Relationship Between Spatial Pattern of Basal Bodies and Membrane Skeleton (Epiplasm) During the Cell Cycle of Tetrahymena: cdaA Mutant and Anti-Membrane Skeleton Immunostaining

Microtubular basal bodies and epiplasm (membrane skeleton) are the main components of the cortical skeleton of Tetrahymena. The aim of this report was to study functional interactions of basal bodies and epiplasm during the cell cycle. The cortex of Tetrahymena cells was stained with anti-epiplasm antibody. This staining produced a bright epiplasmic layer with a dark pattern of unstained microtubular structures. The fluorescence of the anti-epiplasm antibody disappeared at sites of newly formed microtubular structures, so the new basal body domains and epiplasmic layer could be followed throughout the cell cycle. Different patterns of deployment of new basal bodies were observed in early and advanced dividers. In advanced dividers the fluorescence of the epiplasmic layer diminished locally within the forming fission line where the polymerization of new basal bodies largely extincted. In wild type Tetrahymena, the completion of the micronuclear metaphase/anaphase transition was associated with a transition from the pattern of new basal body deployment and epiplasm staining of the early divider to the pattern of the advanced dividers. The signal for the fission line formation in Tetrahymena (absent in cdaA1 Tetrahymena mutationally arrested in cytokinesis) brings about 1) transition of patterns of deployment of basal bodies and epiplasmic layer on both sides of the fission line; and 2) coordination of cortical divisional morphogenesis with the micronuclear mitotic cycle.     

Induction of Blocks in Nuclear Divisions and Overcondensation of Meiotic Chromosomes with Cycloheximide During Conjugation of Tetrahymena thermophila

During conjugation, the micronucleus of Tetrahymena thermophila undergoes five consecutive nuclear divisions: meiosis, third prezygotic division (pregamic mitosis) and two postzygotic mitoses of the synkaryon. The four products of the synkaryon differentiate into macronuclear anlagen and new micronuclei and the old macronucleus is resorbed. The protein synthesis inhibitor cycloheximide, applied during conjugation, induced several developmental blocks. Pairs shifted to the drug during early meiotic prophase (stages I–III) were arrested at prophase. Cycloheximide applied to cells at pachytene (stages IV-VI) to metaphase arrested the conjugants at the stage of modified prometaphase/metaphase with overcondensed, swollen bivalents. In contrast to other systems, in the presence of cycloheximide, separation of chromatids, decondensation of chromosomes and exit from metaphase I were inhibited in both diploid and haploid cells. Pairs shifted to the drug after metaphase I were arrested at postmeiotic interphase after completing one nuclear cycle. The same rule applied to the subsequent cycle; then cells were arrested at the stage of pronuclei, and those pairs with functional pronuclei and synkarya were arrested at the stage of two products of the first postzygotic division (pronuclei were not arrested in nuclear transfer and karyogamy). Only pairs with two products of the first postzygotic division were arrested at the same stage after the cycloheximide treatment. Pairs shifted to cycloheximide during the second postzygotic division were arrested in development of macronuclear anlagen and resorption of old macronuclei. The postmeiotic conjugants pulse-treated with cycloheximide (2 h) yielded heterokaryons retaining parental macronuclei (i.e. they exhibited macronuclear retention).     

Effects of Actinomycin D on Generation Time and Morphogenesis in Paramecium*

SYNOPSIS. The sensitivity of Paramecium tetraurelia (=P. aurelia syngen 4) cells to pulse treatments with various doses of Actinomycin D (AMD) was estimated by comparing the generation times of treated and untreated sister cells. It was found that the delay of division in treated cells depended on the concentration of AMD, on their “age” at the time of the pulse treatment, and on their individual sensitivity. Sensitivity of Paramecium to AMD changes during the cell cycle in a predictable way. About 3 1/2 hr before the normally expected cell fission (total generation time ～ 5 1/2 hr) there is a decrease of sensitivity. Thereafter, the cell enters a new stage with a progressive increase of sensitivity. This 2nd phase ends at the “transition point” (～ 2 hr before cell division), when sensitivity drops abruptly. The division process itself may be altered and slowed down by high concentrations of AMD, even if the drug is applied after the transition point, but this process can never be completely annulled. The impairment of the division mechanism may lead to morphologic anomalies in the offspring. Resorption of oral anlagen in P. tetraurelia probably never occurs during the cell cycle after AMD treatment. The reason for individual variability of the cells, mechanisms controlling development, and the question of an obligate sequence of gene action in each cell cycle are discussed.