Spatio-temporal reorganization of intracytoplasmic microtubules is associated with nuclear selection and differentiation during the developmental process in the ciliateTetrahymena thermophila

Summary Indirect immunofluorescence has revealed various intracytoplasmic microtubular structures, which are transiently polymerized in specific subcellular locations during the developmental process of conjugation in the ciliateTetrahymena thermophila. These structures include: (1) micronuclear spindles, (2) perimicronuclear microtubules, (3) microtubular baskets surrounding migrating pronuclei, and (4) microtubules interconnecting the pronuclei with the conjugants' junctional zone. Furthermore, a peripheral network of intracytoplasmic microtubules related to the cell cortex is present in both vegetative cells and in conjugants. Comparative observations made on cells undergoing normal conjugation and defective conjugation (occurring either spontaneously or induced by taxol) has revealed some rules governing the pattern of deployment of conjugation-specific microtubules. The presence of perinuclear microtubular arrays during early postmeiotic stages of development is strictly limited to more anteriorly located nuclei which includes the selected haploid nucleus that further divides to form the stationary and migratory pronuclei. These perinuclear microtubules may be involved in the positional control of nuclear fates leading to effective nuclear selection. Microtubular bundles associated with pronuclei and connecting the junctional zone are only formed in the presence of functional pronuclei, and may be involved in the guidance of pronuclei leading to their fusion. The mechanism of cytoplasmic control of nuclear differentiation of derivatives of the zygotic nucleus appear to be associated with a coordinate action of two microtubular arrays: spindle microtubules of the second postzygotic division and the peripheral intracytoplasmic network of microtubules, leading to a proper subcortical positioning of the postzygotic nuclei at opposite poles of the cell.Abbreviations MTs Microtubules     

A cytoskeletal system predicts division plane in meiosis ofSelaginella

Summary An ultrastructural investigation of the monoplastidic microsporocytes ofSelaginella arenicola revealed a unique cytoskeletal array that predicts the future division plane before nuclear division takes place. By midprophase of the first meiotic division, the single plastid has divided once and the two plastids lie on opposite sides of the nucleus which is elongated in the plane of the incipient metaphase I spindle. A cytoplasmic structure, the procytokinetic plate (PCP), predicts the division plane of of both plastid and cytoplasm. The PCP consists of a distinct concentration of vesicles lying in the future division plane and an elaborate system of microtubules aligned parallel to the long axis of plastids and nucleus. Microtubules of the axially aligned system appear to terminate in clusters of vesicles in the central zone of the PCP. The PCP with axially aligned microtubules is as predictive of the division plane in these meiotic cells as is the girdling preprophase band of microtubules in mitotic cells.     

Organization of the actin cytoskeleton during pollen development inGasteria verrucosa (Mill.) H. Duval visualized with rhodamine-phalloidin

The three-dimensional organization of the microfilamental cytoskeleton of developingGasteria pollen was investigated by light microscopy using whole cells and fluorescently labelled phalloidin. Cells were not fixed chemically but their walls were permeabilized with dimethylsulphoxide and Nonidet P-40 at premicrospore stages or with dimethylsulphoxide, Nonidet P-40 and 4-methylmorpholinoxide-monohydrate at free-microspore and pollen stages to dissolve the intine.Four strikingly different microfilamentous configurations were distinguished. (i) Actin filaments were observed in the central cytoplasm throughout the successive stages of pollen development. The network was commonly composed of thin bundles ramifying throughout the cytoplasm at interphase stages but as thick bundles encaging the nucleus prior to the first and second meiotic division. (ii) In released microspores and pollen, F-actin filaments formed remarkably parallel arrays in the peripheral cytoplasm. (iii) In the first and second meiotic spindles there was an apparent localization of massive arrays of phalloidin-reactive material. Fluorescently labelled F-actin was present in kinetochore fibers and pole-to-pole fibers during metaphase and anaphase. (iv) At telophase, microfilaments radiated from the nuclear envelopes and after karyokinesis in the second meiotic division, F-actin was observed in phragmoplasts.We did not observe rhodamine-phalloidin-labelled filaments in the cytoplasm after cytochalasin-B treatment whereas F-actin persisted in the spindle. Incubation at 4° C did not influence the existence of cytoplasmic microfilaments whereas spindle filaments disappeared. This points to a close interdependence of spindle microfilaments and spindle tubules.Based on present data and earlier observations on the configuration of microtubules during pollen development in the same species (Van Lammeren et al., 1985, Planta165, 1-11) there appear to be apparent codistributions of F-actin and microtubules during various stages of male meiosis inGasteria verrucosa.Abbreviation DMSO dimethylsulfoxide     

