Formation and degradation of large extrusion bodies in Tetrahymena thermophila: The role of intramacronuclear microtubules in chromatin segregation.

Large extrusion bodies (EBs) in Tetrahymena thermophila were induced by treatment with aphidiocolin (APH), followed by transfer of the cells to a drug free medium. APH induces over-replication of DNA and reversible cell division arrest (Kaczanowski and Kiersnowska, 2011). After treatment the cells were transferred to a drug free medium, and a central granule of chromatin (a prospective EB) surrounded by microtubules developed inside the macronucleus. Subsequently the chromatin of the central granule appeared in the middle region of the dividing macronucleus, and it was later extruded as an EB. The remaining chromatin segregated to opposite ends of the elongating macronucleus and was separated from the chromatin destined for the EB by intramacronuclear microtubules. TUNEL assay (terminal deoxynucleotidyl transferase dUTP nick end labeling]) and in vivo acridine orange (AO) staining showed that the EBs underwent apoptotic-like degradation and autophagy similar to that observed in programmed nuclear death (PND) of the old macronucleus in conjugant cells. Overall, based on the data obtained in this study we proposed the hypothesis that over- replication of DNA induced by APH results in the appearance of defective DNA copies, that are not segregated to the opposite regions of dividing macronucleus and are destined for EBs.

Cohesion of Clonal Life History, Senescence and Rejuvenation Induced by Autogamy of the Histophagous Ciliate Tetrahymena rostrata.

The histophagous ciliate Tetrahymena rostrata was found as a parasite in the renal organs of the land snails Zonitoides nitidus and Cochlicopa lubrica. A starvation medium induced encystment, meiosis, autogamy, and development of new macronuclei. The cell division rate declined linearly with number of divisions from the last autogamy until senescence. The senescing strains were rejuvenated by further encystment-induced autogamy. It is expected, that these processes contribute to genetic variability among the local, small, and isolated T. rostrata populations. Consistent with this expectation, small divergences in the cox-1 sequences appeared even among these strains, which had been isolated from different specimens of the same host species at the same site. The divergences in this gene between our T. rostrata strains from Z. nitidus and other strains from C. lubrica, Helix aspersa, and Deroceras reticulatum in Spain (Segade et al. 2009, Parasitology 136:771-782), were beyond the limits of intra-species variability within the genus Tetrahymena. However there is the lack of inter-strain differences in the life history and cytology among our, the Spanish, and those T.rostrata strains, that are not available for "barcoding" anymore. Therefore, variability in the life history and morphology within T .rostrata is constrained by natural selection.

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.

[Dynamics of symmetric and asymmetric mitosis and cytokinesis]. / Dynamika mitozy i cytokinezy symetrycznej i asymetrycznej.

During cell division the bipolar microtubular mitotic spindle ensures faithful segregation of daughter chromosomes and appearance of the cytokinetic membrane in an equatorial area separating genetically identical daughter cells. This process proceeds in consecutive morphological stages of prophase, metaphase, anaphase and cytokinesis. The progress in embryology and oncology concerns the new data about intervening mechanisms of rotation of a bipolar spindle in prophase and the change of the position of a mitotic spindle in anaphase that result in an asymmetric and differential mitoses. The aim of this review is a discussion of some of molecular and signaling mechanisms which regulate position of mitotic spindles in different types of cells. It turns out that the knowledge of receptor-dependent and receptor-independent molecular mechanisms controlling geometry and localization of cytokinesis in some human cells and in early stages of development of C. elegans opens the new important research fields.

Acquisition of cell polarity during cell cycle and oral replacement in Tetrahymena.

The aim of this study was to search for a mechanism responsible for the acquisition of cell polarity in a ciliate Tetrahymena. Homologs of the mammalian genes coding for CDC42-GSK3beta- MARK/PAR1-MAPs proteins were found in the Tetrahymena genome (Eisen et al., 2006, and this study). These proteins belong to a pathway which controls assembly and disassembly of microtubule bundles and cell polarity in neural cells. In Tetrahymena, there are two types of morphogenesis: divisional and oral replacement (OR). In divisional morphogenesis, an elongation of longitudinal microtubule bundles (LMs) takes place during cell division. In contrast, in OR type morphogenesis, which occurs in starved non-dividing cells, a polar retraction of LMs occurs. In T. pyriformis, the frequency of developmental switch to OR morphogenesis increases in the presence of wortmannin, an inhibitor of the CDC42-GSK3beta-MARK pathway. In contrast, wortmannin when applied to dividing cells does not affect divisional morphogenesis. Using immunostaining with the antibody against mammalian mitotic phosphoproteins (MPM-2) we show that these proteins co-localize with the LMs and are distributed along the anterior-posterior gradient. In addition, we show that during OR type morphogenesis, the fate of LMs correlates with the anterior-posterior gradient of instability of the cortical structures. We used the conditional mouth-less mutant of T. thermophila (Tiedtke et al., 1988) to test if the presence of the oral apparatus is required for the maintenance of cell polarity. We discuss our results in relation to the hypothesis of GSK3-beta-MARK pathway involvement in the acquisition of cell polarity in Tetrahymena.

