Downward regulation of cell size in Paramecium tetraurelia: effects of increased cell size, with or without increased DNA content, on the cell cycle

0 Department of Zoology, University of British Columbia Vancouver , B.C. , Canada V6T 2A9 Two temperature-sensitive cell-cycle mutants were used to generate abnormally large cells (size estimated by protein content) with either normal or increased DNA contents. The first mutant, cd, blocks DNA synthesis, but allows cell growth at the restrictive temperature. The cells do not progress through the cell cycle while at the restrictive temperature, but do recover and complete the cell cycle when returned to permissive conditions. The progeny have increased cell size and normal DNA content. Downward regulation of cell size occurs during the ensuing cell cycle at permissive temperature. Two processes are involved. First, the Gt period is reduced or eliminated. As initial cell size increases there is a progressive shortening of the cell cycle to 75 % of normal. This limit cell-cycle duration is reached when the initial mass of the cell is equal to or greater than that of normal cells at the time of DNA synthesis initiation (0-25 of a cell cycle). Cells with the limit cell cycle begin macronuclear DNA synthesis immediately after fission. The durations of the S period and fission are normal. Second, the rate of cell growth is unaffected by the increase in cell size, and results in the partitioning of excess cell mass between the daughter cells at the next fission. The second mutant, cc2, blocks cell division, but allows DNA synthesis to occur at a reduced rate so that cells with up to about 14c % of the normal initial DNA content and twice the normal cell mass can be produced. The pattern of cell-cycle shortening is the same as in cd. The rates of growth and both the rate and amount of DNA synthesis are proportional to the initial DNA content. This suggests that the rates of growth and DNA synthesis are limited by the transcriptional activity of the macronucleus in both cd and cc2 cells when they begin the cell cycle with experimentally increased cell mass. Increases in both cell size and initial DNA content are required to bring about increases in the rates of growth and DNA accumulation. - In typical eukaryotic cells the mitotic nuclear division mechanism ensures that daughter cells receive chromosomal complements that are quantitatively and qualitatively identical. The mechanism of cytokinesis is not so precise. Sister cells typically differ somewhat in cell mass (e.g. see Killander & Zetterberg, 1965 a). Rates of growth and generation times also vary slightly. Thus, variation in cell mass is introduced during each cell cycle and, if there were no regulatory mechanisms, the variance of cell size within a cell population would increase without limit. This obviously does not occur; cell mass shows only a limited variation (Killander & Zetterberg, 1965a; Killander, 1965; Kimball, 1967; Kimball, Perdue, Chu & Ortiz, 1971; Yen et al. 1975). Consequently, regulation of cell mass must occur in all organisms. This paper examines the kinetics of cell-size regulation in Paramecium and its relation to the cell cycle. Paramecium tetraureUa is a relatively large protist with 50-100 times the volume of a typical diploid eukaryotic cell. It has a large polygenomic macronucleus, which can vary substantially in DNA content without the occurrence of genie imbalance. Cell size can also vary considerably. Although both cell size and macronuclear DNA content vary, there is usually a high correlation between mean cell size and mean DNA content (Kimball, 1967). These observations suggest that Paramecium can regulate cell size and that cell-size regulation and the regulation of macronuclear DNA content are related processes. We have studied two temperature-sensitive (ts) cell-cycle mutants. The ts mutation ecl blocks macronuclear DNA synthesis, and consequently cell division at the restrictive temperature. However, protein synthesis and accumulation continue, so that cells with excess protein content can be produced. A second ts mutation, cc2, also blocks cell division, but allows macronuclear DNA synthesis to continue at the restrictive temperature, although at a reduced rate. The cells show an increase in both protein and DNA content at the restrictive temperature, although DNA content increases at a lower rate than protein content under these conditions. These mutations make it possible to generate cells of increased size experimentally, with or without a concommitant increase in macronuclear DNA content. We were then able to examine the kinetics of downward regulation of cell size, and the roles of macronuclear DNA content and cell size in determining the rates of growth and DNA synthesis, cell-cycle duration and the changes in DNA and protein contents over the course of a recovery cell cycle following the return of the experimental cells to permissive conditions. MATERIALS AND METHODS P. tetraureUa (Sonneborn, 1975), was grown in phosphate-buffered grass medium (Sonneborn, 1970) with Enterobacter aerogenes as the food organism. Two mutant stocks derived from the wild-type stock 51-S were used. Stock d4-ioo2 carries the recessive ts mutation cd, which blocks macronuclear DNA synthesis and cell division (Peterson & Berger, 1976). Stock d4-ioo3 carries the mutation cc2, which blocks cell division but not macronuclear DNA synthesis (Peterson & Berger, 1976). Two groups of approximately 40 hand-synchronized cells were selected prior to fission. One group, designated the control sample, was incubated at the permissive temperature (27 C). This group was used to determine the length of the normal cell cycle, and the protein and DNA content of untreated cells. The other sample, the experimental group, was incubated at 27 C for 0-5 h before the start of heat treatment, to ensure that all cells had completed division before the beginning of the heat shock. Heat treatments were carried out in a water-bath with an electronic temperature controller, which maintained water temperature to 0-02 deg. C. At the end of the heat treatment the cells were returned to the permissive temperature and allowed to reach division. After the first division, one of the daughter cells was fixed, and the other was allowed to progress through the next cycle. After the second, both daughter cells were fixed. The cell cycle that included the heat treatment was designated the first cell cycle and the recovery cell cycle was the second one. Newly divided cells were placed, by micropipette, on albumin-coated microscope slides and most of the culture medium was removed. This ensured maximum flattening of the cells as they dried. After drying the cells were fixed in ethanol/acetic acid mixture (3:1) for 30 min. RNA was removed by acid hydrolysis (5 N-HC1, 45 min, room temperature). Cells were then stained with thefluorochromesacriflavine HC1 and primulin by the two-colour method of Cornelisse & Ploem (1976). Acriflavine stains DNA and primulin stains protein. Quantitativefluorescencemicroscopy Determinations of DNA and pr (...truncated)