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.     

The microtubular cytoskeleton during megasporogenesis in the Nun orchid, Phaius tankervilliae

This study examines the microtubular cytoskeleton during megasporogenesis in the Nun orchid, Phaius tankervilliae . The subepidermal cell located at the terminal end of the nucellar filament differentiates first into an archesporial cell and then enlarges to become the megasporocyte. The megasporocyte undergoes the first meiotic division, giving rise to two dyad cells of unequal size. Immunostaining reveals that microtubules become more abundant as the megasporocyte increases in size. Microtubules congregate around the nucleus forming a distinct perinuclear array and many microtubules radiate directly from the nuclear envelope. In the megasporocyte, prominent microtubules are readily detected at the chalazal end of the cell cytoplasm. After meiosis I, the chalazal dyad cell expands in size at the expense of the micropylar dyad cell. At this stage, new microtubule organizing centres can be found at the corners of the cells. The appearance of these structures is stage-specific and they are not found at any other stages of megasporogenesis. The functional dyad cell undergoes the second meiotic division, resulting in the formation of two megaspores of unequal size. The chalazal megaspore enlarges and eventually gives rise to the embryo sac. As the functional megaspore expands, the microtubules again form a distinct perinuclear array with many microtubules radiating from the nuclear envelope. A defined cortical array of microtubules has not been found in P. tankervilliae during the course of megasporogenesis.     

Mitosis and microtubule organizational changes in isolated generative cells of Allemanda neriifolia

Summary The organizational changes of the microtubules of isolated generative cells of Allemanda neriifolia during division were followed using anti--tubulin and immunofluorescence microscopy. Generative cells were isolated from the pollen tubes after osmotic shock treatment. Immediately after isolation most of the cells remain either in early or late prophase. The shape of the cell changes from spindle to spheroidal. In early prophase the nuclear membrane of the cell appears intact and the cytoplasm full of reticulate microtubules of different shapes and thicknesses. Later, the nuclear membrane breaks up. After the nuclear membrane has broken up, the chromosomes scatter into the cytoplasm and mix with the microtubules. When cells enter metaphase, spindle microtubules form. Afterwards, in anaphase, sister chromatids separate and the spindle disappears. A new array of longitudinally oriented cage microtubules appears. As the cells enter early telophase, the cage microtubules disappear and an array of interpolar microtubules begins to form. Later, in some telophase cells the interpolar microtubules become highly elongated, but in others they soon disappear and become replaced by a thick band(s) (or sheet(s)) of microtubules in the midplane between the two clusters of chromosomes and the cell shape reverts back to spheroidal. In culture no phragmoplast junctions appear in any of the late telophase cells although they are present under the in situ condition (i.e. in pollen tubes).     

Morphology of the microtubule system in mouse kidney epithelial cells]

The cultured mouse kidney cells forming epithelial sheets were studied using an indirect immunofluorescence microscopy with antibodies against tubulin. These cells, as well as fibroblasts, were found to contain a well developed microtubular system sensitive to colcemid. The assembly of microtubules after washing out of colcemid began from one or two perinuclear centers, associated with the cilium-like structure. There were certain differences between the microtubular systems in epithelial cells and fibroblasts: 1) Microtubules in the fibroblasts penetrated the whole cytoplasm including the peripheral lamella whereas in the epithelial cells the lamellar cytoplasm was often free from microtubules. 2) The orientation of microtubules in the epithelial cells, unlike in the fibroblasts, was not correlated with the stable or active state of the cell margin. A possible role of microtubular system in the epithelial cells and fibroblasts is compared and discussed.     

