Pre-meiotic bands and novel meiotic spindle ontogeny in quadrilobed sporocytes of leafy liverworts (Jungermannidae, Bryophyta)

Indirect immunofluorescence and confocal microscopy were used to study the nucleation and organization of microtubules during meiosis in two species of leafy liverworts, Cephalozia macrostachya and Telaranea longifolia. This is the first such study of sporogenesis in the largest group of liverworts important as living representatives of some of the first land plant lineages. These studies show that cytoplasmic quadrilobing of pre-meiotic sporocytes into future spore domains is initiated by girdling bands of γ-tubulin and microtubules similar to those recently described in lobed sporocytes of simple thalloid liverworts. However, spindle ontogeny is not like other liverworts studied and is, in fact, probably unique among bryophytes. Following the establishment of quadrilobing, numerous microtubules diverge from the bands and extend into the enlarging lobes. The bands disappear and are replaced by microtubules that arise from γ-tubulin associated with the nuclear envelope. This microtubule system extends into the four lobes and is gradually reorganized into a quadripolar spindle, each half spindle consisting of a pair of poles straddling opposite cleavage furrows. Chromosomes move on this spindle to the polar cleavage furrows. The reniform daughter nuclei, each curved over a cleavage furrow, immediately enter second meiotic division with spindles now terminating in the lobes. Phragmoplasts that develop in the interzones among the haploid tetrad nuclei guide deposition of cell plates that join with the pre-meiotic furrows resulting in cleavage of the tetrad of spores. These observations document a significant variation in the innovative process of sporogenesis evolved in early land plants.     

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.     

{gamma}-Tubulin and microtubule organization during meiosis in the liverwort Ricciocarpus natans (Ricciaceae)

Extant liverworts are "living fossils" considered sister to all other plants and as such provide clues to the evolution of the microtubule organizing center (MTOC) in anastral cells. This report is the first on microtubule arrays and their γ-tubulin-nucleating sites during meiosis in a member of the Ricciales, a specialized, species-rich group of complex thalloid (marchantioid) liverworts. In meiotic prophase, γ-tubulin becomes concentrated at several sites adjacent to the nuclear envelope. Microtubules organized at these foci give rise to a multipolar prometaphase spindle. By metaphase I, the spindle has matured into a bipolar structure with truncated poles. In both first and second meiosis, γ-tubulin forms box-like caps at the spindle poles. γ-Tubulin moves from spindle poles to the proximal surfaces of telophase chromosomes where interzonal microtubules are nucleated. Although a phragmoplast is organized, no cell plate is deposited, and second division occurs simultaneously in the undivided sporocyte. γ-Tubulin surrounds each of the tetrad nuclei, and phragmoplasts initiated between both sister and nonsister nuclei direct simultaneous cytokinesis. The overall pattern of meiosis (unlobed polyplastidic sporocytes, nuclear envelope MTOC, multipolar spindle origin, spindles with box-like poles, and simultaneous cytokinesis) more closely resembles that of Conocephalum than other marchantiod liverworts.     

Gamma-tubulin in basal land plants: characterization, localization, and implication in the evolution of acentriolar microtubule organizing centers

Although seed plants have gamma-tubulin, a ubiquitous component of centrosomes associated with microtubule nucleation in algal and animal cells, they do not have discrete microtubule organizing centers (MTOCs) comparable to animal centrosomes, and the organization of microtubule arrays in plants has remained enigmatic. Spindle development in basal land plants has revealed a surprising variety of MTOCs that may represent milestones in the evolution of the typical diffuse acentrosomal plant spindle. We have isolated and characterized the gamma-tubulin gene from a liverwort, one of the extant basal land plants. Sequence similarity to the gamma-tubulin gene of higher plants suggests that the gamma-tubulin gene is highly conserved in land plants. The G9 antibody to fission yeast gamma-tubulin recognized a single band of 55 kD in immunoblots from bryophytes. Immunohistochemistry with the G9 antibody clearly documented the association of gamma-tubulin with various MTOC sites in basal land plants (e.g., discrete centrosomes with and without centrioles and the plastid surface in monoplastidic meiosis of bryophytes). Changes in the distribution of gamma-tubulin occur in a cell cycle-specific manner during monoplastidic meiosis in the liverwort Dumortiera hirsuta. gamma-Tubulin changes its localization from the plastid surface in prophase I to the spindle, from the spindle to phragmoplasts and the nuclear envelope in telophase I, and back to the plastid surfaces in prophase II. In vitro experiments show that gamma-tubulin is detectable on the surface of isolated plastids and nuclei of D. hirsuta, and microtubules can be repolymerized from the isolated plastids. gamma-Tubulin localization patterns on plastid and nuclear surfaces are not affected by the destruction of microtubules by oryzalin. We conclude that gamma-tubulin is a highly conserved protein associated with microtubule nucleation in basal land plants and that it has a cell cycle-dependent distribution essential for the orderly succession of microtubule arrays.     

