Respect i ve Rol es of Cent r osomes and Chr omat i n i n t he Conver si on of Mi cr ot ubul e Ar r ays f r om I nt er phase t o Met aphase ERI C KARSENTI , J OHN NEW PORT, and MARC KI RSCHNER Uni ver si t y of Cal i f or ni a, San Fr anci sco, Cal i f or ni a 94143 W e r epor t t he r esul t s of st udi es i n whi ch par t i al l y pur i f i ed cent r osomes, nucl ei , and DNA wer e i nj ect ed i nt o f r og' s eggs, whi ch ar e nat ur al l y ar r est ed i n met aphase or i nt er phase . These r esul t s have l ed t o an i ndependent assessment of t he cont r i but i ons of t he cent r osome and t he chr omat i n t o t he f or mat i on of t he mi t ot i c spi ndl e and suggest a si mpl e expl anat i on f or t he t r ansi t i on f r om i nt er phase t o met aphase mi cr ot ubul e ar r ays . ABSTRACT Mor phogenesi s i n cel l s i s t hought t o be caused by a r ear r angement of cyt oskel et al el ement s i n r esponse t o var i ous i nt er nal or ext er nal si gnal s . Al t hough i n some cases t he si gnal s f or t hese mor phogenet i c changes ar e known, and i n many cases speci f i c component s of t he cyt oskel et on have been i mpl i cat ed, i n no case has i t been possi bl e t o suggest a mechani sm expl ai ni ng pr eci sel y how t he cyt oskel et al el ement s ar e act ual l y r ear r anged . Dur i ng t he past decade, det ai l ed bi ochemi cal and bi ophysi cal st udi es of t he sel f - assembl y of act i n, i nt er medi at e f i l ament s, and mi cr ot ubul es have suggest ed var i ous ways i n whi ch pol ymer i zat i on can be r egul at ed. What i s needed i s some way of under st andi ng how assembl y and di sassembl y mechani sms of t he f i l ament s ar e coor di nat ed spat i al l y t o gi ve t he obser ved or gani zed changes i n cel l shape. A wel l - st udi ed exampl e of a mor phol ogi cal change i s t he t r ansi t i on f r om i nt er phase t o met aphase and back t o i nt er phase . Al t hough t he det ai l s var y consi der abl y i n var i ous pl ant s or ani mal s, t he over al l pat t er n i s t hat t he i nt er phase mi cr ot ubul e di st r i but i on di sappear s t o be r epl aced by a bi pol ar met aphase spi ndl e . I n some ani mal cel l s t he di ssol ut i on of t he i nt er phase mi cr ot ubul e ar r ay i s accompani ed by a r oundi ng- up of t he cel l . A maj or di f f er ence bet ween ani mal s and pl ant s i s t hat i n ani mal cel l s cent r i ol es seem t o pl ay a pr omi nent r ol e i n mi t osi s, wher eas pl ant cel l s do not have cent r i ol es ( except f or some speci es of l ower pl ant s) . Ther e ar e sever al quest i ons r ai sed by t he vol umi nous l i t er at ur e on mi t osi s t hat have not been answer ed by descr i pt i ve st udi es . These i ncl ude t he f ol l owi ng : Howcan t he i nt er phase mi cr ot ubul es di sappear whi l e t her e i s a concomi t ant assembl y of mi cr ot ubul es t o f or mt he spi ndl e i n t he same cel l ? What r ol e does t he cent r i ol e pl ay i n spi ndl e assembl y and what per f or ms i t s f unct i on i n pl ant s? What pr oduces t he shape of t he mi t ot i c spi ndl e? How many speci f i c si gnal s or i nst r uct i ons ar e needed t o br i ng about t he var i ous st at es of spi ndl e assembl y and di sassembl y? A di f f i cul t y i n st udyi ng mi t osi s i s t he r api di t y wi t h whi ch i t pr oceeds and t he di f f i cul t y i n ar r est i ng t he cel l at a speci f i c st age wi t hout i nduci ng physi ol ogi cal i mbal ance . Eggs and oocyt es of var i ous i nver t ebr at e and ver t ebr at e ani mal s ar e nat ur al l y ar r est ed at st ages of mei osi s or mi t osi s and can be made t o pr oceed synchr onousl y t hr ough t he cel l cycl e i n r esponse t o var i ous si gnal s, such as t he addi t i on of hor mones or f er t i l i zat i on . I n addi t i on, many of t hese eggs can be easi l y mi cr oi nj ect ed, and t he behavi or of subcel l ul ar component s can be assayed i n a cyt opl asmi c envi r onment t hat i s at a speci f i c st age of t he cel l cycl e. Usi ng t hi s appr oach, we have l ooked at mi cr ot ubul e assembl y i n Xenopus eggs at met aphase and i n i nt er phase . We have exami ned separ at el y t he r ol e of t he chr omat i n and t he cent r i ol e i n t he est abl i shment of t he met aphase spi ndl e and t he i nt er phase mi cr ot ubul e ar r ays. Our f i ndi ngs suggest , f i r st , t hat a di f f er ence i n t he t hr eshol d concent r at i on f or t ubul i n assembl y may be t he f undament al cont r ol r egul at i ng t he t r ansi t i on bet ween i nt er phase and met aphase, and, second, t hat t he DNA or chr omat i n speci f i cal l y i nf l uences t he l ocal assembl y of mi cr ot ubul es i n met aphase cel l s. Though t he cent r i ol es appear t o cont r i but e t o mi t ot i c spi ndl e assembl y, t he r esul t s r epor t ed her e suggest t hat t he over al l pat hway of spi ndl e f or mat i on i n ani mal s may be ver y si mi l ar t o t hat i n pl ant s . Cel l Cycl e i n Xenopus Eggs Pr oceeds i n t he Absence of t he Nucl eus The unf er t i l i zed Xenopus egg i s ar r est ed i n met aphase of t he second mei ot i c di vi si on wi t h t he spi ndl e i nt act . The THE JOURNAL OF CELL BI OLOGYzyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA " VOLUME 99 No 1 PT. 2 JULY 1984 47s- 54s ® The Rockef el l er Uni ver si t y Pr ess - 0021- 9525/ 84/ 07/ 047s/ 08 $1 . 