Immunofluorescence microscopy of tubulin and microtubule arrays in plant cells. II. Transition between the pre-prophase band and the mitotic spindle

Summary Changing patterns of tubulin immunofluorescence as onion root meristematic cells progress from a mature pre-prophase band (PPB) stage into mitosis are reported here. The PPB reaches its narrowest profile at maturity and then remains the same width throughout the rest of the transition. Concomitant with continuation of chromatin condensation and nucleolar breakdown, both initiated earlier in pre-prophase, alignment of fluorescent fibers along the nuclear envelope (NE) changes. Perinuclear microtubules (MTs), which were parallel to the PPB or randomly arranged when first seen at earlier stages of pre-prophase, assume the orientation of spindle MTS at late preprophase. They lie close to the NE and follow the nuclear contour, ultimately converging upon two focal points directly at the NE surface. MTs also can be seen traversing the cytoplasm between nucleus and cell periphery.As spindle initiation proceeds, PPB fluorescence intensity decreases and eventually is exceeded by the NE-associated fluorescence. PPB and spindle arrays co-exist briefly in the transition period, with spindle MTs typically aligned perpendicular to both the axis of the PPB and its constituent MTs. Total disappearance of the PPB occurs only after chromosome condensation is complete and the nucleus is contained within a spherical or ellipsoid cage of fluorescent fibers comprised of two non-overlapping half-spindles. Like the fully formed prophase spindle which follows, the incipient spindle is neither barrel-shaped nor fusiform, but rather displays MTs radiating from the poles in a smooth arc.     

Actomyosin is Involved in the Organization of the Microtubule Preprophase Band in Arabidopsis Suspension Cultured Cells

The microtubule preprophase bands （PPBs） participate in the sequence of events to position cell plates in most plants. However, the mechanism of PPB formation remains to be clarified. In the present study, the organization of PPBs in Arabidopsis suspension cultured cells was investigated by confocal laser scanning microscopy combined with pharmacological treatments of reagents specific for the cytoskeleton elements. Double staining of F-actin and microtubules （MTs） showed that actin filaments were arranged randomly and no colocalization with cortical MTs was observed in the interphase cells. However, cortical actin filaments showed colocalization with MTs during the formation of PPBs. A broad actin band formed with the broad MT band in the initiation of PPB and narrowed down together with the MT band to form the PPB. Nevertheless, broad MT bands were formed but failed to narrow down in cells treated with the F-actin disruptor latrunculin A. In contrast, in the presence of the F-actin stabilizer phalloidin, PPB formation did not exhibit any abnormality. Therefore, the integrity, but not the dynamics, of the actin cytoskeleton is necessary for the formation of normal PPBs. Treatment with 2, 3-butanedine monoxime, a myosin inhibitor, also resulted in the formation of broad MT bands, indicating that actomyosin may be involved in the rearrangement of MTs to form the PPBs. Double staining of MTs and myosin revealed that myosin concentrated on the PPB region during PPB formation. It is suggested that the actin cytoskeleton at the PPB site may serve as a rack to transport cortical MTs by using myosin when the broad MT band narrows down to form the PPB.     

Comparative immunofluorescence and ultrastructural analysis of microtubule organization in Uronema sp., Klebsormidium flaccidum, K. subtilissimum, Stichococcus bacillaris and S. chloranthus (Chlorophyta)

A detailed comparative examination of microtubule (MT) organization in interphase and dividing cells of Uronema sp., Klebsormidium flaccidum, K. subtilissimum, Stichococcus bacillaris and S. chloranthus was made using tubulin immunofluorescence and transmission electron microscopy (TEM). During interphase all the species bear a well-organized cortical MT system, consisting of parallel bundles with different orientations. In Uronema sp. the cortical MT bundles are longitudinally oriented, whereas in the other species they are in transverse orientation to the axis of the cells. Considerable differences in MT organization were also observed during stages of mitosis, mainly preprophase, as well as cytokinesis. In Uronema sp., a particular radial MT assembly is organized during preprophase-early prophase, which was not observed in the other species. In Stichococcus a fine MT ring surrounded the nucleus during preprophase and prophase. An MT ring, together with single cytoplasmic MTs, was also found associated with the developing diaphragm during cytokinesis in Stichococcus. A phycoplast participates in cytokinesis in Uronema sp., but not in the other species. In Uronema sp. the centrosome functions as a microtubule organizing center (MTOC) during mitosis, but not during interphase and cytokinesis. The phylogenetic significance of these differences is discussed in combination with SSU/ITS sequencing and other, existing molecular data.     

