A Cell Cycle Checkpoint Monitors Cell Morphogenesis in Budding Yeast

8/8/2019 A Cell Cycle Checkpoint Monitors Cell Morphogenesis in Budding Yeast.

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A C ell Cy cle Checkpoint M onitorsCell Morph ogenesis in Budd ing YeastD a n i e l J . L ew * * a n d S t e v e n I . R e e d ** Departm ent of Molecular Cancer Biology, Duke University Medical Center, Durham, North C arolina 27710;and *Departmen t of Molecular B iology, MB 7, The S cripps Research Institute, La Jolla, California 92037

Abstract . Checkpo in t con t ro l s a re r egu l a to ry pa thwaysthat inhibi t ce l l cycle progression in ce l l s tha t have notfa i thful ly comple ted a pr ior s tep in the ce l l cycle . Inthe budd ing yeas t Saccharomyces cerevisiae, D N Arep l i ca t i on and sp ind l e a ssembly a re moni to red bycheckpo in t con t ro l s t ha t p reven t nuc l ea r d iv i s ion i nce l l s t ha t have f a i l ed t o comple t e t he se p rocesse s .Dur ing t he norma l ce l l cyc l e , bud fo rma t ion i s t em-pora l l y co inc iden t wi th DNA rep l i ca t i on and sp ind l ea ssembly , and t he nuc l eus d iv ides a long t hemothe r -bud ax i s i n mi tos i s . In t h i s r epor t , we show

tha t i nh ib it i on o f bud fo rma t ion a l so causes a d rama t i cdelay in nuclear division. This a l lows ce l l s to recoverf rom a t r ans ien t d i s rup t ion o f ce ll po l a r i ty wi thou t be -coming b inuc l ea t e . The de l ay occurs a f t e r DNA rep l i -ca t i on and sp ind l e a ssembly , and re su l t s f rom de l ayedact iva t ion of the mas ter ce l l cycle regula to ry kinase ,Cdc28 . Cdc28 ac t i va t i on i s i nh ib i t ed by phosphory l a -t i on o f Cdc28 on t y ros ine 19, and by de layed accumu-l a t ion o f t he B- type cyc l ins C lb l and C lb2 . T hesere su l ts sugges t the ex i s t ence o f a nove l checkpo in t t ha tmoni to r s ce l l morphogenes i s i n budd ing yeas t .

S UCCESSFUL cell proliferation requ ires th e events ofthe cell cycle to be coordinated so that DNA replica-tion is com pleted before chro mo som e segregation is at-tempted, and chromoso me segregat ion i s comple ted pr ior tocell division. This o rder o f events is controlled by a familyof serine/threonine protein kinases called cyclin-dependentkinases (Cdks ' ; reviewed by Norbury and Nurse , 1992;Na smy th 1993). Cd k activity oscillates during the cell cycle,sequentially triggering DNA replication, chromosome segre-gation, and cytokinesis. If some perturbation delays com ple-t ion of e i ther D NA repl ica tion or assem bly of a microtubulespindle (requ ired for chrom osom e segregation), regulatorypathways called checkpoint controls act to delay cell cycleprogres sion (reviewed by Hartwell and Weinert , 1989; Mur-ray, 1992). This delay is thought to be mediated by regula-tion of Cdk activity.Cdk ac t ivi ty i s dependent upon associa t ion of the kinasecatalytic subun it with posit ive regulatory subunits called cy-clins, and can be regulated by phospho rylation of the cata-lytic subunit, and by association w ith inhibitory proteins(reviewed by Solom on, 1993; Dunphy, 1994; Peter and Her-skowitz, 1994). This allows Cd k activity to respond w ith ex-quisite sensitivity to cues from the cell 's environment, as

A d d r e s s a l l c o r r e s p o n d e n c e to D . J . L e w , D e p a r t m e n t o f M o l e c u l a r C a n c e rB i o l og y , B o x 3 68 6 , D u k e U n i v e r s i t y M e d i c a l C e n t e r , D u r h a m , N C 2 7 71 0 .Tel .: (919) 613-8627. Fax: (919) 613-8605.1 . A b b r e v i a t i o n u s e d i n t h i s p a p e r : C d k , c y c l i n - d e p e n d e n t k i n a s e .

well as signals from within the cell (checkpoint controls).The best-characterized class of checkpoint controls monitorsthe replicative status and general integrity o f the cell'sgenom ic DNA . If the DN A is incompletely replicated ordamaged, these checkpoint cont rols de lay chromosomesegregation (reviewed by Hartw ell and Weinert , 1989; Mu r-ray, 1992). Experiments in the fission yeast Schizosac-charomyces pomb e and the frog Xenopus laevis have sug-gested that this checkpoint prevents entry into mitosis bystimulating the inhibitory phosp horylation of Cdc 2, the Cdkthat promo tes entry into mitosis, on tyro sine 15 (Enoch andNu rse, 1990; Smythe and New port, 1992). An analogous in-hibi tory pa thway tha t phosphoryla tes tyrosine 19 of Cdc28has been described in budding yeast (Russell et al . , 1989;Bo oher et al . , 1993). However, tyrosin e phosphorylation ofCdc28 is not requi red for the opera tion of checkpoint con-trois related to genome integrity in budding yeast , andstudies to d ate have not uncovered any biological ro le for thispathway (Amon et al . , 1992; Sorg er and Murray, 1992; Stue-land et al . , 1993).

Like D NA replication and spindle assembly, the reorgani-zations of the actin-based cytos keleton that shape the bud aretriggered by the cyclin/Cdc28 kinases (Lew and Reed,1993). There were two reasons to suspect the existence ofa checkpoint control that monitored bud formation, anddelayed prog ression to anaphase if budding was defective.First, many environmental stresses, including sudden altera-tions in the temperature or osmolarity of the growth me-dium , result in a transient depo larization of the actin cyto -skeleton in budding yeast (Chowdhury et al . , 1992; Lill ie

T he Rockefeller Universi ty P ress , 0021-9525/95/05/739/11 $2.00The Jou rna l o f Cel l Bio logy , Vo lume 129 , Num ber 3 , May 1995 739-749 739


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and Brown, 1994). The depolar ized period can las t up toan hour , o r 50 -70% of the gene ra t ion t ime , du r ing wh icht ime ce l l s a re unab le to d i rec t g rowth to the bud . Wi thou ta con comi tan t de lay o f the nuc lea r cyc le , many o f the s e ce l lswould becom e binuclea te . However, b inuclea te ce l ls do no tar ise under these condi t ions (Lew, D. , unpubl ished resul ts ;a lso see Fig . 7) . Second, severa l mutant yeas t s t ra ins havebeen d escribed tha t a re specif ica l ly defec t ive in e lemen ts ofthe ac t in cytosk ele ton (Welch e t a l . , 1994). S ome such mu-tants (e .g . , tpm/cel ls , lacking t ropomyos in) have a s ignif i -can t ly longe r gene ra t ion t ime than w i ld type ce l l s bu t a refu l ly v iab le and do no t p roduce s ign i f i c an t numbers o fbinuclea te ce l ls , sugg es t ing tha t the n uclear cycle has s loweddown to ac comm oda te the de fec t i n morphogenes i s (L iu andBretscher , 1992). Since there is no reason to suspect tha tt ropom yos in plays a d irec t ro le in ce l l cycle events o th er thanbud form at ion, th is sugges ts the exis tence of a checkp ointcontrol tha t de tec ts budding defec ts and delays ce l l cycleprogress ion.In contras t , tempera ture-sens i t ive cdc24 mutan t s tha t a recomple te ly unab le to po la r ize the ac t in cy tos ke le ton a t t heres t r ic t ive tempera ture cont inued the nuclear cycle despi tethe inabi l i ty to form a bud (H artwel l e t a l . , 1974; Adam s e ta l . , 1990). In th is report we have reexam ined the effec t ofcdc24 and o the r m u tan t s tha t p reven t budd ing on ce l l cycleprogress ion. W e f ind tha t there is a drama tic ce l l cycle de layin these mutants , and tha t th is de lay is la rgely due to phos-pho ryla t io n of tyros ine 19 of Cdc28. These f indings ident i fya new c lass of checkp oint control respons ive to defec ts in ce l lmorphogenes i s , and s uggest a ro l e fo r Cdc28 ty ros ine phos-phory la t ion in budd ing yeas t .Materials and MethodsYeast StrainsThe yeas t s t ra ins used in th i s s tudy are l i s t ed in Tab le I . S t ra ins 5011-JH01and JPT19-H01 (g i f t s f rom J . Pr ing le , Un iver s i ty o f Nor th C aro l ina , Ch apelHi l l , NC ) are es sen t i a l ly i sogen ic to C276 (Ada ms e t a l . , 1990) . A l l DLYs t ra ins a re in the BF264-15DU background (add, his2, leu2-3, 112, trp1-1%ura3Dns), o r w e r e c o n s t r u c t e d b y b a c k c r o s s i n g cdc24 a l l e l e s ( f r o m t h eC 2 7 6 b a c k g r o u n d ) o r t h e cdc424 al l e l e ( f rom DJTD2-16A; a g i f t f rom D.J o h n s o n , U n i v e r s it y o f V e r m o n t , B u r l i n g to n , V T ) i n t o t h e B F 2 6 4 - 1 5 D Ubackgro und a t l eas t f ive t imes , and are there fo re es sen t i a l ly i sogen ic . The

