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.     

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.     

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.     

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.     

Antimitotic agents and macronuclear division of ciliates

Summary A ciliate protozoan (Tetrahymena pyriformis, amicronucleate strain GL), exposed to 5 mg/ml colchicine to prevent cell division, is able to overcome the initial inhibitory effect of the drug both in exponentially growing and in heat-shock synchronized cultures.Such a regained capability of division is correlated to the reappearance of colchicine-disordered microtubules in the macronuclei. Electron microscopy reveals a tight connection between the inner nuclear membrane and these macronuclear microtubules. Since a decrease of the colchicine titer in the culture medium could not be established the macronuclear microtubules apparently reassemble in the presence of colchicine.     

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.     

A beta-tubulin mutation selectively uncouples nuclear division and cytokinesis in Tetrahymena thermophila

The ciliated protozoan Tetrahymena thermophila contains two distinct nuclei within a single cell-the mitotic micronucleus and the amitotic macronucleus. Although microtubules are required for proper division of both nuclei, macronuclear chromosomes lack centromeres and the role of microtubules in macronuclear division has not been established. Here we describe nuclear division defects in cells expressing a mutant beta-tubulin allele that confers hypersensitivity to the microtubule-stabilizing drug paclitaxel. Macronuclear division is profoundly affected by the btu1-1 (K350M) mutation, producing cells with widely variable DNA contents, including cells that lack macronuclei entirely. Protein expressed by the btu1-1 allele is dominant over wild-type protein expressed by the BTU2 locus. Normal macronuclear division is restored when the btu1-1 allele is inactivated by targeted disruption or expressed as a truncated protein. Immunofluorescence studies reveal elongated microtubular structures that surround macronuclei that fail to migrate to the cleavage furrows. In contrast, other cytoplasmic microtubule-dependent processes, such as cytokinesis, cortical patterning, and oral apparatus assembly, appear to be unaffected in the mutant. Micronuclear division is also perturbed in the K350M mutant, producing nuclei with elongated early-anaphase spindle configurations that persist well after the initiation of cytokinesis. The K350M mutation affects tubulin dynamics, as the macronuclear division defect is exacerbated by three treatments that promote microtubule polymerization: (i) elevated temperatures, (ii) sublethal concentrations of paclitaxel, and (iii) high concentrations of dimethyl sulfoxide. Inhibition of phosphatidylinositol 3-kinase (PI 3-kinase) with 3-methyladenine or wortmannin also induces amacronucleate cell formation in a btu1-1-dependent manner. Conversely, the myosin light chain kinase inhibitor ML-7 has no effect on nuclear division in the btu1-1 mutant strain. These findings provide new insights into microtubule dynamics and link the evolutionarily conserved PI 3-kinase signaling pathway to nuclear migration and/or division in Tetrahymena.     

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.     

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.     

Subcellular localization and role of Ran1 in Tetrahymena thermophila amitotic macronucleus

Amitosis, a direct method of cell division is common in ciliated protozoan, fungi and some animal and plant cells. During amitosis, intranuclear microtubules are reorganized into specified arrays which assist in separation of nucleus, despite lack of a bipolar spindle. However, the regulation of amitosis is not understood. Here, we focused on the localization and role of mitotic spindle assembly regulator: Ran GTPase (Ran1) in macronuclear amitosis in binucleated protozoan Tetrahymena thermophila. HA-tagged Ran1 was localized in the macronucleus throughout the cell cycle of Tetrahymena during vegetative growth, and the accessory factor binding domains of Ran1 contributed to its macronuclear localization. Incomplete somatic knockout of RAN1 resulted in aberrant intramacronuclear microtubule array formation, missegregation of macronuclear chromosomes and ultimately blocked macronuclei proliferation. When the Ran1 cycle was perturbed by overexpression of Ran1T25N (GDP-bound Ran1-mimetic) or Ran1Q70L (GTP-bound Ran1-mimetic), intramacronuclear microtubule assembly was inhibited or multi-micronucleate cells formed. These results suggest that Ran GTPase pathway is involved in assembly of a specialized intramacronuclear microtubule network and coordinates amitotic progression in Tetrahymena.     

The Role of Microtubules in Macronuclear Division of Blepharisma*

SYNOPSIS. The macronucleus of the heterotrich ciliate Blepharisma undergoes a spectacular change in form in preparation for its division. The elongation phase of this cycle has been examined by light and electron microscopy. Coincident with elongation, microtubules appear closely applied to the outer surface of the macronucleus and parallel to the direction of elongation, and disappear as elongation is completed. It is demonstrated that the antimitotic drugs colchicine and podophyllotoxin reversibly block macronuclear elongation but do not entirely inhibit morphogenetic events including cell division. The failure of colchicine-treated macronuclei to begin or continue elongation is correlated with the prevention of formation, disruption, and/or disorientation of the extranuclear microtubules.     

Differential DNA amplification and copy number control in the hypotrichous ciliate Euplotes crassus

During macronuclear development in hypotrichous ciliated protozoans, several thousand macronuclear DNA molecules are amplified several-hundred fold. We investigated the regulation of this amplification by determining the copy numbers of three different macronuclear DNA molecules in the hypotrichous ciliate Euplotes crassus. Two of the macronuclear DNA molecules were present in approximately 1,000 copies per cell, while the third was present in approximately 6,500 copies per cell. These reiteration levels were achieved either during macronuclear development, or shortly thereafter, and were maintained during vegetative growth. The most abundant macronuclear DNA molecule is present as a single-copy sequence in the micronuclear genome. Thus, its high copy number results from differential amplification. These results indicate that DNA amplification during macronuclear development is regulated individually for each macronuclear DNA molecule.     

