INTRACELLULAR POSITIONAL CONTROL OF SURVIVAL OR DEGENERATION OF NUCLEI DURING CONJUGATION IN PARAMECIUM CAUDATUM

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1 J, Cell Sd. 79, (1985) 237 Printed in Great Britain The Company of Biologists Limited 1985 INTRACELLULAR POSITIONAL CONTROL OF SURVIVAL OR DEGENERATION OF NUCLEI DURING CONJUGATION IN PARAMECIUM CAUDATUM AKIRA YANAGI AND KOICHI HIWATASHI Biological Institute, Tohoku University, Sendai 980, Japan SUMMARY The survival or degeneration of nuclei produced after meiosis in Paramecium caudatum depends upon their position in the cytoplasm. The surviving nucleus lies in the paroral region, which is the region around the cytostome. In this study, this was confirmed by quantitative measurements of the location of surviving nuclei in both stained and living conjugating cells. Observation of nuclear behaviour in living cells shows that there is an active mechanism for localizing one of the four meiotic products into the paroral region after the second meiotic division. When this mechanism is inhibited by vinblastine, none of the four nuclei gets into the paroral region and all of them become pycnotic, before degenerating. The results show that migration into the paroral region is essential for survival of the nucleus and that microtubules are involved in this nuclear migration. INTRODUCTION Ciliates have two kinds of nuclei, the somatic macronucleus and the germinal micronucleus. Paramecium caudatum has one of each kind. During conjugation the micronucleus goes through meiosis and four haploid nuclei (meiotic products) are produced. Three of the meiotic products degenerate. The remaining one divides and produces two gametic nuclei. They exchange reciprocally and fertilize to form a synkaryon, which divides three times and differentiates into new macronuclei and a micronucleus. We do not know how all these processes are controlled. But we know at least two stages of the processes in which the fates of nuclei are closely concerned with their position in the cell. One is the stage of differentiation of macro- and micronucleus. At this stage, the position of the spindles of the postzygotic third division is known to determine the micro- and macronuclear differentiation (Mikami, 1980; Grandchamp & Beisson, 1981). The other is the stage of the degeneration of meiotic products. In this stage, three meiotic products lie outside the paroral region and degenerate, but the remaining one lies in the paroral region and survives (Wichterman, 1946; Sonneborn, 1954; Skoblo & Ossipov, 1968). These events pose an interesting problem as to what controls nuclear behaviour and differentiation. Ever since Boveri's (1887) discovery of the difference between somatic and germinal nuclei in Ascaris, many instances of cytoplasmic control of nuclear activity have been found (Bladder, 1958; Bantock, 1961; Illmensee & Mahowald, 1974). Key words: conjugation, intracellular positional control, meiosis, nucleocytoplasmic interaction, Paramecium caudatum.