00 47s Downloaded from http://rupress.org/jcb/article-pdf/99/1/47s/1643001/47s.pdf by guest on 23 October 2023 Depar t ment of Bi ochemi st r y, CCCTTE CTTCRASMI C STATE UNFERTI LI ZED ACTI VATED I NTERPHASE METAPHASE ( G1) ( Mei osi s, M) I NTERPHASE ( 5) CST ARRESTED METAPHASE ( M) ACTI VFTT FI GURE 1 Cyt opl asmi c st at es of t he oocyt e and egg . mei ot i c spi ndl e i n f r ogs i s anast r al . I n mouse ( 14) , and per haps al l ver t ebr at es, i t l acks a cent r i ol e . I n some speci es t he cent r i ol e i s cont r i but ed by t he sper m; i n ot her s i t f or ms spont aneousl y some t i me af t er f er t i l i zat i on . Fer t i l i zat i on or act i vat i on of t he Xenopus egg i ni t i at es compl et i on of mei osi s, and t he egg ent er s i nt er phase . At 60 mi n t he f i r st mi t ot i c di vi si on begi ns, and cl eavage ensues at 90 mi n ( 3) . Ther eaf t er , t he egg di vi des ever y 30 mi n, al t er nat i ng bet ween M and S phases . The Xenopus egg can al so be ar r est ed at t he next mi t ot i c di vi si on by mi cr oi nj ect i on of a smal l quant i t y of cyt opl asm f r om unf er t i l i zed eggs cont ai ni ng t he yet unchar act er i zed f act or cal l ed cyt ost at i c f act or ( 10) . Cyt ost at i c f act or i nj ect i on bl ocks cl eavage and causes ar r est of t he chr omosomes i n a condensed st at e at met aphase on a mi t ot i c spi ndl e ( 11) . Recent l y, Har a et al . ( 6) showed t hat t he cel l cycl e i n ear l y cl eavage i n Xenopus can pr oceed i n t he absence of t he nor mal nucl ear and cyt opl asmi c event s. Enucl eat ed but act i vat ed eggs f ai l t o cl eave but under go per i odi c cont r act i ons of t hei r cor t ex, t i med wi t h t he cel l cycl e . They al so show an osci l l at i on of an M phase- speci f i c cyt opl asmi c f act or ( 4) . The var i ous cyt opl asmi c st at es of t he act i vat ed egg di ct at e t he behavi or of i nj ect ed nucl ei or DNA wi t h r espect t o nucl ear assembl y and di sassembl y, chr omosome condensat i on, and DNA r epl i cat i on ( 5, 7, 9, 12, and f oot not e 1) . Two met aphase st at es can Newpor t , J ., and M. Ki r schner . Regul at i on of t he cel l cycl e dur i ng ear l y Xenopus devel opment . Cel l . I n pr ess . I 48S THE J OURNAL OF CELL BI OLOGY - VOLUME 99, 1984 be st udi ed : t he one r epr esent ed by unf er t i l i zed eggs and t he ot her pr oduced by cyt ost at i c f act or ar r est of f er t i l i zed eggs . I nt er phase st ages can be pr oduced by r el i evi ng t hese mi t ot i c bl ocks ( by pr i cki ng t he unf er t i l i zed eggs or by i nj ect i on of Ca" i nt o cyt ost at i c f act or - ar r est ed eggs [ Fi g . 1] ) . Cent r i ol es Ar e Act i ve i n I nt er phase Cyt opl asm but I nact i ve i n Mi t ot i c Cyt opl asm' Cent r osomes may be i sol at ed f r om mammal i an cel l s and pur i f i ed bet ween 2, 000- and 5, 000- f ol d ( Mi t chi son, T. , and M. Ki r schner , unpubl i shed dat a) . These cent r osomes, f or exampl e, pur i f i ed f r om mouse neur obl ast oma cel l s, cont ai n about 5 % t ubul i n by wei ght , al l of whi ch can be account ed f or by t he t ubul i n i n t he cent r i ol e. I n addi t i on, t hey cont ai n at l east t wo ant i gens char act er i zed as per i cent r i ol ar ( 1, 15) . I n vi t r o, t hese cent r osomes nucl eat e about 50 mi cr ot ubul es, wi t h t he pr oper pol ar i t y ( Mi t chi son, T. , unpubl i shed obser vat i on) . I n ext r act s of f r og' s eggs, t hese cent r osomes wi l l al so ef f i ci ent l y nucl eat e assembl y under condi t i ons under whi ch t her e i s no obser vabl e spont aneous pol ymer i zat i on . They al so suppor t par t henogenet i c devel opment . When i nj ect ed i nt o act i vat ed eggs ( i nt er phase st at e) , t he cent r osomes i nduce t he f or mat i on of l ar ge ast er s wi t hi n 20 Z Most of t he mat er i al s and met hods used i n t hi s wor k have been descr i bed ( 2, 9) . When necessar y, new met hods ar e descr i bed i n t he f i gur e l egends . Si nce t he i nj ect i on event r esumes t he met aphase ar r est and act i vat es t he egg cel l cycl e, t he met aphase st at e of t he cyt opl asm i s not di r ect l y accessi bl e . Act i vat i on was pr event ed, when desi r ed, by addi ng 10 mMEGTA t o t he i nj ect i on medi um. Downloaded from http://rupress.org/jcb/article-pdf/99/1/47s/1643001/47s.pdf by guest on 23 October 2023 2 Cent r osome act i vi t y i n act i vat ed and unact i vat ed eggs . Par af f i n sect i ons . x100 . ( a) Cent r osomes wer e i nj ect ed i n phosphat e buf f er ( 20 mM, pH 7 . 