Embryogenesis in <Emphasis Type="Italic">Sedum acre</Emphasis> L.: structural and immunocytochemical aspects of suspensor development

The changes in the formation of both the actin and the microtubular cytoskeleton during the differentiation of the embryo-suspensor in Sedum acre were studied in comparison with the development of the embryo-proper. The presence and distribution of the cytoskeletal elements were examined ultrastructurally and with the light microscope using immunolabelling and rhodamine-phalloidin staining. At the globular stage of embryo development extensive array of actin filaments is present in the cytoplasm of basal cell, the microfilament bundles generally run parallel to the long axis of basal cell and pass in close to the nucleus. Microtubules form irregular bundles in the cytoplasm of the basal cell. A strongly fluorescent densely packed microtubules are present in the cytoplasmic layer adjacent to the wall separating the basal cell from the first layer of the chalazal suspensor cells. At the heart-stage of embryo development, in the basal cell, extremely dense arrays of actin materials are located near the micropylar and chalazal end of the cell. At this stage of basal cell formation, numerous actin filaments congregate around the nucleus. In the fully differentiated basal cell and micropylar haustorium, the tubulin cytoskeleton forms a dense prominent network composed of numerous cross-linked filaments. In the distal region of the basal cell, a distinct microtubular cytoskeleton with numerous microtubules is observed in the cytoplasmic layer adjacent to the wall, separating the basal cell from the first layer of the chalazal suspensor cells. The role of cytoskeleton during the development of the suspensor in S. acre is discussed.     

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.     

Microtubule organization in germinated pollen of the conifer Picea abies (Norway spruce,Pinaceae)

The organization of microtubules in germinated pollen of the conifer Picea abies (Norway spruce, Pinaceae) was examined using primarily confocal microscopy. Pollination in conifers differs from angiosperms in the number of mitotic divisions between the microspore and the sperm and in the growth rate of the pollen tube. These differences may be orchestrated by the cytoskeleton, and this study finds that there are important functional differences in microtubule organization within conifer pollen compared to the angiosperm model systems. Pollen from P. abies contains two degenerated prothallial cells, a body cell, a stalk cell, and a vegetative cell. The body cell produces the sperm. In the vegetative cell, microtubules form a continuous network from within the pollen grain, out through the aperture, and down the length of the tube to the elongating tip. Within the grain, this network extends from the pollen grain wall to the body and stalk cell complex. Microtubules within the body and stalk cells form a densely packed array that enmeshes amyloplasts and the nucleus. Microtubule bundles can be traced between the body and stalk cells from the cytoplasm of the body cell to the adjoining cell wall and into the cytoplasm of the stalk cell. Body and stalk cells are connected by plasmodesmata. The organization of microtubules and the presence of plasmodesmata suggest that microtubules form a path for intercellular communication by projecting from the cytoplasm to interconnecting plasmodesmata. Microtubules in the elongating tube form a net axial array that ensheathes the vegetative nucleus. Microtubules are enriched at the elongating tip, where they form an array beneath the plasma membrane that is perpendicular to the direction of tube growth. This enriched region extends back 20 μm from the tip. There is an abrupt transition from a net perpendicular to a net axial organization at the edge of the enriched region. In medial sections, microtubules are present in the core of the elongating tip. The organization of microtubules in the tip differs from that seen in angiosperm pollen tubes.     

