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.     

Orientation of cortical microtubules correlates with cell shape and division direction

Summary Cortical microtubules in the epidermis of regeneratingGraptopetalum plants were examined by in situ immunofluorescence. Paradermal slices of tissue were prepared by a method that preserves microtubule arrays and also maintains cell junctions. To test the hypothesis that cortical microtubule arrays align perpendicular to the direction of organ growth, arrays were visualized and their orientation quantified. A majority of microtubules are in transverse orientation with respect to the organ axis early in shoot development when the growth habit is uniform. Later in development, when growth habit is non-uniform and the tissue is contoured, cortical microtubules are increasingly longitudinal and oblique in orientation. Microtubules show only a minor change in orientation at the site of greatest curvature, the transition zone of a developing leaf. To assess the role of the division plane on orientation of arrays, the pattern of microtubules was examined in individual cells of common shape. Cells derived from transverse divisions have predominately transverse cortical arrays, whereas cells derived from oblique and longitudinal divisions have non-transverse arrays. The results show that, regardless of the stage of development, microtubules orient with respect to cell shape and plane of division. The results suggest that cytoskeletal function is best considered in small domains of growth within an organ.Abbrevations DMSO dimethylsulfoxide - EGTA ethylene glycol-bis-(ß-aminoethyl ether)-N, N, N, N-tetra acetic acid - FITC fluorescein isothiocyanate - MTSB microtubule stabilizing buffer - PBS phosphate buffered saline     

Temporal and spatial pattern of differences in microtubule behaviour during Drosophila embryogenesis revealed by distribution of a tubulin isoform

Immunofluorescence staining of Drosophila embryos with a monoclonal antibody specific for acetylated alpha-tubulin has revealed that acetylated and nonacetylated alpha-tubulin isoforms have different patterns of distribution during early development. Acetylated alpha-tubulin was not detected in either interphase or mitotic spindle microtubules during the rapid early cleavage or syncytial blastoderm divisions. Acetylated alpha-tubulin was first observed as interphase lengthened at the end of syncytial blastoderm, and at cycle 14 was localized to a ring of structures clustered around the interphase nuclei. These structures probably represent a set of stable microtubules involved in nuclear elongation. Absence of detectable acetylated alpha-tubulin prior to cellular blastoderm seems to be due to rapid turnover of microtubule arrays rather than to lack of the enzyme required for modification, since acetylated alpha-tubulin appeared in early embryos when micro-tubules were stabilized by taxol treatment or anoxia. Because acetylated alpha-tubulin seems to be characteristic of stable microtubule arrays, the appearance of the antigen at cycle 14 represents a fundamental change in microtubule behaviour in the somatic cells of the embryo. Acetylated alpha-tubulin was not detected in pole cells during the blastoderm or early gastrula stages, indicating that acetylation of alpha-tubulin is not merely a consequence of cellularization. After the onset of gastrulation, interphase microtubule arrays in most cell types contain acetylated alpha-tubulin. However, cells in mitosis lack antibody staining. The resulting unstained patches reveal the stereotyped spatial pattern of cell division during gastrulation. Although the cells that give rise to the amnioserosa have acetylated alpha-tubulin in their interphase arrays at early gastrulation, by germ band elongation these large, plastic cells completely lack staining with anti-acetylated alpha-tubulin. In contrast, differentiated cell types such as neurones, which have arrays of stable axonal microtubules, stain brightly with the specific antibody. Although acetylated and nonacetylated alpha-tubulin are present in roughly equal amounts by the late stages of embryogenesis, acetylated alpha-tubulin is partitioned into the pellet during centrifugation of extracts of embryos homogenized at 4 degrees C.     