Distinctions in meiotic spindle structure and assembly during in vitro and in vivo maturation of mouse oocytes

To better understand the differences in cytoskeletal organization between in vivo (IVO) and in vitro (IVM) matured oocytes, we analyzed remodeling of the centrosome-microtubule complex in IVO and IVM mouse oocytes. Fluorescence imaging revealed dramatic differences in meiotic spindle assembly and organization between these two populations. Metaphase spindles at both meiosis I (M-I) and meiosis II (M-II) in IVO oocytes were compact, displayed focused spindle poles with distinct gamma-tubulin foci, and were composed of acetylated microtubules. In contrast, IVM oocytes exhibited barrel-shaped spindles with fewer acetylated microtubules and gamma-tubulin diffusely distributed throughout the spindle proper. With respect to meiotic progression, IVO oocytes were more synchronous in the rate and extent of anaphase to telophase of M-I and first polar body emission than were IVM counterparts. Furthermore, IVO oocytes showed a twofold increase in cytoplasmic microtubule organizing centers (MTOCs), and constitutive MTOC proteins (gamma-tubulin and pericentrin) were excluded from the first polar body. Inclusion of MTOC constitutive proteins in the polar body and diminished number of cytoplasmic MTOCs was observed in IVM oocytes. These findings were corroborated in IVO oocytes obtained from naturally ovulated and spontaneously cycling mice and highlight a fundamental distinction in the spatial and temporal regulation of microtubule dynamics between IVO and IVM oocytes     

Anastral meiotic spindle morphogenesis: role of the non-claret disjunctional kinesin-like protein

We have used time-lapse laser scanning confocal microscopy to directly examine microtubule reorganization during meiotic spindle assembly in living Drosophila oocytes. These studies indicate that the bipolarity of the meiosis I spindle is not the result of a duplication and separation of centrosomal microtubule organizing centers (MTOCs). Instead, microtubules first associate with a tight chromatin mass, and then bundle to form a bipolar spindle that lacks asters. Analysis of mutant oocytes indicates that the Non-Claret Disjunctional (NCD) kinesin-like protein is required for normal spindle assembly kinetics and stabilization of the spindle during metaphase arrest. Immunolocalization analyses demonstrate that NCD is associated with spindle microtubules, and that the centrosomal components gamma- tubulin, CP-190, and CP-60 are not concentrated at the meiotic spindle poles. Based on these observations, we propose that microtubule bundling by the NCD kinesin-like protein promotes assembly of a stable bipolar spindle in the absence of typical MTOCs.     

苔藓植物孢子发生的研究进展

苔藓植物孢子发生的过程是一个复杂的形态建成的过程,在此过程中,孢子母细胞经过减数分裂的两次精确的核分裂以及细胞质分裂,形成单倍体的四分孢子,再经孢子壁的发育过程,形成成熟的孢子。本文重要介绍了苔藓植物孢子发生过程中细胞质裂片、质体及核的变化、微管系统及纺锤体、胞质分裂和孢子壁形成过程的特点及其研究进展。     

苔藓植物孢子发生的研究进展

苔藓植物孢子发生的过程是一个复杂的形态建成的过程,在此过程中,孢子母细胞经过减数分裂的两次精确的核分裂以及细胞质分裂,形成单倍体的四分孢子,再经孢子壁的发育过程,形成成熟的孢子。本文重点介绍了苔藓植物孢子发生过程中细胞质裂片、质体及核的变化、微管系统及纺锤体、胞质分裂和孢子壁形成过程的特点及其研究进展。     

THE QUADRIPOLAR MICROTUBULE SYSTEM AND MEIOTIC SPINDLE ONTOGENY IN HORNWORTS (BRYOPHYTA: ANTHOCEROTAE)

