JOURNAL OF BACTERIOLOGY,0021-9193/01/$04.0010 DOI: 10.1128/JB.183.7.2372–2375.2001

Apr. 2001, p. 2372–2375 Vol. 183, No. 7

Copyright © 2001, American Society for Microbiology. All Rights Reserved.

Conservative Duplication of Spindle Poles during Meiosisin Saccharomyces cerevisiae

ANDREAS WESP,1 SUSANNE PRINZ,2 AND GERALD R. FINK1,3*

Whitehead Institute for Biomedical Research,1 Center for Cancer Research,2 and Department of Biology,3

Massachusetts Institute of Technology, Cambridge, Massachusetts 02142

Received 21 August 2000/Accepted 11 January 2001

During sporulation in diploid Saccharomyces cerevisiae, spindle pole bodies acquire the so-called meioticplaque, a prerequisite for spore formation. Mpc70p is a component of the meiotic plaque and is thus essentialfor spore formation. We show here that MPC70/mpc70 heterozygous strains most often produce two sporesinstead of four and that these spores are always nonsisters. In wild-type strains, Mpc70p localizes to all fourspindle pole bodies, whereas in MPC70/mpc70 strains Mpc70p localizes to only two of the four spindle polebodies, and these are always nonsisters. Our data can be explained by conservative spindle pole body distributionin which the two newly synthesized meiosis II spindle pole bodies of MPC70/mpc70 strains lack Mpc70p.

In the absence of nitrogen and in the presence of a nonfer-mentable carbon source, Saccharomyces cerevisiae cells of theMATa/MATa constitution undergo meiosis, a process that re-sults in the formation of four haploid spores. In baker’s yeast,both meiotic divisions occur within a single continuous nuclearenvelope. Spore formation begins late during the second mei-otic division (meiosis II). At that stage, the nuclear envelopeassumes a four-lobed structure. One meiosis II spindle polebody is located at each tip of the four lobes and one haploidgenome equivalent is segregated into each of the lobes (9). Theso-called prospore membrane forms on the cytoplasmic side ofeach spindle pole body, extends like a pouch around the lobesof each nucleus, and eventually fuses with itself to enclose ahaploid nucleus (5, 6, 9, 10).

In S. cerevisiae, the spindle pole bodies are functionally equiv-alent to centrosomes in higher eukaryotes. In mitotic cells, thespindle pole bodies coordinate the segregation of chromosomesand nuclear migration through interaction with intra- and ex-tranuclear microtubules, respectively (4, 13, 14). Meiotic spindlepole bodies nucleate the formation of spores in addition to coor-dinating the segregation of chromosomes during both meioticdivisions. This sporulation-specific function of spindle pole bodiesis executed during the second meiotic division. Ultrastructuralstudies have revealed a sporulation-specific modification of spin-dle pole bodies late in meiosis: they acquire the so-called meioticplaque on their cytoplasmic side. This modification of spindlepole bodies is a key prerequisite for spore formation (2, 9). Mu-tations that impair the formation of meiotic plaques result in theabsence of prospore membranes on the respective spindle polebodies (5, 11, 16). Similarly, the formation of two-spored (insteadof four-spored) asci in diploid wild-type cells is a direct conse-quence of the failure to synthesize meiotic plaques on all fourspindle pole bodies (2).

Mpc70p is a meiotic plaque protein and is therefore essen-tial for spore formation (5). We show here that MPC70/mpc70

heterozygotes most often produce two spores instead of fourand that these are always nonsisters. The distribution ofMpc70p to only two of the four spindle pole bodies in MPC70/mpc70 heterozygotes suggests that Mpc70p is present in limit-ing amounts and is distributed to the spindle pole bodies by aconservative mechanism.

Dosage of MPC70 is critical for the efficiency of spore for-mation. Strains used in this study are listed in Table 1. Whena culture of wild-type cells is transferred from rich medium(2% glucose [12]) to sporulation medium (1% potassium ace-tate, 0.02% raffinose), the final sporulation products are, asexpected, primarily tetrads. However, about one-third of theterminal sporulation products in a wild-type cell populationconsist of triads and dyads (Fig. 1A) despite the fact that themajority of cells display a tetranucleate staining pattern duringspore formation (Fig. 1B). To visualize DNA in a sporulatingculture, ethanol-fixed cells were incubated with DAPI (49,69-diamidino-2-phenylindole) (Sigma, St. Louis, Mo.) at a con-centration of 0.4 mg/liter and then viewed with a Nikon TE300inverted microscope equipped with Openlab software. Pictureswere captured using a Hamamatsu digital camera (C4742–95).We dissected triads and dyads of a wild-type culture and foundthat the spores gave rise to haploid progeny and that auxotro-phic markers segregated as expected for a diploid undergoingmeiosis. These data suggest that the triads and dyads found inwild-type culture result from a failure to form spores eventhough the meiotic divisions were typically complete.