The role of microtubules in establishing nuclear spatial patterns in multinucleate green algae

Summary In uninucleate cells, cytokinesis follows karyokinesis, thereby reestablishing a specific nucleus-to-cytoplasm ratio. In multinucleate cells, cytokinesis is absent or infrequent; no plasmalemma boundary defines the cytoplasmic territory of an individual nucleus. Several genera of large multinucleate green algae were examined with epifluorescence light microscopy to determine whether the patterns of cytoplasmic organization establish nuclear cytoplasmic domains. Randomly spaced nuclei, singular mitotic events and cytoplasmic streaming characterize the organization of two genera,Derbesia andBryopsis (Caulerpales). The cells ofValonia, Valoniopsis, Boergesenia, Ventricaria (Siphonocladales), andHydrodictyon (Chlorococcales) display regularly spaced nuclei which undergo synchronous divisions in a stationary cytoplasm. In the cytoplasm of genera with regularly spaced nuclei, microtubules radiate from all nuclei in late telophase-early interphase. These internuclear microtubule arrays are not found in algal genera with randomly spaced nuclei. It is hypothesized that these microtubule arrays play a role in establishing the cytoplasmic domain of each nucleus in genera with regularly spaced nuclei. Loss of microtubule arrays during the events of mitosis correlated positively with the increasing randomization of nuclear patterns in algae grown in microtubule inhibitors. Cytoplasmic domains were maintained when cells were grown in the same media in the dark. This suggests that, after a round of division, regular nuclear spacing in certain multinucleate algae is reestablished by internuclear microtubule arrays, which are not, however, required to maintain spacing during interphase.Dedicated to the memory of Professor Oswald Kiermayer     

Self-organization of interphase microtubule arrays in fission yeast

Microtubule organization is key to eukaryotic cell structure and function. In most animal cells, interphase microtubules organize around the centrosome, the major microtubule organizing centre (MTOC). Interphase microtubules can also become organized independently of a centrosome, but how acentrosomal microtubules arrays form and whether they are functionally equivalent to centrosomal arrays remains poorly understood. Here, we show that the interphase microtubule arrays of fission yeast cells can persist independently of nuclear-associated MTOCs, including the spindle pole body (SPB)--the centrosomal equivalent. By artificially enucleating cells, we show that arrays can form de novo (self-organize) without nuclear-associated MTOCs, but require the microtubule nucleator mod20-mbo1-mto1 (refs 3-5), the bundling factor ase1 (refs 6,7), and the kinesin klp2 (refs 8,9). Microtubule arrays in enucleated and nucleated cells are morphologically indistinguishable and similarly locate to the cellular axis and centre. By simultaneously tracking nuclear-independent and SPB-associated microtubule arrays within individual nucleated cells, we show that both define the cell centre with comparable precision. We propose that in fission yeast, nuclear-independent, self-organized, acentrosomal microtubule arrays are structurally and functionally equivalent to centrosomal arrays.     

The effect of low temperature on the microtubules in root meristem cells of spring and winter cultivars of wheat <Emphasis Type="Italic">Triticum aestivum L.</Emphasis>

We have studied the response of interphase and mitotic microtubule arrays in root meristem cells of spring and winter cultivars of wheat Triticum aestivum L. (Moskovskaya 35 and Moskovskaya 39) to cold stress (1 h at 0°C) and acclimation to cold (3–48 h at 0°C). We show that, in general, interphase microtubules are more resistant to cold then mitotic arrays in both cultivars. During cold stress, no changes are detected in the microtubule system of interphase cells of spring wheat, whereas the density of endoplasmic microtubules increases in interphase cells of winter wheat. During mitosis, the density of the kinetochore fibers of the spindle decreases in the cells of both cultivars, but it is prevailing in the cells of spring cultivar of wheat. During acclimation to cold, the disorganization of the cortical microtubule bundles and the enhanced growth of the endoplasmic microtubule network, which is comprised of microtubule converging centers, are observed in cells of both cultivars. However, the mitotic microtubule systems of winter and spring cultivars respond differently to cold acclimation. During prophase, a diffuse tubulin “halo,”followed by the assembly of microtubule converging centers, accumulate at the perinuclear area in the cells of winter wheat. In cells of spring cultivar, the prophase spindle is only detected during initial stages of cold acclimation. During metaphase, aberrant mitotic spindles, abnormal metaphase plates, and the excessive appearance of microtubule converging centers are observed in cells of both cultivars. Acclimation induces the disorganization of the phragmoplast and the formation of multiple microtubule converging centers during telophase in the cells of both cultivars. Microtubule converging centers are detected at the perinuclear area of daughter cells in winter wheat and in the cortical cytoplasm in spring wheat. The excessive formation of microtubule converging centers suggests the activation of microtubule assembly during prolonged exposure to low temperature. Our data also demonstrates common pathways of microtubule response to cold treatment (0°C).     

