Microtubules and Guidance

8/13/2019 Microtubules and Guidance

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Mcrotubul eBehavi or duri ngGui danceof Pi oneer NeuronGrowthCones i n s i tuJ amesH Sabry, TimothyP OConnor, Loui se Evans, AlmaToroi an-Raymond, *Marc Ki rschner,andDavi d Bent l ey*Depar tment of Bi ochemst ry and Bi ophysi cs, Uni versi ty of Cal i f ornia, San Francisco, Cal i forni a 94143; and *Department ofMol ecul ar and Cel l Bi ol ogy, Uni versi ty of Cal i f ornia, Berkel ey, Cal i forni a94720

Abstract The growt h of an axon t oward i t s targetresul ts fromthe reorgani zat i on of the cyt oskel et on i nresponse to envi ronment al gui dance cues Recent l y de-vel oped i magi ng t echnol ogy makes i t possi bl e to ad-dress the effect of such cues on the neural cytoskel e-ton di rectl y Al t hough hi gh resol ut i on studi es can becarr i ed out on neurons i n vi tr o, t hese ci rcumst ancesdo not recreate the compl exi t y of the natural envi -ronment We report here on the arrangement and dynamcs ofmcr otubul es i n l i v e neurons pathf i ndi ng i n responseto natural gui dance cues i n si tu usi ng the embryoni cgrasshopper l i mb f i l l e t preparati on r ich mcrotu-bul e net work was present w thi n the body of thegrowt h cone and normal l y ext ended i nto the di stalgrowt h cone margi n Compl ex mcr otubul e l oops of tenf ormed tr ansi entl y w thi n the gr owt h cone Br anches

EVELOPJ NG neurons may send thei r processes overgreat di stances and through compl ex envi ronmentst establ i sh contact w th thei r target ce l ls ( Caudyand Bent l ey, 1986b; Dodd and J essel l , 1988 ; Goodman etal . , 1984; Harr i s et al 1987 ; O Leary and Terashi ma, 1988;Tosney and Landmesser, 1985; Westerf i el d and Ei sen,1988) Thi s outgrowth i s not randombut , rather, i s gui dedby envi ronmental cues I t has l ong beenappreci ated that neu-ronal gui dance can essenti al l y be vi ewed as the process bywhi ch the growth cone, thedynamc and expanded t i p of theneuri t e, i s ori ented by envi ronmental cues (Harr i son, 1910) These cues appear to be provi ded byavari ety of factors, i n-cl udi ng subst rate-bound mol ecul es on cel l surf aces and i nthe extracel l ul ar matr i x, anddi f fusib le chemoatt ractant mol -ecules ( Anderson, 1988 ; El ki ns et al . , 1990; Fessl er andFessl er, 1989; Goodman et al . , 1984 ; Rei chardt and Toma-sel l i , 1991 ; Tessi er- Lavi gneandPlaczek, 1991) Thearrange-ment of these factors i n the devel opi ng embr yo presumabl yprovi des the i nf ormati on necessary to gui de the devel opi ngneurons I n order f or growt h cones to reach thei r targets,J H Sabry andT P O Connor cont ri but ed equal l y to the experi ments i nthi s paper

he Rockefel l er Uni versi ty Press, 0021- 9525/ 91/ 10/ 381/ 15 2. 00TheJ ournal of Cel l Bi ol ogy, Vol ume 115, Number 2, October 1991381-395

both w th and w thout mcr otubul es were regul arl y ob-served M crotubul es di d not extend i nto f i l opodi a Dur i ng growt h cone steeri ng events i n response t oi denti f i ed gui dance cues, mcr otubul e behavi our coul dbe moni t ored I n turns towards gui depost cel l s,mcr ot ubul es sel ecti vel y i nvaded branches der i vedfromf i l opodi a that had contacted the gui depost cel l At l i mb segment boundar i es, mcr ot ubul es di spl ayed avari ety of behavi ors, i ncl udi ng sel ecti ve branch i nva-si on, and al so i nvasi on of mul t i pl e branches f ol l owedby sel ecti ve retent i on i n branches or i ented i n the cor-rect di recti on M crot ubul e i nvasi on of mul t i pl ebranches al so was seen i n growt h cones mgrati ng oni nt rasegment al epi t hel i um Both sel ecti ve i nvasi on andsel ecti ve retent i on generate asymmet r i cal mcr otubul earrangements wthin the growt h cone, andmay pl ay akey rol e i n growt h cone steeri ng events

they must al ter thei r di recti onof growt h i n response to thesefactors, a process t ermed steeri ng The process of growt h cone steeri ng f i r s t i nvol ves the ex-pl orati on of t he envi ronment by growth cone f i l opodi a,l amel l i podi a and branches t o l ocate di screte gui dance cuesor to eval uate the spati al di stri buti on of cues i n the regi onaround the growth cone (Bent l ey and Toroi an-Raymond,1986 ; Brayand Hol l enbeck, 1988 ; Gol dberg and Burmei st er,1989 ; M tchi son and Ki rschner, 1988) A subset of f i l opodi a,l amel l i podi a, or branches are then sel ected f or the di recti onof further growth cone extensi on Thi s i s f ol l owed by the i n-tr usi on of cel l ul ar materi al into t he sel ected process, resul t-i ng i n growth cone reori entati on (Al ett a and Greene, 1988;Gol dberg and Burmei st er, 1986; Hei demann et al . , 1984) These cel l ul ar component s are subsequentl y consol i datedi nto a stabl e conf i gurati on characteri sti c of the axon (H ro-kawa et al 1988; Lew s et al . , 1989) Many cel l ul ar components, i ncl udi ng cytoskel etal ele-ment s such as mcr otubul es andacti n f i l aments, and i ntr acel -l ul ar second messenger mol ecul es, are l i ke ly to be i nvol vedi n the di rected growt h of axons ( Gol dberg and Burmei st er,1989; Kater and M l l s , 1991; Lankf ord and Letourneau,1989; Mei ni nger and Bi net , 1989) I t i s not knownwhether

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envi ronmental cues di rect ly affect al l these components, butori ented axonal growt h ul t i matel y must al ter the arr ange-ment of cytoskel etal pol ymers w thi n the axon I ndeed, thegenerati on of an asymmetr i c arrangement of mcr otubul esmay bea key event i n growt h cone steeri ng Thi s can be ac-compl i shed by t wo mechani sms ( a) sel ect i ve mcr otubul ei nvasi on of a l i mted regi on of t he growth cone; or (b) ran-dom nvasi on of many regi ons of the growth cone f ol l owedby sel ecti ve retenti on of mcr otubul es extended i n the di -rect i onof future growth Recentl y, di rect observati ons of m-crotubul es i n l i v ing Xenopus growth cones i n v i t ro havei denti f i ed mcr otubul e behavi ors that may underl i e steeri ngdeci si ons (Tanaka and Ki rschner, 1991) However , i t i s notknown whi ch, i f any, of these behavi ors pl ay a ro le i npathf i ndi ng i n the compl ex envi ronment of the embryoI n order to address t hi s i ssue, we characteri zed t he dy-namc behavi or of mcr otubul es i n response t o i n si t u gui d-ance cues by i magi ng f l uorescentl y l abel ed mcr otubul es i nTi l pi oneer neurons i n l i ve grasshopper embryoni c l i mbf i l l e t s I n the grasshopper embryo, apai r of sibl i ng neurons,t ermed theTi l pi oneers, aret he f i r s t neurons to extendaxonstoward the central nervous system(Bate, 1976; Bent l ey andKeshi shi an, 1982; Ho andGoodman, 1982) They arebornat the di stal t i p of the l i mbbud, emer ge on the basal surf aceof theepi thel i um and extendgrowt h cones proxi mal l y al ongthe l i mb axis As they mgrate, the growth cones contact acompl ex envi ronment consi sti ng of epi thel i al cel l s, basall amna and pre-axonogenesi s neurons t ermed gui depostcel l s ( Anderson and TVcker, 1988 ; Caudy and Bent l ey,1986b; Condi c and Bent l ey, 1989b ; Lef cort and Bent l ey,1987) The pathway taken by the Ti l growth cones i s i l lus-trated i n Fi g Key regi ons of thepathway, wher e di st i ncti vegrowth cone behavi ors occur, are encl osed i n the seri es ofboxes Box 1 showsagrowt h cone i n the f emur, where i t i n-teracts pri mari l y w th i ntrasegmental epi thel i al cel l s and theoverl yi ng basal l amna I n thi s regi on, growth cones mgrateproxi mal l y al ong the l i mb axi s w th f requent smal l coursecorrecti ons Box 2 shows thegrowt h cones spreadi ng on theTrl gui depost cel l at theTr- Cx segment boundary At thi s l o-cat i on, t he growth cones encounter t wo ort hogonal l y ar-ranged bands of l i mb segment boundary cel l s, a di stal bandof hi gh adhesi vi ty ce l ls (see Fi g f i l l ed hexagons) , and aproxi mal band of l ower adhesi vi ty ce l l s shownbythe unf i l l edhexagons i n Fi g ( Basti ani et a l . , 1991; Caudy and Bent l ey,1987 ; Condi c and Bent l ey, 199a) Thegrowth cones extendbranches both dorsal l y (up i n a l l f i gures) and ventr al l y onthef i r s t bandof cel l s, but eventual l y al ways makeaventral turn Box 3 shows the growth cones mgrati ng ventr al l y al ong theTr - Cx segment boundary and approachi ngtheCxl gui depostce l ls Wena growthcone f i l opodi umcontacts t he Cx1 cel l s,thegrowthcone turns al ong i t , by aprocess of f i l opodi al di l a-ti on, t oward the CNS ( O Connor et a l 1990) The T i l pathway i n the embryoni c grasshopper l i mb canberenderedaccessi bl e f or mani pul ati onusi nganopened epi -thel i al f i l l et preparati on (Lefcort andBent l ey, 1987) Thi sexposes the T i l cel l bodi es, but preserves the gui dance i nfor-mati on present i n t he l i mb, and al l ows the growth cones tobe i maged as they mgrate proxi mal l y In the experi mentsreported here, we i nj ected the T i l neuron w th rhodamne-conj ugated bovi ne tubul i n and i maged the l abel ed mcrotu-bul es usi ng a cool ed charge- coupl ed devi ce (CCD) camer asystem (Cast l eman, 1979; H raoka et al . , 1987) The ar-