Ontogeny of the meiotic spindle in hornworts was studied by light microscopy of live materials, transmission electron microscopy, and indirect immunofluorescence microscopy. As in monoplastidic meiosis of mosses and Isoetes, the single plastid divides twice, and the four resultant plastids migrate into the future spore domains where they organize a quadripolar microtubule system (QMS). Additionally, a unique axial microtubule system (AMS) was found to parallel the plastid isthmus at each division in meiosis, much as in the single plastid division of mitosis. This finding is used to make a novel comparison of mitotic and meiotic spindle development. The AMS contributes directly to development of the mitotic spindle, whereas ontogeny of the meiotic spindle is more complex. Nuclear division in meiosis is delayed until after the second plastid division; the first AMS disappears without spindle formation, and the two AMSs of the second plastid division contribute to development of the QMS. Proliferation of microtubules at each plastid results in the QMS consisting of four cones of microtubules interconnecting the plastids and surrounding the nucleus. The QMS contributes to the development of a functionally bipolar spindle. The meiotic spindle is comparable to a merger of two mitotic spindles. However, the first division spindle does not terminate in what would be the poles of mitosis; instead the poles converge to orient the spindle axis midway between pairs of non-sister plastids.     

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)     

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     

Sporogenesis in Bryophytes: Patterns and Diversity in Meiosis

Meiosis in bryophytes retains unusual features that provide clues to the innovation of sporogenesis in early land plants. Sporocytes are typically quadrilobed before nuclear division and the meiotic spindle is quadripolar with poles in the four future spore domains. Whereas seed plants consistently have anastral spindles arising from γ-tubulin in the perinuclear area, bryophytes have spindles organized at POs, plastids, or nuclear envelope. All of these MTOCs are significantly different from centrosomes of the algal ancestors. Mosses and hornworts have quadrilobed sporocytes with meiotic spindles organized at plastids. Meiosis in liverworts is extremely varied. Sporocytes of Jungermanniopsida are deeply quadrilobed and have microtubule bands marking division planes prior to cytoplasmic shaping. Spindles are organized at POs or nuclear envelope. Sporocytes of Marchantiopsida are quadrilobed to apolar with spindles organized at plastids, POs, or nuclear envelope. Pre-meiotic bands have been reported in only one marchantiod, the early divergent Blasia. An atlas of cytological data on 13 liverworts, 3 mosses and 2 hornworts is presented and analyzed.     

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.     

How do Plants Organize Microtubules Without a Centrosome？

A microtubule nucleates from a γ-tubuUn complex, which consists of γ-tubulin, proteins from the SPC971SPC98 family, and the WD40 motif protein GCP-WD. We analyzed the phylogenetic relationships of the genes encoding these proteins and found that the components of this complex are widely conserved among land plants and other eukaryotes. By contrast, the interphase and mitotic arrays of microtubules in land plants differ from those in other eukaryotes. In the interphase cortical array, the majority of microtubules nucleate on existing microtubules in the absence of conspicuous microtubule organizing centers （MTOCs）, such as a centrosome. During mitosis, the spindle also forms in the absence of conspicuous MTOCs. Both poles of the spindle are broad, and branched structures of microtubules called microtubule converging centers form at the poles. In this review, we hypothesize that the microtubule converging centers form via microtubule-dependent microtubule nucleation, as in the case of the interphase arrays. The evolutionary insights arising from the molecular basis of the diversity in microtubule organization are discussed.     

Shifting meiotic to mitotic spindle assembly in oocytes disrupts chromosome alignment

Mitotic spindles assemble from two centrosomes, which are major microtubule‐organizing centers (MTOCs) that contain centrioles. Meiotic spindles in oocytes, however, lack centrioles. In mouse oocytes, spindle microtubules are nucleated from multiple acentriolar MTOCs that are sorted and clustered prior to completion of spindle assembly in an “inside‐out” mechanism, ending with establishment of the poles. We used HSET (kinesin‐14) as a tool to shift meiotic spindle assembly toward a mitotic “outside‐in” mode and analyzed the consequences on the fidelity of the division. We show that HSET levels must be tightly gated in meiosis I and that even slight overexpression of HSET forces spindle morphogenesis to become more mitotic‐like: rapid spindle bipolarization and pole assembly coupled with focused poles. The unusual length of meiosis I is not sufficient to correct these early spindle morphogenesis defects, resulting in severe chromosome alignment abnormalities. Thus, the unique “inside‐out” mechanism of meiotic spindle assembly is essential to prevent chromosomal misalignment and production of aneuploidy gametes.     

Microtubule organization during maturation of Xenopus oocytes: assembly and rotation of the meiotic spindles.

