Microtubules mediate germ-nuclear behavior after meiosis in conjugation of Paramecium caudatum

Microtubule dynamics in Paramecium caudatum were investigated with an anti-alpha-tubulin antibody and a microinjection technique to determine the function of microtubules on micronuclear behavior during conjugation. After meiosis, all four haploid micronuclei were connected by microtubular filaments to the paroral region and moved close to this region. This nuclear movement was micronucleus-specific, because some small macronuclear fragments transplanted from exconjugants never moved to the region. Only one of the four germ nuclei moved into the paroral cone and was covered by microtubule assembly (the so-called first assembly of microtubules, AM-I). This nucleus survived there, while the other three not in this region degenerated. The movement of germ nucleus was inhibited by the injection of the anti-alpha-tubulin antibody. The surviving germ nucleus divided once and produced a migratory pronucleus and a stationary pronucleus. Prior to the reciprocal exchange of the migratory nuclei, microtubules assembled around the migratory pronuclei again (the so-called second assembly of microtubules, AM-II). Then, the migratory pronucleus moved into the partner cell and fused with the stationary pronucleus. Thus, microtubules appear to be indispensable for nuclear behavior: they enable migration of postmeiotic nuclei to the paroral region and they permit the survival of the nucleus at the paroral cone.     

Macronuclear differentiation in conjugating pairs of Tetrahymena treated with the antitubulin drug nocodazole

During Tetrahymena conjugation gamic nuclei (pronuclei) are produced, reciprocally exchanged, and fused in each mate. The synkaryon divides twice; the two anterior nuclei develop into new macronuclei while the two posterior nuclei become micronuclei. The postzygotic divisions were blocked with the antitubulin drug nocodazole (ND). Then pronuclei (gamic nuclei) developed directly into macronuclear anlagen (primordial macronuclei), inducing amicronucleate cells with two anlagen, or, rarely, cells with one anlagen and one micronucleus. ND had a similar effect on cells that passed the first postzygotic division inducing amicronucleate cells with two anlagen, while cells treated with ND at the synkarya stage produced only one large anlage. Different intracytoplasmic positioning of the nuclei treated with ND (pronuclei, synkarya and two products of the first division) shows that most of cell cytoplasm is competent for inducing macronuclear development. Only posteriorly positioned nuclei--products of the second postzygotic division--remain micronuclei. The total cell DNA content, measured cytophotometrically in control and in ND-induced amicronucleate conjugant cells with one and two anlagen, was similar in all three samples at 12 h of conjugation. Eventually, at 24 h this content was about 2 pg (8 C) per anlagen both in nonrefed control and in amicronucleate exconjugants. Therefore "large" nuclei developing in the presence of ND were true macronuclear anlagen.     

Microtubular organization during asymmetrical division of the generative cell inGagea lutea

Video microscopy and conventional or Confocal Laser Scanning Microscopy after DAPI staining and anti-α-tubulin labelling were used to study the asymmetrical division of the generative cell (GC) inGagea lutea. Pollen was cultured for up to 8 hr in a medium containing 10% poly (ethylene glycol), 3.0% to 3.8% sucrose, 0.03% casein acid hydrolysate, 15 mM 2-(N-morpholinoethane)-sulphonic acid-KOH buffer (pH 5.9) and salts. In the pollen grain, the GC had a spherical or ovoid shape and contained a fine network of intermingled microtubules. As the GC entered into the pollen tube, it assumed a cylindrical shape with a length often exceeding 250 μm. A cage of microtubules then developed around the nucleus. The presence of dense and thick microtubular bundles in front of the generative nucleus within the GC coincided with the translocation of the nucleus to the leading end of the GC. No pre-prophase band was ever detected, but a distinct prophase spindle of microtubules was formed. In some GCs a tubulin-rich dot became visible at each pole of the spindle. After nuclear envelope breakdown, the bundles of microtubules spread between the chromosomes and became oriented into parallel arrays. The spindle became shorter at metaphase, and there was no tubulin labelling at the site of the metaphase plate. At anaphase, the microtubular apparatus lost its spindle-shape and a bridge of prominent bundles of microtubules connected the two daughter nuclei. At telophase, the site of the cell plate remained unstained by the anti-α-tubulin antibody, but a distinct phragmoplast of microtubules was formed more closely to the leading nucleus, resulting in the formation of unequal sperm cells (SCs). The leading SC was up to 2.5 times smaller than the following SC and it contained a smaller or equal number of nucleoli.     