The J ournal of Cel l B ol ogy, Vol ume 115, 1991

rangement of mcr otubul es i n growt h cones mgrati ng i n acompl ex envi ronment , and howthey respond duri ng path-f i ndi ng deci si ons, coul d be examned I n part i cul ar, we de-termned where mcr otubul es are present i n growt h cones i nsit u, andhowthei r arrangement changes over t i me as steer-i ngdeci si ons aremade Weal so i nvesti gatedwhether mcro-tubul e rearrangements are di f f erent duri ng steeri ng events atdi f f erent l ocati ons al ong thepathway Thi s approach reveal ednovel aspects of mcr otubul e behavi or that address themech-ani smof growth cone steeri ng Materi al s andMethodsGrasshopper Embr yos andD ssecti onSchi stocerca ameri cana embr yos were obtai ned fromthe Uni versi ty ofCal i forni a at Berkel ey grasshopper col ony Eggs at t he 31-34 stages ofembryoni c devel opment were ster i l i z ed and the embr yos di ssected as previ -ousl y descr i bed (Lefcort and Bentl ey, 1987) Theembryos were transferredto a pol y-L-l ysi ne- coated coversl i p andmai ntai ned i n suppl ementedRPM( Condi c andBentl ey, 1989a) Bri efl y, the embr yos were posi ti onedventralsi de down, thus exposi ng the posteri or aspect of the l i mbbud Thi s surf acewas cut along the l ong axi s of t he l i mb, and t he si des unrol l edand fl attenedout onto t he coversl i p Theexposed i nteri or mesoder mal cel ls were removedusi ng a sucti on pipett e, l eavi ng the basal l amna and epi thel i umderi vedcel l s The Ti l neuronal cel l bodi es werevi sual i zed w th dif ferenti al i nterfer-ence contrast opti cs usi ng a Ni kon i nvert ed compound mcroscope Fl uorescent Label i ngof 7l i bu l i nPuri fi ed bovi ne brai n tubul i n was l abeled w th tetr amethyl - rhodamne aspreviousl y descri bed(Hyman et al . , 1991) Thi s process i nvol ved coval entl yl i nki ng the N- hydroxyl succi ni mdyl ester of tetr amethyl - rhodamne (kC-1171 ; Mol ecul ar Probes, Eugene, OR) t o puri fi ed bovi nebrai ntubul i n Thel abeled tubul i nwas then subj ectedto t wo cycl es of temperature- dependentassembl y/ di sassembl y t o select f or assembl y compet ent tubul i n The l a-bel ed tubul i nwas stored at a concentrat i onof 20- 30 mg/m i n an i nj ect i onbuff er ( 50mMKgl utamate, 0 5 MMMgC12, pH 6 . 5) at - 80CNeuronal Label i ngA Ti l neuron cel l body was i n ected w th rhodamne- conj ugated bovi ne tu-bul i nusi ngapul l ed, bevel l edborosil i cate mcropi pett e Sel ected cel l s wereal so doubl e l abeledw th t he carbocyani ne dye, D O (1 , 1 -di hexadecyl oxa-carbocyani ne perchorate, D OCI 6, ND- 1125 ; Mol ecul ar Probes) as previ -ousl y descr i bed ( O Connor et al . , 1990) Bri efl y, th is i nvol ved ai r dryingt heD Oonto t he ti p of apul l ed mcropi pette, andl abel i ng t he cel l by gentl ytouchi ng i t w th the mcropi pett eM crot ubul e I magi ngandAnal ysi sMost i magi ng, i ncl udi ng the i mages selected f or t he f i gures, was done att he Berkel ey Low l i ght- l evel Vi deo Center usi ng a cool ed charge- coupl eddevi ce (CCD) camer a system (Photometr i cs ; Tuscon, AZ) Thi s systemcompri sed view ng t he f l uorescent ti ssue through a 100X, 1 . 4 N N konobj ecti ve onaNi kon i nvert ed compoundmcroscope w th conventi onal r e-l ay opt i cs connecti ngt he mcroscope t o the Dcamer a The mcroscopeproj ected t he i mage onto a 1320 x 1024 pixel chi p Kodak (KAF- 1400) ,whi ch d ig i t i zedthe i mage andtransferredthe data t o a bul kmemor y storagedevi ce (REO650 erasable opt i cal di sc ; Pinnacl e M cro, I nc . , I rvine, CA) I mage space on the chi pwas0. 06/pi xelhe chi p, l i ght path shutters,and stage focal posi ti on were contr ol l ed byaPercept i cs Bi dVisi on i magi ngsystem(Knoxvi l l e, TN) onaMac I Ncomputer (Appl e Comput ers, Cuper-ti no, CA) I l l umnati onwas provi dedby a 100ercury l amp, generall yi n 300-400m exposures To i mage t he full thi ckness of the growt h cone,f ive t o ei ght opti cal sect i ons were usuall y taken C usters of secti ons wereusuall y takenevery 4 t o 8 mn On occasi on, i mages were taken every3 st o resol ve high f requency events By tr acki ng f l uorescence through multi -ple i mage planes, out-of-focus f l uorescence was i denti fi ed and reducedusi ng Focus soft ware (Vaytek Fai rf i el d, I A) M crotubul es tr aversi ng multi -pl e secti ons were montaged w th Nati onal I nsti tutes of Heal th I mage soft-ware Processed i mages were phot ographed di rect l y froma 1280 x 1024

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Fi gure 1 di agramof the Ti l pi oneer neuronpathway i n the embryoni c grasshopper l i mbbudat t he 35 stage Thepai r of s i bl i ngTi l neurons ari seat the l i mb t i p andthei r growth cones mgratetothe central nervous systemCNS) al ong a stereotyped route Thegrowth cone route refl ects a seri es of steeri ng deci si ons resul t i ngfromencounters w th gui dance cues These i ncl udepreaxonogene-si s gui depost ) neurons Fel , Trl , Cxl hi gh aff in i ty f i l l e dhexa-gons) andl owaffinity unf i l ledhexagons) ci rcumerenti al bands ofepi t hel i al cel l s at l i mb segment boundari es Ti - Fe, Fe-Tr, Tr-Cx),and segmental nerve- root gl i al cel ls SnG Thedashedboxes 1-3)i ndi cate l i mb regi ons where growth cone mcrotubul e behavi or i si l l ustrated i n other f i gures 7F-Fe, t i bi a- f emur boundary Fe-7r,f emur- trochanter boundary Tr- Cx, t rochanter- coxaboundary Thel ength of t he l eg i s approxi matel y 350 , um Dorsal , up; proxi mal ,t o ri ght

pi xel vi deo-scr een E- Machi nes, Beaver t on, OR Usi nga system devel -oped by Drs. J ohn Sedat and Davi d Agard Department of Bi ochemst ry,Uni versi ty of Cal i forni a at San Franci sco), addi ti onal i mages were visual -i zed on an i nverted O ympus I MT2 mcr oscope connected to a Pel ti ercool ed CCD Photometr i cs, Tucson, AZ equi pped w t h a 900 x 900pi xelchi p Texas I nstr uments) , andcontr ol l ed by a Mcrovax I I workstati on D g-i ta l Equi pment Corp Marl boro, MA) MM croscopy

After i magi ng, sel ected embryos were prepared f or EM Thi s i nvol vedf ixing them n2 gl utaral dehyde i n PBSf or 4hat roomemperature Theywere then reacted w t h apri mary serumant i body agai nst HRP that recog-nizes i nsect neurons J anand J an, 1982; Snowet al 1987) Thi s ant i bodywas visual i zed us ing a 10 nmgol d conj ugated secondary ant i body Amer -sham UK) . Aft er asecond fi xati on i n 2 gl utaral dehyde i n PBS, t heti ssuewas ri nsed andosmcatedi n 2 OsO4 i n cacodyl ate buff er for 30mn Thel abel ed ti ssue was then dehydrat ed rapi dl y through an et hanol seri es, andembedded i n ar al di te Grade CY212 Bri ti sh, Pol ysci ences, Warr i ngt on,PA) TheTi l pi oneer neur on was f i r s t i denti fi ed i n thick secti ons at t he l i ghtmcroscopi c l evel by sil ver enhanci ngthegol d part i cles Amersham UK) Thi n secti ons were then cut and observed i n a Phil l ips 400electron mcro-scope 80 kV)

Resul tsTo observe mcrotubul es i n grow ngneurons, we f l att enedout the tubul ar l i mb bud, bathed i t i n cul ture medi um i n-j ected t he Ti l pi oneer neuron, andexposed i t to bri ef pul sesof vi si bl e l i ght Under these condi t i ons, t he route takenbyt he Ti l growth cone was i ndi sti ngui shabl e fromthat of theneuron i n vi vo Thi s suggest s not onl y that natural i n vi vogui dancecues are preserved i n t he f i l l e t , but al so that t he i n-corporati on and subsequent i magi ng of r hodamne-conj u-gatedbovi ne tubul i n has l i t t l e effect on t he pat tern of Ti l ax-onal growh TheTi l pi oneer neuronsare si bl i ngs, andonl yoneof each

Sabry et al M crotubul e Behavi or duri ng Atonal Gui dance

pai r was i nj ected f or these studi es. Al though t hetwoneuronsare str ongl y coupl ed by dye-passi ng j uncti ons Keshi shi anand Bent l ey, 1983 Taghert et al . 1982), no l abel was everf oundto cross t o the noni nj ected c e l l , i mpl yi ng that no un-conj ugated free rhodamnedyewas generatedbyproteol ysi sof the l abel ed monomer The l abel t ravel ed down theaxonandwas f oundto persi st as al owl evel of background f l uores-cence, t hought to represent monomeri c rhodamne- tubul i n,and i n clearl y defi ned l i near tracks i n the axon andgrowthcone n exampl e of these tracks i s i l l ustrated i n Fi g 2whi ch shows a r ich network of l i near tracks i n the growhcone si ngl e track i nvadi ngabranch i s shownby thearrow-head We measured thew dth and i ntensi ty of the tracks i nth is andother cel l s The i magew dthof these tracks, whi chwe w l l refer to as mcrotubul es, was f ound to be very uni -form 0. 24, um SEM 0 01 j , m mean of 15; three measure-ment s per mcr otubul e, three mcr otubul es per cel l i n f i vecel l s at var i ous stages of devel opment) . Thi s val ue i s siml art o publ i shed w dths of f l uorescent i mages of el ectr onmcro-scope-conf i rmed si ngl e mcr otubul es ammak andBori sy,1988) More i mportant l y, al though t he pi xel i ntensity ofthese l i near prof i l es vari ed signi fi cantl y fromcel l to c e l l ,w thi n a gi ven cel l al l prof i l es were of siml ar i ntensi ty Fori nstance, i n t hegrowthcone shown i n Fi g 2, t hemean i nten-s i t y of f i v e tracks f i v e measurements per track) was 91 . 6Uw th anSEM2. 7 2. 9 Thi s represented a val ue i n t hemddl e of the dynamc range of t heCCD chi p whi ch was

