Cortical development associated with conjugation of Paramecium

INTRODUCTION The co ical pa e n o Pa amecium is cha ac e ized by i s asymme y. I displays do so en al and an e opos e io pola i y a bo h whole cell and co ical uni le els. The an e- io and pos e io su u es and he o al appa a us de ine he en al su ace and de e mine he igh and le sides o he cell. The co ex o Pa amecium consis s o plasma memb ane, al eola sys em and epiplasm (a ib ous skele on unde ly- ing he co ical memb anes). I is molded in o idges which o m a pa e n o pa allelog ams o e he cell su ace. Each pa allelog am delimi s a co ical uni , he basic uni o he co ex. Co ical uni s a e a anged in pa allel longi udinal ows. Each co ical uni con ains one o wo basal bodies (1-bb and 2-bb uni s, espec i ely), i s associa ed pos cil- ia y and ans e se mic o ubula ibbons, one cilia y oo - le and one o wo cilia. The a angemen o hese elemen s wi hin he co ical uni es ablishes an asymme y ha is ai ly mani es ed by he cilia y oo le , which a ises om he basal body’s an e io igh quad an and is di ec ed an e- io ly. The dis ibu ion o 1-bb and 2-bb uni s o e he cell su ace is egionalized (Sonnebo n, 1975; I ode e al., 1989). Thus, in he in e phase cell, hey delinea e h ee di - e en ields: he 2-bb ield, whe e e e y uni con ains wo basal bodies, he 1-bb ield, wi h one basal body pe uni , and he mixed ield, whe e andomly dis ibu ed 1-bb and 2-bb uni s coexis . The chie de elopmen al pa hways in Pa amecium a e ege a i e di ision and he sexual p ocess (conjuga ion). These a e he phases when mo phogene ic e en s ake place. Du ing cell di ision, cell componen s mus be dupli- ca ed in o de o p oduce wo comple e daugh e cells. Mo eo e , new mic o ubula s uc u es a e ansien ly equi ed o he mo phogene ic e en s o be p ope ly ca - ied ou (co ical cy ospindle, mic onuclea mi o ic spindle and mac onuclea mic o ubules). Co ical mo phogenesis h oughou cell di ision in Pa amecium has long been s ud- ied (Dippell, 1965, 1968; Kaneda and Hanson, 1974; Fe - nández-Galiano, 1978; Cohen e al., 1982; I ode e al., 1989; Delgado e al., 1990). The in e phase co ical pa e n unde goes impo an modi ica ions du ing ege a i e ep o- duc ion. All o he co ical s uc u es a e eo ganized along wa es sp eading om he o al egion, he ission u ow ac ing as a seconda y induc i e zone. The esponses o he di e en egions o he mo phogene ic wa es depend on hei posi ions on he cell su ace (I ode e al., 1989). Du ing he sexual p ocess o conjuga ion in Pa amecium, as in o he cilia es, cells o complemen a y ma ing ypes mu ually ac i a e and uni e. In conjuga ing pai s, a se ies o nuclea e en s, including mic onuclea meiosis, exchange o p onuclei and syngamy, and agmen a ion o he mac onucleus, occu s. When he cells sepa a e, pos zy- go ic nuclea di isions, di e en ia ion o he new mac o- and mic onuclei and eso p ion o he old mac onucleus con inue in he exconjugan s. Mo eo e , du ing his p ocess, he p eexis ing o al appa a us and o he co ical s uc u es a e eso bed and ebuil . As in di ision mo pho- genesis, ansi o y mic o ubula elemen s appea . Nuclea and s oma ogenic e en s ha e been s udied in de ail (G and- champ and Beisson, 1981; Ng and Mikami, 1981; Ng and Newman, 1984; Ng, 1986; Tam and Ng, 1986; Kwok and Ng, 1989). Howe e , he dynamics o co ical s uc u es o he han he o al appa a us in conjuga ion, has ne e been desc ibed. In his s udy, we ha e ollowed he de elopmen o di - e en co ical componen s in conjuga ion, by using a se o an ibodies ha ecognize cy oskele al s uc u es. Ou obse - 1099 De elopmen 117, 1099-1112 (1993) P in ed in G ea B i ain © The Company o Biologis s Limi ed 1993 The co ical cy oskele on o Pa amecium is cha ac e - ized by a