MOLECULAR AND CELLULAR BIOLOGY, June 2006, p. 4690–4700 0270-7306/06/$08.00⫹0 doi:10.1128/MCB.02315-05 Copyright © 2006, American Society for Microbiology. All Rights Reserved.
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The Condensin Complex Is Essential for Amitotic Segregation of Bulk Chromosomes, but Not Nucleoli, in the Ciliate Tetrahymena thermophila† Marcella D. Cervantes,1,2 Robert S. Coyne,3 Xiaohui Xi,1 and Meng-Chao Yao1,4* Division of Basic Sciences, Fred Hutchinson Cancer Research Center, Seattle, Washington 981091; Molecular and Cellular Biology Program, University of Washington, Seattle, Washington 981952; The Institute for Genomic Research, Rockville, Maryland 208503; and Institute of Molecular Biology, Academia Sinica, Taipei, Taiwan4 Received 4 December 2005/Returned for modification 4 January 2006/Accepted 13 April 2006
The macronucleus of the binucleate ciliate Tetrahymena thermophila contains fragmented and amplified chromosomes that do not have centromeres, eliminating the possibility of mitotic nuclear division. Instead, the macronucleus divides by amitosis with random segregation of these chromosomes without detectable chromatin condensation. This amitotic division provides a special opportunity for studying the roles of mitotic proteins in segregating acentric chromatin. The Smc4 protein is a core component of the condensin complex that plays a role in chromatin condensation and has also been associated with nucleolar segregation, DNA repair, and maintenance of the chromatin scaffold. Mutants of Tetrahymena SMC4 have remarkable characteristics during amitosis. They do not form microtubules inside the macronucleus as normal cells do, and there is little or no bulk DNA segregation during cell division. Nevertheless, segregation of nucleoli to daughter cells still occurs, indicating the independence of this process and bulk DNA segregation in ciliate amitosis. at ⬃45 copies. Genetic data (14, 33, 51) suggest that these chromosomes segregate randomly during amitosis, in contrast to the equal segregation of sister chromatids typical of mitosis. Another interesting feature of the macronucleus is the presence of approximately 90 nucleoli distributed around its periphery (6), each containing approximately 100 copies of the 20-kb chromosome that holds the rRNA genes. During cell division, the nucleoli remain intact and peripheral and segregate with the macronuclear DNA to the daughter cells. Although a bipolar spindle does not form during macronuclear division, microtubules have been shown to be essential for the normal completion of amitosis (26, 34, 50). At the beginning of macronuclear division, ␣-tubulin is distributed diffusely in the macronucleus. The specialized tubulin found at microtubule organizing centers, ␥-tubulin, is also seen within the macronucleus at this time (40). Intramacronuclear and cytoplasmic microtubules become visible as amitosis progresses, extending half the width of the macronucleus and between the macronucleus and the cell cortex, respectively. It is not known whether or how microtubules attach to macronuclear chromatin. The two structural maintenance of chromosomes proteins Smc2p and Smc4p are core proteins of the condensin complexes that are necessary for proper chromosome segregation in meiosis and mitosis of eukaryotes such as budding yeast, fission yeast, nematode worms, insects, and vertebrates (8, 21, 22, 25, 35, 37, 42, 44). The condensin complex was first identified for its role in mitotic chromosome condensation, but its precise functions differ in various organisms. Mutations of the SMC2 and SMC4 homologs of fission yeast result in failure of chromosome condensation and segregation (37), whereas in some vertebrate cells, chromosomes are able to condense, although at a delayed rate, in the absence of condensin (25). In Drosophila and chicken cells lacking SMC2, SMC4, or the non-SMC condensin subunit CAP-D2, localization of topo-
The ciliate Tetrahymena thermophila maintains two nuclei, a germ line nucleus (micronucleus) and a somatic nucleus (macronucleus). Mitosis and meiosis of the micronucleus resemble these processes in other eukaryotes, including equal segregation of chromosomes, and the phosphorylation of histone H3, which is a marker for chromosome condensation during mitosis (48). The macronucleus differs from other eukaryotic nuclei in several respects. It is derived from the micronucleus during conjugation through elaborate genomic rearrangement of the five germ line chromosomes into more than 200 chromosome fragments ranging in size from 20 to more than 3,000 kb (for a review, see reference 52) that have telomeres but no centromeres (7). During vegetative growth, the macronucleus divides by an amitotic mechanism that remains ill defined. Although “amitotic” nuclear division has been documented in a number of organisms, the majority of such observations refer to unequal nuclear division in the absence of cell division, and in fact many of these cases may represent misinterpretations of incomplete mitoses (36). Only in ciliate macronuclei does normal nuclear division occur in the absence of any apparent chromosome condensation or any mechanism for equal segregation of genetic material. Ciliate amitosis is an elaborate process and not a simple constriction of the nucleus into two halves (46), but it lacks many features of mitosis that are conserved in practically all eukaryotes, including chromatin condensation, the formation of a spindle, and the phosphorylation of histone H3 (15). Each non-rRNA gene macronuclear chromosome is present * Corresponding author. Mailing address: Institute of Molecular Biology, Academia Sinica, Taipei, Taiwan. Phone: 886-2782-1436. Fax: 886-2-2788-4177. E-mail:
[email protected]. † Supplemental material for this article may be found at http://mcb .asm.org/. 4690
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isomerase II and passenger protein INCENP to the chromatin scaffold is disturbed and chromosome segregation is disrupted (11, 25, 38). In budding yeast, condensin physically associates with the rRNA gene and has a special role in the proper segregation of nucleoli (13, 16). By studying Tetrahymena SMC4 (referred to here as TtSMC4), we have identified a novel role for this protein in the unusual phenomenon of amitosis. TtSMC4 mutants display an extreme phenotype with a lack of bulk DNA segregation during nuclear division. We find that segregation of nucleoli is not dependent on TtSMC4, suggesting that this event has different functional requirements. In addition to shedding light on the mechanism of amitosis, our work suggests that Tetrahymena may be instrumental in revealing functions of condensin, and possibly other mitotic proteins, that are not as apparent in a mitotic nucleus, where early mutational defects can mask later roles in chromosome segregation, such as recruitment of proteins for microtubule attachment.
