Reorganization of microtubules in the amitotically dividing macronucleus of tetrahymena

We developed a modified immunofluorescence protocol that permitted visualization of microtubules inside the macronucleus of the ciliate Tetrahymena. Although the amitotically dividing macronucleus lacks a spindle, an elaborate system of microtubules is assembled inside the macronucleus and between the macronucleus and the cortex. Microtubules could not be detected inside the interphase macronuclei. The early stage of macronuclear division was associated with the assembly of short macronuclear microtubules that localized randomly. The intramacronuclear microtubules were subsequently organized in a radial manner. During elongation of the macronucleus, the distribution of macronuclear microtubules changed from radial to parallel. During constriction of the macronucleus, dense and tangled macronuclear microtubules were detected at the region of nuclear constriction. In the cytosol, microtubules were linking the macronucleus and cell cortex. During recovery after drug-induced depolymerization, microtubules reassembled at multiple foci inside the macronucleus in close proximity to the chromatin. We propose that these microtubules play roles in chromatin partitioning, macronuclear constriction, and positioning of the macronucleus in relation to the cell cortex.     

The ultrastructure of nuclear division in a suctorian,Tokophrya infusionum

Summary Ultrastructural changes in the micro- and macronucleus throughout division were followed in synchronized cultures of the suctorian, Tokophrya infusionum. After an initial swelling, the micronucleus elongates enormously; microtubules within the micronucleus proliferate and lengthen as the micronucleus elongates. Changes in the macronucleus become visible only after micronuclear division is well underway. The chromatin bodies fuse into long chromatin strands, and the large bundles of microtubules present in the resting macronucleus break up into small groups which parallel the chromatin strands. Colchicine, which prevents reproduction in Tokophrya, seems to block division at a very early stage. The macronucleus appears the same as the resting nucleus of untreated organisms, with numerous microtubules and distinct chromatin bodies. The chromatin in the micronucleus aggregates into large clumps, however, and proliferation of microtubules does not occur.Supported by a Graduate Fellowship at The Rockefeller University.Supported by Grant A1-01407-12 USPHS and Grant A1-08989-01 USPHS.     

Localization of microtubules during macronuclear division in Tetrahymena and possible involvement of macronuclear microtubules in 'amitotic' chromatin distribution

The ciliated protozoa Tetrahymena contains two nuclei, a micronucleus and a macronucleus. In the vegetatively growing cell, the macronucleus divides amitotic while the micronucleus divides by mitosis. It has been indicated that microtubules are involved in macronuclear division and microtubules are observed to exist in the dividing macronucleus. To clarify the localization and the organization of microtubules in the amitotic dividing macronuclei, we used immunofluorescent staining technique. The microtubules were observed in the cytoplasm and macronucleus. The microtubules were organized and dynamically changed their distribution throughout the macronuclear division. We suggest a possibility that these microtubules are involved in 'amitotic' distribution of chromatin throughout the macronuclear division.     

Acineta tuberosa

Zusammenfassung Die Veränderungen im Feinbau des Makronucleus während der ungeschlechtlichen Fortpflanzung von Acineta tuberosa (Suctoria) werden beschrieben. Bei Zeitrafferaufnahmen von Tokophrya lemnarum ist kurz vor der Teilung des Makronucleus eine um mehrere Zentren kreisende Bewegung chromosomaler Fäden beobachtet worden (Heckmann, 1966). Die entsprechenden Stadien bei Acineta wurden nun im elektronenmikroskopischen Bild identifiziert. Von besonderem Interesse ist die Verteilung der Mikrotubuli. Während im Makronucleus älterer Wachstumsstadien und adulter Tiere zahlreiche Bündel von 8 bis 20 Mikrotubuli vorhanden sind, wurden kurz vor und während der Teilung des Makronucleus nur wenige, einzeln liegende Mikrotubuli beobachtet.Wenn auch diese wenigen, einzelnen Mikrotubuli kinetische Strukturen sein mögen, die den kontinuierlichen Fasern der Mitosespindel entsprechen könnten, so zeigen die zahlreichen Tubulibündel vegetativer Zellen keine Beziehung zur Bewegung der chromosomalen Fäden oder zur Streckung des Makronucleus. Es muß angenommen werden, daß die Tubulibündel, die möglicherweise Ausdruck einer besonderen Stoffwechselleistung des somatischen Makronucleus sind, vor der Kernteilung teilweise wieder abgebaut werden.