Microtubules become more dynamic but not shorter during preprophase band formation: a possible "search-and-capture" mechanism for microtubule translocation

The dynamic behavior of the microtubule cytoskeleton plays a crucial role in cellular organization, but the physical mechanisms underlying microtubule (re)organization in plant cells are poorly understood. We investigated microtubule dynamics in tobacco BY-2 suspension cells during interphase and during the formation of the preprophase band (PPB), the cytoskeletal structure that defines the site of cytokinesis. Here we show that after 2 h of microtubule accumulation in the PPB and concurrent disappearance elsewhere in the cortex, the PPB is completed and starts to breakdown exponentially already 20 min before the onset of prometaphase. During formation of the PPB, the dynamic instability, i.e., the stochastic alternating between growing and shrinking phases, of the cortical microtubules outside the PPB increases significantly, but the microtubules do not become shorter. Based on this, as well as on the cross-linking of microtubules in the PPB and the lack of evidence for motor involvement, we propose a "search-and-capture" mechanism for PPB formation, in which the regulation of dynamic instability causes the cortical microtubules to become more dynamic and possibly longer, while the microtubule cross-linking activity of the developing PPB preferentially stabilizes these "searching" microtubules. Thus, microtubules gradually disappear from the cortex outside the PPB and aggregate to the forming PPB.     

Interconversion of metaphase and interphase microtubule arrays, as studied by the injection of centrosomes and nuclei into Xenopus eggs

We have designed experiments that distinguish centrosomal , nuclear, and cytoplasmic contributions to the assembly of the mitotic spindle. Mammalian centrosomes acting as microtubule-organizing centers were assayed by injection into Xenopus eggs either in a metaphase or an interphase state. Injection of partially purified centrosomes into interphase eggs induced the formation of extensive asters. Although centrosomes injected into unactivated eggs (metaphase) did not form asters, inhibition of centrosomes is not irreversible in metaphase cytoplasm: subsequent activation caused aster formation. When cytoskeletons containing nuclei and centrosomes were injected into the metaphase cytoplasm, they produced spindle-like structures with clearly defined poles. Electron microscopy revealed centrioles with nucleated microtubules. However, injection of nuclei prepared from karyoplasts that were devoid of centrosomes produced anastral microtubule arrays around condensing chromatin. Co-injection of karyoplast nuclei with centrosomes reconstituted the formation of spindle-like structures with well-defined poles. We conclude from these experiments that in mitosis, the centrosome acts as a microtubule-organizing center only in the proximity of the nucleus or chromatin, whereas in interphase it functions independently. The general implications of these results for the interconversion of metaphase and interphase microtubule arrays in all cells are discussed.     

FINE STRUCTURE OF THE MALE AND FEMALE GAMETES OF ATRACTOMORPHA PORCATA (SPHAEROPLEACFAE,CHLOROPHYTA) WITH EMPHASIS ON THE DEVELOPMENT AND ABSOLUTE CONFIGURATION OF THE FLAGELLAR APPARATUS1

Gametogenesis in Atractomorpha porcata Hoffman was initiated b the synchronous mitotic division of nuclei within a multinucleate gametangium. Uninucleate gametes were subsequently produced following two series of cytokinetic divisions. The first series involved the formation of phycoplast microtubules (phycoplastic cytokinesis), whereas the second series did not (nonphycoplastic cytokinesis). Centrioles were connected by a rudimentary striated distal fiber by the time they migrated to the planes of division preceding the first series of cytokinetic division. These first divisions produced binucleate gametocytes. A well-developed flagellar apparatus lay near the cell surface in close proximity to each nucleus of the gametocyte prior to the second series of cytokinetic divisions that produced the uninucleate gametes. As seen in apical view, the paired basal bodies were directly opposed, with no lateral displacement of their longitudinal axes. In lateral view, the paired basal bodies diverged from one another at an angle of 130–180° (female) or 170–180° (male) and were connected by an arched, distal striated fiber about 670–750 nm long and 600 nm at its widest part. Four electron-opaque, pyramid-shaped lateral bodies flanked the basal bodies in close contact with their undersurfaces. The flagellar roots demonstrated a cruciate arrangement, with s = 6–9 over 1 (female gametes) or 7–10 over 1 (male gametes) microtubules and d= 2 microtubules. In male gametes, one of the multistranded roots was located close to the eyespot, and a second system of cytoskeletal microtubules was detected internally. Based on gamete ultrastructure, Atractomorpha porcata appears to be the most undifferentiated member of the genus.     