2) , and t he eggs wer e f i xed 2 h l at er . Numer ous ast er s of mi cr ot ubul es ar e vi si bl e ( ar r ows) . ( b) Cent r osomes wer e i nj ect ed i nt o phosphat e buf f er cont ai ni ng 10 mM EGTA, and t he eggs wer e f i xed 2 h l at er . The eggs ar e not act i vat ed ( t he ar r ow i ndi cat es t he mei ot i c spi ndl e) . No ast er s f or med . FI GURE FI GURE 3 Behavi or of kar yopl ast s f r om L 929 cel l s i nj ect ed i nt o t he i nt er phase and met aphase cyt opl asm of Xenopus eggs . Par af f i n sect i ons . x 600 . ( a) Eggs wer e i nj ect ed wi t h kar yopl ast s under act i vat i ng condi t i ons and f i xed 30 mi n l at er ( i nt er phase cyt opl asm) . Nucl ei ar e i nt act ; no mi cr ot ubul es f or m. ( b) Eggs wer e i nj ect ed wi t h kar yopl ast s under nonact i vat i ng condi t i ons and f i xed 1 h l at er ( met aphase cyt opl asm) . Spi ndl es of mi cr ot ubul es f or m ar ound I I I def i ned masses of condensed chr omat i n . phase cyt opl asm suggest s t hat t he egg i n t hi s st at e per haps wi l l not r espond t o exogenous si gnal s f or spi ndl e assembl y . However , i f cr ude nucl ei cont ai ni ng cent r osomes and at t ached cel l ul ar mat er i al ar e i nj ect ed i nt o an unf er t i l i zed egg, t he nucl ear membr ane br eaks down, t he chr omosomes condense, and a bi pol ar spi ndl e f or ms . I n r egi ons of t he egg wher e sever al nucl ei ar e pr esent i n cl ose pr oxi mi t y, mul t i pol ar spi ndl es f or m. Si mi l ar r esul t s ar e obt ai ned i f nucl ei ar e i nj ect ed i nt o eggs ar r est ed at met aphase wi t h cyt ost at i c f act or . Especi al l y i nt er est i ng i s t hat t he i nj ect ed nucl ei under go speci f i c r ear r angement s, possi bl y r el at ed t o t he nor mal pr ophase and pr omet aphase event s bef or e t hey ar e ar r est ed i n a met aphase conf i gur at i on . Dur i ng t hi s ent i r e t i me, t he r esi dent mei ot i c spi ndl e i n t he unf er t i l i zed eggs r emai ns at met aphase. Spi ndl e f i ber s ar e seen i n t he l i ght mi cr oscope t o conver ge on f oci . When t hese f oci ar e exami ned i n t he el ect r on mi cr oscope, i t can be seen t hat some of t hem cont ai n cent r osomes . Most of t he mi cr ot ubul es, however , do not seem t o or i gi nat e at t he cent r osomes . Cr ude nucl ei i nj ect ed i nt o i nt er phase cyt opl asm do not br eak down, but t he associ at ed cent r osomes i nduce ver y l ar ge ast er s . These r esul t s ar e somewhat par adoxi cal . Pur i f i ed cent r osomes ar e act i ve i n i nt er phase but not met aphase cyt opl asm. Cr ude nucl ei , however , f or m spi ndl es i n met aphase cyt opl asm, wher e el ect r on mi cr oscopy demonst r at es t hat t he cent r osomes ar e act i ve . These r esul t s suppor t t he not i on t hat ot her component s i n t he i nj ect ed nucl eus may be r equi r ed f or cent r osome act i vi t y i n t he met aphase st at e but ar e not r equi r ed f or act i vi t y i n t he i nt er phase st at e . I nj ect i on of Cr ude Nucl ei i nt o Met aphase Cyt opl asm I nduces Spi ndl e For mat i on The sur pr i si ng f ai l ur e of cent r osomes t o f unct i on i n met a- 4 Behavi or of l ambda DNA i nj ect ed i nt o unact i vat ed eggs . Eggs wer e i nj ect ed wi t h 2- 5 ng of l ambda DNA i n 20 mM phosphat e buf f er cont ai ni ng 10 mM EGTA and squashed af t er var i ous t i mes i n t he pr esence of t he DNA dye bi sbenzi mi de . Fl uor escence mi cr oscopy . ( a- c) X 900 . ( d) X 220 . ( a) At 5 mi n, t hr eads of DNA wer e spr ead over a l ar ge ar ea i n t he egg cyt opl asm. ( b) Af t er 1 h, t he DNA coal esced i nt o t hi ck f i ber s l ocal i zed i nt o sever al spher i cal st r uct ur es t hat appear ed i n t he cyt opl asm . ( c) Af t er 3 h, t he DNA condensed i nt o smal l st r uct ur es l ooki ng l i ke i l l - def i ned condensed chr omosomes . ( d) Low magni f i cat i on of t he ar ea shown i n c . The condensed DNA i s l ocal i zed i n a l ar ge spher i cal st r uct ur e t hat r esi st ed t he squashi ng pr ocess . FI GURE E. KARSENTI ET AL . Cent r osomes and Chr omat i n i n Mi cr ot ubul e As s embl y 4 9s Downloaded from http://rupress.org/jcb/article-pdf/99/1/47s/1643001/47s.pdf by guest on 23 October 2023 mi n ( Fi g . 2 a) . Appr oxi mat el y one ast er gr ows per cent r osome i nj ect ed . However , when cent r osomes ar e i nj ect ed i nt o unact i vat ed eggs ( met aphase st at e) , no ast er s ar e f or med ( Fi g . 