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)     

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.     

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     

Morphogenetic plastid migration and microtubule organization during megasporogenesis inIsoetes

Summary The large megasporocytes ofIsoetes provide an exceptional system for studying microtubule dynamics in monoplastidic meiosis where plastid polarity assures coordination of plastid and nuclear division by the intimate association of MTOCs with plastids. Division and migration of the plastid in prophase establishes the tetrahedrally arranged cytoplasmic domains of the future spore tetrad and the four plastid-MTOCs serve as focal points of a unique quadripolar microtubule system (QMS). The QMS is a dynamic structure which functions in plastid deployment and contributes directly to development of both first and second division spindles. The nucleation of microtubules at discrete plastid-MTOCs is compared with centrosomal nucleation of microtubules in animal cells where growth of microtubules involves dynamic instability.Abbreviations AMS axial microtubule system - MTOC microtubule organizing center - N nucleus - QMS quadripolar microtubule system - P plastid - PPB preprophase band of microtubules     

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.     

Polar organizers mark division axis prior to preprophase band formation in mitosis of the hepaticReboulia hemisphaerica (Bryophyta)

Summary Changes in the pattern of microtubules during the cell cycle of the hepaticReboulia hemisphaerica (Bryophyta) were studied by indirect immunofluorescence using conventional and confocal laser scanning microscopy (CLSM). The first indication that a cell is preparing for division is fusiform shaping of the nucleus accompanied by the appearance of well-defined polar organizers (POs) at the future spindle poles. Microtubules emanating from the POs ensheath the nucleus and eventually develop into the half-spindles of mitosis. Some of the microtubules from each PO pass tangential to the nucleus and interact in the region of the future mitotic equator. A preprophase band (PPB) forms in this region later in prophase and coexists with the prophase spindle. Thus, the plane of division appears to be determined by interaction of opposing arrays of microtubules emanating from POs. Prometaphase is marked by disappearance of the POs, loss of astral microtubules, and conversion of the fusiform spindle of prophase to a truncated, barrel-shaped spindle more typical of higher plants. Restoration of cortical microtubules in daughter cell occurs on the cell side distal to the new cell plate, but nucleation of microtubules is associated with the nuclear envelope and not with organized POs. At the next division POs appear at opposite poles of preprophase nuclei with no evidence of division and migration that is characteristic of cells with centriolar centrosomes. These data lend additional support for the view that mitosis in hepatics is transitional between green algae and higher plants.Abbreviations AMS axial microtubule system - CLSM confocal laser scanning microscopy - MTOC microtubule organizing center - PO polar organizer - PPB preprophase band of microtubules - QMS quadripolar microtubule system - TEM transmission electron microscopy     

Microtubules in ascidian eggs during meiosis, fertilization, and mitosis

The sequential changes in the distribution of microtubules during germinal vesicle breakdown (GVBD), fertilization, and mitosis were investigated with antitubulin indirect immunofluorescence microscopy in several species of ascidian eggs (Molgula occidentalis, Ciona savignyi, and Halocynthia roretzi). These alterations in microtubule patterns were also correlated with observed cytoplasmic movements. A cytoplasmic latticework of microtubules was observed throughout meiosis. The unfertilized egg of M. occidentalis had a small meiotic spindle with wide poles; the poles became focused after egg activation. The other two species had more typical meiotic spindles before fertilization. At fertilization, a sperm aster first appeared near the cortex close to the vegetal pole. It enlarged into an unusual asymmetric aster associated with the egg cortex. The sperm aster rapidly grew after the formation of the second polar body, and it was displaced as far as the equatorial region, corresponding to the site of the myoplasmic crescent, the posterior half of the egg. The female pronucleus migrated to the male pronucleus at the center of the sperm aster. The microtubule latticework and the sperm aster disappeared towards the end of first interphase with only a small bipolar structure remaining until first mitosis. At mitosis the asters enlarged tremendously, while the mitotic spindle remained remarkably small. The two daughter nuclei remained near the site of cleavage even after division was complete. These results document the changes in microtubule patterns during maturation in Ascidian oocytes, demonstrate that the sperm contributes the active centrosome at fertilization, and reveal the presence of a mitotic apparatus at first division which has an unusually small spindle and huge asters.     

