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     

Microtubules and morphogenesis in ordinary epidermal cells ofVigna sinensis leaves

Summary Undifferentiated ordinary epidermal cells (ECs) ofVigna sinensis leaves possess straight anticlinal walls and cortical microtubules (Mts) scattered along them. At an early stage of EC differentiation cortical Mts adjacent to the above walls form bundles normal to the leaf plane, loosely interconnected through the cortical cytoplasm of the internal periclinal wall. At the upper ends of the Mt bundles, Mts fan out towards the external periclinal wall and form radial arrays. Mt bundles and radial arrays exhibit strict alternate disposition between neighbouring ECs. An identical reticulum of cellulose microfibril (CM) bundles is deposited outside the Mt bundles. Local wall pads rise at the junctions of anticlinal walls with the external periclinal one, where the CM bundles terminate. They display radial CMs fanning towards the external periclinal wall. The CM bundles and radial CM systems prevent local cell bulging, but allow it in the intervening wall areas. In particular, the radial CM systems dictate the pattern of EC waviness by favouring local tangential expansion of external periclinal wall. As a result, ECs obtain an undulate appearance. Constrictions in one EC correspond with protrusions of adjacent ECs. ECs affected by colchicine entirely lose their Mts and do not develop wavy walls, an observation substantiating the role of cortical Mts in EC morphogenesis.Abbreviations CM cellulose microfibril - DTT dithiothreitol - EC epidermal cell - MSB microtubule stabilizing buffer - Mt microtubule - PBS phosphate buffered saline - PMSF phenylmethylsulfonyl fluoride     

Patterns of cortical and perinuclear microtubule organization in meristematic root cells ofAdiantum capillus veneris

Summary The interphase meristematic root cells ofAdiantum capillus venerispossess a well developed cytoskeleton of cortical microtubules (Mts), which disappear at prophase. The preprophase-prophase cells display a well organized preprophase microtubule band (PMB) and a perinuclear Mt system. The observations favour the suggestion that the cell edges included in the PMB cortical zone possess a Mt organizing capacity and thus play an important role in PMB formation. The perinuclear Mts are probably organized on the nuclear surface. The preprophase-prophase nuclei often form protrusions towards the PMB cortical zone and the spindle poles, assuming a conical or rhomboid shape. Mts may be involved in this nuclear shaping.Reinstallation of cortical Mts in dividing cells begins about the middle of cytokinesis with the reappearance of short Mts on the cell surface. When cytokinesis terminates, numerous Mts line the postcytokinetic daughter wall. Many of them converge or form clusters in the cytoplasm occupying the junctions of the new and the old walls. In the examined fern, the cortical Mt arrays seem to be initiated in the cortex of post-cytokinetic root cells. A transitory radial perinuclear Mt array, comparable to that found in post-telophase root cells of flowering plants, was not observed inA. capillus veneris.     

Microtubule organization,mesophyll cell morphogenesis,and intercellular space formation inAdiantum capillus veneris leaflets

Summary Mesophyll cells (MCs) ofAdiantum capillus veneris are elongated and highly asymmetric, bearing several lateral branches and forming a meshwork resembling aerenchyma. Young MCs are polyhedral and display oppositely arranged walls and transverse cortical microtubules (Mts). Their morphogenesis is accomplished in three stages. At first they become cylindrical. Intercellular space (IS) canals, containing PAS-positive material, open through their junctions and expand laterally. During the second stage the cortical Mts form a reticulum of bundles, externally of which an identical reticulum of wall thickenings, containing bundles of parallel cellulose microfibrils, emerges. MCs do not grow in girth in the regions of wall thickenings, where constrictions form and new ISs open. Thus, MCs obtain a multi-lobed form. At the third morphogenetic stage MCs display a multi-axial growth. During this process, additional Mt rings are assembled at the base of cell lobes accompanied by similarly organized wall thickenings-cellulose microfibrils. Consequently, cell lobes elongate to form lateral branches, where MCs attach one another, while the IS labyrinth broadens considerably. Colchicine treatment, destroying Mts, inhibits MC morphogenesis and the concomitant IS expansion, but does not affect IS canal formation. These observations show that: (a) MC morphogenesis inA. capillus veneris is an impressive phenomenon accurately controlled by highly organized cortical Mt systems. (b) The disposition of Mt bundles between neighbouring MCs is highly coordinated, (c) The perinuclear cytoplasm does not appear to be involved in cortical Mt formation. Cortical sites seem to participate in Mt bundling, (d) Although extensive IS canals open before Mt bundling, the Mtdependent MC morphogenesis contributes in IS formation.Abbreviations EM electron microscopy - ER endoplasmic reticulum - IS intercellular space - MC mesophyll cell - MSB microtubule stabilizing buffer - Mt microtubule - PBS phosphate buffered saline     