Fi gure 2 Mcrotubul e arrangements i n a pi oneer growth conespreadi ngongui depost cel l TH Ti l pi oneer neuron has been i n-j ectedw th rhodamne- tubul i nandthegrowth cone i s at theTrl cel l asteri sk) Thi s posi ti on corr esponds to Fi g 1 box 2 Many uni -formcal i ber l i near f l uorescent tracks, whi ch appear t o be si ngl emcrotubul es see text), are observed Al t hough the growth conew l l eventual l y turn ventr al l y at t hi s l ocati on, mcrotubul es arepresent i n branches whi te arr ows) extendingboth dorsal l y andven-t r a l l y al ongtheTr-Cxsegment boundary si ngl e mcrotubul e ar-rowhead) i s present i n a ventr al l y extended branch Bar, 5 i m

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0- 255 U As det ai l ed bel ow, M d e n t i f i e d many s i n g l em crotubul es i n t he d i s t a l growt h cone These data st rongl ysuggest t h a t these f l uorescent t racks represent s i n g l e mcro-t ubul es The l a be l persist edf or t i 7 h, and then sl ow y f aded,probabl y as a r e s u l t of tubul i n turnover TheArrangement of M c r o t ubul e s i n TS1 NeuronsI n 31 embr yos , 31 T i l growt h cones wer e i maged i n whi chm crotubul es wer e c l e a r l y resol ved These i mages wer e ob-t ai ned at var i ous pl aces i n t he pat hway, wi t h mos t observa-t i o n s at t h e tur ni ng deci s i o ns out l i ned by t he boxes i n Fi g To vi s ual i z e t he f u l l extent of t he growt h cone and i t sprocesses, ei ght gr owt h cones wer e si mul t aneousl y l abe ledw t h a l i p o p h i l i c car bocyani ne membrane dye, DOAxons I n a l l neur ons i maged, t he m crotubul es wer ef ound i n t h e axon cyl i nder i n c l o s e l y packed l i nea r a r r a ys Thi s ca n be appreci ated i n Fi g 3 F , wher e t he m crotubul esi n t he upper l e f t of t he panel wer e f ound ori ent ed al ong t hel ong a xi s of t he neuron and wer e c l osel y bundl ed together Thepl asmamembrane was c l osel y apposed t o t he mcrotu-bul e array Ot her exampl es of t he ar r angement of mcrotu-bul es i n axons ca n be seen i n Fi gs 2, 3 E, 4, 7 and 8 Occa-s i ona l l y , a t l ocat i ons of l a t e r a l l y ext ended prot r us i ons , thepl asma membrane was not c l osel y apposed t o t he mcrotu-bul e bundl e, and somem crot ubul e f r e e axopl asmcoul d bevi sual i zed data not shown) Themorphol ogi c boundary bet ween t he axon andgr owt hcone ca n usual l y bedef i nedwhenneurons ar e vi ewed i n v i t r oas t h a t regi on wher e t he axonal cyl i nder abrupt l y i ncreasesi n c al i be r Br ay and Chapman, 1985 ; Gol dber g and Bur-me i s t e r , 1986) However , i n s i t u , t h i s border coul d not e a s i l ybe i d e n t i f i e d by pl asma membranemor phol ogy i n many T i lneur ons I n th es e c a s e s , no abrupt t r a n s i t i o n f r o mt he c y l i n -d r i c al shape of t he axon t o t he gl obul ar shape of t he growt hcone was evi dent However , i n some neurons, the arrange-ment of m crotubul es di d showan unambi guous t r a n s i t i o nfromhe t i g h t l y bundl ed ar r angement c ha r ac t e r i s t i c of axonst o a r i c h net wor k of s i n g l e or smal l bundl es of m crotubul esc h a r a c t e r i s t i c of t he gr owt h cone Fo r exampl e, Fi g 3 Fshows m crotubul es i n a growt h cone of t h i s type ; t he whi t earrow i n d i c a t e s t he t r a n s i t i o n f r o mt he bundl ed m crot ubul ear r ay of t he axon, t o t he l e f t of t he whi t e ar r ow, t o t he moredi spersed ar r angement of t he growt h cone, t o t he r i g h t Growth Cones Al l growt h cones wer e heav i l y i nvest edw t h m crotubul es I n a l l cases, m crotubul es wer e f ound t oi n ha bi t t he d i s t a l regi onof t he growt h cone, up t o t h e pl asmamembrane I n Fi g 2, f or i n s t a n c e, a r i c h net wor k of

m crotubul es ca n be seen i n t he whol e growt h cone I n t h i sf i g u r e , t he edge of t he gr owt h cone was i ndi cat ed by t he back-ground l e ve l of f l uorescence I n Fi g 3 E, m crotubul es i nd i -cated by t he u n f i l l e d arrow) seem t o have ext ended d i r e c t l yunder neat h t he di st al most aspect of t he gr owt h cone pl asmamembrane M crotubul es wer e not r e s t r i c t e d t o t he c e n t r a lport i on of t he growt h cone f or prol onged peri ods of t i me , ashas been descri bed f or sometypes of neur ons i n v i t r o Br i dg-manand Da i l y , 1989 ; Forscher and Smth, 1988) I n c o n t r a s tt o t he axon, growt h cone m crotubul es wer e not r e s t r i c t e dt o t i g ht bundl es, but f requent l y occurred s i n g l y or as smal lbundl es I n s e v e r a l neur ons, i mages wer e t aken every threeseconds ; rapi d shri nkage and r egr owt h of m crotubul es wasnot appar ent I n t h e f i l l e t al t hough t he c e l l body coul d be i d e n t i f i e d byDCo pt i c s , t he growt h cone coul d onl y be vi sual i zed usi ngepi f l uorescence Many prot rusi ons, p a r t i c u l a r l y branches,coul d be i d e n t i f i e d by t he presence of backgr ound f l uo r es -cence ; however , as i t was not cl ear t h a t al l t he f i n e s t r u c t u r eof t he gr owt h cone coul d be s e e n , ei ght growt h cones weredoubl e l abe l ed w t h t he l i p o p h i l i c pl asma membrane dye,DO As has been descr i bed previ ous l y O Connor et al .1990) , weobserved ext ensi ve and var i ed protrusi ve a c t i v i t yf r o m t he growt h cone Types of protr usi ons i ncl uded af i l o p o d i a f i n e , t ubul ar s t r u c t u r e s of a s i ngl e uni f o rmcal i bert h a t a r i s e f r o m t he growt h cone or f r o mbranches ; b v e i l sand l amel l i podi a : t h i n s hee t s t ha t often ext end bet ween e x i s t -i ng f i l o p o d i a ; and c br anches el ongat e processes of v a r i -abl e s i z e and shape t h a t wer e w der than f i l opodi a , and oftentapered f r o m t h ei r base t o t i p

F l i l opodi a The T i l growt h cone i s r i c h i n f i l o p o d i a , whi chwer e abundant i n a l l doubl e- l abel ed gr owt h cones I n t o t a l ,150 f i l o p o d i a wer e i d e n t i f i e d onDO i mages I n Fi g 3 C,many l ong f i l o p o d i a arr owheads) can be seen t o have ar i senat t he di s t a l end of t he growt h cone I n t he i magi ng p l a n e,t he meannumber of f i l o p o d i a per growt h cone was 21 w t ha r ange f r o m 8 t o 34 Themean w dt h was 0 31 j Am w t hanS Mf 0. 0 1 5 4. 8 As was descri bed by O Connor etal 1990) , some f i l o p o d i a were very s t a bl e and unchangi ngi n l engt h or pos i t i on over t ens of m nut es, wher eas otherswer e very dynam c, e x i s t i n g f or a mnute or l e s s None oft he f i l opod i a , even those w t h l ong l i f e t i me s , harbored m -crot ubul es Thi s can be seen i n Fi g 3 D, whi ch i s t he rhoda-m ne- tubul i n i mage of a doubl e-l abel ed growt h cone t heDOi mage i s seen i n Fi g 3 C The u n f i l l e d arrows poi ntt o m crotubul es i n t he body of t he gr owt h cone, and i n abr anch, but none of t he f i l o p o d i a i d e n t i f i e d by arr owheads

F i g ur e 3 M crotubul e arrangement s d ur i n g gr owt h cone m g r a t i o n on a r e l a t i v e l y homogeneous s u b s t r a t e These p os i t i o ns correspondt o Fi g 1 box 1 A and B) growt h cone doubl e l a be l e d w t h a l i p o p h i l i c pl asma membr ane dye D O ; A), and rhodamne t u b u l i n B) Bundl es of m c r o t ubu l e s a r e p r es en t i n branches e x t e n di n g i n d i f f e r e n t , but g en er a l l y p r o xi ma l l y or i e nt e d di r e ct i o ns A and B, u n f i l l e darrows) M crotubul es a r e a bs e nt f r o msome branches A and B, bl ack arrow) Pronounced m crotubul e l oops e x i s t i n t he gr owt h cone A and B, wh i t e arrows) The s i b l i n g neur on was s l i g h t l y l a be l e d w t h D O and t he f a i n t i mage of t he s i b l i n g gr owt h cone can be s e e ni n t he l ower l e f t par t of A Cand D I n a no t h e r d ou bl e - l a be l e d growt h cone i n t h i s r e gi o n, s i ngl e m crotubul es a r e p r es en t i n t he gr owt hcone p er i p he r y D, doubl e arr ow), and can e x t e n d i n t o sma l l branches Cand D u n f i l l e d arrows) Many uni f ormd i ame t e r f i l o p o d i a e x t e n df r o mt he growt h cone C, arrowheads) ; t h e s e do no t c on t a i n m crotubul es E and F) I n a na s c e n t growt h cone whi ch ha s m gr a t e d as h o r t d i s t a nc e f r o mt he c e l l body E , a s t e r i s k =n u c l e u s , m crotubul e b un d l e s f o r m l o op s a g ai n s t t he gr owt h cone p e r i me t e r E , arrow) 34 m n l a t e r t h e s e l o op s have s t r a i g ht e n ed somewhat as t he m crotubul es en t e r a branch formng d o r s a l l y F u n f i l l e d arrow) M crotubul esa l s o e n t e r a ven t r a l branch whi ch was no t s el e ct e d a s t he d i r e c t i o n of growt h cone e x t e n s i o n Thi s growt h cone was s u b s eq ue n t l y i magedby M hown i n F i g s 5 and 6 W t hi n t he body of t he growt h cone , a mar ked t r a n s i t i o n zone F wh i t e arrow) o c c u r s wher e d i s p er s e dm c r o t ubu l e s of t he gr owt h cone p er i p he r y f o r m i n t o a c l o s e l y packed axona l a r r a y Bar i n F r e f e r s t o A-Dand F B a r s , 5 l m