MATERIALS AND METHODS Identification of TtSMC4 and phylogenetic analysis. PCR was performed on a genomic DNA template by using degenerate oligonucleotides targeting the 3⬘ region coding for the conserved C terminus of Smc4p (5⬘-YTNTCHGGTGGT GAAAARAC-3⬘ and 5⬘-ARRGCRGCRTCRAYTTCYTC-3⬘). The resulting 103-bp product was sequenced, and the sequence information internal to the degenerate primers was used to design inverse PCR primers 5⬘-GCAAAAACT AAAGAAAGAGAAC-3⬘ and 5⬘-TACAAGCCAACTCCATTATACTTTATG G-3⬘. Genomic DNA was digested with EcoRI, self-ligated, and used for inverse PCR. A 5-kb inverse PCR product was obtained and sequenced. The sequence was extended to the 5⬘ end of the gene by rapid amplification of cDNA ends-PCR (Invitrogen Corp.). Additional flanking sequence was obtained from the Tetrahymena whole-genome shotgun sequence provided by The Institute for Genomic Research (http://www.tigr.org/tdb/e2k1/ttg/). Positions of introns were mapped by sequencing of reverse transcription (RT)-PCR products spanning the entire gene. RNA for RT-PCR was isolated from cells grown in rich medium. RT-PCR was performed according to the manufacturer’s (Invitrogen Corp.) recommendations. Starting with a multiple sequence alignment of 100 SMC family members (10), a hidden Markov model was generated with the program hmmbuild (http: //hmmer.wustl.edu) and refined with hmmcalibrate. The four Tetrahymena and two Paramecium predicted peptide sequences were incorporated into the alignment by using hmmalign. A neighbor-joining tree was constructed with PAUP (http://paup.csit.fsu.edu/), and the branch lengths were adjusted with Tree-Puzzle (39). Epitope tagging of TtSMC4. By overlapping PCR (54), the hemagglutinin (HA) tag was inserted in front of the stop codon and a neo cassette (18) was inserted 700 bp downstream. A 1.6-kb fragment that included the HA tag followed by the neo cassette and an additional 500 bp of homology on either side was amplified by PCR. The purified fragment was used for biolistic transformation of Tetrahymena (5) and integrated at the macronuclear TtSMC4 locus by homologous recombination. Transformants were selected with paromomycin, and after sufficient growth under selection to allow phenotypic assortment to occur, complete replacement of the endogenous allele was verified by PCR and sequencing. Western analysis. Tetrahymena protein extracts were prepared from growing cells in early log phase. Cells were washed once in 10 mM Tris (pH 7.4) and once in TMS buffer (10 mM Tris [pH 8.0], 1 mM MgCl2, 3 mM CaCl2, 0.24 M sucrose) and then resuspended in TMS to a final concentration of ⬃5 ⫻ 107 cells/ml. NP-40 was added to a final concentration of 0.2%. Cells were lysed by shaking on ice for 15 min. Two volumes of modified buffer C with protease inhibitors (20 mM HEPES [pH 7.9], 0.3 M KCl, 1.5 mM MgCl2, 20% glycerol, leupeptin at 0.5 g/ml, E64 at 10 g/ml, chymostatin at 10 g/ml, antipain at 12.5 g/ml) was added to lysed cells, and the extract was homogenized with 20 strokes with a Dounce homogenizer. Extracts were spun at 42,000 rpm for 45 min, and supernatant was collected. Supernatant was run on a 7.5% sodium dodecyl sulfatepolyacrylamide gel and blotted to nitrocellulose membrane with a semidry electroblotter (Owl Separation Systems). Blots were incubated with mouse
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antibodies against the HA tag (F-2012; Sigma) diluted 1:200 and detected with Amersham anti-mouse horseradish peroxidase conjugate diluted 1:500. TtSMC4 knockouts. The neomycin resistance cassette (18) was inserted into an SMC4 genomic clone between the BglII and EcoRV restriction sites such that it replaced the DNA sequence stretching from the N-terminal conserved domain to the hinge region. The resulting knockout construct was linearized with EcoRI to release the neo cassette flanked with the TtSMC4 genomic sequence prior to transformation. Somatic transformation was performed as previously described (5). Heterozygous germ line transformants were carried through genomic exclusion as previously described (53) to create heterokaryons that are homozygous knockouts in the germ line nucleus and contain a wild-type macronucleus. Correct integration was verified by PCR and Southern analysis. FISH. Fluorescence in situ hybridization (FISH) was carried out as described by Loidl and Scherthan (29), with a few exceptions. Plasmid pD5H8, containing the rRNA gene, was digested with XhoI and NotI before labeling. After labeling of 2 g of probe, 20 g of salmon sperm DNA was added and DNA was precipitated and resuspended at 100 ng of labeled DNA/l. Unlabeled Tetrahymena DNA was not added to the probe. After hybridization, 2 g/ml 3,4⬘,6⬘diamidino-2-phenylindole (DAPI) was added to the first of three 10-min washes. Immunofluorescence assay. For immunofluorescence assay with antibodies against Nopp52 and the HA tag, cells were washed in 10 mM Tris (pH 7.4) and resuspended in 0.5 ml of 10 mM Tris (pH 7.4), to which was added 2 ml of 1% paraformaldehyde in PHEM buffer (45). The cells were incubated at room temperature for 6 min, washed twice in phosphate-buffered saline (PBS), resuspended in PBS, and dropped onto coverslips. Coverslips were allowed to dry at room temperature and then stored at 4°C. Before use, coverslips were warmed to room temperature, soaked in PBS for 45 min, and then incubated with primary antibody (diluted in PBS at 1:200 for anti-Nopp52 and anti-Pdd1p and 1:100 for anti-HA). Coverslips were incubated with primary mouse antibody at 37°C for 2 h, soaked in PBS for 45 min, and then incubated in secondary antibody diluted 1:100 in PBS for 30 min at 37°C and washed as before. Coverslips were inverted onto 7 l of Vectashield (Vector Laboratories) with 1 g/ml DAPI. Immunofluorescence with antibodies against ␣-tubulin was done as described by Fujiu and Numata (17). Mating cells were prepared as described by Numata et al. (34). The ␣-tubulin antibody was obtained from the University of Iowa Hybridoma Bank and used at a 1:25 dilution in PBS. The secondary anti-mouse fluorescein isothiocyanate-conjugated antibody was obtained from Sigma and used at a 1:100 dilution in PBS.