Immunofluorescence and electron microscopic studies of microtubule organization during the cell cycle ofDictyota dichotoma (Phaeophyta,Dictyotales)

Summary Interphase cells ofDictyota dichotoma (Hudson) Lamour. lack cortical microtubules (Mts) but display an impressive network of cytoplasmic microtubules (c-Mts). These are focussed on two opposed perinuclear centriolar sites where centrin or a centrin-homologue is localized. Some of the Mts surround the nucleus, but the majority traverse the cytoplasm as bundles variously directed towards the plasmalemma. In apical cells, and to a lesser extent in the square or slightly elongated meristematic cells, Mts are more or less evenly arranged. In elongated cells they form thick bundles longitudinally traversing the cytoplasm; a pattern maintained in differentiated cells. In early prophase the non-perinuclear Mts disappear but by late prophase a bi-astral arrangement of short Mts is observed. They enter polar nuclear depressions and attach to differentiated regions of the nuclear envelope where polar gaps open. By metaphase the spindle Mts converge on the centrioles at the polar gaps. At anaphase, interzonal Mts are evident and the asters start to reassemble. After telophase disruption of the interzonal Mts, the daughter nuclei approach each other, but move apart again before cytokinesis. The latter movement keeps pace with the development of two interdigitating Mt systems, ensheathing both daughter nuclei. The partition membrane bisects this Mt cage. Between telophase and cytokinesis the centrosomes separate, finally occupying opposed perinuclear sites. New Mts arise at the new centrosomes, some terminating on the consolidating partition membrane. Our data show thatD. dichotoma vegetative cells display a prominent cytoplasmic Mt cytoskeleton, which undergoes continual, but definite, change in organization during the cell cycle.     

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     

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     

Microtubular fir-trees in mitotic spindles of onion roots

Summary The organization of kinetochore fibers was examined inAllium root cells processed for tubulin immunocytochemistry. Metaphase fibers consist of a core or trunk of Mts to which are attached numerous branches, yielding a bottle-brush of fir-tree pattern similar to that reported inHaemanthus endosperm cells. Many of the branches cross the midzone and extend into the opposite half-spindle. In addition, branch Mts associate with more than one kinetochore fiber. During anaphase, branch Mts elongate while the trunks shorten and fuse into polar caps. Our results are discussed in terms of spindle fiber organization and Mt polarity.Abbreviations K Kinetochore - Mt microtubule     

Tubulin conformation in microtubule-free cells ofVigna sinensis

Summary The primary leaf, epicotyl, and root cells ofVigna sinensis seedlings grown continuously in a 0.08% colchicine solution, become microtubule-free and polyploid. In meristematic root cells a tubulin transformation is detected 1–3 h after the treatment had begun. Tubulin strands are organized at the positions of the pre-existing microtubules. Frequently, the strands converge on or are organized in the cortical cytoplasmic zone where in normal cells the preprophase microtubule band (PMB) is assembled. In meristematic root cells subjected to a 6–12 h colchicine treatment, the tubulin strands become perinuclear, entering the cortical cytoplasm at regions close to the nucleus. One day after the onset of the treatment, tubulin generally forms a continuous reticulum of interconnected strands in all the organs examined. In most cells this reticulum surrounds the nucleus partly or totally or lies close to it, exhibiting variable configurations in different cells. After prolonged treatments, the organization of the tubulin reticulum changes further. Now this consists of crystal-like structures interconnected by thin strands.On thin sections of fixed tissue the tubulin strands consist of paracrystalline material. The distribution of this material in the affected cells coincides with that of tubulin reticulum visualized by immunofluorescence. In transverse planes each strand exhibits circular subunits arranged close to one another in a hexagonal pattern but in longitudinal ones variable images were observed. The paracrystalline material persists in root cells subjected to an 8-day continuous colchicine treatment. The immunolabeled strands seem to be composed of tubulin-colchicine complexes and not pure tubulin.     