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     

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.     

Preprophase bands in a suspension culture of the monocot Spartina pectinata

Continuous suspension cultures of the marsh grass Spartina pectinata grow as either unorganized colonies or files of cells. Immunofluorescence of tubulin revealed microtubule (MT) structures similar to those encountered in meristematic cells, including cortical microtubule (MT) bands in some interphase cells and in all prophase cells. These MT bands were judged to be preprophase bands (PPBs) on the basis of their temporal appearance in the cell cycle and their position and orientation relative to division planes. Although PPBs are widely thought to be associated with organized tissues and polarized divisions, there are reports of PPBs in suspension cultures of four dicot species. This is the first report of a PPB in suspension cultures of a monocot species.     

Preprophase bands in cultured multinucleate soybean protoplasts

Summary Multinucleate cells derived from soybean protoplasts were used to investigate the effect of increased nuclear number on the development and frequency of preprophase bands (PPBs) of microtubules (MTs). The results do not support the assumption that one nucleus establishes one PPB because the majority of multinucleate cells had only one large PPB. However, nuclear number or ploidy level has some influence on PPB development since double PPBs occurred more often in multinucleate than uninucleate cells. Double (divergent) PPBs were present at early and late stages of PPB development, suggesting that they are not a transient stage. PPBs in multinucleate cells developed in a similar fashion to those in uninucleate cells. In multinucleate cells, each dividing nucleus had its own spindle and phragmoplast. Subsequent phragmoplast development was frequently uncoupled from PPB distribution. Most multinucleates contained a single large PPB but at telophase, multiple phragmoplasts oriented in different planes.Abbreviations MT microtubule - MtSB microtubule stabilizing buffer - PBS phosphate buffered saline - PNF perinuclear fluorescence - PPB preprophase band     

Monoclonal antibodies to a fern spermatozoid detect novel components of the mitotic and cytokinetic apparatus in higher plant cells

Summary The aim of this study was to search for uncharacterized components of the plant cytoskeleton using monoclonal antibodies raised against spermatozoids of the fernPteridium (Marc et al. 1988). The cellular distribution of crossreacting immunoreactive material during the division cycle in wheat root tip cells was determined by immunofluorescence microscopy and compared to the fluorescence pattern obtained with antitubulin. Five antibodies are of special interest. Pas1D3 and Pas5F4 detect a diffuse cytoplasmic material, which, during mitosis, follows the distribution of microtubules (MTs) at the nuclear surface and in the preprophase band (PPB), spindle and phragmoplast. The immunoreactive material codistributes specifically with MT arrays of the mitotic apparatus and does not associate with interphase cortical MTs. Pas5D8 is relevant to the PPB and spatial control of cytokinesis. It binds in a thin layer at the cytoplasmic surface throughout the cell cycle, except when its coverage is transiently interrupted by an exclusion zone at the PPB site and later at the same site when the phragmoplast fuses with the parental cell wall.Pas2G6 reacts with a component of basal bodies and the flagellar band in thePteridium spermatozoid and recognizes irregularly shaped cytoplasmic vesicles in wheat cells. During interphase these particles form a cortical network.Pas6D7 binds to dictyosomes and dictyosome vesicles. At anaphase the vesicles accumulate at the equator and subsequently condense into the cell plate.Abbreviations MT microtubule - PPB preprophase band     

Aluminium effects on microtubule organization in dividing root-tip cells of Triticum turgidum. I. Mitotic cells