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Acineta tuberosaII. The distribution of microtubules in the macronucleus during asexual reproduction

Summary Ultrastructural changes in the macronucleus during the complete cycle of asexual reproduction of Acineta tuberosa (Suctoria) are described. Using time-lapse photography Heckmann (1966) demonstrated that in Tokophrya lemnarum just prior to macronuclear division thread-like chromatin strands rotate around several centres. Corresponding stages have now been identified in electron micrographs of Acineta.Of considerable interest is the distribution of microtubules during cell cycle. Numerous straight bundles of 8 to 20 microtubules are present in the macronucleus of more advanced metamorphosing stages and adult suctorian animals. On the other hand only a very few separate microtubules were observed in predivisional and divisional stages. Although these few microtubules may represent kinetic elements, similar to continuous fibers of the mitotic spindle the numerous bundles of microtubules of non-reproducing cells show no relation to the movement of chromatin strands or to the macronuclear elongation prior to division. It is assumed that these masses of microtubules might be the expression of a special physiological activity of the somatic macronucleus, and that at least part of them become depolymerized before macronuclear division starts.

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Über einen Teil der Ergebnisse wurde auf der 150. Konferenz der British Society for Experimental Biology, Bristol, 26. 3.–29. 3. 68, berichtet (Bardele, 1968 b).

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Mit Unterstützung durch die Deutsche Forschungsgemeinschaft.     

The effect of trifluoroperazine on microtubules,nuclear division and the nuclear membranes of the ciliate Nyctotherus ovalis

Exposure of the dividing ciliate Nyctotherus ovalis to the tranquilizer trifluoroperazine (TFP; 10 M) leads to the complete disassembly of kinetochore microtubules in the metaphase micronucleus. Interpolar microtubules located underneath the micronuclear envelope at anaphase and telophase stembody microtubules are more resistant to TFP. However, stembodies of drug-exposed ciliates are much shorter than in the controls. In their centre they contain only a reduced number of widely separated microtubules, indicating that assembly of new tubules or elongation of existing microtubules at this site, which appears essential for further separation of the future daughter nuclei, is blocked by TFP. Although microtubules polymerized in the macronucleus during its elongation include a set of tubules made up of more than 13 protofilaments, comparable to the micronuclear stembody microtubules, they are much more sensitive towards drug treatment. Macronuclear tubules become completely depolymerized resulting in failure of nuclear stretching. Already elongated macronuclei can still become constricted in their centre which suggests that microtubules are not involved in this process. Disassembly and higher sensitivity of macronuclear compared with micronuclear microtubules may be explained by a different composition and behaviour of nuclear membranes towards TFP in the two types of nuclei. While the micronuclear envelope may be only partially destroyed where it is facing the macronucleus, the inner membrane of the macronuclear envelope is severely affected by drug treatment. It shows a multitude of infoldings accompanied by attachment of chromatin to it. Cytoplasmic microtubules which proved resistant to other depolymerizing drugs become partly disassembled during TFP treatment.     

The Nuclear Apparatus of the Ditransversal Ciliate Homalozoon vermiculare (Ciliophora,Rhabdophora) during Interphase and Division. I. The Macronucleus1

ABSTRACT. The nuclear apparatus of Homalozoon vermiculare consists of a single moniliform macronucleus and about 25 micronuclei. The number of macronuclear segments depends (i) on the number of divisions of individual segments during the interphase and (ii) on the number of segments that arise prior to cytokinesis from the (temporary) filiform macronucleus. Precytokinetic changes of the macronucleus involve the fusion of individual segments followed by contraction and subsequent elongation of the entire macronucleus. The chromatin bodies uncoil into fine fibrils during macronuclear contraction. At the time when the division furrow appears, the macronucleus starts to renodulate. The interphase segment contains a more or less reticulated chromatin body partly attached to the nuclear envelope and about 30 polymorphous nucleoli. The latter consist of the pars granulosa, the pars fibrosa, and an additional fibrillar component. The nucleoli undergo drastic changes prior to division and the granular component disappears completely during macronuclear condensation. On the average, the macronucleus contains a 3,400-fold amount of DNA compared with a haploid micronucleus, but the intraspecific differences in the DNA content of the entire macronucleus are extremely large. In contrast, DNA content and size of an individual segment of the macronucleus are precisely regulated during interphase.     