Gamma-tubulin colocalizes with microtubule arrays and tubulin paracrystals in dividing vegetative cells of higher plants

Summary The distribution of -tubulin throughout cell division is studied in several taxa of higher plants. -Tubulin is present along the whole length of microtubules (Mts) in every cell stage-specific Mt array such as the preprophase band, the preprophase-prophase perinuclear Mts, the kinetochore Mt bundles, the phragmoplast, and the telophase-interphase transition Mt arrays. -Tubulin follows with precision the Mt pattern, being absent from any other, Mt-free, cell site. In cells treated with anti-Mt drugs, -tubulin is present only on degrading or on reappearing Mt arrays, while it is totally absent from cells devoid of Mts. -Tubulin is also present in tubulin paracrystals, which are formed in colchicine-treated cells. These observations support the view that in higher plants -tubulin may not be a microtubule-organizing-center-specific protein, but it may play a certain structural and/or functional role being related to - and -tubulin.Abbreviations Mt microtubule - MTOC microtubule-organizing center - PPB preprophase band     

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.     

Aluminium Effects on Microtubule Organization in Dividing Root-Tip Cells of Triticum turgidum. II. Cytokinetic Cells

Triticum turgidum were examined, using tubulin immunolabeling and electron microscopy. In cells, which at the beginning of the treatment were at a transitional stage between anaphase and telophase, the transformation of the interzonal microtubule (Mt) system into a phagmoplast was delayed. In cells treated at a telophase/ cytokinetic stage, the lateral phragmoplast expansion towards the cell periphery was delayed or inhibited. Besides, in cells entering telophase through an abnormal mitosis, Al inhibited phragmoplast formation and induced the organization of atypical tubulin bundles. The latter formed a network around the reassembling polyploid nucleus. The Al-effects resulted in the disturbance of cytokinesis and the formation of binuclear or polyploid cells, which lacked typical Mts. Instead of them, the post-telophase cells displayed atypical tubulin aggregations. In addition, Al affects cell plate development. Dividing cells, encompassing early interphase daughter nuclei, contained incomplete, atypical cell plates. The latter were quite thick, wavy and perforated, showing large “islands”, which contained electron transparent material. In some cells, the atypical cell plates gave rise to incomplete daughter walls, but in some others they were dismantled. The aberrant cell plates as well as the young daughter cell walls fluoresced intensely after aniline blue staining, an observation suggesting that they contain significant quantities of callose. The above findings combined with those derived from the study of the Al-effects on the mitotic spindle show that Mt cytoskeleton is a target site of Al toxicity in dividing cells. Received 24 October 2000/ Accepted in revised form 19 January 2001     

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.     

Reorganization of the microtubule array in prophase/prometaphase requires cytoplasmic dynein-dependent microtubule transport

When mammalian somatic cells enter mitosis, a fundamental reorganization of the Mt cytoskeleton occurs that is characterized by the loss of the extensive interphase Mt array and the formation of a bipolar mitotic spindle. Microtubules in cells stably expressing GFP-alpha-tubulin were directly observed from prophase to just after nuclear envelope breakdown (NEBD) in early prometaphase. Our results demonstrate a transient stimulation of individual Mt dynamic turnover and the formation and inward motion of microtubule bundles in these cells. Motion of microtubule bundles was inhibited after antibody-mediated inhibition of cytoplasmic dynein/dynactin, but was not inhibited after inhibition of the kinesin-related motor Eg5 or myosin II. In metaphase cells, assembly of small foci of Mts was detected at sites distant from the spindle; these Mts were also moved inward. We propose that cytoplasmic dynein-dependent inward motion of Mts functions to remove Mts from the cytoplasm at prophase and from the peripheral cytoplasm through metaphase. The data demonstrate that dynamic astral Mts search the cytoplasm for other Mts, as well as chromosomes, in mitotic cells.     

Centrosome and microtubule dynamics in apical cells ofSphacelaria rigidula (Phaeophyceae) treated with nocodazole