The effects of aluminium (Al) on dividing root-tip cells of Triticum turgidum were investigated with tubulin immunolabelling and electron microscopy. Aluminium affects the mechanisms controlling the organization of microtubule (MT) cytoskeleton, as well as tubulin polymerization, and induces the following aberrations in mitotic cells. (1) It delays the MT disassembly during mitosis, resulting in the persistence of preprophase MT bands in the late prophase cells, the presence of prophase spindles in prometaphase cells, and a disturbance in the shortening of kinetochore MT bundles in anaphase cells. (2) It interferes with the self-organization process of MTs into bipolar systems, inhibiting the formation of prophase and metaphase spindles. (3) Aluminium induces the formation of atypical MT arrays, which in the immunofluorescent specimens appear as ring-like tubulin aggregations in the cortical cytoplasm of the preprophase/prophase cells and as endoplasmic tubulin bundles in prophase and metaphase/anaphase cells; abnormal preprophase MT bands are assembled, consisting of atypical cortical and endoplasmic MT bundles, the latter clearly lining the nuclear envelope on the preprophase MT band plane. (4) It disorders the chromosome movements carried out by the mitotic spindle. In addition, after prolonged Al treatments chromatin condensation is inhibited. The outcome is greatly disturbed organization and function of the mitotic apparatus, as well as inhibition of cells from entering mitosis. This study shows that the MT cytoskeleton is a target site of Al toxicity in mitotic root-tip cells of T. turgidum . The possible mechanisms by which Al exerts its toxicity on MT organization and function are discussed.     

Microtubule reorganization accompanying preprophase band formation in guard mother cells ofAvena sativa L.

Summary In order to study developmental changes in microtubule organization attending the formation of a longitudinally oriented preprophase band, the guard mother cells ofAvena were examined using a new procedure for anti-tubulin immunocytochemistry on large epidermal segments. We found that the interphase band (IMB) of transverse cortical microtubules present in these cells following asymmetric division is replaced after subsidiary cell formation by mesh-like to radial microtubules that extend throughout the cytoplasm. Many of the Mts are also grouped in bundles. Gradually, this intermediate array is succeeded by longitudinal elements of the PPB. Thus, preprophase band formation is accompanied by a 90° shift in Mt orientation, with a radial arrangement serving as an intermediate stage. The micrographs are most consistent with the rearrangement of intact Mts, although changes in Mt assembly are possible as well. The role of the IMB in guard mother cells is also discussed.Abbreviations GMC guard mother cell - IMB interphase microtubule band - Mt microtubule - PPB preprophase band     

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.     

Localization of the Functional p34cdc2 Homolog of Maize in Root Tip and Stomatal Complex Cells: Association with Predicted Division Sites

We have used an antibody against the functional homolog of the cdc2 kinase of maize to localize the p34cdc2 protein within dividing cells of the root apex and the stomatal complex of leaf epidermis. The microtubule cytoskeletal structure of plant cells was visualized concomitantly with a monoclonal antibody specific for [alpha]-tubulin. We found that the cdc2 protein is localized mainly to the nucleus in plant cells at interphase and early prophase. This finding contrasts markedly with the predominantly cytoplasmic staining obtained using antibody to the PSTAIRE motif, which is common to cdc2 and numerous cdc2-like proteins. In a subpopulation of root cells at early prophase, the p34cdc2 protein is also distributed in a band bisecting the nucleus. Double labeling with the maize p34cdc2Zm antibody and tubulin antibody revealed that this band colocalizes with the preprophase band (PPB) of microtubules, which predicts the future division site. Root cells in which microtubules had been disrupted with oryzalin did not contain this band of p34cdc2 protein, suggesting that formation of the microtubule PPB is necessary for localization of the p34cdc2 kinase to the plane of the PPB. The p34cdc2 protein is also localized to the nucleus and PPB in cells that give rise to the stomatal complex, including those cells preparing for the highly asymmetrical divisions that produce subsidiary cells. Association of the p34cdc2 protein with the PPB suggests that the cdc2 kinase has a role in establishing the division site of plant cells and, therefore, a role in plant morphogenesis.     