Differential DNA Amplification and Copy Number Control in the Hypotrichous Ciliate Euplotes crassus

ABSTRACT. During macronuclear development in hypotrichous ciliated protozoans, several thousand macronuclear DNA molecules are amplified several-hundred fold. We investigated the regulation of this amplification by determining the copy numbers of three different macronuclear DNA molecules in the hypotrichous ciliate Euplotes crassus. Two of the macronuclear DNA molecules were present in approximately 1,000 copies per cell, while the third was present in approximately 6,500 copies per cell. These reiteration levels were achieved either during macronuclear development, or shortly thereafter, and were maintained during vegetative growth. The most abundant macronuclear DNA molecule is present as a single-copy sequence in the micronuclear genome. Thus, its high copy number results from differential amplification. These results indicate that DNA amplification during macronuclear development is regulated individually for each macronuclear DNA molecule.     

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.     

Micronuclear genome organization in Euplotes crassus: a transposonlike element is removed during macronuclear development.

After mating, hypotrichous ciliated protozoa transform a set of their micronuclear chromosomes into thousands of short, linear DNA molecules that form the macronuclear genome. To examine micronuclear genome organization in the hypotrich Euplotes crassus, we have analyzed two cloned segments of micronuclear DNA as well as the macronuclear DNA molecules that are derived from them. E. crassus was found to display a number of features characteristic of other hypotrich genomes, including (i) clustering and close spacing of the precursors of macronuclear DNA molecules, (ii) the frequent occurrence of internal eliminated sequences within macronuclear precursors, (iii) overlapping macronuclear precursors, (iv) lack of telomeric repeats at the ends of macronuclear precursors, and (v) alternative processing of the micronuclear chromosome to yield multiple macronuclear DNA molecules. In addition, a moderately repetitive, transposonlike element that interrupts the precursors of two macronuclear DNA molecules has been identified and characterized. This transposonlike element, designated Tec1, is shown to be reproducibly removed from one of the macronuclear precursors during independent episodes of macronuclear development.     

Organization of gene and non-gene sequences in micronuclear DNA of Oxytricha nova.

In order to study the derivation of the macronuclear genome from the micronuclear genome in Oxytricha nova micronuclear DNA was partially digested with EcoRI, size fractionated, and then cloned in the lambda phage Charon 8. Clones were selected a) at random b) by hybridization with macronuclear DNA or c) by hybridization with clones of macronuclear DNA. One group of these clones contains only unique sequence DNA, and all of these had sequences that were homologous to macronuclear sequences. The number of macronuclear genes with sequences homologous to these micronuclear clones indicates that macronuclear sequences are clustered in the micronuclear genome. Many micronuclear clones contain repetitive DNA sequences and hybridize to numerous EcoRI fragments of total micronuclear DNA, yielding similar but non-identical patterns. Some micronuclear clones containing these repetitive sequences also contained unique sequence DNA that hybridized to a macronuclear sequence. These clones define a major interspersed repetitive sequence family in the micronuclear genome that is eliminated during formation of the macronuclear genome.     

The processing of macronuclear-destined DNA sequences microinjected into the macronuclear anlagen of the hypotrichous ciliate Stylonchia lemnae.

We describe the construction of a vector carrying the micronuclear versions of two macronuclear DNA molecules, one of which was modified by the insertion of a polylinker sequence. This vector was injected into the polytene chromosomes of the developing macronucleus of Stylonychia and its processing during further macronuclear development and its fate in the mature macronucleus were analyzed. In up to 30% of injected cells the modified macronuclear DNA sequence could be detected. While the internal eliminated sequences (IES) present in the macronuclear precursor DNA sequence are still retained in the mature macronucleus, the modified macronuclear DNA sequence is correctly cut out from the vector, telomeres are added de novo and it is stably retained in the macronucleus during vegetative growth of the cells. This vector system represents an experimental system that allows the identification of DNA sequences involved in the processing of macronuclear DNA sequences during macronuclear development.     

Studies on macronuclear DNA from Paramecium aurelia

Macronuclear DNA was isolated from purified macronuclei of Paramecium aurelia and the size distribution was determined with regard to growth phase and method of extraction. DNA molecules as long as 105 microns and as short as 0.2 microns were observed. It was concluded that the method of extraction affected the observed length of DNA extracted and that macronuclear DNA isolated from cells in balanced growth was less susceptible to nuclease degradation than was DNA isolated from cells in stationary phase. Renaturation studies were performed on macronuclear DNA and a kinetic complexity of 22-times E. coli DNA was determined. This value was similar to those values reported for Tetrahymena and Stylonychia macronuclear DNA. Correcting for GC base content yielded a kinetic complexity for Paramecium macronuclear DNA of 11-times E. coli DNA which corresponded to 3 X 10(10) daltons. There would be about 1400 copies of a unit genome of this complexity within each newly replicated macronucleus. Density gradient analysis indicated that the genes coding for ribosomal RNA had a greater density in CsCl than the bulk DNA. Molecular hybridization studies indicated that the genes coding for 25 S RNA represented 0.14 percent of the total macronuclear DNA. Correcting for GC base content, this corresponded to 30-35 25 S RNA genes per unit genome. These results on Paramecium are discussed in relationship to other ciliate macronuclear DNA.