complex, pola ized and asymme ical o gani- za ion. In o de o analyse he co ical de elopmen o Pa amecium e au elia du ing he sexual p ocess o con- juga ion, di e en an ibodies ha e been used o ollow he de elopmen o i e co ical componen s: basal bodies, cilia y oo le s, ou e la ice, epiplasm and cy ospindle. This s udy demons a es ha he co ex o Pa amecium unde goes an elabo a e de elopmen p ocess ollowing conjuga ion. Some o he co ical s uc u es ha a e no enewed a any o he momen o he cell cycle, a e eso bed a e conjuga ion and eplaced by o he newly o med ones. The eo ganiza- ion o he co ical componen s occu s acco ding o di - e en mo phogene ic wa es ha sp ead o e he cell su ace. The o al sys em and he p eo al su u e line ac as mo phogene ic epicen es. Key wo ds: Pa amecium, conjuga ion, co ical mo phogenesis SUMMARY Co ical de elopmen associa ed wi h conjuga ion o Pa amecium M. Rosa io Rome o and An onio To es Depa amen o de Mic obiologia, Facul ad de Biologia, Uni e sidad de Se illa, Apdo. 1095. 41080 Se illa, Spain 1100 a ions indica e ha conjuga ion is a p ocess in which no only nuclei and he o al appa a us a e enewed, bu also he o he co ical componen s unde go mo phogene ic changes. This mo phogene ic p ocess includes eg ession and neo- o ma ion wa es sp eading o e he whole cell su ace and o igina ing om he o al sys em and he an e io su u e line. MATERIAL AND METHODS S ains and g ow h condi ions This s udy has been ca ied ou on s ain d4-2 o Pa amecium e au elia, ma ing ypes VII and VIII. The cells we e g own a 27°C acco ding o he Sonnebo n p ocedu e (1970a) in Ce ophyl medium bac e ized he day be o e wi h Klebsiella pneumoniae and supplemen ed wi h 0.4 µg/ml β-si os e ol. Sampling Pos au ogamous cells o complemen a y ma ing ypes we e g own in ubes o h ee days and mildly s a ed o induce ma ing eac- i i y a 27°C. The wo ma ing ypes we e mixed in Pe i dishes, and abou 1.5 hou s a e agglu ina ion he cells we e ed wi h medium o e mina e conjuga ion o loose pai s and o p e en u he o ma ion o new pai s, so ha synch onized samples could be ob ained. Abou h ee hou s a e mixing, igh pai s we e col- lec ed. Abou h ee hou s la e he pai s began o sepa a e. Excon- jugan cells we e collec ed, pe meabilized and ixed a di e en imes, anging om 5 o 45 minu es a e sepa a ion. Immuno luo escence Exconjugan cells we e pe meabilized o 5 minu es in he mic o- ubule s abilizing bu e PHEM (Schliwa and Van Ble kom, 1981) con aining 1% T i on X-100. Then cells we e ixed in eshly p e- pa ed 2% pa a o maldehyde in PHEM bu e o 1 hou . A e washing wi h T is-bu e ed saline (TBS) o phospha e-bu e ed saline (PBS) con aining 0.3% Tween 20 and 3% bo ine se um albumin (BSA), cells we e incuba ed o 1-2 hou s in he p ima y an ibody dilu ed in he same washing solu ion. Then hey we e washed se e al imes and incuba ed o 1 hou in he seconda y an ibody dilu ed 1:200 in he same bu e . A e washing, cells we e moun ed in glyce ol con aining 2% N-p opyl galla e and obse ed unde a Zeiss III epi luo escen mic oscope and pho- og aphed wi h Kodak T i-X ilm. The same esul s we e ob ained using he same me hod wi hou ixa ion. In his case, PHEM bu e was used h oughou he p ocess. An ibodies The p ima y an ibodies used in his s udy, as well as he expe i- men al condi ions, sou ces and speci ici ies, a e shown in Table 1. The second an ibodies we e FITC-labelled goa an i- abbi an i- body, FITC-labelled an i-mouse Ig (Pas eu P oduc ion, Pa is) o bio inyla ed sheep an i-mouse Ig, de ec ed by Texas Red-labelled s ep a idin (Ame sham). RESULTS Basal bodies The monoclonal an ibody 1A2, an i- y osyla ed α- ubulin was used as he main ma ke o basal bodies. In Pa ame - cium isualized by immuno luo escence, his an ibody only