. Taken together, these results show that Glu MTs localize IFs in migrating 3T3 fibroblasts and suggest that detyrosination of tubulin acts as a signal for the recruitment of vimentin IFs to MTs.     

The Katanin Microtubule Severing Protein in Plants

Katanin is a heterodimeric microtubule （MT） severing protein that uses energy from ATP hydrolysis to generate internal breaks along MTs. Katanin p60, one of the two subunits, possesses ATPase and MT-binding/severing activities, and the p 80 subunit is responsible for targeting of katanin to certain subcellular locations. In animals, katanin plays an important role in the release of MTs from their nucleation sites in the centrosome. It is also involved in severing MTs into smaller fragments which can serve as templates for further polymerization to increase MT number during meiotic and mitotic spindle assembly. Katanin homologs are present in a wide variety of plant species. The Arabidopsis katanin homolog has been shown to possess ATP-dependent MT severing activity in vitro and exhibit a punctate localization pattern at the cell cortex and the perinuclear region. Disruption of katanin functions by genetic mutations causes a delay in the disappearance of the perinuclear MT array and results in an aberrant organization of cortical MTs in elongating cells. Consequently, katanin mutations lead to defects in cell elongation, cellulose microfibril deposition, and hormonal responses. Studies of katanin in plants provide new insights into our understanding of its roles in cellular functions.     

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.     

Microtubule organizing centers in plant cells: localization of a 49 kDa protein that is immunologically cross-reactive to a 51 kDa protein from sea urchin centrosomes in synchronized tobacco BY-2 cells

Summary A 49 kDa protein in tobacco BY-2 cells has been found to be cross-reactive with antibodies raised against a 51 kDa protein that was isolated from sea urchin centrosomes and identified as a microtubule-organizing center (MTOC) in animal cells. Tracing the fate of the 49 kDa protein during progression of the cell cycle in highly synchronized tobacco BY-2 cells revealed that this protein was colocalized with plant microtubules (MTs): the location of the 49 kDa protein coincided with preprophase bands (PPBs), mitotic spindles and phragmoplasts. Furthermore, between the M and G₁ phases, the 49 kDa protein was observed in the perinuclear regions, in which the initials of MTs are organizing to form cortical MTs. At the G₁ phase the location of the 49 kDa protein in the cell cortex coincided with that of the cortical MTs. It appeared that the 49 kDa protein in the cell cortex was transported as granules from the perinuclear regions. Thus, it is highly probable that the 49 kDa protein, which reacts with antibodies against the 51 kDa protein in sea urchin centrosomes, plays the role of an MTOC in plant cells. Thus, the mechanisms for organizing MTs in higher organisms appear to share a common protein, even though the organization of MTs is superficially very different in plant and animal cells.Abbreviations DAPI 4,6-diamidino-2-phenyl indole - MT microtubule - MTOC microtubule-organizing center - PAGE polyacrylamide gel electrophoresis - PBS phosphate-buffered saline - PPB preprophase band - SDS sodium dodecylsulfate     

Development of the preprophase band from random cytoplasmic microtubules in guard mother cells of Allium cepa L.

The organization of microtubule (MT) arrays in the guard mother cells (GMCs) of A. cepa was examined, focussing on the stage at which a longitudinal preprophase band (PPB) is established perpendicular to all other division planes in the epidermis. In the majority of young GMCs, including those seen just after asymmetric division, MTs are distributed randomly throughout the cortex and inner regions of the cytoplasm. Few MTs are associated with the nuclear surface. As the GMCs continue to develop, MTs cluster around the nucleus and a PPB appears as a wide longitudinal band. Microtubules also become prominent between the nucleus and the periclinal and transverse walls, while they decrease in number along the radial longitudinal walls. The PPB progressively narrows by early prophase, and a transversely oriented spindle gradually ensheaths the nucleus. These observations indicate that the initial, broad PPB is organized by a rearrangement of the random cytoplasmic array of MTs. Additional reorganization is responsible for MTs linking the nucleus and the cortex in the future plane of the cell plate, and for narrowing of the PPB.Abbreviations GMC guard mother cell - MT microtubule - PPB preprophase band     

A planar microtubule-organizing zone in guard cells of Allium: experimental depolymerization and reassembly of microtubules

