Organization of the mitotic apparatus during generative cell division inNicotiana tabacum

Summary In order to gain a more complete understanding of the organization of the mitotic apparatus (MA) in the generative cells (GCs) of flowering plants, pollen tubes ofNicotiana tabacum were examined using tubulin immunocytochemistry and Hoechst fluorescence. The observations were then compared with previously published information onTradescantia GCs and the MA of somatic cells. At the onset of division, the prominent microtubule (Mt) bundles characteristic of GCs are reorganized into a more random Mt network. At late prophase/prometaphase, kinetochores appear to interact with this network, resulting in the formation of K-fibers that frequently link in tree-like aggregates. The GC MA takes the form of a distinct spindle and often has pointed, focused poles; the metaphase plate is usually oblique. Karyokinesis involves both anaphase A and B; lengthening of interzonal Mts is accompanied by elongation of the spindle. In late anaphase/early telophase, phragmoplast Mts are formed in association with the proximal face of the sperm nuclei. The phragmoplast remains prominent for some time, so that its Mts as well as another population generated from the distal face of the sperm nuclei constitute the initial sperm cytoskeleton. Comparisons indicate that the spindle in tobacco GCs falls on a continuum of organization between that of somatic cells and the MA ofTradescantia GCs.Abbreviations GC generative cell - MA mitotic apparatus - Mt microtubule     

Relationship between the generative cell and vegetative nucleus in pollen tubes of Nicotiana tabacum

Summary Fluorescence microscopy was used to visualize microtubules (Mts) and chromatin in an effort to further clarify the relationship between the generative cell (GC) and vegetative nucleus (VN) in pollen tubes of tobacco. Prominent Mt bundles are present in one or more GC extensions that can be finger-like or lamellar in form. While the VN is positioned distal to the GC in most cases, it can also straddle the cell or lie proximal to it. In all cases, however, extensions embrace, penetrate or clasp the VN. GC Mts are reorganized during the formation of the mitotic apparatus, and cell extensions are fully or partially withdrawn. By telophase in many pollen tubes, the VN shifts to a more proximal position and appears to adhere to the region of the GC containing the phragmoplast. Application of oryzalin leads to the disorganization of Mts, changes in cell shape, including the loss or alteration of cell extensions, and separation of the GC and VN in some cases. However, the position and polarity of the VN is maintained in most pollen tubes. The results indicate that GC Mts and cell extensions play a role in the association with the VN. However, the relationship appears to be controlled by other factors as well. Attention should now be directed at potential interactions involving the VN envelope, vegetative plasma membrane, GC plasma membrane and extracellular matrix.Abbreviations GC Generative cell - MGU male germ unit - Mt microtubule - VN vegetative nucleus     

Cytoskeletal changes during generative cell division and sperm formation inTradescantia virginiana

Summary Cytoskeletal organization and chromosome behavior were studied inTradescantia generative cells prior to and during sperm formation using in vitro grown pollen tubes and fluorescence staining methods. Before pollen germination, the crescent-shaped generative cell contains a reticulate microtubule (Mt) system. The cell elongates dramatically after germination, and its Mts assume a helical to longitudinal arrangement. Chromosome condensation is evident approximately 3hr after germination. Kinetochores appear as dark interruptions in the Mt array, and thus seem to attach directly to interphase fibers. No metaphase plate typical of other cells is observed with either DAPI or anti-tubulin staining. Instead, the chromosomes adopt a twisted or braided arrangement, with kinetochores distributed along the length of the cell and kinetochore fibers linked to each other and to surrounding fibers. Anaphase is characterized by a staggered, overlapping separation of chromosomes and by elongation of Mt branches connecting opposing kinetochore fibers. Cytokinesis appears to utilize a furrowing process; a phragmoplast or cell plate was never seen. As a result of these events, the sperm directly inherit their cytoskeleton from generative cell Mts involved in division. No actin fibers are observed at any stage using rhodamine-phalloidin staining. The results are discussed in terms of other reports on sperm formation, possible mitotic and cytokinetic mechanisms, and past distinctions between Mt arrays in higher plant somatic cells.Abbreviations CD cytochalasin D - DAPI 46-diamidino-2-phenyl-indole - DMSO dimethylsulfoxide - K-fiber kinetochore fiber - Mf microfilament - Mt microtubule - PPB preprophase Mt band - RP rhodamine phalloidin     