The endomembrane system and mechanism of membrane flow in the green alga,Gloeomonas kupfferi (Volvocales,Chlorophyta) I. An ultrastructural analysis

Summary The endomembrane system of the chlamydomonad flagellate,Gloeomonas kupfferi (Chlorophyta), is complex. It consists of a proliferating ER network, a perinuclear complex of 14–18 dictyosomes and 8–12 vacuoles and an anterior contractile vacuole complex. The ER network extends from the nuclear envelope outwards, ensheafhs a dictyosome, extends out through a lobe of the chloroplast and terminates in the thin zone of peripheral cytoplasm between the chloroplast and plasmamembrane. The individual dictyosome is polar with distinct cis- and trans-faces. The cis-face is closely associated with transition vesicles emerging from the adjacent ER. Large vesicles emerge from peripheral swellings of terminal cisternae. The dictyosome-associated ER is connected to the peripheral vacuolar system. During cell division and cytokinesis, changes in the endomembrane system occur. Dictyosomes divide and quickly separate to form perinuclear complexes around the daughter nuclei. Each dictyosome undergoes morphological changes during this wall precursor-producing stage. ER lines the furrow zone and is closely associated with phycoplast microtubules. A discussion of the endomembrane system in membrane flow mechanics is provided.Abbreviations ER endoplasmic reticulum - OsFeCN Osmium ferricyanide     

鹤顶兰胚囊发育过程中微管变化的共焦显微镜观察

光镜的观察确定了鹤顶兰（Ｐｈａｉｕｓ ｔａｎｋｅｒｖｉｌｌｉａｅ （Ａｉｔｏｎ） Ｂｌ．）胚囊发育属单孢子蓼型。应用免疫荧光标记技术及共焦镜观察了胚囊发育过程中微管分布的变化。当孢原细胞初形成时,细胞内的微管呈网状分布。之后,孢原细胞体积增大发育为大孢子母细胞。大孢子母细胞延长,进入减数分裂Ⅰ。微管由分裂前的网状分布变为辐射状排列。二分体的两个细胞内的微管分布一样,呈辐射状。四分体的近珠孔端的３ 个大孢子解体,细胞内的微管消失。靠合点端的功能大孢子内有许多微管呈网状分布。当功能大孢子进入第一次有丝分裂时,细胞内的微管由网状变为辐射状,从核膜伸展至周质。再经两次有丝分裂形成八核胚囊。在核分裂之前微管一般是呈网状分布并紧包围着核。在分裂期间二核和四核胚囊都呈极性现象,微管系统也呈极性分布。微管在八核胚囊内的分布变化情形特别复杂。首先,八核分别作不同程度的移动,其中两个核移向胚囊中央,珠孔端和合点端的３ 个核分别互相靠拢,形成３ 个区,即中央区、反足区和卵器区。胚囊未形成区时,８ 个核都被网状分布的微管包围着。当胚囊明显分成区时,反足区内的微管仍作网状分布。中央区的微管分布则趋疏松,形成篮形结构,包围着液泡和两个极核。在     

Time-course analysis of nuclear events during conjugation in the marine ciliate Euplotes vannus and comparison with other ciliates (Protozoa,Ciliophora)

Ciliates represent a morphologically and genetically distinct group of single-celled eukaryotes that segregate germline and somatic functions into two types of nuclei and exhibit complex cytogenetic events during the sexual process of conjugation, which is under the control of the so-called “mating type systems”. Studying conjugation in ciliates may provide insight into our understanding of the origins and evolution of sex and fertilization. In the present work, we studied in detail the sexual process of conjugation using the model species Euplotes vannus, and compared these nuclear events with those occurring in other ciliates. Our results indicate that in E. vannus: 1) conjugation requires about 75 hours to complete: the longest step is the development of the new macronucleus (ca. 64h), followed by the nuclear division of meiosis I (5h); the mitotic divisions usually take only 2h; 2) there are three prezygotic divisions (mitosis and meiosis I and II), and two of the eight resulting nuclei become pronuclei; 3) after the exchange and fusion of the pronuclei, two postzygotic divisions occur; two of the four products differentiate into the new micronucleus and macronucleus, respectively, and the parental macronucleus degenerates completely; 4) comparison of the nuclear events during conjugation in different ciliates reveals that there are generally three prezygotic divisions while the number of postzygotic divisions is highly variable. These results can serve as reference to investigate the mating type system operating in this species and to analyze genes involved in the different steps of the sexual process.     