Microtubule Cycle in Root Tip Cells of Allium fistulosum L.

Using immunofluorescent localization techniques and TEM methods, the organization of microtubule arrays during the cell cycle of root tip cells of Allium fistulosum L. was studied. There are four basic types of microtubule organization, namely, interphase cortical microtubule, pre-prophase band microtubule, spindle microtubule and phragmoplast microtubule, which constitute the typical microtubule cycle in dividing cells of higher plants. The fluorescent figures of microtubules observed under fluorescent microscope were explained and analysed by the ultrastractural informations of microtubules obtained from TEM.     

Drosophila katanin is a microtubule depolymerase that regulates cortical-microtubule plus-end interactions and cell migration

Regulation of microtubule dynamics at the cell cortex is important for cell motility, morphogenesis and division. Here we show that the Drosophila katanin Dm-Kat60 functions to generate a dynamic cortical-microtubule interface in interphase cells. Dm-Kat60 concentrates at the cell cortex of S2 Drosophila cells during interphase, where it suppresses the polymerization of microtubule plus-ends, thereby preventing the formation of aberrantly dense cortical arrays. Dm-Kat60 also localizes at the leading edge of migratory D17 Drosophila cells and negatively regulates multiple parameters of their motility. Finally, in vitro, Dm-Kat60 severs and depolymerizes microtubules from their ends. On the basis of these data, we propose that Dm-Kat60 removes tubulin from microtubule lattice or microtubule ends that contact specific cortical sites to prevent stable and/or lateral attachments. The asymmetric distribution of such an activity could help generate regional variations in microtubule behaviours involved in cell migration.     

TONNEAU2/FASS regulates the geometry of microtubule nucleation and cortical array organization in interphase Arabidopsis cells

Organization of microtubules into ordered arrays involves spatial and temporal regulation of microtubule nucleation. Here, we show that acentrosomal microtubule nucleation in plant cells involves a previously unknown regulatory step that determines the geometry of microtubule nucleation. Dynamic imaging of interphase cortical microtubules revealed that the ratio of branching to in-bundle microtubule nucleation on cortical microtubules is regulated by the Arabidopsis thaliana B' subunit of protein phosphatase 2A, which is encoded by the TONNEAU2/FASS (TON2) gene. The probability of nucleation from γ-tubulin complexes localized at the cell cortex was not affected by a loss of TON2 function, suggesting a specific role of TON2 in regulating the nucleation geometry. Both loss of TON2 function and ectopic targeting of TON2 to the plasma membrane resulted in defects in cell shape, suggesting the importance of TON2-mediated regulation of the microtubule cytoskeleton in cell morphogenesis. Loss of TON2 function also resulted in an inability for cortical arrays to reorient in response to light stimulus, suggesting an essential role for TON2 and microtubule branching nucleation in reorganization of microtubule arrays. Our data establish TON2 as a regulator of interphase microtubule nucleation and provide experimental evidence for a novel regulatory step in the process of microtubule-dependent nucleation.     

Microtubule dynamics: Patronin, protector of the minus end

It has long been surmised that cellular microtubules are capped at the minus ends to prevent their depolymerization. A recent study provides the first definitive identification of a minus-end-specific capping protein, termed Patronin, which protects the microtubule arrays of both mitotic and interphase cells.     

How do Plants Organize Microtubules Without a Centrosome？

A microtubule nucleates from a γ-tubuUn complex, which consists of γ-tubulin, proteins from the SPC971SPC98 family, and the WD40 motif protein GCP-WD. We analyzed the phylogenetic relationships of the genes encoding these proteins and found that the components of this complex are widely conserved among land plants and other eukaryotes. By contrast, the interphase and mitotic arrays of microtubules in land plants differ from those in other eukaryotes. In the interphase cortical array, the majority of microtubules nucleate on existing microtubules in the absence of conspicuous microtubule organizing centers （MTOCs）, such as a centrosome. During mitosis, the spindle also forms in the absence of conspicuous MTOCs. Both poles of the spindle are broad, and branched structures of microtubules called microtubule converging centers form at the poles. In this review, we hypothesize that the microtubule converging centers form via microtubule-dependent microtubule nucleation, as in the case of the interphase arrays. The evolutionary insights arising from the molecular basis of the diversity in microtubule organization are discussed.     

