Gamma-tubulin colocalizes with microtubule arrays and tubulin paracrystals in dividing vegetative cells of higher plants (original) (raw)
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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.
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.
Two γ -tubulin isoforms are differentially expressed during development in Helianthus annuus
Physiologia Plantarum, 2001
The cytoskeleton is involved in major developmental events in munoblotting, with bands at 52 and 58 kDa. The larger plant cell growth and differentiation. Nucleation events play a key role in the dynamic and organization of the microtubule -tubulin (58 kDa) is present in all the sunflower tissues tested and is associated with the nucleus. The smaller -tubulin (52 (Mt) cytoskeleton. Among many proteins involved in Mt kDa), differing from the former at the carboxy-terminal end, nucleation, -tubulin has been identified as an essential com-
-Tubulin Is Essential for Microtubule Organization and Development in Arabidopsis
THE PLANT CELL ONLINE, 2006
The process of microtubule nucleation in plant cells is still a major question in plant cell biology. g-Tubulin is known as one of the key molecular players for microtubule nucleation in animal and fungal cells. Here, we provide genetic evidence that in Arabidopsis thaliana, g-tubulin is required for the formation of spindle, phragmoplast, and cortical microtubule arrays. We used a reverse genetics approach to investigate the role of the two Arabidopsis g-tubulin genes in plant development and in the formation of microtubule arrays. Isolation of mutants in each gene and analysis of two combinations of g-tubulin double mutants showed that the two genes have redundant functions. The first combination is lethal at the gametophytic stage. Disruption of both g-tubulin genes causes aberrant spindle and phragmoplast structures and alters nuclear division in gametophytes. The second combination of g-tubulin alleles affects late seedling development, ultimately leading to lethality 3 weeks after germination. This partially viable mutant combination enabled us to follow dynamically the effects of g-tubulin depletion on microtubule arrays in dividing cells using a green fluorescent protein marker. These results establish the central role of g-tubulin in the formation and organization of microtubule arrays in Arabidopsis.
Microtubule Components of the Plant Cell Cytoskeleton
Plant physiology, 1994
Corresponding author; fax 1-612-625-5754. Abbreviations: MAP, microtubule-associated protein; MTOC, microtubule organizing center; PPB, preprophase band. Copyright Clearance Center: 0032-0889/94/l04/0001/06. LITERATURE CITED Asada T, Sonobe S, Shibaoka H j1991) Microtubule translocation in the cytokinetic apparatus of cultured tobacco cells. Nature 350: 238-241 Brown RC, Lemmon BE (1992) Polar organizers in monoplastidic mitosis of hepatics (Bryophyta). Cell Motil Cytoskel22 72-77 Caplow M (1992) Microtubule dynamics. Curr Opin Cell Biol 4 Carpenter JL, Kopczak SD, Snustad DP, Silflow CD (1993) Semiconstitutive expression of an Arabidopsis thaliana a-tubulin gene. Plant Mo1 Biol21: 937-942 Carpenter JL, Ploense SE, Snustad DP, Silflow CD (1992) Preferential expression of an a-tubulin gene of Arabidopsis in pollen. Plant Cell4 557-571 Chu B, Snustad DP, Carter JB (1993) Alteration of 0-tubulin gene expression during low-temperature exposure in leaves of Arabidopsis thaliana. Plant Physiol 103 371-377 Cleary AL, Gunning BES, Wasteneys GO, Hepler PK (1992) Microtubule and F-actin dynamics at the division site in living Tradescanfia stamen hair cells. J Cell Sci 103 977-988 Colasanti J, Cho S-O, Wick S, Sundaresan V (1993) Localization of the functionalp34'd'2 homologue of maize in dividing cells of the root tip and stomatal complex: association with the predicted division site in premitotic cells. Plant Cell5 1101-1 11 1 Cyr RJ, Palevitz BA (1989) Microtubule-binding proteins from carrot. Planta 177: 245-260 Fosket DE, Morejohn LC (1992) Structural and functional organization of tubulin. Annu Rev Plant Physiol Plant Mo1 Biol 43: Fulton C, Simpson PA (1976) Selective synthesis and utilization of flagellar tubulin. The multi-tubulin hypothesis. In R Goldman, T Pollard, J Rosenbaum, eds, Cell Motility. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, pp 987-1005 Giddinge TH Jr, Staehelin LA (1991) Microtubule-mediated control of microfibril deposition: a re-examination of the hypothesis. In
Nuclear γ-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. z
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.