Cortical Control over the Direction of Macronuclear Elongation in the Heterotrich Ciliate Stentor coeruleus1

Earlier experimental work involving macronuclear implants in Stentor coeruleus has shown that the cytoplasmic cortex of the nuclear site 1) attracts the macronucleus and 2) holds it in place during interphase. Now experiments indicate macronuclei transferred with overlying cortex elongate in the direction of the transferred cortical pigment stripes, whether or not the transferred stripes realign in the direction of the host stentor's stripes. Therefore the third function of the cortex is to determine the direction of elongation and thus assure that both daughter cells at division receive part of the macronucleus.     

A cytological study of micronuclear elongation during conjugation in Tetrahymena

Micronuclear elongation is the first major event in a series of nuclear changes occurring during the sexual stage of the life cycle of Tetrahymena. Beginning at about one hour after cells of complementary mating types have conjugated, the micronucleus leaves its recess in the macronucleus and swells slightly. This is accompanied by a reorganization of its chromatin from a reticular to a solid body. In the next stage the micronucleus assumes an egg shape, a development concomitant with the appearance of microtubules. While the chromatin spins out from the dense body, and microtubules increase in number, the nucleus assumes a spindle shape. During the elongation, which increases the length of the nucleus some fifty fold, microtubules are prominent in clusters just internal to the nuclear membrane, and parallel to the longitudinal axis of the nucleus. When elongation is completed the nucleus is curved around the macronucleus. Internally, partially condensed strands of chromatin are located off-center, towards the macronuclear side, and the density of the microtubules is diminished. At all the stages, DNA is located throughout the nucleus; neither discrete chromosomes nor synaptonemal complexes are seen. Occasionally cytoplasmic membrane systems are seen fused to the nuclear envelope which retains the typical appearance of a double membrane with pores.     

Electron Microscopy of the Nuclear Events During Binary Fission in Paramecium multimicronucleatum

SYNOPSIS. From the interphase to the early stage of binary fission in Paramecium multimicronucleatum , when the micro-nuclei are situated close to the macronucleus, the microtubules in the cytoplasm seem to connect the nuclear pores of macro- and micronuclei. During the 1st half of macronuclear division, the microtubules are formed outside the macronucleus, while during the latter half of division, numerous microtubules appear inside it. Chromatin bodies and nucleoli remain unchanged during macro-nuclear division, but the latter show temporory irregularity in shape. In late prophase of micronuclear division, spindle micro-tubules are formed, and a polar structure, composed of randomly dispersed twisting filaments, is formed at each pole of the micro-nucleus at anaphase. Spindle microtubules terminate on the surface of this structure. The nuclear envolope of the macro-and micronuclei remains intact thruout division. The envelope of the daughter micronuclei is derived from the pre-existing one.     

Macronuclear division and cytokinesis in Tetrahymena

Tetrahymena contains a micronucleus and a macronucleus. The micronucleus divides with typical mitosis, while the macronucleus divides amitotically. Although the mechanism responsible for macronuclear division was previously unknown, we clarified the organization of microtubules during macronuclear division. The macronuclear microtubules dynamically changed their distribution in an organized way throughout the macronuclear division. The macronuclear microtubules and the cytoplasmic microtubules cooperatively carried out the macronuclear division. When the micronuclear division was finished, p85 appeared at the presumptive division plane prior to the cytokinesis. The p85 directly interacted with calmodulin in a Ca(2+)-dependent manner, and p85 and CaM colocalized to the division furrow during cytokinesis. Moreover, the Ca(2+)/CaM inhibitor, W7, inhibited the direct interaction between p85 and CaM, the localization of both proteins to the division plane, and the formation of the division furrow. Thus, Ca(2+)/CaM and p85 have important roles in initiation and progression of cytokinesis in Tetrahymena.     

Cytology of an Indian Race of Blepharisma undulans (Stein)

SYNOPSIS. The Indian race of Blepharisma undulans described in this paper measures 150 μ in length. The macronucleus consists of 5–7 nodes, all of equal size. During binary fission, condensation of macronucleus is followed by its elongation and a thinning of the middle region which breaks with the division of the animal. It later attains the typical vegetative form.
During conjugation 7 or 8 micronuclei pass through the first pregamic division, 5 to 7 through the second pregamic division and one product of the second division takes part in the third division. The rest degenerate. At the same time, the macronucleus also starts degenerating. After the synkaryon has divided twice, the conjugating pairs separate. Of the 4 products, 3 become macronuclear anlagen and one, micronuclear anlage.
The micronuclei divide asynchronously both during binary fission and during conjugation. There is apparently considerable diversity in the structure and behaviour of the macronucleus and micronuclei in the different races of Blepharisma undulans.     