Katanin controls mitotic and meiotic spindle length

Accurate control of spindle length is a conserved feature of eukaryotic cell division. Lengthening of mitotic spindles contributes to chromosome segregation and cytokinesis during mitosis in animals and fungi. In contrast, spindle shortening may contribute to conservation of egg cytoplasm during female meiosis. Katanin is a microtubule-severing enzyme that is concentrated at mitotic and meiotic spindle poles in animals. We show that inhibition of katanin slows the rate of spindle shortening in nocodazole-treated mammalian fibroblasts and in untreated Caenorhabditis elegans meiotic embryos. Wild-type C. elegans meiotic spindle shortening proceeds through an early katanin-independent phase marked by increasing microtubule density and a second, katanin-dependent phase that occurs after microtubule density stops increasing. In addition, double-mutant analysis indicated that gamma-tubulin-dependent nucleation and microtubule severing may provide redundant mechanisms for increasing microtubule number during the early stages of meiotic spindle assembly.     

Preprophasic microtubule systems and development of the mitotic spindle in hornworts (Bryophyta)

Summary Studies of monoplastidic mitosis in hornworts (Bryophyta) using transmission electron microscopy and indirect immunofluorescence staining of microtubules have revealed that two mutually perpendicular microtubule systems predict division polarity in preprophase. Events of cytoplasmic reorganization in preparation for division occur in the following order: migration of the single plastid to a position perpendicular to the division site, constriction of the plastid where its midpoint intersects the division site, development of an axial system of microtubules parallel to the elongating plastid isthmus, and appearance of an atypical preprophase band of microtubules (PPB). The PPB is asymmetrical with a tight band of microtubules on the side over the plastid isthmus and a broad band of widely spaced microtubules over the nucleus. The axial system contributes directly to development of the spindle. In prometaphase, the axial system separates at the equator and additional microtubule bundles project from polar regions, creating two opposing halfspindles. The PPB is still present during asymmetrical organization of the spindle and microtubules extending from the broad portion of the PPB to poles appear to be incorporated into the developing spindle. Dynamic changes in the microtubular cytoskeleton demonstrate (1) intimate relationship of plastid and nuclear division, (2) contribution of preprophase/prophase microtubule systems to spindle development in monoplastidic cells, and (3) dynamic reorientation of microtubules from one system to another.     

Microtubule distribution in dv, a maize meiotic mutant defective in the prophase to metaphase transition

Microsporogenesis in Zea mays, the meiotic reduction of diploid sporocytes to haploid microspores, proceeds through a well-defined developmental sequence. The ability to generate mutants that affect the process makes this an ideal system for elucidating the role of the cytoskeleton during plant development. We have used immunofluorescence microscopy to compare microtubule distribution in wild-type and mutant microsporocytes. During normal meiosis the distribution of microtubules follows a specific temporal and spatial pattern that reflects the polar nature of microspore formation. Perinuclear microtubule staining increases and the nucleus elongates in the future spindle axis during late prophase I. Metaphase I spindles with highly focused poles align along the long axis of the anther locule. Cytokinesis occurs perpendicular to the spindle axis. The second division axis shifts 90 degrees with respect to the first division plane, thereby yielding an isobilateral tetrad of microspores. Microtubule distribution patterns during meiosis suggest that a nuclear envelope-associated microtubule organizing center (MTOC) controls the organization of cytoplasmic microtubules and contributes to spindle formation. The meiotic mutant dv is defective in the transition from a prophase microtubule array to a metaphase spindle. Instead of converging to form focused poles, the metaphase spindle poles remain diffuse as in prometaphase. This defect correlates with several abnormalities in subsequent developmental events including the formation of multinucleate daughter cells, multiple microspindles during meiosis II, multiple phragmoplasts, polyads of microspores, and cytoplasmic microtubule foci. These results suggest that dv is a mutation that affects MTOC organization.     

Kinesin‐14 is Important for Chromosome Segregation During Mitosis and Meiosis in the Ciliate Tetrahymena thermophila

Ciliates such as Tetrahymena thermophila have two distinct nuclei within one cell: the micronucleus that undergoes mitosis and meiosis and the macronucleus that undergoes amitosis, a type of nuclear division that does not involve a bipolar spindle, but still relies on intranuclear microtubules. Ciliates provide an opportunity for the discovery of factors that specifically contribute to chromosome segregation based on a bipolar spindle, by identification of factors that affect the micronuclear but not the macronuclear division. Kinesin‐14 is a conserved minus‐end directed microtubule motor that cross‐links microtubules and contributes to the bipolar spindle sizing and organization. Here, we use homologous DNA recombination to knock out genes that encode kinesin‐14 orthologues (KIN141, KIN142) in Tetrahymena. A loss of KIN141 led to severe defects in the chromosome segregation during both mitosis and meiosis but did not affect amitosis. A loss of KIN141 altered the shape of the meiotic spindle in a way consistent with the KIN141's contribution to the organization of the spindle poles. EGFP‐tagged KIN141 preferentially accumulated at the spindle poles during the meiotic prophase and metaphase I. Thus, in ciliates, kinesin‐14 is important for nuclear divisions that involve a bipolar spindle.     

