I nhi bi t i on of Neuri t e I ni t i at i on and Growt h by Taxol PAUL C. LETOURNEAU and ALI CE HARJ U RESSLER Depart ment of Anat omy, Uni ver si t y of M i nnesot a, M i nneapol i s, M i nnesot a 55455 ABSTRACT We cul t ur ed sensor y neur ons f r om chi ck embr yos in medi a cont ai ni ng t he al kal oi d t axol at concent r at i ons f r om 7 X 10 -9 to 3 .5 X 10-6 M . When pl at ed at t axol concent r at i ons above 7 X 10 -8 Mf or 24 h, neur ons have shor t br oad ext ensi ons t hat do not el ongat e on t he cul t ur e subst r at um . Whèn act i vel y gr owi ng neur i t es ar e exposed to t hese l evel s of t axol , neur i t e gr owt h st ops i mmedi at el y and does not r ecommence . The br oad pr ocesses of neur ons cul t ur ed 24 h with t axol cont ai n densel y packed ar r ays of m i cr ot ubul es t hat l oop back at t he ends of t he pr ocess . Neurof i l ament s ar e segr egat ed f r om m i cr ot ubul es i nt o bundl es and t angl ed mas s es in t hese t axol - t r eat ed neur ons . At t he ends of neur i t es t r eat ed f or 5 m in with t axol , m i cr ot ubul es al so t ur n and l oop back abnormal l y t owar d t he per i kar yon . In t he pr esence of 7 X 10 -9 Mt axol neur i t es do gr ow, al t hough t hey ar e br oader and l ess br anched t han normal l y . The neur i t es of t hese cel l s appear to have nor mal st r uct ur e except f or a l ar ge number of m i cr ot ubul es . Taxol pr obabl y st i mul at es m i cr ot ubul e pol ymeri zat i on in t hese cul t ur ed neur ons . At hi gh l evel s of t he dr ug, t hi s act i on i nhi bi t s neur i t e i ni t i at i on and out growt h by removi ng f r ee t ubul i n f r om t he cyt opl asm and dest r oyi ng t he nor mal cont r ol of m i cr ot ubul e assembl y in gr owi ng neur i t es . The r api d i nhi bi t i on suggest s t hat m i cr ot ubul e assembl y may occur at neur i t e t i ps . At l ower concent r at i ons, t axol may sl i ght l y enhance t he mechani sms of m i cr ot ubul e assembl y in neur ons, and t hi s al t er at i on of nor mal pr ocesses changes t he morphogenet i c pr oper t i es of t he gr owi ng neur i t es . Many cel l ul ar m i cr ot ubul es ar e in a dynam ic equi l i br i um with unpol ymer i zed t ubul i n and associ at ed pr ot ei ns ( 1, 13, 17, 28, 40) . Sever al dr ugs (e .g ., col chi ci ne) bi nd to t ubul i n, and promot e t he di sassembl y of t hese m i cr ot ubul es ( 25) . Thi s act i on has been used to pr obe t he i nvol vement of l abi l e m i cr ot ubul es in sever al act i vi t i es, i ncl udi ng m i t osi s and cel l motility ( 16, 42) . Taxol , anot her pl ant al kal oi d, bi nds to t ubul i n and l owers t he cr i t i cal concent r at i on r equi r ed f or pol ymeri zat i on of pur i f i ed t ubul i n ( 32, 35) . The t axol - con- t ai ni ng m i cr ot ubul es ar e r esi st ant to col d t emper at ur e and concent r at i ons of Ca" t hat normal l y depol ymer i ze m i cr ot u- bul es . Addi t i on of t axol to cel l s i nduces unusual ar r ays of m i cr ot ubul es, and pr oduces ef f ect s t hat i ncl ude abnor mal myof i bri l l ogenesi s and i nhi bi t i on of m i t osi s, cel l pr ol i f er at i on, and cel l m i gr at i on ( 2, 3, 10, 29, 30, 33, 34, 39, 43) . Thus, it appear s t hat promot i on and st abi l i zat i on of m i cr ot ubul es can di sr upt cel l f unct i ons, j ust as m i cr ot ubul e depol ymer i zat i on does . M i cr ot ubul es have sever al r ol es in neur i t e out gr owt h . Sen- si t i vi t y t o col chi ci ne has i mpl i cat ed m i cr ot ubul es in t he out - gr owt h and st abi l i zat i on of neur i t es, as wel l as in t he nor mal suppr essi on of f i l opodi al act i vi t y al ong neur i t es, proxi mal to t he gr owt h cone ( 8, 9, 47) . Taxol has been appl i ed t o expl ant s of mous e sensor y gangl i a, and unusual ar r ays of m i cr ot ubul es THE J OURNAL OF CELL BI OLOGYzyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA " VOLUME 98 APRI L 1984 1355- 1362 0 The Rockef el l er Uni ver si t y Pr ess - 0021- 9525/ 84/ 04/ 1355/ 08 $1 . 00 wer e r epor t ed in neur onal s omas and neur i t es ( 29) . However , ef f ect s of t axol on neur i t e out gr owt h wer e not ment i oned in t he paper . We have f ound t hat neur i t e out gr owt h in vitro f r omsensor y neur ons of chi ck embr yos is i nhi bi t ed by t axol ina dose- dependent manner . At hi gh concent r at i ons neur i t e pr oduc- t i on is lim i t ed to shor t st out meganeuri t es . At t he l ow concen- t r at i ons l onger , t hi ck neur i t es ar e f ormed, but t hey have abnormal l y f ew br anches . Exi st i ng neur i t es cease gr owt h im- medi at el y when exposed to hi gh concent r at i ons of t axol . Al t er at i on in pr ot r usi ve act i vi t y at neur i t e t i ps is al so pr oduced by t axol . Neur ons t r eat ed f r om begi nni ng of cul t ur e with hi gher concent r at i ons of t axol cont ai n bundl es of cl osel y packed m i cr ot ubul es t hat excl ude neurof i l ament s and ot her or ganel l es . These f i ndi ngs i ndi cat e t hat an abnor mal enhance- ment of m i cr ot ubul e assembl y and st abi l i t y di sr upt s t he mec h- ani sm of neur i t e el ongat i on . We will di scuss t he r el at i onshi ps of t hese r esul t s to t he dynam i cs of m i cr ot ubul es in gr owi ng neur i t es . MATERI ALS AND METHODS Cel l CUl t or e : Dor sal r oot gangl i a wer e di ssect ed f r om 9- 11- d- ol d chi ck embr yos and di ssoci at ed with cr ude t r ypsi n ( 20) . The r esul t ant cel l suspensi on was pl aced i nt o a cul t ur e medi umcont ai ni ng 40%F12 nut r i ent m i xt ur e, 10% 1355