RESULTS Identification of the Tetrahymena SMC4 homolog. Smc4p is a core protein of the condensin complex and is conserved throughout the eukaryotic kingdom. We initially obtained the complete gene sequence of TtSMC4 by degenerate PCR with oligonucleotides targeting the C-terminal conserved domain, followed by inverse PCR. Introns were mapped by sequencing of RT-PCR products. Subsequently, the T. thermophila macronuclear whole-genome shotgun sequence and preliminary gene annotations were released (http://www.tigr.org/tdb/e2k1/ttg; http://www.ciliate.org). Searches of the currently available predicted gene annotations have revealed four Tetrahymena SMC homologs, with gene identification numbers 211.m00035, 27.m00251, 138.m00121, and 51.m00273. The latter of these is the gene identified in our analysis. To confirm its identification as an SMC4 ortholog, we incorporated our experimentally confirmed coding sequence, as well as the predicted peptide sequences of the other three Tetrahymena sequences and two predicted SMC homologs that have been identified in the related ciliate Paramecium tetraurelia (GenBank accession numbers CAI39061 and CAI39060) into an alignment of 100 SMC sequences (all 92 eukaryotic sequences plus 8 prokaryotic sequences) generated by Cobbe and Heck (10) (see Fig. S1 in the supplemental material). A neighbor-joining phylogenetic tree of the resulting alignment (Fig. 1 shows a representative subset of taxa) reveals that each of the four Tetrahymena sequences falls into a distinct clade corresponding to one of the four SMC sub-
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FIG. 2. TtSmc4p is present in both nuclei during vegetative growth. (A and B) Immunofluorescence of cells in vegetative growth shows TtSmc4p-HA in the germ line micronucleus and in the somatic macronucleus during interphase. (C and D) TtSmc4p-HA is also present in both nuclei during cell division. At this stage of cell division, the macronucleus (mac) has begun the process of division and the micronucleus (mic) is in S phase. TtSmc4p-HA, green; DNA, DAPI, blue.
FIG. 1. The Tetrahymena genome encodes four identifiable SMC family members. A neighbor-joining phylogenetic tree of representative eukaryotic, bacterial, and archaeal members of the SMC family is shown with bootstrap values (100 replicates) at the nodes. The four T. thermophila homologs (TT; in red) fall into the previously defined subfamilies of SMC1 through SMC4. Abbreviations: BSUB, Bacillus subtilis; RBCT, Rhodobacter sphaeroides; HALO, Halobacterium sp.; PRCL, Prochlorococcus marinus; PYRF, Pyrococcus furiosus; HS, Homo sapiens; OS, Oryza sativa; DM, Drosophila melanogaster; SC, S. cerevisiae; SP, Schizosaccharomyces pombe; CE, Caenorhabditis elegans; EC, Encephalitozoon cuniculi; PT, P. tetraurelia.
families, with our gene sequence in the SMC4 subgroup. No identifiable ortholog of SMC5 or SMC6 has been found in T. thermophila. The TtSMC4-HA protein is localized to both nuclei during vegetative growth. To study the localization of TtSmc4p during vegetative growth, we constructed a strain where the endogenous macronuclear gene was replaced with the TtSMC4 gene containing an HA epitope at the 3⬘ end, allowing the tagged gene to be under the control of the native promoter (in Tetrahymena, all protein synthesis depends on the activity of macronuclear genes). Because the endogenous macronuclear gene could be completely replaced with the epitope-tagged allele, we conclude that the tag did not interfere with normal functioning of the protein. In Western blot assays of vegetative extracts with antibodies against the HA epitope, the major band detected has the expected size for the tagged protein of ⬃175 kDa (see Fig. S2 in the supplemental material). The anti-HA antibody did not generate a background signal in
immunofluorescence analysis on untransformed cells (data now shown), in agreement with previous use of the HA tag in Tetrahymena (40). Knowing that the primary activities of Smc4p in most organisms occur during meiosis and mitosis, we expected the tagged protein TtSmc4p-HA to be seen specifically in the germ line nucleus, which divides by mitosis during vegetative growth. Cellular localization of TtSmc4p-HA was examined by indirect immunofluorescence. As expected, during vegetative growth the TtSmc4-HA protein is found in the germ line micronucleus. The protein is also localized in the somatic macronucleus that divides by amitosis (Fig. 2A and B). The fluorescence signal in all cells of the asynchronous culture was roughly the same, indicating that TtSmc4p-HA is as evident in both nuclei during cell division (Fig. 2C and D) as in the rest of the cell cycle. Localization to both nuclei during vegetative growth suggests that Smc4p may be involved in amitotic, as well as mitotic, nuclear division. Starvation of cells results in gradual loss of TtSmc4p. After 24 h of starvation, 55% of the cells showed a significantly reduced amount of fluorescent signal compared with vegetatively growing cells (Fig. 3, arrowhead). The signal was lost more rapidly from micronuclei than from macronuclei (Fig. 3, arrow). After 48 h of starvation, 45% of the cells had no detectable TtSmc4p-HA. The signal was rapidly restored after a return to growth medium; 7 h later, 97% of the cells had TtSmc4p in both nuclei. Immunofluorescence was performed with conjugating cells containing the tagged gene only in the somatic macronucleus. During conjugation, the parent cell’s macronucleus is de-
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FIG. 3. During starvation, many cells have an undetectable amount of TtSmc4p-HA. After 24 h in starvation medium, 55% of the cells had a reduced amount of the tagged TtSmc4 protein (arrowhead). Other cells still had significant amounts of the tagged protein but less in the micronuclei (arrow). The number of cells without detectable TtSmc4p-HA increases with longer starvation. A, DNA, DAPI, blue; B, TtSmc4p-HA, green; mac, macronucleus; mic, micronucleus.
