Nuclear cytoplasmic domains,microtubules and organelles in microsporocytes of the slipper orchid Cypripedium californicum A. Gray dividing by simultaneous cytokinesis

Microsporocytes of the slipper orchidCypripedium californicum A. Gray divide simultaneously after second meiosis. The organization and apportionment of the cytoplasm throughout meiosis are functions of nuclear-based radial microtubule systems (RMSs) that define domains of cytoplasm - a single sporocyte domain before meiosis, dyad domains within the undivided cytoplasm after first meiosis, and four spore domains after second meiosis. Organelles migrate to the interface of dyad domains in the undivided cytoplasm after first meiotic division, and second meiotic division takes place simultaneously on both sides of the equatorial organelle band. Microtubules emanating from the telophase II nuclei interact to form columnar arrrays that interconnect all four nuclei, non-sister as well as sister. Cell plates are initiated in these columns of microtubules and expand centrifugally along the interface of opposing RMSs, coalescing in the center of the sporocyte and joining with the original sporocyte wall at the periphery to form the tetrad of microspores. Organelles are distributed into the spore domains in conjunction with RMSs. These data, demonstrating that cytokinesis in microsporogenesis can occur in the absence of both components of the typical cytokinetic apparatus (the preprophase band of microtubules which predicts the division site and the phragmoplast which controls cell-plate deposition), suggest that plant nuclei have an inherent ability to establish a domain of cytoplasm via radial microtubule systems and to regulate wall deposition independently of the more complex cytokinetic apparatus of vegetative cells.     

MICROTUBULES ASSOCIATED WITH SIMULTANEOUS CYTOKINESIS OF COENOCYTIC MICROSPOROCYTES

Microtubule arrays associated with simultaneous cytokinesis in the coenocytic microsporocytes of Lonicera japonica and Impatiens sultani were studied by indirect immunofluorescence. The future division planes are not predicted prior to meiosis by either a preprophase band of microtubules or cytoplasmic lobing. Cleavage planes appear to be determined by position of the four haploid nuclei and the development of postmeiotic microtubule systems. Perpendicular second division spindles in Lonicera result in tetrahedrally arranged tetrads while parallel spindles in Impatiens result in tetragonal arrangement. Immediately following meiosis bands of microtubules, the secondary spindles, develop between both sister and nonsister nuclei. These arrays give way to systems of microtubules that radiate equally from each of the four nuclei in the coenocytic sporocyte. Simultaneous cytokinesis is initiated by centripetal wall deposition at the periphery of the sporocyte and proceeds along planes marked by interaction of the opposing arrays of nuclear-based microtubules.     

Polar organizers and girdling bands of microtubules are associated with γ-tubulin and act in establishment of meiotic quadripolarity in the hepatic Aneura pinguis (Bryophyta)

Summary. Meiosis in Aneura pinguis is preceded by extensive cytoplasmic preparation for quadripartitioning of the diploid sporocyte into a tetrad of haploid spores. In early prophase the four future spore domains are defined by lobing of the cytoplasm and development of a quadripolar prophase spindle focused at polar organizers (POs) centered in the lobes. Cells entering the reproductive phase become isolated and, instead of hooplike cortical microtubules, have endoplasmic microtubule systems centered on POs. These archesporial cells proliferate by mitosis before entering meiosis. In prophase of each mitosis, POs containing a distinct concentration of γ-tubulin appear de novo at tips of nuclei and initiate the bipolar spindle. Cells entering meiosis become transformed into quadrilobed sporocytes with four POs, one in each lobe. This transition is a complex process encompassing assembly of two opposite POs which subsequently disperse into intersecting bands of microtubules that form around the central nucleus. The girdling bands define the future planes of cytokinesis and the cytoplasm protrudes through the restrictive bands becoming quadrilobed. Two large POs reappear in opposite cleavage furrows. Each divides and the resulting POs migrate into the tetrahedral lobes of cytoplasm. Cones of microtubules emanating from the four POs interact to form a quadripolar microtubule system (QMS) that surrounds the nucleus in meiotic prophase. The QMS is subsequently transformed into a functionally bipolar metaphase spindle by migration of poles in pairs to opposite cleavage furrows. These findings contribute to knowledge of microtubule organization and the role of microtubules in spatial regulation of cytokinesis in plants. Correspondence and reprints: Department of Biology, University of Louisiana-Lafayette, Lafayette, LA 70504-2451, U.S.A.     