The perinuclear microtubule system in the green algaAcetabularia: anchor or motility device?

Summary Migrating secondary nuclei inAcetabularia are tightly associated with actin bundles and possess a comet-like tail composed of microtubules. When secondary nuclei begin to settle in preparation for cyst morphogenesis, the tails expand into radially symmetrical arrays of microtubules. Concomitantly, nuclei become gradually dissociated from the actin bundles and eventually stop moving, even though the actin bundles remain intact and persist through this stage. If, however, the radial perinuclear microtubule arrays are destroyed by inhibitors, the nuclei reassociate with the actin bundles and regain their motile activity. Because this movement is sensitive to Cytochalasin D, we propose that actin is required for nuclear movements, whereas microtubules most likely function as a trailing anchor that begins to act as a braking device above a certain threshold in the number and length of perinuclear microtubules.Dedicated to Prof. Dr. Dr. h.c. Eberhard Schnepf on the occasion of his retirement     

Involvement of 14-nm Filament-Forming Protein and Tubulin in Gametic Pronuclear Behavior during Conjugation in Tetrahymena

We have studied in detail the immunofluorescence localizations of Tetrahymena 14-nm filament-forming protein (49-kDa protein) in relation to tubulin in conjugating wild-type Tetrahymena thermophila (B strain) pairs and in pairs between B strain and star strains with defective micronuclei. The results suggest that germ nuclear behavior during conjugation may involve the following cytoskeletal structures: (1) during meiosis, microtubule structures are involved in micronuclear elongation and meiotic division; (2) at the postmeiotic stage, 49-kDa protein network structures that are formed independently of the existence of pronuclei are involved in the selection and the survival of one of four meiotic products; (3) during the third prezygotic division, gametic pronuclear transfer, and zygote formation, a cytoskeletal structure in which the 49-kDa protein colocalizes with microtubules and which is dependent on the existence of a normal gametic pronucleus is involved in gametic pronuclear behavior, and (4) during the postzygotic divisions, the microtubules are involved in nuclear behavior.     

Structure and function of the microtubular cytoskeleton during endosperm development in wheat: an immunofluorescence study

Summary The three-dimensional structure of the microtubular cytoskeleton of developing wheat endosperm was investigated immunocytochemically. Semi-thin sections were prepared from polyethylene glycol embedded ovaries. At the free-nuclear stage the endosperm cytoplasm with regularly distributed nuclei surrounded a large central vacuole and exhibited an extensive network of fluorescent labelled microtubular assemblies radiating from each nucleus. As was found in other coenocytes, this particular and nuclear-dependent cytoskeletal configuration functions in the arrangement of nuclei and in the stabilization of the nuclear positions. At the beginning of cellularization of the endosperm the formation of vacuoles altered the radiating networks. It is likely that the radiating microtubular arrays function in the formation of phragmoplasts, independent of nuclear divisions. The formation of anticlinal cell walls, giving rise to openended cell cylinders, coincides with the occurrence of phragmoplast microtubular arrays which were demonstrated during the period of cell wall elongation. The microtubular system radiating from the nuclei in these cell cylinders anchored the nuclei in stage- and locus-specific positions. During the development of aleurone and inner endosperm cells, cell morphogenesis was related to earlier demonstrated types of microtubular configurations in the cortical cytoplasm. This suggests that a general mechanism is involved.Abbreviations A alveolus - AL aleurone layer - CE central endosperm - CV central vacuole - DAP days after pollination - END endosperm - FITC fluorescein isothiocyanate - GAR-FITC goat anti-rabbit antibodies conjugated with FITC - I integument - IE PC inner epidermis pericarp - II inner integument - N nucleus - NC nucellus cells - NE nucellar epidermis - NUC nucellus - OI outer integument - PBS phosphate buffered saline - PC pericarp - PEG polyethylene glycol - V vacuole     

The architecture of microtubular network and Golgi orientation in osteoclasts--major differences between avian and mammalian species

