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The FEBS Journal Jul 2012Amitosis, a direct method of cell division is common in ciliated protozoan, fungi and some animal and plant cells. During amitosis, intranuclear microtubules are...
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.
Topics: Amino Acid Sequence; Binding Sites; Blotting, Western; Cell Division; Cytoplasm; Gene Knockout Techniques; Guanosine Diphosphate; Guanosine Triphosphate; Hydrolysis; Macronucleus; Microscopy, Confocal; Mitosis; Models, Biological; Molecular Sequence Data; Mutation; Protozoan Proteins; Sequence Homology, Amino Acid; Tetrahymena thermophila; ran GTP-Binding Protein
PubMed: 22594798
DOI: 10.1111/j.1742-4658.2012.08634.x -
The Journal of Eukaryotic Microbiology 2014Only a limited number of studies exist on the life cycles of nonmodel ciliates such as Chilodonella uncinata (Cl: Phyllopharyngea). The handful of papers on this taxon...
Only a limited number of studies exist on the life cycles of nonmodel ciliates such as Chilodonella uncinata (Cl: Phyllopharyngea). The handful of papers on this taxon indicate the presence of a heteromeric macronucleus, marked by separate DNA-rich and DNA-poor regions. Here, we study the life cycle of C. uncinata using confocal laser scanning microscopy with 4',6-diamidino-2-phenylindole staining, which allows us to differentiate nuclear dynamics of the micronucleus and the macronucleus during life-cycle stages. We photo-documented various stages and confirmed aspects of the development of the new macronucleus previously characterized by electron microscopy. We further reveal the heteromeric structure of the macronucleus with Z-stacks and three-dimensional (3D) reconstructions. We find no evidence for the presence of an endosome at the center of the macronucleus during vegetative growth. In addition to illustrating the life cycle of this ciliate, the approaches developed for this study will enable additional comparative analyses of nuclear dynamics using fluorescence microscopy.
Topics: Ciliophora; Imaging, Three-Dimensional; Life Cycle Stages; Macronucleus; Microscopy, Confocal
PubMed: 24547950
DOI: 10.1111/jeu.12109 -
Cell Death and Differentiation Mar 2001A key characteristic of apoptosis is its regulated nuclear degradation. Apoptosis-like nuclear degradation also occurs in the ciliated unicellular organism, Tetrahymena...
A key characteristic of apoptosis is its regulated nuclear degradation. Apoptosis-like nuclear degradation also occurs in the ciliated unicellular organism, Tetrahymena thermophila. Chromatin of the macronucleus undergoes massive condensation, a process that can be blocked by caspase inhibitors. The nucleus becomes TUNEL-positive, and its DNA is cleaved into nucleosome-sized fragments. In a matter of hours the macronucleus is completely degraded, and disappears. The condensed nucleus sequesters acridine orange, which means that it might become an acidic compartment. We therefore asked whether lysosomal bodies fuse with the condensed macronucleus to form an autophagosome. We monitored acid phosphatase (AP) activity, which is associated with lysosomal bodies but is not found in normal nuclei. We find that after the macronucleus condenses AP activity is localized in cap-like structures at its cortex. Later, after the degrading macronucleus loses much of its DNA, acid phosphatase deposits appear deeper within the nucleus. We conclude that although macronuclear elimination is initiated by an apoptosis-like mechanism, its final degradation may be achieved through autophagosomy.
Topics: Acid Phosphatase; Animals; Apoptosis; Autophagy; Chromatin; Conjugation, Genetic; Kinetics; Lysosomes; Macronucleus; Tetrahymena thermophila
PubMed: 11319612
DOI: 10.1038/sj.cdd.4400807 -
Developmental Genetics 1992The macronucleus of Tetrahymena contains a large number of DNA molecules of subchromosomal size. They belong to about 270 species each one occurring at an average number...
The macronucleus of Tetrahymena contains a large number of DNA molecules of subchromosomal size. They belong to about 270 species each one occurring at an average number of 45 copies. Macronuclei divide unequally and nothing is known of segregation control. This and the elimination and degradation of DNA during macronuclear amitosis make the clonal stability of macronuclei a problem of qualitative and quantitative control on a subchromosomal level. We studied the contribution of DNA elimination to the quantitative composition of the macronucleus cytophotometrically in single cells of different strains. This was done under standard conditions and under conditions known to influence the amount of macronuclear DNA. The following results were found: Elimination of DNA occurs at almost every division. The size of the elimination body is highly variable but still positively correlated with the macronuclear DNA content. In T. thermophila the amount of eliminated DNA is 2.5% of the G2 content and is not dependent on the growth state. It varies with species, amounting to as much as 8% in T. pigmentosa. During conditions which increase the macronuclear DNA content, very little DNA is eliminated. On the other hand, large amounts are eliminated under other conditions causing the macronuclear DNA content to decrease. DNA to be eliminated at division is synthesized at the same time as bulk DNA. We developed a computer program which helps us study the effects of DNA elimination and unequal divisions upon the copy numbers of subchromosomal DNA classes.(ABSTRACT TRUNCATED AT 250 WORDS)
Topics: Animals; Cell Division; Cell Nucleus; Computer Simulation; DNA, Protozoan; Tetrahymena
PubMed: 1499151
DOI: 10.1002/dvg.1020130203 -
Protist Oct 2006The three-dimensional (3D) organization of nucleoli in the somatic nuclei (macronuclei) of recently fed and starved Didinium nasutum was reconstructed on the basis of...