Localization of a 170 kDa myosin heavy chain in plant cells

Summary A polyclonal antibody directed against a 170 kDa myosin heavy chain from lily pollen tubes was employed to (a) assess the cellular distribution of the polypeptide using immunofluorescence methods, and (b) ascertain if similar polypeptides are present in pollen tubes and somatic cells of other species. Fluorescence is associated with particles of various size as well as an amorphous component, and is concentrated in the apical cytoplasm of lily and tobacco pollen tubes. Apical fluorescence is more extensive in lily than in tobacco, which may be related to different streaming patterns and apical zonation seen at the ultrastructural level. In suspension cells of tobacco andArabidopsis, fluorescence is concentrated around the nuclei. Dual localizations indicate that anti-myosin fluorescence may be associated with the presence of actin. Little or no staining was seen in controls consisting of either pre-immune serum or mono-specific IgG that had been preadsorbed with the 170 kDa polypeptide. Immunoblots show that a 170 kDa immunoreactive polypeptide is present in pollen tubes of tobacco andTradescantia virginiana in addition to lily, and in suspension culture cells of tobacco andArabidopsis and extracts of wholeArabidopsis seedlings. Our results show that a conserved 170 kDa myosin heavy chain is present in a variety of monocot and dicot cells. They are also consistent with the presence of multiple myosins in plants in general and pollen tubes in particular.Abbreviations BSA bovine serum albumin - IgG immunoglobulin G - Mf microfilament - Mt microtubule - PBS phosphate-buffered saline - PME 50 mM Pipes, 5mM EGTA - 2mM MgSO₄, pH6.9.     

Some events of mitosis and cytokinesis in the generative cell of Ornithogalum virens L.

The organization of the microtubule (Mt) cytoskeleton during mitosis and cytokinesis of the generative cell (GC) in Ornithogalum virens L. (bicellular pollen type, chromosome number, n = 3) from prophase to telophase/sperm formation was investigated by localization of -tubulin immunofluorescence using a conventional fluorescence microscope and a confocal laser scanning microscope. Chromosomes were visualized with DNA-binding fluorochrome dyes (ethidium bromide and 46-diamino-2-phenyl-indole). The GC of O. virens is characterized by G2/M transition within the pollen grain and not in the pollen tube as occurs in the majority of species with bicellular pollen. It was found that prophase in the GC starts before anthesis and prometaphase takes place after 10 min of pollen germination. The prophase Mts are organized into three prominent bundles, located near the generative nucleus. The number of these Mt bundles is the same as the number of GC chromosomes, a relation which has not previously been considered in other species. The most evident feature in the prophase/ prometaphase transition of O. virens GC is a direct rapid rearrangement of Mt bundles into a network which appears to interact with kinetochores and form a typical prometaphase Mt organization. The metaphase chromosomes are arranged into a conventional equatorial plate, and not in tandem as is thought to be characteristic of GC metaphase. The metaphase spindle consists of kinetochore fibres and a few interzonal fibres which form dispersed poles. Anaphase is characterized by a significant elongation of the mitotic spindle concomitant with the extension of the distance between the opposite poles. At anaphase the diffuse poles converge. Cytokinesis is realized by cell plate formation in the equatorial plane of the GC. The phragmoplast Mts between two future sperm nuclei appear after Mts of the mitotic spindle have disappeared.Abbreviations DAPI 46-diamino-2-phenyl-indole - GC generative cell - GN generative nucleus - Mt microtubule This research was made possible in part due to TEMPUS Programme and Global Network for Cell and Molecular Biology UNESCO grants to Magorzata Bana. The experimental part of the work was done in Siena University. M. Banas is very grateful to Prof. Mauro Cresti and his group for scientific interest, offering the excellent laboratory facilities, and kind reception.     

