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.     

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.     

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.     

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.     

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.     

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.     

Macro- and Micronuclei of Tetrahymena pyriformis: A Model System for Studying the Structure and Function of Eukaryotic Nuclei*†

The macro- and micronucleus of Tetrahymena pyriformis are formed from a common diploid synkaryon during conjugation. Shortly after the 2nd postzygotic division, distinct morphologic and physiologic differences develop between the 2 nuclei. Micronuclei remain small, presumably diploid, and electronmicroscopic observations indicate that micronuclear DNA is contained in a dense, fibrous, chromosome-like coil. Macronuclei contain considerably more DNA than micronuclei, and the DNA of the macronucleus is found largely in the chromatin bodies typical of ciliate nuclei. The functional differences between macro- and micronuclei in vegetative cells also are striking. The template activity of DNA in the micronucleus is highly restricted compared to that in the macronucleus. Micronuclei synthesize and contain little RNA, and do not contain either nucleoli or ribonucleoprotein granules. Macronuclei, on the other hand, synthesize and contain large amounts of RNA and have many nucleoli and ribonucleoprotein granules. Macro- and micronuclei also have distinct differences in the timing of DNA synthesis during the cell cycle and in the timing and mechanism of nuclear division. Finally, during conjugation the macronucleus becomes pycnotic and disappears while the micronucleus undergoes meiosis and fertilization, ultimately giving rise to new macro- and new micronuclei. In short, the macro- and micronuclei of Tetrahymena provide an excellent system for studying the molecular mechanisms by which the same (or related) genetic information is maintained in different structural and functional states. Methods have been devised to isolate and purify macro- and micronuclei of Tetrahymena in the hope of correlating differences in the nucleoprotein composition of these nuclei with differences in their structure and function. The DNAs of macro- and micronuclei have been found to differ markedly in their content of a methylated base, N⁶-methyl adenine, and major differences in the histones of the 2 nuclei have been observed. Macronuclei contain histones similar to those found in vertebrate nuclei, while 2 major histone fractions seem to be missing in micronuclei. In addition, histone fraction F2A1 which is found in multiple, acetylated forms in macronuclei, is present only as a single, unacetylated form in micronuclei.     

Nuclear Behavior of Euplotes woodruffi During Conjugation

SYNOPSIS. During conjugation of E. woodruffi , the micro-nucleus divides repeatedly four times prior to synkaryon formation and twice thereafter. The first division resembles an ordinary somatic mitosis, resulting in the formation of two daughter nuclei in each conjugant. Both products of this division enter the second division which corresponds to the heterotypic division of other ciliates, characterized by a parachute stage. Following this stage sixteen bivalents appear and separate into dyads and pass to the poles. During the following divisions individualized chromosomes do not appear but only certain chromatin elements comparable to those seen in the somatic and preliminary divisions. These divide and pass to the poles. All daughter nuclei of the second division enter and complete the third division. Only two of the products of the third division enter the final pregamic division while the rest degenerate. Exchange of pronuclei and their fusion leads to synkaryon formation. The conjugants then separate and in each exconjugant the synkaryon divides twice in rapid succession. Of the four products one condenses to become the functional micronucleus, another enlarges rapidly to become the macronuclear anlage while the remaining two degenerate and disintegrate. The old macronucleus breaks into irregular and polymorphic bodies. As the macronuclear anlage enlarges the remnants of the old macronucleus reorganize and fuse with the macronuclear anlage to form a characteristic vegetative macronucleus.     

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.     

Ultrastructural changes in vitro of rat liver nucleoli in response to polyamines

Summary Structural alterations of the nucleoli of rat liver cells were noted when these nuclei were isolated with spermidine or spermine rather than magnesium. When 5–10 mM spermidine or spermine were used to isolate the nuclei, the nucleoli were a) larger, b) contained numerous and sometimes large lacunae, and c) were less aggregated and had prominent chromatin caps. These chromatin caps gave the nucleolus a ring-shaped appearance in the light microscope. These findings, coupled with physiological data that indicate that polyamines enhance nucleolar RNA polymerase activity (Russell et al., 1971), suggest that spermidine and spermine may be involved in the control of ribosomal RNA synthesis. To our knowledge, this is the first instance of the direct stimulation of ribosomal RNA synthesis during nuclear isolation.Supported by USPHS Grant NS-07934.     