A new type of microtubular cytoskeleton in microsporogenesis of Lavatera thuringiaca L.

Summary. In Lavatera thuringiaca, kariokinesis and simultaneous cytokinesis during the meiotic division of microsporogenesis follow a procedure similar to that which takes place in the majority of members of the class Angiospermae. However, chondriokinesis occurs in a unique way found only in species from the family Malvaceae. Chondriokinesis in such species is well documented, but the relationship between the tubulin cytoskeleton and rearrangement of cell organelles during meiosis in L. thuringiaca has not been precisely defined so far. In this study, the microtubular cytoskeleton was investigated in dividing microsporocytes of L. thuringiaca by immunofluorescence. The meiotic stages and positions of cell organelles were identified by staining with 4′,6-diamidino-2-phenylindole. We observed that, during prophase I and II, changes in microtubular cytoskeleton configurations have unique features, which have not been described for other plant species. At the end of prophase I, organelles (mostly plastids and mitochondria) form a compact envelope around the nucleus, and the subsequent phases of kariokinesis take place within this arrangement. At this point of cell division, microtubules surround the organelle envelope and separate it from the peripheral cytoplasm, which is devoid of plastids and mitochondria. In telophase I, two newly formed nuclei are tightly surrounded by the cell organelle envelopes, and these are separated by the phragmoplast. Later, when the phragmoplast disappears, cell organelles still surround the nuclei but also move a little, starting to occupy the place of the disappearing phragmoplast. After the breakup of tetrads, the radial microtubule system is well developed, and cell organelles can still be observed as a dense envelope around the nuclei. At a very late stage of sporoderm development, the radial microtubule system disappears, and cell organelles become gradually scattered in the cytoplasm of the microspores. Using colchicines, specific inhibitors of microtubule formation, we investigated the relationship between the tubulin cytoskeleton and the distribution of cell organelles. Our analysis demonstrates that impairment of microtubule organization, which constitutes only a single component of the cytoskeleton, is enough to disturb typical chondriokinesis in L. thuringiaca. This indicates that microtubules (independent of microfilaments) are responsible for the reorganization of cell organelles during meiotic division. Correspondence: D. Tchórzewska, Department of Plant Anatomy and Cytology, Maria Curie-Skłodowska University, Akademicka 19, 20-033 Lublin, Poland.     

Pollen development in orchids 1. Cytoskeleton and the control of division plane in irregular patterns of cytokinesis

Summary The cytokinetic apparatus in microsporogenesis lacks a preprophase band of microtubules and the selection of cytokinetic planes is dependent upon disposition of nuclei which define cytoplasmic domains via post-meiotic radial systems of microtubules. Meiotic cytokinesis was investigated in hybrid moth orchids (Phalaenopsis) exhibiting irregular patterns of cytokinesis. In these polliniate orchids, spindle orientation is imprecise, and the tetrad nuclei (therefore the microspores) may be in rhomboidal, tetrahedral or linear arrangement. The hybrid Sabine Queen (section Phalaenopsis) regularly undergoes simultaneous cytokinesis, as is common in orchids. The hybrid Vista Rainbow (section Amboinenses) produces either a complete dyad wall, a partial wall, or no wall after first nuclear division. In all cases, a first division phragmoplast is initiated in the interzonal region and expands centrifugally into the peripheral cytoplasm. Fluorescence microscopy shows that the phragmoplast consists of fusiform bundles of microtubules and Factin bisected by a non-fluorescent zone. If a cell plate fails to form, a band of organelles polarized in the equatorial region effectively divides the cell into two domains. The organelles disperse when a dyad wall is complete, but tend to remain polarized around an incomplete wall. In four-nucleate coenocytes, the usual interzonal microtubules between sister nuclei (primary) form slightly in advance of secondary arrays between non-sister nuclei. Phragmoplasts are initiated in sites defined by the post-meiotic microtubule arrays.Abbreviations CLSM confocal laser scanning microscope/microscopy - DMSO dimethylsulfoxide - FITC fluorescein isothiocyanate - PPB preprophase band of microtubules - TEM transmission electron microscope/microscopy     

