Amicronucleate Cells Research Articles

Conjugation in Euplotes raikovi (Protista, Ciliophora): New Insights into Nuclear Events and Macronuclear Development from Micronucleate and Amicronucleate Cells.

Ciliates form a distinct group of single-celled eukaryotes that host two types of nuclei (micro and macronucleus) in the same cytoplasm and have a special sexual process known as conjugation, which involves mitosis, meiosis, fertilization, nuclear differentiation, and development. Due to their high species diversity, ciliates have evolved different patterns of nuclear events during conjugation. In the present study, we investigate these events in detail in the marine species Euplotes raikovi. Our results indicate that: (i) conjugation lasts for about 50 h, the longest stage being the development of the new macronucleus (ca. 36 h); (ii) there are three prezygotic micronuclear divisions (mitosis and meiosis I and II) and two postzygotic synkaryon divisions; and (iii) a fragment of the parental macronucleus fuses with the new developing macronucleus. In addition, we describe for the first time conjugation in amicronucleate E. raikovi cells. When two amicronucleate cells mate, they separate after about 4 h without evident nuclear changes; when one amicronucleate cell mates with a micronucleate cell, the micronucleus undergoes regular prezygotic divisions to form migratory and stationary pronuclei, but the two pronuclei fuse in the same cell. In the amicronucleate cell, the parental macronucleus breaks into fragments, which are then recovered to form a new functional macronucleus. These results add new information on the process of conjugation in both micronucleate and amicronucleate Euplotes cells.

Colchicine-induced degeneration of the micronucleus during conjugation in Tetrahymena.

ABSTRACTOne of the most dramatic examples of nuclear morphogenesis occurs during conjugation in Tetrahymena when the micronucleus elongates to a size longer than the cell itself. After contraction to a spherical shape, the nucleus moves directly to chromosome separation in the first meiotic division. Here we investigate the consequences of interrupting the elongation process. Colchicine, a microtubule inhibitor, caused retraction of elongated structures. With time, cells began to lose their micronuclei, and by five hours more than half of the paired cells had at least one cell missing a micronucleus. After reversing the colchicine block, existing micronuclei did not undergo elongation again, nor did meiosis occur. These observations indicate that micronuclear elongation is critical to subsequent meiotic division. Further, nuclear elimination occurs, which could be due to meiotic failure or possibly a problem downstream from meiosis. An analysis of the process of colchicine-induced micronuclear degeneration indicated that it was regulated by a caspase-dependent mechanism, characteristic of apoptosis, and then resorbed by a lysosome-dependent autophagic mechanism. Amicronucleate cells failed to grow when returned to nutrient medium, likely because of a lesion in the post-conjugation reconstruction of a functioning oral apparatus. The ease by which a large number of nuclei are induced to “self-destruct” may make this system useful in investigating the link between colchicine treatment and nuclear death in Tetrahymena, and in investigating how nuclear death could be regulated in living cells more generally. Finally, we note that this phenomenon might relate to the evolution of amicronucleate species of Tetrahymena.

Inactivation of a Macronuclear Intra-S-phase Checkpoint in Tetrahymena thermophila with Caffeine Affects the Integrity of the Micronuclear Genome

Aphidicolin (APH), an inhibitor of DNA polymerase α, arrested cell divisions in Tetrahymena thermophila. Surprisingly, low concentrations of APH induced an increase of macronuclear DNA content and cell size in non-dividing cells. In spite of the cell size increase, most proliferation of basal bodies, ciliogenesis and development of new oral primordia were prevented by the APH treatment. The division arrest induced by APH was partly overridden by caffeine (CAF) treatment, which caused the fragmentation ("pulverization") of the chromosomes in G2 micronuclei. Somatic progeny of dividers with pulverized micronuclei (APH+CAF strains) contained aneuploid and amicronucleate cells. The amicronucleate cells, after losing their oral structures and most of their cilia, and undergoing progressive disorganization of cortical structures, assumed an irregular shape ("crinkled") and were nonviable. "Crinkled" cells were not formed after APH + CAF treatment of the amicronuclear BI3840 strain, which contains some mic-specific sequences in its macronucleus. Most of the APH +CAF strains had a typical "*"- like conjugation phenotype: they did not produce pronuclei, but received them unilaterally from their mates and retained old macronuclei. However, 4 among 100 APH+CAF clones induced arrest at meiotic metaphase I in their wt mates. It is likely that the origin of such clones was enhanced by chromosome pulverization.