2 238 A. Yanagi and K. Hiwatashi In ciliates, Sonneborn (1954) suggested that nuclear behaviour and differentiation in Paramecium aurelia were controlled by the cytoplasm. Nanney (1953) also suggested cytoplasmic control of nuclear behaviour in Tetrahymena. The process of degeneration of meiotic products in ciliates provides an excellent model for the study of nucleocytoplasmic interactions. In this study we describe the behaviour of meiotic products in P. caudatum and discuss the role of a particular region of cytoplasm, the paroral region, in determining the survival or degeneration of the nucleus. MATERIALS AND METHODS Stocks and culture methods Stocks used were mating type V and VI of 27aG3 in Paramecium caudatum, syngen 3. The culture medium was fresh lettuce juice diluted with Dryl's (1959) solution (DS), ph7-0, and inoculated with Klebsiella pneumoniae one day before use (Hiwatashi, 1968). Measurement of the position of meiotic products When conjugating pairs entered the stage of the degeneration of three of the meiotic products, they were air-dried, fixed in Carnoy's acetic acid/ethanol (1:3, v/v) for lomin and hydrolysed in 6M-HC1 for 30min at room temperature (18-6 C). They were then stained by the Feulgen method and counterstained with 0-5 % (w/v) Fast green F.C.F. Photomicrographs of the stained cells were taken and the relative positions of the meiotic products with respect to the longitudinal and transverse axes of the cells were measured on these. Continuous observation of micronuclear behaviour Micronuclear behaviour was observed in living cells by Nomarski differential-interferencecontrast microscopy. Cells were put between a coverglass and agar (0-35 %) on a depression slide so as to stop their motion (Sugai, personal communication). The agar gel contained 1 mm-cacl2, 1 mm-kcl and 5 mm-hepes, ph 7-0. Continuous observation of living cells after vinblastine treatment Conjugating cells were observed essentially by the same method as described above but with a slight modification. A glass slide with plastic strips was used instead of the depression slide (Fig. 1). Before placing the cells on the agar, they were put into 7 mgml" 1 bovine serum albumin (Knowles, 1974; Harumoto & Hiwatashi, 1982) in DS or K-DS (in which KH Z PO 4 is substituted for NaH2PO4 in DS). As shown in Fig. 1, vinblastine (Sigma) solution (100^gml" 1 in DS) was injected into the space between the coverslip and the slide. After 30min, vinblastine solution was withdrawn using a small piece of filter paper. It was replaced by DS, which was removed with filter paper. This operation was repeated three times to remove vinblastine completely. Finally, cells were mounted in DS and observation of micronuclear behaviour was continued for about 4h. RESULTS Position of meiotic products When mating reactive cells of complementary mating types are mixed and induced to conjugate, micronuclei in conjugating pairs enter meiosis. At 25 C the first meiotic division occurs about h and the second division about h after mixing mating types. About 1 h after the second meiotic telophase, three of the four meiotic division products become pycnotic and begin to degenerate. Conjugating pairs at this stage were fixed and stained, and relative positions of the

3 Positional control of nuclear fate 239 degenerating and surviving nuclei were measured on photomicrographs of the stained preparations (Fig. 2). The distribution of meiotic products in relation to the longitudinal and transverse axes of the cell is shown in Fig. 3. As seen in the figure, most surviving nuclei lie in the paroral region. Though some of the surviving nuclei lie some distance from the paroral region, this may be an artifact induced by airdrying and fixation, because in living cells the surviving nuclei always lie in the paroral region, as mentioned in the next paragraph (Fig. 5). Pycnotic nuclei on the way to degeneration seem to be randomly distributed. They appear to float in the cytoplasm and are more free-moving than nuclei are before pycnosis has begun. The nuclei probably become free from cytoskeletal restraint during pycnosis. Therefore the position of pycnotic nuclei is different from that of the nuclei just before pycnosis. Behaviour of meiotic products To know by what mechanism one of the meiotic products is brought to the paroral region, the behaviour of the four meiotic products was observed in living conjugating cells continuously from the onset of the second meiotic division to the stage of degeneration of three meiotic products. It was possible to follow the migration and eventual fate (survival or degeneration) of each of the four nuclei. At telophase of the second meiotic division and at the stage just before the degeneration of the three meiotic products, the location of the nuclei was recorded on photomicrographs and the relative distance of each nucleus from the centre of the cytostome was measured Fig. 1. Diagram of equipment for the observation of living cells. A. Cross-section of B at the site of the arrows; B, upper view.

4 240 A. Yanagi and K. Hiwatashi Fig. 2. Photomicrograph of conjugating cells during degeneration of nuclei. Cells were stained by the Feulgen reaction and counterstained with 0-5 % (w/v) Fast green F.C.F. Arrows indicate the surviving nuclei at the paroral region. Arrowheads indicate pycnotic nuclei on the way to degeneration. The space between the asterisks is the paroral region. Bar, 50 /im. on these. Degenerating nuclei were distinguished by their small size and round shape from the larger, spindle-shaped surviving nuclei. Figs 4 and 5 show the distributions of prospective surviving and degenerating nuclei over various relative distances from the centre of the cytostome at the telophase of the second meiotic division and at the stage just before nuclear degeneration begins, respectively. As shown in Fig. 4, about 60 % (from nos 1-7) of the cells have prospective surviving nuclei more or less near the paroral region at telophase of the second meiotic division. However, those nuclei are further from the paroral region than those lying in the paroral region just before the onset of nuclear degeneration (Fig. 5). Fig. 6 contains photomicrographs of conjugating cells at telophase of the second meiotic division and at the stage just before nuclear degeneration. When the two pictures are compared, it is clear that the prospective surviving nuclei just before nuclear degeneration are closer to the cytostome than those at the second meiotic telophase. The remaining 40% are grouped into two cases. In one case (no. 8) none of the meiotic products are in the paroral region. In the other case (nos 9 12), even if some nuclei are in the position nearest to the cytostome, they are not prospective surviving nuclei. In both cases,