A kinesin-like protein, KatAp, in the cells of arabidopsis and other plants.

The kinesin-like proteins (KLPs) are a large family of plus- or minus-end-directed microtubule motors important in intracellular transport, mitosis, meiosis, and development. However, relatively little is known about plant KLPs. We prepared an antibody against two peptides in the microtubule binding domain of an Arabidopsis KLP (KatAp) encoded by the KatA gene, one of a family of genes encoding KLPs whose motor domain is located near the C terminus of the polypeptide. Such KLPs typically move materials toward the minus end of microtubules. An immunoreactive band (Mr of 140,000) corresponding to KatAp was demonstrated with this antibody on immunoblots of Arabidopsis seedling extracts. During immunofluorescence localizations, the antibody produced weak, variable staining in the cytoplasm and nucleus of interphase Arabidopsis suspension cells but much stronger staining of the mitotic apparatus during division. Staining was concentrated near the midzone during metaphase and was retained there during anaphase. The phragmoplast was also stained. Similar localization patterns were seen in tobacco BY-2 cells. The antibody produced a single band (Mr of 130,000) in murine brain fractions prepared according to procedures that enrich for KLPs (binding to microtubules in the presence of AMP-PNP but not ATP). A similar fraction from carrot suspension cells yielded a cross-reacting polypeptide of similar apparent molecular mass. When dividing BY-2 cells were lysed in the presence of taxol and ATP, antibody staining moved rapidly toward the poles, supporting the presence of a minus-end motor. Movement did not occur without ATP, with AMP-PNP, or with ATP plus antibody. Our results indicate that the protein encoded by KatA, KatAp, is expressed in Arabidopsis and is specifically localized to the midzone of the mitotic apparatus and phragmoplast. A similar protein is also present in other species.     

γ-Tubulin in tobacco pollen tubes: association with generative cell and vegetative microtubules

-Tubulin was localized in tobacco pollen tubes using an antibody raised against a peptide conserved in all known -tubulins. Antibody staining occurs in a primarily punctate pattern along the length of the microtubule bundles in generative cells and along cortical microtubules in the vegetative cytoplasm. During generative cell division, -tubulin is localized in the forming mitotic apparatus. By metaphase, it is present along kinetochore fibers except at their plus ends located at the kinetochores. By telophase, staining is observed in the phragmoplast, where it again avoids the plus ends of microtubules at the cell plate. -Tubulin is also present at the periphery of the sperm nuclei. A patch of intense staining on the distal side of each nucleus marks the site of assembly of a new population of sperm microtubules. No specific fluorescence is present in control pollen tubes treated with preimmune IgG. These localization patterns bear similarities to those seen in somatic cells and in addition may help explain changes in microtubule arrays between generative cells and sperm.     

21-kDa polypeptide,a low-molecular-weight cyclophilin,is released from pollen of higher plants into the extracellular medium in vitro