Identification of mRNAs expressed differentially between micronucleates and amicronucleates of Pseudourostyla cristata during starvation

The evident character of amicronucleates of Pseudourostyla cristata, as compared with the normal micronucleates, was that a substantial proportion of cells exhibited an abnormal AZM. Moreover, the percentage of abnormal AZMs in amicronucleates during starvation was much higher. To understand how the molecular events of the micronucleus govern this mode, we employed suppression subtractive hybridization (SSH) to identify candidate genes which express in micronucleate cells but not in amicronucleate cells during starvation. A total of 284 transcripts were screened by cDNA array and 17 clones were confirmed to be differentially expressed among micronucleate cell. All 17 ESTs were analyzed by blaster matching in GeneBank at deduced amino acid sequence: 10 clones, no homologous proteins; 4 clones, myoglobin protein; 1 clone, cytochrome P450 monooxygenase; 1 clone, cytosolic malate dehydrogenase; the last clone, homologs of hsp90, which is interesting. Traditionally, heat shock proteins are known for their function during cellular stress as molecular chaperones responsible for the folding, refolding and transport of newly synthesized protein. Why was it significantly enhanced in micronucleates but not in amicronucleates during starvation? Further research including RACE, immunoblotting and gel mobility shift will be carried out. This work was supported by a grant from NSFC (No: 30370210) to L. P. JIN.

Involvement of active cellular mechanisms on the disorganization of oral apparatus in amicronucleate cells in Tetrahymena thermophila.

Ciliated protozoa, a group of unicellular eukaryotes, have two kinds of nuclei, a macronucleus (somatic nucleus) and a micronucleus (germinal nucleus) in a single cell. We previously reported that amicronucleate cells of Tetrahymena thermophila induced by nocodazole gradually lost their oral apparatus (OA) and ciliary rows but that amacronucleate cells did not. Since the macronucleus is responsible for the gene expression in the vegetative phase, the effects of actinomycin D and cycloheximide on the disorganization of the OA in amicronucleate cells induced by nocodazole were investigated. These inhibitors prevented the disorganization of the OA in amicronucleate cells. Amicronucleate cells did not grow even in the medium supplemented with high concentration of Fe, Cu and folinic acid which allow cells to grow without formation of food vacuoles. The results suggest that the macronucleus in the amicronucleate cells plays an active role in the induction of disorganization of the OA and malfunctions of nutrient uptake from the cell surface and/or in the fundamental cell division mechanisms, resulting in the death of amicronucleate cells.

The vegetative micronucleus has a critical role in maintenance of cortical structure in Tetrahymena thermophila.

The vegetative role of the germinal micronucleus of Tetrahymena thermophila was studied. Amicronucleate cells that had spontaneously arisen in star strains (very aged cones with defective micronuclei) were observed in B* as well as in A* and in C* and were found to lack the oral apparatus and to have disordered ciliary rows. To reduce uncertainty given the very small micronucleus and possible effects of aging factors, we induced amicronucleate cells in a young clone by treatment with the antitubulin drug, nocodazole, and observed their cortical structure and nuclei. Amicronucleate cells gradually lost their oral apparatus and then their ciliary rows became disordered, even without cell division, and they became crinkled cells. It can be concluded that the vegetative micronucleus appears to play a critical role in the maintenance of the cortical structure, especially of the oral apparatus.

Role of Germ Nuclei in Conjugation of Paramecium caudatum: (Conjugation/conjugation-specific antigen/germ nuclei/monoclonal antibody/Paramecium caudatum).

Ciliated protozoa have two distinct nuclei, germinal micronuclei and somatic macronuclei. Macronuclei are transcriptionally active, but micronuclei are almost inert. To know whether micronuclei have any function in conjugation of Paramecium caudatum, expression of the conjugation-specific nuclear antigen, CSN-1, in amicronucleate cells during conjugation was examined. Micro- (mic) and amicronucleate (amic) cells were conjugated in three combinations (mic-mic, mic-amic and amic-amic). In conjugation of two amicronucleate cells, the cells were negative against the CSN-1 antibody by indirect immunofluorescence staining and underwent abnormal macronuclear fragmentation. These were not observed in conjugation of micronucleate cells. In conjugation between micro- and amicronucleate cells, the amicronucleate mates were negative against the antibody in the beginning, but after receiving a pronucleus from the micronucleate mates, they became positive. This indicates that the CSN-1 antigen or factors involved in the expression of the antigen can hardly pass across the fused cell membrane between conjugating cells before pronuclear exchange. The present results suggest that micronuclei have some function in expression of the CSN-1 antigen and the macronuclear fragmentation, both of which are specific events in conjugation.