Table L Yeast Strains

cdc28::LEU2 d i s r u p t io n w a s c o n s t r u c t e d b y r e p l a c i n g a X h o l / S a c I f r a g -m e n t o f C d c 2 8 ( c o n t a i n in g 1 /2 t h e c o d i n g r e g i on ) w i t h a S a l I / S a c I f r a g m e n tc o n t a i n i n g L E U 2 ( p r e v i o u s l y c l o n e d a s a S a l I / X h o I f r a g m e n t i n t o t h ep U C l 9 S a l I s i t e ) . GALI::CLBa n d GALI::CLN2p l a s m i d s w e r e i n t e g r a te dat t he LEU 2 locus as descr ib ed (Ghiara e t a l . , 1991 ; Lew e t a l . , 1991 ; S tue-l and e t a l . , 1993). pGALI::M1HI and pGALl::weel (g i f t s f rom P. Russe l l ,T h e S c r i p p s R e s e a r c h I n s t it u t e , L a J o i l a , C A ) c o n t a i n t h e MIH1 o r weelgenes (Russe l l e t a l . , 1989) c loned in to YCpG2. pCDC28ngp (a g i f t f romP . S o r ge r , M I T , C a m b r i d g e , M A ) i s a T R P l - m a r k e d c e n t r o m e r i c p l a sm i ddescr ib ed by Sorger and M urray (1992) . S t ra ins were cons t ru c ted us ings t a n d a r d y e a s t g e n e ti c p r o c e d u r e s ( S h e r m a n e t a l . , 1 9 82 ) .C e l l Sy nc hrony and F low C y tome t ryC e l l s w e r e g r o w n i n y e a s t n i t r o g e n b a s e ( D i f c o L a b o r a t o r i e s I n c . , D e t r o i t,M I ) s u p p l e m e n t e d w i th 2 % s u c r o s e o r r a t ti n o s e a n d 0 .5 % c a s a m i n o a c i d s ,to a dens i ty o f 5 -10 106 ce l l s /ml a t 25C. 1 -3 l i te rs o f ce l l s were ch i l l edon i ce , son ica ted , and e lu t r i a t ed as descr ib ed (Lew and Reed , 1993) . G1d a u g h t e r c e l l s w e r e h a r v e s t e d b y c e n t r if u g a t io n a n d r e s u s p e n d e d i n Y E P(1% yeas t ex t rac t , 2% b acto -p ep ton e , 0 .005% aden ine , 0.005% urac i l ) sup -p l e m e n t e d w i t h 2 % d e x t r o s e ( Y E P D ) o r g a l a c t o s e (Y E P G ) a s i n d i c a t e d , t oa f ina l dens i ty o f 2 - -4 106 ce l l s /ml . A f t er i ncubat ion , c e l l s were f ixed ,s t a i n e d w i t h p r o p i d iu m i o d i d e , a n d a n a l y z e d b y f l o w c y t o m e t r y e x a c t l y a sd e s c r i b e d ( L e w e t a l . , 1 9 92 ) . P r o p o r t i o n o f c e l l s w i t h G 1, S , a n d G 2 D N Acon ten t s were es t im ated us ing LYSYS I I so f tware in two ways : wi th h i s to -g r a m m a r k e r s o n F L 3 h i s t o g r a m s , o r w i t h r e g i o n s o n F L 3 v e r s u s F S C d o tp lo t s ( see Lew e t a l . , 1992). T hese gave iden t i ca l resu l t s . Fo r the exper im en to f F ig . 7 , c e l l s w e r e g r o w n o n Y E P S ( Y E P + 2 % s u c r o s e ) a t 3 0 C , a n da- fac to r was added to a f ina l concen t ra t ion o f 50 ng /ml . 1 .5 h l a t e r ga lac tosew a s a d d e d t o 2 % , a n d t h e c e l l s w e r e i n c u b a t e d f o r o n e m o r e h o u r . T h e c e l l sw e r e t h e n h a r v e s t e d b y c e n t ri f u g a t io n a n d r e s u s p e n d e d i n f r e s h Y E P S +2 % galac tose wi th no o r - fac to r ( t ime zero ) . DA PI s t a in ing was as desc r ibedby McKinney e t a l . (1993) .Qua nt i ta t ion o f Chro mos ome Segregationand C el l Viabi l i tyC e l l s s t a i n e d w i t h p r o p i d i u m i o d i d e w e r e o b s e r v e d u s i n g a n A x i o p h o t p h o -tom icrosc ope wi th a 1 00x ob jec t ive (Car l Zei s s , Inc . ) . A t l eas t 200 ce l l swere sco red fo r each t imepo in t as fo llows : (a ) wi ld type ce l l s : f i r s t , t he per -c e n t o f c e l l s w i t h s e g r e g a t e d c h r o m o s o m e s ( tw o e q u a l p r o p i d i u m s t a i n e dm a s s e s , i n m o t h e r a n d b u d ) w a s q u a n t i ta t e d . N e x t , t h e p e r c e n t o f c e l ls t h a th a d u n d e r g o n e n u c l e a r d i v i s io n a n d c e l l s e p a r a t i o n w a s d e t e r m i n e d b ycoun t ing ce l l numbers ( fo r t h i s , s epara t e samples were ad jus t ed to 3 .7%f o r m a l d e h y d e , a n d t h e n u m b e r o f c e l l s /m l w a s c o u n t e d u s i n g a h e m o c y t o m -e t e r ) a c c o r d i n g t o t h e f o r m u l a :

No . o f ce l l s ( t) - No . o f ce l l s (0 )Percen t d iv ided ce l l s ( t ) = No . o f ce l l s (0 )T h e c u m u l a t iv e p e r c e n t c h r o m o s o m e s e g r e g a t i o n w a s t h e n c a l c u l a te d b ya d d i n g t h e p e r c e n t o f c e i ls w i t h s e g r e g a t e d c h r o m o s o m e s t o t h e p e r c e n t o f

Strain Relevant Genotype SourceC 2 7 6 M A T a / M A T ~ t J . P r i n g le5 0 1 1 - J H 0 1 M A T a / M A T a , cdc24-1/cdc24-1 J . P r i n g l eJ P T 1 9 - H O 1 M A T a / M A T a , cdc24-4/cdc24-4 J . P r i n g l eD L Y 6 64 M A T a M A T a , cdc24-4/cdc24-4 T h i s s t u d yD L Y 6 68 M A T a / M A T a , cdc24-4/cdc24-4, GALI::CLBI(LEU2) T h i s s t u d yD L Y 6 69 M A T a / M A T a , cdc24-4/cdc24-4, GALI::CLB2(LEU2) T h i s s t u d yD L Y 6 7 0 M A T a / M A T c ~ , cdc24-4/cdc24-4, GALl::CLB3(LEU2) T h i s s t u d yD L Y 6 72 M A T a / M A T u , cdc24-4/cdc24-4, GALI::CLN2(LEU2) T h i s s t u d yD L Y 6 7 3 M A T a / M A T t ~ , cdc24-4/cdc24-4, pGALI::MIH1 T h i s s t u d yD L Y 6 8 5 M A T a / M A T c ~ , cdc24-4/cdc24-4, cdc28::LEU2/cdc28::LEU2 pCDC28 mr T h i s s t u d yD L Y 1 2 9 M A T a / M A T ~ , cdc28::LEU2/cdc28::LEU2, pCDC28 r~gr T h i s s t u d yD L Y 6 8 1 M A T a / M A T c e , cdc42-1/cdc42-1 T h i s s t u d yD L Y 5 M A T a / M A T c ~ D . L e wD L Y 1 M A T a , barl D . L e wD L Y 3 4 4 M A T a , barl GALI : :CLBI (LEU2) D. LewD L Y I 0 0 6 M A T a / M A T u , pGALl::weel D. Lew

