novel mad2 alleles isolated in a schizosaccharomyces pombe … · mad2 functions required for...

Copyright � 2007 by the Genetics Society of AmericaDOI: 10.1534/genetics.106.061309

Novel mad2 Alleles Isolated in a Schizosaccharomyces pombe g-Tubulin MutantAre Defective in Metaphase Arrest Activity, but Remain Functional for

Chromosome Stability in Unperturbed Mitosis

Yoshie Tange and Osami Niwa1

Kazusa DNA Research Institute, Kisarazu, Chiba 292-0818, Japan

Manuscript received May 25, 2006Accepted for publication January 18, 2007

ABSTRACT

A previously isolated fission yeast g-tubulin mutant containing apparently stabilized microtubulesproliferated at an approximately identical rate as wild type, yet the mutant mitosis spindle dynamics wereaberrant, particularly the kinetochore microtubule dynamics. Progression through mitosis in the mutant,however, resulted in mostly accurate chromosome segregation. In the absence of the spindle assemblycheckpoint gene, mad21, the spindle dynamics in the g-tubulin mutant were greatly compromised, leading toa high incidence of chromosome missegregation. Unlike in wild-type cells, green fluorescent protein (GFP)-tagged Mad2 protein often accumulated near one of the poles of an elongating spindle in the g-tubulinmutant. We isolated novel mad2 mutants that were defective in arresting mitotic progression upon grossperturbation of the spindle formation but remained functional for the viability of the g-tubulin mutant.Further, the mad2 mutations did not appreciably destabilize minichromosomes in unperturbed mitoses.When overexpressed ectopically, these mutant Mad2 proteins sequestered wild-type Mad2, preventing itsfunction in mitotic checkpoint arrest, but not in minichromosome stability. These results indicated that theMad2 functions required for checkpoint arrest and chromosome stability in unperturbed mitosis aregenetically discernible. Immunoprecipitation studies demonstrated that GFP-fused mutant Mad2 proteinsformed a Mad1-containing complex with altered stability compared to that formed with wild-type Mad2,providing clues to the novel mad2 mutant phenotype.

ACCURATE segregation of replicated chromosomesin mitosis is essential for cell proliferation. In all

eukaryotes, this process is dependent on the mitoticapparatus, the spindle, which is transiently formed in themitotic phase of the cell-division cycle. The spindle is amicrotubule (MT)-based bipolar structure primarilydevoted to chromosome segregation. A crucial event inchromosome segregation is sister-chromatid separation,which occurs at the onset of the mitotic anaphase, after allof the sister kinetochores are properly linked to theirrespective spindle poles in a bi-oriented manner. Theanaphase promoting complex (APC)-mediated ubiquiti-nation and degradation of securin, which trigger theactivation of separase protease and the subsequent dis-ruption of sister-chromatid cohesion, constitute keyevents in sister-chromatid separation. Sister chromosomesets are then separated by the combined mechanisms ofanaphase A and B, that is, the shrinkage of the pole-to-kinetochore MTs (kMTs) and the elongation of thepole-to-pole distance, respectively. Sister-chromatid sepa-ration occurs on individual chromosomes, but must betemporally and spatially regulated so that all chromo-

somes are simultaneously split into sister chromosomesand faithfully distributed into each of the two daughternuclei. The spindle assembly checkpoint (SAC) isthought tobeessential for thiscoordinatedchromosomebehavior in mitosis and to prevent aneuploidy (Cleve-

land et al. 2003; Lew and Burke 2003; Kwon andScholey 2004; Pinsky and Biggins 2005).

SAC genes, such as BUB, MAD, and MPS1, wereoriginally identified in budding-yeast mutants that weredefective in arresting in M-phase when the spindleassembly was impaired with MT poisons such as benomyl(Hoyt et al. 1991; Li and Murray 1991; Weissand Winey

1996). It was subsequently determined that the SACgenes are conserved among a wide range of species(Cleveland et al. 2003; Lew and Burke 2003; Pinsky andBiggins 2005), including Schizosaccharomyces pombe (He

et al. 1997, 1998; Bernard et al. 1998; Millband andHardwick 2002; Kim et al. 2003). SAC functions includemonitoring the lack of kMT attachment to the kineto-chore or the absence of tension at the kinetochore andblocking mitotic progression, primarily through produc-ing diffusible APC inhibitors. Even a single affected kinet-ochore can produce the inhibitors (Rieder et al. 1995;Kerscher et al. 2003), thereby allowing time for the aff-ected kinetochores to correct their linkage to kMTs andensuring coordinated chromosome segregation. Mad2

1Corresponding author: Kazusa DNA Research Institute, 2-6-7 Kazusa-kamatari, Kisarazu, Chiba 292-0818, Japan. E-mail: [email protected]

Genetics 175: 1571–1584 (April 2007)

is a key molecule in SAC that binds to Cdc20 (Slp1 infission yeast) to block APC activation. According to acurrently accepted theory (Musacchio and Hardwick

2002; De Antoni et al. 2005; Hardwick 2005; Yu 2006),Mad2 forms a complex with Mad1 and is recruited tokinetochores that are not occupied by kMTs; the boundcomplex then functions to activate free Mad2 to form theMad2–Cdc20 complex. This activation process is thoughtto include a specific interaction with Mad2 that requires asegment in Mad2 distinct from the C-terminal segmentinvolved in binding to both Mad1 and Cdc20. Thus, at thephysiological level of Mad2 expression, its activity isdependent on Mad1, and the major role of Mad1 inSAC is thought to be the activation of Mad2.

The fission yeast is useful for investigating the molec-ular mechanisms and regulation of mitosis. As in otherorganisms (Kwon and Scholey 2004), proper formationof the spindle in fission yeast requires factors that interactwith MTs. Among them are a kinesin family protein, Cut7[a homolog of BimC, kinesin-5, according to the standardnomenclature (Lawrence et al. 2004)], which is requiredfor the formation of the bipolar spindle and spindleelongation (Hagan and Yanagida 1990, 1992; Troxell

et al. 2001); Pkl1 and Klp2 (homologous to Kar3, kinesin-14), which are required for proper spindle length (Klp2probably has a role in antagonizing Cut7) (Pidoux et al.1996; Troxellet al. 2001); and Klp5 and Klp6 (belongingto the Kip3-KinI-family, kinesin-8), which are required forproper metaphase spindle formation and chromosomesegregation, probably through the regulation of kMTstability at the kinetochore (Garcia et al. 2002; West et al.2002). Nonkinesin-like proteins, including Dis1 and itshomolog Alp14/Mtc1, the TOG/XMAP215-family pro-teins that affect the MT stability and the kinetochore–kMT interaction (Nakaseko et al. 2001; Garcia et al.2002), Ase1 for MT bundling (Loiodice et al. 2005;Yamashita et al. 2005), and kinetochore and its interact-ing proteins (Cleveland et al. 2003; Lew and Burke 2003;Sanchez-Perez et al. 2005) are also implicated in spindledynamics.