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Fi gure 4 Transi ent mcrotubul e l oop f ormati on i n growth cones Thi s t i me- l apse sequence of agrowth cone i n the f emur shows t hetr ansi ent f ormati on A , col l apse B , and re- f ormat i on C ofmcr otubul e l oops unf i l l edarr ows Ti meA, 0mn B, 52mn C,86 mn Bar , 5 1m

The J ournal of Cel l Bi ol ogy, Vol ume 115, 1991

i nFi g 3harboredmcrotubul es l owi ntensi ty and l ack ofuni f ormcal i ber i ndi cate that the f ai nt si gnal i n the f i l opodi ai n Fi g 3 represents background f l uorescenceBranches I n al l growth cones, branches were observedAs menti oned above, we operati onal l y def i ned branches asany el ongate protrusi on l arger i n cal i ber than a f i l opodi umTheyusual l y hadataperi ng prof i le so t hat thebasew dthwasgreater than theapexw dth Unl i ke f i l opodi a, branches oftenharbored mcr otubul es We examned the number of mcro-tubul e-i nvadedbranches i ngrowth cones si mul taneousl y l a-bel edw thD Oand rhodamne- tubul i n I n si x growth conessoexamned, mcr otubul es werepresent i n57 of branches range 40-100 Thi s i s i l l ust r at ed i n Fi g 3 A and C ,where unf i l l ed arrows i ndi cate branches seen by D Ol abel -i ng Themcrotubul es i n those branches can be seen i n Fig3 Band D , respecti vel y The bl ack arrows i nFi g 3 AandB i ndi cate a branch i denti f i ed by D O staini ng Fi g 3 A ,whi ch does not contai n mcrotubul es Fig 3 B I n twocases, short branches were seen to forml ong before the m-crotubules sel ecti vel y i nvaded the structure 41 and 185mn Thi s i mpl i es that the f ormati on and mai ntenance ofsome short branches does not requi re nearby mcrotubul es,and that branchexi stencedoes not i mpl y i nevi tabl emcrotu-bul e i nvasi on I ndeed, sel ecti ve and non- randombranch i n-vasi on seems to be a mechani smused by growth cones tosteer at gi ven pathway deci si ons see bel owM crotubul es FormCompl exLoops i n theGrowthConeI n 14 growth cones, mcrotubul es were f ound i n compl exl oop structures Thi s was usual l y seen as the growth conewas mgrati ng proxi mal l y i nthe f emur Fig 1 box 1 or wasencounteri ngthe trochanter- coxasegment boundary Fig 1box 2 An exampl e of the f ormer i s showni n Fig 3 A andB The whi te arrowi n Fi g 3Bi ndi cates theapex of agroupof mcrotubul e l oops The correspondi ngmembrane i mage,i l l ustrated i n Fi g 3 A, shows that these l oops formw thi nthe confi nes of a smooth growth cone thei r presence i s notapparent i n the shape of the cel l i tself Another exampl e ofmcrotubul e l oops canbe seen i nFi g 3E Here, themcrotu-bul esare i ndi catedby the unf i l l ed arrow and can beseen t ol oop j ust under the di stal surf aceof the growth cone Fi g 3F i s an i mage of t he same growth cone shown i n Fi g 3 E34 mn l ater The mcrotubul e l oops can s t i l l be seen, andthe mcr otubul es i ndi cated by t he unf i l l ed arrow have i n-vaded a branch I nmany i nstances, t he l oops were f ound t obe tr ansi ent Thi s i s i l l ustrated i nFi g 4, whi ch shows a temporal sequence of i mages of the same growth cone Themcrotubul e l oops are i ndi catedby t he unf i l l ed arrows Theyexi st i n Fi g 4A, col l apse i n Fi g 4 B, and then reformi nFi g 4 CEMConf i rms M crotubul e Ar rangements To examnemcrotubul e arrangements and l oops at the ul tr astr ucturall evel , growth cones were i maged, and thenprepared f or EM

Thi s al l owed the correlati on of mcrotubul e organi zati on i nthe i magi ng study w th an el ectr on mcroscopi c anal ysi s oft he same growth cone The growth cone shown i n Fi g 3 was f i xed short l y after this i mage was taken, and examnedby EMFig 5 Thesurf aceof thegrowth cone i s i mmunol a-bel ed w th gol d part i cles seeMethods and Materi al s Thetwobranches seen i n Fig 3F can be seen i n theupper ri ghtand l ower left regi ons of themcrograph The mcrotubul esi ndi cated by the unf i l l ed arrow i n Fi g 3 correspond i nconf i gurati on and l ocati ont o those i ndi catedby the l arge ar-

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Fi gure S El ectr onmcrographof agrowthconegrowngi n t he f emur Thegrowthconeshown i n Fi g was prepared f or Mt o conf i rmt hearrangement of mcrotubul es seen i n the i magi ng study The Ti l growth cone was i denti fi edby i mmunogol d visual i zati on of a neuron-specif i c pri mary ant i body The curvedmcrotubul es seen i n Fig 3are l ocated i n the center of thegrowth cone arrow Several i ndi vi dualmcr otubul es can be seen i n t hedorsal branch l arge arrowheads The mcrotubul e i ndi cated by t he smal l arrowheads i s shown i n detai li n Fi g 6 Bar j mrowheads i n Fi g 5 Note that they are f ound as si ngl emcrotubul es Loopedmcrotubul es were al so f ound at theel ectr on mcroscopi c l evel and are i ndi cated by the arrowFi g 6 shows a hi gher power i mage of the same growth coneas i n Fi g 5 Loopedmcrotubul es, i ndi catedby thesmal l ar-rowheads i nFi g 5 are i ndi catedby the l argearrowheads i nFi g 6 Al t hough the l oopedmcrotubul e was not cont i nuousat theMl evel i t di d showbends w th siml ar curvature t othat i n the f l uorescent i mages G ven that each secti onwas

Sabry et al M crotubul e Behavi or duri ng Axonal ui dance

umi n thi ckness, i t was not cl ear whether the l oopedmcrotubul e was cont i nuous i n seri al secti onsTheDynamcs of M crotubul eMovementduri ngSteeri ngEventsPi oneer growth cone steeri ng behavi or differs i n di f f e rentl i mb r egi ons dependi ng upon the nature of the substratew th whi ch the growth cone i s i n contact Several generalcl asses of steeri ng behavi ors, i ncl udi ng vei l extensi on or

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Fi gure El ectr on mcr ographshow ngdetai l of mcrotubul e bend-i ng A hi gher magni f i cati on of t he mcrograph shown i n Fi g 5i l lustratingthe curvature of mcrotubul es seen i n t he f l uorescent i mages shown i n Fi g 3 F Bar, 1 um

retracti on, branch extensi onor retracti on and f i l opodi al di l a-ti onhavebeendi sti ngui shed O Connor et al . 1990) We ex-amned mcrotubul e behavi or at vari ous l i mb l ocat i onswher e these threemai n types of steeri ng events occur Fi rst , on i ntrasegmental epi thel i al cel l s i n the md-f emurregi on Fi g 1 box 1 the growt h cone constant l y makessmal l course corr ecti ons to keep i t grow ng proxi mal l y al ongt he l i mb axis O Connor et al . 1990) Fi ve growt h coneswere i maged i n this regi on Themaj or morphol ogi c formofgrowt h cone mgrati on was by vei l extensi on in i t ial vei l ex-tr usi on between f i l opodi awas f ol l owed by engor gement oft he vei l by gr owt h cone cytopl asm Thi s type of growt h i ss im l a r to that of PC 2ce l ls and Apl ysi a neurons i maged i nv i t ro Al ett a and Greene, 1988; Gol dberg and Burmei st er,1986) Branches wer e normal l y present i n growt h cones i nthis regi on, andthey of ten harbored mcr otubul es Fi g 3 AandB shows a growt h cone i n the f emur, and the branchescontai ni ngmcrotubul es are i ndi catedby theunf i l l ed arrows

si ngl e branch w t hout mcr otubul es i s shownby t he bl ackarrow Usual l y more than one branch harbored mcrotu-bul es, al thoughonl yonewas ori ented i n thefuturedi recti onof growt h cone extensi on I n thi s si tuati on, mcr otubul es i n-vadedmorethan one branch, but wer e retai ned i nonl y oneFor i nstance, the growt h cone shown i n Fi g 3Fwas i n t hef emur, and mcr otubul es were present i n both the dorsalbranch, i ndi cated by the unf i l l ed arrow and the ventralbranch, seen i n the l ower ri ght of the i mage Al though bot hbranches were general l y ori ented proxi mal l y, that i s to theri ght, the ventral branch was eventual l y w t hdrawn and thegr owt h cone extended al ong the dorsal branch data notshown) Hence, i n this regi on of t he l i mb, asymmetr i cmcr otubul e arrangements wer e set up by sel ecti ve retenti onof mcr otubul es i n specif i c branches