The Journal of Cell Biology, Volume 129, 1995 740


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d i v i d e d c e l l s . T h i s i n d e x b e c o m e s a n o v e r e s t i m a t e a t la t e r t i m e p o i n t s w h e ns o m e c e l ls s t a r t t o e n t e r m i t o s i s f o r t h e s e c o n d t i m e , s o d a t a f o r w i l d t y p ec e l ls a r e n o t p l o t t ed b e y o n d 9 0 - 1 2 0 m i n . ( b ) M o r p h o g e n e s i s m u t a n t s : s i n c et h e s e c e l l s d o n o t f o r m a b u d o r d i v i d e , t h e p e r c e n t c h r o m o s o m e s e g r e g a t i o ni s s i m p l y c a l c u l a t e d b y t h e f r e q u e n c y o f b i n u c l e a t e c e i ls . A t l a t e r t i m e p o i n t si n s o m e e x p e r i m e n t s ( w h e r e t h e c h e c k p o i n t w a s o v e r r i d d e n ) , s i g n i f i c a n tn u m b e r s o f t e t r a n u c l e a t e c e l l s w e r e o b s e r v e d . S i n c e t h e p e r c e n t c h r o m o -s o m e s e g r e g a t i o n i s a c u m u l a t i v e m e a s u r e , t h e s e w e r e i n c l u d e d . T h e r e a r et h r e e p o t e n t i a l s o u r c e s o f e r r o r i n t h i s m e a s u r e m e n t : ( i ) I f c h r o m o s o m es e g r e g a t i o n o c c u r s p e r p e n d i c u l a r t o t h e p l a n e o f f o c u s , a b i n u c l e a t e c e l lm i g h t b e s c o r e d a s m o n o n u c l e a t e . B e c a u s e t h e s e c e l l s t e n d to g r o w l a r g ea n d t h e c h r o m o s o m e s a r e g e n e r a l l y w e l l s e p a ra t e d , s u c h i n s t a n c e s w e r e e a s -i l y d e t e c te d b y f o c u s s i n g u p a n d d o w n , s o t h i s s o u r c e o f e r r o r i s p r o b a b l ynegl ig ib le . ( i i ) A t l a t e r t i m e s c e ll l y s i s o c c u r r e d i n m a n y c a s e s . C e l l b o d i e sw i t h o u t n u c l e i w e r e n o t c o u n t e d i n o u r s c o r i n g , s o i f l y s i s p re f e r e n ti a l l ya f f e ct e d m o n o - o r b i n u c l e a t e c e l l s , t h i s w o u l d s i g n i f i c a n t l y s k e w o u r e s t i -m a t e s . W e d i d , i n f a c t, o b s e r v e t h a t i n m a n y i n s t a n c e s t h e p e r c e n t c h r o m o -s o m e s e g r e g a t i o n d e c r e a s e d a t v e r y l a t e t i m e s , s u g g e s t i n g t h a t b i n u c l e a t ec e l l s w e r e p r e f e r en t i a l l y l y s i n g . ( i i i ) T h e t h i r d s o u r c e o f e r r o r c o m e s f r o mt h e f a c t t h a t t h e s t a r l i n g e l u t r i a t e d p o p u l a t i o n s w e r e c o n t a m i n a t e d w i t h2 - 1 0 % b u d d e d c e l l s, d e p e n d i n g o n t h e e x p e r i m e n t . W e e x c l u d e d b u d d e dc e l l s f r o m o u r c o u n t s , b u t a l a t e r t i m e p o i n t s ( f o l l o w in g c e l l d i v i s i o n o f t h e s eb u d d e d c e l l s ) t h e s e w o u l d c o n t r i b u t e a n e x c e s s o f m o n o n u c l e a t e d c e l l s . T ot r y t o a v o i d s i g n i f i c a n t e r r o r s , e l u t r i a t i o n s w e r e r e p e a t e d t o o b t a i n t h e p u r e s ts t a r l i n g p o p u l a t i o n s p o s s i b l e ( < 5 % b u d d e d c e l l s ) .

T h e s e s o u r c e s o 1~ r r o r ( p a r t i c u l a r ly l y s i s ) w o u l d l e a d t o u n d e r e s t i m a t e so f t h e p e r c e n t c h r o m o s o m e s e g r e g a t i o n a t l a t e t i m e s . I n c a s e s w h e r e t h e p e r -c e n t c h r o m o s o m e s e g r e g a t i o n d e c r e a s e d a t l a t e t i m e s t h e d a t a w e r e d e e m e du n r e l i a b l e a n d w e r e n o t p l o t t ed . E v e n s o , i t s h o u l d b e b o r n e i n m i n d t h a ta l t h o u g h t h e c u m u l a t i v e p e r c e n t c h r o m o s o m e s e g r e g a t io n r a r e ly r o s e a b o v e7 0 - 8 0 % b y o u r m e a s u r e , t h e t r u e e f f ic i en c y o f c h r o m o s o m e s e g r e g a t io n w a sp r o b a b l y h i g h e r .Ce l l v iab i l i ty w as a ssayed a s fo l lows: 7 tz l of the ce l l cu l ture was d i lu tedi n t o 2 m l o f Y E P D o n i c e a n d m i x e d b y v o r t e x in g . S i n c e t h e c e l l s h a d b e e ns o n i c a t e d p r i o r t o e l u t r i a t i o n , t h i s s t e p w a s n o t r e p e a t e d . 0 . 1 m l o f t h ed i l u t e d c u l t u r e w a s p l a t e d o u t o n t o Y E P D - a g a r , a n d t h e p l a t e s w e r e k e p tf o r 2 d a t r o o m t e m p e r a t u r e . D u p l i c a t e d i l u t i o n s a n d p l a t i n g s w e r e p e r -f o r m e d f o r e a c h t i m e p o i n t . C o l o n y n u m b e r s p e r p l a te w e r e c o u n t e d , a n dt h e a v e r a g e s w e r e n o r m a l i z e d t o t h e n u m b e r s a t t i m e z er o , d e s i g n a t e d1 0 0 % .Analysis of RNA and Protein LevelsP r o t o c o l s f o r R N A e x t r a c t i o n , f o r m a l d e h y d e - a g a r o s e g e l s , a n d N o r t h e r nb l o t t i n g w e r e a s d e s c r i b e d ( R e e d e t a l . , 1 9 8 2 ; E l d e r e t a l . , 1 9 8 3 ; S a m b r o o ke t a l . , 1 9 8 9 ) . P r o b e s ( D N A f r a g m e n t s c o n t a i n i n g t h e e n t i r e c o d i n g r e g i o n so f C L B 1 - 3 , C L N 2 , o r A C / / ) w e r e l ab e l e d u s i n g ra n d o m p r i m e r l a b e li n g k i tsa c c o rd i n g t o t h e m a n u f a c t u r e r 's r e c o m m e n d a t i o n s ( B o e h r i n g e r M a n n h e i mB i o c h e m i c a l s , I n d i a n a p o l i s , I N ) .

C e l l e x t r a c t s w e r e m a d e a s d e s c r i b e d ( S t u e l a n d e t a l . , 1 9 9 3 ) , a n d t o t a lp r o t e i n w a s q u a n t i t a t e d u s i n g t h e B i d R a d a s s a y ( B i d R a d L a b o r a t o r i e s ,R i c h m o n d , C A ) . 5 0 # g o f p r o t e in w a s l o a d e d p e r l a n e o n t o 1 1% p o l y a c r y l -a m i d e g e l s a n d b l o t t e d a s d e s c r i b e d ( S t u e l a n d et a l . , 1 9 9 3 ) . A n t i b o d i e s w e r ea s f o l l o w s : c t - P S T A I R E , m o u s e m o n o c l o n a l , u s e d a t 1 : 2 0 , 0 0 0 d i l u t i o n( Y a m a s h i t a et a l . , 1 9 9 2 ) ; a - C l b 2 , a f f i n it y - p u r if i e d r a b b i t p o l y c l o n a l , u s e da t 1 : 2 0 0 d i lu t i o n ( G r a n d i n a n d R e e d , 1 9 9 3 ) ; a - C I b 3 , a f f i n it y - p u r if i e d r a b b i tp o l y c l o n a l , u s e d at 1 : 2 0 0 d i lu t i o n ( G r a n d i n a n d R e e d , 1 9 9 3 ) ; a - p h o s p h o -t y r o s i n e , m o u s e m o n o c l o n a l 4 G 1 0 ( U p s t a t e B i o t e c h n o l o g y I n c . , L a k eP l a c i d , N Y ) , u s e d a t 1 : 1, 00 0 d i l u t io n . H R P - c o n j u g a t e d a - m o u s e o r t ~ - ra b b i ts e c o n d a n t i b o d i e s w e r e u s e d a t 1 : 3, 00 0 d i l u t i o n . E C L k i t s ( A m e r s h a mC o r p . , A r l i n g t o n H e i g h t s , I L ) w e r e u s e d a c c o r d i n g t o m a n u f a c t u r e r ' s i n -s t r u c t i o n s f o r d e t e c t i o n o f H R P a c t iv i t y .p 1 3 b e a d s w e r e p r e p a r e d a s d e s c r i b e d ( D u n p h y e t a l . , 1 9 8 8 ) , a n d i n -c u b a t e d w i t h 1 .3 m g o f e x t r a ct f o r 3 0 m i n a t r o o m t e m p e r a t u r e . E x t r a c t i o nb u f f e r s a n d w a s h i n g c o n d i t i o n s w e r e e x a c t l y a s d e s c r i b e d ( K o r n b l u t h e t a l . ,1992) .Ana lys is of His tone H1 Kinase Ac t iv i tyand cdc25 TreatmentI m m u n o p r e c i p i t a ti o n a n d h i s t o n e H 1 k i n a s e a s s a y s w er e p e r f o r m e d a s d e -s c r i b e d ( S t u e l a n d e t a l . , 1 9 9 3 ) , s t a r l i n g w i t h 1 0 0 / ~ g o f c e l l e x t ra c t . H i s t o n eb a n d s w e r e e x c i s e d f r o m t h e g e l s a n d r a d i o a c t i v i t y w a s q u a n t i t a t e d b yC e r e n c o v c o u n t i ng . R e c o m b i n a n t S . p o m b e c d c 2 5 p r o t e i n ( a g i f t f r o m C . H .