We previously isolated a cold-sensitive g-tubulin mu-tant, gtb1-93, in fission yeast and demonstrated that it hasstrong genetic interactions with Pkl1, Klp5/6, and Dis1(Tange et al. 2004). At a restrictive temperature, themutant produced an abnormally elongated spindle thatfailed to properly segregate chromosomes (Paluh et al.2000). It contained apparently stabilized cytoplasmicMT bundles at both restrictive and permissive temper-atures. Although the mutant proliferated at permissivetemperatures at a rate nearly identical to that of wild type(Tangeet al. 2004), as we demonstrate in this study, closeexamination of the mutant mitoses revealed peculiarabnormalities. More specifically, the kMT dynamics inmetaphase through anaphase A appeared strikinglydifferent from that of wild-type mitosis. Despite the ab-normal mitosis, nuclear division ended with near-normalchromosome segregation. Near-normal mitosis in the

g-tubulin mutant required the SAC genes in varyingdegrees. By isolating new novel mutations in the mad2gene, we demonstrated that the function of Mad2 re-quired for the g-tubulin mutant mitosis were geneticallydiscernible from the canonical SAC function. Further-more, the new mad2 mutants, unlike a complete loss-of-function mutation of the mad2 gene, did not appreciablydestabilize chromosome stability in unperturbed mitosis.

MATERIALS AND METHODS

Strains and general genetic methods: Yeast strains used arelisted in supplemental Table 1 at http://www.genetics.org/supplemental/. Strains carrying disrupted mad2 (mad2D) andmad1D, both by ura41 insertion, as well as a strain with the mad21-GFP, were obtained from T. Matsumoto (Ikui et al. 2002). Strainswith sid41-GFP were described in Tangeet al. (2004). The greenfluorescent protein (GFP)-tagged atb21 gene expressed underthe control of the nda2 promoter at the lys1 locus was kindlyprovided by H. Masuda (for construction, see Masuda et al.2006). GFP-tagged cdc131 was obtained from M. Yanagida via theYeast Genetic Resource Center. Gene disruption with the G-418-resistant gene was performed according to Bahler et al. (1998).YE containing G-418 (Sigma Chemical, St. Louis) at a concen-tration equivalent to 100 mg/ml was used for this selection.Strains carrying the lacO sequence at cen2 or ade6 togetherwith the lacI-GFP expression system were obtained from A.Yamamoto. Rich media, YE, supplemented YE (YES), and YPD,and a synthetic medium, EMM2, were used (Alfa et al. 1993).For culture of the mad2D gtb1 double mutant or for athiabendazole (TBZ)-sensitivity assay, 5 or 20 mg/ml TBZ,respectively, was added to the medium. For the minichromo-some stability assay, YE or EMM2 (supplemented with 5 mg/mladenine sulfate) were used. EMM2 was used when selectivepressure for the plasmid was needed.

Cytologic methods: For the experiment shown in Figure 1or other experiments where indicated, 10 mm hydroxyurea(HU) was added to a logarithmic phase of culture at 33� for 3hr, and cells were collected and washed on a glass filter,resuspended in fresh media (EMM2 or YE), and incubated for75 to 90 min. Methanol fixation was performed as follows:Yeast cells were harvested by a glass filter, soaked in chilledmethanol, and kept at �80� for 2 hr. Fixed cells wereresuspended in PEMS buffer (100 mm PIPES, 1 mm EGTA, 1mm MgCl2, 1.2 m sorbitol, pH 6.9) and digested with zymolyasebefore staining with 49,6-diamidino-2-phenylindole (DAPI).Condensed chromosomes in the cut7 mutant and in carben-dazim (CBZ)-treated cells were observed with DAPI stainingafter glutaraldehyde fixation. CBZ (50 mg/ml) was added tolog-phase cultures in YES medium at 30� for the indicatedtime. Fluorescence in situ hybridization (FISH) was performedaccording to Goto et al. (2001), using the probes cos1228(chromosome 2) and cos1322 (chromosome 3) (Mizukami

et al. 1993), which were mapped, respectively, at�50 and 150 kbfrom the respective centromere (The Wellcome Trust SangerInstitute, S. pombe Genome Project). Microscopic observation ofliving cells was performed using the DeltaVision system(Applied Precision, Issaquah, WA) placed in an air-conditionedroom set at 30� 6 0.5�. Relevant parameters for image acqui-sition are described in the legends to Figures 2–4 and 6. Imagespresented in this work were obtained by assembling the pixelswith the highest signal intensities through all images fromdifferent focal planes acquired at the same time point withoutthe computational removal of out-of-focus information. Spin-dle length, presented in Figure 2, A and B, was measured bytaking the difference in focal plane into account.

1572 Y. Tange and O. Niwa

Isolation of new mad2 alleles: A polymerase chain reaction(PCR)-based mutagenesis of the mad21 gene was performedusing the Diversify PCR random mutagenesis kit (CLONTECH,Palo Alto, CA), and the fragments produced were cloned in amulticopy vector (pKD10; Shimanuki et al. 1997) and trans-formed into W986 (mad2D). Each transformant was first testedfor TBZ sensitivity on YE solidified with agar (YEA) containing20 mg/ml TBZ at 33�. The transformants were crossed with astrain in which the G-418-resistant marker (Bahler et al. 1998)was inserted 600 bp upstream of the start codon of the gtb1-93mutant gene. Spores from this cross were spread on EMM2plates (to select for Ura1 and Leu1 colonies) and incubatedat 33� for 3 days followed by replica plating onto G418 platesto select for the gtb1-93 mutation linked to the drug-resistantmarker. After incubating for 2 days, the number of largecolonies growing on the drug-containing plates was comparedwith control plates (see Figure 7). Two strains displayed TBZhypersensitivity and many large colonies on the G-418 plateswere selected (see text). To create strains containing singlyintegrated copies of the mad2 alleles, we first used PCR togenerate a 6-kb-long genomic DNA fragment containingthe mad2 gene near the center of the fragment, in which theG-418-resistant gene (Bahler et al. 1998) was inserted 136 bpdownstream from the termination codon of the mad2 gene.This fragment was transformed into HM713 (h� leu1 ura4mad2Tura41), and G418-resistant and Ura� transformantswere selected. Correct integration was confirmed by sequenc-ing the relevant genomic DNA segment. For GFP tagging ofthese mad2 alleles, a GFP-tagging cassette with a nourseothricin-resistant (natr) marker was used (a kind gift from T. Toda) (Sato

et al. 2005). Nourseothricin (clonNAT) was purchased fromWerner BioAgents (Jena, Germany) and used at 100 mg/ml.