Calcium ions play a key role in the elongation and orientation of pollen tubes. We found that significant amounts of 21-kDa polypeptide were specifically released into the extracellular medium when pollen grains of lily, Lilium longiflorum Thunb., were incubated in the presence of EGTA or at low concentrations of Ca²⁺. This phenomenon was also dependent on pH and on the concentrations of MgCl₂ in the medium; the release of 21-kDa polypeptide from pollen was suppressed by increasing the MgCl₂ concentration and by lowering pH. Germination of pollen grains was inhibited in the medium into which the 21-kDa polypeptide had been released. This inhibition was irreversible; germination did not occur on transfer of the pollen grains into basal culture medium. Immuno-electron microscopy using an antibody against 21-kDa polypeptide showed that this polypeptide was present in the cytoplasm, vegetative nucleus and generative cell. When the pollen was treated with a medium containing EGTA, the density of 21-kDa polypeptide in the cytoplasm significantly decreased, but its density in vegetative nuclei and the generative cell did not, suggesting that only cytoplasmic 21-kDa polypeptide was released into the extracellular medium. The 21-kDa polypeptide was also present in the pollen of other higher-plant species, such as Tradescantia virginiana L., Nicotiana tabacum L. (angiosperms), and Cryptomeria japonica D. Don. (gymnosperm), and was also released into the medium in the presence of EGTA. In the case of C. japonica, however, it was released from pollen at alkaline pH above 8.5. The expression of 21-kDa polypeptide was not pollen-specific, because 21-kDa components immunoreactive with the anti-21-kDa polypeptide serum also existed in vegetative organs and cells of lily or tobacco. However, the 21-kDa polypeptide was not released into the extracellular medium from cultured tobacco BY-2 cells, even in the presence of EGTA. Amino acid sequences of two peptide fragments derived from 21-kDa polypeptide matched well those of low-molecular-weight cyclophilin (CyP). The antiserum against 21-kDa polypeptide recognized the CyP A from calf thymus and that in A431 carcinoma cells. The 21-kDa polypeptide fraction purified from lily pollen possessed peptidyl-prolyl cis-trans isomerase activity, which was suppressed by cyclosporin A (CsA), an inhibitor of enzyme activities of CyPs. From these results, we concluded that the 21-kDa polypeptide is a low-molecular-weight CyP. The present study showed that CyP in the pollen of higher plants is released into the extracellular matrix under unfavorable conditions.Abbreviations CaM Calmodulin - CBB Coomassie-brilliant-blue - CsA Cyclosporin A - CyP Cyclophilin     

The effects of a triazole fungicide,propiconazole, on pollen germination,tube growth and cytoskeletal distribution in Tradescantia virginiana

The effects of propiconazole on germination and tube growth of Tradescantia virginiana pollen when incorporated in germination media at 0, 102, 136, or 170 l l^–1 were evaluated using light microscopy and immunocytochemistry. Propiconazole inhibited pollen germination, cytoplasmic streaming, and tube elongation. Treatments also induced abnormal tube morphology and cytoskeletal distribution. Tubes treated with propiconazole displayed weaker microfilament (Mf) signals along the pollen tubes, with amorphous staining. Microtubule (Mt) distribution was also severely affected. In treated tubes, the proximal portions had characteristically fragmented Mts. Fewer Mt bundles were seen in the subapical region, and these were located further from the apex. Propiconazole effects were generally concentration dependent. The results indicate that propiconazole affects both Mfs and Mts; however, the effects may be an indirect result of the drug's influence on membranes.     

Dynein-related polypeptides in pollen and pollen tubes

 Microtubules in pollen tubes are evident within the vegetative and generative cell cytoplasm. This observation led to the formulation of several hypotheses regarding the role of microtubules in cytoplasmic movement and the migration of the vegetative nucleus/generative cell along the pollen tube. The study of microtubular motor proteins in pollen tubes followed the discovery and characterization of an immunoreactive homolog of mammalian kinesin in tobacco pollen tubes. Recent identification of dynein-related polypeptides in pollen tubes of Nicotiana tabacum and pollen of Ginkgo biloba is a significant step in the definition of the role of microtubule function within pollen and pollen tubes. Received: 31 May 1996 / Revision accepted: 26 July 1996     

Pollen mitosis in the slipper orchid Cypripedium fasciculatum

Pollen mitosis in the slipper orchid Cypripedium fasciculatum was studied using correlated methods of immunofluorescence and transmission electron microscopy. Unlike the more highly evolved orchids, the cypripedioid orchids shed pollen as monosulcate monads. Prior to pollen mitosis, the microspore nucleus migrates to a proximal position opposite the aperture, as is typical of monocotyledons. There is no distinct generative pole microtubule system (GPMS) like that recently reported in development of pollen polarity in the vandoid moth orchid Phalaenopsis. Instead, microtubules in early prophase are concentrated around the nucleus and extend into the cytoplasm toward the future generative pole. Once the nucleus has migrated to the continuous surface opposite the aperture, microtubules surround the nucleus evenly and show no tendency to be more concentrated in the generative domain. The mitotic spindle, which develops from the perinuclear microtubules, is asymmetrically placed in the microspore and is cone-shaped. The generative pole is broad and closely appressed to the continuous spore surface, while the vegetative pole is pointed and located in the interior of the microspore. As the chromosomes move poleward, microtubules proliferate in the interzone and a phragmoplast develops. The phragmoplast expands in a hemispherical path beyond the interzone following an array of microtubules that radiates from the generative nucleus. Data from this study indicate that evolution of pollen in orchids includes a shift in location of the generative cell from proximal to distal and the evolution of a GPMS, in addition in the well-known trend toward increased pollen aggregation and loss of exine.     