In the present study, we analyze multinuclear osteoclasts obtained from several avian and mammalian species and describe the reorganization of their microtubular architecture and Golgi complex orientation during osteoclast differentiation and activation for bone resorption. In nonresorbing quail and chicken multinuclear osteoclasts, microtubules radiate from multiple centrosomal microtubule-organizing centers (MTOCs), whose number is equal to the number of nuclei. However, centrosomal MTOCs disappear at the time of cell activation for bone resorption and the Golgi membranes redistribute to circumscribe nuclei. In contrast to avian osteoclasts, both resorbing and nonresorbing rat, rabbit, and human osteoclasts have no or few centrosomal MTOCs. Instead, after cold-induced depolymerization, regrowing microtubules nucleate from the perinuclear area where immunofluoresce and immunoelectron scanning microscopy reveal pericentriolar matrix protein pericentrin associated with vimentin filaments. Furthermore, the circumnuclear reorganization of MTOCs and the Golgi is a result of mammalian osteoclast maturation and occur before any resorptive activity of the mononuclear osteoclasts and their fusion into multinucleated cells. Our results show that unlike previously suggested, the nuclear surfaces of mammalian osteoclasts act as the microtubule anchoring sites similarly to nuclear surfaces in multinucleated myotubes and suggest the role of perinuclear intermediate filament network in orchestrating the microtubular cytoskeleton.     

Mitosis of the free-living flagellate Bodo saltans strain Ps+ (Kinetoplastidea, Bodonida)

Mitosis of the free-living flagellate Bodo saltans of the Ps+ strain characterized by the presence of prokaryotic cytobionts in the perinuclear space was studied. Division of B. saltans Ps+ nuclei occurs by the closed intranuclear type of mitosis without condensation of chromosomes. At the initial stages of nuclear division, consecutive anlage of two spatially separated microtubular spindles begins. The spindle containing about 20 microtubules appears first, then, at an angle of 30–40° to it, the second spindle containing half as many microtubules is formed. The microtubules of the first spindle are associated with 4 pairs of kinetochores, the microtubules of the second one—with 2 pairs. The kinetochores of B. saltans Ps+ have a pronounced laminar structure. Both spindles rest with their ends directly on the internal membrane of the nuclear envelope and form 4 well-pronounced poles. The equatorial phase of mitosis in B. saltans Ps+ is not revealed. The divergence of sister kinetochores towards the poles occurs independently in each spindle. At the elongation phase of mitosis, the poles of both spindles are united in pairs to form a single bipolar structure composed of two loose bundles of microtubules. At this stage of nuclear division, the kinetochores reach the poles of the subspindles and cease to be visible. At subsequent nuclear division stages the nucleus acquires a dumbbell shape. During the reorganization phase the sister nuclei are separated. In the perinuclear space of the interphase nuclei of B. saltans Ps+, 1–2 prokaryotic cytobionts are present. In the course of mitosis, these organisms divide intensively, such that their number can reach 20 and more per nucleus. During separation of sister nuclei, the “excessive” cytobionts are released into the cytoplasmic vacuoles formed by external membranes of the nuclear envelope.     

New arrays of cytoplasmic microtubules in the fission yeastSchizosaccharomyces pombe

Summary New arrays of microtubules in the fission yeastSchizosaccharomyces pombe, which distribute in the cell in a cell cycle-dependent manner, were characterized using conventional and confocal laser scanning immunofluorescence microscopy. During the interphase and prophase, we observed abundant cytoplasmic microtubules between cell poles, a peripheral network of randomly and helically distributed cortical microtubules, and perinuclear microtubules surrounding the nucleus. At the anaphase and telophase, an equatorial ring containing tubulin was visualized. This ring colocalized with an actin contractile ring, suggesting that they may control the plane of cell division cooperatively.Abbreviations MT(s) microtubule(s) - cMT(s) cytoplasmic microtubule(s) - CLSM confocal laser scanning microscopy - DAPI 4,6-diamidino-2-phenylindole     

Dynamic behaviour of stationary pronuclei during their positioning in Paramecium caudatum

During conjugation of Paramecium caudatum, there are two well-known stages when nuclear migration occurs. What happens to the nuclei is closely related to their localisations in cells. The first of these stages is the entrance of one meiotic product into the paroral region. This nucleus survives, while the remaining three outside this area degenerate. The second stage is the antero-posterior localisation of eight synkaryon division products. Four posterior nuclei are differentiated into macronuclear anlagen, whereas four anterior nuclei remain as the presumptive micronuclei. In this experiment, the process of the third prezygotic division of P. caudatum was studied with the help of protargol staining. Here, a third nuclear migration was discovered. By two spindle turnings and two spindle elongations, stationary pronuclei were positioned near migratory pronuclei. This positioning of stationary pronuclei could shorten the distance for transferred migratory pronuclei to recognise and reach the stationary pronuclei. This fosters the synkaryon formation of P. caudatum.     