mpc70/mpc70 mutants complete the meiotic divisions, butno spores are formed (15; our unpublished observations). Aculture of the MPC70/mpc70 heterozygote, however, routinelyyielded more than 70% of the terminal sporulation products asdyads (Fig. 1A), corresponding to a fivefold increase in dyadformation compared to wild-type cells. The increased forma-tion of dyads was not a consequence of failed meiotic divisions,because the vast majority of MPC70/mpc70 cells displayed atetranucleate staining pattern during spore formation (Fig.1B). Consistent with this, spores from dyads and triads of aculture of MPC70/mpc70 cells gave rise to haploid progeny,and the auxotrophic markers in the cross segregated as ex-pected for a diploid undergoing meiosis. Spores recovered

* Corresponding author. Mailing address: Whitehead Institute forBiomedical Research, Nine Cambridge Center, Cambridge, MA02142-1479. Phone: (617) 258-5215. Fax: (617) 258-9872. E-mail: fink@wi.mit.edu.

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from dyads of MPC70/mpc70 cultures contained either wild-type MPC70 or the mutant mpc70 allele. Thus, MPC70 ishaploinsufficient for tetrad formation but does not act in aspore-autonomous way.

Using the centromere-linked trp1 auxotrophic marker, wefound that dyads from both wild-type and MPC70/mpc70 cul-tures consisted of nonsister spores (one TRP1 and one trp1).

Chi-square analysis was performed with the null hypothesisbeing that dyads were generated randomly (two-thirds shouldbe nonsister dyads and one-third should be sister dyads). Theresulting P value of less than 0.01 indicates that dyads gener-ated in wild-type and MPC70/mpc70 heterozygotes are mostlikely not the result of random failure of spore formationamong four nuclei in any given ascus. Together, our resultsindicate that Mpc70p is a limiting component during sporeformation and that reduced levels of MPC70 result in an in-creased frequency of nonsister dyad formation. However, wefound that the introduction of additional copies of MPC70 in awild-type strain did not increase the efficiency of tetrad forma-tion. Therefore, MPC70 is necessary but not sufficient for theefficient packaging of all four spores.

Mpc70p is distributed to the spindle pole bodies by a con-servative mechanism. To determine when in the sequence ofmeiotic divisions Mpc70p is recruited to spindle pole bodies,we localized both Mpc70p and Tub4p (gamma tubulin) insporulating wild-type strains. Colocalization would signal re-cruitment of Mpc70p to the spindle pole bodies because yeastgamma tubulin is an intrinsic component of this organelle (7,15). Diploids with one genomic copy of wild-type MPC70 re-placed by an MPC70-HA allele, where a triple hemagglutininantigen (HA) tag was fused in-frame 249 nucleotides down-stream of the start codon of MPC70, were sporulated andprocessed for immunofluorescence (17). Anti-gamma tubulin(Tub4p [3]) and anti-Mpc70p (HA) antibodies were added andvisualized using rhodamine- and fluorescein isothiocyanate(FITC)-coupled secondary antibodies (Jackson ImmunoRe-search Laboratories, Inc., West Grove, Pa.), respectively. Nocross-reactivities of the secondary antibodies were observed,and no FITC signal was detected in cells lacking theMPC70-HA construct. DNA was visualized using DAPI con-tained within the mounting medium (Vectashield, Burlingame,Calif.).

Prior to the first meiotic division, we detected Tub4p but notMpc70p on the spindle pole bodies. This is consistent with theabsence of MPC70 mRNA early in meiosis (1). As the spindlepole bodies duplicated and the meiosis I spindles formed, wefrequently detected mononucleate and binucleate cells withMpc70p on both meiosis I spindle pole bodies. As meiosisproceeded, Mpc70p was associated with all spindle pole bodiesof binucleate cells both before and after the two meiosis Ispindle pole bodies duplicated (Fig. 2A). After completion ofthe second meiotic division (as indicated by tetranucleatecells), Mpc70p remained associated with all four spindle polebodies (Fig. 2A). Thus, in the majority of wild-type cells,Mpc70p localized to all spindle pole bodies throughout bothmeiotic divisions.

FIG. 1. The level of MPC70 is critical for the efficiency of sporeformation. Wild-type (1/1) and MPC70/mpc70 (1/2) cultures weresubjected to sporulation for 32 h. (A) The numbers of two-, three-, andfour-spored terminal sporulation products were determined micro-scopically and expressed as percentages of the total (sum of dyads,triads, and tetrads). At least 540 individual terminal products wereconsidered. Wild-type cells mainly produced tetrads as terminal sporu-lation products, while MPC70/mpc70 cells mainly yielded dyads. (B)DAPI staining of wild-type (MPC70/MPC70) and heterozygous(MPC70/mpc70) mutants subjected to sporulation medium for 7 h. Themajority of cells in both cultures displayed a tetranucleate stainingpattern. Bar, 10 mm.