Transformations of the Pleiomorphic Plant MTOC during Sporogenesis in the Hepatic Marchantia polymorpha

Sporogenesis in the hepatic Marchantia polymorpha L. provides an outstanding example of the pleiomorphic nature of the plant microtubule organizing center （MTOC）. Microtubules are nucleated from γ-tubuUn in MTOCs that change form during mitosis and meiosis. Following entry of cells into the reproductive pathway of sporogenesis, successive rounds of mitosis give rise to packets of 4-16 sporocytes. Mitotic spindles are organized at discrete polar organizers （POs）, a type of MTOC that is unique to this group of early divergent land plants. An abrupt and radical transformation in microtubule organization occurs when sporocytes enter meiosis： POs are lost and γ-tubulin is closely associated with surfaces of two large elongated plastids that subsequently divide into four. Migration of the four plastid MTOCs into a tetrahedral arrangement establishes the future spore domains and the division polarity of meiosis. As is typical of many bryophytes, cones of microtubules from the four plastid MTOCs initiate a quadripolar microtubule system （QMS） in meiotic prophase. At this point a transformation in the organization of the MTOCs occurs. The γ-tubulin detaches from plastids and forms a diffuse spheroidal pole in each of the spore domains. The plastids, which are no longer MTOCs, continue to divide. The diffuse MTOCs continue to nucleate cones of microtubules during transformation of the QMS to a bipolar spindle. Following meiosis I, γ-tubulin is associated with nuclear envelopes, and the spindles of meiosis II are organized from diffuse MTOCs at the tetrad poles. At simultaneous cytokinesis, radial microtubule systems are organized at nuclear envelope MTOCs in each of the tetrad members.     

Microfilament Distribution in Maize Meiotic Mutants Correlates with Microtubule Organization

Microtubules and microfilaments often codistribute in plants; their presumed interaction can be tested with drugs although it is not always clear that these are without side effects. In this study, we exploited mutants defective in meiotic cell division to investigate in a noninvasive way the relationship between the two cytoskeletal elements. By staining unfixed, permeabilized cells with rhodamine-phalloidin, spatial and temporal changes in microfilament distribution during maize meiosis were examined. In wild-type microsporocytes, a microtubule array that radiates from the nucleus disappeared during spindle formation and returned at late telophase. This result differed from the complex cytoplasmic microfilament array that is present at all stages, including karyokinesis and cytokinesis. During division, a second class of microfilaments also was observed in the spindle and phragmoplast. To analyze this apparent association of microtubules and microfilaments, we examined several meiotic mutants known to have stage-specific disruptions in their microtubule arrays. Two mutations that altered the number or form of meiotic spindles also led to a dramatic reorganization of F-actin. In contrast, rearrangement of nonspindle, cytoplasmic microtubules did not lead to concomitant changes in F-actin distribution. These results suggested that microtubules and microfilaments interact in a cell cycle-specific and site-specific fashion during higher plant meiosis.     

Division polarity,development and configuration of microtubule arrays in bryophyte meiosis

Summary First and second division spindles and the three cell plates of moss meiosis are oriented in accordance with polarity established during meiotic prophase. Plastids are located at the second division poles and cytoplasmic infurrowing marks the planes along which the cytoplasm will cleave into four spores. Anaphase I spindles that terminate in two focal points of microtubules straddling opposite cleavage furrows reflect the unusual tetrahedral origin of the functionally bipolar spindle. The organelles (except for the plastids which remain in the four cytoplasmic lobes) are polarized in the first division equatorial region at the time of phragmoplast microtubule assembly and remain in a distinct band after microtubule disassembly. Prophasic spindles appear to be directly transformed into metaphase II spindles in the predetermined axes between mutually perpendicular pairs of plastids. Cell plates form by vesicle coalescence in the equatorial regions of the two sets of second division phragmoplasts at approximately the same time as a cell plate belatedly forms in the organelle band. The cytoplasmic markers (plastid migration, cytoplasmic lobing and infurrowing) that predict poles and cleavage planes in free cells lacking a preprophase band strongly strengthens the concept that division sites are capable of preserving preprogrammed signals that can be triggered later in the process of cell division.     