deco a es basal bodies, pa o he con ac ile acuole oo le s and pa o he pos o al ib es. In in e phase cells, immuno luo escence wi h an i- y e eals exac ly he same pa e n o basal body dis ibu ion as ha deduced by I ode e al. (1989) om sil e -s ained images (Fig. 1). Ou esul s show ha he in e phase pa e n o basal bodies is al e ed du ing conjuga ion by eso p ion o one basal body in e e y uni o he 2-bb ield. Fu he mo e, he igh side o he o al es ibulum is diso ganized because o he cell usion and i s basal bodies a e la e ea anged in o ows in he exconjugan (Figs 3, 4A, 5A). In pai s a an ad anced phase o conjuga ion, as well as in ea ly excon- jugan s, e e y uni in he 2-bb ield o he co ex has los one basal body and so displays only a single basal body (Figs 2, 3, 38B). Basal body eso p ion con inues in he exconjugan ollowing an an e opos e io sequence on bo h he en al and do sal su aces. This eso p ion wa e sp eads o e he mixed ield as well, whe e no mally 1-bb and 2-bb uni s coexis . Mos o he 2-bb uni s lose one o hei basal bodies, so, as he eso p ion wa e p og esses, he mixed ie l d is educed and he zone wi h single basal bodies is inc eased. The do sal su ace (Figs 6-8) is he las egion in aded by he eso p ion wa e. A he end o his p ocess, eso p ion o one basal body has occu ed in e e y 2bb uni . Reso bed basal bodies a e eplaced by new ones ha econs uc he no mal pa e n o basal body dis ibu ion. When he eso p ion wa e eaches he do sal su ace o he cell, basal body duplica ion has al eady begun on he en- al su ace (Figs 4A, 7, 38B). Basal body duplica ion begins a he basal body ows closes o he buccal open- ing. This duplica ion p ocess apidly p opaga es o he adja- cen ows on bo h he igh and le . Duplica ion s a s om he an e io su u e line and p og esses pos e io ly along each ow o basal bodies (Fig. 9), adding one basal body in he uni s ha no mally display wo basal bodies in a ma u e ege a i e cell (Fig. 5A,B). Du ing basal body duplica ion in exconjugan Pa ame - cium, he an e io and he pos e io basal bodies o a co - ical uni show di e en eac i i ies owa ds some an i- ubulin an ibodies, he an e io one being he mos s ongly deco a ed by uni e sal an i- ubulin an ibodies such as an i- β- ubulin (da a no shown) and by he monoclonal an ibody 1A2 agains α- y ubulin (Fig. 4B). Howe e , he pos e- io basal body, ain ly deco a ed by hese an ibodies, is M. R. Rome o and A. To es Table 1. An ibodies used in his s udy An ibody Speci ici y Re e ences Dilu ion Bu e An i-kd Cilia y oo le Spe ling, 1991 1/500 PBS I4B7Epiplasm Adou e, Cle o 1/5 PBS I3D3Ou e la ice Adou e, Cle o 1/5 PBS 1A2Ty osinyla ed α- ubulin K eis, 1987 1/400 PBS 6-11B-1 Ace yla ed α- ubulin Pipe no, 1985 1/5 TBS, PBS MPM-2 Mi o ic phosphop o eins Da is, 1983 1/50 PBS An i-β- ubulin β- ubulin Ame sham 1/100 TBS, PBS 1101Co ical de elopmen in conjugan Pa amecium mo e s ongly s ained wi h an i-PA ubulin han he an e- io one (da a no shown). These basal body s aining pa - e ns obse ed in exconjugan s du ing basal body duplica- ion exac ly coincide wi h hose seen du ing basal body p oli e a ion in di ision (Adou e e al., 1991; ou unpub- lished obse a ions). Cy ospindle The cy ospindle is a ansi o y co ical s uc u e consis ing o bundles o 4 o 20 longi udinal mic o ubules ha un om pole o pole on he igh side o he cilia y ows, a he op o he longi udinal idges o he co ex (Kaneda and Hanson, 1974; Sunda a aman and Hanson, 1976; Cohen e al., 1982). This mic o ubula a ay appea s on he cell co ex du ing di ision and sexual p ocesses. Cy ospindle assembly in exconjugan cells (Fig. 38A) begins abou 5 minu es a e pai sepa a ion. A his ime mic onuclei a e in me aphase and agmen s o he old mac onucleus a e obse ed in he cy oplasm (Fig. 13). The i s mic o ubula bundles o he cy ospindle appea a ound he o al ca i y, ini ially on he igh side (Fig. 10) and hen on he le (Fig. 11). The cy ospindle bundles g adually inc ease hei leng h and hickness. Longi udinal g ow h occu s a bo h ends o he bundles, un il hey each he an e- io and pos e io su u e lines (Figs 11, 12, 16). A he same ime, new mic o ubula bundles appea , adjacen o he p e- exis ing ones, ollowing a ans e se sequence which s a s om he o al opening and sp eads o he igh and le owa ds he do sal su ace o he cell (Figs 14, 15). In his manne , e e y longi udinal idge unning be ween ows becomes occupied by a mic o ubula bundle (Figs 16, 17). Cy ospindle assembly is comple ed by bidi ec ional elon- ga ion o all he bundles, which i s each he an e io pole and hen he pos e io pole o he cell. The p ocess o cy ospindle o ma ion akes abou 10-15 minu es. Du ing his ime, mic onuclea mi osis con inues and, when he cy ospindle is comple ed, mic onuclei a e in elophase. Reso p ion o old mac onuclea agmen s p o- g esses du ing his ime as well. A la e phases o cy ospin- dle assembly mos o he agmen s o he old mac onu- cleus ha e been eso bed. The cy oplasmic mic o ubula ne wo k a his phase has ewe mic o ubules han no mal (Fig. 18). When he cy ospindle eaches he pos e io end o he cell i immedia ely begins o disassemble (Figs 19-23, 38A). The eg ession o each mic o ubula bundle s a s om he an e io and pos e io su u e lines, i.e. bundles sho en om bo h ends un il hey disappea . The bundles closes o he o al appa a us a e he i s ones o be disassembled (Fig. 19). The cy ospindle disassembly wa e p og esses om he buccal opening o he igh and le sides o he cell, his p og ession being as e on he le side. Mo eo e , he sh inkage o he mic o ubula bundles is as e a he pos- e io ex eme han a he an e io one. The le en al su - ace is he i s zone o lose he cy ospindle (Fig. 20), ol- lowed by he igh en al su ace. A la e s ages o he disassembly p ocess, sho bundles o mic o ubules, sh unk a hei ex emes, emain on he do sal an e io su ace o he exconjugan cell (Fig. 21). In exconjugan pa amecia, cy ospindle assembly and de elopmen o he new o al appa a us o e lap. As conju- Fig. 1. Immuno luo escence image o an in e phase Pa amecium ( en al su ace) labelled wi h he an ibody 1A2. This an ibody e eals he pa e n o basal body dis ibu ion o e he cell su ace. Black lines delimi he h ee di e en ields exis ing in in e phase: he 1-bb ield, in which e e y co ical uni con ains a single basal body, he 2-bb ield, whe e e e y uni con ains wo basal bodies and he mixed ield, whe e 1-bb and 2-bb uni s coexis . R, igh side o he cell; L, le side. ×650. Fig. 2. Immuno luo escence image o a la e conjuga ing pai deco a ed by he an ibody 1A2. One basal body o each co ical uni in he 2-bb ield has been eso bed (a owheads). ×800. 1102 ga ing cells sepa a e, he new o al appa a us is poo ly de el- oped. I is small and C-shaped (s age 4, Ng and Newman, 1984; Figs 3, 10). When he cy ospindle has o med, lying along he en i e cell, he o al appa a us has a ained i s ma u e pa e n (s age 6, Ng and Newman, 1984), bu i s ill lacks he pos o al ib es ha we e also disassembled du ing conjuga ion (Fig. 18). The pos o al ib es g ow while he cy ospindle eg esses. Thus, a he end o he disassembly p ocess he pos o al ib es ha e a ained hei no mal leng h (Fig. 24). A his s age (s age 8, Ng and Newman, 1984) exconjuga ing cells eco e hei abili y o eed. Cilia y oo le s Cilia y oo le s (kine odesmal ib es) a e s ia ed ib ils a is- M. R. Rome o and A. To es 1103Co ical de elopmen in conjugan Pa amecium ing om he basal bodies (in co ical uni s wi h wo basal bodies i a ises om he pos e io one). They un s aigh an e io