Immunoprecipitation analysis: Strains containing GFP-tagged Mad2 were cultivated for logarithmic growth at 30� inYPD medium and harvested by centrifugation. When required,10 or 12 mm HU was added to the culture, followed by a 3-hrincubation for S-phase arrest. To release the cells from theS-phase arrest, the cells were incubated for 75 min in theabsence of HU. For strains carrying the cut7 ts or nda3 cs

mutations, cells were incubated at 36� for 3 hr or at 18� for 6hr, respectively, to obtain mitotically arrested cells. Cells werewashed once with TE (10 mm Tris–HCl, 1 mm EDTA, pH 7.5),frozen in liquid nitrogen, and stored at �80� until use. Immu-noprecipitation was performed according to Saitoh et al.(2005) with modifications. To the frozen cell pellet, an equalamount of extraction buffer [50 mm HEPES–Na, pH 7.5, 10%glycerol, 0.5 mm dithiothreitol, 125 mm NaCl, 0.2% NP-40, 1%proteinase inhibitor cocktail (P8215, Sigma), 1 mm Pefabloc SC(Roche, Nutley, NJ)] was added and cells were disrupted withglass beads using Multi-Beads Shocker (Yasui Kikai, Osaka,Japan). The soluble fraction of the extracts was obtained bycentrifuging at 1500 3 g for 5 min and then at 14,000 3 g for 10min. An aliquot of the soluble extract containing 2 mg of pro-tein was brought to 100 ml with extraction buffer and incubatedat 4� for 1 hr with or without 0.15% of sodium dodecyl sulfate(SDS). After incubation, the SDS concentration was adjusted to0.05% by dilution, and the soluble extract was further incubatedat 4� for 1 hr with 25 ml Dynabeads M-280 (Dynal Biotech ASA,Oslo, Norway) conjugated with sheep anti-mouse IgG, whichwas preincubated with 2 mg of mouse anti-GFP antibody (RocheDiagnostics). After washing three times with the extractionbuffer, bound proteins were recovered in a loading buffer forSDS–polyacrylamide gel electrophoresis by heating at 100� for 5min. Western blot analysis was performed with anti-Mad1 rabbitpolyclonal antibody (a generous gift from T. Matsumoto) andwith the anti-GFP antibody. For the reciprocal immunoprecip-itation experiments, we attempted to create appropriately taggedMad1 protein, but failed to obtain functionally active constructs.

Cdc2 kinase assay: Cdc2 kinase activity in cell extracts wasmeasured essentially as described in Moreno et al. (1989), butactivity was assayed in the crude extracts. Histone H1 waspurchased from Roche Applied Science (Tokyo). After the reac-tion, H1 histone was separated on a 15% SDS–polyacrylamidegel, and incorporated 32P was quantified using the Bio-Imaginganalyzer system (Fuji Photo Film, Tokyo).

RESULTS

Aberrant mitosis in the g-tubulin mutant: As pre-viously reported, the cold-sensitive g-tubulin mutant(gtb1-93) doubles at a rate almost identical to that of wildtype at permissive temperatures (Tangeet al. 2004). Therewere several aberrant features, however, associated withits mitosis.

Strains with or without the gtb1-93 mutation containingthe GFP-tagged a2-tubulin (Masuda et al. 2006) for visualiz-ing spindles were treated with HU to synchronize cellcultures. After release from the S-phase block, mitoticcells appeared at approximately the same time in bothstrains. Methanol-fixed cells were stained with DAPI andexamined for spindle length and chromosome separa-tion. The spindle length in cells synchronized by HU-block release was longer than that in untreated cells.Nevertheless, in gtb11 cells, when spindle length was $6mm, two equally sized chromosomal masses became wellseparated [Figure 1, A and E (blue)]. In the gtb1 mutant,however, chromosomes were frequently not well sepa-rated in a spindle, even when spindle length exceeded7 mm (Figure 1B), and there were lagging chromosomesin 11 of 85 mitoses (Figure 1, C and F). Despite thisaberrancy, mutant cells in late anaphase or telophase(when the chromosomes nearly reached the ends of thecell) contained two equally sized chromosomal massesin 166 of 171 cells [the remaining cells contained largeand small chromatin regions (n ¼ 2) and lagging chro-mosomes (n ¼ 3)]. Wild-type cells contained normallysegregated chromosomes in all 191 cases examined.Thus, replicated chromosomes seemed to be distributedinto daughter cells in near-normal fidelity at the per-missive temperature in the mutant.

Live analysis of spindle dynamics in the g-tubulinmutant: To examine the mutant mitosis in more detail,we performed live analyses of spindle dynamics and chro-mosome behavior. Spindle dynamics in wild-type fissionyeast are divided into three phases (Nabeshima et al.1998; Mallavarapu et al. 1999). In phase 1, a bipolarspindle is formed; in phase 2, spindle length remainsunchanged or only slightly elongates; and in phase 3, thespindle elongates to fully separate the chromosomes.Phases 2 and 3 correspond to prometaphase/metaphaseand anaphase B, respectively. Anaphase A occurs at theend of phase 2, but occupies only a small part of it.

The spindle dynamics were determined using GFP-tagged Sid4, whose localization is strictly confined to thespindle pole body (SPB) throughout the cell cycle(Chang and Gould 2000). The distance between the

Novel mad2 Mutants in Fission Yeast 1573

two separating Sid4 signals was measured. Although theonset of phase 2 was not clear, the timing of the onset ofphase 1 as well as that of phase 3 could be unambiguouslydetermined. We ascertained in a separate experimentthat, consistent with previous reports (Yanagida et al.1999; Decottignies et al. 2001), Cdc13 (B cyclin)-GFPdisappeared from the spindle and accumulated along thenuclear periphery at approximately the onset of phase 3in both wild-type and mutant cells (data not shown).Spindle length at the onset of anaphase B was in the rangeof 2.2–3.6 mm (2.82 6 0.39 mm; n¼ 21) in wild type, whilethat in the gtb1 mutant was between 3.0 and 5.0 mm (3.936 0.45 mm; n ¼ 24). Thus the metaphase spindle lengthin the mutant was 39% longer than that in the wild type.We then determined the duration of phases 1 and 2: 8.5 6

1.8 min in the wild type and 8.9 6 3.0 min in the mutant.This indicated that there was no significant difference inthe average length of the duration from the start of phase1 to the end of phase 2, although there was more vari-ability in the mutant. There was little or no linear corre-lation between the duration of phases 1 and 2 and thespindle length at the onset of phase 3 in either the wild-type or the gtb1 mutant (supplemental Figure 3 at http://www.genetics.org/supplemental/). In Figure 2, A and B,all of the spindle elongation profiles were assembled in asingle figure for each genotype by setting the anaphase Bonset as time 0. The speed of SPB separation during phase1 was faster in the mutant (�1.7-fold) than in the wild type,but it was almost identical in phase 3 (Figure 2, A and B).

Anaphase A kMT dynamics were impaired in the gtb1mutant: We observed chromosome behavior by using thekinetochore protein Mis6 tagged with GFP (Saitoh et al.1997). Unlike in wild-type cells, chromosome segregationof all chromosomes (anaphase A) did not occur simulta-neously near the center of the spindle, but occurred atapparently more scattered positions along the spindle(data not shown). Because of the weak signal intensity ofthe Mis6-GFP and the swift movement of the signals, wewere unable to trace individual kinetochores in this anal-ysis. Therefore, we used the lacO/lacI-GFP system, whichallowed for fluorescent marking of unique chromosomalloci. The loci that we used in this study were the cen2 (5 kbfrom the centromere) and the ade6 locus on chromo-some 3 (Nabeshima et al. 1998; Yamamoto and Hiraoka

2003). Sid4-GFP was also used simultaneously to deter-mine spindle length. We measured the distances betweenSid4 and Sid4, Sid4 and cen2, and cen2 and cen2 at eachtime point. Representative results in the wild type as wellas in the mutant are shown in Figure 3.