Spline and flagellar microtubules are resistant to mitotic disrupter herbicides

Summary Microtubule arrays in developing spermatogenous cells of pteridophytes have unique microtubule organizing centers and post-translation modifications of tubulin. Sensitivity of these arrays to the microtubule-destabilizing effects of the mitotic disrupter herbicides was examined by immunofluorescence, transmission and immunogold electron microscopy. Acetylated, stabilized arrays, such as the spline, and microtubules of the basal bodies and flagella are formed after the final mitotic division and are resistant to these herbicides. Non-acetylated, dynamic arrays that exist prior to the final mitosis, such as interphase and mitotic arrays, are eliminated by all of these herbicides, with symptomology (arrested prometaphase, lobed nuclei, irregular cell plate formation) similar to that observed in other land plants. The only exception to the instability of these mitotic microtubule arrays are the few microtubules that are collected by kinetochores into short tufts. The presence of structurally-distinguishable MTOCs, such as the blepharoplast, did not confer resistance, despite the anchoring of the minus ends of the microtubules. Simultaneous treatment with herbicide and 5-bromodeoxyuridine (BrdU), with subsequent detection with anti-BrdU of cells that had gone through S-phase during the BrdU incubation, reveals that only acetylated arrays formed prior to herbicide treatment are resistant. These data indicate that only actively polymerizing, dynamic microtubule arrays are sensitive to the destabilizing effects of the mitotic disrupter herbicides.Abbreviations MTOC microtubule organizing center - BrdU 5-bromodeoxyuridine     

The fission yeast transforming acidic coiled coil-related protein Mia1p/Alp7p is required for formation and maintenance of persistent microtubule-organizing centers at the nuclear envelope

Microtubule-organizing centers (MTOCs) concentrate microtubule nucleation, attachment and bundling factors and thus restrict formation of microtubule arrays in spatial and temporal manner. How MTOCs occur remains an exciting question in cell biology. Here, we show that the transforming acidic coiled coil–related protein Mia1p/Alp7p functions in emergence of large MTOCs in interphase fission yeast cells. We found that Mia1p was a microtubule-binding protein that preferentially localized to the minus ends of microtubules and was associated with the sites of microtubule attachment to the nuclear envelope. Cells lacking Mia1p exhibited less microtubule bundles. Microtubules could be nucleated and bundled but were frequently released from the nucleation sites in mia1Δ cells. Mia1p was required for stability of microtubule bundles and persistent use of nucleation sites both in interphase and postanaphase array dynamics. The γ-tubulin–rich material was not organized in large perinuclear or microtubule-associated structures in mia1Δ cells. Interestingly, absence of microtubules in dividing wild-type cells prevented appearance of large γ-tubulin–rich MTOC structures in daughters. When microtubule polymerization was allowed, MTOCs were efficiently assembled de novo. We propose a model where MTOC emergence is a self-organizing process requiring the continuous association of microtubules with nucleation sites.     

MICROTUBULE ORGANIZATION 1 regulates structure and function of microtubule arrays during mitosis and cytokinesis in the Arabidopsis root

MICROTUBULE ORGANIZATION 1 (MOR1) is a plant member of the highly conserved MAP215/Dis1 family of microtubule-associated proteins. Prior studies with the temperature-sensitive mor1 mutants of Arabidopsis (Arabidopsis thaliana), which harbor single amino acid substitutions in an N-terminal HEAT repeat, proved that MOR1 regulates cortical microtubule organization and function. Here we demonstrate by use of live cell imaging and immunolabeling that the mor1-1 mutation generates specific defects in the microtubule arrays of dividing vegetative cells. Unlike the universal cortical microtubule disorganization in elongating mor1-1 cells, disruption of mitotic and cytokinetic microtubule arrays was not detected in all dividing cells. Nevertheless, quantitative analysis identified distinct defects in preprophase bands (PPBs), spindles, and phragmoplasts. In nearly one-half of dividing cells at the restrictive temperature of 30 degrees C, PPBs were not detected prior to spindle formation, and those that did form were often disrupted. mor1-1 spindles and phragmoplasts were short and abnormally organized and persisted for longer times than in wild-type cells. The reduced length of these arrays predicts that the component microtubule lengths are also reduced, suggesting that microtubule length is a critical determinant of spindle and phragmoplast structure, orientation, and function. Microtubule organizational defects led to aberrant chromosomal arrangements, misaligned or incomplete cell plates, and multinucleate cells. Antiserum raised against an N-terminal MOR1 sequence labeled the full length of microtubules in interphase arrays, PPBs, spindles, and phragmoplasts. Continued immunolabeling of the disorganized and short microtubules of mor1-1 at the restrictive temperature demonstrated that the mutant mor1-1(L174F) protein loses function without dissociating from microtubules, providing important insight into the mechanism by which MOR1 may regulate microtubule length.     