M c G o w a n , T h e S c r i p p s R e s e a r c h I n s t i t u t e, L a J o l l a , C A ) w a s p r e p a r e d a n du s e d a s d e s c r i b e d ( M i l l a r e t a l ., 1 9 91 ). M o c k t r e a t m e n t w a s i d e n t i c a l e x c e p tt h a t n o c d c 2 5 w a s a d d e d .

Resul t sA G2 Delay in the Cell Cycle of Mutants Defective inCell MorphogenesisT o exami ne t he e f f ec t s o f de l ayed budd i ng on ce l l cyc l ep r ogr es s i on w e m ade use o f t emper a t u r e s ens i t i ve cdc24 an dcdc42 strains. CDC42 e n c o d e s a r a s -r e l a te d G T P a s e o f t h eR ho / R ac f am i l y ( Johnson and Pr i ng l e , 1990) and CDC24en-c o d e s a G D P / G T P e x c h a n g e f a c t o r fo r C d c 4 2 ( Z h e n g e t a l . ,1994) . T he mu t an t s a r e unab l e t o po l a r i ze t he ac t i n cy t oske l -e t on o r f o r m a bud a t t he r e s t r ic t i ve t emper a t u r e ( S l oa t e t a l .,198 t ; A dams e t a l . , I 990) . T he se mut an t s t ra i ns and i so -gen i c w i l d t ype con t r o l s w er e g r ow n a t t he pe r mi s s i ve t em-pe r a t u r e , and sma l l G 1 daugh t e r ce l l s w er e i so la t ed by cen-t r if uga l e l u t r ia t i on . T h e sync hr on i zed G 1 popu l a t i ons w er et hen i ncuba t ed a t t he r e s t r i c t i ve t emper a t u r e , and ce l l cyc l ep r o g r e s si o n w a s m o n i t o r e d b y f lo w c y t o m e t r y a n d f l u o r e s-cence mi c r osc opy o f p r op i d i um i od i de s t a ined ce l ls . C o m-pa r ed t o t he w i l d t yp e con t r o l s , t he b udd i ng- de f ec t i ve s t r a insw er e de l ayed i n ce l l cyc l e p r ogr es s i on , w i t h a de l ay o f abou tt w o h o u r s b e tw e e n c o m m e n c i n g D N A r e p l ic a t io n a n d c o m -p l e t ing ch r o mo som e seg r ega t ion ( F i g . 1 A ) .T o de t e r mi ne t he na t u r e o f t he ce l l cyc l e de l ay i n mor e de -t a i l, G 1 pop u l a t i ons o f w i l d t ype and i sogen i c cdc24 cel l sw er e i so l a t ed on t he s ame day us i ng i den t i ca l e l u t r i a t i onpa r ame t e r s , and m oni t o r ed a s above . T h ese s t r a i ns i n it i a tedand compl e t ed D N A r ep l i ca t i on w i t h i nd i s t i ngu i shab l e k i -ne t i c s , bu t ch r omosome seg r ega t i on w as de l ayed f o r t w ohour s i n t he cdc24 cel l s (Fig. 1 B) . Sim i lar resul t s were ob-ta ined wi th i sogenic wi ld type and cdc42 strains (not shown).I m m u n o f l u o r e s c e n c e m i c r o s c o p y o f t h e cdc24 cel l s us ingan t i - ~ - t ubu l i n an t i body show ed t ha t du r i ng t he de l ay mos tce l l s ( > 80%) had a s sembl ed a shor t i n t r anuc l ea r sp i nd l echa r ac t e r i s t ic o f G 2 ce l ls , con f i r mi ng t he r e su l t s o f A damset a l . (1990) . Th us , cdc24 and cdc42 cel l s di splay a speci f icG2 delay in the cel l cycle .T he G 2 de l ay obse r ved i n t hese mut an t s cou l d be due t oan i ndependen t r o l e f o r t he C dc24 and C dc42 p r o t e i ns i nchr om osom e seg r ega t ion , i n add i t ion t o t he i r r o l e i n gene r a t-i ng ce i l po l a r it y . T o a sk w he t h e r C dc2 4 p l ayed such a r o l e ,w e d e t e r m i n e d w h e t h e r b u d d e d cdc24 cel l s a l so delayedchr o mo som e seg r ega ti on a t the r e s t r i c t ive t emper a t u r e . W i l dt y p e a n d cdc24ce l l s w er e a r r e s t ed a t t he pe r mi s s i ve t emper -a t u r e us i ng t he d r ug hydr oxyur ea . T h i s d r ug i nh i b i t s D N Ar ep l i ca t i on , and t he ce l l s a r r e s t w i t h f u l l y f o r med ma t u r ebuds . T he ce l l s w er e t hen sh i f t ed t o t he r e s t r ic t i ve temp er a -t u r e , and one hour l a t e r t he d r ug w as w ashed ou t . U nde rt hese cond i t i ons , cdc24ce l l s p r ogr es sed t h r ough D N A r ep l i -ca t i on and ch r omosome seg r ega t i on w i t h t he s ame k i ne t i c sa s t he w i l d t ype ce l ls , show i ng t ha t C dc24 i s no t r equ i r ed f o rt i me l y ch r omosome seg r ega t i on i n budded ce l l s ( F i g . 2 ) .S i mi l a r r e su l t s w er e ob t a i ned w i t h cdc42 mut an t s ( no tshow n) . T h i s sugges ts t ha t t he G 2 de l ay obse r ved i n t he un -budded mut an t s ( F i g . 1 ) w as no t an i ndepende n t e f f ec t o f t hemut a t i ons , bu t r a t he r w as due t o t he depo l a r i zed cy t oske l e -t on o r t he absence o f a bud .

Lew and Reed A M o r p h o g e n e s i s C h e c k p o i n t i n Y e a s t 74 1


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Figure 1. A G2 delay in thecell cycle of mutants defectivein cell morphogenesis. (A) (31daughter cells from diploidwild type (WT-1, C276; WT-2,DLY5), cdc24-1 (5011-JH01,isogenic to WT-1), and cdc42-1(DLY681, isogenic to WT-2)strains were isolated by centri-fugai elutriation as describedin Materials and Methods,and inoculated into YEPD at36C (C276, 50 11-JH01) or37.5C (DLY5, DLY681). Ali-quots were taken at the indi-cated times, stained with pro-pidium iodide, and analyzedby flow cytometry or fluores-cence microscopy. (e) Per-centage of cells that had initi-ated DNA replication (DNAcontent >2C by flow cytome-try). (o ) Percentage of cells that had completed chro mosom e segregation (quantitated as described in Materials and Methods). (B) Iso-genic wild type (C276: o) and cdc244 (5011-JH0h e) cells were analyzed as above. (Top)Percenta ge of cells that had initiated DN A repli-cation. (Middle)Percentage of cells that had com pleted DN A replication. (Bottom)Percentage of cells that had completed chromosomesegregation. Synchrony experiments were pe rformed a minimum of three times for each strain, with consistent results.

Figure 2. Cdc24 is not required for timely chromoso me segregationin budded cells. Wild type (C276: o) and isogenic cdc24-1 (5011-JH01: *) cells were incubated in YEPD supplemented with 0.2 Mhydroxyurea for 3 h at 25C, and then shifted to 36C fo r I h, asindicated in the d iagram (top). At this point, 95 % of the wild ty pecells and 93 % of the cdc24cells had large buds and a single, un-divided mass of DNA (assessed by propidium iodide staining). Thecells were then harvested by centrifugation and resuspended in pre-warmed YEPD at 36C (time zero in the graph at bottom). Aliquotswere taken at the indicated times, stained with p ropidium iodide,and analyzed by flow cytometry o r fluorescence microscopy. Flowcytometry revealed that wild type and cdc24 cells (>90%) repli-cated their DNA within 45 min (shaded bar),and then underwentchromosome segregation with similar kinetics. This experimentwas performed twice, with consistent results.

