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).