2b) . Si mi l ar l y, cent r osomes i nj ect ed i nt o f er t i l i zed eggs ar r est ed at met aphase wi t h cyt ost at i c f act or f ai l t o f or m ast er s . Mor eover , ast er s t hat f i r st f or med i n t he i nt er phase cyt opl asm ( act i vat ed eggs) and t hat t hen wer e r et ur ned t o t he met aphase st at e ( by cyt ost at i c f act or i nj ect i on) di sappear . These r esul t s show cl ear l y t hat cent r osomes, whi ch ar e f ul l y compet ent t o act as mi cr ot ubul e- or gani zi ng si t es, ar e act i ve i n i nt er phase cyt opl asm and i nact i ve i n met aphase cyt opl asm. I t i s easi l y shown t hat t he cent r osomes ar e not dest r oyed or i r r ever si bl y i nhi bi t ed. Cent r osomes can be i nj ect ed i nt o unf er t i l i zed eggs under condi t i ons under whi ch t he egg r emai ns i n t he met aphase st at e . Af t er sever al hour s t he eggs can be act i vat ed by pr i cki ng, at whi ch t i me ast er s f or m. Cent r osomes can even be i nj ect ed i nt o i mmat ur e oocyt es, whi ch ar e t hen al l owed t o mat ur e i n vi t r o. When t hey ar e ar r est ed nat ur al l y at mei ot i c met aphase, no ast er s f or m. However , af t er act i vat i on, ast er s appear and t hei r f or mat i on depends compl et el y on t he pr evi ous i nj ect i on of cent r osomes . Ther ef or e, cent r osomes ar e act i ve i n i nt er phase and i nact i ve i n met aphase, and t hi s act i vi t y i s r ever si bl y expr essed. I nj ect i on of Kar yopl ast Nucl ei i nt o Met aphase Cyt opl asm I nduces Spi ndl e For mat i on I nj ect i on of Bot h Kar yopl ast Nucl ei and Cent r osomes i nt o Met aphase Cyt opl asm I nduces Pol ar Spi ndl es The spi ndl es f or med wi t h cr ude nucl ei di f f er f r om t hose Behavi or of l ambda DNA i nj ect ed i nt o t he i nt er phase and met aphase cyt opl asm of Xenopus eggs . Par af f i n sect i ons . ( a, b, d) x 600 . ( c) x 100 . ( a) 1 h af t er i nj ect i on i nt o unact i vat ed eggs ( met aphase cyt opl asm) . Lar ge bundl es of mi cr ot ubul es ( bl ack ar r ows) f or m ar ound t he DNA, whi ch has not yet f ul l y condensed ( whi t e ar r owheads) . ( b) At t he same t i me, t he DNA i nj ect ed i nt o act i vat ed eggs al so has par t l y condensed ( whi t e ar r owheads) . No mi cr ot ubul es f or m. Some nucl ei st ar t t o appear ( bl ack ar r owheads) . ( I nset ) One of t he numer ous nucl ei f ound i n eggs f i xed 3 h af t er i nj ect i on . ( c) 3 hour s af t er i nj ect i on i nt o unact i vat ed eggs al l t he DNA has condensed i nt o smal l st r uct ur es i ncl uded i n spher es excl udi ng t he yol k and cont ai ni ng a l ar ge number of mi cr ot ubul es . ( Ar r ow) I nj ect i on si t e . ( d) Hi gh magni f i cat i on of t he st r uct ur e shown i n c . The condensed DNA ( whi t e ar r owheads) i s sur r ounded by a l ar ge amount of f i br ous mat er i al ( mi cr ot ubul es) . FI GURE 5 5 0S THE J OURNAL OF CELL BI OLOGY - VOLUME 99, 1984 Downloaded from http://rupress.org/jcb/article-pdf/99/1/47s/1643001/47s.pdf by guest on 23 October 2023 To t est t he ef f ect of t he nucl eus pr oper on t he act i vi t y of cent r osomes i t was necessar y t o r ender nucl ei f r ee of cont ami nat i ng cent r osomes. Kar yopl ast s, whi ch ar e nucl ei sur r ounded by a pl asma membr ane but f r ee of most cyt opl asmi c cont ami nant s, can be pr epar ed f r om cul t ur ed cel l s by t r eat ment wi t h cyt ochal asi n B and cent r i f ugat i on . About 99% of a gi ven pr epar at i on of kar yopl ast s ar e f r ee of cent r i ol es ( 16) . We have conf i r med t hi s by st ai ni ng wi t h an ant i body speci f i c f or per i cent r i ol ar mat er i al . The kar yopl ast s l acki ng cent r osomes wer e l ysed wi t h l ysol eci t hi n and i nj ect ed i nt o eggs . I n act i vat ed eggs ( i nt er phase cyt opl asm) t he kar yopl ast nucl ei swel l , but t her e i s no si gn of mi cr ot ubul e assembl y or ast er f or mat i on, i ndi cat i ng t hat f unct i onal l y as wel l as st r uct ur al l y no cent r osomes ar e pr esent ( Fi g. 3 a) . I n unact i vat ed eggs ( met aphase cyt opl asm) , t he nucl ear envel ope of t he kar yopl ast nucl ei appar ent l y di sappear s, t he chr omosomes condense, and a l ar ge number of f i ber s can be seen t o gr ow ar ound t he chr omat i n ( Fi g . 3 b) . These f i ber s r ear r ange t o f or m spi ndl es l acki ng a shar p f ocus. The r ear r angement of t he f i ber s and chr omosomes i s si mi l ar t o t hat descr i bed f or t he pl ant spi ndl e, as exempl i f i ed by wor k wi t h Haemant hus ( 13) . When sever al nucl ei ar e i n pr oxi mi t y t o each ot her , a r i ng of f i ber s f i r st sur r ounds t he gr oup of nucl ei . I n 30 mi n t hi s r ear r anges i t sel f i nt o a ci r cl e, wi t h t he chr omosomes on t he i nsi de and br oad mi cr ot ubul e f oci at i nt er val s ar ound t he ci r cl e . These ar r angement s ar e si mi l ar t o t he cor r espondi ng st r uct ur es seen wi t h cr ude nucl ei but have much br oader f oci , wi t h no f i ber s r adi at i ng t owar d t he cyt opl asm. These r esul t s cl ear l y demonst r