Activation o f Clb2/Cdc28 and Clb3/Cdc28 Kinases IsDelayed in cdc24 CellsThe G2 delay in budding-defect ive mutants could ar i se as acheckpoint cont rol of cell cycle progress ion, or a l ternat ivelyas a "subst rate-product" dependency (Har twel l and Weiner t ,1989) . For example, the depolar ized act in cytogkeletoncould resul t in a mechan ical defect in spindle e longat ion. Inthis second model , Cdc28 would t r igger anaphase wi th nor-mal kinet ics , but t ranslat ion of this s ignal into vis ible chro-mos om e segregat ion would be delayed because of a defect inspindle funct ion resul t ing f rom the depolar ized act in cyto-skeleton. To determine w hether the delayed chr omo som esegregat ion in cdc24 mutants was associated wi th Cdc28regulat ion, w e f i rs t examined the kinet ics of act ivat ion ofCdc28 assoc iated wi th Clb2. S ince nuclear divis ion i s t r ig-gered by the Clbl and Clb2 pai r of cycl ins (of which Clb2is more impo r tant : F i tch et a l . , 1992; Richardson et a l . ,1992) , a G2 delay could come about by inhibi t ion ofClbl ,2/Cdc28 act ivat ion. Wild type and cdc24 G1 cells wereisolated by cent r i fugal e lut r ia t ion as before and incubated atthe res t r ic tive temperature . Cel l ext racts were prepar ed f ro msamples harvested at 30-rain intervals , and Clb2/Cdc28complexes were immunoprecipi ta ted us ing Clb2-speci f ic an-t ibodies . The kinase act ivi ty associated wi th these com-plexes was then assayed in vi t ro us ing the s tandard tes t sub-s t ra te , his tone H1. In wi ld type cell s Clb2/Cdc 28 kinase wasinduced to a peak at 2 .5 h, shor t ly af ter DNA repl icat ion(Fig. 3, A and B) . However , Clb2/Cdc 28 kinase induct ion incdc24 cel l s was delayed by two hours (F ig. 3 B) . This delayin kinase induct ion closely matched the delay in chromo-some segregat ion (F ig. 1 B) .

We also examined the kinet ics of induct ion of Clb3/Cdc 28kinase act ivi ty. Al though the Clb3 and Clb4 p ai r of cycl insare not essent ia l , they can become impor tant for cel l cycleprogress ion when other Clb cycl ins are compromised (F i tch



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et al., 1992; Richardson et al., 1992). In agreement withprevious studies (Grandin and Reed, 1993), we found thatClb3/Cdc28 kinase activity accumu lated slightly earlier thanClb2/Cd c28 kinase activity in wild type cells, peaking at 2 h(Fig. 3 C). In cdc24 cells, however, induction of Clb3/Cdc 28kinase activity was delayed, although n ot to the same deg reeas Clb2/Cdc28 kinase activity (compare Fig. 3, B and C).Clb/Cdc28 Activit y Is Inhibit ed by Transcriptional andPosttranslationalMechanismsTo determ ine the basis for the delayed induction o f kinase ac-t ivi ty, we monitored the accumulat ion of Clb2 and Clb3 pro-teins in cdc24 cells. Since w ild type cells enter a secon d cellcycle fol lowing nuclear divis ion, we cannot direct ly com parekinase activity in wild type (seco nd cycle) versus cdc24 (firstcycle) cells at late time points. T herefo re, wild type cells thathave been elutriated and incu bated for the same perio d in thepresence of the ant i -microtubule agent nocodazole were usedas a control. These cells accumulate in mitosis of the firstcycle, allowing comparison with cdc24 cells delayed in thefirst cycle, cdc24 cells accumulated less Clb2 protein thanthe controls , which accounted for at leas t part of the ob-served lack of Clb2/Cdc28 kinase act ivi ty during the G2delay (Fig. 4 A). Comparison of CLB2 mRNA levels re-vealed that there was also a delay in CLB2 mRNA induct ion(Fig. 4 B: scanning densitometry indicated that the CLB2/ACT/ratio reached similar levels at 2 h in control cells and3 h in cdc24 cells). Although delayed mRNA induction ac-counts for a part of the delay in Clb2-associated kinase in-duction, it probably does no t account for all of it since mR NAaccumulat ion was only delayed by one hour (Fig. 4 B),while kinase induction was delayed by two hours (Fig. 3 B).

In contrast to the results with Clb2 , the levels of Clb3 pro -tein (Fig. 4 A) and mRNA (not shown) were not lower incdc24 than control cells. Thus, the approximately twofoldlower level of Clb3-associated kinase activity in these cellsmust be due to posttranslational regulation. Since inhibitorytyros ine phosphorylat ion of Cdks has been implicated incheckpoint controls in other organisms (see Introduction),we speculated that such a mechanism might account for theinhibition of Clb3-associated kinase activity, and the residualinhibition of Clb2-associated kinase activity, in cdc24 cells.To determine whether Cdc28 tyros ine phosphorylat ion oc-curred in cdc2,1cells, wild type or cdc24 cells were shiftedto the restrictive temperature for 2 or 3 h. Cdc28 was en-riched using pl 3-Sepha rose beads (often used as an affinityreagent for purifying cdc2 homologs: Dunphy et a l . , 1988),and analyzed by Western blot t ing with ant i -phosphotyros ine

Figure 3. Activation of Clb2/Cdc28 and Clb3/Cdc28 kinases isdelayed in cdc24cells. (A-C) Wild type (C276: o) and isogeniccdc24-1 (5011-JH01: o) GI daughter cells were isolated by centrifu-gal elutriation as described in Materials and Methods, and inocu-lated into YEPD at 36C. A liquots taken at the indicated times wereprocessed for flow cytometry, RNA analysis, or protein analysis asdescribed in M aterials and M ethods. (A) The percent of cells with

4C DNA content as measured by flow cytometry is show n. (B)Clb2/Cdc28 complexes were immunoprecipitated from cell ex-tracts as described in Materials and Methods, and kinase activitywas assayed in vitro with the test su bstrate historic H1. Autorad iog-raphy o f the 32P-labeled histone (which runs as a smeary doubleton 11% SDS-PAGE) s shown below, and the historic-assoc iated ra-dioactivity is quantitated in the graph above. (C) Clb3/Cdc28 com-plexes were immunoprecipitated and kinase activity was assayed asin R Extracts and kinase assays were performed with duplicate cellpellets from a single experiment, and gave identical results. Consis-tent data were obtained in independent experiments with lessdetailed time-courses.

L e w a n d R e e d A Morphogenesis Checkpoint in Yeast 7 4 3


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Figure 4. Clb/Cdc28 activityis inhibited by transcriptionaland posttranslational mecha-nisms. (A) Wild type (C276)and isogenic cdc244 (5011-JH01) G1 daughter cells wereisolated by centrifugal elutria-tion and incubated as in Fig. 3except that nocodazole wasadded to the w ild type cells toa fina l concentration of 15/~g/mL, from a 10 mg/mLstock in DMSO (this was donein parallel with the experi-ment of Fig. 3). The cellswere incubated at 36C for2.5 h (at which time all thecells had a 4C DNA content),and processed for proteinanalysis as described in Ma-terials and Methods. Clb2/Cdc28 (left) and Clb3/Cdc28(right) complexes were immu-noprecipitated from 100 t~g ofcell extract and assayed forhistone H1 kinase activity(top). The percentages shownreflect the radioactivity incor-porated into historic, normal-ized to the nocodazole treated wild typ e samples (100% = 300,376 cpm for Clb2, 208,193 cpm for Clb3). I n parallel, 50/zg o f cell extractwere separated by SDS-PAGE and imm unoblotted with c~-Clb2(middle, left), a-Clb3 (middle, right), or c~-PSTAIRE(bottom) antibodiesas described in Materials and Methods. Th e latter recognizes both Cdc28 (lowerband) and Pho85 (upperban d) and serves as a normaliza-tion control. (B) RN A was extracted from the same experiment as above, and processed as described in Materials an d Methods. N orthernblots were probed with radiolabeled CLB2 or AC T/(to control for RNA loading) probes, as indicated. (C) H istone HI kinase activityin Clb2/Cdc28 or Clb3/Cdc28 complexes from the same samples as in A was assayed following mock (- ) treatment or cdc25 phosphatasetreatment (+) as described in Materials and Methods. The A numbers reflect the percent change in kinase activity resulting from cdc25treatment for each sample. (D) Wild type (DLY5), cdc24 (DLY664), and cdc24 CDC28ngr (DLY685) cells were grown on YEPD andshifted to 36C for 2 or 3 h, as indicated. Cdc28 was enriched using p13 beads (Materials and Methods), separated by SDS-PAGE, andimmunoblotted with a-phosphotyrosine (top) or t~-PSTAIRE(bottom)antibodies. As a positive control, Cdc2 8 from cells (DLYI006) in-duced t o overexpress we.el for 3 h in galactose m edium is show n in the right hand lane. T his experiment was repeated using four different~-phosphotyrosine antibodies. Ap art from variability in signal strength and background bands, all antibodies gave the same result.

ant ibodies (Fig. 4 D) . As a posi t ive control , extracts wereprepared f rom cells that overexpressed the S. pombe wee1 ki-nase previously shown to phosph orylate cdc2 at tyros ine 15(corresponding to Cdc28 tyros ine 19) . As shown in Fig. 4D, Cdc28 f r om cdc24 cells gave an elevated s ignal with thephosph otyrosine antibody. This s ignal was abolished in con-trol cdc24 cells that contained a CD C2 8 r~grmutation, sug-gest ing that the s ignal arose f rom phosphorylat ion of tyro-s ine 19. The cdc25 ph osphata se of S. pombe has been shownto dep hospho rylate tyros ine 15 of edc2 an d act ivate i ts ki-nase (Dunphy and Kumagai , 1991; Gautier e t a l . , 1991;Millar e t a l . , 1991). Assumin g that this phosphatase wouldalso be ef fective on tyros ine 19-phosphorylated Cdc28, wet r ea ted the immunopr ec ip i ta ted Clb3/Cdc28 complexes withrecombinant cdc25 protein and then reassayed the his toneH1 kinase act ivi ty. This t reatm ent had no ef fect on the k inasef rom nocodazole- treated wild type cel ls (or asynchronouswild type cel ls : c dc25- treated k inase levels were within 5 %of untreated controls in s ix independent exper iments) , butthe Clb3/Cdc28 kinase f rom cdc24 cells were activated2-fold, to levels compa rable to the no codazole control (Fig.