Plastid polarity and meiotic spindle development in microsporogenesis ofSelaginella

Summary Microsporogenesis inSelaginella was studied by fluorescence light microscopy and transmission electron microscopy. As in other examples of monoplastidic meiosis the plastids are involved in determination of division polarity and organization of microtubules. However, there are important differences: (1) the meiotic spindle develops from a unique prophase microtubule system associated with two plastids rather than from a typical quadripolar microtubule system associated with four plastids; (2) the division axes for first and second meiotic division are established sequentially, whereas as in all other cases the poles of second division are established before those of first division; and (3) the plastids remain in close contact with the nucleus throughout meiotic prophase and provide clues to the early determination of spindle orientation. In early prophase the single plastid divides in the plane of the future division and the two daughter plastids rotate apart until they lie on opposite sides of the nucleus. The procytokinetic plate (PCP) forms in association with the two slender plastids; it consists of two spindle-shaped microtubule arrays focused on the plastid tips with a plate of vesicles at the equatorial region and a picket row of microtubules around one side of the nucleus. Second plastid division occurs just before metaphase and the daughter plastids remain together at the spindle poles during first meiotic division. The meiotic spindle develops from merger of the component arrays of the PCP and additional microtubules emanating from the pair of plastid tips located at the poles. After inframeiotic interphase the plastids migrate to tetrahedral arrangement where they serve as poles of second division.Abbreviations AMS axial microtubule system - FITC fluorescein isothiocyanate - MTOC microtubule organizing center - PCP procytokinetic plate - QMS quadripolar microtubule system - TEM transmission electron microscope (microscopy)     

TETRASPORE encodes a kinesin required for male meiotic cytokinesis in Arabidopsis

A key step in pollen formation is the segregation of the products of male meiosis into a tetrad of microspores, each of which develops into a pollen grain. Separation of microspores does not occur in tetraspore (tes) mutants of Arabidopsis thaliana, owing to the failure of male meiotic cytokinesis. tes mutants thus generate large 'tetraspores' containing all the products of a single meiosis. Here, we report the positional cloning of the TES locus and details of the role played by the TES product in male cytokinesis. The predicted TES protein includes an N-terminal domain homologous to kinesin motors and a C-terminus with little similarity to other proteins except for a small number of plant kinesins. These include the Arabidopsis HINKEL protein and NACK1 and two from tobacco (Nishihama et al., 2002), which are involved in microtubule organization during mitotic cytokinesis. Immunocytochemistry shows that the characteristic radial arrays of microtubules associated with male meiotic cytokinesis fail to form in tes mutants. The TES protein therefore is likely to function as a microtubule-associated motor, playing a part either in the formation of the radial arrays that establish spore domains following meiosis, or in maintaining their stability.     

Microtubular configurations during meiosis and megasporogenesis in Gasteria verrucosa and Chamaenerion angustifolium

Summary In Gasteria and Chamaenerion, microtubular configurations were visualized immunocytochemically during meiosis and megasporogenesis in order to study their relationship to cell development, meiotic divisions and selection of the functional megaspore. In Chamaenerion, the intensity of the fluorescence found in megaspores was weaker than that found in Gasteria. Both plants exhibited concentrations of microtubules around the meiocyte nuclei during pachytene-diplotene. Preprophase bands were not observed. In Chamaenerion, cytoplasmic microtubules radiating from meiocyte nuclei were found at late prophase, the dyad stage and in the functional megaspore; in Gasteria, they were observed only at the dyad stage and in the functional megaspore. During the second meiotic division of Gasteria, dividing cells and their nuclei exhibited differences in volumes. Also, the two microtubular spindles of the dyad cells had different widths. Fluorescence indicating the presence of the cytoskeleton diminished during maturation of the large functional megaspores in both plants, whereas in the three degenerating smaller megaspores, fluorescence intensity persisted. Our conclusion is that only an indirect relationship exists between the organization of the microtubular cytoskeleton and selection of the functional megaspore.     