The J oumal of Cel l Bi ol ogy, Vol ume 115, 1991

Anot her type of steeri ng event occurs wher e the Ti lgrowt h cone encounters theepi thel i al ce l ls that formtheTr-Cx l i mb segment boundary Fi g 1 box 2) The epi thel i alce l ls at the boundary are knowntodi f f er i n shape and surf aceproperti es fromthei r i ntrasegmental counterparts Basti aniet al 1991 Caudy and Bentl ey, 196a; Condi c and Bent l ey,199a) Thegrowt h cones encounter anorthogonal i nterf acebetween a di stal band of hi ghl y adhesi ve epi t hel i a l cel l s shownas abandof f i l l e dhexagons i n Fi g 1 and anadj acentproxi mal band of l ower- af fi ni ty ce l ls shown as a band ofunf i l l ed hexagons i n Fi g 1 At th is i nterf ace, branches aresent i n both dorsal up i n al l f i gures and ventral di rec-ti ons The relati ve size of these branches i s qui te vari abl eand ranges froml arge dorsal branches and smal l ventralbranches, t hrough symmet r i c branch si ze, t o l arge ventraland smal l dorsal branches However , regardl ess of the in i t ialbranch si ze, thegrowt h cone i nvari abl y makes a ventral turnal ong the boundaryFi ve growt h cones wer e i maged as they made this ventralturnat thet rochanter- coxasegment boundary Al l f ive repre-sented si tuati ons wher e branches wer e sent bot hdorsal l y andventr al l y Two cl asses of mcr otubul e movement s wer e seen Inthe f i r s t cl ass three cases , mcr otubul es i ni t i al l y i nvadedboth the dorsal and ventral branches Thi s i s i l l ustrated i nFi g 2, whi ch shows a growt h cone spreadi ng at the Tr - Cxboundary t he l ocati on of theTrl gui depost cel l i s i ndi catedby the asteri sk Note that the ventral l ower) and dorsalbranches, i ndi cated by the whi te arrows, bot h contai nmcr otubul es, even t hough the growt h cone w l l eventual l yturn ventr al l y Wt h t i me, more mcr otubul es wer e seen t oi nvade the ventral branch datanot shown) I n al l cases, thedorsal branch and i t s mcr otubul es persi sted dur i ng thi sper i od Other st udi es have shown that this dorsal branch i seventual l y resor bed Caudyand Bent l ey, 196b ; O Connoret al . 1990) Thi s turni ng mechani smdi d not seemto i n-vol ve sel ecti ve branch i nvasi on by mcr otubul es, but rathersel ecti ve retent i on of those mcr otubul es i n t he ventralbranch

I n contrast, t he second cl ass two cases) of mcr otubul emovements at thi s segment boundary turn di d i nvol ve sel ec-t ive ventral branch i nvasi on, and i s portrayed i nFi g 7 Thi sf i gure shows a seri es of i mages of the same growt h cone asi t makes the ventral turnat theTr - Cx segment boundary Thestage was moved s l ight ly after the i mage i n Fi g 7to fol l owthe growt h cone The growt h cone has mgrated t o the Trlcel l , whi ch i s i ndi cat ed by the bl ack arrow i n Fi g 7A Atthis poi nt , several f i l opodi a and smal l branches extend bothdorsal l y and ventr al l y al ong the segment boundary, at theri ght of each of the panel s Themcr otubul es formacl osel ypacked bundl e i n the growt h cone i n Fi g 7 C, but are notori ented dorsoventral l y al ong the boundary I n Fi g 7 D,however , the bundl e shows a sl i ght ventral ori entati on Notethat no mcr otubul es have entered the dorsal or proxi malbranches seen i n theupper ri ght regi onof Fi g 7D Theven-tral branch, whi ch i s al so voi d of mcr otubul es at t hi s t i me,i s shown by the arrowheads i n Fi g 7 D I n Fi g 7 E, themcr otubul es have cl earl y entered the ventral branch i ndi -cated by the arrowheads) The dorsal branches persi stedf orthe durat i on of the i magi ng, but they never harbored mcro-tubul es No morphol ogi c di f f erences wer e apparent betweengrowt h cones that used thi s type of turni ng and those i n t hef ormer cl ass Al t hough these t wo cl asses of mcr otubul emovements di f f ered i n whet her the dorsal branch harbored

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Fi gure 7 Sel ecti vemcrotubul e i nvasi on dur i ngagrowth cone steeri ng event at a l i mb segment boundary A Ti me, mn Theposi ti onof this rhodamne-t ubul i n- l abel ed pi oneer neuron growth cone i s shown i n Fi g 1 box 2 pronounced mcrotubul e l oop i s present atthe FeTr segment boundary whi te arrow smal l bundl e of mcrotubul es unfi l led arrow extends al ong the process which contacts theTrl cel l bl ack arrow l ocated at the Tr- Cx segment boundary B Ti me, h 31 mn The process extendi ng t o the Tr - Cx boundary hasthi ckened unf i l l ed arrow The l oop at the FeTr boundary whi t e arrow has strai ghtened, and the axon di stal t o this boundary thati s left of thewhi tearrow has enl arged C Ti me, h 6mn Theprocess extendingt o t he Tr- Cx boundary cont i nues to enl arge unf i l l edarrow D Ti me, 4 h mn The process extendi ng t o the Tr- Cx boundary has expanded to nor mal axonal cal i ber unf i l l ed arrowThe Tr- Cx segment boundary i s i ndi cated by the tr i angl e Mcrotubul es end abruptl y at t he boundary, al though f i l opodi a andbrancheshave extended i n several di recti ons i ncl udi ng ventr al l y arrowheads E Ti me, 7 h 9mn Mcrotubul es arrowheads have sel ecti vel yi nvadedthebranches extendingventr al l y i n thedi recti onthegrowth cone w l l take Mcrotubul es di dnot i nvadebranches extendeddorsal l yor proxi mal l y Bar umSabry et al M crotubul e Behavi or duri ng Axonal Gui dance 8


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Fi gure 8 Sel ecti ve mcrotubul e i n-vasi on duri ng growt h cone steeri ngevents at the Cxl gui depost cel ls.Three rhodamne- tubul i n- l abel edgrowth cones (A- B, C-D, andE-Fare i maged as they reori ent abruptl yat t he Cxl gui depost cel ls ( F i g 1box 3) (A) Ti me, 0 mn growthcone mgrat i ng ventr al l y al ong t heTr - Cx segment boundary ( shown bytr i angl e) extends asi ngl e f i l opodi um(whi tearrow) t o contact t heCxl cel ls(unf i l ledarrow) (B) Ti me, 28 mnThe f i l opodi um now has a branchmorphol ogy, and has been i nvadedby mcrotubul e(s) (whi te arrow) M-crotubul es have not i nvaded otherregi ons of the growth cone (C) Ti me,0 mn Anot her growt h cone at as l i ght l y l ater stagehasabroadl amel -l umon theTr- Cx segment boundary(i ndi cated by the tr i angl e) smal lnumber of mcrotubul es (whi te ar-row are present i n t h e process cross-i ng fromthe segment boundary t othe Cxl cel l s ( unf i l l ed arrow) (D)Ti me, 51mn Thel amel l umhaswth-drawn f romthe boundary, and t henascent axon (whi te arrow) crossesfromthe boundary t o the Cxl cel l(unfi l led arrow) ( E) Another growthcone, extendi ng ventr al l y al ong t heTr- Cx boundary ( tr i angl e) , al so hasa process (whi te arrow) extendi ngacross theboundary t o theCxl cel ls(unf i l led arrow) (F) I n an enl argedvi ew of t he branch poi nt shown i nE, i t appears that mcr otubul es di -verge from three di f f erent mcrotu-bul e bundl es present al ong theTr - Cxboundary t o enter t he branch (whi tearrow) crossi ng t o the Cx1 cel ls ( un-f i l l e d arrow) Some mcrotubul es (ar-rowhead) conti nue al ong t he bound-ary past t he branch poi nt I n suchcases, the growth cone mayhave ad-vanced al ong the boundary past theCxl cel ls before t he f i r s t f i l opodi alcontact w th those cel ls Bars ( A- E)5 I , m (F) 1 l m


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m crot ubul es, both t u r n s event ual l y i nvol ved t he streamngof m crotubul es i n t o t h e v e n t r a l branch Hence, at t he s t e e r -i ng deci si on made at t he Tr- Cx segment boundary, both se-l e c t i v e i nvasi on and s el ec t i ve r e t ent i o n wer e seen as mec ha-ni sms of generat i ng asymmet r i c m crotubul e ar r angement s The t h i r d , and perhaps mos t s t r i k i n g, of t he s t e er i ng mech-ani sms used by t he T i l pi oneer neur ons i s e l i c i t e d by an i n t e r -act i on bet ween t he Ti l gr owt h cone and gui depost c e l l s Three gui depost c e l l s ar e found i n t he T i l pat hway F el , T r l ,and t he Cxl pai r maj or growt h cone reor i entat i on occursat t he Cxl turn shown i n Fi g 1 box 3) Thi s turn was i m-aged three t i mes, and t he smal l er angl e Tr l t ur n was i magedt w c e I n al l f i v e c a s e s , thet urns wer e accompl i shed by s e l e c -t i v e i nvasi on of a s i n g l e gr owt h cone branch by m crotubul es Fi g 8 A and B shows a gr owt h cone maki ng t he t ur n t ot he Cxl s The gr owt h cone has m gr a t ed v e n t r a l l y al ong t hesegment boundar y, whi ch i s i ndi cat ed by t he t r i a n g l e i n Fi g 8 A The l o c a t i o n of t he Cxl c e l l s i s i ndi cated by t he u n f i l l e da r ro w I n Fi g 8 A and B) , note t h a t f i l o p o d i a and br anchesextend f r o m t he gr owt h cone i n both proxi mal t o t he r i g h tand d i s t a l t o t he l e f t di rect i ons s i n g l e f i l opodi umcan beseen ext endi ng t o t he Cxl s , as shownby t he whi t e a r ro w Fi g 8 Bshows t hesamegr owt h cone 28mn l a t e r m crotubul e s)have s e l e c t i v e l y i nvaded t he branch whi ch has f or med f r o mt he f i l opodi umext endi ng t o t he Cxl c e l l s Fi g 8 whi t e ar -r ow Theref ore, m crotubul es had s e l e c t i v e l y ext ended i nt he d i r e c t i o n of gr owt h cone m gr a t i on Thi s s e l e c t i v i t y oc-curred when there was onl y one branch p r e s e n t , ext endi ngt o t he Cxl c e l l s , and al so when addi t i onal proxi mal or d i s t a lbr anches wer e present as i n Fi g 8 A and B Fi g 8 CandD shows anot her gr owt h cone at a l a t e r s t a g e i n t he turn The i n i t i a l l y smal l number of m crotubul es t h a t have enteredt he branch ext endi ng t o t he gui depost c e l l have become t henascent axon m crotubul e f a s c i c l e as t he t ur n i s compl et ed Previ ous s t u d i e s of t h i s turn i n d i c a t e t h a t oc cas i o nal l y apr oxi mal l y extended f i l opodi ummay not make contact w t ha Cxl c e l l un t i l a f t e r the l eadi ng edge of t he growt h cone hasmgra ted v e n t r a l l y past t he c e l l Caudy and B en t l e y , 19866 O Connor e t al 1990) Wei maged one growt h cone wher et h i s pattern of gr owt h apparent l y occurred I n t h i s s i t u at i o n,t he branch contacti ng t he Cxl gui depost c e l l harboredm crot ubul es t h a t appear ed t o or i g i na t e d i r e c t l y f r o m t hemai n gr owt h cone bundl es Thi s i s shown i n Fi g 8 E, andi n hi gher magni f i cat i on i n Fi g 8 F The l oc at i on of t he Cxlc e l l s i s i ndi cat ed by t he u n f i l l e d arrow and t he t r i a n g l e i nFi g 8 Eshows t he l ocat i on of t he Tr - Cx segment boundary Not e t h a t four m cr ot ubul e bundl es seem o a r i s e f r o m l a r g erbundl es w thi n t he gr owt h cone, and en t e r t he branch extend-i n g t o t he Cxl c e l l Fi g 8 whi t e arrow The ar r owhead i nFi g 8Fs hows m crotubul es i n the d i s t a l r egi on of the gr owt hcone, whi ch had mgrated past the Cxl c e l l s Befor e gr owt h cones make t he t ur n at t he t rochant er- coxaboundar y Fi g 1 box 2 , a gui depost c e l l medi at ed turn oc-cur s at t he Tr l c e l l A s i n gl e f i l opod i a l contact w t h the Tr lgui depost c e l l ser v es t o r eor i ent t he growt h cone by f i l o p o -d i a l d i l a t i o n W observed t wo growt h cones at t h i s l ocat i onwher e a smal l number of m crotubul es s e l e c t i v e l y entered abranch i n contac t w t h the Tr l c e l l The l a t e r stages of onesuch tu rn i s shown i n t he f i r s t t h r e e panel s of Fi g 7 The Trlgui depost c e l l i s i ndi cated by t he bl ack arrow andt he branchcontact i ng i t by t he u n f i l l e d arr ow Al t hough ot her f i l o p o d i aand smal l br anches werep r e s e n t , m crotubul es onl y entered