Assembly of the meiotic spindles during progesterone-induced maturation of Xenopus oocytes was examined by confocal fluorescence microscopy using anti-tubulin antibodies and by time-lapse confocal microscopy of living oocytes microinjected with fluorescent tubulin. Assembly of a transient microtubule array from a disk-shaped MTOC was observed soon after germinal vesicle breakdown. This MTOC-TMA complex rapidly migrated toward the animal pole, in association with the condensing meiotic chromosomes. Four common stages were observed during the assembly of both M1 and M2 spindles: (1) formation of a compact aggregate of microtubules and chromosomes; (2) reorganization of this aggregate resulting in formation of a short bipolar spindle; (3) an anaphase-B-like elongation of the prometaphase spindle, transversely oriented with respect to the oocyte A-V axis; and (4) rotation of the spindle into alignment with the oocyte axis. The rate of spindle elongation observed in M1 (0.7 microns min-1) was slower than that observed in M2 (1.8 microns min-1). Examination of spindles by immunofluorescence with antitubulin revealed numerous interdigitating microtubules, suggesting that prometaphase elongation of meiotic spindles in Xenopus oocytes results from active sliding of antiparallel microtubules. A substantial number of maturing oocytes formed monopolar microtubule asters during M1, nucleated by hollow spherical MTOCs. These monasters were subsequently observed to develop into bipolar M1 spindles and proceed through meiosis. The results presented define a complex pathway for assembly and rotation of the meiotic spindles during maturation of Xenopus oocytes.     

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     

Acentriolar microtubule organization centers and Ran‐mediated microtubule formation pathways are both required in porcine oocytes

Mammalian oocytes lack centrioles but can generate bipolar spindles using several different mechanisms. For example, mouse oocytes have acentriolar microtubule organization centers (MTOCs) that contain many components of the centrosome, and which initiate microtubule polymerization. On the contrary, human oocytes lack MTOCs and the Ran‐mediated mechanisms may be responsible for spindle assembly. Complete knowledge of the different mechanisms of spindle assembly is lacking in various mammalian oocytes. In this study, we demonstrate that both MTOC‐ and Ran‐mediated microtubule nucleation are required for functional meiotic metaphase I spindle generation in porcine oocytes. Acentriolar MTOC components, including Cep192 and pericentrin, were absent in the germinal vesicle and germinal vesicle breakdown stages. However, they start to colocalize to the spindle microtubules, but are absent in the meiotic spindle poles. Knockdown of Cep192 or inhibition of Polo‐like kinase 1 activity impaired the recruitment of Cep192 and pericentrin to the spindles, impaired microtubule assembly, and decreased the polar body extrusion rate. When the Ran^GTP gradient was perturbed by the expression of dominant negative or constitutively active Ran mutants, severe defects in microtubule nucleation and cytokinesis were observed, and the localization of MTOC materials in the spindles was abolished. These results demonstrate that the stepwise involvement of MTOC‐ and Ran‐mediated microtubule assembly is crucial for the formation of meiotic spindles in porcine oocytes, indicating the diversity of spindle formation mechanisms among mammalian oocytes.     

Involvement of Polo-like kinase 1 in MEK1/2-regulated spindle formation during mouse oocyte meiosis

Our recent studies have shown that MEK1/2 is a critical regulator of microtubule organization and spindle formation during oocyte meiosis. In the present study, we found that Plk1 colocalized with p-MEK1/2 at various meoiotic stages after GVBD when microtubule began to organize. Also, Plk1 was able to coimmunoprecipitate with p-MEK1/2 in metaphase I stage mouse oocyte extracts, further confirming their physical interaction. Taxol-treated oocytes exhibited a number of cytoplasmic asters, in which both Plk1 and p-MEK1/2 were present, indicating that they might be complexed to participate in the acentrosomal spindle formation at the MTOCs during oocyte meiosis. Depolymerization of microtubules by nocodazole resulted in the complete disassembly of spindles, but Plk1 remained associated with p-MEK1/2, accumulating in the vicinity of chromosomes. More importantly, when p-MEK1/2 activity was blocked by U0126, Plk1 lost its normal localization at the spindle poles, which might be one of the most vital factors causing the abnormal spindles in MEK1/2-inhibited oocytes. Taken together, these data indicate that Plk1 and MEK1/2 regulate the spindle formation in the same pathway and that Plk1 is involved in MEK1/2-regulated spindle assembly during mouse oocyte meiotic maturation.