4 C) . S imilar ly, Clb2-associated kinase f rom cdc24 cells(but not wild type cel ls ) could be s t imulated 53% by cdc25treatment, a l though given the very low Clb2 abundance inthese cel ls the s ignif icance of this result is less c lear (Fig. 4C) . S ince the ef fic iency of Cdc28 d ephosph orylat ion by theS. pombe cdc25 phosphatase is unknown, these results rep-resent minim um est imates of the degree of inhibit ion causedby tyros ine 19 phosphorylat ion in cdc24 ceils . Th ese exper i-ments show tha t Cdc28 tyr os ine phosphor y la t ion takes p lacein cdc24 cells , and accou nts for a t leas t par t of the delay inClb/Cdc28 act ivat ion. However , they do not show thatCdc28 tyros ine phospborylat ion is impor tant for generat ingthe G2 delay in these cel ls. No te that Cdc28 phosp horylat ionwas previously show n to oc cur in hydroxyurea- treated cei ls,but was no t impor ta nt for the cel l cycle block in those cel ls(Amon et a l . , 1992) .I t has been shown that Cdc28 tyros ine pbosphorylat ioncan inhibi t Clb2/Cd c28 com plexes but not C1rd/Cdc28 com-plexes (Booher et a l . , 1993) . Our ob servations extend thesef indings by showing that Clb3/Cdc28 complexes are alsosubject to this form of regulat ion.

The Journal of Cell Biology, Volum e 129, 1995 744


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Override o f the Delay in Chromosome SegregationTh e r esu lt s p r esen t ed ab o v e a r e co n s i s t en t w i th t h e h y p o th e-s i s t h a t t h e G2 d e l ay i n mu tan t s t h a t can n o t fo rm a b u d i sd u e t o t h e d e l ay ed i n d u c t i o n o f C lb /Cd c2 8 k in ases . Ho wev er ,i t i s a lso p o ss ib l e t h a t t h e G2 d e l ay i s cau sed i n d ep en d en t l y(e .g . , b y mech an i ca l p ro b l em s wi th sp in d l e fu n c t io n , a s a r -g u ed ab o v e) . T o r eso lv e t h is q u es t i o n we co n s t ru c t ed a se to f i so g en i c cdc24 s t r a in s ca r ry in g v ar i o u s cy c l i n g en esd r iv en b y t h e r eg u l a tab l e GA L/p ro m o te r ( see M ate r i a l s an dM eth o d s ) . Th i s p ro m o te r i s o ff wh en ce l l s a r e g ro wn in me-d iu m l ack in g g a l ac to se , b u t i s s t ro n g ly i n d u ced u p o n t r an s fe rt o g a i ac to se co n t a in in g med iu m. No te t h a t i n t h i s s t r a i nb ack g ro u n d (BF 2 6 4 -1 5 DU) , a so mewh at l o n g er (3 -4 h ) G2d e l ay was o b se rv ed i n cdc24 ce l l s (F ig . 5 ) . We r easo n ed t h a ti f i n h ib i ti o n o f cy c l i n accu mu la t i o n w ere r esp o n s ib l e fo r t h eG2 d e lay , t h en i n d u c t i o n o f h ig h l ev e l s o f v a ri o u s C lb s u s in gth e G A/ .2 p ro m o te r m ig h t e l imin a t e t h e G2 d e lay. In co n -t r as t , i f t h e d e l ay r esu l t ed f ro m a mech an i ca l d e fec t i n sp in -d l e e l o n g a t i o n , i t wo u ld n o t b e a f f ec t ed b y m an ip u l a t io n o fClb l ev e l s . S y n ch ro n o u s G1 p o p u l a t i o n s o f i so g en i c wi ldty p e , cdc24, and cdc24 GALI::CLB s t r a in s were i so l a ted asb efo re an d i n cu b a t ed a t t h e r es t r ic t i v e t emp era tu re i n g a l ac -t o se -co n t a in in g med ia . As sh o wn in F ig . 5 A , i n d u ced ex -p res s io n o f C lb l , C lb 2 , o r C lb 3 r esu l t ed i n t h e co mp le t ee l imin a t i o n o f t h e G2 d e l ay , so t h a t cdc24 ce l l s u n d erwen tch ro m o so m e seg reg a t io n a t th e sam e t ime as w i ld t y p e cel l s.In co n t r as t , i n d u ced ex p ress io n o f th e G1 cy c l i n C ln 2 h ad n oef f ec t, a s ex p ec t ed . T h ese r esu l t s imp ly t h a t t h e G2 d e l ay i sa co n seq u e n ce o f Cd c2 8 r eg u l a t i o n r a th e r th an a mec h an i ca ld e fec t in sp in d l e fu n c t i o n , an d s t ro n g ly su p p o r t t h e h y p o th e-s i s th a t t h e d e l ay co mes a b o u t t h ro u g h a r eg u l a to ry ch eck -p o in t co n t ro l r esp o n d in g t o so m e asp ec t o f th e mo rp h o g en e-s is defect .S in ce i n d u c t i o n o f CLB t r an sc r i p t i o n co u ld o v er r i d e t h eG2 d e l ay co mp le t e ly , o n e mig h t sp ecu l a t e t h a t t h e p red o mi -n an t cau se o f t h e G2 d e l ay was t h e d e l ay ed accu mu la t i o n o fCLB1 a n d CLB2 t ranscr ip ts . However , the level o f Clb syn-t h es i s f r o m t h e G A L / p r o m o t e r e x c e e d s th e l e v el n o r m a l lyat tained dur ing the cel l cycle , and i t i s possib le that sucho v erex p ress io n wo u ld l ead t o C lb l ev e l s h ig h en o u g h t o o v er -wh e lm o th e r fo rms o f r eg u l a t io n , su ch as t y ro s in e p h o s -p h o ry l a t i o n o f Cd c2 8 . In d eed , t h e f ac t t h a t i n d u c t io n o f h ig hlevels o f CLB3 mRNA co u ld o v er r i d e t h e G2 d e l ay (F ig . 5A) suggests that th is i s the case, s inc e Clb3 acc umu lat ion wasn o t d e l ay ed i n cdc24 cei l s (F ig . 4 A) . In add i t ion , recen tf ind ings ind icate that induct ion of CLB1 and CLB2 t r an -sc r i p t s i s l a rg e ly d u e t o a p o s i t i v e f eed b ack l o o p wh ereb yClb l ,2 /Cd c2 8 k in ase ac t i v i ty s t imu la tes CLB1,2 m R N A s y n-th es i s (Am o n e t a l . , 1 9 9 3 ). T h i s su g g est s t h a t t h e d e lay ed ac -cu mu la t i o n o f CLB2 t r an sc r i p t s co u ld o ccu r as an i n d i r ec tef fect o f post - t ranslat ional inh ib i t ion of Clb /Cd c28 k inases .To d e t e rmin e wh e th er Cd c2 8 t y ro s in e p h o sp h o ry l a t i o nco u ld acco u n t fo r a s i g n i f ican t p a r t o f t h e G2 d e l ay , we co n -s t ru c t ed s t r a ins i n w h ich t h e MIH1 g en e ( t h e Saccharomycescerevisiae h o m o l o g y o f the S. pombe cdc25 gene; Russel l e ta l . , 1 9 8 9 ) was ex p ressed f ro m th e GA L/p ro mo te r . We r ea -so n ed t h a t M ih l o v erex p ress io n wo u ld r esu l t i n d ep h o sp h o r -y l a t i o n o f Cd c2 8 t y ro s in e 1 9 , an d t h a t i f th i s was im p o r t an tfo r t h e G2 d e l ay, ch ro m o so m e seg reg a t io n wo u ld b e acce l e r -a t ed . Th i s was i n d eed t h e case (F ig . 5 B) : t h e majo r i t y o ft h e G2 d e l ay was ab o l i sh ed b y M ih l i n d u c t i o n . To en su re t h a t

A7 5 .