Sabry et al Mcrotubul eBehavior duri ngAxonal Guidance

t he branch ext endi ng toTr l Subsequent l y, addi t i onal m c r o -t ubul es accrued w t hi n t h i s branch unt i l i t reached t he c a l i be rof t he nascent axon Fi g 7D These r e s u l t s suggest t h a t atal l gui depost c e l l s , s t e er i ng by s el e c t i v e f i l opod i a l d i l at i on i sacc ompani ed by s el ec t i v e m crotubul e i nvasi on D scussi onI n t he embr yoni c gras shopper l e g, t he m g rat i on of t he T i lpi oneer gr owt h cones f o l l o ws a stereotyped pat hway F i g 1f r o m t he t i b i a t o t he c e n t r a l nervous system a path l engt hof -0 5mmBa te , 1976 Bent l ey and Caudy, 1983 Caudyand B en t l e y , 19866 Ho andGoodman 1982) Gui danceover t h i s compl ex pat hway i s accompl i shed by many gr owt hc o ne - c e l l i nt e r a c t i o ns i nc l udi ng those i nvol v ing i n t r a s eg -ment al e pi t h el i a l c e l l s , segment boundar y ep i t h el i a l c e l l s , andpreaxonogenesi s neur ons cal l ed gui depost c e l l s Caudy andBe nt l e y , 19866 Condi c and Bent l ey, 19896 ; Keshi shi anand Be nt l e y, 1983 Lefcort and Bent l ey, 1987 O Connor eta l 1990) Gui dance f e a t u r e s whi ch medi at e nor mal gr owt hcone m gra t i on ar e preserved i n a l i mb f i l l e t pr eparat i onwher e i ndi vi dual c e l l s ar e accessi bl e f or observati on and ex -peri ment al mani pul ati on Lefcort andBe nt l e y , 1987 O Con-nor et al 1990) W observed m crotubul e ar r angement sduri ng gr owt h cone m gr a t i on i n response t o t hese i n s i t ugui dance cues by i n j e c t i n g t he T i l neuron w t h r hodam ne-l abel ed tubul i n Label ed bovi ne tubul i n r a p i d l y i ncorporated i n t o l i n eart r a c k s i n t he axon and growt h cone Thi s i s consi st ent w t ht he hi ghdegree of homol ogy bet ween bovi neand i n s ec t t ubu-l i n Rudol ph et al 1987) Project i on of t he i mage onto ahi gh resol ut i onD h i p , deconvol uti on of mul t i pl e i magep l a n e s , and use of convent i onal comput er i mage enhance-ment sof t ware pr ovi ded adequat e resol ut i on of l i n ea r t r a c k s I ndi v i dual t r acks wer e uni fo r m i n w d t h and i nt e ns i t y al ongt h ei r whol e l engt h Furt her mor e, w t h i n t he same gr owt hcone, d i f f e r e nt tracks wer e uni f orm i n w d t h and i n t e n s i t y Wdths cor r esponded c l osel y t o those reported f or f l u o r e s -c e n t l y l abel ed m crotubul es whose uni ty and i d e n t i t y wer esubsequent l y confi r med i n el ect r o n m cr ogr aphs Sammakand Bo r i s y , 1988) El ectr on m cr ogr aphs of Ti l gr owt hcones f i x e d i mmedi at el y a f t e r i magi ng reveal ed i ndi vi dualm crotubul es s i m l a r i n l ocat i on and shape to f l uorescentt r a c k s previ ousl y i maged i n t he same gr owt h cone F i g s 5and 6 ar r owheads) These r e s u l t s str ongl y suggest t h a t t heuni t a r y f l uor escent t r a cks wer e i ndi vi dual m crotubul es These ar e t he f i r s t observat i ons of i ndi vi dual m crotubul edi sposi t i on and behavi or i n gr owt h cones m gra t i ng ands t e er i ng on t he i n s i t u s u b s t r a t e TheArrangement of M cr o t ubul es i n TF1 NeuronsAxons I n both f i x e d and l i v e neur ons i n v i t r o , m crotubul esi n t he axon ar e arranged i n a cl os el y packed, l i ne ar bundl e Bl ack et al 1989 ; Br ady et al 1984 Bray and Hol l en-beck, 1988 Hei demann et a l 1984 Hi r okawa et al 1988 ;Kei t h, 1990 Mei ni nger and Bi n et , 1989) Thi s ar r angementwas a l s o seen i n Ti l axons i n s i t u s ee Fi gs 3 Fand4) Al -t hough axonal m crotubul es wer e gener a l l y i n t h i s confi gura-t i o n, occasi onal l y t hey coul d be di spl aced t o one s i d e ors p l a y out i n t o smal l bundl es, especi al l y at t he l o c at i o ns ofl a t e r a l prot rusi ons I n neurons vi ewed i n v i t r o , t he border bet ween t he gr owt h

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Fi gure 9 A di agramof features of mcrotubul e arrangements i npi oneer growthcones i n s i t u 1 Bundl edmcrotubul es i n theaxon 2 Mcrotubul e l oops seen at axonal branch poi nts and i n t hegrowthcone 3 Bundl es of mcr otubul es extendi ng i nto abranch4 Absence of mcr otubul es i n fi l opodi a S Regi ons of thegrowthconeperi phery canbedevoi dof mcr otubul es 6 Where a fi l opo-di umexpands into a branch, mcrotubul e i nvasi on i s often ob-served 7 Mcrotubul es often extend to t he growth cone per i ph-ery 8 Mcrotubul es sel ecti vel y i nvade a branch f ormed f romaf i l opodi umwhi chhascontacted a gui depost cel l 9 Somebranchesare devoid of mcrotubul es

coneand axon i s def i nedas that regi onwhere theaxonal cyl -i nder abrupt l y i ncreases i n cal i ber Bray and Chapman1985 Gol dberg and Burmei ster, 1986) At thi s border therei s a transi t i on inthe ar rangement of both act i n andmcrotu-bul es Forscher and Smth, 1988 Mtchi son and Ki rschner,1988) I n thi s regi on, themcrotubul earrangement changesfromt he hi ghl y bundl ed formi n the axon to the spl ayed outpattern i n t he growthcone I n some Ti l neurons, thi s changewas easi l y i denti f i abl e Furthermore, the l ocati on of thechange i n mcrotubul e arrangement coul d bedi spl aced fromthe change i n cel l shape at t he border between the growthcone and axon Fi g 3 F I t woul d seemthat t he f ormati onof the cl osel y packed bundl e of mcrotubul es characteri sti cof the axon can occur centr al l y wt hi n t he growth cone Thi ssuggests that t he f ormat i on of an axonmay f i r s t i nvol ve t hef ormati on of a ti ght bundl e of mcrotubul es i n the growthcone, and subsequentl y thecol l apse of t he pl asmamembranearound that bundl e Thi s process has been seen i n l i v e Xenopus neurons vi ewed i nt i me l apse i n v i t ro TanakaandKi rsch-ner, 1991) I t i s l i ke ly that theaxonal bundl i ng i n thegrowthcone refl ects the act i vi ty of mcrotubul e associ ated protei ns Matus, 1990) , suggesti ng that the acti vi ty of suchmol ecul esi s spat i al l y l ocal i zed to the axon and central growth coneGrowthCones The grasshopper Ti l growthcones i n si t uhave a r i ch i nvestment of mcrotubul es Thi s and other fea-tures of the Ti l growth cone are shown i n schemati c formi nFi g 9 An i ssueonwhi ch previ ous observat i ons have vari edi s the degree towhi ch mcrotubul es areconf i ned to t he cen-

tral core area of thegrowth cone I n some earl y el ectronm-croscope studi es Bunge, 1973 I senberg and Smal l , 1978),and i n l i veApl ysi abag cel l growthconeswhi ch are i mmobi -l i zed on a hi ghl y adhesi ve substrate Forscher and Smt h,1988) , mcr otubul es rarel y extend t o the growth cone mar-