Re-organization of microtubules during preprophase band development inAdiantum protonemata

Summary Microtubule organization during preprophase band development was investigated using immunofluorescence microscopy in filamentous protonemal cells (approx. 600 m in length, 20 m in width) ofAdiantum capillus-veneris L. Protonemata pre-cultured under red light were transferred to continuous blue light or total darkness to induce synchronous cell division. Preprophase bands were found under both light conditions. In an early stage of development, the preprophase band which is transverse to the cell axis overlapped with an interphase cortical array of microtubules which is random or parallel to the cell axis. The interphase cortical array disappeared thereafter. While the width of the preprophase band became narrow during development under dark conditions, under blue light conditions it did not.Spatial and temporal aspects of the disappearance of the interphase cortical array of microtubules were also investigated. The interphase cortical array began to disappear at nearly the same time as the beginning of preprophase band formation. Under blue light, the disruption of cortical microtubules started at approx. 150 m from the tip (approx. 120 m from the nucleus), and spread toward the tip as far as the nuclear region and toward the base to an area approx. 300–400 m from the tip. Cortical microtubules remained in the basal part of the protonema. The pattern of disappearance between the tip and nucleus could not be determined. Under dark conditions, the pattern of the disappearance of cortical microtubules was somewhat different in many cells from that encountered with exposure to blue light. Microtubules first re-oriented from longitudinal to transverse, and then gradually disappeared. In some cells, the pattern of disappearance was similar to that observed under blue light.Abbreviations DAPI 4, 6-diamidino-2-phenylindole - ICM interphase cortical microtubules - PBS phosphate buffered saline - PPB preprophase band - MT microtubule     

The microtubular cytoskeleton during development of the zygote,proembryo and free-nuclear endosperm inArabidopsis thaliana (L.) Heynh

The microtubular cytoskeleton has been studied during development of the zygote, proembryo and free-nuclear endosperm inA. thaliana using immunofluorescence localization of tubulin in enzymatically isolated material. Abundant micro tubules (MTs) are found throughout proembryogenesis. Microtubules in the coenocytic endosperm are mainly internal. By contrast, there is a re-orientation of MTs to a transverse cortical distribution during zygote development, predominantly in a subapical band which accompanies a phase of apical extension. The presence of these cortical arrays coincides with the elongation of the zygote. Cortical arrays also accompany elongation of the cylindrical suspensor. Extensive networks of MTs ramify throughout the cytoplasm of cells in the proembryo proper. Perinuclear arrays are detected in a number of cell types and MTs contribute to typical mitotic configurations during nuclear divisions. Preprophase bands of MTs are absent throughout megasporogenesis and embryo-sac development and do not occur in endosperm cell divisions. We have observed MTs throughout the first division cycle of the zygote. By placing the observed stages in a most probable sequence, we have identified this cell cycle as the point during embryogenesis at which a preprophase band is reinstated as a regular feature of cell division. Preprophase bands were observed to predict planes of cytokinesis in cell divisions up to the octant stage.Abbreviations DIC differential interference contrast optics - MT microtubule - PPB preprophase band of microtubule We thank Ms. Margaret Travers for her helpful English translation of Yakovlev and Alimova (1976) and Mr. James Whitehead for preparation of Fig. 11. M.C.W. was supported by an Australian Postgraduate Research Award.     

Microtubule organization during development of stomatal complexes inLolium rigidum