The microtubular cytoskeleton during development of the zygote,proembryo and free-nuclear endosperm inArabidopsis thaliana (L.) Heynh

The microtubular cytoskeleton has been studied during development of the zygote, proembryo and free-nuclear endosperm inA. thaliana using immunofluorescence localization of tubulin in enzymatically isolated material. Abundant micro tubules (MTs) are found throughout proembryogenesis. Microtubules in the coenocytic endosperm are mainly internal. By contrast, there is a re-orientation of MTs to a transverse cortical distribution during zygote development, predominantly in a subapical band which accompanies a phase of apical extension. The presence of these cortical arrays coincides with the elongation of the zygote. Cortical arrays also accompany elongation of the cylindrical suspensor. Extensive networks of MTs ramify throughout the cytoplasm of cells in the proembryo proper. Perinuclear arrays are detected in a number of cell types and MTs contribute to typical mitotic configurations during nuclear divisions. Preprophase bands of MTs are absent throughout megasporogenesis and embryo-sac development and do not occur in endosperm cell divisions. We have observed MTs throughout the first division cycle of the zygote. By placing the observed stages in a most probable sequence, we have identified this cell cycle as the point during embryogenesis at which a preprophase band is reinstated as a regular feature of cell division. Preprophase bands were observed to predict planes of cytokinesis in cell divisions up to the octant stage.Abbreviations DIC differential interference contrast optics - MT microtubule - PPB preprophase band of microtubule We thank Ms. Margaret Travers for her helpful English translation of Yakovlev and Alimova (1976) and Mr. James Whitehead for preparation of Fig. 11. M.C.W. was supported by an Australian Postgraduate Research Award.     

Microtubule configurations and nuclear DNA synthesis during initiation of suspensor-bearing embryos from Brassica napus cv. Topas microspores

In the new Brassica napus microspore culture system, wherein embryos with suspensors are formed, ab initio mimics zygotic embryogenesis. The system provides a powerful in vitro tool for studying the diverse developmental processes that take place during early stages of plant embryogenesis. Here, we studied in this new culture system both the temporal and spatial distribution of nuclear DNA synthesis places and the organization of the microtubular (MT) cytoskeleton, which were visualized with a refined whole mount immunolocalization technology and 3D confocal laser scanning microscopy. A ‘mild’ heat stress induced microspores to elongate, to rearrange their MT cytoskeleton and to re-enter the cell cycle and perform a predictable sequence of divisions. These events led to the formation of a filamentous suspensor-like structure, of which the distal tip cell gave rise to the embryo proper. Cells of the developing pro-embryo characterized endoplasmic (EMTs) and cortical microtubules (CMTs) in various configurations in the successive stages of the cell cycle. However, the most prominent changes in MT configurations and nuclear DNA replication concerned the first sporophytic division occurring within microspores and the apical cell of the pro-embryo. Microspore embryogenesis was preceded by pre-prophase band formation and DNA synthesis. The apical cell of the pro-embryo exhibited a random organization of CMTs and, in relation to this, isotropic expansion occurred, mimicking the development of the apical cell of the zygotic situation. Moreover, the apical cell entered the S phase shortly before it divided transversally at the stage that the suspensor was 3–8 celled.     

Reassembly of microtubules in protoplasts ofSaccharomyces cerevisiae after nocodazole-induced depolymerization

Summary By following microtubule neoformation after their complete destruction by nocodazole, we analyzed the pattern of microtubule nucleation in protoplasts ofSaccharomyces cerevisiae. Using immunofluorescence, the drug was shown to induce rapid and complete disassembly of both cytoplasmic and spindle microtubules and to selectively block protoplast nuclear division at a defined stage of the cell cycle. Treated protoplasts placed in a drug-free environment recovered a more abundant microtubular system. The majority of microtubules re-formed at SPBs whereas a minority of free-ended microtubules nucleated in the cytoplasm of the protoplasts without any detectable association with recognizable nucleation sites. Random nucleation of free microtubules might be induced by high amounts of unpolymerized tubulin likely to be present in the protoplasts at the moment of drug release.Abbreviations MT microtubule - NOCO nocodazole - SPBs spindle pole bodies - PMSF phenylmethylsulfonyl fluoride - BSA bovine serum albumine - sMT spindle microtubule - cMT cytoplasmic microtubule - MTOC microtubule organizing center     