graded and replaced with a new macronucleus that contains only untagged TtSMC4. Thus, the only tagged TtSmc4p found in progeny cells was residual protein derived from the gene in the old, parental macronucleus. During conjugation, tagged TtSmc4p became undetectable in developing new macronuclei at a relatively early stage (10 to 12 h after initiation of mating) but remained detectable in newly formed zygotic micronuclei (data not shown). We found that the tagged parental protein was still detectable in progeny cells that had divided once following conjugation, but only in the micronuclei of these cells (Fig. 4).
TtSMC4 is essential for viability in Tetrahymena. The presence of TtSmc4p-HA in both nuclei during cell division suggests that it may play a role in both micronuclear mitosis and macronuclear amitosis. To determine if TtSMC4 is essential for mitosis or amitosis, the gene was disrupted by the replacement of an internal 1.3-kb region with the neomycin resistance gene cassette (referred to hereafter as neo). We obtained somatic TtSMC4 gene “knockdown” strains where approximately 50% of the macronuclear wild-type gene copies were replaced with the smc4-1::neo allele, but we were unable to exceed ⬃50% replacement (data not shown). These somatic knockdown
FIG. 4. Parental TtSmc4p-HA is detectable in the micronuclei of progeny. Strains carrying tagged TtSMC4-HA only in the macronucleus were taken through conjugation. During conjugation, the parental macronucleus with TtSMC4-HA is degraded and a new macronucleus with only the endogenous, untagged TtSMC4 gene is created. Parental TtSmc4p-HA is detectable in the micronuclei of progeny karyonide cells, which have divided once following conjugation. A, DNA, DAPI, blue; B, TtSmc4p-HA, green; C, merge; mac, macronucleus; mic, micronucleus.
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TABLE 1. Mating of TtSMC4 homozygous germ line knockout heterokaryons (mic⫺/⫺) does not produce viable progeny a Strains
Wild type ⫻ wild type Wild type ⫻ TtSMC4 mic⫺/⫺ mutant TtSMC4 mic⫺/⫺ mutant ⫻ TtSMC4 mic⫺/⫺ mutant
% Mating pairs
% Viable progeny
84 81 88
70 69 0
a Mating of heterokaryons with wild-type strains produced a similar number of progeny as a wild-type mating. Mating of two TtSMC4 mic⫺/⫺ heterokaryons did not produce viable progeny.
strains showed defects during macronuclear division. The cell division cleavage furrow cut into the macronucleus before segregation of DNA was complete, and many cells had extranuclear DNA bodies (data not shown). These “extrusion bodies” resembled those reported for mutants of a class I histone deacetylase (49). Micronuclear mitosis appeared normal (data not shown). Since we were unable to obtain a complete somatic knockout cell population, the protein is likely essential for vegetative growth. To further study the function of TtSmc4p, we constructed germ line knockout heterokaryon strains. In such strains, although the micronucleus is a homozygous knockout, the macronucleus remains wild type; thus, the cells are phenotypically wild type during vegetative growth. However, because the wildtype macronucleus is degraded during conjugation and replaced with a new macronucleus derived from a mitotic sister of the zygotic micronucleus, mating of these heterokaryons produces progeny that do not contain the TtSMC4 gene in either nucleus but may have residual TtSmc4p protein from the parental cells. The HA tagging experiment described above suggests that most or all parental TtSmc4p would be found in the micronuclei of progeny. Mating a wild-type strain with an smc4-1::neo heterokaryon strain produces the same number of viable progeny as a control wild-type mating (Table 1). Thus, the mutant strain is able to mate normally and the resulting heterozygous progeny are viable, as expected for a recessive mutation. The knockdown experiments described above indicate that a 50% reduction in wild-type TtSMC4 gene copies may be deleterious to growth, but by the process of phenotypic assortment, many of the heterozygous progeny produced by this mating probably assort rapidly toward a higher proportion of wild-type gene copies. In the mating of two knockout heterokaryons, cytological observation showed that progression through conjugation was identical in timing and appearance to mating between two wild-type strains. Localization of Pdd1p, which is required for DNA rearrangement, was also identical to that in the wild type (data not shown). To determine whether genomic rearrangement occurred normally, single conjugating pairs were isolated and allowed to progress until pair separation had occurred and conjugation would normally be complete. The separated cells of each pair were then reisolated for analyses: completion of conjugation was confirmed by cytological staining in one cell, while single-cell PCR was performed on the other cell with primers spanning the M deletion element (12). Deletion of the M element occurred normally in these cells (data not shown). However, despite apparently successful completion of conju-