[An involvement of polokinases in control of progress of the cell-cycle--the mechanism of transient translocation and formation of an activated protein-protein complexes during mitosis]. / Rola polo-kinaz(y) w regulacji cyklu komórkowego--mechanizm translokacji i tworzenia kompleksów bialkowych przez polokinazy.

Polokinases are a subfamily of the mitotic serine/threonine kinases involved in coordination of a run of mitosis of eukaryotic cells. The main polo-like-kinase 1p (PLK1) is a passenger protein transiently localized to centrosomes, kinetochores and central spindle during mitosis and is required for bi-orientation of the normal metaphase spindle. Its activity is regulated at the level of protein stability and by action of upstream kinases, so that it peaks in metaphase and drops as cells exit mitosis. Regulation of location and activity of Plk1p is bi-phasic: the COOH terminal polo box domain binds to an array of mitotic phosphoproteins and followed by an allosteric conformation is activated to phosphorylate many its substrates. These mode of action involves polokinases into critical transitions of the cell cycle phases, and in control at some checkpoints of this cycle.

Isolation of a Tetrahymena thermophila strain which induced metaphase I meiotic arrest: new pathway of abortive conjugation.

A hypodiploid strain of Tetrahymena thermophila has been obtained that shows arrest at the stage of condensed nuclei, corresponding to metaphase I of normal conjugants and induced arrest at meiotic metaphase I (i.e. at the stage of condensed, bivalent chromosomes) in its wt partner mate. The metaphase I arrested conjugants retained their old macronuclei and most of them underwent cell fusion, instead of separation of exconjugants. The doublets were viable and cortically integrated. When the arrest inducing strain was crossed to the haploid tester strain, the haploid micronuclei were arrested in the meiotic metaphase I as the diploid ones had been; the monovalent, chromosomes were condensed, the arms of sister chromatids were not separated, and they were not segregated. Separation of the arms of sister chromatids and disjunction of bivalent chromosomes were not prerequisite for the formation of microtubular spindles in those cells that were arrested in meiotic metaphase I. After re-feeding, the doublet cells resumed cell divisions, segregating two macronuclei and micronuclei at random. One macronucleus was derived from the arrest inducing strain and the other from the tester strain. Heterokaryon strains with macronuclei derived from the parental arrest inducing strain and with the micronucleus derived from the parental wt tester strain were obtained. Surprisingly, these heterokaryons did not induce meiotic arrest. Thus, the arrest in the melotic metaphase I was induced by the micronucleus and not by the macronucleus of the arrest inducing strain.

The fenestrin antigen in submembrane skeleton of the ciliate Tetrahymena thermophila is proposed as a marker of cell polarity during cell division and in oral replacement.

Tetrahymena thermophila cells have two types of polarized morphogenesis: divisional morphogenesis and oral reorganization (OR). The aim of this research is the analysis of cortical patterns of immunostaining during cell division and in OR using previously characterized antibodies against fenestrin and epiplasm B proteins. During cell division, the anarchic field of basal body proliferation of the new developing oral apparatus (AF) showed concomitant strong binding of the fenestrin antigen and withdrawal of a signal of the epiplasm B antigen. At a specific stage, the fenestrin antigen also appeared as a character of the anterior cortex pole, with a co-localized decrease in the detected epiplasm B antigen. The fenestrin antigen also showed a polarity of duplicating basal bodies in ciliary rows. Indirect immunofluorescence and immunogold labeling experiments were performed in the absence and presence of an inhibitor of activity of serine/threonine kinases, 6-dimethylaminopurine (6-DMAP) as an inducer of the oral replacement process. In the presence of 6-DMAP, one class of cells started OR, and some others were trapped and affected in cell division. Both types of cells showed an instability of oral structures and formed enlarged primordial oral fields. These anarchic fields (AFs) bind the fenestrin antigen, with disappearance of epiplasmic antigen staining. Only one protein (about 64 kDa) is detected in western blots by the anti-fenestrin antibody and it accumulated in 6-DMAP-treated cells that are involved in uncompleted morphogenetic activity. At a defined stage of oral development, both during cell division and in OR, the fenestrin antigen served as a marker of polarity of the cell of the anterior pole character.

Correlation between the Shortened Period of Cell Pairing during Genomic Exclusion and the Block in Posttransfer Nuclear Development in Tetrahymena thermophia.: (conjugation/cell contact/star strains/nuclear transfer/Tetrahymena).

The duration of pairing in crosses of the micronuclearly defective strain A* V and the amicronuclear strain BI3840 of Tetrahymena thermophila is shorter than that in normal conjugation. In controls, pairing takes about 620 min at 30°C and 60 min more at 28°C. In contrast pairing of crosses of the A* strain took 470 min at 30°C and 490 min at 28°C, and that of crosses of BI3840 strain took 440 min at 30°C. The course of nuclear development in the tester strain crossed with the A* strain was similar to that in the control until the 3rd prezygotic division. Unilateral transfer of the pronuclei occurred later than on reciprocal transfer in control crosses; posttransfer divisions in hemikarya was completely blocked in over 90% of the cases examined. Defective cell contact in crosses of the A* V strain and an amicronuclear BI3840 strain may be correlated with block in nuclear division and in macronuclear development, since unfertilized cells from triplet conjugation remain haploid and develop normally.