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

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

Programming of the macronucleus of Paramecium during asexual and sexual reproduction: A further study with cytidine analogues, dimethylsulfoxide, L-ethionine and N-butyric acid

The control of the function of the macronucleus of Paramecium is studied, in connection with its role in the compensation for the asexual somatic function of the micronucleus. Following removal of the micronuclei, amicronucleate cell lines as a rule suffer a transient period of growth and developmental depression in the initial phase of asexual propagation. But they gradually recover to near-normal. Previous studies of treatment of amicronucleate cells with cytidine analogues have implicated the macronucleus in compensating for the somatic function of the micronucleus following the loss of the micronucleus, and the activation of this macronuclear function probably involves DNA-demethylation. The present study further tests this notion, by treating micronucleate cells with agents known to promote demethylation of 5-methylcytosine. After treatment, the cells were vegetatively propagated, and then enucleated to give rise to amicronucleate cell lines. Treatments with dimethylsulfoxide, L-ethionine, and 5-aza-2'-deoxycytidine promoted recovery in amicronucleate cell lines thus derived. Cells treated with 6-azacytidine did not produce such an effect. Hence, the compensatory mechanism, presumably residing in a repressed state in the macronucleus, can be activated or primed to activate by demethylating agents even before the loss of the micronucleus, and once established the new macronuclear programme perpetuates in succeeding asexual cell generations. This shows that during asexual propagation the macronuclear programme can be altered to 'pre-adapt' the cells for amicronuclearity. Treatment of micronucleate conjugants with 5-azacytidine, when the macronuclear anlagen develop, produced clones that had become similarly pre-adapted. There were also some indication of persistence of such effects of the analogue into the next clonal cycle following autogamy. The notion of macronuclear DNA-demethylation as a basis for the activation and maintenance of the compensatory mechanism is discussed.

Induction of reinitiation of meiosis in amphibian Bufo oocytes by injection of ciliate Paramecium extracts

Germinal vesicle breakdown (GVBD) of amphibian Bufo oocytes can be induced if Paramecium extracts were injected into them. The activity of meiosis-reinitiation-inducing factor (MRIF) appeared in premeiotic G 1 cells, then the activity fluctuates according to the degrees of micronuclear chromatin condensation in meiosis. Proliferating micronucleate and amicronucleate cells also showed the same activity. MRIF differed from MPF (M phase promoting factor), because MRIF appeared not only in M phase cells but also in premeiotic interphase cells and its action on the induction of GVBD was inhibited by cycloheximide. Preliminary experiments showed that MRIF was a heat-labile soluble protein.

Identification of a spindle-associated protein in ciliate micronuclei

ABSTRACT Ciliated protozoa display a nuclear dualism, with germinal micronuclei and a somatic macronucleus. During mitosis, which proceeds without disruption of the nuclear envelope, a spindle is organized within the micronucleus from, presumably, intranuclear microtubule-organizing centres (MTOCs). In order to characterize these MTOCs, monoclonal antibodies generated against human centrosomes were screened on several ciliates and particularly on Paramecium tetraurelia. In this ciliate, the monoclonal antibody CTR 532, which decorates centrosomal and spindle-associated components in mammalian cells, specifically labelled the micronuclei during interphase. At the electron-microscope level, it stained a fibrous material surrounding microtubules localized on the inner face of the nuclear envelope. During mitosis this decoration extended all over the metaphase spindle. At all stages of the cell cycle, the decoration remained specific to the micronucleus and was absent not only from all of the various cytoplasmic and cortical microtubule arrays but also from the macronuclei, even at early stages of their development from the zygotic nucleus. CTR 532 recognizes a single 170xl(rMr polypeptide in the cytoskeletal fraction that contains micronuclei and this polypeptide is absent in the cytoskeletal fraction of amicro-nucleate cells.

Cytidine Analogues and Stomatogenic Recovery in Amicronucleate Paramecium tetraurelia and Paramecium jenningsi