Ultrastructural studies on plastids of generative and vegetative cells inLiliaceae 3. Plastid distribution during the pollen development inGasteria verrucosa (Mill.) Duval

Summary This paper describes the development of pollen grains ofGasteria verrucosa from the late microspore to the mature two-cellular pollen grain. Ultrastructural changes and the distribution of plastids as a result of the first pollen mitosis have been investigated using light and electron microscopy. The microspores as well as the generative and the vegetative cell contain mitochondria and other cytoplasmic organelles during all of the observed developmental stages. In contrast, the generative cell and the vegetative cell show a different plastid content. Plastids are randomly distributed within the microspores before pollen mitosis. During the prophase of the first pollen mitosis the plastids become clustered at the proximal pole of the microspore. The dividing nucleus of the microspore is located at the distal pole of the microspore. Therefore, the plastids are not equally distributed into both the generative and the vegetative cell. The possible reasons for the polarization of plastids within the microspore are briefly discussed. The lack of plastids in the generative cell causes a maternal inheritance of plastids inGasteria verrucosa.     

Interactions between microtubules and the nuclear envelope during mitosis in a fern

Summary The relationships between microtubules and the nuclear membrane have been studied during the cell division cycle in adventitious roots ofDryopteris filixmas. At prophase, the equatorial band of microtubules hitherto only reported for higher plant cells is present in the cytoplasm. The occurrence of the band is correlated with a striking change in the shape of the nucleus. Microtubules leave the peripheral cytoplasmic band in groups and are found to partially encircle the nucleus. At the same time pole-to-pole fibres also lie close to the nuclear envelope. A specific function for some of these fibres is proposed, and the results are discussed in the light of previous work on the cytoplasmic features of prophase. The behaviour of the nuclear membrane during mitosis and its reformation around the daughter nuclei are described. These results are related to the concept of microtubule organizing centres.     

洋葱小孢子母细胞减数分裂过程中微管分布变化

应用间接免疫荧光标记技术和激光共聚焦扫描显微镜成像技术观察洋葱小孢子母细胞减数分裂过程中微管分布变化。减数分裂之前,小孢子母细胞中的微管较短,呈辐射状,由细胞核表面向四周扩散。减数分裂开始后,细胞质中的一部分微管蛋白聚集成纺锤体微管,控制染色体的分布。进入减数分裂I后期,纺锤体微管变为牵引染色体移向两极的着丝粒微管和连接纺锤体两极的极丝微管。之后,所有微管集中在两个核之间,构成成膜体。然后,微管解聚成微管蛋白弥散在细胞质中。减数分裂I完成后,二分体2个子细胞中的微管蛋白又聚集成2个纺锤体微管,开始减数分裂II过程。经过减数分裂II中期,2个二分体细胞中的微管再次集中在2个细胞核之间形成成膜体,隔离2个细胞核。此后,微管蛋白解聚,弥散分布在小孢子细胞质中。     

Ultrastructural Features of Mitosis in Anisonema sp. (Euglenida)1

The fine structure of stages in mitosis in a colorless euglenoid, Anisonema sp., reveals that chromosomes remain condensed throughout the life cycle and are attached to the nuclear envelope at interphase. The onset of mitosis is marked by the anterior migration of the nucleus towards the base of the reservoir and by elongation of the nucleolus. The nuclear envelope persists throughout mitosis. Microtubules are generated in the peripheral nucleoplasm adjacent to the envelope and attach to the chromosomes while they are still associated with the envelope. The region of microtubular contact develops into a distinct layered kinetochore as the developing spindle with attached chromosomes separates from the nuclear envelope and moves into the nucleoplasm. The mature spindle consists of a number of subspindles each containing about 8–10 microtubules and a few associated chromosomes. Both chromosomal and non-chromosomal microtubules are present in each subspindle and extend towards the envelope terminating at or near the nuclear pores. Chromosomal segregation is concomitant with nuclear elongation. By late division, an interzonal spindle develops in the dumbbell-shaped nucleus and nucleolar separation occurs. Continued invagination of the nuclear envelope in the region of the interzonal spindle eventually separates the daughter nuclei. A remnant of the interzonal spindle persists in the cytoplasm until cytokinesis.     