at e t hat i n met aphase cyt opl asm t he spi ndl e can assembl e i n t he absence of cent r osomes and t hat t he r esul t i ng spi ndl e r esembl es t hat of pl ant s . f or med wi t h kar yopl ast nucl ei i n t he l ack of ast r al f i ber s i n t he f or mer and t he f ocused nat ur e of t he spi ndl e f i ber s i n t he l at t er . To see i f t he cr ude nucl ear di st r i but i on i nvol ved st r uct ur al el ement s ot her t han nucl ei and cent r osomes, we at t empt ed t o r econst i t ut e t hi s di st r i but i on by si mul t aneousl y i nj ect i ng kar yopl ast nucl ei and cent r osomes . When bot h nucl ei and cent r osomes wer e i nj ect ed i nt o met aphase cyt opl asm, some mul t i pol ar spi ndl es wer e f ound t o have wel l - f ocused pol es wi t h smal l r adi at i ng ast er s . Ast er s wer e seen onl y i n ver y cl ose pr oxi mi t y t o t he condensed chr omat i n . As expect ed, no ast er s wer e pr esent i n r egi ons of t he cyt opl asm f r ee of chr omat i n . These r esul t s suggest t hat i n met aphase cent r osomes ar e act i ve i n t he r egi on near t he nucl eus or chr omat i n . These exper i ment s l eave uncl ear whet her i t i s t he chr omat i n, ot her nucl ear component s, or cyt opl asmi c mat er i al adher i ng t o t he kar yopl ast nucl eus t hat i s r equi r ed f or spi ndl e assembl y . Hi gh Mol ecul ar Wei ght DNA I nj ect ed i nt o Met aphase Cyt opl asm Pr omot es Mi cr ot ubul e Assembl y Recent l y, For bes et al . ( 2) have shown t hat DNA f r om var i ous sour ces, i ncl udi ng bact er i ophage l ambda, wi l l spon- 7 Fr agment s of bact er i ophage T4 DNA pr epar ed by soni cat i on . T4 DNA ( 0 . 5 mg/ ml ) was soni cat ed at l ow i nt ensi t y wi t h a Br ansoni c 350 ( Br anson Soni c Power Co . , Danbur y, CT) f or var i ous l engt hs of t i me . The r esul t i ng DNA f r agment s wer e anal yzed on an 0 . 8% agar ose gel i n Tr i s- bor at e, EDTA buf f er at 100 V f or 3 . 5 h . Et hi di um br omi de st ai ni ng . ( a) T4 DNA. ( b) Soni cat ed f or 5 s . Hi gher mol wt = 23 kb . ( c) Soni cat ed f or 10 s . Hi gher mol wt = 10 kb . ( d) Soni cat ed 20 s . Hi gher mol wt = 6 kb . ( e) Soni cat ed 30 s . Hi gher mol wt = 3 kb . ( f ) Mol ecul ar wei ght mar ker s ( Hi nd I I I cut of l ambda DNA) . FI GURE 6 The f i br ous ar eas sur r oundi ng t he condensed DNA cont ai n numer ous mi cr ot ubul es . Tr ansmi ssi on el ect r on mi cr oscopy . ( a) x 25, 000 . ( b) x 45, 000 . ( See r ef er ence 2 f or t echni cal det ai l s . ) ( a) The st ar i ndi cat es t he cyt opl asmi c ar ea wher e t he DNA i s l ocal i zed . Mi cr ot ubul es gr ow al ong t hi s cyt opl asmi c domai n . Not e t hat t he ar ea cont ai ni ng t he mi cr ot ubul es i s ver y r i ch i n r i bosomel i ke par t i cl es ( bot t om par t of t he pi ct ur e) . ( b) Mi cr ot ubul es at hi gher magni f i cat i on . FI GURE E. KARSENTI ET AE . Cent r os omes and Chr omat i n i n Mi cr ot ubul e As s embl y 5 1s Downloaded from http://rupress.org/jcb/article-pdf/99/1/47s/1643001/47s.pdf by guest on 23 October 2023 t aneousl y assembl e i nt o nucl eusl i ke st r uct ur es when i nj ect ed i nt o i nt er phase eggs. The same DNA i nt r oduced i nt o t he met aphase cyt opl asm of unact i vat ed eggs does not pr omot e t he, f or mat i on of nucl ei . Fi ve mi nut es af t er i nj ect i on, l ong t hr eads of DNA can be vi sual i zed i n squashes of eggs st ai ned wi t h t he DNA dye bi sbenzi mi de ( Hoechst ) ( Fi g. 4a) . The DNA i s t hen packaged i nt o smal l st r uct ur es l ooki ng l i ke i r r egul ar l y condensed chr omat i n . Thi s packagi ng t akes over 3 h ( Fi g . 4 b and c) . The condensed DNA i s al ways f ound i n l ar ge ( 50- 200 um) spher i cal st r uct ur es excl udi ng t he yol k ( Fi g. 4 d) . These st r uct ur es cont ai n a l ar ge amount of f i br ous mat er i al or gani zed ar ound t he condensed DNA, as i s shown i n t he par af f i n sect i ons of Fi g. 5 c and d. One hour af t er i nj ect i on i nt o act i vat ed eggs ( i nt er phase cyt opl asm) , most of t he DNA has f or med chr omat i n, but onl y a par t of i t has assembl ed i nt o nucl ei ( Fi g . 5 b) . No f i ber s ar e appar ent ar ound t he f r ee chr omat i n or t he nucl ei ( Fi g. 5b) . I n cont r ast , af t er t he same i ncubat i on t i me, a l ar ge amount of f i ber s have al r eady assembl ed ar ound t he DNA i nj ect ed i nt o unact i vat ed eggs ( met aphase cyt opl asm) ( Fi g . 