Summary Treatment of young thalli ofSphacelaria rigidula with 0.04 g of nocodazole (Nz) per ml for up to 36 h affects microtubules (Mts) only slightly, but blocks a large number of mitotic cells in metaphase, without disruption of the metaphase plate. Higher concentrations of Nz (0.1 g/ml) depolymerize interphase Mts. Only a few perinuclear and some short Mts resist and remain associated with the centrosomes. Fragmented Mts or groups of Mts sometimes remain in the apical dome. After treatment with 0.1 g of Nz per ml, prometaphase cells are blocked at metaphase, while post-metaphase cells become binuclear, due to the failure of cytokinesis. With anticentrin immunofluorescence, a positive centrin signal is always observed in the centrosome area. Centrosome duplication is not affected by Nz, but separation is disturbed. After recovering for 2–4 h, most of the blocked metaphases proceed normally. In such cells duplicated centrosomes are seen in different stages of separation. In some cells independent aster-like microtubule configurations appear in the apical dome, occasionally displaying centrin at their centre. During recovery various configurations of bimitosis or multipolar mitosis were found. The multipolar spindles may share common centrosomes. Up to four centrosomes may accompany each nucleus. In some 24 h treated cells, as well as in cells recovering for 2 h, the centrin-positive structure is rod-like, extending in opposite directions from the usual position to the poles. Electron microscopical examination of thin sections revealed that the growth pattern of the apical cells is disrupted after Nz treatment. The observations show that: (a) the Mt cytoskeleton is involved in maintaining the polarity and growth pattern of apical cells, (b) mitosis is blocked by low concentrations of Nz without significant depolymerization of Mts, (c) the centrosome cycle is independent of the nuclear cycle, (d) centrosome separation and differentiation are disturbed by Nz treatment, (e) during recovery from Nz treatment, centrosomal material that may have separated from the centrosomes, as well as Mt fragments that resisted depolymerization, may operate as Mt nucleation centres.Abbreviations DIC differential interference contrast - EM electron microscope - Mt microtubule - MTOC microtubule-organizing center - Nz nocodazole - NBBC nucleus-basal body connector     

Anaphase chromosome separation in dividing generative cells ofTradescantia

Summary In dividing generative cells ofTradescantia, kinetochore pairs do not line up on a typical metaphase plate, but instead are distributed along the length and depth of the cell prior to anaphase onset. Kinetochore (K) fibers are linked to each other and to a system of axial microtubule (Mt) bundles in an arrangement that makes discrete half spindles, if present, not immediately obvious. Because such arrangements may have important implications for the conduct of the remainder of division, anaphase events were closely scrutinized using a combination of tubulin and kinetochore immunocytochemistry (the latter with CREST serum). Anaphase appears to consist of three principal processes. Around the time of anaphase onset, K-fibers and surrounding Mts become reorganized into two large superbundles. To each superbundle is attached a set of nonfilial kinetochores bound for one end of the cell. The K-fibers then appear to shorten to varying degrees; in many cases, kinetochores become linked directly to the superbundles. The superbundles then separate in an anaphase B-like process, further moving the kinetochores toward opposite ends of the cell. The superbundles themselves shorten, and distances within the bundles also decrease, such that the kinetochores cluster closer together. The results indicate that reorganization of Mts into superbundles (and the consequential manifestation of bipolarity) is important for orderly chromosome separation.     

Calmodulin and wound healing in the coenocytic green alga Ernodesmis verticillata (Kützing) Børgesen

The involvement of calmodulin (CaM) in wound-induced cytoplasmic contractions in E. verticillata was investigated. Indirect immunofluorescence of CaM in intact cells showed a faint, reticulate pattern of fluorescence in the cortical cytoplasm. Diffuse fluorescence was evident deeper within the cytoplasm. In contracted cells, CaM co-localizes with actin in the cortical cytoplasm in extensive, longitudinal bundles of microfilaments (MFs), and in an actin-containing reticulum. No association of CaM with tubulin was ever observed in the cortical cytoplasm at any stage of wound-healing. When contraction rates in wounded cells are measured, a lag period of 2 min is followed by a rapid, steady rate of movement over the subsequent 10 min. The delay in the initiation of longitudinal contraction corresponds to the time necessary for the assembly of the longitudinal MF bundles. Cytoplasmic motility was inhibited in a dose-dependent manner by CaM antagonists. In these inhibited cells, MF bundles did not assemble, or were poorly formed. In the latter case, CaM was always found associated with MFs. These results indicate a direct spatial and temporal correlation between CaM and actin, and a potential role for CaM in regulating the formation of functional MF bundles during wound-induced cytoplasmic contraction in Ernodesmis.Abbreviations CaM calmodulin - DMSO dimethyl sulfoxide - EGTA ethylene glycol-bis(-aminoethyl ether)-N,N,N,N-tetraacetic acid - MF(s) microfilament(s) - MT(s) microtubule(s) - TFP trifluoperazine - w-5 N-(6-aminohexyl)-1-naphthalenesulfonamide - W-7 N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide We are especially grateful to: Dr. J.A. West (University of California, Berkeley) for the original algal isolates; Dr. L. Van Eldik (Vanderbilt University School of Medicine) and Dr. J.L. Lessard (University of Cincinnati College of Medicine) for graciously providing CaM and actin antibodies, respectively; Dr. S.J. Roux (University of Texas, Austin) for the gift of purified oat CaM; Dr.H. Green (Smith, Kline and French Laboratories, Philadelphia, Penn., USA) for providing the trifluoperazine; and M.E.T. Scioli for assistance with the statistical analyses. Portions of this work were supported by National Science Foundation grant DCB 8402345 and U.S. Department of Agriculture grant 87-CRCR-1-2545 to J.W.L.     