The J ournal of Cel l Bi ol ogy, Vol ume 115, 1991

gi n I n other studi es, on f i xed and extracted chi ck sensoryand reti nal neuron, or rat sympathet i c growth cones Bri dg-man and Dai l ey, 1989 Letourneau, 1983 Letoumeau andShatt uck, 1989 Tsui et al . , 1984), and l i v e Xenopus growthcones i n v i t r o Thnakaand Ki rschner, 1991), mcrotubul esregul arl y extend t o thedi stal margi n of the growth cone I nTi l neurons i n sit u, the usual si tuati on i s f or a dense arrayof mcrotubul es tobe extended ri ght upto thel eadi ngmargi nof the growth cone Fig 2 Thus, thedi stal growth cone re-gi on of Ti l pi oneer neurons appears to be more heavi l y i n-vested w th mcrotubul es than has been observed i n somegrowth cones i n v i t r oFi l opodi a extendi ng fromTi l growth cones i n si t u neverharboredmcrotubul es Fi gs 3, CandDand 9 These i ns i t u observati ons are i n accordance w th previ ous i n v i t rostudi es, at both the l i ght and el ectr on mcroscope l evel s, i n-di cati ng that f i l opodi a arecomposedpri mari l y of F-act i n andl ackmcrotubul es Bri dgmanandDai l ey, 1989 Letourneau,1983 Smt h, 1988) I n neurons i n vi tro, and i n Ti l neuronsi nvi vo, cytochal asi n- i nduceddi sassembl y of f i l amentous ac-t i n resul ts i n t he l oss of f i l opodi a, suggest i ng that act i n i snecessary f or f i l opodi al extensi onandmai ntenance Bentl eyand Toroi an- Raymond, 1986 Marsh and Letourneau,1984) Unl i ke f i l opodi a, branches extending fromthe Ti l growthcone oft en di d harbor mcrotubul es Figs 2 and 9) However, approximatel y hal f of thegrowthconebranchesdi dnotcontai n mcrotubul es seeFi g 3, AandB Thi s i s i n accor-dance w th observat i ons of f i xed neurons i n vi tr o, wherebranches bothw thandw thout mcrotubul es have beenob-served Tsui etal . , 1984) Furthermore, many short branchesf ormed and exi st ed w thout nearby mcrotubul es We con-cl ude that mcrotubul es are not necessary for the i n i t ia t ionofbranch f ormati on, nor f or themai ntenance of short branches Branches can be f ormed frompreexisti ng f i l opodi a Thi si s perhaps best appreci ated i n thosebranches that f ormf romf i l opodi a i n contact w th gui depost ce l ls Af ter the f i l opo-di umcontacts a gui depost cel l , i t i ncreases i n cal i ber and

eventual l y converts to a branch O Connor et al . , 1990) Such branches al ways acqui redmcrotubul es However , theconversi onof the f i l opodi umto a branch al ways occurred i nadvance of t he acqui si ti on of mcrotubul es I n Fi g 8 A, f orexampl e, a f i l opodi umcontacted the C gui depost cel l s However , mcr otubul es di d not i nvade the structure unti l i thas undergone a conversi on to a branch some 30mn l ater Fig 8B Thus, f i l opodi a di d not appear to be enl arged bythe process of mcrotubul e i ntr usi on rather, mcrotubul esal ways i nt ruded i nto a space that was al ready present M crotubul eMovement dur i ngSteer i ngEventsThe f i ndi ng that many mcr otubul es are normal l y present i nthe growth cone and i n branches of Ti l growth cones i n si t urai ses the possi bi l i ty that theymay pl ay a di r ect r ol e i n cer -ta in phases of steeri ng Theprocess of growth cone steeri ngcan be f ormal l y di vi ded i nto three phases The f i r s t phasecompri ses expl orati onor sampl i ng of theenvi ronment i nthevi c ini ty of the growth cone I t appears to bemedi atedby therandomprotr usi on of f i l opodi a there i s no evi dence thatf i l opodi al extensi on i s preferenti al l y di rectedtowards the tar-get Bef ore a turn i s made, however , one or a f ewof thebranches or f i l opodi a must be chosen as the future track of

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t he extendi ng gr owt h cone Thi s second phase of s t e er i ng i scal l ed ori ent at i on The cyt oskel etal r equi r ements f o r ori en-t a t i o n are not known The f i n a l phase of s t e er i ng i s consol i -dat i on and conversi on of t he arr angement s of cytoskel etaland ot her mol ecul ar f e a t u r e s of t he growt h cone t o t h e chemi c a l l y and mechani cal l y more s t a bl e confi gurati ons f ound i nt he nascent axon Whi l e t he absence of m crot ubul es f r o m f i l o p o d i a i n d i -c a t e s t h a t m crot ubul es ar e unl i kel y t o be i mport ant i n t heexpl orati on phase, t h e i r di sposi t i on appears t o be a key e l e -ment of t he ori ent at i on phase Thi s can be seen i n Fi g 8 Aand B At t he begi nni ng of t h i s phase Fi g 8 A) , t h e i r di spo-s i t i o n i s not o r i e nt e d and at i t s end Fi g 8 B t h e i r pl ace-ment def i nes wher e consol i dat i on i nt o t he nascent axon canoccur Consol i dat i on i s shown i n Fi g 8 Cand D Hence,we suggest t h a t t he endpoi nt of ori ent at i on i s t he generat i onof an asymmet r i c arr angement of m crot ubul es i n t he gr owt hcone There are t wo general mechani sms by whi ch t h i s canbe accompl i shed One i s t h a t m crot ubul es coul d i nvade onl yt hose branches t h a t are f ound i n t he f u t u r e di rect i on ofgrowt h cone ext ensi on t e r m t h i s s el ec t i v e m crot ubul ei nvasi on An a l t e r na t i v e mechani smwoul d be t h a t mcrotu-bul es r andom y i nvade l l ext ant branches, and are subse-quent l y s t a b i l i z e d i n t hose ext endi ng i n the preferred d i r e c -t i o n Thi s we t e r m s e l e c t i v e m crot ubul e r e t e n t i o n Them crot ubul e i magi ng i n t h i s st udy al l ows one t o addressd i r e c t l y whi ch mechani sm s operati ng at a gi ven turn At gui depost c e l l i nduced t u r n s such as t hose at Trl andCxl , s e l e c t i v e m crot ubul e i nvasi on was seen i nvari abl y Fi gs 8 and 9 I n a dd i t i o n t h i s s el ec t i v e mechani smwasal soseen i n t wo of f i v e turns observed at t he t rochanter- coxase gment boundar y Fi g 7) I n c o n t r a s t t o t h i s t h r e e of f i v eturns at t he segment boundary wer e made usi ng s e l e c t i v er e t e n t i o n to generate an asymmet r i c m crot ubul e arrange-ment m crotubul es i nvade bot h t he v e n t r a l and d o r s a lbranches Fi g 2) I ntr us ion of m crot ubul es i n t o some, butnot l l branches was al s o s een i n gr owt h cones mgrat i ngproxi mal l y t hrough t he f emur Fi g 3) Theref or e, bot hs el ec t i v e m crot ubul e i nvasi on and s el ec t i v e m crot ubul er e t e n t i o n appear t o underl i e s t e e r i n g deci si ons, dependi ngon t he gr owt h cone l ocat i on i n t he l i mbThemechani smused t o generat e t he asymmet r i c mcrotu-bul e arr angement i s l i k e l y t o be de te rm ned by bot h t he h e t -erogenei t y and nat ure of gui dance i nformat i on conf ront ed byt he gr owt h cone duri ng t he expl orat ory phase O Connor etal 1990) At gui depost cel l - medi at ed t u r n s t he observeds el e ct i v e m crot ubul e i nvasi onmay r e f l e c t a l a r ge di f f erencei n t he s i g na l provi ded by a s i n gl e hi ghl y l o ca l i z ed hi gha f f i n i t y gui depost c e l l and t he surroundi ng c e l l s Se l e c t i v em crot ubul e r e t e n t i o n on t he ot her hand, may occur wher et he di f f erences bet ween t he substr at es encount er ed by T i lf i l o p o d i a are not so di ssi ml ar Bot h at d i f f e r e n t l o c at i o nsw t h i n t he f emur, and al ong t he se gment boundary, t he spa-t i l r a t e of change of subst rat e a f f i n i t y may be qui te l ow I nt h i s more ambi guous s i t u a t i o n t he sequence f r o m f i l o p o d i a lcontact w t h an accept abl e s u b s t r a t e t o branch f ormati on,t o accrual of successi ve m crot ubul es i n t o t he branch, t oconsol i dati on of a l arge bundl e of m crot ubul esmayproceedt hrough s e v e r a l s t e p s bef ore one branch pr e va i l s Thus s e l e c -t i v e m crot ubul e i nvasi on of a s i n gl e branch and s el ec t i v em crot ubul e r e t e n t i o n f ol l ow ng mul t i - branch i nvasi on seeml i k e l y t o be ext r emes of a range of m crot ubul e behavi or