5 Positional control of nuclear fate 241 prospective surviving nuclei eventually migrated into the paroral region from the position at the second meiotic telophase (Figs 4 and 5). These results show that in all of the observed cells, the prospective surviving nucleus migrates into the paroral region after the second meiotic division. Observation of living cells shows that the surviving nuclei always lie in the paroral region just before nuclear degeneration (Fig. 5). During observation of nuclear behaviour in living cells, conjugating cells occasionally showed repeated back-and-forth movements in the space between the coverglass and agar. In such cases, organelles, such as the meiotic products, macronucleus, etc., tend to be held in the original position in the field of view by the pressure between the coverglass and the agar, so that they move to-and-fro relative to the cytoplasm in the opposite direction to the movement of the cell as a whole. But the prospective surviving nucleus in the paroral region did not move within the cytoplasm and remained stationary there. This observation suggests that the surviving nucleus at the paroral region is anchored to the cell cortex or the cell membrane o Anterior Ventral Posterior 8 x o 50 oo 1 11' 8 s '? 50 B/AxlO0(%) 100 Fig. 3. Relative position of surviving nucleus () and pycnotic nucleus on the way to degeneration (O). A is the length of the cell and B is the distance from the anterior end of the cell to the nucleus. C is the width of the cell and > is the distance from the ventral side of the cell to the nucleus. The rectangle near 50 % on the abscissa shows the location of the cytostome.

6 242 A. Yanagi and K. Hiwatashi Inhibition of nuclear localization to the paroral region It is well known that in many organisms movement of the nuclei is controlled by microtubules (Oakley & Morris, 1980; Bestor & Schatten, 1981; Vogelmann, Bassel & Miller, 1981). It has recently been suggested that nuclear exchange at conjugation in Tetrahymena also depends on microtubules (Orias, Hamilton & Orias, 1983). In order to know whether microtubules are involved in the migration of the meiotic product to the paroral region, conjugating pairs in three different stages were treated with 100 /ig m!" 1 vinblastine for 30 min and observed continuously for about 4 h after the treatment. As shown in Table 1, when conjugating cells in metaphase of the first meiotic division were treated with vinblastine, the normal nuclear changes were not disturbed and the micronucleus underwent meiosis. When the cells at the stage after migration of one meiotic product to the paroral region were treated, the normal process of degeneration of three meiotic products and survival of one was also not disturbed and the surviving nucleus divided. However, when cells in the second meiotic anaphase were treated, no nucleus entered the paroral region and all four meiotic products became pycnotic and degenerated in 10 cells out of 14 observed. In three of the remaining cells, nuclear migration was not inhibited and one of the B/AxlO0(%) i...,, rhh-t 1 x HJ-H * Anterior ^ * * 5 Posterior.,.. Ah«I ,.,. HH = 7 J o.,.... ^H 8 «x $ ^H 9 i...,. ^H 10 f I,,.,, r-ff-. 11 xx x 1...,. rj(-h 12 x x Fig. 4. Relative position of prospective surviving nucleus () and degenerating nucleus () in the cell at the second meiotic telophase. A is the distance from the anterior end of the cell to the centre of the cytostome; B is the distance from the nucleus to the centre of the cytostome. The number on the left indicates the number of the sample.