Phragmoplasts in the Absence of Nuclear Division

All land plants (embryophytes) use a phragmoplast for cytokinesis. Phragmoplasts are distinctive cytoskeletal structures that are instrumental in the deposition of new walls in both vegetative and reproductive phases of the life cycle. In meristems, the phragmoplast is initiated among remaining non-kinetochore spindle fibers between sister nuclei and expands to join parental walls at the site previously marked by the preprophase band of microtubules (PPB). The microtubule cycle and cell cycle are closely coordinated: the hoop-like cortical microtubules of interphase are replaced by the PPB just prior to prophase, the PPB disappears as the spindle forms, and the phragmoplast mediates cell plate deposition after nuclear division. In the reproductive phase, however, cortical microtubules and PPBs are absent and cytokinesis may be uncoupled from the cell cycle resulting in multinucleate cells (syncytia). Minisyncytia of 4 nuclei occur in microsporocytes and several (typically 8) nuclei occur in the developing megagametophyte. Macrosyncytia with thousands of nuclei may occur in the nuclear type endosperm. Cellularization of syncytia involves formation of adventitious phragmoplasts at boundaries of nuclear-cytoplasmic domains (NCDs) defined by radial microtubule systems (RMSs) emanating from non-sister nuclei. Once initiated in the region of microtubule overlap at interfaces of opposing RMSs, the adventitious phragmoplasts appear structurally identical to interzonal phragmoplasts. Phragmoplasts are constructed of multiple opposing arrays similar to what have been termed microtubule converging centers. The individual phragmoplast units are distinctive fusiform bundles of anti-parallel microtubules bisected by a dark mid-zone where vesicles accumulate and fuse into a cell plate.     

Division polarity and plasticity of the meiosis I spindle inCypripedium californicum (Orchidaceae)

Summary Establishment of division polarity and meiotic spindle organization in the lady's slipper orchidCypripedium californicum A. Gray was studied by immunocytochemistry, confocal and transmission electron microscopy. Prior to organization of the spindle for meiosis I, the cytoplasmic domains of the future dyad and spindle polarity are marked by: (1) constriction of the prophase nucleus into an hourglass shape; (2) reorganization of nuclear-based radial microtubules into two arrays that intersect at the constriction; and (3) redistribution of organelles into a ring at the boundary of the newly defined dyad domains. It is not certain whether the opposing microtubule arrays contribute directly to the anastral spindle which is organized in the perinuclear areas of the two hemispheres. By late prophase each half-spindle consists of a spline-like structure from which depart the kinetochore fibers. This peculiar spindle closely resembles the spline-like spindle of generative-cell mitosis in certain plants where the spindle is distorted by physical constraints of the slender pollen tube. In the microsporocyte, the elongate spindle of late prophase/metaphase is curved within the cell so that the poles are not actually opposite each other and chromosomes do not form a plate at the equator. By late telophase the poles of the shortened halfspindles lie opposite each other. Plasticity of the physically constrained plant spindle appears to be due to its construction from multiple units terminating in minipoles. Cytokinesis does not follow the first meiosis. However, the dyad domains are clearly defined by radial microtubules emanating from the two daughter nuclei and the domains themselves are separated by a disc-like band of organelles.     