Consistent with previous reports (Nabeshima et al.1998; West et al. 2002), the pair of cen2 signals remainedaround the center of the spindle in wild-type phase 2before the separation, although in some cases the pair ofsignals occasionally moved off the center and separatedthere. The duration of anaphase A determined by thismethod (from the separation of the sister-chromatidpair to the complete shortening of kMTs) was generally,45 sec. The onset of anaphase B (phase 3) occurred

Figure 1.—Separation of chro-mosomes on the mitotic spindle.Synchronized cells with GFP-tagged a-tubulin at 30� in EMM2medium were fixed with methanoland stained with DAPI. In A–D, mi-crotubules (green) and chromo-somes (red) (color panels) andchromosomes in black-and-whiteimages are shown. Cells containinga spindle .�5 mm were chosen,and then the spindle length andthe segregation figures of chromo-somes were recorded. The figureswere classified as equal (represen-tative image as in A and in blueboxes in E–G), incomplete separa-tion (B, red), lagging chromosomenear the spindle center (C, pink)orclosetothespindlepole(green),and unequal (D, yellow). Each boxin E–G represents one cell. Strainsused were YT417 (wild type),YT413 (gtb1-93), and YT415 (gtb1-93 mad2D). See supplementalTable 1 (at http://www.genetics.org/supplemental/) for strains.


around the end of anaphase A (Figure 3, A and B). Incontrast, there were several aberrant features in themutant mitoses (Figure 3, C–F). First, sister centromerepairs tended not to be positioned near the spindlecenter, but to oscillate within the range of the spindle,and the separation of the pairs occasionally occurredaway from the spindle center. In some cases, the sep-aration occurred at or near the spindle pole (Figure 3F).Second, the kMTshrinkage rate in anaphase A was oftendifferent within pairs (for example, see Figure 3, D andE). Remarkably, one of each pair of kMTs sometimeseven seemed to elongate while the other was shortening,even after the onset of anaphase A (Figure 3E). Probablyrelated to this phenomenon, we encountered caseswhere sister-chromatid separation paused for a period[45 sec in Figure 3E, and 1 min in Figure 4 (right panels,green triangles)]. Third, the anaphase A movement ofindividual centromeres sometimes lasted far into ana-phase B (see, for example, Figure 3, C and F). Almostidentical results were obtained when using the ade6marker (data not shown).

We then observed the cen2 and ade6 loci simulta-neously in living cells. In wild-type cells, both of the locibegan to separate almost simultaneously (within 15 sec)in 12 of 17 cases, but in other cases the separation timingwas different by 30–50 sec (for example, see Figure 4, leftpanels). The differential separation appeared in ap-proximately the same frequency in the mutant; in 11 of14 mitoses, sister chromatids of chromosomes 2 and 3separated simultaneously (Figure 4, right panels), whilein others it occurred at a 30- to 45-sec interval. Thus,

temporal coordination of sister-chromatid separationamong chromosomes was not altered in the mutant,although spatial coordination was impaired. Thus, themutant mitosis was characterized by a longer metaphasespindle, off-centered positioning of sister-chromatidseparation, uncoordinated shrinkage of each kMT, andlagging chromosomes due to incomplete anaphase Ain the middle of anaphase B. These features mightexplain the unusual distribution of chromosomes de-scribed in Aberrant mitosis in the g-tubulin mutant.

Some, but not all, spindle checkpoint genes arerequired for ensuring near-normal proliferation of theg-tubulin mutant: Given the aberrancy in spindle andchromosome dynamics in the mutant, we examinedwhether SAC function was involved in ensuring the near-normal chromosome segregation. We combined indi-vidual checkpoint mutants with the gtb1 mutation andexamined the colony size of the double mutants grownat permissive temperatures for the gtb1 mutant (Figure5). The combination of mad2D, mad3D, or bub1D muta-tions with the gtb1 mutation led to the formation of smallcolonies, containing many dead cells. The mph1D

mutation seemed to exert a more deleterious effect. Incontrast, on the basis of colony size, the bub3D mutationhad little synergetic effect with the gtb1 mutation.Intriguingly, the mad1D gtb1-93 double mutant did notform colonies from spores, indicating that this check-point gene has some functions different from those ofMad2 in the gtb1 mutant. The mad1D gtb1 double-mutantcells made extremely small colonies when a low concen-tration of TBZ (5 mg/ml) was added to the medium,

Figure 2.—Live observation ofspindle dynamics. Spindle lengthwas determined as the distance be-tween two separating Sid4-GFP sig-nals. Living cells were observedevery 20 sec, and at each time point12 images were taken at serial focalplanes at 0.2-mm steps (A and B).These parameters were changedto 30 sec, 6 images, and 0.4-mmsteps inCandD.Thetimeoftheon-set of anaphase B (phase 3) was setas time 0 and all figures obtainedfor each strain are assembled in asingle figure. (A) Wild type (strainP18, number of cells (n) ¼ 21).(B) gtb1-93 (P168, n ¼ 24). (C)mad2D (YT522, n ¼ 10). (D) gtb1-93 mad2D (YT519, n ¼ 14).


which relieves the gtb1 defect (Tange et al. 2004), butfailed to form colonies when the cells were transferred toa TBZ-free medium. This indicated that the doublemutant was not solely defective in germination fromspores but in fact was lethal in the absence of the drug.The double mutant had a deregulated septation defect,which was not observed in the mad2D gtb1 double mutant(Y. Tange and O. Niwa, unpublished results).

Mad2 is required for near-normal chromosomesegregation in the g-tubulin mutant mitosis: As describ-ed above, the gtb1-93 mad2D double mutant was viable butproduced very small colonies at permissive temperaturesfor the gtb1 mutation (see Figure 5). Cells composing thesmall colonies (excluding apparently dead cells) had�20% plating efficiency. To determine the reason for thislow viability, we examined chromosome segregation as

shown in Figure 1G. Two notable features were apparentin the double mutant compared to the gtb1 singlemutant. One was the reduction of ‘‘incomplete separa-tion’’ (shown in red in Figure 1) in spindles .7 mm, andthe other was the high incidence of unequal nucleardivision (Figure 1D and yellow in Figure 1G) and laggingchromosomes (Figure 1C and green and pink in Figure1G), both indicating errors in chromosome segregation.FISH analysis using probes for chromosomes 2 and 3revealed a high frequency of nondisjunction in thedouble mutant (supplemental Figure 1 at http://www.genetics.org/supplemental/). Thus, the low viability ofthe double mutant was accounted for by the high in-cidence of errors in chromosome segregation.