γ-Tubulin is associated with a cortical-microtubule-organizing zone in the developing guard cells of Allium cepa L.

A key event in the differentiation of elliptically shaped guard cells such as those in Allium is the formation of a radial array of cortical microtubules (Mts) which, by controlling the orientation of wall microfibrils, plays an important role in cell shaping. Previous experiments strongly indicated that the array is nucleated in a zone adjacent to the new ventral wall soon after cytokinesis. In order to further clarify the function of this zone, we performed dual immunolocalizations on Allium guard cells with anti--tubulin, to detect Mts, and an antibody to -tubulin, a protein known to be present at Mt-organizing centers in other species and recently identified in plants as well. -Tubulin antibody stained the cortical zone adjacent to the ventral wall, while little or no fluorescence was present elsewhere along the radial Mt array or at other sites in the cell. The antibody also stained the mitotic poles and phragmoplast in guard mother cells, as it does in other material. No staining was seen when the primary antibody was omitted. The results are consistent with nucleation of the radial array at a cortical-Mt-organizing zone next to the ventral wall, and set the stage for more in-depth studies on the spatial and temporal control of Mt formation in differentiating cells.Abbreviations CLSM confocal laser scanning microscope - FITC fluorescein isothiocyanate - Mt microtubule - MTOC microtubule-organizing center This work was supported by National Science Foundation grant DCB-9019285 to B.A.P., National Institutes of Health (NS30009) and American Cancer Society (CD6255) grants to H.C.J., and a University of Georgia Graduate School Assistantship to B.L. We thank Dr. Mark Farmer and the University of Georgia Center for Advanced Ultrastructural Research for the use of the confocal microscope.     

Telophase-interphase transition in taxol-treatedTriticum root cells: cortical microtubules appear without the prior presence of a radial perinuclear array

Summary Taxol stabilizes phragmoplast microtubules (Mts) in cytokinetic root cells ofTriticum, causing a delay in the rate of cytokinesis. As a result, the daughter nuclei acquire interphase appearance in mid- to late-cytokinetic taxol-affected cells much earlier than in control cells. Cortical Mts in such cells appear directly in the cell cortex, without the prior organization of a radial perinuclear Mt array as in control cells. These observations suggest that: (a) Whether perinuclear Mt assembly occurs or not in post-telophase cells is a matter of timing between the nuclear cycle and cytokinesis, (b) Mt organizing activity on the daughter nuclei surface is temporal, (c) Cortical Mts can be in situ assembled in the cortex of post-telophase cells of flowering plants without any participation of perinuclear Mts.Abbreviations Mt microtubules - MTOC microtubule organizing centre - DMSO dimethyl sulfoxide - EM electron microscope     

The organization of F-actin in root tip cells ofAdiantum capillus veneris throughout the cell cycle