gation, mating pairs did not produce viable smc4-1::neo mutant progeny (Table 1). Therefore, we conclude that TtSMC4 is essential for vegetative growth in Tetrahymena. TtSMC4 is necessary for amitotic division of the macronucleus. We observed the first few cell divisions after conjugation of heterokaryons to determine what defects were responsible for the inviability of the smc4-1::neo progeny. At the end of conjugation (24 h after mixing of cells), pairs normally separate to form two exconjugants. The percentage of mutant exconjugants with two new macronuclei and one micronucleus per cell (the normal configuration of an exconjugant) was the same as in wild-type exconjugants (86% for the wild type versus 88% for heterokaryons; n ⫽ 100). Therefore, the TtSMC4 homozygous germ line knockouts did not arrest at the two-macronucleus–two-micronucleus stage during conjugation, as do other mutants defective in new macronucleus formation (12, 30). After feeding, exconjugants divide to produce progeny cells, or karyonides. This division of exconjugants requires mitosis of the micronucleus but not macronuclear division. The division of karyonides is the first division after conjugation that requires amitosis. As a control, we followed 44 wild-type pairs; 14% remained paired (did not successfully finish conjugation), while 86% produced at least 40 cells by 24 h, suggesting that the progeny had completed four or more divisions. In parallel, we followed 57 smc4::neo mutant pairs to determine how many cell divisions the progeny were able to complete (Fig. 5). After
FIG. 5. Progeny of smc4-1::neo heterokaryons do not divide more than twice. By 24 h in growth medium, wild-type progeny (black bars) have divided more than five times to produce 40 or more cells. For knockout heterokaryon pairs (gray bars), ⬃28% remained paired; ⬃21% had one exconjugant divide once to produce three cells; in 26% of the pairs, both exconjugants divided to produce four karyonides; in 25% of the pairs, both exconjugants divided once or twice to produce between five and eight cells.
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24 h in growth medium, 28% of the pairs remained paired and 21% of the pairs separated and had one exconjugant divide once to produce three cells, while 26% had both exconjugants divide once, producing four karyonides. The remaining 25% of the pairs produced four karyonides, some of which divided another time before ceasing to divide (thus producing five to eight cells). In summary, none of the knockout progeny was able to divide more than twice. To follow division of the progeny, cells were examined by immunofluorescence with antibodies against the micronucleusspecific linker histone (1). The smc4-1::neo mutant karyonides underwent micronuclear division (mitosis) normally, possibly because of the presence of residual parental TtSmc4p in the micronucleus (Fig. 4). Although mitosis was completed normally, there was no apparent macronuclear division. The macronuclear DNA remained on one side of the cleavage furrow during cell division. This unequal segregation did not appear to be the result of a failure of the macronucleus to migrate to the center of the cell, as has been hypothesized to account for unequal macronuclear segregation in a paclitaxel-hypersensitive -tubulin mutant (41). In addition, the macronucleus of the knockout cells was not directly cut by the cleavage furrow, as was observed in the 50% knockdown cells, as noted above. The cells continued through cytokinesis, producing one cell apparently lacking macronuclear DNA, as judged by DAPI staining. By 10 h after being returned to growth medium, 15% of the cells had no DAPI-detectable macronuclear DNA (n ⫽ 100) versus 0% for wild-type progeny (n ⫽ 100; Fig. 6). Thus, smc4-1::neo mutant progeny are able to successfully complete mitosis and cytokinesis but fail to properly divide the macronucleus. TtSMC4 is not necessary for nucleolar segregation in Tetrahymena. Studies of yeast, in which the nucleolus remains intact throughout cell division, have shown that the condensin complex is necessary for proper nucleolar segregation (16, 27). There are approximately 90 free nucleoli in Tetrahymena, located around the periphery of the macronucleus. Each nucleolus contains ⬃100 copies of the rRNA genes. The rRNA genes are found on free linear molecules that are not physically linked to the rest of the macronuclear chromatin. To determine if nucleolar distribution or segregation was disturbed in the smc4-1::neo mutant cells, we performed an immunofluorescence assay with antibodies against nucleolar protein Nopp52, which has been previously shown to localize to nucleoli and bind to the rRNA gene (32). Nucleoli are distributed around the periphery of the macronucleus in wildtype cells (Fig. 7A). In the portion of smc4-1::neo mutant cells that lack any DAPI-detectable macronuclear DNA, Nopp52 staining is nonetheless found near the micronucleus (Fig. 7B). This association of nucleoli with the micronucleus is similar to the normal positioning of the micronucleus in an indentation on the macronucleus during interphase in wild-type cells. FISH of the rRNA gene confirmed that the rRNA gene segregates along with the nucleoli in smc4-1::neo mutant cells, as in wildtype cells (Fig. 7C and D). Electron microscopic examination of wild-type cells (Fig. 8A) shows the electron-dense micronucleus and, within the macronucleus, peripheral nucleoli and dispersed heterochromatic chromatin bodies, which have been shown to be enriched in a homolog of heterochromatin protein 1 (23, 24). In comparison, smc4-1::neo
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FIG. 6. Macronuclear DNA does not segregate during cell division of smc4-1::neo mutant cells. (A) In dividing wild-type cells, the macronucleus begins to elongate after the micronucleus has completed mitosis and the two mitotic products have moved to opposite poles of the cell. The arrow points to the cytokinetic furrow of a dividing cell. All cells in the population have macronuclear DNA. (B) smc4-1::neo mutant cells fail to segregate macronuclear DNA during cell division, resulting in some cells with DNA only in the micronucleus (arrowhead). Mitosis is completed as in wild-type cells, but the macronuclear DNA does not elongate or move past the cleavage furrow (arrow). Cytokinesis is completed, producing one cell without DAPI-detectable macronuclear DNA. Micronucleus-specific linker histone (MLH), green; DNA, DAPI, blue.