5 a) . Thi s occur s al t hough t he DNA has not r eached i t s f i nal l evel of condensat i on ( Fi g. 4) . No f i br ous mat er i al has been obser ved ar ound t he condensed DNA i n unact i vat ed eggs i ncubat ed f or 2 h i n 10- pg/ ml nocodazol e . Thi s st r ongl y suggest s t hat t he f i ber s obser ved i n t he absence of nocodazol e ar e pr i mar i l y mi cr ot ubul es. Mor eover , t hi n sect i ons made t hr ough t he f i br ous ar eas and obser ved by el ect r on mi cr oscopy cont ai n a l ar ge amount of mi cr ot ubul es ( Fi g. 6) . DNA of bact er i ophage T4 and E. col i or i gi n pr omot es a l ocal pol ymer i zat i on of mi cr ot ubul es when i nj ect ed i nt o unact i vat ed eggs . However , t he pl asmi d PBR322 and t he si ngl e- st r anded or r epl i cat i ve f or mof t he phage M 13 f ai l t o i nduce any vi si bl e l ocal assembl y of mi cr ot ubul es i n t he met aphasi c cyt opl asm. Thi s i s r el at ed t o t hei r smal l si ze, as we shal l see. T, DNA was soni cat ed f or var i ous per i ods of t i me t o pr oduce f r agment s of DNA of var i ous si zes ( Fi g . 7) . I t was f ound t hat t he ext ent of l ocal mi cr ot ubul e assembl y decr eases wi t h t he aver age si ze of t he DNA. DNA smal l er t han 10 kb f ai l s t o pr omot e any det ect abl e l ocal mi cr ot ubul e assembl y ( Fi g . 8a- c) . The r ever se exper i ment has been done wi t h PBR322 . The ci r cul ar doubl e- st r anded DNA ( 4. 3 kb) has been cut by r est r i ct i on enzymes ( Eco RI and Hi nd I I I ) and r el i gat ed by t he T, l i gase t o pr oduce pol ymer s l ar ger t han 10 kb ( Fi g . 9) . Al t hough PBR322 i s t ot al l y unabl e t o pr omot e mi cr ot ubul e assembl y i n unact i vat ed eggs, t he l i gat ed f or m i s a st r ong pr omot er of l ocal pol ymer i zat i on ( Fi g. 10) . Even hi gh concent r at i on of l ow mol ecul ar wei ght DNA ( 100 ng/ egg) f ai l s t o i nduce assembl y . Var i ous ani oni c pol ymer s such as f r og' s l i ver RNA, dext r an sul f at e ( 500 kd) , or hepar i n have no act i vi t y i n i nduci ng any ki nd of mi cr ot ubul e pol ymer i zat i on i n t he eggs . The r esul t s r epor t ed her e suggest t hat i t i s not t he ani oni c pr oper t i es of DNA t hat pr omot e mi cr ot ubul e assembl y . Rat her i t i s t he assembl y of DNA i nt o chr omat i n, whi ch i n t he met aphase st at e accumul at es and/ or act i vat es component s, t hat pr omot es l ocal pol ymer i zat i on . I nt er est i ngl y, t hi s does not have t o come about by t he pr i or assembl y of an i nt er phase nucl eus but can occur di r ect l y by f or mat i on of met aphase chr omat i n . I t i s not cl ear why smal l DNA does not pr omot e mi cr ot ubul e assembl y . I n any case, t he pr esent r esul t s suggest t hat i n a gi ven cyt opl asmi c envi r onment t her e i s a r el at i onshi p bet ween t he l ocal mass of chr omat i n and t he number of mi cr ot ubul es assembl ed ar ound i t . I f ver i f i ed, t hi s may pr ove t o be i mpor t ant i n expl ai ni ng t he si ze of t he spi ndl e i n r el at i on t o t he number of chr omosomes . 5 2S THE JOURNAL OF CELL BI OLOGY - VOLUME 99, 1984 Met aphase and I nt er phase Cyt opl asm Di f f er i n Thei r Abi l i t y t o I nduce Mi cr ot ubul e Assembl y As an i ndi r ect measur e of t he pol ymer i zabi l i t y of t ubul i n i n eggs, we exami ned t hei r r esponse t o di f f er ent concent r at i ons of D2 0 . As r epor t ed ( 8) , D20 i nduces mi cr ot ubul e assembl y i n unf er t i l i zed eggs but not i n oocyt es ( 8) . The assembl y t akes t he f or mof smal l f oci of pol ymer i zat i on, whi ch di spl ace yol k and ot her vesi cl es and whi ch ar e r eadi l y obser ved wi t h t he ai d of t he l i ght mi cr oscope . When t hi s assay was appl i ed t o eggs i n met aphase or i nt er phase, a consi st ent and si gni f i cant di f f er ence was obser ved i n t he D2 0 concent r at i on necessar y t o i nduce spont aneous pol ymer i zat i on . Thi r t y per cent D20 i s r equi r ed t o i nduce assembl y i n act i vat ed eggs i n i nt er phase. Unact i vat ed eggs, at met aphase, r equi r e 40%. When we i nj ect ed cent r osomes i nt o met aphase cyt opl asm ( unact i vat ed eggs) and i ncubat ed t he eggs i n var i ous concent r at i ons of D2 0, we f ound t hat ast er s f or med i n 20% D20 . These dat a suggest di mi ni shed pol ymer i zabi l i t y of t ubul i n i n t he met aphase st at e, because hi gher D2 0 concent r at i ons wer e r equi r ed t o i nduce spont aneous assembl y . I n addi t i on t hey demonst r at e t hat D2 0 can over come t he i nhi bi t i on of cent r osome- dependent ast er assembl y i n eggs ar r est ed i n a met aphase st at e. Mechani sm of t he Mi t ot i c- l nt er phase Tr ansi t i on of Mi cr ot ubul e Ar r ays The exper i ment s r epor t ed her e suggest some new appr oaches t o t he quest i ons r ai sed ear l i er on how t he i nt er phase mi cr ot ubul e ar r ay i s conver t ed t o t he spi ndl e, howt he spi ndl e get s i t s shape, what r ol e t he cent r i ol e has i n spi ndl e assembl y, and how many si gnal s ar e r equi r ed t o assembl e t he spi ndl es . Downloaded from http://rupress.org/jcb/article-pdf/99/1/47s/1643001/47s.pdf by guest on 23 October 2023 Mi cr ot ubul e assembl y i s not pr omot ed by shor t bact er i ophage