ly, close o he igh side o he basal body ow. They ex end o a dis ance o se e al co ical uni s, o e - lapping o he s in he same longi udinal ow. The o e lap- ping ib es cons i u e longi udinal bundles unning he ull leng h o he cell (Dippell, 1964). Cilia y oo le s ha e been isualized by means o immuno luo escence wi h he an i-kd an ise um. In conju- ga ion o Pa amecium, he i s modi ica ion o he cilia y oo le pa e n is obse ed when cells sepa a e. As men- ioned abo e, he igh es ibulum is diso ganized in he new exconjugan s, wi h diso ganized basal bodies, which lack cilia y oo le s (Fig. 25A,B, 38C). A his s age, cil- ia y oo le s on he es o he co ex ha e no been modi- ied ye . They a e s ill long ib es cons i u ing longi udinal bundles (Fig. 25A). As he basal bodies on he igh es ibu- lum a e being a anged in ows, small ib es s a g owing om hem (Figs 26A,B, 27A,B). Ou obse a ions o immuno luo escence wi h he an i-kd an ise um indica e ha cilia y oo le s a e enewed o e he en i e co ex (Fig. 38C): he old ib es eg ess and new ones eplace hem. Fi s , old ib es unde go a pa ial eg ession, becoming sho e and hinne . Soon hey de ach om hei basal bodies, o be comple ely disassembled in he in e nal cy oplasm. A new cilia y oo le hen de elops in e e y uni o eplace he old ones (Figs 26B, 27B). De ached old ib e s accumula e in he cy oplasm and a e clea ly isualized by i m m u n o luo escence wi h he an i-kd an ibody. An i-kd dec- o a ion inside he cell changes h oughou he cilia y oo - le emodelling p ocess. A an ea ly s age, as in in e phase cells, his an ibody does no ecognize any s uc u e wi hin he cy oplasm (Fig. 28). Howe e , when some o he co i- cal uni s ha e los hei old cilia y oo le s, many sca e ed ib ils a e seen in he cy oplasm (Fig. 29). La e , as new cil- ia y oo le s g ow, he numbe o inne ib ils dec eases un il e en ually hey disappea . As men ioned abo e, he new exconjugan cells keep hei old cilia y oo le s o e he en i e co ex, excep on he igh side o he o al appa a us. A his ime, a wa e o eg ession and pos e io de achmen o he old ib es begins. This wa e commences in he basal body ows closes o he o al opening (Fig. 25B), om whe e i ex ends o he adja- cen ows on he igh and le , un il i eaches he do sal su ace. Wi hin each ow, i s a s om he an e io su u e line and p og ess pos e io ly. Thus, he i s ib es o sho en a e he ones closes o he p eo al su u e, which no longe ex end ac oss i (Fig. 26B). Then, he emodelling wa e ex ends pos e io ly along he ows, mo e quickly on he le side han on he igh (Fig. 27A). A he end o his p ocess all cilia y oo le s o he exconjugan will ha e been eplaced (Figs 30, 31). Spe ling e al. (1991) epo ed ha he deco a ion o cil- ia y oo le s wi h he monoclonal an ibody MPM-2, speci ic o phosphop o eins, jus p ecedes ib e disassembly. They also obse ed MPM-2 s aining in exconjugan cells. In o de o ind ou whe he his s aining is co ela ed wi h cil- ia y oo le eo ganiza ion (as i occu s du ing di ision), immuno luo escence wi h MPM-2 was pe o med on exconjugan cells (Figs 32, 33). We obse ed ha MPM-2 s aining in exconjugan Pa amecium s a s in he basal body ows closes o he o al appa a us and p oceeds in an an e o- pos e io wa e o igina ing om he an e io su u e line wi hin each ow. I p ecedes ib e disassembly and ollows exac ly he spa ial sequence obse ed o cilia y oo le emodelling. Ou e la ice and epiplasm Co ical uni s in Pa amecium a e delinea ed by meshes ha cons i u e a egula co ical ne wo k, he ou e la ice (Von Gelei, 1937; Pa ducz, 1962; Eh e and MacA dle, 1974; Cohen el al., 1987). Unde lying he co ical memb anes he e is a ib ous laye , he epiplasm. The epiplasm is o ga- nized as indi idual scales wi hin each co ical uni , i.e. wi hin