CELL STRUCTURE AND FUNCTION 20: 239-244 (1995) © 1995 by Japan Society for Cell Biology The Vegetative Micronucleus has a Critical Role in Maintenance of Cortical Structure in Tetrahymena thermophila Tomomi Haremaki1#, Toshiro Sugai1, and Mihoko Takahashi2t department of Biology, Ibaraki University, Mito 310, Japan and institute of Biological Sciences, University of Tsukuba, Tsukuba 305, Japan Keywords: Tetrahymena/amicronucleate/ciliary rows/oral structure ABSTRACT.The vegetative role of the germinal micronucleus of Tetrahymena thermophila was studied. Amicronucleate cells that had spontaneously arisen in star strains (very aged clones 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 in- duced 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. Tetrahymena thermophila, a ciliated protozoan, has There is a possibility that the inertness of the micronu- two nuclei which differ in structure and function: the cleus might be due to the difference of DNAcontent be- diploid (2N= 10) (19) micronucleus, which is germinal, tween the micronucleus and the macronucleus. To elimi- and the polygenomic macronucleus which is somatic, nate the possible gene dosage effect, Mayoand Orias being transcriptionally active and responsible for vir- (13) constructed a heterokaryon that has a galactoki- tually all phenotypes of the cell. During conjugation, nase gene (galA+) in the micronucleus and a galAl mu- the micronucleus becomes transcriptionally active in tant gene in the macronucleus. They reported that assay meiotic prophase I (20). After meiosis, gametic nuclei for the galactokinase activity of soluble cell extracts pre- are formed, and after nuclear exchange and fertiliza- pared from the heterokaryon showed no activity at- tion, the newmicro- and macronucleus are formed tributable to the micronuclear gene. from products of the fertilized nucleus (15, 19). A com- In vegetative growth, the micronucleus is generally plex series of molecular events occurs during the de- thought to be inert and has no role except its ownre- velopment of the new macronucleus; polyploidization production. In several species of ciliates, however, a of the micronuclear genome to the 45 C level, amplifica- cell line which has no micronucleus (amicronucleate) tion of rDNA,DNAsequence rearrangement, and elimi- showed some abnormalities (for a review see Ref 18). In nation of 10-20% of the micronuclear DNAsequences Paramecium, Mikami (14) reported that an amicronu- (for a review see Ref 8). The sequences eliminated from cleate cell line induced by enucleation had a small oral the micronuclear genomeare collectively referred to as apparatus (OA), resulting in slow growth. Organization mic-specific DNA. of the OAin amicronucleate cell lines is a major focus Genie activity of the micronucleus in vegetative of study in several ciliates (for a review see Ref 18). In growth phase has not been detected by autoradiography T. thermophila, the vegetative micronucleus is not usu- (9) nor by a more sensitive approach using heterokar- ally dispensable because an amicronucleate cell line yons in which the macronucleus and micronucleus have has almost never been established despite repeated at- different alleles at specific loci. The phenotype of these tempts. Anexception is the mutant amicronucleate cell heterokaryoncells alwaysdependson the genotypeof line BI3840 isolated by Kaney and Speare (10). The the macronucleus and not of the micronucleus (1, 2, 5). BI3840 cell contains some mic-specific DNAsequences # Present address: Institute of Biological Sciences, University of in the macronucleus (12). Whether the sequences are re- Tsukuba, Tsukuba 305, Japan sponsible for the vegetative micronuclear function is r To whomreprint requests should be addressed. not known. Abbreviations: OA;oral apparatus, PYG;proteose peptone-yeast Whatis the essential role of the micronucleus for extract-glucose, NKC;NaCl KC1 CaCl2. vegetative growth in T. thermophila! Very aged clones, 239 T. Haremaki et al. star strains, often have small and genetically defective pie alkaline Giemsamethod stains both the cortical structure micronuclei which have abnormal chromosomal consti- and the nucleus. To determine the presence or absence of the tution. This meansthat the intact micronucleus is not al- micronucleus, 2%orcein in 45%acetic acid was used, as this ways necessary. In cell populations of star strains, C* staining showed the micronucleus, even whenit overlapped or A*, abnormal shaped cells, so-called crinkled cells the macronucleus. spontaneously appear. These cannot give rise to viable clones whenisolated. The crinkled cell has been shown RESULTS to be amicronucleate (16) and lacks the OA(3), suggest- ing a critical morphogenetic