TABLE 1. Strains used in this study

Strain Genotype

WAY627 ..........................ura3/ura3 leu2::hisG/LEU2 his3::hisG/his3::hisG TRP1/trp1::hisG lys2/lys2 ho::LYS2/ho::LYS2WAY622 ..........................MATa ura3 leu2::hisG his3::hisG lys2 ho::LYS2WAY690 ..........................ura3/ura3 leu2::hisG/LEU2 his3::hisG/his3::hisG TRP1/trp1::hisG lys2/lys2 ho::LYS2/ho::LYS2 mpc70::his5/mpc70::his5WAY683 ..........................ura3/ura3 leu2::hisG/LEU2 his3::hisG/his3::hisG TRP1/trp1::hisG lys2/lys2 ho::LYS2/ho::LYS2 mpc70::his5/MPC70WAY1022 ........................ura3/ura3 leu2::hisG/LEU2 his3::hisG/his3::hisG TRP1/trp1::hisG lys2/lys2 ho::LYS2/ho::LYS2 MPC70:3HA/mpc70::his5WAY1030 ........................ura3/ura3 leu2::hisG/LEU2 his3::hisG/his3::hisG TRP1/trp1::hisG lys2/lys2 ho::LYS2/ho::LYS2 MPC70:3HA/MPC70

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Since Mpc70p is essential for spore formation and a reducedlevel of MPC70 results in the formation of nonsister dyads (Fig.1), we determined the Mpc70p localization pattern in sporu-lating MPC70/mpc70 heterozygotes carrying an HA-tagged al-lele as the sole functional copy of MPC70. Cells were processedfor immunolocalization of Mpc70-HAp and Tub4p as de-scribed above. We found that Mpc70p localizes to both meiosisI spindle pole bodies in both mononucleate and binucleatecells. As meiosis proceeds, in the majority of MPC70/mpc70cells, unlike the situation in wild-type cells, Mpc70p is associ-ated with only two of the four meiosis II spindle pole bodies(Fig. 2B). Remarkably, the two Mpc70p-positive spindle polebodies were always associated with nonsister genomes, whichwas inferred from comparison of the spindle orientation withMpc70p staining (Fig. 2B). This observation is consistent withour genetic findings that a culture of MPC70/mpc70 heterozy-gous mutants predominantly forms nonsister dyads. Moreover,it demonstrates directly that only Mpc70p-positive spindle polebodies are competent for spore formation.

Mpc70p is limiting for spore formation. Although a popu-lation of wild-type cells yields primarily asci with four spores(tetrads) as the final sporulation product, dyads are alwaysrecovered from such cultures. Previous studies have shown thatgeneration of these dyads in wild-type culture is not the resultof random death of spores (2). Rather, the spores recoveredfrom those dyads always contained nonsister genomes. That is,only one haploid genome of each meiosis II spindle was recov-ered and only one spindle pole body of each of the meiosis IIspindles carried a meiotic plaque. On the basis of these obser-vations, it has been proposed that duplication of spindle polebodies occurs in a conservative manner (that is, preexistingspindle pole bodies serve as templates for the synthesis of newspindle pole bodies) and that certain proteins essential formeiotic plaque formation may be limiting in wild-type cells (2).

Mpc70p is a meiotic plaque protein (5). We show here thatMPC70/mpc70 strains that have only one of the two copies ofMPC70 yield primarily dyads and that those dyads are alwayscomposed of nonsister spores. Thus, the molecular basis fordyad formation in MPC70/mpc70 mutants is likely the samenonrandom process that yields dyads in wild-type culture: thepresence of only one meiotic plaque per meiosis II spindle.This conclusion is supported by the finding that in the majorityof MPC70/mpc70 heterozygotes, Mpc70p localized to only twoof the four meiosis II spindle pole bodies and, importantly,those spindle pole bodies were associated with nonsister ge-nomes. Thus, only Mpc70p-positive spindle pole bodies arecompetent for spore formation.

What is the function of Mpc70p on the spindle pole bodies?Mpc70p may provide a meiosis-specific scaffold for the assem-bly of other proteins on spindle pole bodies, which themselvesmay assist prospore membrane assembly. Consistent with this,Mpc70p, like other structural components of the spindle polebody, contains a coiled-coil domain that can engage in bothhomotypic and heterotypic interactions (5; our unpublishedobservations). Alternatively, as Mpc70p is a component of themeiotic plaques of spindle pole bodies, it may directly bindprospore membrane vesicles and thereby control prosporemembrane formation on spindle pole bodies.