The three-dimensional (3D) organization of nucleoli in the somatic nuclei (macronuclei) of recently fed and starved Didinium nasutum was reconstructed on the basis of serial ultra-thin sections. It was shown that nucleoli, looking on the single sections like individual separate structures, appeared to be parts of the large complicated branchy nucleolar networks. A 30 h starvation did not lead to disintegration of this network, but stimulated formation of numerous vacuoles in the granular component of nucleoli, which becomes more condensed. Unlike starved D. nasutum, in fed ciliates numerous holes appeared in the fibrillar component located at the periphery of nucleoli. These holes may presumably serve as channels for transporting newly synthesized rRNA. To our knowledge, this is the first report of a 3D reconstruction of the nucleolar apparatus in ciliates.
Topics: Animals; Cell Nucleolus; Ciliophora; DNA, Ribosomal; Imaging, Three-Dimensional; Microscopy, Electron; Nucleolus Organizer Region
PubMed: 16904938
DOI: 10.1016/j.protis.2006.06.001 -
Nucleic Acids Research Mar 2003Dramatic DNA reorganization and elimination processes occur during macronuclear differentiation in ciliates. In this study we analyzed whether cytosine methylation of...
Dramatic DNA reorganization and elimination processes occur during macronuclear differentiation in ciliates. In this study we analyzed whether cytosine methylation of specific sequences plays a functional role during DNA rearrangement. Three classes of sequences, macronuclear-destined sequences (MDSs, pCE7), members from a large family of transposon-like elements and micronuclear-specific sequences (pLJ01), differing in their structure and future destiny during nuclear differentiation, were studied in the micronucleus, the developing macronucleus and, when present, in the mature macronucleus. While the MDSs become processed to a 1.1 and 1.3 kb gene-sized macronuclear DNA molecule, the family of transposon-like elements represented by MaA81 becomes removed late in the course of polytene chromosome formation. The micronuclear-specific sequence pLJ01 is eliminated together with bulk micronuclear DNA during degradation of polytene chromosomes. No methylated cytosine could be detected in the vegetative macronucleus and no difference in methylation pattern was observed either between micronucleus and developing macronucleus in MDSs or in a micronuclear-specific sequence. However, a significant percentage of the cytosines contained in the transposon-like element becomes methylated de novo in the course of macronuclear differentiation. This is the first demonstration that cytosine methylation in specific sequences occurs during macronuclear differentiation and may provide a first step towards understanding epigenetic factors involved in DNA processing.
Topics: Animals; Cell Nucleus; Chromatography, High Pressure Liquid; Cytosine; DNA Methylation; DNA, Protozoan; Hypotrichida; Polymerase Chain Reaction
PubMed: 12595545
DOI: 10.1093/nar/gkg233 -
Journal of Cell Science Nov 1983The gram-negative bacterium Holospora obtusa is an endonuclear symbiont of Paramecium caudatum, which is incorporated into the host cells via the food vacuoles and...
The gram-negative bacterium Holospora obtusa is an endonuclear symbiont of Paramecium caudatum, which is incorporated into the host cells via the food vacuoles and infects their macronucleus exclusively, but never the micronucleus. Since these two kinds of nuclei originate from a fertilization nucleus, it is assumed that the macronucleus acquires a property necessary for it to be recognized by the bacterium at a certain time during the nuclear differentiation process. We found that this property is acquired by four of the eight postzygotic nuclei as soon as the four nuclei differentiate morphologically into the macronuclear anlagen.
Topics: Animals; Cell Nucleus; Gram-Negative Bacteria; Paramecium; Symbiosis; Time Factors; Vacuoles
PubMed: 6662855
DOI: 10.1242/jcs.64.1.137 -
The Journal of Eukaryotic Microbiology Sep 2022This review addresses nine areas of knowledge revealed by micromanipulations performed with Paramecium. Microinjection has shown that sexual maturation and senescence of... (Review)
Review
This review addresses nine areas of knowledge revealed by micromanipulations performed with Paramecium. Microinjection has shown that sexual maturation and senescence of Paramecium caudatum is a programmed process conducted by a specific gene and its product protein. In Paramecium tetraurelia, autogamy was revealed to depend on the number of DNA syntheses rather than the number of cell divisions in clonal aging. The cytoplasmic complementarity test established that microinjection of wild-type cytoplasm can correct genetic defects of mutants. The concept of complementarity together with protein chemistry revealed compounds that control membrane excitability. In non-Mendelian inheritance, noncoding small RNAs made from the parental micronucleus regulate the rearrangement of the progeny's macronuclear DNA. The macronucleus has the potential to be used as a factory for genetic engineering. The development and differentiation of progeny's nuclei in mating pairs are controlled by the parental macronucleus. The chemical reaction processes associated with exocytosis have been revealed by microinjection of various enzymes and antibodies. Using the fusion gene of histone H2B and yellow-fluorescence protein, it was revealed that the fusion gene-mRNA is transferred between cells during mating. Experiments with endosymbiotic bacteria and the host shed light on the conditions needed to establish sustainable symbiotic relationships.