We then performed live analysis of the spindledynamics in mad2D gtb1-93 double mutants and also in

Figure 3.—Live analysis of sister-chromatid sep-aration. Fission yeast cells carrying both Sid4-GFPand cen2-GFP were observed every 15 sec with 10steps (0.2 mm) along the z-axis at each time point.Cells in late phase 1 or in phase 2 were chosen forobservation. Distances (d1–d4), as defined in thefigure (Sid4-GFP: green dots; cen2-GFP: orangestars), were measured and plotted. (A and B) Wildtype (P172). (C–F) gtb1-93 (P171).


mad2D single mutants (Figure 2, C and D). As antici-pated from its nearly normal growth, the mad2D singlemutant had spindle dynamics comparable to the wildtype (Figure 2C), although the duration of phase 1 plusphase 2 appeared to be less than in wild-type cells (7.5 6

1.7 min; n ¼ 8), which was �1 min shorter than in thewild type. Spindle length at the onset of anaphase B(2.4 6 0.27 mm) might also be shorter than that of wild-type spindles. In sharp contrast, there was a drasticchange in the double mutant (Figure 2D). It was diffi-cult to determine the timing of anaphase B onset in thedouble mutant, even with the aid of Cdc13 localizationanalysis. Thus, the results shown in Figure 2D are onlyapproximate. Nevertheless, it was clear that in thedouble mutant the length of spindles at the onset ofanaphase B were highly variable. Also, the duration ofphase 2 was very short, and in some cells it was almostmissing. Live observation of sister-chromatid separationwith the cen2 and ade6 loci revealed unequal chromo-some segregation in 7 of 30 mitoses in the double mutant(four nondisjunction and three lagging chromosomes).These live analyses demonstrated that in the absence offunctional Mad2 in the gtb1 mutant, the duration of theprogression through phases 1 and 2 was greatly short-ened and the formation of the prometaphase/meta-phase spindle was impaired. This in turn suggested thatanaphase B tended to start without prior properformation of the bipolar metaphase spindle that wouldbe required for accurate chromosome segregation.

As reported previously (Tange et al. 2004), the cold-sensitive growth phenotype of the g-tubulin mutant wassuppressed by an a2-tubulin mutant, which itself washypersensitive to the MT-destabilizing drug TBZ, orpartially suppressed by the addition of a low concentra-tion of TBZ into the medium. These MT-destabilizingconditions alleviated the harmful effects of the mad2mutation on the gtb1 mutant growth (data not shown),

Figure 4.—Timing of sister-chromatid separation of differ-ent chromosomes. Cells containing cen2-GFP and ade6-GFP aswell as Sid4-GFP were observed in living cells every 15 sec. Ateach time point, 10 images were taken with a single z-axis stepof 0.2 mm. P175 (WT) and P174 (gtb1) were used. In wild type,one of the sister-chromatid pairs was already separated in thefourth frame, but the other remained unseparated until thesixth frame. In the mutant, both pairs appeared to separate si-multaneously (third frame from the top), but one of them(marked by green triangle) stopped separating for .1 minwhile the other (red triangle) continued to separate.

Figure 5.—Differential requirements of SAC genes forgrowth of the g-tubulin mutant. Approximately the sameamount of cells freshly grown on a YE plate at 33� was streakedout on the same medium and incubated at 30� for 3 days. Strainsused are HM123 (WT), W604 (gtb1), HM713 (mad2), YT578(mad2 gtb1), YT406 (mad3), YT410 (mad3 gtb1), YT405 (bub1),YT409 (bub1 gtb1), YT407 (bub3), YT411 (bub3 gtb1), YT408(mph1), and YT412 (mph1 gtb1).


suggesting that the occurrence of improperly stabilizedMTs affects the spindle formation so that it becomesheavily dependent on Mad2 (and probably also on someother checkpoint genes) to proceed with near-normaldynamics.

Mad2 localization is altered in the mutant: Becausethe near-normal mitosis in the gtb1 mutant was verydependent on Mad2, we examined whether the localiza-tion of the protein was altered. We used GFP-tagged Mad2(Ikui et al. 2002) and observed it in living cells. Consistentwith previous reports (Ikui et al. 2002; Saitoh et al. 2005)during interphase of wild-type cells, Mad2-GFP waslocalized to the nuclear periphery as well as to thechromatin region, and during the prophase to prometa-phase it was highly accumulated at kinetochores, whichwere often clustered (Figure 6, A and B). After the properattachment of kinetochores to spindle MTs was estab-lished in metaphase, the intense kinetochore signalsdiminished. Thus, on the phase 2 spindles, Mad2-GFPlocalization was observed only near the poles and veryfaintly along the spindle (Figure 6C). The localization ofMad2 was altered in the mutant in two aspects. One wasthat phase 2 spindles had brighter Mad2 signals at theends of the spindle, often at only one of the poles (Figure6E). Moreover, phase 2 spindles in the mutant sometimescontained a bright dot on the spindle between the poles,which was moving along the spindle (Figure 6H). From itsbehavior, we assumed that the bright dot between thespindle poles was a pair of kinetochores, but this was notverified. Less intense dot signals sometimes coexistedwith the bright dot on the same spindle. Mad2 localiza-tion was also altered in phase 3. In wild type there wereonly faint signals at the poles of the spindles (Figure 6D),but in the mutant, brighter signals were often observed,generally near one of the poles (Figure 6, F and G). Thesignals were not dot-like in shape but usually elongatedtoward the spindle center, and in some cases we notedmultiple dots moving inward from the pole. The phase 3spindles in the mutant occasionally contained multipledot signals between the poles, which were moving to thespindle end (see supplemental Figures 4–7 at http://www.genetics.org/supplemental/ for movies). The mutant-specific Mad2 localization on the metaphase–anaphasespindle might reflect the aberrant kMT dynamics duringthese phases. As has been suggested in other organisms(Buffin et al. 2005), Mad2 protein might flow from thekinetochores to the spindle poles during anaphase infission yeast. In the mutant, there might be an increasedchance of detachment of kMTs from kinetochores thatwould lead to Mad2 recruitment to the kinetochores andsubsequent transfer toward the pole. Also, because of theprolonged existence of kMTs in the mutant, more Mad2proteins might be transferred to the pole and accumulatethere. The possibility that the localization of Mad2protein along the spindle and at the poles was indepen-dent from the kinetochore, however, could not be ruledout. According to Saitoh et al. (2005), in a subset of

kinetochore mutants in which Mad2 localization to un-attached kinetochores was reduced, the accumulation ofMad2 at the spindle pole was rather enhanced.

Isolation of mad2 mutants that are defective ingeneral SAC but retain the function for g-tubulinmutant viability: The differential requirements of SACgenes, particularly of Mad1 and Mad2, for the g-tubulinmutant led us to examine whether the function of Mad2for ensuring near-normal mitosis in the mutant differsfrom the function to arrest mitotic progression respond-ing to gross perturbation of spindle formation [for thesake of simplicity, we hereafter use gtb1-CS (for chromo-some stability) and general SAC to distinguish Mad2

Figure 6.—Dynamic localization of Mad2-GFP in mitoticcells. (A–D) Wild type (W949). (E–H) gtb1 (W948). (A) Pro-phase. (B) Prometaphase. (C) Metaphase (phase 2). (D) Ana-phase (phase 3). (E–G) Early anaphase to late anaphase. (H)Metaphase (phase 2): sequential images showing dynamicmovement of intense Mad2-GFP dot along the spindle. Orig-inal images are available as movies in supplemental Figures 4and 5 (wild type) and supplemental Figures 6 and 7 (gtb1) athttp://www.genetics.org/supplemental/. Images were takenevery 15 sec, nine images with 0.3-mm steps in the z-axis ateach time point.


functions required in these different conditions]. Toaddress this directly, we attempted to isolate mad2mutants that were defective in general SAC functionbut retained gtb1-CS activity. We isolated two mad2 mutantgenes (mad2-56 and mad2-64) on a multicopy plasmid.These mad2 alleles supported the growth of the gtb1mutant as well as the wild-type mad2 (Figure 7A).