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.     

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.     

Division of cell nuclei, mitochondria, plastids, and microbodies mediated by mitotic spindle poles in the primitive red alga Cyanidioschyzon merolae

To understand the cell cycle, we must understand not only mitotic division but also organelle division cycles. Plant and animal cells contain many organelles which divide randomly; therefore, it has been difficult to elucidate these organelle division cycles. We used the primitive red alga Cyanidioschyzon merolae, as it contains a single mitochondrion and plastid per cell, and organelle division can be highly synchronized by a light/dark cycle. We demonstrated that mitochondria and plastids multiplied by independent division cycles (organelle G1, S, G2 and M phases) and organelle division occurred before cell–nuclear division. Additionally, organelle division was found to be dependent on microtubules as well as cell–nuclear division. We have observed five stages of microtubule dynamics: (1) the microtubule disappears during the G1 phase; (2) α-tubulin is dispersed within the cytoplasm without forming microtubules during the S phase; (3) α-tubulin is assembled into spindle poles during the G2 phase; (4) polar microtubules are organized along the mitochondrion during prophase; and (5) mitotic spindles in cell nuclei are organized during the M phase. Microfluorometry demonstrated that the intensity peak of localization of α-tubulin changed in the order to spindle poles, mitochondria, spindle poles, and central spindle area, but total fluorescent intensity did not change remarkably throughout mitotic phases suggesting that division and separation of the cell nucleus and mitochondrion is mediated by spindle pole bodies. Inhibition of microtubule organization induced cell–nuclear division, mitochondria separation, and division of a single membrane-bound microbody, suggesting that similar to cell–nuclear division, mitochondrion separation and microbody division are dependent on microtubules.     

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.     

Meiosis-Specific Stable Binding of Augmin to Acentrosomal Spindle Poles Promotes Biased Microtubule Assembly in Oocytes

In the oocytes of many animals including humans, the meiotic spindle assembles without centrosomes. It is still unclear how multiple pathways contribute to spindle microtubule assembly, and whether they are regulated differently in mitosis and meiosis. Augmin is a γ-tubulin recruiting complex which “amplifies” spindle microtubules by generating new microtubules along existing ones in mitosis. Here we show that in Drosophila melanogaster oocytes Augmin is dispensable for chromatin-driven assembly of bulk spindle microtubules, but is required for full microtubule assembly near the poles. The level of Augmin accumulated at spindle poles is well correlated with the degree of chromosome congression. Fluorescence recovery after photobleaching shows that Augmin stably associates with the polar regions of the spindle in oocytes, unlike in mitotic cells where it transiently and uniformly associates with the metaphase spindle. This stable association is enhanced by γ-tubulin and the kinesin-14 Ncd. Therefore, we suggest that meiosis-specific regulation of Augmin compensates for the lack of centrosomes in oocytes by actively biasing sites of microtubule generation within the spindle.     

ULTRASTRUCTURE OF SPOROGENESIS IN THE MOSS,AMBLYSTEGIUM RIPARIUM I. MEIOSIS AND CYTOKINESIS

Emphasis is placed on three aspects of meiosis in the moss Amblystegium riparium (Hedw.) BSG: 1***) nature of the sporogenous layer; 2) prophasic microtubules and polarity; and 3) cleavage pattern. Spore tetrads develop while still encased by archesporial cell walls. The cellular nature of the sporogenous layer differs from the more usual occurrence of free sporocytes released into a common spore sac. Two important events mark the establishment of sporocyte polarity during meiotic prophase: 1) migration of the four plastids to the distal tetrad poles (telophase II poles); and 2) ingrowth of the sporocyte wall in eventual cleavage planes between the tetrad poles. An extensive, plastid-based microtubule system is associated with organelle migration during the establishment of sporocyte polarity in meiotic prophase. Disruption of the nuclear envelope in prometaphase I occurs at sites opposite the four plastids where microtubules extend from plastid envelope to nuclear envelope. Formation of a cell plate following the first meiotic division results in a dyad, whereas in many mosses meiosis is completed in the undivided sporocyte and is followed by simultaneous cleavage into a spore tetrad. Spore cleavage is accomplished by vesicular coalescence resulting in septa that coincide with the prophasic wall ingrowths.     