each mesh o he ou e la ice. Epiplasm and ou e la ice de elopmen a e he e o e s ic ly coo dina ed (Fig. 38D). When cells sepa a e ollowing conjuga ion, he basal body ows su ounding he o al ca i y a e diso ganized and co ical uni s in his egion a e no well de ined, especially on he igh side o he o al appa a us. I3D3 and I4B7 dec- o a ion in his a ea o he co ex is e y ain in ea ly excon- jugan s (Figs 34, 35). Du ing conjuga ion he cells become sho e , so ha he ou e la ice meshes in he exconjugan a e smalle han in in e phase cells. Figs 3-9. Immuno luo escence images o exconjugan s labelled by he an ibody 1A2, showing he eo ganiza ion o he basal body pa e n. Fig. 3. A an ea ly s age, he an e io egion o he cell (ou lined zone) displays a single basal body in each co ical uni . The 2-bb ield and pa o he mixed ield ha e been a ec ed by he basal body eso p ion wa e. The pa e n o basal bodies o he igh o he o al appa a us is diso ganized (a ow). as, an e io su u e; ps, pos e io su u e. ×875. Fig. 4. A la e s age, when basal body duplica ion is aking place. (A) View o he en i e cell. Basal bodies o he igh o he o al appa a us a e s ill diso ganized (a ows). ×875. (B) Enla gemen o a po ion o he an e io egion o his cell. Basal bodies ha e jus been duplica ed. The an e io basal body o each 2-bb uni appea s mo e s ongly s ained by he an ibody 1A2 han he pos e io one. ×1600. Fig. 5. A la e s age o basal body duplica ion. (A) View o he en i e cell. Basal bodies on he igh o he o al appa a us ha e been a anged in ows. The en al su ace o he exconjugan has eco e ed he no mal pa e n o basal bodies. ×875. (B) Enla gemen o he an e io egion o he cell in A. The wo basal bodies o each uni show he same eac i i y owa ds he an ibody. ×1600. Figs 6-8. Do sal iews o he cells in Figs 3, 4 and 5, espec i ely. ×875. Fig. 6. The basal body eso p ion wa e has only a ec ed he an e io pole, which con ains 1-bb pe uni . The equa o ial egion s ill displays 1-bb and 2-bb uni s andomly dis ibu ed. Fig. 7. Mos o he 2-bb uni s on he do sal su ace ha e eso bed one basal body. Fig. 8. Basal bodies in he an e io pole ha e been duplica ed, bu on he es o he do sal su ace mos o he uni s s ill con ain one single basal body. Fig. 9. A po ion o he an e io egion o an exconjugan cell showing he beginning o he basal body duplica ion wa e. The uni s closes o he an e io su u e line (as) in e e y basal body ow al eady con ain wo basal bodies (a ows), whe eas he mo e pos e io uni s o each ow s ill display one single basal body (as e isks). oa, o al appa a us. ×1600. 1104 Ou esul s wi h he monoclonal an i-ou e la ice an i- body, I3D3, indica e ha , a e conjuga ion, his s uc u e is a leas pa ially eo ganized. A p og essi e appea ance o new ans e se pa i ions wi hin some o he p eexis ing co - ical uni s is obse ed in exconjugan s (Figs 36, 37). These pa i ions always appea close o he an e io bounda y o M. R. Rome o and A. To es Figs 10-15. Immuno luo escence images o h ee exconjugan s a sucessi e s ages o cy ospindle o ma ion. Figs 10, 11, 12, 14 and 15 show 6-11B-1 deco a ion. oa, o al appa a us. ×980. Fig. 10. Ini ial s age o cy ospindle assembly. The i s mic o ubula bundles appea on he igh side o he o al appa a us (a owhead). Fig. 11. A la e s age. Cy ospindle bundles can be seen on he igh and le (a owheads). They a e g owing in hickness and leng h. The mic o ubula bundles closes o he o al appa a us a e hicke and hey ha e eached he an e io su u e line (a ows). Fig. 12. A mo e ad anced s age o cy ospindle o ma ion. New mic o ubule bundles ha e been o med and he p eexis ing ones ha e g own (a owheads). Many o hem ha e eached he an e io su u e, bu s ill no he pos e io one. Fig. 13. In e io iew o he cell in Fig. 10, deco a ed by an i-β- ubulin an ibody. F agmen s o he old mac onucleus emain in he cy oplasm (ma). The mic onuclei (mi) a e in he 2nd pos zygo ic me aphase. Many cy oplasmic mic o ubules (cm) a e obse ed in o he cell. Fig. 14. Do sal su ace o he cell in Fig. 11. Cy ospindle assembly wa e has no in aded he do sal su ace ye . Fig. 15. Do sal su ace o he cell in Fig. 12. Mic o ubula bundles ha e appea ed on he do sal su ace o he exconjugan (a owheads). 