7 Positional control of nuclear fate B//lxl00(%) j 6 a 7 x x x 8 * x Fig. 5. Relative position of prospective surviving nucleus () and degenerating nucleus () in the cell just before nuclear degeneration. The abscissa and the number on the left are as in Fig. 4. Observed cells are also as in Fig. 4. meiotic products entered the paroral region and survived. In the last one nuclear migration was inhibited, but one of meiotic products survived outside the paroral region. We cannot explain why this occurred. However, undue.stress should not be placed on this isolated result. DISCUSSION It is known from qualitative observations that in meiosis of Paramecium, the surviving nucleus comes to live in the paroral region and degenerating nuclei are elsewhere the cytoplasm (Wichterman, 1946; Sonneborn, 1954; Skoblo & Ossipov, 1968). This has been confirmed by quantitative study of stained samples (Fig. 3) and also by observation of nuclear behaviour in living cells (Fig. 5). In P. aurelia, Sonneborn (1954) reported that in an abnormal strain (d59), the meiotic products of which never get into the paroral region, no nuclei survived, and concluded that survival of the nucleus depends on localization in the paroral region and the nuclei that fail to get in are destined to degenerate. However, this result cannot exclude the possibility that in that abnormal strain all of the meiotic products are destined to degenerate and for this reason they did not enter the paroral region. We do not know

8 244 A. Yanagi and K. Hituatashi Fig. 6. Position of meiotic products in the conjugating cells at telophase of the second meiotic division (A), and at the stage just before nuclear degeneration (B). A and B are the same sample but at different stages of conjugation. Arrows indicate meiotic products. Note that one of meiotic products (d) in the left cell of B is degenerating and very small. The space between the asterisks is the paroral region; s, prospective surviving nucleus. Bar, 50 fan. whether the surviving nucleus is predetermined and then becomes localized in the paroral region or whether it survives because of its localization in the paroral region. This was examined by the study of inhibition of nuclear localization at the paroral region. When migration of meiotic products was inhibited by treatment with Table 1. Degeneration of meiotic products after treatment with vinblastine Stage of the treatment* 1st meiotic metaphase 2nd meiotic anaphase One meiotic product in the paroral region For details, see the text. No. of cells observed No. of cells with all nuclei pycnotic

9 Positional control of nuclear fate 245 vinblastine and they could not reach the paroral region, all of them became pycnotic, before degenerating (Table 1). This result shows that the paroral region has an essential role in the survival of the nucleus. What mechanism is involved in bringing a prospective surviving nucleus to the paroral region? There may be at least two mechanisms: one is that spindles of the second meiotic division bring the surviving nucleus into the paroral region; and the other is that there is an active mechanism for nuclear localization in the paroral region after the second meiotic division. In the differentiation of micro- and macronuclei after conjugation, Mikami (1980) reported that the position of spindles directly determines the fate of nuclei. In the determination of the surviving meiotic product, however, the second mechanism is supported by the observation of nuclear behaviour in living cells (Fig. 4). This indicates that there is a mechanism at work that allows one of the four meiotic products to move to the paroral region after the second meiotic division and that the position of the second meiotic spindles does not directly determine the fate of meiotic products. Since vinblastine prevents the nucleus from migrating into the paroral region (Table 1), microtubules may be involved in the nuclear migration. It has recently been reported that intermediate filament protein is involved in micronuclear behaviour during conjugation in Tetrahymena (Numata, Sugai & Watanabe, 1985). The same kind of protein may also be concerned with nuclear migration in P. caudatum. According to Sonneborn (1954), enlargement of the macronucleus may play an important role in localization of a prospective surviving nucleus in the paroral region in P. aurelia. In this species, the initially dense macronucleus opens up into a complicated skein and occupies a very large part of the cell while the micronuclei go through meiosis. The meiotic products, positioned between the enlarging macronucleus and the cell surface, are carried closer and closer to the cell surface by the enlargement of the macronucleus. This may play an important role in the migration of a micronucleus into the paroral region. But in P. caudatum this is not the case, because the enlargement of the macronucleus by skein formation does not occur when the micronucleus is undergoing meiosis but occurs at about the stage of the postzygotic third division of the synkaryon. From observations of nuclear behaviour in living cells, the prospective surviving nucleus seemed to be anchored in the paroral region. The anchoring of the nucleus may be important for the survival of the nucleus, but we do not know why this should be so. Further analysis of this phenomenon will be important for understanding the mechanism of nuclear survival. It is also not certain whether any of the four meiotic products can enter the paroral region or whether the nucleus entering the paroral region is predetermined before nuclear migration. This problem will be clarified by enucleation of meiotic products by micromanipulation, which is now under way.

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