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- D - -3 4 5Time ( h )

M I H fW T, . W T y , ~ / 9 . . ~ : ', : : ; , H A Y , -

: / YEgN cdc24

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Figure 5. Override of he delay in chromosom e segregation. (A) Iso-genic wild type (DLY5, e), cdc24-4 (DLY664, a) , cdc24-4GAI_J::CLB1 (DLY668, o), cdc24-4 GALI::CLB2 (DLY669, o),cdc24-4 GA1.J::CLB3 (DLY670, - ) , and cdc24-4 GALI::CLN2(DLY672, zx) G1 daughter cells w ere isolated by centrifugal elutri-ation as described in Materials and Methods, and inoculated intoYEPD at 36C. Aliquots taken at the indicated times were stainedwith propidium iodide and the percent chromosome segregationwas quantitated as described in Materials and M ethods. (B ) Wildtype (DLY5, o), cdc24-4 (DYL664, a), cdc24-4 GALI::MIHI(DLY673, zx), CDC28ngF (DLY129, o), and cdc24-4 CDC28noF(DLY685, -) were analyzed as in A. Flow cytometry show ed thatthe CDC28ngFcells replicated their DNA about 15 min earlierthan the other cells: variations of this ma gnitude in the length ofG1 were not uncomm on between different experiments. Each syn-chrony experiment was performed at least twice, with consistentresults.t h is e f f ec t was d u e t o d e p h o sp h o ry l a t i o n o f Cd c2 8 t y ro s in e1 9, r a t h e r t h an so me o th er p u t a t i v e M ih l su b s t r a te , we co n -s t ru t t ed an i so g en ic cdc24 s t rain in which the CDC28 g en ewas r ep l aced wi th a m u tan t t h a t ch an g ed t h e t y ro s in e 1 9 r es i -d u e t o p h en y l a l an in e (CDC28ngr), t h u s r en d er in g Cd c2 8resis tan t to inh ib i t ion by th is pathway. Again , the major i tyo f t h e G2 d e l ay (2 .5 -3 h ) was ab o l i sh ed b y t h i s mu ta t i o n(F ig . 5 B ) . Th e r es id u a l G2 d e l ay ( ab o u t 4 5 m in r e l a t iv e t owi ld t y p e) seen i n t h ese s t r a i n s mu s t b e d u e t o so me o th eri n h ib i t o ry mech an i sm (p erh ap s t h e d e l ay ed CLB1,2 mRNAinduct ion ; see D iscussion) . T ogether , these resu l t s show thatd efec t s in b u d fo rmat io n t r i g g er a ch ec k p o in t co n t ro l t h a t d e -l ays ce l l cy c l e p ro g ress io n t h ro u g h Cd c2 8 r eg u l a t i o n , i nl a rg e p a r t b y p h o sp h o ry l a t i o n o f Cd c2 8 t y ro s in e 1 9.

Lew and Reed A Morphogenesis Checkpoint in Yeast 74 5


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c d c 2 4 C e ll s R e m a i n V i a b le a n d A b l e t o B u d d u r i n g t h eG 2 D e l a y

Bud form ation normally begins at about the G1/S transi t ion,not in G2 . I f the checlqaoint- induced G 2 delay is to be anef fect ive defence agains t condit ions that i rans iently incapaci-ta te cytoskeletal polar izat ion, then cel ls should be able toini t ia te bud formation in G2 once budding capacity is re-s tored. To assess the abil i ty of cel ls to form a bud fol lowinga transient bloc k, G1 cells of a cdc24 s train were incubatedfor var ious per iods at the res tr ic t ive temperature and thenshif ted down to the permiss ive temperature . This exper im entrevealed that cel ls that were shif ted down du r ing the G 2 de-lay per iod (1-3 h of incubation) budded remark ably rapidly,efficiently and sync hron ously (Fig. 6, A and B). If the cellswere main tained at the res tr ic tive temperature pas t the t im eof chromo som e segregation, however , only a f ract ion of thecells formed buds , and budding was s lower and less syn-chronous (Fig. 6, A and B) . This suggests that the checkpointdoes indeed m aintain the cel ls in a s ta te that is highly favor-able for ef f ic ient bud formation upon recovery f rom theblock.The cel ls that budded af ter nuclear divis ion (4-5 h shif t-down) were f ixed and s tained with DAPI 1-2 h af ter shif t-down (not shown) . In most cases , both of the nuclei ( i .e .,DAPI - s ta ined masses ) had d iv ided a long the mother - budaxis , yielding te tranucleate cel ls with binucleate mothersand buds . S ignificant minor i t ies of cel ls with two nuclei inthe mother and none in the bud, and with three nuclei in the

mother and one in the bud, were also observed.The result that G2 cel ls were able to bud ef f ic iently wassomew hat surpr ising, s ince we had previously found that theG1 cyclins, and par t icularly Clnl and Cln2, were impor tantto promot e cytoskeletal polar izat ion (Lew and Reed, 1993).Given the normal per iodici ty of Clnl ,2 exp ress ion, these cy-

cl ins would not be expected to b e present in (32 cells . To ex-amine this more closely, we mon itored CLN2 mRN A leve lsin synchronized cdc24 cultures incubated at the res tr ic t ivetemperature . Whereas wild type control cel ls t reated withnocodazole underwent a cycle of CLN2 express ion andrepress ion, CLN2 mR NA leve ls in cdc24 ceils were inducedand continued to r ise dur ing the G2 delay (Fig. 6 C), Thus,G1 cyclins that promote cyto skeletal polar izat ion accumulateto high levels dur ing the checkpoint- induced G2 delay, amechan ism that may help to maintain the readiness of thesecells to bud up on recovery f rom the cdc24 blo ck (see Fig. 8).We then exam ined cel l viabil i ty during incubation of syn-chronous cdc24 populat ions at the res tr ic t ive temperature .As expected, the cel ls remained viable dur ing the G 2 delay(Fig. 6 D ) . A f ter several hours viabil i ty was gradually los t ,but this loss occurred about an hour af ter the cel ls final ly un-derwent chromosome segregation, indicat ing that chromo-some segregation was not in i tself a le thal event . The cau seof the loss in viability is unclear, but it was accelerated byMIH1 overexpress ion (Fig. 6 D) or the CDC 28 ~gr mutation(not shown). Thus , some aspect o f continued cel l cycleprogress ion is le thal to cel ls that are unable to polar ize theactin cytoskeleton, and one ef fect of the checkpoint is to pro-tect cells against this lethal event.

Figure 6. Recovery of cdc24ceils from a transient arrest.(.4) cdc24-4 (JPT19-H01) G1daughter cells were isolatedby centrifugal elutriation asdescribed in Materials andMethods, and inoculated intoYEPD at 36C. At the indi-cated times, aliquots wereshifted down to 25C andbudding was monitored mi-croscopically at 20-rain inter-vals (o, t2, percent of cellsbudded). Chromosome segre-gation (e) was also moni-tored in the cells at 36C.(B) The same results as inA were replotted on an ex-panded time axis, with timezero corresponding to thetime of temperature shift. T henumber of hours at 36C priorto shift-down is indicated bythe numbers next to eachcurve. (C) The same North-ern blots as in Fig. 4 B wereprobed with CLN2 or AC//probes as indicated. Quantita-tion of these samples and sam-pies from an independent ex-periment indicated that at latetimes the cdc24-arrested cells accumulated 6- t o 20-fold higher levels of CLN2 RNA (normalized to the ACH control) than the peak levelsin wild type ceils. (D) cdc24-4 (DLY664, e) and cdc24-4 GALI::MIH1 (DLY673, o) G1 cells were incubated at 36C and analyzed todetermine cell viability as described in Materials and Methods. This experiment was performed three times, with consistent results.



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Osmotic Shock and Cell Cycle ProgressionThe experiments described above were al l performed withmuta nt strains that had defects in cytoskeletal polarity. To in-vestigate the role of the check point in wild type cells, we ex-amined the effects of osmotic shock on the cell cycle. Chowd-hury et al. (1992) previously demonstrated that addition ofsorbi tol (or other solutes) to the growth medium caused atrans ient depolarizat ion of the act in cytoskeleton. We con-firmed this observation, and found that this depolarizationdelayed bud formation by one h our in a populat ion of cel lssynchron ized by mating phero mon e (Fig. 7 A). This was ac-comp anied by a cell cycle delay so that the ceils did not un-dergo nuclear divis ion unt i l a bud had been formed, and nobinucleate cells were observed (300 ceils counted). If this

Figure 7. Recovery of wild type cells from osmotic shock. (A) Wildtype (DLY1) cells were grown in YEPS, arrested with m atingpheromone, and released into galactose-containing medium (timezero in graph: see Materials and Methods). The percentage of cellsthat had formed a b ud was quantitated (200 ceils counted) in sam-ples fixed at the indicated times. At 1 h, sorbitol was added to onehalf of the culture (e) to 0.72 M final concentration. GALI::CLB1cells (DLY34 4) treated identically budded w ith indistinguishablekinetics and showe d the same response to osmotic shock. (B)GALI::CLB1cells (DLY344) that had undergone an osm otic shockfrom the same experiment as in A were fixed at 3 h following phero-mone release and stained with DAPI. Show n is a picture taken withcombined fluorescence and transmitted light, illustrating the highfrequency of binucleate cells generated by this treatment. Similarresults were obtained in two independent experiments.