Summary The patterns of F-actin in relation to microtubule (Mt) organization in dividing root tip cells ofAdiantum capillus veneris were studied with rhodamine-phalloidin (RP) labelling and tubulin immunofluorescence. Interphase cells display a well organized network of cortical/subcortical, endoplasmic and perinuclear actin filaments (AFs), not particularly related to the interphase Mt arrays. The cortical AFs seem to persist during the cell cycle while the large subcortical AF bundles disappear by preprophase/prophase and reappear after cytokinesis is completed. In some but not all of the preprophase cells the cortical AFs tend to form a band (AF-PPB) coincident with the preprophase band of Mts (Mt-PPB). In metaphase and anaphase cells AFs are localized in the cell cortex, around the spindle and inside it coincidently with kinetochore Mt bundles. During cytokinesis AFs are consistently found in the phragmoplast. In oryzalin treated cells neither Mt-PPBs, spindles and phragmoplasts exist, nor such F-actin structures can be observed. In cells recovering from oryzalin, AF-PPBs, AF kinetochore bundles and AF phragmoplasts reform. They show the same pattern with the reinstating respective Mt arrays. In contrast, in cells treated with cytochalasin B (CB), AFs disappear but all categories of Mt arrays form normally.These observations show that F-actin organization in root tip cells ofA. capillus veneris differs from that of root tip cells of flowering plants examined so far. In addition, Mts seem to be crucial for F-actin organization as far as it concerns the PPB, the mitotic spindle, and the phragmoplast.Abbreviations AF actin filament - CB cytochalasin B - MBS m-male-imidobenzoyl-N-hydroxysuccinimide ester - MSB microtubule stabilizing buffer - Mt microtubule - PBS phosphate buffered saline - PPB preprophase band - RP rhodamine phalloidin     

Localization of a kinesin-like calmodulin-binding protein in dividing cells of Arabidopsis and tobacco

The cloning and characterization of a novel kinesin-like protein ( k inesin-like c almodulin- b inding p rotein, KCBP) from Arabidopsis and other plants has recently been described. Unlike all other known kinesin-like proteins, KCBP interacts with calmodulin in the presence of micromolar calcium. An antibody specific to KCBP was raised using a calmodulin-binding synthetic peptide that is unique to KCBP. The KCBP antibody detected a single protein of about 140 kDa in Arabidopsis and tobacco, the size predicted from cDNA sequences. In synchronized cell cultures, the amount of KCBP was abundant during M-phase and very low in interphase. To get some insight into the function of this novel motor protein, KCBP in Arabidopsis and tobacco cells was localized by indirect immunofluorescence microscopy using affinity-purified anti-KCBP antibody. The KCBP was localized to the pre-prophase band, the mitotic spindle and the phragmoplast. The association of KCBP with microtubule arrays in dividing cells suggests that this minus-end-directed microtubule motor protein is likely to be involved in the formation of these microtubule arrays and/or functions associated with these structures.     

Clathrin is involved in organization of mitotic spindle and phragmoplast as well as in endocytosis in tobacco cell cultures

Summary. We previously identified a 175 kDa polypeptide in Lilium longiflorum germinating pollen using a monoclonal antibody raised against myosin II heavy chain from Physarum polycephalum. In the present study, the equivalent polypeptide was also found in cultured tobacco BY-2 cells. Analysis of the amino acid sequences revealed that the 175 kDa polypeptide is clathrin heavy chain and not myosin heavy chain. After staining of BY-2 cells, punctate clathrin signals were distributed throughout the cytoplasm at interphase. During mitosis and cytokinesis, clathrin began to accumulate in the spindle and the phragmoplast and then was intensely concentrated in the cell plate. Expression of the C-terminal region of clathrin heavy chain, in which light chain binding and trimerization domains reside, induced the suppression of endocytosis and the formation of an aberrant spindle, phragmoplast, and cell plate, the likely cause of the observed multinucleate cells. These data strongly suggest that clathrin is intimately involved in the formation of the spindle and phragmoplast, as well as in endocytosis. Correspondence and reprints: Department of Life Science, Graduate School of Life Science, University of Hyogo, Harima Science Park City, Hyogo 678-1297, Japan. Present address: RIKEN Plant Science Center, Yokohama, Kanagawa, Japan.     