Both mad2-56 and mad2-64 were isolated on the basis oftheir hypersensitivity to TBZ, indicative of the deficiencyin general SAC (Figure 7B). We verified this finding bytwo different criteria. The first criterion was the ability toblock mitotic progression in the cut7 ts mutant; the exis-tence of hypercondensed chromosomes was consideredto be an indication of general SAC activity (Kim et al.1998). With the mad21 allele, 44.0% cells contained hy-percondensed chromosomes, while with mad2-56 andmad2-64 this value was reduced to 4.6 and 6.7%, re-spectively, values only slightly higher than the controlvalue with the vector (Table 1). Importantly, when themutant alleles were introduced into mad21 cells with themulticopy plasmid, they had a clear dominant-negativeeffect over the mad21 gene, suggesting that they seques-tered the active Mad2 product, preventing its checkpointfunction. The second criterion used was the accumula-tion of Cdc2 kinase activity after mitotic arrest (He et al.1997). In both nda3 cs and cut7 ts mutants, the mad21-dependent increase in Cdc2 kinase activity was significantlyreduced in the newly isolated mad2 mutants (supplemen-tal Figure 2 at http://www.genetics.org/supplemental/).Taken together, we concluded that these mad2 alleleswere not functional in blocking mitotic progressionwhen spindle assembly was grossly compromised, butremained functional for gtb1-CS.

Mutant Mad2 proteins formed a Mad1-containingcomplex with altered stability: Mutation sites in the newmad2 alleles were determined. In mad2-56, Ser at 185 waschanged to Arg and in mad2-64, Met52 and Asp177 were

changed to Ile and Val, respectively. Either singlemutation in the mad2-64 allele alone did not confer anextensive SAC defect, although the D177V mutationdisplayed slightly reduced SAC activity (Table 1).

Because both of the mutant proteins had an aminoacid exchange in the carboxyl terminal segment ofMad2, which is implicated in the stable complexformation with Mad1 (Luo et al. 2002; Musacchio and

Figure 7.—Genetic properties of new mad2mutants. (A) The G-418 plate assay. Coloniesgrown on EMM2 plates from spores producedfrom crosses between P5 (kanr linked to thegtb1 mutation) and W986 (mad2) carrying a plas-mid with mad21 (a), empty vector alone (b),mad2-56 (c), or mad2-64 (d) were replica platedonto YE with or without G418 (see text for de-tails). (B) The TBZ-sensitivity assay. HM713(mad2D) carrying a plasmid with the indicatedmad2 alleles grown on EMM2 medium wasstreaked on YE plates with or without TBZ and in-cubated at 33� for 3 days.

TABLE 1

Mad2-dependent mitotic arrest assessed in the cut7 mutant

mad2 allelein host cell

mad2 alleleon plasmid

% of cells with condensedchromosomes

(total no. of cells)

mad2D mad2 1 44.0 (184)None 2.2 (180)mad2-56 4.6 (151)mad2-64 6.7 (180)mad2-64 M52I 42.9 (163)mad2-64 D177V 31.6 (190)

mad2 1 mad2 1 67.2 (119)None 35.6 (174)mad2-56 7.8 (255)mad2-64 7.8 (218)

mad2 1 No plasmid 52.7 (163)mad2D 1.7 (241)mad21Tkanr 53.2 (171)mad2-56Tkanr 5.2 (271)mad2-64Tkanr 6.5 (245)

Strains containing plasmid were incubated at 36� for 4 hr inminimal medium, but those without plasmid were incubatedin YPD medium at 36� for 3 hr. Cells were fixed with glutar-aldehyde and stained with DAPI. Cells that had hypercon-densed chromosomes were scored. Yeast strains used wereP43 (mad2D) and HM783 (mad21) for the tests with plasmids,and P43, HM783, YT541 (mad21Tkanr), YT542 (mad2-56Tkanr), and YT543 (mad2-64Tkanr) for chromosomalmad2 alleles.


Hardwick 2002; Sironi et al. 2002; De Antoni et al.2005), we tested whether the amount and/or stability ofthe Mad1-containing complex was altered. GFP-taggedMad2 proteins were expressed under the control of anative mad2 promoter. Cell extracts were immunopreci-pitated with anti-GFP antibody, and the amounts of co-immunoprecipitated Mad1 were compared by Westernblot analysis (see materials and methods). In a controlexperiment, we ascertained that the immunoprecipita-tion of Mad2-GFP and Mad1 was dependent on addedanti-GFP-antibody and on the GFP-tagging of Mad2(data not shown). Comparable amounts of Mad2-GFPwere recovered in both wild type and mutants (Figure 8).The mobility of GFP-fused mad2-64 mutant proteins wasslower in the electrophoresis, although it is not clearwhy. The amounts of co-immunoprecipitated Mad1were not appreciably different between wild type andmutants. They were not affected by increasing the saltconcentration to 0.5 m NaCl (data not shown). However,when the extracts were incubated in the presence of alow concentration of the detergent SDS, prior to theaddition of the antibody, the amount of Mad1 was greatlyreduced in the mutant extracts, but not in the wild-typeextract (Figure 8). The reduction of Mad1 by SDStreatment in the mutant extracts was not due todegradation of the protein possibly induced by theSDS treatment, because there was no reduction of theprotein in SDS-treated whole extracts (Figure 8). Theseresults strongly suggested that the mutant Mad2 pro-teins form a Mad1/Mad2 complex that has a differentstability from that formed with wild-type Mad2. Wethen addressed whether the amount of the SDS-resistantform of the complex changes in a cell-cycle-relatedmanner or in response to SAC-inducing conditions(see materials and methods), but failed to see anynotable changes at least in the soluble cell extracts (datanot shown).

We also examined whether the localization propertiesof mutant Mad2 were altered from wild-type protein,

using the GFP-tagged genes. There was no notable dif-ference in the localization profile under any experimen-tal conditions that we tested when using singly integratedgenes (i.e., in mitotically arrested cells induced by thenda3cs mutation and by CBZ treatment, and in wild-typecells that were synchronized by HU arrest and release). Itremains to be examined, however, whether there are anydifferences in the amount and/or in the dynamic natureof the localization. In cells that expressed the GFP-taggedMad2 proteins from the multicopy plasmid, wild-typeprotein accumulated at a site in the interphase nucleus,presumed to be at the SPB or at the clustered centro-meres, whereas there was very little accumulation of themutant proteins (data not shown).