1105Co ical de elopmen in conjugan Pa amecium he uni . Fo ma ion o ans e se pa i ions i s occu s in he ows closes o he o al appa a us, and hen ex ends o he do sal su ace. As newly o med ans e se sep a appea , deg ada ion o some o he old ones akes place (Fig. 37), so ha he numbe o co ical uni s is main ained. Thus, he posi ion o ans e se pa i ions o he ou e la ice changes and hey now de ine di e en uni s, mixing ma e ial coming om old adjacen uni s. Likewise, new sep a sepa a e he ma e ial o an old uni in o wo new ones. This phenomenon o co ical uni eo ganiza ion is only obse ed in he mixed ield o he co ex. Ou e la ice meshes in 2-bb and 1-bb ields emain unal e ed. DISCUSSION In his pape we desc ibe he mo phogene ic e en s occu - ing in he co ex o Pa amecium du ing conjuga ion. The majo cy oskele al s uc u es ha e been s udied by means o immunological p obes. The mo phogene ic s a egy used by Pa amecium o ep oduce i s o al and soma ic basal body a ays du ing cell di ision is signi ican ly di e en om o he e olu ionally dis an cilia es. Whe eas some cilia es such as Oxy ichids eso b and eo ganize mos o hei basal bodies a e e y di ision, Pa amecium conse es all basal bodies, and he new ones a e a anged ollowing he p eexis ing pa e n (Sonnebo n, 1963; Beisson and Sonnebo n, 1965). Like- wise, while mos o he cilia es de elop hei new o al appa- a us independen ly o he old one and can egene a e i , Pa amecium de elops i s new o al appa a us close o he old one du ing di ision and no mally canno egene a e i (Ta a , 1954; Sonnebo n, 1963). The sexual p ocesses o au ogamy and conjuga ion he e o e a e he only occasions in he cell cycle when Pa amecium enews i s o al sys em. Ou obse a ions indica e ha conjuga ion is also he mo - phogene ic p ocess by which o he co ical elemen s, con- se ed du ing di ision, unde go a emodelling p ocess. The de elopmen o he basal body pa e n in conjuga - ing cells has been ollowed mos ly by using he monoclonal an ibody 1A2 as a basal body ma ke . The esul s ob ained wi h his an ibody we e con i med by using o he an i- ubu- lin an ibodies, such as an i-α- and an i-β- ubulin, 6-11B-1 and an i-axonemal ubulin o Pa amecium (Cohen e al., 1982). Basal body eso p ion occu s in co ical uni s con aining wo basal bodies. The ques ion a ises as o whe he he an e- io o he pos e io basal body is he one disappea ing. Immuno luo escence images lead us o hink ha he an e- io basal body is he one eso bed while he pos e io one emains. When s ained wi h an i- ubulin an ibodies, ea ly exconjugan cells display, in e e y uni , only he ib ils no mally associa ed wi h he pos e io basal body, i.e. ans- e se and pos cilia y ibbons and cilia y oo le s. Du ing basal body duplica ion, he an e io and pos e io basal bodies show di e en eac i i ies owa ds an i- ubu- lin an ibodies. Since duplica ing basal bodies in exconju- gan cells show he same s aining pa e n seen in di iding cells (see esul s), i may be concluded ha new basal bodies in exconjugan Pa amecium, as in di ide s (Dippel, 1968), appea an e io o he old ones. Remodelling o ans e se pa i ions o he ou e la ice implies a modi ica ion o he p eexis ing co ical uni s. New ans e se segmen s a e o med and he epiplasmic ma e - ial unde lying each uni is edis ibu ed in o new