γ-Tubulin and microtubule organization during microsporogenesis in Ginkgo biloba

This is the first report on -tubulin and microtubule arrays during microsporogenesis in a gymnosperm. Meiosis in Ginkgo biloba is polyplastidic, as is typical of the spermatophyte clade, and microtubule arrays are organized at various sites during meiosis and cytokinesis. In early prophase, a cluster of -tubulin globules occurs in the central cytoplasm adjacent to the off-center nucleus. These globules diminish in size and spread over the surface of the nucleus. A system of microtubules focused on the -tubulin forms a reticulate pattern in the cytoplasm. As the nucleus migrates to the center of the microsporocyte, -tubulin becomes concentrated at several sites adjacent to the nuclear envelope. Microtubules organized at these foci of -tubulin give rise to a multipolar prophase spindle. By metaphase I, the spindle has matured into a distinctly bipolar structure with pointed poles. In both first and second meiosis, -tubulin becomes distributed throughout the metaphase spindles, but becomes distinctly polar again in anaphase. In telophase I, -tubulin moves from polar regions to the proximal surface of chromosome groups/nuclei where interzonal microtubules are organized. No cell wall is deposited and the interzonal microtubules embrace a plate of organelles between the two nuclear cytoplasmic domains (NCDs) of the dyad. Following second meiosis, phragmoplasts that form between sister and non-sister nuclei fuse to form a complex six-sided structure that directs simultaneous cytokinesis. -Tubulin becomes associated with nuclei after both meiotic divisions and is especially conspicuous in the distal hemisphere of each young microspore where an unusual encircling system of cortical microtubules develops.     

The effect of taxol onTriticum preprophase root cells: preprophase microtubule band organization seems to depend on new microtubule assembly

Summary To examine whether preprophase microtubule band (PPB) organization occurs by rearrangement of pre-existing, or by assembly of new microtubules (Mts), we treated root cells ofTriticum turgidum with taxol, which stabilizes pre-existing Mts by slowing their depolymerization. With taxol early preprophase cells failed to form a normal PPB and PPB narrowing was prevented in cells that had already formed a wide one. The PPB became persistent in prometaphase cells and the formation of multipolar prophase-prometaphase spindles was induced. These data favour the suggestion that PPB formation and narrowing, as well as prophase spindle development, are dynamic processes depending on continuous Mt assembly at the PPB site and in the perinuclear cytoplasm.Abbreviations Mt microtubule - MTOC microtubule organizing centre - PPB preprophase microtubule band - DMSO dimethyl sulfoxide     

Kinetochore behavior during generative cell division inTradescantia virginiana

Summary Previous observations indicate that division of the generative cell inTradescantia virginiana is characterized by several unusual features, including persistence of surrounding microtubule (Mt) bundles during karyokinesis, lack of a distinct metaphase plate and direct contribution by mitotic Mts to the cytoskeleton of young sperm. We have further probed karyokinesis in these cells using additional antitubulin and chromosome staining, as well as kinetochore visualizations with CREST serum. The CREST antibodies reveal kinetochores as paired and single fluorescent dots similar to those seen in other species stained with this preparation. Double localizations show that the dots are located at the ends of Mt bundles previously identified as kinetochore fibers (Palevitz and Cresti 1989). Before anaphase, paired kinetochores are distributed along the length of the cell. They also tend to be located at the cell periphery or are directly connected to peripheral Mt bundles by their kinetochore (K)-fibers. Twelve pairs of dots can be counted per cell, equal to the expected number of chromosomes. During anaphase, kinetochore separation starts at various positions along the length of the cell, producing single, relatively uniformly distributed kinetochores in the crotches of forks formed by K-fiber trunks and elongating Mt branches attached to the base of the trunks. Eventually, K-fibers with attached kinetochores aggregate in stepwise fashion on thick Mt bundles at both ends of the cell. This pattern is reflected in the cytoskeleton of young sperm. These results further document the unusual distribution of chromosomes and kinetochores inTradescantia generative cells and the origin of the Mt cytoskeleton in sperm cells.Abbreviations CREST Calcinosis, Raynaud's phenomenon, Esophageal dysmotility, Sclerodactyly, Telangiectasia - K-fiber kinetochore fiber - Mt microtubule Dedicated to the memory of Professor Oswald Kiermayer