Minichromosome stability in unperturbed mitosiswas only marginally affected by the new mad2mutations: As another way to access Mad2 activity, wemeasured minichromosome stability by the half-sectormethod (Allshire et al. 1995). In the presence of thewild-type mad2 allele, mitotic loss occurred approximatelyonce/3 3 103 cell divisions (Table 2). In the absence ofMad2, minichromosome instability increased 6-fold.When cells carried the mad2-56 or mad2-64 alleles, theminichromosome remained stable, and the stability wasonly 1.3- or 1.1-fold different from that of wild type. Whenthe same cells were challenged with 7.5 mg/ml TBZ toperturb the spindle assembly, minichromosome stabilitywas greatly reduced in cells carrying either the mad2-56 ormad2-64 alleles (11- and 6-fold, respectively), but it wasonly slightly affected in mad21-carrying cells (1.8-fold).This is consistent with the fact that the new mad2 alleleswere defective in general SAC.

With regard to the dominant-negative effect of the newmad2 mutants over wild-type mad2 in cut7 mutants(described above), we tested minichromosome stabilityin wild-type cells that carried the plasmid with differentmad2 alleles. Stability was not altered in these cells(Table 2). Further, the growth of the gtb1 mutant wasnot affected by the multicopy plasmid bearing the newmad2 alleles (data not shown). These results also demon-strated that the functions of Mad2 required for generalSAC and for gtb1-CS and minichromosome stability aregenetically discernible.

Activity of a single copy of the new mad2 alleles:Because the new mad2 alleles were first isolated inplasmids and subsequent tests (described above) wereperformed with the plasmid-borne genes, we createdstrains containing a single integrated copy of the mad2allele (see materials and methods). Using these mad2alleles, we first tested whether these alleles are recessive tothe wild type. Diploid cells heterozygous at the mad2 locus(mad21/mad2-56, mad21/mad2-64, and mad21/mad2-null)had TBZ resistance similar to that of homozygousmad21/mad21 wild-type diploid cells (data not shown).This suggested that both of the mad2 mutants wererecessive to wild type with regard to general SACfunction. Thus, the observed dominant-negative effects

Figure 8.—Immunoprecipitation analysis of the mutantMad2 proteins. Strains containing mad21-GFP (YT579),mad2-56-GFP (YT580), and mad2-64-GFP (YT581) were used.Immunoprecipitated proteins were detected by Western blotanalysis using the indicated antibodies. IP, immunoprecipi-tated. WCE, whole-cell extracts. Protein size markers are onthe right: open triangle (80 kDa) and closed triangle (50kDa). See materials and methods for experimental details.


of the plasmid-borne mad2 alleles (see Table 1) werelikely due to overproduction of the mutant proteins. Wethen tested these single-copy mad2 alleles for TBZsensitivity (Figure 9A), the activity to support the growthof the g-tubulin mutant (Figure 9, C and D), thecheckpoint function to arrest mitosis in the cut7 mutantand in CBZ-treated cells (Table 1, Figure 9B), and theeffect on minichromosome stability (Table 2). The resultsindicated that the single copy of the new mad2 mutantshad activities that were comparable to those observedwith multicopy plasmids. The results demonstratedthat the differential activities of the new mad2 mu-tants displayed in various aspects of Mad2-requiringcellular processes were not solely due to the high genedosage.

DISCUSSION

The defective mitotic phenotype in the g-tubulinmutant was strikingly similar to that described in theklp5/klp6 mutant in several aspects (Garcia et al. 2002;West et al. 2002). First, both had longer metaphasespindles and lagging chromosomes until the middle ofanaphase B. Second, sister-chromatid separation occurred

at scattered sites on the metaphase spindle. Third, thespindle elongation rates in anaphase B as well as thechromosome segregation in late anaphase were normalor nearly normal. These properties might be explainedby deregulated kMT dynamics or by generally slowershrinkage rates of kMTs. The Klp5/6 kinesins localizenear the kinetochores and are implicated in thedisassembly of the kMTs at the plus ends to generatethe force for the bipolar attachment of sister kineto-chores. The lack of Klp5/6 might give rise to theincreased stability of kMTs and/or defects in thekinetochore–kMT interaction (Garcia et al. 2002; West

et al. 2002; Sanchez-Perez et al. 2005). The gtb1-93mutant contained apparently stabilized cytoplasmic MTbundles, which were very similar in shape to those in theklp5/6 mutant (Paluh et al. 2000; Tange et al. 2004); thecold sensitivity of the gtb1-93 mutant was enhanced inthe presence of a TBZ-resistant b-tubulin (Yamamoto

1981; Y. Tange and O. Niwa, unpublished data); anddefects in the gtb1-93 mutant were relieved undervarious MT-destabilizing conditions (Tange et al. 2004;this study). These findings suggest that the peculiarspindle dynamics in the g-tubulin mutant, particularlythe defective anaphase A, are due to stabilized MTs.Kinetochore MTs formed in the mutant might somehow

TABLE 2

Effect of mad2 mutations on minichromosome stability

mad2 allele atchromosomal locus mad2 allele on plasmid TBZ Total no. of colonies % half-sectored colonies

mad2Da mad21 0 mg/ml 247,165 0.032None 250,158 0.19mad2-56 260,861 0.047mad2-64 266,571 0.029mad21 7.5 mg/ml 41,076 0.054None 7,066 1.8mad2-56 40,922 0.43mad2-64 36,916 0.21

mad21 b mad21 0 mg/ml 38,422 0.005None 36,545 0.012mad2-56 44,037 0.014mad2-64 36,188 0.006

mad21Tkanr c NAd 0 mg/ml 72,048 0.011mad2D 53,651 0.15mad2-56Tkanr 33,853 0.035mad2-64Tkanr 64,886 0.020mad21Tkanr 7.5 mg/ml 66,316 0.030mad2D 6,170 0.91mad2-56Tkanr 16,994 0.78mad2-64Tkanr 31,399 0.26

a h� leu1 ade6-210 mad2D Ch16 (P112; see supplemental Table 1 at http://www.genetics.org/supplemental/) was transformedwith a plasmid carrying the indicated mad2 alleles. Minichromosome stability assay was performed on synthetic medium selectivefor the plasmid marker.

b Same as in footnote a, but P113 was used (h� leu1 ade6-210 Ch16).c h� leu1 ade6-210 Ch16 with the indicated mad2 alleles at the chromosomal loci were used to determine minichromosome sta-

bility on YE plates. Strains used were P224, P112, P225, and P227.d NA, not applicable.


resist disassembly of the plus-end MTs, which is requiredfor the movement in anaphase A. Analogous g-tubulinmutants in Aspergillus also have an anaphase A defect(Prigozhina et al. 2004; Li et al. 2005). It is not wellunderstood how the altered form of g-tubulin affectsMTstability, although it is most likely at the plus end (seeZimmerman and Chang 2005). Despite the apparentsimilarity in spindle dynamics, the requirement of theklp5/6 mutant for SAC genes is different from that of thegtb1 mutant. The klp5/6 mutant is heavily dependent onBub1, but less dependent on Mad2 and Mph1 (West

et al. 2002). This fact suggests that underlying mecha-nistic defects that require SAC gene functions aredifferent between these mutants. Further studies areneeded to examine this issue.