The condensin complex is essential for amitotic segregation of bulk chromosomes, but not nucleoli, in the ciliate Tetrahymena thermophila

The macronucleus of the binucleate ciliate Tetrahymena thermophila contains fragmented and amplified chromosomes that do not have centromeres, eliminating the possibility of mitotic nuclear division. Instead, the macronucleus divides by amitosis with random segregation of these chromosomes without detectable chromatin condensation. This amitotic division provides a special opportunity for studying the roles of mitotic proteins in segregating acentric chromatin. The Smc4 protein is a core component of the condensin complex that plays a role in chromatin condensation and has also been associated with nucleolar segregation, DNA repair, and maintenance of the chromatin scaffold. Mutants of Tetrahymena SMC4 have remarkable characteristics during amitosis. They do not form microtubules inside the macronucleus as normal cells do, and there is little or no bulk DNA segregation during cell division. Nevertheless, segregation of nucleoli to daughter cells still occurs, indicating the independence of this process and bulk DNA segregation in ciliate amitosis.     

The Dynamics of Micronuclear Mitosis in the Living Ciliate Spirostomum teres as Revealed by Polarization Microscopy1

ABSTRACT. Micronuclear mitosis in living Spirostomum teres has been studied by sensitive polarization microscopy, and the dynamic aspects of micronuclear division are described. The small, spherical, interphase micronuclei lie in form-fitting depressions in the macronuclear surface. Nuclear division begins with the rounding and slight swelling of the macronucleus and, coincidentally, the micronuclei move out of the depressions and away from the macronucleus, increase in size, and become weakly birefringent. As mitosis proceeds, the micronuclei increase in uniaxial birefringence and elongate to form irregular ovoids that convert to angular structures displaying principal axes of positive birefringence so divergent as to appear oriented at a right angle to one another. Micronuclei maintain this appearance for as long as 60 min and then abruptly change to rectangular-shaped structures, increase in uniaxial birefringence, and begin anaphase elongation. The somewhat dumbbell-shaped micronuclei lengthen at the constant rate of 2.0 μm/min to reach lengths >70 μm. It appears that little half-spindle shortening occurs during spindle elongation. Accompanying the changes in micronuclear spindle length are changes in birefringence. Just before elongation begins, presumably metaphase, the micronucleus is uniformly and intensely birefringent. At the magnifications employed, a chromosome plate is not clearly visible as a region of reduced birefringence. As elongation begins, the putative half-spindles are more birefringent than is the interzone, a condition that is maintained until the spindles have achieved ～30% elongation, at which time a region of increased birefringence develops at the center of the interzone. This pattern persists for a very short time, then gives way to a uniform birefringence of the entire separation spindle that is maintained until elongation is completed. The rate of micronuclear spindle elongation, changes in micronuclear dimensions, and corresponding changes in birefringence are discussed with respect to possible mechanisms of mitosis.     

Apical growth and mitosis are independent processes in Aspergillus nidulans

Summary. It is well established that cytoplasmic microtubules are depolymerized during nuclear division and reassembled as mitotic microtubules. Mounting evidence showing that cytoplasmic microtubules were also involved in apical growth of fungal hyphae posed the question of whether apical growth became disrupted during nuclear division. We conducted simultaneous observations of mitosis (fluorescence microscopy) and apical growth (phase-contrast microscopy) in single hyphae of Aspergillus nidulans to determine if the key parameters of apical growth (elongation rate and Spitzenkörper behavior) were affected during mitosis. To visualize nuclei during mitosis, we used a strain of A. nidulans, SRS27, in which nuclei are labeled with the green-fluorescent protein. To reveal the Spitzenkörper and measure growth with utmost precision, we used computer-enhanced videomicroscopy. Our analysis showed that there is no disruption of apical growth during mitosis. There was no decrease in the rate of hyphal elongation or any alteration in Spitzenkörper presence before, during, or after mitosis. Our findings suggest that apical growth and mitosis do not compete for internal cellular resources. Presumably, the population of cytoplasmic microtubules involved in apical growth operates independently of that involved in mitosis.Present address: Department of Plant Sciences, University of Oxford, Oxford, United Kingdom.     