Summary Microtubule (MT) arrays in stomatal complexes ofLolium have been studied using cryosectioning and immunofluorescence microscopy. This in situ analysis reveals that the arrangement of MTs in pairs of guard cells (GCs) or subsidiary cells (SCs) within a complex is very similar, indicating that MT deployment is closely coordinated during development. In premitotic guard mother cells (GMCs), MTs of the transverse interphase MT band (IMB) are reorganized into a longitudinal array via a transitory array in which the MTs appear to radiate from the cell edges towards the centre of the walls. Following the longitudinal division of GMCs, cortical MTs are reinstated in the GCs at the edge of the periclinal and ventral walls. The MTs become organized into arrays which radiate across the periclinal walls, initially from along the length of the ventral wall and later only from the pore site. As the GCs elongate, the organization of MTs and the patterns of wall expansion differ on the internal and external periclinal walls. A final reorientation of MTs from transverse to longitudinal is associated with the elongation and constriction of GCs to produce mature complexes. During cytokinesis in the subsidiary mother cells (SMCs), MTs appear around the reforming nucleus in the daughter epidermal cells but appear in the cortex of the SC once division is complete. Our results are thus consistent with the idea that interphase MTs are nucleated in the cell cortex in all cells of the stomatal complex but not in adjacent epidermal cells.Abbreviations GMC guard mother cell - GC guard cell - IMB interphase microtubule band - MT microtubule - PPB preprophase band - SMC subsidiary mother cell - SC subsidiary cell     

Pb/Cu effects on the organization of microtubule cytoskeleton in interphase and mitotic cells of Allium sativum L.

The effects of lead and copper on the arrangement of microtubule (MT) cytoskeleton in root tip cells of Allium sativum L. were investigated. Batch cultures of garlic were carried out under defined conditions in the presence 10⁻⁴ M Pb/Cu of various duration treatments. With tubulin immunolabelling and transmission electron microscopy (TEM), we found four different types of MT structures depending on the cell cycle stage: the interphase array, preprophase band, mitotic spindle and phragmoplast were typical for the control cells. Pb/Cu affected the mechanisms controlling the organization of MT cytoskeleton, and induces the following aberrations in interphase and mitotic cells. (1) Pb/Cu induced the formation of atypical MT arrays in the cortical cytoplasm of the interphase cells, consisting of skewed, wavy MT bundles, MT fragments and ring-like tubulin aggregations. (2) Pb/Cu disordered the chromosome movements carried out by the mitotic spindle. The outcome was chromosome aberrations, for example, chromosome bridges and chromosome stickiness, as well as inhibition of cells from entering mitosis. (3) Depending on the time of exposure, MTs disintegrated into shorter fragments or they completely disappeared, indicating MT depolymerization. (4) Different metals had different effects on MT organization. MTs were more sensitive to the pressure of Cu ions than Pb. Moreover, TEM observations showed that the MTs were relatively short and in some places wavy when exposed to 10⁻⁴ M Pb/Cu solutions for 1–2 h. In many sections MTs were no longer visible with increasing duration of treatment (>4 h). Based on these results, we suggested that MT cytoskeleton is primarily responsible for Pb/Cu-associated toxicity and tolerance in plants.     

Polarized endoplasmic reticulum aggregations in the establishing division plane of protodermal cells of the fern <Emphasis Type="Italic">Asplenium nidus</Emphasis>

The determination of the division plane in protodermal cells of the fern Asplenium nidus occurs during interphase with the formation of the phragmosome, the organization of which is controlled by the actomyosin system. Usually, the phragmosomes between adjacent cells were oriented on the same plane. In the phragmosomal cortical cytoplasm, an interphase microtubule (MT) ring was formed and large quantities of endoplasmic reticulum (ER) membranes were gathered, forming an interphase U-like ER bundle. During preprophase/prophase, the interphase MT ring and the U-like ER bundle were transformed into a MT and an ER preprophase band (PPB), respectively. Parts of the ER-PPB were maintained during mitosis. Furthermore, the plasmalemma as well as the nuclear envelope displayed local polarization on the phragmosome plane, while the cytoplasm between them was occupied by distinct ER aggregations. These consistent findings suggest that Α. nidus protodermal cells constitute a unique system in which three elements of the endomembrane system (ER, plasmalemma, and nuclear envelope) show specific characteristics in the establishing division plane. Our experimental data support that the organization of the U-like ER bundle is controlled on a cellular level by the actomyosin system and intercellularly by factors emitted from the leaf apex. The possible role of the above endomembrane system elements on the mechanism that coordinates the determination of the division plane between adjacent cells in protodermal tissue of A. nidus is discussed.     