MONOPLASTIDIC CELL DIVISION IN LOWER LAND PLANTS

In many bryophytes and vascular cryptogams mitosis and/or meiosis takes place in cells containing a single plastid. In monoplastidic cell division plastid polarity assures that nuclear and plastid division are infallibly coordinated. The two major components of plastid polarity are morphogenetic plastid migration and microtubule organization at the plastids. Before nuclear division the plastid migrates to a position intersecting the future division plane. This morphogenetic migration is a reliable marker of division polarity in cells with and without a preprophase band of microtubules (PPB). The PPB, which predicts the future division plane before mitosis, is a characteristic feature of land plants and its insertion into the cytokinetic apparatus marks the evolution of a cortical microtubule system and a commitment to meristematic growth. Microtubule systems associated with plastid division, the axial microtubule system (AMS) in mitosis and the quadripolar microtubule system (QMS) in meiosis, contribute to predictive positioning of plastids and participate directly in spindle ontogeny. Division polarity in monoplastidic sporocytes is remarkable in that division sites are selected prior to the two successive nuclear divisions of meiosis. Plastid arrangement prior to meiosis determines the future spore domains in monoplastidic sporocytes, whereas in polyplastidic sporocytes the spore nuclei play a major role in claiming cytoplasmic domains. It is hypothesized that predivision microtubule systems associated with monoplastidic cell division are early forming components of the mitotic apparatus that serve to orient the spindle and insure equal apportionment of nucleus and plastids. “Can it be supposed that cytoplasm would be intrusted with so important a task as the preparation of a chloroplast for each of the four nuclei that are later to preside over the spores before there is any indication that such nuclear division is to take place?” Bradley Moore Davis, 1899     

Endosperm Development in Barley: Microtubule Involvement in the Morphogenetic Pathway

An immunofluorescence study of sectioned barley endosperm imaged by confocal laser scanning microscopy provided three-dimensional data on the relationship of microtubules to the cytoplasm, nuclei, and cell walls during development from 4 to 21 days after pollination (DAP). Microtubules play an important role throughout endosperm ontogeny. The syncytium is organized into units of nuclear-cytoplasmic domains by nuclear-based radial microtubule systems that appear to control the pattern of the first anticlinal walls at 5 to 6 DAP. After 7 DAP, phragmoplasts of two origins (interzonal and cytoplasmic) guide wall formation. Large compartments formed by the "free growing" walls in association with cytoplasmic phragmoplasts formed adventitiously at interfaces of opposing microtubule systems are subsequently subdivided by interzonal phragmoplast/cell plates to give rise to the starchy endosperm. During development of the aleurone layer from 8 to 21 DAP, the microtubule cycle is typical of plant histogenesis; cortical microtubules are hooplike, and preprophase bands of microtubules predict the division plane.     

A differential phenotypic expression of a divergent spindle mutation in interspecific Brachiaria hybrids

Several mutations are known to alter the normal progression of meiosis and can be correlated with defects in microtubule distribution. The dv mutation affects the spindle organization and chromosomes do not converge into focused poles. Two Brachiaria hybrids presented the phenotypic expressions of dv mutation but exhibited many more details in the second division. Bivalents were distantly positioned and spread over a large metaphase plate and failed to converge into focused poles. Depending on the distance of chromosomes at the poles, telophase I nuclei were elongated or the chromosomes were grouped into various micronuclei of different sizes in each cell. The first cytokinesis occurred. However, when there were micronuclei, a second cytokinesis immediately took place dividing the prophase II meiocytes into three or four cells. In each meiocyte, meiosis progressed to the second division. Slightly elongated nuclei or micronuclei were recorded in telophase II. After a third cytokinesis, hexads or octads were formed. Pollen grains of different sizes were generated. One of these hybrids presented a higher frequency of abnormal cells than when previously analyzed. The fate of these hybrids as genitors or as candidates for cultivars in the Brachiaria breeding program is discussed.     

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

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

γ-Tubulin,microtubule arrays,and quadripolarity during sporogenesis in the hepatic<Emphasis Type="Italic"> Aneura pinguis</Emphasis> (Metzgeriales)

This is the first report on the organization of a quadripolar microtubule system (QMS) in polyplastidic meiosis of a hepatic with polar organizers (POs). Unlike the monoplastidic sporocytes of mosses and hornworts, in which meiotic quadripolarity can be traced to plastid division and migration, sporocytes of Aneura pinguis are polyplastidic and tetrahedrally lobed before the QMS is organized. Whereas the QMS in mosses and hornworts is plastid-based, the QMS of A. pinguis is focused at four POs where gamma tubulin (-tubulin) is concentrated. An aster of microtubules emanates from each PO centered in the four cytoplasmic lobes and the opposing radial microtubules interact to form the QMS that envelops the nucleus. A functionally bipolar spindle is gradually formed as the four poles converge in pairs on either side of opposite cleavage furrows. The resulting spindle remains quadripolar. Although -tubulin is most concentrated in the deeply concave poles straddling cleavage furrows, it also extends into the spindle itself. Telophase groups of chromosomes curve around the polar cleavage furrows and a phragmoplast that originates in the interzonal region guides a cell plate that extends to the equatorial cleavage furrows. Discrete POs are reformed at opposite tips of the elongated dyad nuclei in prophase II and microtubules radiating from them give rise to the spindles of second meiosis. Spindles remain sharply focused and -tubulin extends into distal portions of the spindle. Interzonal phragmoplasts that expand to join with pre-established cleavage furrows mediate cytokinesis resulting in a tetrad of spores. Each young tetrad member has a radial microtubule system emanating from the nucleus.     