Topics: Cytoplasm; Macronucleus; Micromanipulation; Paramecium; Paramecium tetraurelia
PubMed: 35318763
DOI: 10.1111/jeu.12909 -
Cytoskeleton (Hoboken, N.J.) Apr 2011Myo1 is a class XIV Tetrahymena myosin involved in amitotic elongation and constriction of the macronucleus into two subnuclei at cell division. Elongation of the...
MyTH4, independent of its companion FERM domain, affects the organization of an intramacronuclear microtubule array and is involved in elongation of the macronucleus in Tetrahymena thermophila.
Myo1 is a class XIV Tetrahymena myosin involved in amitotic elongation and constriction of the macronucleus into two subnuclei at cell division. Elongation of the macronucleus is accompanied by elongation of an intramacronuclear microtubule array, which is oriented parallel to the axis of nuclear elongation. Elongation of the macronucleus often fails to occur or is only partially completed in a MYO1 knockout, and division of the macronucleus is frequently uncoupled from cytokinesis. Myo1 contains a myosin tail homology 4 (MyTH4) and a band 4.1, ezrin, radixin, moesin homology (FERM) domain. Recently, we used green fluorescent protein (GFP) fusions to demonstrate that the entire FERM domain, independent of MyTH4, is essential for localization of FERM to the cytoskeleton and does not appear to directly affect nuclear division. Antiactin coprecipitates GFP-FERM, tubulin, actin, and Myo1. The immunoprecipitated GFP-FERM cosediments with either exogenous F-actin or exogenous microtubules. Here, we show that overexpressed GFP-MyTH4 colocalized with antitubulin to intramacronuclear microtubules. Ninety percent of overexpressing cells assembled intramacronuclear microtubules that did not become organized into a parallel array. Amitosis did not advance in the absence of the parallel array of intramacronuclear microtubules. Five percent of overexpressing cells organized the parallel array, but the microtubules and the macronucleus did not achieve full elongation. Partially elongated macronuclei constricted without cytokinesis. Antiactin coprecipitated GFP-MyTH4, tubulin, and actin. AntiGFP pulled down GFP-MyTH4, tubulin, and actin. GFP-MyTH4 cosedimented with either exogenous microtubules or exogenous F-actin. A novel finding from this study is that MyTH4 and FERM have overlapping and distinct roles in the function of a myosin.
Topics: Actins; Amino Acid Sequence; Blotting, Western; Cell Nucleus; Fluorescent Antibody Technique; Green Fluorescent Proteins; Immunoprecipitation; Macronucleus; Microtubules; Molecular Sequence Data; Myosin Heavy Chains; Myosins; Protein Binding; Protein Structure, Tertiary; Protozoan Proteins; Sequence Homology, Amino Acid; Tetrahymena thermophila; Tubulin
PubMed: 21387572
DOI: 10.1002/cm.20506 -
Methods in Cell Biology 2012The ciliate Tetrahymena thermophila can be said to undergo a variety of developmental programs. During vegetative growth, cells coordinate a variety of cell-cycle... (Review)
Review
The ciliate Tetrahymena thermophila can be said to undergo a variety of developmental programs. During vegetative growth, cells coordinate a variety of cell-cycle operations including macronuclear DNA synthesis and a-mitotic fission, micronuclear DNA synthesis and mitosis, cytokinesis and an elaborate program of cortical morphogenesis that replicates the cortical organelles. When starved, cells undergo oral replacement, transformation into fast-swimming dispersal forms or, when encountering cells of a complementary mating type, conjugation. Conjugation involves a 12 hour program of meiosis, mitosis, nuclear exchange and karyogamy, and two postzygotic divisions of the fertilization nucleus. This chapter reviews experimental data exploring the developmental dependencies associated with both vegetative and conjugal development.
Topics: Cell Cycle; Cell Cycle Checkpoints; Cell Nucleus Division; Conjugation, Genetic; Cyclins; DNA Damage; DNA Repair; DNA Replication; DNA, Protozoan; Eukaryotic Cells; Genes, Protozoan; Macronucleus; Micronucleus, Germline; Reproduction, Asexual; Tetrahymena thermophila
PubMed: 22444146
DOI: 10.1016/B978-0-12-385967-9.00007-4