F-actin redistributions at the division site in livingTradescantia stomatal complexes as revealed by microinjection of rhodamine-phalloidin

Summary Microinjection of rhodamine-phalloidin into living cells of isolatedTradescantia leaf epidermis and visualisation by confocal microscopy has extended previous results on the distribution of actin in mitotic cells of higher plants and revealed new aspects of actin arrays in stomatal cells and their initials. Divisions in the stomatal guard mother cells and unspecialised epidermal cells are symmetrical. Asymmetrical divisions occur in guard mother precursor cells and subsidiary mother cells. Each asymmetrical division is preceded by migration of the nucleus and the subsequent accumulation of thick bundles of anticlinally oriented actin filaments localised to the area of the anticlinal wall closest to the polarised nucleus. During prophase, in all cell types, a subset of cortical actin filaments coaligns to form a band, which, like the preprophase band of microtubules, accurately delineates the site of insertion of the future cell wall. Following the breakdown of the nuclear envelope, F-actin in these bands disassembles but persists elsewhere in the cell cortex. Thus, cortical F-actin marks the division site throughout mitosis, firstly as an appropriately positioned band and then by its localised depletion from the same region of the cell cortex. This sequence has been detected in all classes of division inTradescantia leaf epidermis, irrespective of whether the division is asymmetrical or symmetrical, or whether the cell is vacuolate or densely cytoplasmic. Taken together with earlier observations on stamen hair cells and root tip cells it may therefore be a general cytoskeletal feature of division in cells of higher plants.Abbreviations GMC guard mother cell - MT microtubule - PPB preprophase band - Rh rhodamine - SMC subsidiary mother cell     

Cell cycle-dependent targeting of a kinesin at the plasma membrane demarcates the division site in plant cells

Eukaryotic cells have developed different mechanisms to establish the division plane. In plants, the position is determined before the onset of mitosis by the preprophase band (PPB). This ring of microtubules surrounds the nucleus and disappears completely by prometaphase. An unknown marker is left behind by the PPB, providing the necessary spatial cues during cytokinesis. At the position of the PPB, cortical actin is removed or modified to generate an actin-depleted zone that was proposed to provide the structural means for phragmoplast guidance. Here, we identify a plasma membrane domain that emerges at the onset of mitosis and persists until the end of cytokinesis. The narrow band in the plasma membrane corresponds to the position of the PPB and is prevented from accumulation of a GFP-tagged kinesin GFP-KCA1; hence, it is called the KCA-depleted zone (KDZ). The KDZ demarcates the cortical division site independent from the mitotic cytoskeleton. Cell divisions in the absence of a KDZ resulted in misplaced cell plates, suggesting that the PPB transmits a signal to the plasma membrane required for correct cell plate guidance and vesicular targeting to the cortical division site.     

Rearrangement of the actin filament and microtubule cytoskeleton during induction of microspore embryogenesis inBrassica napus L. cv. Topas

Summary Changes in the actin filament and microtubule cytoskeleton were examined during heat- and cytochalasin D-induced embryogenesis in microspores ofBrassica napus cv. Topas by rhodamine phalloidin and immunofluorescence labelling respectively. The nucleus was displaced from its peripheral to a more central position in the cell, and perinuclear actin microfilaments and microtubules extended onto the cytoplasm. Heat treatment induced the formation of a preprophase band of microtubules in microspores; preprophase bands are not associated with the first pollen mitosis. Actin filament association with the preprophase band was not observed. The orientation and position of the mitotic spindle were altered, and it was surrounded with randomly oriented microfilaments. The phragmoplast contained microfilaments and microtubules, as in pollen mitosis I, but it assumed a more central position. Cytoskeletal reorganisation also occurred in microspores subjected to a short cytochalasin D treatment, in the absence of a heat treatment. Cytochalasin D treatment of microspores resulted in dislocated mitotic spindles, disrupted phragmoplasts, and symmetric divisions and led to embryogenesis, confirming that a normal actin cytoskeleton has a role in preventing the induction of embryogenesis.Abbreviations CD cytochalasin D - MF actin microfilament - MT microtubule - PPB preprophase band