The generation of the unique radial array of microtubules (MTs) in stomatal guard cells raises questions about the location and activities of relevant MT-organizing centers. By using tubulin immunofluorescence microscopy, we studied the pattern of depolymerization and reassembly of MTs in guard cells of Allium cepa L. Chilling at 0°C reduces the MTs to small remnants that surround the nuclear surface of cells in the early postcytokinetic stage, or form a dense layer along the central portion of the ventral wall in older guard cells. A rapid reassembly on rewarming restores either MTs extending from the nuclear surface randomly throughout the cytoplasm in very young cells, or an array of MTs radiating from the dense layer at the ventral wall later in development. A similar pattern of depolymerization and reassembly is achieved by incubation with 100 M colchicine followed by a brief irradiation with ultraviolet (UV) light. Incubation with 200 M colchicine leads to a complete depolymerization that leaves only a uniform, diffuse cytoplasmic fluorescence. Nonetheless, UV irradiation of developing guard cells induces the regeneration of a dense layer of MTs at the ventral wall. The layer is again positioned centrally along the wall, even if the nucleus has been displaced by centrifugation in the presence of cytochalasin D. Neither the regenerated layer nor the perinuclear MTs seen earlier are related to the staining pattern of serum 5051, which reportedly binds to centrosomal material in animal and plant cells. The results support the view that, soon after cytokinesis, a planar MT-organizing zone is established in the cortex along the central portion of the ventral wall, which then generates the radial MT array.Abbreviations GC guard cell - MT microtubule - MTOC microtubule-organizing center - UV ultraviolet To whom correspondence should be addressed.     

Kinetochore microtubules in PTK cells.

We have analyzed the fine structure of 10 chromosomal fibers from mitotic spindles of PtK1 cells in metaphase and anaphase, using electron microscopy of serial thin sections and computer image processing to follow the trajectories of the component microtubules (MTs) in three dimensions. Most of the kinetochore MTs ran from their kinetochore to the vicinity of the pole, retaining a clustered arrangement over their entire length. This MT bundle was invaded by large numbers of other MTs that were not associated with kinetochores. The invading MTs frequently came close to the kinetochore MTs, but a two-dimensional analysis of neighbor density failed to identify any characteristic spacing between the two MT classes. Unlike the results from neighbor density analyses of interzone MTs, the distributions of spacings between kinetochore MTs and other spindle MTs revealed no evidence for strong MT-MT interactions. A three-dimensional analysis of distances of closest approach between kinetochore MTs and other spindle MTs has, however, shown that the most common distances of closest approach were 30-50 nm, suggesting a weak interaction between kinetochore MTs and their neighbors. The data support the ideas that kinetochore MTs form a mechanical connection between the kinetochore and the pericentriolar material that defines the pole, but that the mechanical interactions between kinetochore MTs and other spindle MTs are weak.     

A calmodulin-sensitive interaction between microtubules and a higher plant homolog of elongation factor-1 alpha.

The microtubules (MTs) of higher plant cells are organized into arrays with essential functions in plant cell growth and differentiation; however, molecular mechanisms underlying the organization and regulation of these arrays remain largely unknown. We have approached this problem using tubulin affinity chromatography to isolate carrot proteins that interact with MTs. From these proteins, a 50-kD polypeptide was selectively purified by exploiting its Ca(2+)-dependent binding to calmodulin (CaM). This polypeptide was identified as a homolog of elongation factor-1 alpha (EF-1 alpha)--a highly conserved and ubiquitous protein translation factor. The carrot EF-1 alpha homolog bundles MTs in vitro, and moreover, this bundling is modulated by the addition of Ca2+ and CaM together (Ca2+/CaM). A direct binding between the EF-1 alpha homolog and MTs was demonstrated, providing novel evidence for such an interaction. Based on these findings, and others discussed herein, we propose that an EF-1 alpha homolog mediates the lateral association of MTs in plant cells by a Ca2+/CaM-sensitive mechanism.     

Formation of division spindles in higher plant meiosis

Depolymerisation of the MT cytoskeleton during late prophase makes it impossible to follow the cytoskeleton cycle in centrosomeless plant meiocytes. This paper describes rearrangements of the MT cytoskeleton during plant meiotic spindle formation in normally dividing pollen mother cells in various higher plant species and forms in which the cytoskeleton does not depolymerise at prophase. In such variants of the wild-type, cytoskeleton rearrangements can be observed at late prophase/early prometaphase. Radial MT bundles coalesce in the meridian plane, reorientate tangentially, curve and give rise to a developed ring-shaped perinuclear cytoskeleton system at the meridian. During nuclear envelope breakdown this ring disintegrates and splits into a set of free MT bundles. Three sub-stages of prometaphase are indicated: early prometaphase (disintegration of perinuclear ring and invasion of MTs into the former nuclear area), middle prometaphase or chaotic stage (formation of bipolar spindle fibres), and late prometaphase (formation of bipolar spindle). Analysis of a range of abnormal phenotypes (disintegrated, multiple, polyarchal, chaotic spindles) reveals two previously unknown processes during late prometaphase: axial orientation and consolidation of the spindle fibres.     