Self-organization of microtubule bundles in anucleate fission yeast cells

Self-organization of cellular structures is an emerging principle underlying cellular architecture. Properties of dynamic microtubules and microtubule-binding proteins contribute to the self-assembly of structures such as microtubule asters. In the fission yeast Schizosaccharomyces pombe, longitudinal arrays of cytoplasmic microtubule bundles regulate cell polarity and nuclear positioning. These bundles are thought to be organized from the nucleus at multiple interphase microtubule organizing centres (iMTOCs). Here, we find that microtubule bundles assemble even in cells that lack a nucleus. These bundles have normal organization, dynamics and orientation, and exhibit anti-parallel overlaps in the middle of the cell. The mechanisms that are responsible for formation of these microtubule bundles include cytoplasmic microtubule nucleation, microtubule release from the equatorial MTOC (eMTOC), and the dynamic fusion and splitting of microtubule bundles. Bundle formation and organization are dependent on mto1p (gamma-TUC associated protein), ase1p (PRC1), klp2p (kinesin-14) and tip1p (CLIP-170). Positioning of nuclear fragments and polarity factors by these microtubules illustrates how self-organization of these bundles contributes to establishing global spatial order.     

Mini spindles, the XMAP215 homologue, suppresses pausing of interphase microtubules in Drosophila

Drosophila Mini spindles (Msps) protein belongs to a conserved family of microtubule-associated proteins (MAPs). Intriguingly, this family of MAPs, including Xenopus XMAP215, was reported to have both microtubule stabilising and destabilising activities. While they are shown to regulate various aspects of microtubules, the role in regulating interphase microtubules in animal cells has yet to be established. Here, we show that the depletion or mutation of Msps prevents interphase microtubules from extending to the cell periphery and leads to the formation of stable microtubule bundles. The effect is independent of known Msps regulator or effector proteins, kinesin-13/KinI homologues or D-TACC. Real-time analysis revealed that the depletion of Msps results in a dramatic increase of microtubule pausing with little or no growth. Our study provides the first direct evidence to support a hypothesis that this family of MAPs acts as an antipausing factor to exhibit both microtubule stabilising and destabilising activities.     

Microtubule reorganization in tobacco BY-2 cells stably expressing GFP-MBD

 Microtubule organization plays an important role in plant morphogenesis; however, little is known about how microtubule arrays transit from one organized state to another. The use of a genetically incorporated fluorescent marker would allow long-term observation of microtubule behavior in living cells. Here, we have characterized a Nicotiana tabacum L. cv. Bright Yellow 2 (BY-2) cell line that had been stably transformed with a gfp-mbd construct previously demonstrated to label microtubules (J. Marc et al., 1998, Plant Cell 10: 1927–1939). Fluorescence levels were low, but interphase and mitotic microtubule arrays, as well as the transitions between these arrays, could be observed in individual gfp-mbd-transformed cells. By comparing several attributes of transformed and untransformed cells it was concluded that the transgenic cells are not adversely affected by low-level expression of the transgene and that these cells will serve as a useful and accurate model system for observing microtubule reorganization in vivo. Indeed, some initial observations were made that are consistent with the involvement of motor proteins in the transition between the spindle and phragmoplast arrays. Our observations also support the role of the perinuclear region in nucleating microtubules at the end of cell division with a progressive shift of these microtubules and/or nucleating activity to the cortex to form the interphase cortical array. Received: 2 June 1999 / Accepted: 13 August 1999