ABSTRACTRemoval of the micronuclei of Paramecium tetraurelia and Paramecium jenningsi by micropipetting generates amicronucleate cell lines. These cell lines go through a period of growth depression for several dozen fissions, but they gradually recover. Amicronucleate cells in the depression period characteristically exhibit abnormal oral development, particularly reduction in the length of the buccal cavity and an abnormal pattern of the oral membranelles. To test the notion that the macronucleus is involved in the recovery of amicronucleate cell lines, DNA demethylation drugs were administered to amicronucleates in the depression period. After at least 4 fissions, the treated amicronucleates were assessed for their progress in recovery by scoring the proportion of cells with normal oral membranelles. Cvtidine analogues which demethylate cytosine specifically at the 5 position, namely 5‐azacytidine, 5‐aza‐2′‐ deoxycytidine and 5‐fluoro‐2′‐deoxycytidine. promoted recovery of the amicronucleates. Cytidine, 6‐azacytidine, 2′‐fluoro‐2′‐deoxy‐cytidine and cytosine‐β‐D‐arabinofuranoside did not. These results suggest that (i) 5‐methylcytosine is present in the macronucleus of these Paramecium species, probably in small amounts and (ii) recovery of amicronucleates involves demethylation of macronuclear DNA. This implies that in normal cells the micronuclei are involved in maintaining the macronuclear DNA in a methylated state and hence the inactivation of the macronuclear sequences that are to be employed for stomatogenic recovery. A general mechanism for the control of gene expression may therefore be employed for the regulation of specific sequences.

The role of the micronucleus in stomatogenesis in sexual reproduction of Paramecium tetraurelia: laser microbeam irradiation of the micronucleus.

Fifteen amicronucleate cell lines and 22 cell lines with defective micronuclei were obtained following selective laser microbeam irradiation of the micronucleus. The amicronucleate cell lines showed reduced growth rate and formed abnormal oral apparatuses in asexual reproduction, and failed to produce any oral apparatus in autogamy, in agreement with previous observations on amicronucleate cells obtained by micropipetting. The 22 cell lines with defective micronucleus exhibited various abnormalities of the oral apparatus newly formed during autogamy. These abnormalities included the arrest of membranelle assembly, reduction in the length of the buccal cavity and oral membranelles, disruption of the organization of the membranelles, quadrulation of the dorsal peniculus, and failure of addition of membranellar basal body rows. Hence the micronucleus plays multiple roles in sexual stomatogenesis, deciding early steps of oral membranelle assembly and affecting their subsequent patterning. Our results agree with the notion that the micronucleus acts during a critical period between the second meiotic division and up to the formation of the zygotic nucleus to control the early stage of oral membranelle assembly. Laser microbeam irradiation might have created recessive mutations and/or chromosomal aberrations, which were expressed during this critical period with the formation of abnormal postmeiotic nuclei.

Etude Biométrique et Détermination des Teneurs en ADN Nucléaire des Populations de Ciliés Astomes du Type Almophrya mediovacuolataNgassam, 1983

A biometric study and a cytophotometric analysis of micronuclear DNA content of populations of Astome ciliates Almophrya mediovacuolata parasitizing the same host enable to distinguish 4 cell types: small cells (Pm) with 2 micronuclei (DNA content: 0.5 units), amicronucleate cells (Mm) having an intermediate size, large cells (Gm) with either 1 (Gml) or 2 (Gm2) micronuclei of which DNA contents are respectively 34.5 and 14.1 units. Macronuclear DNA content/micronuclear DNA content ratios in the three populations Pm, Gml and Gm2 are respectively 1100, 45 and 114. The distribution of macronuclear DNA between daughter cells is unequal.

Micronucleus-specific DNA sequences in an amicronucleate mutant of Tetrahymena.

DNA from the amicronucleate Tetrahymena cell line BI3840 was probed for DNA sequences which are limited to the micronucleus in wild-type cells. Four micronucleus-specific DNA sequences were not detectable in DNA from the amicronucleate cell line. Two of the six micronucleus-specific DNA sequences tested hybridized to DNA from amicronucleate cells. Both the number of fragments homologous to these sequences and the intensity of hybridization were reduced in the DNA from the amicronucleate cells relative to DNA from a wild-type cell line, indicating that less than one micronucleus equivalent of the micronucleus-specific DNA sequences was retained in the amicronucleate cell line. Thus many micronucleus-specific DNA sequences were eliminated from the developing macronucleus of BI3840 as they are in wild-type cells, but in at least two cases the elimination was incomplete. In situ hybridization suggested that the DNA sequences which are limited to the micronucleus in wild-type cells are present in the macronucleus of the amicronucleate cell line. Southern blots of DNA from the amicronucleate cell line were also probed with DNA sequences which are retained in the macronucleus. At least two types of genome rearrangements occurred in the BI3840 macronucleus as they do in wild-type cells. No spurious rearrangements were observed.