The effects of mpc70 mutation differ from other parametersand mutations that alter the number of spores resulting from

meiosis. For example, nutrient availability and temperaturecan influence the ratio between tetrads, triads, and dyads (8).In addition, mutations can lead to the formation of only dyads,even though meiotic divisions are complete. For example, onesuch mutation (hfd1-1) produces dyads consisting of nonsisterspores (11) whereas other mutations (cyr1-1, spo3) mainly pro-duce dyads consisting of random spores (16). It is important torealize, however, that in those cases the dyads were formed ata high frequency only if the mutation was present in a homozy-gous configuration. This behavior is unlike that observed forMPC70, where nonsister dyads are formed preferentially incultures of MPC70/mpc70 heterozygous mutants and mpc70/mpc70 mutants fail to form spores altogether.

FIG. 2. Localization of Mpc70p to only one spindle pole body ofeach meiosis II spindle in MPC70/mpc70 mutants and colocalization ofMpc70p with Tub4p to spindle pole bodies during meiotic divisions.Cells were sporulated, fixed, and processed for immunofluorescence ofMpc70p and Tub4p. DNA was visualized with DAPI. (A) In wild-typecells, Mpc70p localizes to all spindle pole bodies of binucleate cellsboth before and after the duplication of meiosis I spindle pole bodies,as well as to all four spindle pole bodies of tetranucleate cells. (B) Inheterozygous MPC70/mpc70 mutants, however, Mpc70p localizes toonly two of the four spindle pole bodies. Importantly, the Mpc70p-positive spindle pole bodies are always associated with nonsister ge-nomes. Bar, 5 mm.

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The behavior of MPC70/mpc70 heterozygotes is consistentwith a conservative distribution of Mpc70p. The localization ofMpc70p in MPC70/MPC70 diploids and in MPC70/mpc70 dip-loids suggests that the sequence of conservative spindle polebody duplication and modification is fully sufficient to explainthe formation of dyads if Mpc70p is limiting (Fig. 3). Mpc70plocalized to spindle pole bodies only at a time when the meiosisI spindle had already formed. We later observed Mpc70p on allfour meiosis II spindle pole bodies in wild-type cells. Thissequence suggests that spindle pole body duplication andMpc70p-dependent plaque formation alternate during bothmeiotic divisions and that, under normal circumstances, newlysynthesized meiosis II spindle pole bodies without meioticplaques are short-lived. As a consequence, in wild-type cells, allfour spindle pole bodies carry meiotic plaques and thus tetradsare formed.

However, if a meiotic plaque protein such as Mpc70p be-comes limiting prior to (or at) the duplication of meiosis Ispindle pole bodies, cells end up with two newly synthesizedmeiosis II spindle pole bodies without meiotic plaques andwith two (parental) meiosis II spindle pole bodies carryingmeiotic plaques (Fig. 3). Our results suggest that this scenariooccurs in the majority of MPC70/mpc70 cells: Mpc70p is con-sumed for meiotic plaque formation on the meiosis I spindlepole bodies and therefore no Mpc70p is available for meioticplaque formation on the newly synthesized meiosis II spindlepole bodies. As a consequence, nonsister dyads are prevalentas terminal sporulation products.

This work was supported by NIH grant GM35010. G.R.F. is anAmerican Cancer Society Professor of Genetics. A.W. was supportedby grants from the Roche Research Foundation, the Novartis Fondszur Forderung der Wissenschaft, and the Schweizerischer National-fonds.

This work was conducted at the W. M. Keck Foundation BiologicalImaging Facility of the Whitehead Institute for Biomedical Research.

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FIG. 3. Model of alternate spindle pole body duplication and modification. The model reinforces the assumption that spindle pole bodiesduplicate in a conservative fashion during meiosis (2). That is, preexisting spindle pole bodies serve as templates for the synthesis of new spindlepole bodies. Early in meiosis, the sole spindle pole body duplicates to form the meiosis I spindle and Mpc70p is recruited to the two meiosis Ispindle pole bodies (A and B) to form meiotic plaques. Novel unmodified meiosis II spindle pole bodies (A9 and B9) without meiotic plaques arethen synthesized in a conservative way. In wild-type cells, Mpc70p is then recruited to the newly synthesized meiosis II spindle pole bodies (A9 andB9) to form the meiotic plaques. As a result, prospore membranes form on all four meiosis II spindle pole bodies. In MPC70/mpc70 mutants,however, all of the Mpc70p is consumed for meiotic plaque formation at the two meiosis I spindle pole bodies; therefore, no Mpc70p is left to berecruited to the newly synthesized meiosis II spindle pole bodies (A9 and B9). As a consequence, only one spindle pole body each from the twomeiosis II spindles is modified (A and B), and only nonsister dyads form. Open and filled circles represent spindle pole bodies without and withmeiotic plaques, respectively.

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