Spindle formation and dynamics of gamma-tubulin and nuclear mitotic apparatus protein distribution during meiosis in pig and mouse oocytes

This work focuses on the assembly and transformation of the spindle during the progression through the meiotic cell cycle. For this purpose, immunofluorescent confocal microscopy was used in comparative studies to determine the spatial distribution of alpha- and gamma-tubulin and nuclear mitotic apparatus protein (NuMA) from late G2 to the end of M phase in both meiosis and mitosis. In pig endothelial cells, consistent with previous reports, gamma-tubulin was localized at the centrosomes in both interphase and M phase, and NuMA was localized in the interphase nucleus and at mitotic spindle poles. During meiotic progression in pig oocytes, gamma-tubulin and NuMA were initially detected in a uniform distribution across the nucleus. In early diakinesis and just before germinal vesicle breakdown, microtubules were first detected around the periphery of the germinal vesicle and cell cortex. At late diakinesis, a mass of multi-arrayed microtubules was formed around chromosomes. In parallel, NuMA localization changed from an amorphous to a highly aggregated form in the vicinity of the chromosomes, but gamma-tubulin localization remained in an amorphous form surrounding the chromosomes. Then the NuMA foci moved away from the condensed chromosomes and aligned at both poles of a barrel-shaped metaphase I spindle while gamma-tubulin was localized along the spindle microtubules, suggesting that pig meiotic spindle poles are formed by the bundling of microtubules at the minus ends by NuMA. Interestingly, in mouse oocytes, the meiotic spindle pole was composed of several gamma-tubulin foci rather than NuMA. Further, nocodazole, an inhibitor of microtubule polymerization, induced disappearance of the pole staining of NuMA in pig metaphase II oocytes, whereas the mouse meiotic spindle pole has been reported to be resistant to the treatment. These results suggest that the nature of the meiotic spindle differs between species. The axis of the pig meiotic spindle rotated from a perpendicular to a parallel position relative to the cell surface during telophase I. Further, in contrast to the stable localization of NuMA and gamma-tubulin at the spindle poles in mitosis, NuMA and gamma-tubulin became relocalized to the spindle midzone during anaphase I and telophase I in pig oocytes. We postulate that in the centrosome-free meiotic spindle, NuMA aggregates the spindle microtubules at the midzone during anaphase and telophase and that the polarity of meiotic spindle microtubules might become inverted during spindle elongation.     

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

Summary Immunofluorescence and TEM studies of meiosis in two mosses (Bryophyta) provide evidence that the prophasic tetrahedral system of microtubules contributes directly to the metaphase I spindle. Intense staining of tubulin, conspicuously absent around the nuclear envelope, is first seen associated with plastids. By mid-prophase, microtubules radiate from the plastids to the nuclear envelope and become organized into six bands that interconnect the four plastids, forming a tetrahedral cytoskeleton surrounding the nucleus. During transition of prophase to metaphase, the four poles of the tetrahedral microtubule system converge in pairs toward opposite cleavage furrows. Opposite furrows occupy mutually perpendicular planes and the pair of microtubule focal points straddling one furrow lies at right angles to the pair straddling the opposite furrow. Additional microtubules terminate in numerous small clusters in the concave polar regions arching over the cleavage furrows. By early anaphase, the microtubule focal points lie very close to the division axis. We conclude that microtubules recruited from the prophasic quadripolar system are incorporated into the mature metaphase I spindle and the two principal focal points at each pole are those derived from poles of the prophasic quadripolar system.     

Characterization of Polo-like kinase-1 in rat oocytes and early embryos implies its functional roles in the regulation of meiotic maturation,fertilization, and cleavage

Polo-like kinase 1 (Plk1) is a family of serine/threonine protein kinases that play important regulatory roles during mitotic cell cycle progression. In this study, Plk1 expression, subcellular localization, and possible functions during rat oocyte meiotic maturation, fertilization, and embryonic cleavages were studied by using RT-PCR, Western blot, confocal microscopy, drug-treatments, and antibody microinjection. Both the mRNA and protein of this kinase were detected in rat maturing oocytes and developing embryos. Confocal microscopy revealed that Plk1 distributed abundantly in the nucleus at the germinal vesicle (GV) stage, was associated with spindle poles during the formation of M-phase spindle, and was translocated to the spindle mid-zone at anaphase. In fertilized eggs, Plk1 was strongly stained in the cytoplasm between the apposing male and female pronuclei, from where microtubules radiated. Throughout cytokinesis, Plk1 was localized to the division plane, both during oocyte meiosis and embryonic mitosis. The specific subcellular distribution of Plk1 was distorted after disrupting the M-phase spindle, while additional aggregation dots could be induced in the cytoplasm by taxol, suggesting its intimate association with active microtubule assembly. Plk1 antibody microinjection delayed the meiotic resumption and blocked the emission of polar bodies. In conclusion, Plk1 may be a multifunctional kinase that plays pivotal regulatory roles in microtubule assembly during rat oocyte meiotic maturation, fertilization, and early embryonic mitosis.