Mitochondrial distribution and microtubule organization in fertilized and cloned porcine embryos: implications for developmental potential

Mitochondrial distribution and microtubule organization were examined in porcine oocytes after parthenogenesis, fertilization and somatic cell nuclear transfer (SCNT). Our results revealed that mitochondria are translocated from the oocyte's cortex to the perinuclear area by microtubules that either constitute the sperm aster in in vitro-fertilized (IVF) oocytes or originate from the donor cell centrosomes in SCNT oocytes. The ability to translocate mitochondria to the perinuclear area was lower in SCNT oocytes than in IVF oocytes. Sperm-induced activation rather than electrical activation of SCNT oocytes as well as the presence of the oocyte spindle enhanced perinuclear mitochondrial association with reconstructed nuclei, while removal of the oocyte spindle prior to sperm penetration decreased mitochondrial association with male pronuclei without having an apparent effect on microtubules. We conclude that factors derived from spermatozoa and oocyte spindles may affect the ability of zygotic microtubules to translocate mitochondria after IVF and SCNT in porcine oocytes. Mitochondrial association with pronuclei was positively related with embryo development after IVF. The reduced mitochondrial association with nuclei in SCNT oocytes may be one of the reasons for the low cloning efficiency which could be corrected by adding yet to be identified, sperm-derived factors that are normally present during physiological fertilization.     

Minispindles and cytoplasmic domains in microsporogenesis of orchids

Summary Changes in the microtubular cytoskeleton during meiosis and cytokinesis in hybrid moth orchids were studied by indirect immunofluorescence. Lagging chromosomes not incorporated into telophase nuclei after first meiotic division behave as small extra nuclei. Events in the microtubular cycle associated with these micronuclei are similar to and synchronous with those of the principal nuclei. During second meiotic division the micronuclei trigger formation of minispindles which are variously oriented with respect to the two principal spindles. After meiosis, radial systems of microtubules measure cytoplasmic domains around each nucleus in the coenocyte. Cleavage planes are established in regions where opposing radial arrays interact and the cytoplasm cleaved around micronuclei is proportionately smaller than that around the four principal nuclei. These observations clearly demonstrate that nuclei in plant cells are of fundamental importance in microtubule organization and provide strong evidence in support of our recently advanced hypothesis that division planes in simultaneous cytokinesis following meiosis are determined by establishment of cytoplasmic domains via radial systems of nuclear-based microtubules rather than by division sites established before nuclear division.Abbreviations DMSO dimethylsulfoxide - FITC fluorescein isothiocyanate - MTOC microtubule organizing center - PBS phosphate buffered saline - PPB preprophase band of microtubules     

The structure of the flagellar apparatus of the swarm cells ofPhysarum polycephalum

Summary Flagellation ofPhysarum polycephalum amoebae (Myxomycete) involves the formation around the two kinetosomes of a flagellar apparatus leading to a modification in the shape of the amoeba and its nucleus. A tridimensional ultrastructural model of the flagellar apparatus is proposed, based upon observation of the isolated nucleo-flagellar apparatus complex. The flagellar apparatus is composed of a non-microtubular structure (the posterior para-kinetosomal structure), five microtubular arrays and two flagella: a long anterior flagellum and a short flagellum directed backwards. The asymmetry of the flagellar apparatus is due mainly to the presence of the posterior para-kinetosomal structure on the right side of the posterior kinetosome and of the two asymmetrical microtubular arrays 3 and 4. Thus, the flagellar apparatus is right-handed. This asymmetry implies also some spatial constraints on two other microtubular arrays (2 and 5). Except in the case of the microtubular array 1 which links the proximal end of the anterior kinetosome to the nuclear membrane, the number of microtubules of each microtubular array seems to be well defined: 39, 5–6, 7–9, and 2+2 for the microtubular arrays 2, 3, 4, and 5 respectively. All the elements of the nucleo-flagellar apparatus complex are linked either directly or indirectly through bridges. Furthermore, the microtubules which composed the microtubular array 3 are linked through bridges while the microtubules of the microtubular arrays 2, 3, and 4 seem to be linked through a reticulate material. All these spatial relationships lead to a great cohesion of the nucleo-flagellar apparatus complex which appears to be a well defined structure. This suggests thatPhysarum amoebal flagellation can be a promising system to study the morphogenesis of an eucaryotic cell.Abbreviations PIPES Piperazine-N,N-bis [2-ethane-sulfonic acid] - EGTA [Ethylenebis(oxyethylenenitrile)]tetraacetic acid - DMSO Dimethyl sulfoxide     