A beta-tubulin mutation selectively uncouples nuclear division and cytokinesis in Tetrahymena thermophila

The ciliated protozoan Tetrahymena thermophila contains two distinct nuclei within a single cell-the mitotic micronucleus and the amitotic macronucleus. Although microtubules are required for proper division of both nuclei, macronuclear chromosomes lack centromeres and the role of microtubules in macronuclear division has not been established. Here we describe nuclear division defects in cells expressing a mutant beta-tubulin allele that confers hypersensitivity to the microtubule-stabilizing drug paclitaxel. Macronuclear division is profoundly affected by the btu1-1 (K350M) mutation, producing cells with widely variable DNA contents, including cells that lack macronuclei entirely. Protein expressed by the btu1-1 allele is dominant over wild-type protein expressed by the BTU2 locus. Normal macronuclear division is restored when the btu1-1 allele is inactivated by targeted disruption or expressed as a truncated protein. Immunofluorescence studies reveal elongated microtubular structures that surround macronuclei that fail to migrate to the cleavage furrows. In contrast, other cytoplasmic microtubule-dependent processes, such as cytokinesis, cortical patterning, and oral apparatus assembly, appear to be unaffected in the mutant. Micronuclear division is also perturbed in the K350M mutant, producing nuclei with elongated early-anaphase spindle configurations that persist well after the initiation of cytokinesis. The K350M mutation affects tubulin dynamics, as the macronuclear division defect is exacerbated by three treatments that promote microtubule polymerization: (i) elevated temperatures, (ii) sublethal concentrations of paclitaxel, and (iii) high concentrations of dimethyl sulfoxide. Inhibition of phosphatidylinositol 3-kinase (PI 3-kinase) with 3-methyladenine or wortmannin also induces amacronucleate cell formation in a btu1-1-dependent manner. Conversely, the myosin light chain kinase inhibitor ML-7 has no effect on nuclear division in the btu1-1 mutant strain. These findings provide new insights into microtubule dynamics and link the evolutionarily conserved PI 3-kinase signaling pathway to nuclear migration and/or division in Tetrahymena.     

The quadripolar microtubule system in lower land plants

The quadripolar microtubule system (QMS) is a complex array that is associated with predivision establishment of quadripolarity in sporocytes of lower plants (bryophytes and lycopsids). The QMS unerringly predicts the polarity of the two meiotic divisions and plays a central role in development of both the mitotic apparatus (MA) and cytokinetic apparatus (CA) which together accomplish quadripartitioning of the sporocyte into four haploid spores. The QMS is typically, but not exclusively, associated with monoplastidy and precocious quadrilobing of the cytoplasm. In early meiotic prophase the single plastid divides and the resultant plastids migrate so that either the tips of two plastids or the four plastids resulting from a second division are located in the future spore domains. Microtubules that emanate from the plastid tips or from individual plastids in the spore domains interact in the future planes of cytokinesis and give rise to the QMS. The QMS, which encages the prophase nucleus, consists of at least four and usually six (when spore domains are in tetrahedral arrangement) bipolar spindle-like arrays of microtubules presumably with minus ends at plastids in spore domains and plus ends interacting in the future plane of cytokinesis. Each of the six arrays is essentially like the single axial microtubule system (AMS) that intersects the division site and is transformed into the spindle in monoplastidic mitosis in hornworts. As comparative data accumulate, it appears that the AMS is not unique to monoplastidic cell division but instead represents a basic microtubule arrangement that survives as spindle and phragmoplast in cell division of higher plants.