The calmodulin-stimulated ATPase of maize coleoptiles is a 140000-Mr polypeptide

The calmodulin-stimulated ATPase of maize (Zea mays L.) coleoptiles has been purified by calcium-dependent binding to a calmodulin affinity column. In the presence of protease inhibitors (phenylmethylsulfonylfluoride and chymostatin) a polypeptide of relative molecular mass (M_r) 140000 (±10000) is obtained on sodium-dodecylsulphate polyacrylamide gels. This polypeptide is recognised specifically by an affinity-purified polyclonal antibody to mammalian calmodulin-stimulated calcium-pumping ATPases and is of similar M_r to the erythrocyte-membrane calcium pump (138000 M_r).Abbreviations EGTA ethylene glycol-bis(-aminoethylether)-N,N,N,N-tetraacetic acid - M_r apparent molecular mass - SDS sodium dodecyl sulphate     

The anti-centrosome monoclonal antibody 6C6 reacts with a plasma membrane-associated polypeptide of 77 kDa fromNicotiana tabacum pollen tubes

Summary In the pollen and pollen tube of higher plants, the distribution of the microtubular cytoskeleton has been extensively studied. Even though the pattern of microtubules is known, one of the most remarkable deficiencies is the absence of data on the localization of microtubule-nucleation sites in the pollen tubes. In order to get insights about the localization of centrosome-like structures in the pollen tube ofNicotiana tabacum L., we have used the monoclonal antibody 6C6 to search for pericentriolar antigen(s). The antibody was initially raised against a component of animal centrosomes and has been already employed to locate centrosomal structures in other plant cell types. By immunoblotting analysis, a polypeptide of Mr 77,000 was identified specifically in the membrane-associated protein fraction of the pollen tube, and is absent from the soluble protein pool. Immunofluorescence observations have shown the polypeptide to be located in the apical part of the pollen tube (about 40–50 m from the tip) in association with the cortical area. A purified plasma membrane fraction from the growing pollen tubes has been obtained, using H⁺-ATPase activity as an organelle marker. The plasma membrane fraction was shown to be enriched in the M_r 77,000 polypeptide, which can be extracted from membranes by treatment with the detergent CHAPS at a concentration of 0.5%. These data open new research perspectives on the localization and analysis of putative cortical microtubule nucleation sites in the pollen tube.Abbreviations ATP adenosine-5-triphosphate - CHAPS 3-[(3-cholamidopropyl)-dimethylammonio]-propanesulfonate - DTT dithiothreitol - EDTA ethylenediaminetetracetic acid - EGTA ethylene glycolbis(-amino-ethyl ether) N,N,N,N-tetraacetic acid - HEPES 4-(2-hydroxyethyl)-1-piperazineethane sulphonic acid - MES 2-(N-morpholino)ethane sulphonic acid - MT microtubule - SDS-PAGE sodium-dodecyl-sulphate polyacrylamide gel electrophoresis - PMSF phenylmethyl-sulphonyl-fluoride - TAME tosyl-arginine-methylester     

The mitotic apparatus during unequal microspore division observed by a confocal laser scanning microscope

Summary The structure of the mitotic apparatus during the microspore division ofTradescantia paludosa, which has a distinctively unequal division of large vegetative and small generative cells, was studied using -tubulin immunofluorescence methods and confocal laser scanning microscopy. Mitotic apparatuses began to develop asynchronously during early prophase at the vegetative pole (VP) and during prometaphase at the generative pole (GP). Both, however, reached completion together at the same time during metaphase. At the VP from prophase to prometaphase, microtubules (MTs) did not converge on the pole, and there was a circular area containing only a few MTs. The prophase spindles on the VP side were in the form of domes or cones that lacked the top. In the metaphase, however, the MTs concentrated at the pole to form a representative cone-shaped half-spindle. At the GP from prometaphase to metaphase, the MTs did not concentrate, and a circular area existed that lacked MTs. The half-spindles formed truncated cones. When the phragmoplast developed and curved around the generative nucleus during the telophase. it first grew toward the long axis of the ellipsoidal-shaped microspore; and after it arrived at the inner membrane of the microspore, it again curved past the generative nucleus toward the short axis. In conclusion, it was found that the mitotic apparatus ofT. paludosa microspores with its asynchronous growth and asymmetrical spindle structure and with its three dimensional growth of phragmoplasts had a peculiar developmental manner related to unequal division.