Cell division in the marine slime mold,Labyrinthula sp., and the role of the bothrosome in extracellular membrane production

Summary Electron microscopic observations of vegetative cell division inLabyrinthula indicate that the specialized invaginations of the cell surface called bothrosomes arisede novo between newly divided daughter cells and function in the production of the membrane-bound extracellular matrix or slimeways. Protocentrioles are formed before each division and persist through cell separation but are not found in interphase cells. Cytokinesis begins after the completion of mitosis and occurs by vesicle accumulation and fusion, an unusual cytokinetic mechanism reminiscent of zoospore cleavage. Cell elongation after cytokinesis is accompanied by elongation of the Golgi apparatus and the appearance of non-spindle microtubules.     

The organization of microtubules during generative-cell division inConvallaria majalis

Summary The organization of the microtubule cytoskeleton in the generative cell ofConvallaria majalis has been studied during migration of the cell through the pollen tube and its division into the two sperm cells. Analysis by conventional or confocal laser scanning microscopy after tubulin staining was used to investigate changes of the microtubule cytoskeleton during generative-cell migration and division in the pollen tube. Staining of DNA with 4,6-diamidino-2-phenylindole was used to correlate the rearrangement of microtubules with nuclear division during sperm cell formation. Before pollen germination the generative cell is spindle-shaped, with microtubules organized in bundles and distributed in the cell cortex to form a basketlike structure beneath the generative-cell plasma membrane. During generative-cell migration through the pollen tube, the organization of the microtubule bundles changes following nuclear division. A typical metaphase plate is not usually formed. The generative-cell division is characterized by the extension of microtubules concomitant with a significant cell elongation. After karyokinesis, microtubule bundles reorganize to form a phragmoplast between the two sperm nuclei. The microtubule organization during generative-cell division inConvallaria majalis shows some similarities but also differences to that in other members of the Liliaceae.Abbreviations CLSM confocal laser scanning microscopy - EM electron microscopy - GC generative cell - GN generative nucleus - MT microtubule - SC sperm cell - SN sperm nucleus - VN vegetative nucleus     

Elimination of DNA sequences during macronuclear differentiation in Tetrahymena thermophila,as detected by in situ hybridization

Previous studies have indicated that certain sequences in the micronuclear genome are absent from the somatic macronucleus of Tetrahymena (Yao and Gorovsky, 1974; Yao and Gall, 1979; Yao, submitted). The present study used in situ hybridization to follow the elimination process during the formation of the new macronucleus. Micronuclear-specific DNA cloned in recombinant plasmids was labelled with ³H and hybridized to cytological preparations of T. thermophila at various stages of conjugation. Despite a smaller size and lower DNA content, the micronucleus has more hybridization than the mature macronucleus. Hybridization initially increased in the anlage (newly developing macronucleus) to reach a maximal level right after the old macronuclei had disappeared. The hybridization in the anlage then decreased to a significant extent prior to the first cell division. The results suggest that the micronuclear-specific sequence is first replicated a few rounds before it is eliminated from the anlage, and the elimination process occurs without nuclear division.     

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

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

A UNIQUE MITOTIC VARIATION IN THE MARINE DINOFLAGELLATE OXYRRHIS MARINA (PYRROPHYTA)1

Mitosis is described in the flagellate Oxyrrhis marina Dujardin and is compared in related genera. Dense plaques develop in the nuclear envelope at prophase and give rise to an intranuclear spindle. Some of the microtubules associate with the chromosomes while others extend across the nucleus. The basal bodies migrate toward the poles early in division and retain a position lateral to the nuclear poles throughout mitosis. Microtubules are not present between the nucleus and the basal bodies. The nucleolus is persistent and elongates throughout anaphase and telophase. Chromosomal separation is accomplished by sliding of non-chromosomal microtubules and by elongation of the nuclear envelope rather than by shortening of the spindle microtubules. The nuclear envelope begins to constrict in the center early in anaphase. Continued constriction of the envelope and elongation of the nucleus leads to the formation of a dumbbell-shaped nucleus by late telophase. Mitosis culminates by the constriction of the nucleus into two daughter nuclei. The taxonomic position of Oxyrrhis marina is discussed in light of these findings.