Nuclear γ-Tubulin during Acentriolar Plant Mitosis (original) (raw)
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Nuclear g-Tubulin during Acentriolar Plant Mitosis
The Plant Cell, 2000
Neither the molecular mechanism by which plant microtubules nucleate in the cytoplasm nor the organization of plant mitotic spindles, which lack centrosomes, is well understood. Here, using immunolocalization and cell fractionation techniques, we provide evidence that ␥ -tubulin, a universal component of microtubule organizing centers, is present in both the cytoplasm and the nucleus of plant cells. The amount of ␥ -tubulin in nuclei increased during the G 2 phase, when cells are synchronized or sorted for particular phases of the cell cycle. ␥ -Tubulin appeared on prekinetochores before preprophase arrest caused by inhibition of the cyclin-dependent kinase and before prekinetochore labeling of the mitosis-specific phosphoepitope MPM2. The association of nuclear ␥ -tubulin with chromatin displayed moderately strong affinity, as shown by its release after DNase treatment and by using extraction experiments. Subcellular compartmentalization of ␥ -tubulin might be an important factor in the organization of plant-specific microtubule arrays and acentriolar mitotic spindles.
Nuclear gamma-Tubulin during Acentriolar Plant Mitosis
THE PLANT CELL ONLINE, 2000
Neither the molecular mechanism by which plant microtubules nucleate in the cytoplasm nor the organization of plant mitotic spindles, which lack centrosomes, is well understood. Here, using immunolocalization and cell fractionation techniques, we provide evidence that ␥-tubulin, a universal component of microtubule organizing centers, is present in both the cytoplasm and the nucleus of plant cells. The amount of ␥-tubulin in nuclei increased during the G 2 phase, when cells are synchronized or sorted for particular phases of the cell cycle. ␥-Tubulin appeared on prekinetochores before preprophase arrest caused by inhibition of the cyclin-dependent kinase and before prekinetochore labeling of the mitosis-specific phosphoepitope MPM2. The association of nuclear ␥-tubulin with chromatin displayed moderately strong affinity, as shown by its release after DNase treatment and by using extraction experiments. Subcellular compartmentalization of ␥-tubulin might be an important factor in the organization of plant-specific microtubule arrays and acentriolar mitotic spindles. z
Higher plant cells: Gamma-tubulin and microtubule nucleation in the absence of centrosomes
Microscopy Research and Technique, 2000
The assembly of the higher plant cytoskeleton poses several fundamental questions. Since different microtubule arrays are successively assembled during the cell cycle in the absence of centrosomes, we can ask how these arrays are assembled and spatially organized. Two hypotheses are under debate. Either multiple nucleation sites are responsible for the assembly and organization of microtubule arrays or microtubule nucleation takes place at one site, the nuclear surface. In the latter case, microtubule nucleation and organization would be two distinct but coregulated processes. During recent years, novel approaches have provided entirely new insights to understand the assembly and dynamics of the plant cytoskeleton. In the present review, we summarize advances made in microscopy and in molecular biology which lead to novel hypotheses and open up new fields of investigation. From the results obtained, it is clear that the higher plant cell is a powerful model system to investigate cytoskeletal organization in acentrosomal eukaryotic cells.
Association of gamma-tubulin with kinetochore/centromeric region of plant chromosomes
The Plant Journal, 1998
Monoclonal antibodies raised against a phylogenetically conserved peptide from the C-terminal domain of γ-tubulin molecule were used for immunofluorescence detection of γ-tubulin in acentriolar mitotic spindles of plant cells. The antibodies stained kinetochore fibres along their whole length, including the close vicinity of kinetochores. After microtubule disassembly by the antimicrotubular drugs amiprophos-methyl, oryzalin and colchicine, γ-tubulin was found on remnants of kinetochore fibres attached to chromosomes. In cells recovering from the amiprophosmethyl treatment, γ-tubulin was localized with the regrowing kinetochore microtubule fibres nucleated or captured by kinetochore/centromeric regions. On isolated chromosomes, γ-tubulin co-localized with α-tubulin in the kinetochore/centromeric region. The data presented suggest that in acentriolar higher plant cells γ-tubulin might be directly or indirectly involved in modulation and/ or stabilization of kinetochore-microtubule interactions.
Protoplasma, 1994
The localization in higher plant cells of phosphorylated proteins recognized by the monoclonal antibody MPM-2 was investigated, with particular attention to putative microtubule organizing centres (MTOCs). Immunofluorescence and immunogold electron microscopy showed that MPM-2 did not localize with most putative MTOCs in cells and protoplasts of the gymnosperm Picea glauca and in cells of the angiosperm Vicia faba. The distribution of phosphoproteins detected by MPM-2 was similar during mitosis in both species. At late interphase and early prophase MPM-2 preferentially labelled nucleoli and the region around the condensing chromosomes but not the cytoplasm. General labelling of the cytoplasm followed dissolution of the nuclear envelope and by prometaphase centromeres stained strongly. At metaphase and very early anaphase kinetochores stained strongly by immunofluorescence but only weakly using immunogold; spindle microtubules (MTs) showed little staining. Kinetochore staining disappeared during anaphase and by telophase centromeres and loose regions of chromatin in reforming nuclei were labelled. Treatment with the anti-microtubular drug amiprophosmethyl (APM) showed that the phosphorylation/dephosphorylation cycle detected by MPM-2 proceeded independently of the mitotic spindle. Staining of centromeres/kinetochores with MPM-2 suggests that phosphorylation and dephosphorylation of this region of mitotic chromosomes may be involved in chromosome organization, chromatid separation and MT nucleation and/or attachment.