We demonstrated that the quasi-normal chromosomesegregation in the g-tubulin mutant is heavily depen-dent on Mad2. Some observations that we made in thisstudy might provide a clue to Mad2 dependency. Theabsence of Mad2 had only a subtle effect on the spindledynamics in otherwise wild-type mitosis, but it had aprofound effect in the g-tubulin mutant: mitotic pro-gression was accelerated and the formation of theprometaphase/metaphase spindle was almost abol-ished. The g-tubulin mutant contains MTs that are morestable than those in wild type, which might enhance thebundling of MTs to form bipolar spindles and spindleelongation, whereas the kinetochore–kMT attachmentas well as the regulation of kMT depolymerization at thekinetochore might be impaired, which would result inthe lack of tension at the kinetochore or in the loss of aproper attachment of the kMTs to the kinetochoreduring metaphase/anaphase A. The presence of suchaffected kinetochores might be monitored by a check-point system that includes Mad2 that would allow timefor the proper attachment of kMTs to the kinetochores.Such a monitoring system, if present, should not beidentical with general SAC, because it did not requireBub3 and was not dependent on the Mad2 activity that isrequired for general SAC, as demonstrated in this study.Another possibility is that Mad2 has a direct role inchromosome segregation, perhaps for the attachmentof kinetochores to kMTs. In this case, the requirementfor the hypothesized activity of Mad2 in chromosomesegregation would be much greater in the g-tubulinmutant than in the wild type. Both of these possibilitiesare compatible with the observation that Mad2-GFPoften remained on the metaphase/anaphase spindles asbright dots. It should be noted that Vanoosthuyse et al.

Figure 9.—Activity of a single copy of the new mad2 alleles.mad21 (#) indicates the normal wild-type mad2 allele, whilemad21, mad2-56, and mad2-64 indicate that these alleles werereplaced with the mad2D allele together with the kanr markergene (see materials and methods). mad2D denotes themad2 allele disrupted by insertion of the ura41 gene, the kanr

marker was not used here. (A) TBZ sensitivity test. YE contain-ing 20 mg/ml TBZ was used together with a control plate. In-cubation was at 33� for 3 days. Strains used were HM123,HM713, YT547, YT549, and YT553. (B) Activity of mitotic ar-rest. Strains used were the same as in A. CBZ (50 mg/ml) wasadded to a logarithmic-phase culture and incubated for theindicated time periods. Percentage of cells containing hyper-condensed chromosomes was measured. mad21(#) (solidcircles); mad21 (open circles); mad2D (crosses); mad2-56(open triangles); mad2-64 (open squares). More than 200cells were scored for each strain. (C) Capability of differentmad2 alleles to support the growth of gtb1 mutant. Incubationwas at 33� or 30� for 3 days. Strains used were W604, YT578,YT555, YT556, and YT559. (D) Patterns of chromosome seg-regation in late anaphase. Strains with indicated mad2 allelesin the gtb1-93 background (same as in C) were incubated at30� in YE for 1.5 hr after the treatment with HU and stainedwith DAPI. More than 170 mitoses were scored for each strain.


(2004) reported a separation-of-function mutation infission yeast bub1, in which the SAC activity is lost but thatfor chromosome stability is retained, analogous to themad2 mutants isolated in this study.

In this study, we isolated novel mad2 alleles that weredefective in general SAC but retained activity for gtb1-CSand for minichromosome stability in unperturbed mito-sis. These alleles were in fact leaky mutants; that is, neitherof them had completely lost the activity for general SAC,and they had slightly reduced activity for gtb1-CS and forminichromosome stability. Thus, as the first approxima-tion, it might be that general SAC function requires ahigh level of Mad2 activity, while for the gtb1-CS andminichromosome stability, low activity of Mad2 is suffi-cient. The new mad2 mutants were not merely low-activitymutants, however, as they did not have additive activitywhen ectopically overexpressed; they robustly seques-tered active Mad2 from general SAC function. Impor-tantly, the same high level of expression of the mutantsdid not have harmful effects either on the growth of thegtb1 mutant or on minichromosome stability. Thus, wesuggest that the new mad2 mutants were rather specifi-cally impaired in the activity that is required for generalSAC but not for gtb1-CS and minichromosome stability.Because an attempt to isolate mad2 mutants that arespecifically defective in gtb1-CS was unsuccessful, it re-mains to be determined whether the activity required forgtb1-CS is also required for general SAC.

Both alleles have a missense mutation in the C-terminal segment of Mad2, which is implicated in theformation of a stable complex with Mad1 and with Slp1/Cdc20 (Luo et al. 2002; Musacchio and Hardwick

2002; Sironi et al. 2002; De Antoni et al. 2005). In thisregard, it is interesting that the mad2-64 mutant has anadditional mutation in the N-terminal region; neitherof the two mutations induced a significant defect ingeneral SAC function. The mutated amino acids in themad2-64 mutant might cause a conformational changein the Mad2 protein, so that they affect the activation ofMad2. The results obtained in this study suggest that themutant Mad2 proteins form a complex with Mad1 withaltered stability. A stable Mad1/Mad2 complex is re-quired for the activation of Mad2 in the formation of theMad2/Cdc20 complex, although the precise mecha-nism involved in the activation is still a matter of debate(Yu 2006). It is possible that the mutant Mad2 proteinforms an unstable complex with Mad1, which wouldcause inefficient production of the Mad2/Cdc20 com-plex. Further, because the same structural motif of Mad2is used for the formation of the Mad2/Cdc20 complex, itis likely that the stability of Mad2/Cdc20(Slp1) is alsoaffected in the new mad2 mutants. It will be interesting toexamine if the Mad2/Slp1(Cdc20) complex formationis affected in the new mad2 mutant.

We fortuitously found that Mad1 has an essentialfunction in the survival of the g-tubulin mutant. Althoughfurther detailed investigation is required, a defective phe-

notype of the mad1D gtb1-93 double mutant was clearlyrelated to a septation defect. This observation is reminis-cent of the fact that fission yeast Mad1 was identified as asuppressor of a septation mutant (Kim et al. 2003). In addi-tion, in the absence of Mad1, minichromosome instabilityis greatly increased, independent of Mad2. Our study (Y.Tange and O. Niwa, unpublished data) also showed thatminichromosome stability is highly variable between dif-ferent SAC gene mutants. Together, the findings of thisstudy clearly show the need to elucidate the function ofindividual SAC genes for the fidelity of chromosometransmission during unperturbed mitosis.

We are grateful to T. Matsumoto for the anti-Mad1 serum and yeaststrains. We also thank M. Yanagida, A. Yamamoto, and T. Toda for theyeast strains and plasmids and A. Kurabayashi for FISH analysis. Thiswork was supported by the Kazusa DNA Research Institute and partlyby a grant (no. 16370083) from the Japan Society for the Promotion ofScience to O.N.

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Communicating editor: P. Russell


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