Preprophase band formation and cortical division zone establishment: RanGAP behaves differently from microtubules during their band formation

Correct positioning of the division plane is a prerequisite for plant morphogenesis. The preprophase band (PPB) is a key intracellular structure of division site determination. PPB forms in G2 phase as a broad band of microtubules (MTs) that narrows in prophase and specializes few-micrometer-wide cortical belt region, named the cortical division zone (CDZ), in late prophase. The PPB comprises several molecules, some of which act as MT band organization and others remain in the CDZ marking the correct insertion of the cell plate in telophase. Ran GTPase-activating protein (RanGAP) is accumulated in the CDZ and forms a RanGAP band in prophase. However, little is known about when and how RanGAPs gather in the CDZ, and especially with regard to their relationships to MT band formation. Here, we examined the spatial and temporal distribution of RanGAPs and MTs in the preprophase of onion root tip cells using confocal laser scanning microscopy and showed that the RanGAP band appeared in mid-prophase as the width of MT band was reduced to nearly 7 µm. Treatments with cytoskeletal inhibitors for 15 min caused thinning or broadening of the MT band but had little effects on RanGAP band in mid-prophase and most of late prophase cells. Detailed image analyses of the spatial distribution of RanGAP band and MT band showed that the RanGAP band positioned slightly beneath the MT band in mid-prophase. These results raise a possibility that RanGAP behaves differently from MTs during their band formation.     

Origin of cortical microtubules organized at M/G1 interface: recruitment of tubulin from phragmoplast to nascent microtubules

The origin of cortical microtubules (CMTs) was investigated in transgenic BY-2 cells stably expressing a GFP (green fluorescent protein) -tubulin fusion protein (BY-GT16). In a previous study, we found that CMTs were initially organized in the perinuclear regions but then elongated to reach the cell cortex where they formed bright spots, and that the appearance of parallel MTs from the bright spots was followed by the appearance of transverse MTs (Kumagai et al., Plant Cell Physiol. 42, 723-732, 2001). In this study, we investigated the migration of tubulin to the reorganization sites of CMTs at the M/G1 interface. After synchronization of the BY-GT16 cells by aphidicolin, the localization of GFP-tubulin was monitored and analyzed by deconvolution microscopy. GFP-tubulin was found to accumulate on the nuclear surface near the cell plate at the final stage of phragmoplast collapse. Subsequently, GFP-tubulin accumulated again on the nuclear surface opposite the cell plate, where nascent MTs elongated to the cell cortex. The significance of these observations on the mode of CMT organization is discussed.     

Fate of nascent microtubules organized at the M/G1 interface, as visualized by synchronized tobacco BY-2 cells stably expressing GFP-tubulin: time-sequence observations of the reorganization of cortical microtubules in living plant cells

Transgenic BY-2 cells stably expressing a GFP (green fluorescent protein)-tubulin fusion protein (BY-GT16) were subcultured in a modified Linsmaier and Skoog medium. The BY-GT16 cells could be synchronized by aphidicolin and the dynamics of their microtubules (MTs) were monitored by the confocal laser scanning microscopy (CLSM). We have succeeded in investigating the mode of reorganization of cortical MTs at the M/G1 interface. The cortical MTs were initially organized in the perinuclear regions and then they elongated to reach the cell cortex, forming the bright spots there. Subsequently, the first cortical MTs rapidly elongated from the spots and they were oriented parallel to the long axis towards the distal end of the cells. Around the time when the tips of the parallel MTs reached the distal end, the formation of transverse cortical MTs followed in the cortex near the division site, as we had previously suggested [Hasezawa and Nagata (1991) Bot. Acta 104: 206, Nagata et al. (1994) Planta 193: 567]. It was confirmed in independent observations that the appearance of the parallel MTs was followed by the appearance of the transverse MTs in each cell. We found that the transverse MTs spread through the whole cell cortex within about 20-30 min, while the parallel MTs disappeared. The significance of these observations on the mode of cortical MT organization is discussed.     

Formation and function of phragmoplast during successive cytokinesis stages in higher plant meiosis

Cytoskeletal rearrangements were studied during meiotic telophase in a number of monocotyledonous plant species. Wild type and abnormal meiosis (in wide cereal hybrids, meiotic mutants and allolines) was analyzed. It was found that central spindle fibers that move centrifugally, along with newly-formed MTs, are the basis of phragmoplast formation and function in PMCs of monocotyledonous plant species with successive cytokinesis stages. A model for centrifugal movement of the meiotic phragmoplast is proposed; this model is a modification of the corresponding process during B-anaphase.     

Microtubule patterns during meiosis in two higher plant species

Summary A monoclonal antibody to higher plant tubulin was used to trace microtubule (MT) structures by immunofluorescence throughout mitosis and meiosis in two angiosperms,Lycopersicon esculentum andOrnithogalum virens. Root tip cells showed stage specific MT patterns typical of higher plant cells. These included parallel cortical interphase arrays oriented perpendicular to the long axis of the cell, preprophase band MTs in late interphase through prophase, barrelshaped spindles, and finally phragmoplasts. Pollen mother cell divisions exhibited randomly oriented cortical MT arrays in prophase I, pointed spindles during karyokinesis, and elongate phragmoplasts. A preprophase band was not observed in either meiotic division. MT initiation sites were seen as broad zones associated with the nuclear envelope.