Confocal Microscopic Observations on Microtubular Cytoskeleton Changes During Megasporogenesis and Megagametogenesis in Phaius tankervilliae (Aiton) Bl.

In nun orchid (Phaius tankervilliae (Alton) B1. ) embryo sac development follows the monosporic pattern. Changes in the pattern of organization of the microtubular cytoskeleton during megasporogenesis and megagametogenesis in this orchid were studied using the immunofluorescence technique and eonfocal microscopy. At the initial stage of development the microtubules in the arehesporium were randomly oriented into a network. Later the archesporial cell elongated to form the megasporocyte. The cytoskeleton in the elongated megasporoeyte was radially organized in which microtubules extending from the nuclear envelope to the peripheral region of the cell. The megasporoeyte then underwent meiosis 1 to form a dyad. The dyad cell at the chalazal end was larger than the cell at the micropylar end. Microtubules in the dyad cell were radially oriented. The dyad underwent meiosis to give rise to a linear array of four megaspores (i. e. tetrad formation). The chalazal-far most megaspore survived and became the functional megaspore, which contained a set of randomly oriented microtubules. The microtubules in the other 3 megaspore disappeared as the cells degenerated. The functional megaspore then underwent mitotic division giveing rise to a 2 nucleate embryo sac. The nuclei of the 2-nucleate embryo sac were separated by a set of longitudinally oriented microtubules which ran parallel to the long axis of the embryo sac. Each nucleus in the embryo sac was surrounded by a set of perinuelear microtubules. The gnucleate embryo sac again underwent mitotic division to form a 4-nucleate embryo sac. The division of the two nuclei was synchronous. But the orientation of the division plan of the two spindles was different (i. e. the spindle microtubules at the chalazal end ran parallel with the long axis of the embryo sac and those at the mieropylar end ran at right angle to the axis of the embryo sac). The 4 nuclei of the 4-nucleate embryo sac were all tightly surrounded by randomly oriented microtubules. Later the paired nuclei at the micropylr end and at the chalazal end as well underwent mitotic division in seguence. At this time when the embryo sac had reached the 8-nucleate embryo sac stage. The pattern of organization of the microtubules was very complex. Initially the nuclei were surrounded by a set of randomly oriented microtubules, but after the two polar nuclei had moved to the central region of the embryo sac, three different organizational zones of microtubules appeared, viz: a randomly oriented set of microtubules surrounding each nucleus in the chalazal zone: a set (in the form of a basket) of cortical microtubules which surrounded the vacuoles and the two polar nuclei in the central zone and a loosely knitted network of microtubules surrounding the nucleus that later became the egg cell nucleus in the micropylar zone. The two nuclei that would become the nuclei of the synergids were surrounded by a set of more densely packed mierotubules. Towards far the most micropylar end some microtubules formed thick bundles. The site of appearance of these thick bundles coincided with the site of development of the filiform apparatus. The pattern of microtubule organization after cellularization (i. e. at the beginning of embryo sac maturation) did not change much. The author's results indicated that various patterns of microtubule organization observed in the developing embryo sac of nun orchid reflected the complexity and dynamism of the embryo sac.     

Microtubule-driven nuclear movements and linear elements as meiosis-specific characteristics of the fission yeasts Schizosaccharomyces versatilis and Schizosaccharomyces pombe