Sabry et al M crotubute Behavi or dur i ng Axonal Gui dance

whi ch i s dependent upont he d i s p a r i t y bet ween envi r onment -dependent i nt r a c el l u l a r s i gna l s i n d i f f e r e n t gr owt h coneregi ons What sort of s i gna l s may be i nvol ved? The generati on ofhet erogeneous m crot ubul e arr angement s i s c e n t r a l t omanyc e l l u l a r processes, i ncl udi ng yeast bud f ormat i on Adamand P r i n gl e 1984) , pi gment granul e aggr egat i on i n t e l e os tr e t i n a Trout t and Burnsi de, 1988) , e p i t h e l i a l c e l l responset o wounds, and spi ndl e f ormat i on duri ng m t osi s Ki rschnerand M t chi son, 1986 ; M t chi son and Ki rschner, 1985) Ot h e s e t h e mechani smby whi ch t he m crot ubul es are a r -ranged has been i n ve s t i g at e d i n t he most d et a i l i n spi ndl e f o r -mat i on Here, i t appears t h a t duri ng f ormat i on of t he s p i n dl em crot ubul es r andom y assembl e, and are s el ec t i v el y s t a b i -l i z e d by cont act w t h t he ki net ochore Cassi meri s et l 1990 Hayden et al 1990 ; M t chi son et al 1986) A s i m -l a r s i t u a t i o n coul d underl i e s el ec t i v e r e t e nt i on of m c r ot u -bul es i n s e l e c t e d gr owt h cone regi ons or branches Where s el e ct i v e i nvasi on occurs, i t see ms l i k e l y t hat c er -t a i n r egi ons of t he gr owt h cone are more r ecept i ve t om crot ubul e i nvasi on or assembl y Themol ecul ar and b i o -physi cal basi s f o r s e l e c t i v e m crot ubul e i nvasi on i s unknown t appears t h a t under basel i ne condi t i ons, m crot ubul e i n va -si onmaybe pr event ed by t he physi cal presence of t he peri ph-e r a l a c t i n net work Thi s i s suggested by ul t r a st r uc t u r a l ob-servat i ons Let our neau, 1983) , exper i ment al pert urbat i onsof a c t i n i n growt h cones i n v i t r o Forscher and Smth, 1988 ;Sm t h, 1988) , and by bi ophysi cal consi derat i ons of d i f f u s i o nc o e f f i c i e n t s i n a c t i n gel s Luby- Phel ps et al 1988) I f t hi si s t he c a s e then cont act w t h hi gh a f f i n i t y cues mus t l o c a l l ya l t e r t h i s s i t u a t i o n Thi s a l t e r a t i o n coul d be e f f e c t e d by spa-t i a l l y r e s t r i c t e d changes i n t ensi on Br ay and Hol l enbeck,1988 ; Hei demann et l 1990 Let our neau, 1975) , and/ ort he concentr at i on of second mess enger s mol ecul es Bent l eyet al 1991 ; Bi xby, 1989 ; Forscher, 1989 Kat er and Mi l l s1991 ; Lankf ord and Let ourneau, 1989 ; Let our neau andShat t uck, 1989) t i s al so possi bl e t h a t these e f f e c t o r mol e-c u l e s change a chem cal i n t e r a c t i o n bet ween a c t i n andm crot ubul es t h a t promot es m crot ubul e i nvas i on Gos l i n et l 1989 Let our neau, 1983 Moral es and Fi f kova, 1989) Anot her i s s ue rai sed by our f i ndi ngs i s t he nat ure of t hemechani smby whi ch m crot ubul es i nvade growt h conebranches Thi s i nvasi onmay represent newm crot ubul e as -sembl y f r o m t he monomeri c tubul i n pool ont o t he d i s t a lpl us ends of t he m crot ubul es Baas and Bl ack, 1990 ; Bamburg et al 1986) Al t e r n at i v el y m crot ubul es coul d bet ransl ocat ed i n t o branches One appr oach t o di s t i ngui shi ngthese a l t e r n at i v es woul d be t o makea smal l f l uorescent markon t he m crot ubul es, and t hen observe t he movement of t hemark as m crot ubul es i nvade t he gr owt h cone Thi s t y pe ofst udy has been carri ed out ; i t was f ound t h a t m crot ubul esmarked i n t he axon near t he gr owt h cone move di s t al l y i n t ot he growt h cone, even i n t he absence of axonal el ongat i on Rei nsch et a l 1991) Thi s suggest s t h a t m crot ubul e t r a n s -l o c a t i o n may cont ri but e t o branch i nvasi on One of t he mos t s t r i k i n g f e at u r es of t he T i l gr owt h conesi n s i t u was the regul ar occurrence of t r a ns i e nt m crot ubul el oops Loopi ng m crot ubul es have been descri bed bef ore i nf i x e d permeabi l i zed growt h cones i n v i t r o Lankf or d andKl ei n, 1990 ; Tsui et a l 1984) They have a l s o been seen t of o r mand col l apse i n r e a l t i me i magi ng of l i v e Xenopus neu-rons i n v i t r o Tanaka and Ki rschner, 1991) Thei r presence

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i n t i me-l apse i mages of m grat i ng Ti l growt h cones, subse-quent l y conf i r med by el ectr on m cr oscopy, i n di c at e s t h a tt hey ar e nor mal f e a t u r e s i n process outgr owt h Al t houghm crotubul e pol ymer s have been descri bed as bi ophysi cal l yr i g i d whencomparedt o other cytoskel etal pol ymer s M zu-shi ma- Sugano et al . 1983) , t he f or ces r equi r ed t o bendm crotubul es coul d be suppl i edby t heGTPhydrol ysi s asso-c i ated w t h assembl y , or by t he ATPhydrolys i s assoc i atedw t h m crot ubul e based motor s Hi l l , 1987) What ever t hesource, t h i s force coul d provi de t he energy needed t o al l owm crotubul es t o i nvade s el ec t i v e r egi ons of t he gr owt h cone schemat i c show ng t he maj or types of m crotubul e ar -r angement s f ound i n t he T i l pi oneer gr owt h cone i n s i t u shown i n Fi g 9 Many of these f e a t u r e s may cont r i but e tos t r a t e gi e s underl yi ng gr owt h cone steeri ng Further s t u d i e son t he mol ecul ar nature of m crot ubul e s t a b i l i z a t i o n and i n-t e r a ct i o n w t h other cytoskel etal and second mes senger e l e -ment s i n t he gr owt h cone wi l l i ncr ease our underst andi ng ofhowneurons s t e e r t owar d t h ei r t a r g e t , and generat e t he con-nect i ons of the adul t nervous systemWt hank E Tanaka, S Rei nsch, andF Lefcort f or sti mulati ng comment son t heexperi ments ; T M tchi son, R Val e, andK Lankford f or comment son t he manuscr i pt ; and L Rei chardt f or al l ow ng use of hi s i magi ng s y s -t em Support f o r t hi s workwas provi dedby Medi cal Research Counci l ofCanada fel l owshi ps t o J H Sabry andT P O Connor , Nati onal Sc i enceFoundat i on Biol ogi cal F a c i l i t i e s Center Grant BBS-8714246t o t he Uni ver-s i t y of Cal i forn ia at Berkel ey, L u c i l l e Markey Foundat i on Awardt o t heUniversi ty of Cal i forni a at SanFranci sco I magi ng Center , Nati onal I n s t i -t u t e s of Heal t h J a v i t s AwardNS09074-22 t oBentl ey and a Nati onal I n-s t i t u t e of Genera l Medi ca l Sci ences grant t oKi rs chner Recei ved f or publ i cat i on 17May 1991 and i n revised f o rm24 J une 1991 ReferencesAdams , A EM and J R Pr i ngl e 1984 R el a t i o n s hi p of a c t i n and t u bu l i ndi s t r i b ut i o n i n w l d- t ype and morphogeneti c mut ant Sacchar omyces c e r e v i -s i a e J . C el l B io l 98 : 934- 945 A l e t t a , J M andL A Gr eene 1988 Grow h cone confi gurati on and advance at i me - l a ps e st udyusi ngvi deo- enhanced d i f f e r e n t i a l i n t e r f e r e nc e c o nt r a s t m -croscopy J Neurosci 8 : 1425- 1435 Anderson, H 1988 Drosophi l a adhesi on mol ecul es and neu ra l devel opment Trends Neurosci 11 : 472-475 Anderson, H and R P Tucker 1988 Pi oneer neurons us e bas a l l amna asa substratumf or outgr owt h i n t h e embryoni c gras shopper l i mb Devel opmentComb 104 : 601- 608 Baas, PandMBl ack 1990 I ndi vi dual m crotubules i n t he axon con-s i s t of domai ns t hat di f f e r i n both composi t i on and s t a b i l i t y J Cel l Biol 11 1 : 495- 509 Bambur g, J R D Br a y, and K Chapman 1986 Ass embl y of m crotubulesat t he t i p of grow ng axons Nature Land 321 : 778- 790 B as t i a ni ,J . HGdeCouet , J MA Qui nn, K J K ot r l a , K J Kar l st r omC S Goodman, and E E B al l 1991 Transi en t and dynam c expressi onof megprotei n duri ng devel opment o f t he grass hopper embr yo Dev Biol I n pr es s B a t e , CM1976 Pi oneer neurones i n an i n s ec t embr yo Natur e Zond 260 : 54- 56 Bentl ey, D andCaudy . 1983 Navi gat i onal s u b s t r a t e s f o r p er i p he r al pi -oneer growt h cones l i mb- segment boundari es, and gui depost neurons . Col dSpri ngHar bor Symp Quant Biol 48 : 573- 585Bentl ey, D and H Keshi shi an 1982 P at h f i n di n g by p e r i p h er a l pi oneer neu-rons i n grasshoppers Sci ence Wash DC 218 : 1082- 1088 Bentl ey, D and A Tor oi an- Raymond 1986 D sori ented pathfi ndi ngby pi -oneer neurone growt h cones depri vedof f i l o p o d i a by c y t o c ha l a s i n t r eatment Natur e 323 : 712-715 Bentl ey, D P B Guthri e, and S B Kat er 1991 Calc i umon d i s t r i b u t i o ni n nascent pi oneer axons and coupl ed preaxonogenesi s neurons i n s i t u J Neur osci 11 : 1300- 1308 Bi xby, J L 1989 Prote i n ki naseC s i nvolved i n l amn i n s t i mu l a t i o n of n e u r i t eoutgr owt h Neur on 3 : 287-297 Black,M P Baas, and S Humphr i es 1989 Dynam cs of a l p ha - t u b ul i nd ea c et y l a t i o n i n i n t a ct neurons J Neurosci 9 : 358- 368 Brady, S . T MT y e l l , and R J Lasek 1984 Axona t u bu l i n and axonal

The J ournal of C el l Biol ogy, Vol ume 115, 1991

mcr otubul es bi ochem cal evi dence f or c o l d - s t a b i l i t y J C el l B io l 99 : 1716-1724 Bray, D and K Chapman 1985 Anal ysi s of m crospike movement s on t heneuronal growt h cone J Neurosci 5: 3204- 3213 Bray, and P J Hol l enbeck . 1988 Gr owt h cone mo t i l i t y and gui dance Annu Rev. C el l Biol 4: 43- 61 .Br i dgman, P C andME Dai l ey 1989 The organi zati on of myosi n and a c t i ni n r a pi d frozen nerve growt h cones J C el l Biol 108: 95- 109 Bunge, MB 1973 Fi ne s t r u c t u r e of nerve f i b er s and growt h cones of i s o l a t e dsympatheti c neurons i n c ul t u r e J C el l Biol 56 : 713- 735Cassi meri s, L CL Ri eder, GRupp, and E D Sal mon 1990 Stabi l i tyofm cr ombul e at t achment t o met aphase ki net ochores i n PtKI c e l l s J Cel l Sc i 96 :9 -15 Cast l eman, K R 1979 Di g i t a l I mage Processi ng P r en t i c e- H al l , I n c . Engl e-wood C l i f f s , NJ . 429 pp Caudy, M and D Bentl ey 1986x. 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