Nuclear transplant studies of the determination of mating type in germ nuclei of Paramecium tetraurelia

The micronucleus from vegetative cells of one mating type (O or E) in Paramecium tetraurelia was transplanted by micropipet into amicronucleate cells of opposite mating type (E or O). When autogamy was induced in the recipient cells, they developed new macronuclei and micronuclei derived from the transplanted micronucleus and usually expressed the same mating type as the recipients. The results indicate that micronuclei in the asexual phase may be undetermined for mating type. Recipient E cells in which the macronucleus had been previously removed were transplanted with a whole macronucleus from an O cell. Their mating type was soon transformed E to O before the occurrence of autogamy, and remained O after autogamy. This demonstrates that the transplanted macronucleus determined the O cytoplasmic state to determine the developing zygotic macronucleus for mating type O. It is unlikely that the micronucleus is determined for mating type in O or E cell during the asexual cycle.

Analysis of germinal aging in Paramecium caudatum by micronuclear transplantation

The role of the micronucleus in the age-dependent increase in mortality after conjugation in Paramecium has been investigated using micronuclear transplantation. The clone of Paramecium caudatum used for this study had a lifespan of about 750 fissions. In this clone, the fission rate began to decrease about 450 fissions after conjugation. Mortality after selfing conjugation also began to appear at about 450 fissions and gradually increased with clonal age. Cells at about 650 fissions showed 10–70% survival after selfing conjugation but when their micronuclei were transplanted into amicronucleate cells of about 450 fissions, the progeny survival increased to 70–90%. When micronuclei from cells 700–750 fissions old were transplanted into amicronucleate cells of 100–150 fissions, however, increase in progeny survival was very rare. The results indicate that micronuclei in cells up to the age of 650 fissions can function normally if the cytoplasmic environment is young.

Transplantation of germ nuclei in Paramecium caudatum: III. Role of germinal micronucleus in vegetative growth

By using the technique of nuclear transplantation in Paramecium [1], amicronucleate and renucleate clones were prepared in P. caudatum. The major differences between amicronucleate and micronucleate cells in the vegetative stage are elongation of cell cycle time, decrease in food vacuole formation, and shortening of the buccal cavity in the amicronucleate cells. These characteristics of amicronucleate cells are closely related with the absence of micronucleus, because all of these abnormalities were cured when the micronucleus was transplanted again into the amicronucleate. It is evident that the germinal micronucleus plays an important role not only during the sexual cycle but also in vegetative growth. Elongation of the cell cycle time in amicronucleates was also observed in P. bursaria and P. jenningsi.

GENETIC ANALYSIS OF MATING TYPE DIFFERENTIATION IN PARAMECIUM TETRAURELIA. II. ROLE OF THE MICRONUCLEI IN MATING-TYPE DETERMINATION

The two complementary mating types, O and E, of Paramecium tetraurelia are normally inherited cytoplasmically. This property has generally been interpreted to indicate the presence of cytoplasmic factors that determine macronuclear differentiation towards O or E. In these macronuclear-cytoplasmic interactions, the micronuclei were held to be unbiased and the determination to be established in the course of macronuclear development. In order to ascertain whether the micronuclei were actually neutral, amicronucleate clones were needed and a method to produce them was developed. In crosses between amicronucleate clones and normal micronucleate clones, we have observed regular deviations from cytoplasmic inheritance: the commonest deviation is that most O amicronucleate cells become E when they receive a micronucleus from an E partner. The data can be interpreted by assuming that the micronuclei are predetermined and that the apparent "cytoplasmic" inheritance of the two mating types is due, in E cells, to E-determining factors present in the cytoplasm and in the nucleus; and, in O cells, to O-determining factors present only or mainly in the nucleus.

Limiting conditions for conjugation in Tetrahymena: Cellular development and factor active in conjugation

AbstractConjugation in Tetrahymena thermophila is a three component system requiring starved cells of two complementary mating types and an extracellular macromolecular factor active in conjugation (FAC). Because the rate of cell pairing is related to the concentration of FAC in the extracellular medium, an assay for FAC was developed using standard test cells. The rate of release of FAC into the medium was measured in cells transferred from growth medium. It was found that a) FAC production is rapid, being detectable within 15 min after shift down; b) within 1 hr, sufficient FAC is released by the cells to support near maximal pairing activity among test cells; and c) cycloheximide completely blocks release of FAC. It was further demonstrated that addition of high‐titer FAC to freshly shifted down cells did not shorten the delay period before conjugation. Therefore, the starvation period is a time during which a) FAC is released into the medium, and b) cells develop receptivity to mating interactions, with the latter being rate‐limiting for conjugation. FAC is produced by sexually immature cells, by cells in growth medium, and by cells starving in high ionic strength buffer, but not by nonmating amicronucleate cells of T. pyriformis. Cell interactions during mating are modulated by FAC, but only among developmentally receptive cells.