uni s. Changes in he ou e la ice meshes only occu s in he mixed ield o he co ex, whe e 1-bb and 2-bb uni s appea andomly dis ibu ed. Mo eo e , new ans e se pa i ions Figs 16-18. Exconjugan cell wi h a comple e cy ospindle, deco a ed by an i-β- ubulin an ibody. ×875. Fig. 16. Ven al su ace. The cy ospindle bundles un all along he basal body ows om he an e io o he pos e io su u es. Fig. 17. Do sal su ace. E e y longi udinal idge a his s age is occupied by a mic o ubula bundle unning om pole o pole o he cell. Fig. 18. In e io iew o he cell. The o al appa a us (oa) has a ained i s ma u e pa e n, bu he pos o al ib es has no been o med ye . A his s age, he cell has ewe cy oplasmic mic o ubules (cm). Mic onuclei a e in elophase. Long mic onuclea mi o ic spindles a e obse ed (mi). 1106 M. R. Rome o and A. To es Figs 19-24. Exconjugan cells a di e en s ages o cy ospindle disassembly. All images a e om 6-11B-1 deco a ion, excep Fig. 24 which shows an i-β- ubulin s aining. Fig. 19. An ea ly s age. Ven al su ace. Mic o ubula bundles ha e begun o sho en om he su u e lines (a ows), hose closes o he o al appa a us being he ones mo e disassembled. ×900. Fig. 20. A la e s age. Ven al su ace. The wa e o disassembly p og esses (a ows). Mic o ubula bundles on he igh side o he cell ha e al eady been eso bed. ×900. Fig. 21. A he las s ages o cy ospindle disassembly sho agmen s o mic o ubule bundles emain on he an e io do sal su ace (a owhead). ×875. Fig. 22. Do sal su ace o he cell in Fig. 19. The wa e o disassembly is beginning o in ade he do sal su ace om bo h poles o he cell (a ows). ×900. Fig. 23. Do sal iew o he cell in Fig. 20. Mic o ubula bundles disassembly p og esses om he an e io and pos e io poles (a ows). ×900. Fig. 24. In e io iew o a cell a a s age co esponding o ha o Fig. 21, deco a ed by an i-β- ubulin an ibody. The pos o al ib es (p ) ha e g own and he cy oplasmic ne wo k is eco e ing i s no mal numbe o mic o ubules (cm). ×825. 1107Co ical de elopmen in conjugan Pa amecium Figs 25-29. Immuno luo escence images o exconjugan s deco a ed by he an i-kd an ise um. Fig. 25. Ea ly exconjugan . The zone on he igh o he o al ca i y (a owhead) lacks cilia y oo le s, while he es o he co ex keeps he old ib es. (A) View o he whole cell. ×765. (B) Enla gemen o a egion o he cell showing he o al ca i y and pa o he an e io su u e. The mos an e io cilia y oo le s o he basal body ows closes o he o al appa a us ha e begun o sho en (a ow). ×1500. Fig. 26. (A) The wa e o cilia y oo le emodelling ex ends o he adjacen basal body ows on he igh and le , s a ing om he an e io su u e line (a ows). As he basal bodies on he igh o he o al opening a e being a anged in ows, small cilia y oo le s s a g owing om hem (a owhead). ×875. (B) Enla gemen o pa o he cell in Fig. 26A. The la ge a ows ma k pa ially disassembled old ib es, de ached om hei ancho ing poin s. The small a ows ma k small new ib es which will eplace he old ones. ×1870. Fig. 27. A la e s age o he cilia y oo le emodelling. The emodelling wa e has sp ead pos e io ly om he an e io su u e line o e he zone delimi ed by he a ows. On he es o he co ex he old ib es s ill emain. (A) ×1000. (B) Enla gemen o he cen al egion o he same cell. La ge a ows ma k old de aching cilia y oo le s; small a ows ma k new de eloping ib es. ×1375. Fig. 28. In e io iew o he ea ly exconjugan o Fig. 25. The an i-kd an ise um does no ecognize any ib ila s uc u e in he in e nal cy oplasm. Fig. 29. In e io iew o an exconjugan a an in e media e s age o he cilia y oo le eo ganiza ion. Many sca e ed ib ils a e ecognized by he an ise um in he in e nal cy oplasm (a ows). They p esumably co espond o old ib es de ached om hei basal bodies (compa e o he image o Fig. 28).