T, DNA f r agment s i n met aphase eggs . Par af f i n sect i ons . x 600 . T, DNA soni cat ed f or var i ous l engt hs of t i me ( see Fi g. 7) was i nj ect ed i n 10 mM EGTA i nt o Xenopus eggs ( unact i vat ed) . The eggs wer e f i xed 2 h l at er . ( a) T, DNA soni cat ed f or 5 s ( maxi mum si ze, =25 kb) . Smal l ar eas cont ai ni ng mi cr ot ubul es of t en conver gi ng t o a f ocus wer e obser ved . ( b) T, DNA soni cat ed f or 10 s ( maxi mum si ze, =10 kb) . A f ew bundl es of mi cr ot ubul es or gani zed l i ke hal f - spi ndl es or ast er s ar e f ound i n t he eggs . ( c) Ta DNA soni cat ed f or 20 s ( maxi mum si ze, =6 kb) . No mi cr ot ubul es wer e f ound . The DNA i s i mpossi bl e t o see i n t hese bl ack- and- whi t e pi ct ur es . On col or pr i nt s, i t i s vi si bl e i n a and b but not i n c . The pr esence and decr easi ng si ze of t he DNA was, however , cl ear l y vi sual i zed by Hoechst bi sbenzi mi de st ai ni ng of egg squashes i n al l t hr ee cases . FI GURE 8 Her e we r epor t f our key obser vat i ons : ( a) I sol at ed cent r osomes ar e act i ve i n i nt er phase and i nact i ve i n met aphase cyt opl asm; ( b) i sol at ed kar yopl ast cyt opl asm, suggest i ng t hat t ubul i n pol ymer i zabi l i t y i s gr eat er i n i nt er phase t han i n met aphase. nucl ei as wel l as pur i f i ed DNA have These obser vat i ons suggest t hat an over al l def i ci ency i n no ef f ect on mi cr ot ubul e assembl y i n i nt er phase cyt opl asm mi cr ot ubul e assembl y i n met aphase i s over come l ocal l y i n but i nduce mi cr ot ubul e assembl y i n met aphase cyt opl asm; t he egg, di r ect l y or i ndi r ect l y, by condensed chr omat i n . Thi s ( c) cent r osomes pl us nucl ei gl obal r educt i on of mi cr ot ubul e pol ymer i zabi l i t y dur i ng met aphase woul d r esul t i n t he depol ymer i zat i on of t he i nt er phase r econst i t ut e essent i al l y nor mal ast r al spi ndl es i n met aphase cyt opl asm; and ( d) i t i s easi er t o i nduce spont aneous pol ymer i zat i on wi t h D2 0 i n i nt er phase ast er . The l ocal pr omot i on of mi cr ot ubul e assembl y i n t he vi ci ni t y of t he chr omat i n woul d al l ow f or t he assembl y of t he spi ndl e at t he s ame t i me . I t i s not cl ear how chr omat i n i nduces l ocal i zed mi cr ot ubul e pol ymer i zat i on, but i t i s cl ear t hat t he DNA does not r equi r e speci f i c sequences f ound onl y i n eukar yot i c or gani sms. The cent r i ol e i s cl ear l y not essent i al t o t he assembl y of t he spi ndl e . However , cent r osomes i n t he vi ci ni t y of t he chr omat i n ar e act i vat ed and i nf l uence t he over al l mi cr ot ubul e di st r i but i on . The det er mi nat i on of t he shape of t he spi ndl e i s pr obabl y compl ex, but i t cer t ai nl y must i nvol ve, as a l ar ge component , t he asymmet r i c di st r i but i on of a mi cr ot ubul e- pr omot i ng act i vi t y act i vat ed by t he met aphase The mi cr oi nj ect i on exper i ment s wer e desi gned pr i mar i l y t o assay c omponent s r equi r ed f or spi ndl e f or mat i on . Yet t hese exper i ment s may al so cont r i but e t o our under st andi ng of t he st eps of spi ndl e f or mat i on. The met aphase st at e, whet her i n t he unf er t i l i zed egg or i n cl eavi ng eggs ar r est ed wi t h cyt ost at i c f act or , i s st abl y ar r est ed at a uni que poi nt i n t he cel l cycl e . Never t hel ess, cel l nucl ei , cent r osomes, or DNA i nj ect ed i nt o a i n t hi s ar r est ed cyt opl asmi c st at e wi l l i ni t i at e sever al di scr et e event s l eadi ng t o t he f or mat i on of a met aphase spi n9 Li gat i on of PBR322 . The pl asmi d PBR322 ( 500 l Ag/ ml ) was cut ( 1 h at 37° C) and l i gat ed wi t h t he T4 DNA l i gase f or 5 and 10 mi n at 16° C. Af t er phenol ext r act i on and et hanol pr eci pi t at i on, i t was r esuspended i n 20 mMphosphat e, 20 mM EGTA ( pH 7 . 2) , and sampl es wer e anal yzed on an 0 . 8% agar ose gel i n Tr i s- bor at e, EDTA buf f er at 100 V f or 5 h . Et hi di um br omi de st ai ni ng . ( a) Mol ecul ar wei ght mar ker . Hi nd I I I cut of l ambda DNA. ( b) Nat i ve PBR 322 . ( c) EcoRl - Hi nd I I I cut of PBR322 . ( d) Af t er 5 mi n of l i gat i on by t he T4 DNA l i gase . ( e) Af t er 10 mi n of l i gat i on . dl e, condensed chr omat i n, and a di sper sed nucl ear envel ope . FI GURE The r ear r angement s of t he mi cr ot ubul es l eadi ng t o a met aphase st at e seem ver y si mi l ar t o t hose of ear l y pr ophase and pr omet aphase, suggest i ng t hat i n t he met aphase- ar r est ed egg, al l t he i nf or mat i on necessar y f or t he ear l y mi t ot i c event s i s pr esent . I f t hi s i s t r ue, i t may suggest t hat t he mi t ot i c- i nt er phase conver si on i s a t wo- st ep pr ocess, wi t h al l of t he det ai l s of spi ndl e assembl y " har d wi r ed" i nt o t he i ndi vi dual c omponent s, whi ch mer el y r espond t o t he new envi r onment . Recent exper i ment s conduct ed i n our l abor at or y suggest t hat DNA synt hesi s may be r egul at ed by a si mi l ar t wo- st at e mec hani s m. ' W e woul d l i ke t o acknowl edge t he Nat i onal I nst i t ut e of Gener al Medi cal Sci ences and t he Amer i can Cancer Soci et y f or t hei r suppor t of t hi s wor k . We al so acknowl edge t he i nt el l ect ual and exper i ment al cont r i but i on of Ti m Mi t chi son and Ri char d Hubbl e. We t hank Cynt hi a Cunni ngham- Her nandez f or pr epar i ng t he manuscr i pt . REFERENCES 1 . Cal ar co- Gi l l am, P. D. , M. C. Si eber t , R. Hubbl e, T . Mi t chi son, and M. Ki r schner . 