mutant cells (Fig. 8B) contain a cluster of nucleoli adjacent to the micronucleus but very few chromatin bodies. Although the nuclear envelope is difficult to discern in both wild-type and mutant cells, the clustering of nucleoli in the mutant cells suggests that they are likely to be physically contained within the nuclear envelope of a greatly reduced macronucleus. These results indicate that the smc4-1::neo mutant cells did not completely fail to divide the macronucleus. Nucleoli and the rRNA gene segregate to both daughter cells, but the bulk of the macronuclear DNA segregates to only one. The organization of intramacronuclear microtubules is disrupted during macronuclear division of smc4-1::neo mutant cells. In addition to failure of nucleolar segregation, spindle defects have also been reported for condensin mutants (3). Although a spindle does not form during macronuclear division, microtubules are involved in amitosis. Microtubule inhibitors disrupt macronuclear division in both Tetrahymena pyriformis (26, 50) and T. thermophila (26). It was reported (26) that, in the presence of colchicine, about one-third of T. thermophila cell divisions resulted in one amacronucleate daugh-
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FIG. 7. smc4-1::neo mutant cells segregate nucleoli without bulk DNA segregation. (A) An immunofluorescence assay with antibodies against nucleolar protein Nopp52 shows progeny of a wild-type mating have a normal macronucleus with nucleoli at the periphery. No nucleoli are present in the transcriptionally inactive micronucleus. (B) smc4-1::neo mutant progeny do not have obvious DNA within the macronucleus but still have nucleolar staining. FISH of the rRNA gene confirms that it is segregated to the daughter cells during division of wild-type (C) or smc4-1::neo mutant (D) progeny. nucleolar protein Nopp52, red; rRNA gene, green; DNA, DAPI, blue.
ter. In these previous studies, it was not determined whether the nucleoli segregated without the chromatin. We investigated the organization of microtubules in dividing smc4-1::neo mutant cells. In wild-type cells, macronuclear division begins with microtubule nucleation (34), which is marked by the diffuse appearance of ␣-tubulin within the macronucleus. Nucleation is followed by elongation of microtubules within the macronucleus and from the nucleus to the cell
cortex. At cytokinesis, a bundle of microtubules is seen at the nuclear constriction (Fig. 9C) and nucleoli and macronuclear DNA have segregated to the daughter cell, with the nucleoli remaining around the periphery of the macronucleus (Fig. 9A and B). In dividing smc4-1::neo mutant cells, the nucleoli are pulled into the two daughter cells while the majority of macronuclear DNA remains on one side of the cleavage furrow (Fig. 9D). In these cells, macronuclear microtubules do not
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that the failure of macronuclear DNA segregation in null mutants may occur after microtubule nucleation and that condensin may aid in intranuclear microtubule stability or attachment, even in the absence of centromeres. DISCUSSION
FIG. 8. Fine structure of smc4-1::neo mutant cells after unequal nuclear division. An electron micrograph of a wild-type cell (A) shows peripheral nucleoli (black arrowheads) and numerous heterochromatic chromatin bodies (white arrowheads) spread throughout the macronucleoplasm. The micronucleus is not visible in this section. In smc4-1::neo mutant cells (B), nucleoli cluster around the micronucleus (MIC) but few chromatin bodies are present.
pass the stage of nucleation, even after the cleavage furrow has begun to form (Fig. 9E and F). Cytoplasmic microtubules are visible in the mutant cells in the vicinity of the segregating nucleoli (Fig. 9D and E, yellow arrows). These results suggest
In studying a homolog of SMC4, we have found surprising roles for condensin in Tetrahymena amitosis. We first found that tagged TtSmc4p localized to both the macronucleus and the micronucleus during vegetative growth and that cells lacking TtSMC4 are not viable, with an apparent macronuclear segregation defect. Unlike the mitotic missegregation seen in condensin-depleted cells of other species, the TtSMC4-null cells did not segregate the bulk of their DNA during amitosis. We found that the nucleoli segregate into the daughter cells even without bulk DNA segregation. This extreme phenotype is likely due, at least in part, to the lack of microtubule elongation inside the macronucleus during amitosis, in agreement with earlier studies showing highly unequal macronuclear division in T. thermophila cells dividing in the presence of the microtubule-destabilizing drug colchicine (26). These results reveal a role for condensin in bulk chromatin segregation, possibly through microtubule interactions, that is independent of the hallmarks of mitosis: chromatin condensation, equal segregation mediated by a spindle, and phosphorylation of histone H3. In contrast, the role of TtSMC4 in mitotic division of the micronucleus is unclear. Following conjugation, TtSmc4p shows a preferential localization or stability in the micronucleus, and therefore no conclusions can be drawn from the knockout phenotype regarding whether its function is essential for mitosis. In the somatic knockdown experiment, micronuclear mitosis appeared normal but it is possible that, under these conditions as well, the protein remained at functional levels of abundance in micronuclei, although not in macronuclei. The TtSMC4 knockout mutant shows a remarkable phenotype of nucleolar segregation without bulk DNA segregation, indicating the independence of these processes, at least in amitosis. The unique properties of nucleoli have led to special adaptations for their segregation in other eukaryotes (43). In vertebrates, nucleoli disassemble at each mitosis, whereas in budding yeast cells, which divide much more rapidly, a special mechanism, involving condensin, allows nucleolar segregation without disassembly. Such a role for condensin has apparently not evolved (or been maintained) in Tetrahymena because of its unusual rRNA gene structure and amitotic nuclear division. Segregation of nucleoli in Tetrahymena may occur primarily through their association with the nuclear envelope. The question of what role condensin has in amitosis is an intriguing one. The macronucleus does not have discernible chromosome condensation at any time in the cell cycle. However, the sensitive FISH methods required to detect chromosome condensation in Saccharomyces cerevisiae (19), which has chromosomes similar in size to Tetrahymena macronuclear chromosomes, are difficult to apply to the polyploid macronucleus of Tetrahymena. Although a modest reduction in mitotic chromosome condensation is detectable in S. cerevisiae condensin mutants (44), the resolution of comparable FISH meth-
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FIG. 9. Intranuclear microtubules are disrupted inside the dividing macronuclei of smc4-1::neo mutant cells, although cytoplasmic microtubules are seen in the area of nucleolar segregation. (A and B) In a dividing wild-type cell, the micronucleus has completed mitosis and the macronuclear DNA and nucleoli are segregating equally to the daughter cells. Microtubules are visible within the dividing macronucleus. (C) Enlargement of the macronucleus. The arrowheads in panels B and C point to the same position, in the constriction of the macronucleus. (D and E) In smc4-1::neo mutant cells, the micronucleus has completed mitosis and nucleoli are segregating to the daughter cells but the macronuclear DNA remains to one side of the cleavage furrow. Only cytoplasmic microtubules (e.g., yellow arrows) are visible; the ␣-tubulin inside the macronucleus remains diffuse. Apparent differences in cortical microtubule arrangement between the wild type and mutant are only the result of the confocal microscopy plane of focus (F) enlargement of the macronucleus. The oral apparatus (OA) is near the center of the dividing cell in panel E. ␣-Tubulin, green; nucleolar protein Nopp52, red; DNA, DAPI, blue.