Ultrastructure of the nuclear apparatus of the infusorian Loxodes magnus. I. The macronuclei, macronuclear anlagen and the interphasic micronuclei]

The nuclear apparatus of Loxodes magnus Stokes (Holotricha) consists of numerous macronuclei which belong to the diploid type and never divide, and of numerous micronuclei. No nuclear groups exist; individual nuclei often lie in cytoplasmic islets surrounded by large lacunae of the smooth endoplasmic reticulum. Interphasic micronuclei have two-membraned envelopes with numerous pores, usually lined at the cytoplasmic side with a layer of vacuoles, channels, or flattened vesicles of the smooth endoplasmic reticulum. The chromatin of the micronuclei consists of anastomosing threads, 0.1--0.2 mum wide, between which several nucleolus-like bodies of microfibrillar structure occur. Adult macronuclei have a similar nuclear envelope and a similar system of vacuoles, channels, and flattened agranular cisternae outside it. The macronucleus contains a single large composite nucleolus with 3 or 4 fibrillar cores inside the common granular cortex. The fibrillar cores are pierced by channels containing nucleolar organizers in the form of strands of condensed chromatin. The peripheral zone of the macronucleus is filled with decondensed chromatin fibrils and contains a number of small chromocenters and several aggregates of RNP granules. No protein inclusions (spheres) have been observed in Loxodes macronuclei. The macronuclear anlagen, developing in the cycle of every cell division, show progressive decondensation of the chromosomes and formation of several nucleoli, each with its own organizer. Later on, the nucleoli fuse into a single nucleolus. The small chromocentres are the last to form.     

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.     

Fine structure of the macronucleus during the cell division cycle of Euplotes eurystomus,a Ciliate Protozoon

This paper reports new observations obtained from a study of macronuclear fine structure throughout various stages of the cell division cycle of Euplotes. Study of the ultrastructural organization of the macronuclear chromatin indicates that much of the chromatin is organized into continuous masses, portions of which appear to be attached to the nuclear envelope. The macronuclear envelope appears unchanged in the region of a replication band, and apparent attachments of the chromatin to the inner membrane of the nuclear envelope are maintained in the reticular and diffuse zones. Intranuclear helices were never observed in the diffuse zone. During macronuclear division, linear elements (fibrils or microtubules) were observed in close association with both chromatin bodies and nucleoli. The ultrastructural data suggest that the intranuclear linear fibrils have two functions: elongation of the dividing nucleus, and attachment of chromatin bodies and nucleoli to the envelope. The significance of these observations for macronuclear division and chromatin segregation is considered.     

The relationship between microtubules and chromatin in spermatids ofAcrididae

Summary A frequent coincidence of perinuclear microtubules and chromatin strands at nuclear membrane level was observed in spermatids ofChorthippus apicalis andOedipoda coerulescens. More developed spermatids also show apparent continuity between these chromatin strands and microtubules. We would suggest the importance of this relationship in the arrangement adopted by these strands.     

The Fine Structure of the Phoront of the Apostomatous Ciliate,Hyalophysa chattoni*

The phoront of the apostomatous ciliate, Hyalophysa chattoni, is an encysted stage that is carried on the exoskeleton of its crustacean host until the ecdysis of the host. At molting the phoront rapidly metamorphoses to the feeding stage, excysts, and immediately begins to feed on exuvial fluid trapped in the cast-off exoskeleton. The fine structure of the resting phoront resembles that of the preceding migratory stage, the tomite. A prominent ventral tuft of cilia, the ogival field, has vanished, and the trichocysts that paralleled the kinetics have disappeared. The dense inclusion bodies that were concentrated around the mouth and falciform fields have dispersed and greatly decreased in number. The cytoplasm and its membranous organelles do not appear visibly condensed or altered from the preceding stage in the life cycle. The phoront is merely quiescent instead of dormant. Unlike the few ciliate cysts previously examined by electron microscopy, the phoront's cyst is not divisible into separable layers. It resembles the loricae of certain suctoria in being formed principally of a fibrous substance, the outer surface of which has a paracrystalline pattern. The peduncle attaching the cyst to the crab's gill is a continuation of the cyst wall although its structure is somewhat modified. The most conspicuous innovation in the phoront's fine structure is the massive tracts of microtubules that run longitudinally through the macronucleus. The microtubules are in intimate contact with Feulgen-positive chromatin masses which are crowded toward the periphery of the macronucleus.     