1994
In most eukaryotic cells, microtubules (MTs) are assembled at identified nucleating sites, such as centrosomes or spindle pole bodies. Higher plant cells do not possess such centrosome-like structures. Thus, the fundamental issues of where and how the intracellular plant MTs are nucleated remain highly debatable. A large body of evidence indicates that plant MTs emerge from the nuclear periphery. In this study, we developed an in vitro assay in which isolated maize nuclei nucle- ate MT assembly at a tubulin concentration (14 pM of neurotubulin) that is not efficient for spontaneous MT assembly. No MT-stabilizing agents, such as taxo1 or dimethyl sulfoxide, were used. Our model provides evidence that the nuclear surface functions as a MT-nucleating site in higher plant cells. A monoclonal antibody raised against a pericentriolar antigen immunostained the surface of isolated nuclei, and a 100-kD polypeptide in 4 M urea-treated nuclear extracts was detected.
Microtubule-dependent microtubule nucleation based on recruitment of γ-tubulin in higher plants
Nature Cell Biology, 2005
Despite the absence of a conspicuous microtubule-organizing centre, microtubules in plant cells at interphase are present in the cell cortex as a well oriented array 1,2 . A recent report suggests that microtubule nucleation sites for the array are capable of associating with and dissociating from the cortex 3 . Here, we show that nucleation requires extant cortical microtubules, onto which cytosolic γ-tubulin is recruited. In both living cells and the cell-free system, microtubules are nucleated as branches on the extant cortical microtubules. The branch points contain γ-tubulin, which is abundant in the cytoplasm, and microtubule nucleation in the cell-free system is prevented by inhibiting γ-tubulin function with a specific antibody. When isolated plasma membrane with microtubules is exposed to purified neuro-tubulin, no microtubules are nucleated. However, when the membrane is exposed to a cytosolic extract, γ-tubulin binds microtubules on the membrane, and after a subsequent incubation in neuro-tubulin, microtubules are nucleated on the pre-existing microtubules. We propose that a cytoplasmic γ-tubulin complex shuttles between the cytoplasm and the side of a cortical microtubule, and has nucleation activity only when bound to the microtubule.
The Plant Cell, 2004
Although seed plants have ␥-tubulin, a ubiquitous component of centrosomes associated with microtubule nucleation in algal and animal cells, they do not have discrete microtubule organizing centers (MTOCs) comparable to animal centrosomes, and the organization of microtubule arrays in plants has remained enigmatic. Spindle development in basal land plants has revealed a surprising variety of MTOCs that may represent milestones in the evolution of the typical diffuse acentrosomal plant spindle. We have isolated and characterized the ␥-tubulin gene from a liverwort, one of the extant basal land plants. Sequence similarity to the ␥-tubulin gene of higher plants suggests that the ␥-tubulin gene is highly conserved in land plants. The G9 antibody to fission yeast ␥-tubulin recognized a single band of 55 kD in immunoblots from bryophytes. Immunohistochemistry with the G9 antibody clearly documented the association of ␥-tubulin with various MTOC sites in basal land plants (e.g., discrete centrosomes with and without centrioles and the plastid surface in monoplastidic meiosis of bryophytes). Changes in the distribution of ␥-tubulin occur in a cell cycle-specific manner during monoplastidic meiosis in the liverwort Dumortiera hirsuta. ␥-Tubulin changes its localization from the plastid surface in prophase I to the spindle, from the spindle to phragmoplasts and the nuclear envelope in telophase I, and back to the plastid surfaces in prophase II. In vitro experiments show that ␥-tubulin is detectable on the surface of isolated plastids and nuclei of D. hirsuta , and microtubules can be repolymerized from the isolated plastids. ␥-Tubulin localization patterns on plastid and nuclear surfaces are not affected by the destruction of microtubules by oryzalin. We conclude that ␥-tubulin is a highly conserved protein associated with microtubule nucleation in basal land plants and that it has a cell cycle-dependent distribution essential for the orderly succession of microtubule arrays.
The Journal of Cell Biology, 1983
The development of the preprophase band (PPB) of microtubules (MT) in meristematic plant cells was studied by using antibodies to pig brain tubulin and indirect immunofluorescence microscopy. The PPB is first visible as a wide band of MT that are arranged only slightly more densely than flanking MT of the cortical interphase array. MT progressively become more tightly packed together, and other cortical MT are no longer seen as the PPB matures. The surface of the nuclear envelope (NE) displays no tubulin fluorescence during interphase but begins to fluoresce in the early stages of PPB development, and its intensity progressively increases thereafter. The pattern at the NE is usually diffuse at first, suggesting the presence of nonpolymerized tubulin, but fibers along the NE can be resolved at later stages.