ods has thus far been insufficient to detect a difference between the wild type and smc4::neo mutants (our unpublished results). Thus, it remains possible that condensation of macronuclear chromosomes does occur and is essential for their proper segregation, perhaps because coupling of condensation with topoisomerase II activity is necessary to untangle the mass of chromatin (19). Genetic evidence (14) indicates that there is no chromosome pairing or equal segregation after macronuclear DNA replication. The chromosome fragments do not have centromeres (7), and our study shows that condensin is not required for nucleolar segregation. These facts exclude other roles attributed to condensin in various organisms, such as release of sister chromatid cohesion, kinetochore organization, and nucleolar segregation (2–4, 8, 11, 13, 16, 20, 28, 35, 38). Yet TtSmc4p is necessary for bulk chromatin segregation during amitosis, a phenotype that has not been seen in other organisms. Condensin mutants of budding yeast and fission yeast fail to segregate the nucleolus during mitosis but continue to segregate the bulk of the chromatin (16, 27). Metazoan condensin mutants show
defects in chromosome segregation but also still segregate the bulk of the chromatin (4, 20, 25). Thus, our study reveals a new role for condensin in segregating chromosomes that lack centromeres. There are two other known roles of condensin that should be considered with regard to macronuclear division. Firstly, condensin has been linked to DNA repair in fungi (9). We did not investigate the possibility that DNA repair was affected in the TtSMC4 mutants; however, the study of RAD51 in Tetrahymena has shown that a disruption in DNA repair does not result in a complete block in macronuclear DNA segregation. A portion of the macronuclear DNA is cut by the cleavage furrow, but the majority of the DNA does segregate (31). Thus, the SMC4 mutant phenotype observed here is not likely due to a defect in DNA repair. Secondly, condensin plays a role in maintaining the structure of the chromatin scaffold (25). Disruption of the Tetrahymena chromatin scaffold, which may be essential for microtubule attachment, could be partly responsible for the phenotype of the TtSMC4 mutants.
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Although we do not know whether condensin interacts directly with a microtubule binding protein, it is clear that condensin is required for proper microtubule organization during amitosis. The TtSMC4 mutation appears to specifically disrupt intranuclear microtubule elongation and attachment, while cytoplasmic microtubules are still able to form. The mechanism of amitosis may be viewed from the perspective of a closed mitosis, where astral and spindle microtubules coordinate the division of the nucleus through pulling and pushing forces on the nuclear envelope (for a review, see reference 47). In Tetrahymena, cytoplasmic microtubules appear to connect the nuclear envelope and the cell cortex during cell division, possibly pulling on the nuclear envelope in a manner resembling the action of astral microtubules. Intranuclear microtubules are nucleated within or around the chromatin, eventually reaching from the chromatin to the nuclear envelope, pushing the nuclear envelope in a manner resembling the action of spindle microtubules (17, 40). In TtSMC4 mutants, ␣-tubulin spots are seen throughout the macronuclear chromatin rather than at the periphery, supporting the idea that these microtubules are nucleated within the macronuclear chromatin. The lack of microtubule elongation in these mutants suggests that microtubules may not attach to chromatin, leading to depolymerization of microtubules and failure of DNA segregation while still allowing cytoplasmic microtubules to exert pulling forces on the nuclear envelope. It is possible that TtSMC4 is required for the recruitment of proteins that attach intranuclear microtubules even in the absence of a centromere and kinetochore. Identifying the proteins interacting with condensin in the macronucleus may help us identify functions of condensin related to microtubule organization during amitosis. It should be noted, however, that earlier studies of T. thermophila exposed to colchicine (26), while documenting frequent highly unequal macronuclear division, also showed the capacity of most macronuclei to divide (without being cut by the cytokinetic furrow) in the complete absence of intranuclear microtubules. Perhaps the imbalance of intranuclear and cytoplasmic microtubules in the TtSMC4 knockout cells leads to their extreme phenotype. In addition, if severe inequality of division were the major phenotypic consequence of the TtSMC4 knockout, we might expect the daughter cell that had received the bulk of the chromatin to continue dividing, which it generally does not do. This suggests the possibility of another defect in the knockout cells, perhaps involving the triggering of a cell division checkpoint. Within the Tetrahymena genome sequence, we have identified matches to the condensin subunits found in Drosophila. Only one homolog each of SMC2, SMC4, and XCAP-G was identified, although two copies of the non-SMC condensin subunits XCAP-D2 and XCAP-H were identified, suggesting that, like certain higher eukaryotes (35), Tetrahymena may have two condensin complexes containing different non-SMC subunits. TtSmc4p would presumably be common to both complexes, and disruption of TtSMC4 would disrupt the function of both. It will be interesting to determine if there are indeed two condensin complexes functioning in Tetrahymena and if they are distributed differently between the two nuclei. Our studies of TtSMC4 have revealed unexpected mutant phenotypes, as well as surprising features of nuclear division.