1983 . Cent r osome devel opment i n ear l y mouse embr yos as def i ned by an aut oant i body agai nst per i cent r i ol ar mat er i al . Cel l . 35 : 621- 629. 2 . For bes, D . J ., M . Ki r schner , and J. Newpor t . 1983. Spont aneous f or mat i on of nucl eusl i ke st r uct ur es ar ound bact er i ophage DNA mi cr oi nj ect ed i nt o Xenopus eggs . Cel l . 34 : 1323 . 3 . Ger har t , J . C. 1980. Mechani sms r egul at i ng pat t er n f or mat i on i n t he amphi bi an egg and ear l y embr yo . I n Bi ol ogi cal Regul at i on and Devel opment . R. F. Gol dber ger , edi t or . Pl enum Pr ess, New Yor k . 4 . Ger har t , J . C. , M. Wu, and M. Ki r schner . 1984 . Cel l cycl e dynami cs of an M- phase speci f i c cyt opl asmi c f act or i n Xenopus l aevi s oocyt es and eggs. J Cel l Bi ol . 98 : 12471255 . 5 . Gur don, J . B. , and H. R. Woodl and . 1968. The cyt opl asmi c cont r ol of nucl ear act i vi t y i n ani mal devel opment . Bi ol . Rev . Camb. Phi l os . Soc. 43 : 233- 267. 6 . Har a . K. , P. Tydeman, and M. Ki r schner . 1980 . A cyt opl asmi c cl ock wi t h t he same per i od as t he di vi si on cycl e i n Xenopus eggs . Pr oc. Nai l . Ac ad Sci . USA. 77 : 462- 466. 7 . Har l and, R. M., and R. A. l . askey . 1980. Regul at ed r epl i cat i on of DNA mi cr oi nj ect ed i nt o eggs of Xenopuss l aevi s. Cel l . 21 : 761- 771 . 8 . Hei demann, S. R. , and M . Ki r schner . 1975 . Ast er f or mat i on i n eggs of Xenopus l aevi s : i nduct i on by i sol at ed basal bodi es. J. Cel l Bi oL 67 : 105- 117 . 9 . Kar sent i , E. , J . Newpor t , R. Hubbl e, and M. Ki r schner . 1984 . The i nt er conver si on of met aphase and i nt er phase mi cr ot ubul e ar r ays, as st udi ed by t he i nj ect i on of cent r osomes and nucl ei i nt o Xenopus eggs . J . Cel l Bi ol . 98: 1730- 1745 . 10 Li gat ed PBR322 DNA pr omot es mi cr ot ubul e assembl y i n met aphase eggs . PBR322 l i gat ed f or 5 mi n by t he T4 DNA l i gase as descr i bed i n t he l egend t o Fi g . 9 was i nj ect ed i nt o eggs ( nonact i vat ed) i n t he pr esence of 10 mM EGTA . 2 h l at er , t he eggs wer e f i xed . Par af f i n sect i on . X 600 . ( Ar r owheads) Condensed DNA . ( Ar r ows) Mi cr ot ubul e bundl es . Not e t hat mi cr ot ubul es do not seem t o be i n di r ect cont act wi t h t he DNA. FI GURE E. KARSENTI ET AL . Cent r osomes and Chr omat i n i n Mi cr ot ubul e As s embl y 5 3s Downloaded from http://rupress.org/jcb/article-pdf/99/1/47s/1643001/47s.pdf by guest on 23 October 2023 chr omat i n . 10 . Masai , Y. 1974 . A cyt ost at i c f act or i n amphi bi an oocyt es: i t s ext r act i on and par t i al char act er i zat i on . J. Exp. Zool . 187 : 141- 147. 11 . Meyer hof , P . G., and Y. Masui . 1979. Chr omos ome condensat i on act i vi t y i n Rana pi pi ens eggs mat ur ed i n vi vo and i n bl ast omer es ar r est ed by cyt ost at i c f act or ( C. S. F. ) . Ex p. Cel l Res. 123: 345- 353. 12 . Mi ake- Lye, R. , J . Newpor t , and M. Ki r schner . 1983. Mat ur at i on pr omot i ng f act or i nduces nucl ear envel ope br eakdown i n cycl ohexi mi de- ar r est ed embr yos of Xenopus l aevi s . J. Cel l Bi ol . 97: 81- 91 . 13 . Mol e- Baj er , J . , and A. S. Baj er . 1983 . Act i o n of t axol on mi t osi s: modi f i cat i on of mi cr ot ubul e ar r angement s and f unct i on of t he mi t ot i c spi ndl e i n Haer namhus endosper m. J. Cel l Bi ol . 96 : 527- 540 . 14, Szol l osi , D. , P. Cal ar co, and P. Donahue . 1972 . Absence of cent r i ol es i n t he f i r st and second mei ot i c spi ndl es of mouse oocyt es. J . Cel l Sci . 11 : 521- 541 . 15, Tuf f anel l i , D. L . , F . McKeon, D. Kl ei nsmi t h, T. K. Bur ham, and M. Ki r schner . 1983 . Ant i cent r osome and ant i cent r i ol e ant i bodi es i n t he scl er oder ma spect r um. Ar ch . Der mal ol . 119 : 560- 566. 16 . Zor n, G. A. , J . Lucas, and J . R. Kat es . 1979. Pur i f i cat i o n and char act er i zat i on of r egener at i ng mouse L929 kar yopl ast s . Cel l . 18: 659- 672 . Downloaded from http://rupress.org/jcb/article-pdf/99/1/47s/1643001/47s.pdf by guest on 23 October 2023 54s THE JOURNAL OF CELL BI OLOGY - VOLUME 99, 1984