role of the micronucleus. Three types of abnormal cells in starstrains. Weob- Since it is rather difficult to determine the presence or served the cortical structure of B*VI, which has not absence of the micronucleus of the star strains because been studied so far, during log phase. Percentages of of its small size, in the present study we induced amicro- morphologically abnormal cells varied greatly from cul- nucleate cells from young cells with diploid micronu- ture to culture (0.1-20%). Although the crinkled cells, clei. They were easily induced by treatment with an anti- having disordered ciliary rows and no OA, were ob- tubulin drug, nocodazole. In the amicronucleate cell its served in them (see Fig. 2F), we found two other types OA is lost and then even its ciliary rows are disinte- of abnormal cells. Cells of both types were normal in grated. Loss of the OAoccurred without cell division. shape but lacked OAs. One type had normal ciliary These results suggest that a critical vegetative function rows (see Fig. 2D) but the other had disordered ciliary of the micronucleus is to maintain the cortical struc- rows (see Fig. 2E). Similar results were obtained on ob- ture, especially the OA. servation of A*III and C*III (data not shown). The morphologically abnormal cells seemed to be amicronu- MATERIALS AND METHODS cleate. But it was difficult to determine unambiguously whether all of these abnormal cells lacked the micronu- Strains and growth conditions. The strain B21922 used in cleus, because of the very small size of the micronucleus this study is derived from the inbred strain B1868 of T. ther- of the star strains. mophila. Fertility of B21922 is more than 80%. Star strains Amicronucleate cells obtained by nocodazole treat- A*III, B*VI and C*III are very aged clones with defective ment ofayoung strain. To eliminate error in judging micronuclei and are genetically sterile. The star strains were amicronucleates, we induced amicronucleates in the kindly provided by P. Bruns (Cornell University). Cells were young strain B21922 which has a clearly visible diploid grown axenically in PYGmedium (2% proteose peptone (Difco), 1% yeast extract (Difco), 1% glucose) at 26°C. Induction of the amicronucleate cell. To induce amicronu- 1 ) 8 - cleate cells, the antitubulin drug nocodazole (methyl 5-2-thion- o ylcarbonyl, l-benzimidazole-2-yl carbamate; Janssen), was a > CS added to a dividing cell population (J. Gaertig, pers. com- 2 > 6 - - 6 0 r n o c 3 V - d o mun.). Nocodazole was stored as a 2 mg/ml stock solution in o ca dimethylsulfoxide at 4°C. To obtain a semi-synchronously O 4 - - 4 0 - E dividing cell population, late-log phase cells were pelleted by 0 5 o o hand centrifugation. An equal volume of fresh PYGmedium £ 蝣20 ^ was added to the cell pellet. This cell suspension was madeas shallow as possible and kept overnight at 15°C in a plastic petri dish, and then diluted to about 1/100 by fresh PYGmedi- 10 12 14 16 18 20 um containing nocodazole (5 fig/ml) and kept at 26°C. Divid- hours ing cells began to appear about 150 minutes after the dilution. Fig. 1. Increase of cells without OAafter nocodazole treatment. The division index at peak was 10-20%. Around 200 minutes B21922cells were treated with nocodazole and samples were obtained from beginning of the treatment, the cells were washed with at 2 hour intervals after transfer to NKCsolution. The percentage of NKCsolution (0.2% NaCl, 0.008% KC1, 0.012% CaCl2) to re- the amicronucleate cells did not change very muchthroughout the 20 move the drug and were resuspended in NKCsolution, or iso- hours. The percentage of the amicronucleate cells lacking OAgradual- lated into glucose-rich medium (0.25% proteose peptone, ly increased in time but amicronucleate cells and empty cells were 0.25% yeast extract and 5% glucose). slow to lose their OAs(only show at 20 hours). Staining ofcorticalstructure and nucleus. Cells were fixed Horizontal axis; time (hours) after the end of treatment with nocoda- with 3%formalin for 30 minutes and transferred to methanol zole. Left vertical axis; the percentage of amicronucleate cells ( å ). acetic acid fixative (3 : 1). The cells were spread on a glass Right vertical axis; the percentage of OAlacking cells in amicronucle- ate cells (O), in amacronucleate cells (à") and in empty cells (A). Ver- slide, air-dried, treated with 5 N HC1for 30 minutes and tical bars indicate standard error of five independent experiments. stained with 3% Giemsa (Merk) in 0.02 M Na2HPO4. This sim- Sample size was 500 cells. 240 Vegetative Role of Tetrahymena Micronucleus micronucleus. For the induction of amicronucleates, no- zole treatment. With regard to the nuclei, four cell types codazole was used.