Meiotic prophase in Schizosaccharomyces pombe is characterized by striking nuclear movements and the formation of linear elements along chromosomes instead of tripartite synaptonemal complexes. We analysed the organization of nuclei and microtubules in cells of fission yeasts undergoing sexual differentiation. S. japonicus var. versatilis and S. pombe cells were studied in parallel, taking advantage of the better cytology in S. versatilis. During conjugation, microtubules were directed towards the mating projection. These microtubules seem to lead the haploid nuclei together in the zygote by interaction with the spindle pole bodies at the nuclear periphery. After karyogamy, arrays of microtubules emanating from the spindle pole body of the diploid nucleus extended to both cell poles. The same differentiated microtubule configuration was elaborated upon induction of azygotic meiosis in S. pombe. The cyclic movements of the elongated nuclei between the cell poles is reflected by a dynamic and coordinated shortening and lengthening of the two microtubule arrays. When the nucleus was at a cell end, one array was short while the other bridged the whole cell length. Experiments with inhibitors showed that microtubules are required for karyogamy and for the elongated shape and movement of nuclei during meiotic prophase. In both fission yeasts the SPBs and nucleoli are at the leading ends of the moving nuclei. Astral and cytoplasmic microtubules were also prominent during meiotic divisions and sporulation. We further show that in S. versatilis the linear elements formed during meiotic prophase are similar to those in S. pombe. Tripartite synaptonemal complexes were never detected. Taken together, these findings suggest that S. pombe and S. versatilis share basic characteristics in the organization of microtubules and the structure and behaviour of nuclei during their meiotic cell cycle. The prominent differentiations of microtubules and nuclei may be involved in the pairing, recombination, and segregation of meiotic chromosomes.     

Induction of Blocks in Nuclear Divisions and Overcondensation of Meiotic Chromosomes with Cycloheximide During Conjugation of Tetrahymena thermophila

During conjugation, the micronucleus of Tetrahymena thermophila undergoes five consecutive nuclear divisions: meiosis, third prezygotic division (pregamic mitosis) and two postzygotic mitoses of the synkaryon. The four products of the synkaryon differentiate into macronuclear anlagen and new micronuclei and the old macronucleus is resorbed. The protein synthesis inhibitor cycloheximide, applied during conjugation, induced several developmental blocks. Pairs shifted to the drug during early meiotic prophase (stages I–III) were arrested at prophase. Cycloheximide applied to cells at pachytene (stages IV-VI) to metaphase arrested the conjugants at the stage of modified prometaphase/metaphase with overcondensed, swollen bivalents. In contrast to other systems, in the presence of cycloheximide, separation of chromatids, decondensation of chromosomes and exit from metaphase I were inhibited in both diploid and haploid cells. Pairs shifted to the drug after metaphase I were arrested at postmeiotic interphase after completing one nuclear cycle. The same rule applied to the subsequent cycle; then cells were arrested at the stage of pronuclei, and those pairs with functional pronuclei and synkarya were arrested at the stage of two products of the first postzygotic division (pronuclei were not arrested in nuclear transfer and karyogamy). Only pairs with two products of the first postzygotic division were arrested at the same stage after the cycloheximide treatment. Pairs shifted to cycloheximide during the second postzygotic division were arrested in development of macronuclear anlagen and resorption of old macronuclei. The postmeiotic conjugants pulse-treated with cycloheximide (2 h) yielded heterokaryons retaining parental macronuclei (i.e. they exhibited macronuclear retention).     

Ultrastructure of the mitotic nuclei in Leishmania mexicana ssp. Tridimensional reconstruction of the mitotic spindle and dense plaques

The ultrastructure of mitotic nuclei of the promastigote Leishmania mexicana ssp. was studied by serial thin sections and three-dimensional reconstructions of each divisional stage. At the beginning of nuclear division (equatorial stage), a set of six dense plaques located about the equatorial region of the nucleus and a microtubular spindle develops in the two opposing poles of the nucleus (two sets of polar microtubules). The microtubular mitotic spindle is entirely intranuclear with the nuclear membrane persisting through mitosis. The polar spindle consists of a discrete bundle of about 50 microtubules and the equatorial spindle is formed by about 100 microtubules. The spindle may contain several continuous microtubules, but no microtubular organizing centres were observed in association with the spindle. The plaques and hemiplaques are associated with microtubular bundles; some of the spindle microtubules converge on kinetochore-like plaques. It is suggested that the spindle has a special significance in the physiology of mitosis. The two sets of hemiplaques may guide the separation of the daughter genomes. At the beginning of the elongational stage the mitotic plaques split into halves and each set of half-plaques migrates to one pole. It is concluded that the dense plaques play a kinetochore-like role and thus Leishmania mexicana ssp. may have six chromosomal units. Mitotic events of this species are essentially similar to those of Trypanosoma cruzi.