Distribution of microtubules during the breakdown of the nuclear envelope of the Xenopus oocyte: an immunocytochemical study

Xenopus oocytes were stained by anti-tubulin and anti-MAP1 antibodies during the first meiotic cell division. In the prophase-blocked oocytes, only few microtubules are present around the upper part of the nuclear envelope. These microtubules are resistant to cold, calcium and antimitotic drug treatments. At this stage, monoclonal anti-MAP1 antibody and polyclonal anti-centrosome antibody reveal punctate staining of the nucleus and nucleoli. During the progesterone-induced maturation, a microtubular network appears at the basal part of the disrupting nucleus. Anti-MAP1 and anti-centrosome antibodies stain a dense layer at the basal part of this microtubular array. Microtubules present in this array are cold, calcium- and antimitotic drug sensitive. Anti-MAP1 and anti-tubulin antibodies stain the whole metaphase II spindle, whereas only the poles of the metaphase II spindle are stained by the anti-centrosome antibody.     

Dynamic changes in microtubule organization during division of the primitive dinoflagellate Oxyrrhis marina

The marine dinoflagellate Oxyrrhis marina has three major microtubular systems: the flagellar apparatus made of one transverse and one longitudinal flagella and their appendages, cortical microtubules, and intranuclear microtubules. We investigated the dynamic changes of these microtubular systems during cell division by transmission and scanning electron microscopy, and confocal fluorescent laser microscopy. During prophase, basal bodies, both flagella and their appendages were duplicated. In the round nucleus situated in the cell centre, intranuclear microtubules appeared radiating toward the centre of the nucleus from densities located in some nuclear pores. During metaphase, both daughter flagellar apparatus separated and moved apart along the main cell axis. Microtubules of ventral cortex were also duplicated and moved with the flagellar apparatus. The nucleus flattened in the longitudinal direction and became discoid-shaped close to the equatorial plane. Many bundles of microtubules ran parallel to the short axis of the nucleus (cell long axis), between which chromosomes were arranged in the same direction. During ana-telophase, the nucleus elongated along the longitudinal axis and took a dumbbell shape. At this stage a contractile ring containing actin was clearly observed in the equatorial cortex. The cortical microtubule network seemed to be cut into two halves at the position of the actin bundle. Shortly after, the nucleus divided into two nuclei, then the cell body was constricted at its equator and divided into one anterior and one posterior halves which were soon rebuilt to produce two cells with two full sets of cortical microtubules. From our observations, several mechanisms for the duplication of the microtubule networks during mitosis in O. marina are discussed.     

Spatial congruence between exine pattern,microtubules and endomembranes in Vigna pollen

The role of microtubules and endomembranes in pollen wall pattern formation in Vigna vexillata L. was examined using fluorescence laser scanning confocal microscopy. Indirect immunofluorescence using anti--tubulin antibodies revealed that the arrangement of the cortical microtubular cytoskeleton in microspores resembled the reciprocal of the reticulate ektexine ornamentations of mature V. vexillata pollen. Patches of microtubules in cortical cytoplasm corresponded in location with the lumina of the exine reticulum and with apertural sites. Microtubules were absent from cytoplasm under muri (ridges) of the exine reticulum. Labeling of microspores during the mid-tetrad stage with the endomembrane-specific fluorochrome DiOC₆ produced a pattern similar to that of the microtubules; i.e., DiOC₆ staining was localized in cytoplasm underlying lumina and absent from cortical cytoplasm underlying sites of muri. This report represents the first observation of congruence of the pattern of occurrence of any subcellular organelles with exine pattern and, in particular, the congruence of both microtubules and endomembranes in cortical cytoplasm with the lumina of the reticulate exine.