Microtubule dynamics in living dividing plant cells: confocal imaging of microinjected fluorescent brain tubulin (original) (raw)
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Plant and Cell Physiology, 2000
Transgenic Arabidopsis thaliana, stably expressing a GFP-TUA6 fusion protein, were subcultured in B5 medium supplemented with 2,4-D and BA. In the cell suspensions, the microtubular changes in the mitotic cells could be monitored by time-sequence observations using a timelapse system of fluorescence microscopy. We have succeeded in following the microtubule (MT) dynamics in living cells throughout mitosis, from the late G 2 phase to early d phase, and found that, at the M/Gi interface, the cortical MTs were firstly reorganized in the perinuclear regions and then in the cortex, as we had previously suggested (Hasezawa and Nagata 1991, Nagata et al. 1994). The significance of this observation on the origin of cortical MTs is discussed.
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
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.
Development
There are two conflicting ideas about the site of reassembly of cortical microtubules following cytokinesis. Some observations indicate that microtubules (MTs) radiate from the surface of the postcytokinetic nuclear envelope, before becoming organized at the cortex. On the other hand, results of regrowth experiments, following MT depolymerization by drugs, suggest that the array may assemble directly upon the cortex. In this study, we have taken advantage of the significant separation between nucleus and cortex, in large, vacuolated epidermal cells, to determine which of these two potential sites supports the earliest stages of regrowth in the undrugged, native state. MTs in stem epidermis of Datura stramonium L. were stained by indirect immunofluorescence. This was performed on hand-cut sections of tissue in which the cells were not separated by enzymes or distorted by air- drying. Epidermal cells with these sheets were optically sectioned by confocal laser scanning microscopy and ...
Journal of Cell Science, 1994
Microtubule (MT) turnover within the four principal MT arrays, the cortical array, the preprophase band, the mitotic spindle and the phragmoplast, has been measured in living stamen hair cells of Tradescantia that have been injected with fluorescent neurotubulin. Using the combined techniques of confocal laser scanning microscopy and fluorescence redistribution after photobleaching (FRAP), we report that the half-time of turnover in spindle MTs is t 1/2 = 31 +/- 6 seconds, which is in excellent agreement with previous measurements of turnover in animal cell spindles. Tradescantia interphase MTs, however, exhibit turnover rates (t 1/2 = 67 +/- seconds) that are some 3.4-fold faster than those measured in interphase mammalian cells, and thus are revealed as being highly dynamic. Preprophase band and phragmoplast MTs have turnover rates similar to those of interphase MTs in Tradescantia. The spatial and temporal aspects of the fluorescence redistribution after photobleaching in all fou...
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.
The Journal of Cell Biology, 1981
Microtubules participate as morphogenetic tools in two basic processes by which plants develop their characteristic forms : (a) production of new cells in specific sites and with specific initial shapes by partitioning of parental cells, and (b) further shaping of the progeny during their expansion and differentiation . In respect of (a), microtubules are present in the mitotic spindle, where they develop in the absence of centrioles (14). Immediately before the division cycle they are deposited as a transitory "pre-prophase band" (PPB) , which in its positioning predicts the site and plane of the future cytokinesis. At telophase another microtubule system contributes to the organization of the phragmoplast, which contains the new partitioning wall, or cell plate. In respect of(b), there are many instances of congruence between the orientation of microtubules in the cell cortex during interphase ("interphase cortical arrays") and the orientation of currently deposited microfibrils of cell wall material (see 12 and 15 for recent summaries) . The inference is that the cell exerts geometrical control over its expansion by setting up specifically oriented microtubule arrays. These in turn guide wall deposition, thereby regulating the mechanical properties of the wall and determining its spatial reaction to the turgor forces that drive cell expansion.
1985
SUMMARY Monoclonal antibodies to yeast tubulin have been used to visualize the distribution of microtubules in the intact filamentous protonemata of the moss Physcomitrella patens. Protonemata were prepared for immunofluorescence by fixation in formaldehyde and cells were made permeable with Driselase. Extensive cell files were preserved by 'blotting' the moss onto glutaraldehydederivatized coverslips. Problems due to fluorescence from chloroplasts were obviated by extraction with dimethyl sulphoxide and the non-ionic detergent, Nonidet NP40. These improvements allowed us to determine that microtubules were present throughout the cell cycle in the apical dome of caulonemal tip cells, that there was a pronounced association of microtubules with the nucleus, that 'astral' microtubules were associated with the mitotic spindle and during anaphase may be involved in reorientation of the spindle before an oblique cytokinesis in caulonemata and that the cytokinetic phragmoplast appeared identical to the structure described for higher plants. Microtubules appeared to converge at the very tip of apical caulonemal cells and this was studied further by treating cells with CIPC — a drug that is known to produce multiple microtubuleorganizing centres — and which here produces multiple foci for microtubules at the tip. These observations emphasize the involvement of microtubules in tip growth, alignment of the cell plate and nuclear migration - processes that are fundamental to the morphogenesis of filamentous organisms