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By studying conserved members of mitotic complexes in amitosis, we can uncover new roles for these proteins and elucidate the mechanism of acentric chromatin segregation. ACKNOWLEDGMENTS We are grateful to Rachel Howard-Till, Harmit Malik, Hisashi Tanaka, and the anonymous reviewers for critical review of the manuscript, which led to many improvements; to the Yao lab and Gerald Smith’s lab for helpful discussions; to Nancy Schult for assistance in cloning and sequencing TtSMC4; to Peter Bruns for providing lab facilities and encouragement to R.S.C.; to Elizabeth Greene and Jonathan Badger for assistance with protein sequence and phylogenetic analysis; and to the ciliate community for support. We thank Kathy Collins, Emily Wiley, and David Allis for antibodies. Preliminary sequence data for the T. thermophila scaffolds were obtained from The Institute for Genomic Research. This research was supported by National Institutes of Health grant GM26210 (to M.C.Y.), National Science Foundation grant MCB-0096270 (to R.S.C.), and National Institutes of Health training grant fellowship T32HD007183-26 (to M.D.C.). REFERENCES 1. Allis, C. D., C. V. Glover, J. K. Bowen, and M. A. Gorovsky. 1980. Histone variants specific to the transcriptionally active, amitotically dividing macronucleus of the unicellular eucaryote, Tetrahymena thermophila. Cell 20:609– 617. 2. Aono, N., T. Sutani, T. Tomonaga, S. Mochida, and M. Yanagida. 2002. Cnd2 has dual roles in mitotic condensation and interphase. Nature 417: 197–202. 3. Bhalla, N., S. Biggins, and A. W. Murray. 2002. Mutation of YCS4, a budding yeast condensin subunit, affects mitotic and nonmitotic chromosome behavior. Mol. Biol. Cell 13:632–645. 4. Bhat, M. A., A. V. Philp, D. M. Glover, and H. J. Bellen. 1996. Chromatid segregation at anaphase requires the barren product, a novel chromosomeassociated protein that interacts with topoisomerase II. Cell 87:1103–1114. 5. Bruns, P. J., and D. Cassidy-Hanley. 2000. Biolistic transformation of macroand micronuclei. Methods Cell Biol. 62:501–512. 6. Bruns, P. J., A. L. Katzen, L. Martin, and E. H. Blackburn. 1985. A drugresistant mutation in the ribosomal DNA of Tetrahymena. Proc. Natl. Acad. Sci. USA 82:2844–2846. 7. Cervantes, M. D., X. Xi, D. Vermaak, M. C. Yao, and H. S. Malik. 2006. The CNA1 histone of the ciliate Tetrahymena thermophila is essential for chromosome segregation in the germline micronucleus. Mol. Biol. Cell 17:485– 497. 8. Chan, R. C., A. F. Severson, and B. J. Meyer. 2004. Condensin restructures chromosomes in preparation for meiotic divisions. J. Cell Biol. 167:613–625. 9. Chen, E. S., T. Sutani, and M. Yanagida. 2004. Cti1/C1D interacts with condensin SMC hinge and supports the DNA repair function of condensin. Proc. Natl. Acad. Sci. USA 101:8078–8083. 10. Cobbe, N., and M. M. Heck. 2004. The evolution of SMC proteins: phylogenetic analysis and structural implications. Mol. Biol. Evol. 21:332–347. 11. Coelho, P. A., J. Queiroz-Machado, and C. E. Sunkel. 2003. Condensindependent localisation of topoisomerase II to an axial chromosomal structure is required for sister chromatid resolution during mitosis. J. Cell Sci. 116:4763–4776. 12. Coyne, R. S., M. A. Nikiforov, J. F. Smothers, C. D. Allis, and M. C. Yao. 1999. Parental expression of the chromodomain protein Pdd1p is required for completion of programmed DNA elimination and nuclear differentiation. Mol. Cell 4:865–872. 13. D’Amours, D., F. Stegmeier, and A. Amon. 2004. Cdc14 and condensin control the dissolution of cohesin-independent chromosome linkages at repeated DNA. Cell 117:455–469. 14. Doerder, F. P., J. C. Deak, and J. H. Lief. 1992. Rate of phenotypic assortment in Tetrahymena thermophila. Dev. Genet. 13:126–132. 15. Flickinger, C. J. 1965. The fine structure of the nuclei of Tetrahymena pyriformis throughout the cell cycle. J. Cell Biol. 27:519–529. 16. Freeman, L., L. Aragon-Alcaide, and A. Strunnikov. 2000. The condensin complex governs chromosome condensation and mitotic transmission of rDNA. J. Cell Biol. 149:811–824. 17. Fujiu, K., and O. Numata. 2000. Reorganization of microtubules in the amitotically dividing macronucleus of tetrahymena. Cell Motil. Cytoskelet. 46:17–27. 18. Gaertig, J., L. Gu, B. Hai, and M. A. Gorovsky. 1994. High frequency vector-mediated transformation and gene replacement in Tetrahymena. Nucleic Acids Res. 22:5391–5398. 19. Guacci, V., E. Hogan, and D. Koshland. 1994. Chromosome condensation and sister chromatid pairing in budding yeast. J. Cell Biol. 125:517–530.
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