A Cytological Study and Reclassification of Trichophrya collini (Saedeleer & Tellier)1

ABSTRACT. Trichophrya collini has a polygonal, dorsoventrally flattened body (up to 75 μm diam.), with capitate tentacles arranged in 1–3 rows within peripheral fascicles. There is a central polymorphic macronucleus, an associated micronucleus, and numerous peripheral contractile vacuoles with ventral discharge pores. The cell has a multilayered cortex and the cytoplasm contains suctorian organelles such as crescentic bodies, elongate dense bodies, and haptocysts. The highly contractile tentacles have an axoneme with an outer ring of 24 microtubules separated into six groups and an inner ring of six curved lamellae, each with five microtubules. The lamellae at the distal and proximal ends of the axoneme are arranged in a helix, and the outer ring microtubules are joined in a distal connective sheath. In the apical knob of the tentacle, the haptocysts are borne on a central capsule, Reproduction is by endogenous budding to produce a single oval-shaped swarmer, with equatorial ciliature, which metamorphoses within 3 h. These observations suggest that this organism, previously known as Heliophrya collini Saedeleer & Tellier, is synonymous with Platophrya rotunda Gönnert, Craspedophrya rotunda Rieder, and Heliophrya rotunda Matthes. Its endogenous mode of budding assigns it to the genus Trichophrya. but it is distinct from Trichophrya rotunda Hentschel, and should be reclassified to Trichophrya collini (Saedeleer & Tellier).     

Ultrastructural features of mitosis inLeishmania adleri

Summary The interphase nucleus ofLeishmania adleri has clumps of chromatin associated with the nuclear envelope and a large centrally located nucleolus. Prior to mitosis the basal bodies replicate at the cell anterior. Subsequently, dense plaques appear in the equatorial region of the nucleus at the time of spindle development. Microtubules appear in the nucleus adjacent to the nuclear envelope and embedded in the matrix of the plaques. A central spindle composed of a single bundle of microtubules develops and spans the nucleus. Plaques and nucleolar components laterally associate with the spindle and migrate towards the poles. The central spindle elongates to three to four times its original length separating the forming daughter nuclei and producing an interzonal spindle. A remnant of the interzonal spindle remains attached to each of the daughter nuclei until late into cytokinesis. The kinetoplast does not divide until after the completion of mitosis.     

Phosphoglucomutase isozymes of red cells in Mus musculus: Additional polymorphism at PGM-1 compared by two electrophoretic systems

Phosphoglucomutase (PGM) of red cells was examined in 15 inbred strains of mice, using two different starch gel electrophoretic buffer systems. Two new alleles, Pgm-1 ^c and Pgm-1 ^d, were discovered at the Pgm-1 locus. Pgm-1 ^c was first identified in strain C3H/HeNWe and Pgm-1 ^d in 129/ReWl. No variation was observed at the Pgm-2 locus.Supported in part by USPHS Pre-doctoral Fellowship No. 5 F1 GM-32,680, USPHS Research Resources Grant No. 5-PO6 RR 00343-05, USPHS (National Cancer Institute) Chemotherapy Contract 71-2010, and Biomedical Sciences Support Grant FR 07037 to the University of Kansas.     

A New Type of Manchette Structure in Spermatids of Culex tigripes(Culicidae)

Abstract In young spermatids of Culex tigripes, microtubules organize a manchette which surrounds the nucleus. When the nucleus elongates, 1–5 expansions appear on the wall of the microtubules. They grow and branch out while the nucleus elongates and chromatin condenses. Expansions are straight or curved in shape. They have the same thickness as the microtubule walls, but they rarely constitute links between microtubules. The manchette disappears naturally at the end of spermiogenesis. The action of colchicine on spermatids leads to the complete disappearance of the microtubules and expansions, and inhibits the lengthening of the nucleus.