cell cycle delay was generated in the same way as in cdc24cells, then elevated expression of CLB2 should el iminate thedelay (see Fig. 5 A). To test this, a GALl::CLB2 strain wasarres ted with mating pher omon e and t ransferred to galactosemedium. Cel ls were released from pheromone arres t andsubjected to an osmo tic shock as before, and at 3 h after re-lease the cells were fixed and stained with DAPI to visual-ize nuclei. As shown in Fig. 7 B, many of these cells (28 %:300 cells counted) were binucleate. Most of the binucleatecel ls were unbudded or had small buds (40% of cells withbuds


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{ C L B I , 2mRNA

c d c 4 2 Imp a i r e d / S w e l M i h lC e l lm y o 2 ~ Polarity / ~

Oemot lc /ShockP

\ _ CLN1 ,2mRNAAccumulat ion

G2Delay

Recove r yof Cel lPolarityFigure 8. Summ ary of the etfects of the morphogenesis checkpointon C dc28 and c yclins. See text for details.

r e a l ly u s e a m orphogenes i s checkpo in t to s low the ce l l cyc ledur ing recove ry f rom s uch pe r tu rba t ions , t hen ove r r id ing thecheckpo in t s hou ld re s u l t i n the g ene ra t ion o f b inuc lea te ce i l sas the ce l l cycle cont inues unfe t tered in ce i ls tha t canno t bud.Indeed , a l a rge f rac tion o f w i ld type ce i l s expos ed to an os -mot i c s h i f t became b inuc lea te upon ov e r r ide o f the check-point (Fig . 7) . Since osmo tic shock provides a very differen tway o f pe r tu rb ing morphogenes i s ( compared to cdc24 o rcdc42 mutants ) , th is resul t va l ida tes our conclus ions fromthe m utan t s tud ie s.T he molecu la r na tu re o f the de fect ( s) s ens ed by the nove lcheckpo in t i s unknown . I t s eems l ike ly tha t e i the r the p re s -ence o f a bud , the s i ze o f the bud , o r s ome o the r man i fe s t a -t ion o f c e l l po la r i ty i s mon i to red . Howeve r, we canno t ru leou t the pos s ib i l it y tha t the checkpo in t mon i to r s s ome ve ryindirec t consequen ce of the dis turbed ce l l polar i ty . Conceiv-ably , such ind irec t e ffec ts mig ht even encom pass defec ts inch romos ome s t ruc tu re o r s p ind le p rope r t i e s , bu t the s ede fec ts mus t be d i s t inc t f rom thos e mon i to red by the o the rknown checkpoints , s ince the effec ts of t r iggering thesecheckpo in t s on C dc28 ac t iv i ty a re v e ry d i f fe ren t (s ee be low) .The (32 delay observed in cdc24 and cdc42 mutan t s i st rans ient , despi te the fac t tha t the morph ogene s is defec t (a tthe res t r ic t ive tempera ture) is perm anent . I t seems l ike ly tha tthe checkpo in t func t ions in w i ld type ce l l s t o ad jus t fo r t r an -s ient lapses in ce l l polar i ty , which (a t leas t in lab condi t ions)rare ly las t longer than an hour. Thu s , the 2--4-h checkpointde lay may be more than s u f f i c ien t fo r th i s pu rpos e .T he ex i s t ence o f a mech an i s m tha t c an mo n i to r s om e a s -pec t o f the ac t in cy tos ke le ton o r c e l l morpho logy has longbeen pos tu la t ed fo r mammal ian ce l l s (C ur t i s and Seeha r ,1978). S ignals ar ising from such a "mechan ica l" (ra ther than" chemica l" ) s enso r a re though t to gene ra te im por tan t inpu t sinto prol i fera t ive and different ia t ion decis ions in m any ce l ltypes durin g developm ent (Ingber , 1993). I t i s tempting tospecula te tha t the molecular bas is for such mechanica l s ig-na l l ing pa thways in mam mals cou ld be re l a t ed to the yeas t

morphogenes is checkpoint ident i f ied here . The avai labi l i tyo f power fu l mo lecu la r gene t i c too l s in the yeas t s ys t ems hou ld make i t pos sib le to t e s t t h i s hypo thes i s in the no t toodis tant fu ture .A Role for Cdc28 ~/rosine Phosphorylationin Budding YeastA regu la to ry pa thway tha t cou ld inh ib i t Cdks by ty ros inephosphoryla t ion was f i rs t described in the f is s ion yeas t S.pombe (Gou ld and Nurse , 1989), an d shown to play a cr i t ica lrole in regula t ing entry in to mitos is (Dunphy, 1994). Fiss ionyeas t c el l s t ha t a re unab le to phos phory la t e cdc2 a t t y ros ine15 enter mitos is premature ly , even i f the DNA is not fu l lyrepl ica ted, leading to "mitot ic ca tas t rophe" (Enoch andNurse , 1990; Lund gren e t a l . , 1991). This regula to ry path-way has now been cha rac te r ized in m any s pec ie s inc lud ingf rogs , mamm als , f ru i t f l ie s , and budd ing yeas t ( rev iewed inDunphy , 1994). S tudies in metazoan s have confi rm ed the im-por t ance o f th is pa thway in regu la ting en t ry in to mi tos i s , bu tun t i l now budd ing yeas t ha s s eemed to p rov ide the excep t ionto the ru le. M uta t ion o f CDC28tyrosine 19 to pheny la lan inehad no e f fec t on ce l l cyc le p rog re s s ion even unde r cond i t ionstha t induced DNA damage (Am on e t a l ., 1992 ; So rge r andMu rray, 1992), ra is ing the ques t ion of wh at role , i f any, wasp layed by the Cdc28 ty ros ine phos phory la t ion pa thway inbudd ing yeas t .In th is paper , we show ed tha t ce lls tha t were unab le to po-lar ize the ac t in cytoske le ton or form a bud inhibi ted ac t iva-t ion o f C lb /Cdc28 k ina s e s , t hus de lay ing ch romos om e s eg re -ga t ion . M uta t ion o f CDC28 yros ine 19 to phen y la lan ine , o rove rexpre s s ion o f the M ih l phos pha ta s e , abo l i s hed mos t o fthe de lay in ch romos ome s eg rega t ion , demons t ra t ing tha tty ros ine phos phory la t ion o f Cdc28 w as impor tan t fo r the de -l ay in v ivo . T hes e da ta s how tha t phos phory la t ion o f Cdc28ty ros ine 19 does indeed p lay an imp or tan t ro l e in regu la t ingcel l cycle progress ion, in ce l ls tha t experience defec ts inmorphogenes is (Fig . 8) .

In add i t ion to ty ros ine phos phory la t ion o f Cdc28 , c e l l sw i th m orphogenes i s de fec ts d i s p layed a de layed induc t ion o fCLB2 mR NA , and a s us t a ined hype r induc t ion o f CLN2mR NA (F ig . 8 ). R ecen t s tud ie s have s hown tha t CLB2 t ran-scr ipt ion is subjec t to a pos i t ive fe~lback loop wherebyClb /Cdc28 k ina s e ac t iv i ty s t imu la te s CLB2m R N A a c c um u -la t ion (Am on e t a l . , 1993). Thus , i t i s poss ible tha t the inhi-b i t ion o f C lb /Cdc28 k ina s e by ty ros ine phos phory la t ion wasrespons ible for the de lay in CLB2mR NA induc t ion . Fu r the r ,Clb/Cd c28 kin ase ac t iv i ty was shown to play a role in repres -s ion of CLN2 mR NA in G2 (Am on e t a l . , 1993) . T hus , t hes us ta ined induc t ion o f CLN2 m R N A c o u l d a l so b e d u e t o t h einh ib it ion o f C lb /Cdc28 k ina s e by ty ros ine phos phory la t ion .However, the fac t tha t revers ing the inhibi t ion due to Cdc2 8tyros ine phosphoryla t ion s t i l l le f t a res idual G2 delay (ofabou t 45 m in ) ind ica te s tha t t he morphogenes i s checkpo in tdoes no t work s o le ly by th i s mechan i s m.T he e f fec ts o f the morphogenes i s checkpo in t on Cdc28 andcycl ins are very different f rom the effects of the other k now ncheckpo in ts . T rea tmen t o f c e l ls w i th hydroxyurea (wh ich in -h ib i ts D NA rep l ica t ion ) o r nocodazo le (wh ich inh ib i t s as -s embly o f the m ic ro tubu le s p ind le ) c aus e s a l a rge -budded a r -re s t w i th und iv ided nuc le i and m ode ra te (hydroxyurea ) o rhigh (nocod azle) Clb/Cdc 28 kina se ac tiv i ty . To date , no m a-n ipu la t ion o f Cdc28 o r cyc l in s ha s ove rcome the a r re s t

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