Cal modul i n-M i cr ot ubul e Associ at i on in Cul t ured Mammal i an Cel l s 904 W I LLI AM J . DEERY, ANTHONY R. MEANS, and B . R. BRI NKLEY Depart ment of Cel l Bi ol ogy, Bayl or Col l ege of Medi ci ne, Texas Medi cal Cent er , Houst on, Texas 77030 ABSTRACT A Tr i t on X- 100- l ysed cel l syst em has been used to i dent i f y cal modul i n on t he cyt oskel et on of 3T3 and t r ansf or med SV3T3 cel l s . By i ndi r ect i mmunof l uorescence, cal modul i n was f ound to be associ at ed with bot h t he cyt opl asm icm i cr ot ubul e compl ex and t he cent r o- s omes . A number of cyt opl asm ic m i cr ot ubul es mor e r esi st ant to di sassembl y upon ei t her col d ( 0- 4°C) or hypot oni c t reat ment , as wel l as f ol l owi ng di l ut i on have been i dent i f i ed . Mos t of t he st abl e m i cr ot ubul es appear ed to be associ at ed wi t h t he cent r osome at one end and with t he pl asma membr ane at t he ot her end . These m i cr ot ubul es coul d be i nduced to depol ymer- i ze, however , by m i cromol ar Ca" concent r at i ons . These dat a suggest t hat , by i nt er act i ng di r ect l y with t he m i cr ot ubul e, cal modul i n may i nf l uence m i cr ot ubul e assembl y and ensur e t he Ca" - sensi t i vi t y of bot h m i t ot i c and cyt opl asm ic m i cr ot ubul es . Si nce t he di scover y t hat Ca" i nhi bi t s m i cr ot ubul e assembl y in vitro ( 36) , subsequent st udi es have i ndi cat ed t hat sever al f act or s can i nf l uence t he degr ee of Ca" - sensi t i vi t y . These f i ndi ngs have i mpl i cat ed i nt r acel l ul ar Ca" i n t he r egul at i on of m i cr ot ubul e assembl y in vi vo . Tubul i n possesses hi gh i nt r i nsi c sensi t i vi t y to m i cromol ar Ca" concent r at i ons ( 1, 16) and t hi s sensi t i vi t y can decr ease t o t he m illimolar r ange f or t ubul i n- cont ai ni ng m i cr ot ubul e- associ at ed pr ot ei ns ( MAPS) ' t hat co- pur i f y with t ubul i n f ol l owi ng cycl es of as s em- bl y- di sassembl y ( 20, 26) . As s embl y in cr ude br ai n ext r act s exhi bi t s hi gh Ca" - sensi t i vi t y ( 12, 29) and addi t i on of cr ude ext r act to cycl e- pur i f i ed t ubul i n r est or es t he sensi t i vi t y ( 25) . A cl ass of pr ot ei ns t hat co- pur i f i es withm i cr ot ubul es f r om r at br ai n and t hat gr eat l y i ncr eases m i cr ot ubul e st abi l i t y in vitro ( 14, 21, 35) ( st abl e t ubul e- onl y pr ot ei ns [ STOPS] ), has r ecent l y been i dent i f i ed . Pol ymeri zed m i cr ot ubul es cont ai n- i ng concent r at i ons of STOPS subst oi chi omet r i c to t hat of t ubul i n ar e st abl e at 0- 4°C or in t he pr esence of m illimolar Ca" , condi t i ons t hat normal l y woul d r api dl y depol ymer i ze m i cr ot ubul es ( 13) . However , addi t i on of ATP or t he Ca" - dependent r egul at or y pr ot ei n, cal modul i n, r ender s t hese m i- cr ot ubul es l abi l e t o col d or Ca" , r espect i vel y . The decr eased Ca" - sensi t i vi t y of m i cr ot ubul es st abi l i zed by MAPs and STOPS, f or exampl e, mus t be modul at ed by s ome f act or ( s) pr esent in cr ude ext r act s . Cal modul i n is one ' Abbr evi at i ons used i n t hi s paper : CMTC, cyt opl asm ic m i cr ot ubul e compl ex ; MAPs , m i cr ot ubul e- associ at ed pr ot ei ns ; PEG, pol yet hyl ene gl ycol ; RB, r eassembl y buf f er ; STOPS, st abl e t ubul e- onl y pr ot ei ns . candi dat e because it was i ni t i al l y demonst rat ed t hat cal mo- dul i n ant i bodi es speci f i cal l y st ai n col d- st abl e ki net ochor e- t o- pol e and i nt er zonal m i cr ot ubul es in t he m i t ot i c appar at us ( 38) . In addi t i on, it was suggest ed t hat Ca++/ cal modul i n coul d medi at e m i cr ot ubul e di sassembl y in vi t r o, al bei t at hi gh cal - modul i n to t ubul i n mol ar r at i os ( 19, 20, 24, 27) . Fur t her suppor t f or t hi s hypot hesi s c omes f r om t he obser vat i ons t hat MAP2 ( 28) and STOPS ( 14) will bi nd to a cal modul i n af f i ni t y col umn in a Ca" - dependent f ashi on . Fi nal l y, col d- st abl e m i cr ot ubul es r esi st ant to Ca" al one, r eadi l y depol ymeri ze at subst oi chi omet r i c cal modul i n concent r at i ons in vitro ( 13) . However , despi t e t he above f i ndi ngs, to dat e, t he pr esence of cal modul i n on i nt er phase cyt opl asm ic m i cr ot ubul es has not been demonst rat ed . Fol l owi ng i ncubat i on of cel l s in Tr i t on X- 100, mos t of t he pl asma membr ane is sol ubi l i zed and unpol ymer i zed t ubul i n is r eadi l y di l ut ed and ext r act ed, cr eat i ng a permeabi l i zed cel l syst em f or st udyi ng t he m i cr ot ubul e- subuni t di ssoci at i on r e- act i on ( 2, 30) . The cel l s ar e al so wel l ext r act ed f or subsequent i mmunof l uorescent st udi es . In t he pr esent r epor t , we have empl oyed such a Tr i t on- l ysed cel l syst em to st udy t he l ocal i - zat i on of cal modul i n upon di sassembl y of t he cyt opl asm ic m i cr ot ubul e compl ex ( CMTC) and f ol l owi ng m i cr ot ubul e r eassembl y . We obser ve a number of cyt opl asm ic m i cr ot u- bul es t hat do not di sassembl e upon ei t her col d t reat ment or var i ous di l ut i on condi t i ons but do di sassembl e in t he pr esence of m i cromol ar Ca" concent r at i ons . I ndi r ect i mmunof l uores- cence st udi es i ndi cat e t hat cal modul i n is associ at ed with cyt opl asm ic m i cr ot ubul es and t he cent r osomes . THE J OURNAL OF CELL BI OLOGYzyxwvutsrqponmlkjihgfedcbaZYX " VOLUME 98 MARCH 1984 904- 910 0 The Rockef el l er Uni ver si t y Pr ess - 0021- 9525/ 84/ 03/ 0904/ 07 $1 . 00 on October 20, 2015 jcb.rupress.org Downloaded from Published March 1, 1984