Cortical development associated with conjugation of Paramecium

INTRODUCTION The cortical pattern of Paramecium is characterized by its asymmetry. It displays dorsoventral and anteroposterior polarity at both whole cell and cortical unit levels. The anterior and posterior sutures and the oral apparatus define the ventral surface and determine the right and left sides of the cell. The cortex of Paramecium consists of plasma membrane, alveolar system and epiplasm (a fibrous skeleton underlying the cortical membranes). It is molded into ridges which form a pattern of parallelograms over the cell surface. Each parallelogram delimits a cortical unit, the basic unit of the cortex. Cortical units are arranged in parallel longitudinal rows. Each cortical unit contains one or two basal bodies (1-bb and 2-bb units, respectively), its associated postciliary and transverse microtubular ribbons, one ciliary rootlet and one or two cilia. The arrangement of these elements within the cortical unit establishes an asymmetry that is fairly manifested by the ciliary rootlet, which arises from the basal body’s anterior right quadrant and is directed anteriorly. The distribution of 1-bb and 2-bb units over the cell surface is regionalized (Sonneborn, 1975; Iftode et al., 1989). Thus, in the interphase cell, they delineate three different fields: the 2-bb field, where every unit contains two basal bodies, the 1-bb field, with one basal body per unit, and the mixed field, where randomly distributed 1-bb and 2-bb units coexist. The chief developmental pathways in Paramecium are vegetative division and the sexual process (conjugation). These are the phases when morphogenetic events take place. During cell division, cell components must be duplicated in order to produce two complete daughter cells. Moreover, new microtubular structures are transiently required for the morphogenetic events to be properly carried out (cortical cytospindle, micronuclear mitotic spindle and macronuclear microtubules). Cortical morphogenesis throughout cell division in Paramecium has long been studied (Dippell, 1965, 1968; Kaneda and Hanson, 1974; Fernández-Galiano, 1978; Cohen et al., 1982; Iftode et al., 1989; Delgado et al., 1990). The interphase cortical pattern undergoes important modifications during vegetative reproduction. All of the cortical structures are reorganized along waves spreading from the oral region, the fission furrow acting as a secondary inductive zone. The responses of the different regions to the morphogenetic waves depend on their positions on the cell surface (Iftode et al., 1989). During the sexual process of conjugation in Paramecium, as in other ciliates, cells of complementary mating types mutually activate and unite. In conjugating pairs, a series of nuclear events, including micronuclear meiosis, exchange of pronuclei and syngamy, and fragmentation of the macronucleus, occurs. When the cells separate, postzygotic nuclear divisions, differentiation of the new macroand micronuclei and resorption of the old macronucleus continue in the exconjugants. Moreover, during this process, the preexisting oral apparatus and other cortical structures are resorbed and rebuilt. As in division morphogenesis, transitory microtubular elements appear. Nuclear and stomatogenic events have been studied in detail (Grandchamp and Beisson, 1981; Ng and Mikami, 1981; Ng and Newman, 1984; Ng, 1986; Tam and Ng, 1986; Kwok and Ng, 1989). However, the dynamics of cortical structures other than the oral apparatus in conjugation, has never been described. In this study, we have followed the development of different cortical components in conjugation, by using a set of antibodies that recognize cytoskeletal structures. Our obser1099 Development 117, 1099-1112 (1993) Printed in Great Britain © The Company of Biologists Limited 1993 The cortical cytoskeleton of Paramecium is characterized by a complex, polarized and asymmetrical organization. In order to analyse the cortical development of Paramecium tetraurelia during the sexual process of conjugation, different antibodies have been used to follow the development of five cortical components: basal bodies, ciliary rootlets, outer lattice, epiplasm and cytospindle. This study demonstrates that the cortex of Paramecium undergoes an elaborate development process following conjugation. Some of the cortical structures that are not renewed at any other moment of the cell cycle, are resorbed after conjugation and replaced by other newly formed ones. The reorganization of the cortical components occurs according to different morphogenetic waves that spread over the cell surface. The oral system and the preoral suture line act as morphogenetic epicentres. Key words: Paramecium, conjugation, cortical morphogenesis SUMMARY Cortical development associated with conjugation of Paramecium M. Rosario Romero and Antonio Torres Departamento de Microbiologia, Facultad de Biologia, Universidad de Sevilla, Apdo. 1095. 41080 Sevilla, Spain 1100 vations indicate that conjugation is a process in which not only nuclei and the oral apparatus are renewed, but also the other cortical components undergo morphogenetic changes. This morphogenetic process includes regression and neoformation waves spreading over the whole cell surface and originating from the oral system and the anterior suture line. MATERIAL AND METHODS Strains and growth conditions This study has been carried out on strain d4-2 of Paramecium tetraurelia, mating types VII and VIII. The cells were grown at 27°C according to the Sonneborn procedure (1970a) in Cerophyl medium bacterized the day before with Klebsiella pneumoniae and supplemented with 0.4 µg/ml β-sitosterol. Sampling Postautogamous cells of complementary mating types were grown in tubes for three days and mildly starved to induce mating reactivity at 27°C. The two mating types were mixed in Petri dishes, and about 1.5 hours after agglutination the cells were fed with medium to terminate conjugation of loose pairs and to prevent further formation of new pairs, so that synchronized samples could be obtained. About three hours after mixing, tight pairs were collected. About three hours later the pairs began to separate. Exconjugant cells were collected, permeabilized and fixed at different times, ranging from 5 to 45 minutes after separation. Immunofluorescence Exconjugant cells were permeabilized for 5 minutes in the microtubule stabilizing buffer PHEM (Schliwa and Van Blerkom, 1981) containing 1% Triton X-100. Then cells were fixed in freshly prepared 2% paraformaldehyde in PHEM buffer for 1 hour. After washing with Tris-buffered saline (TBS) or phosphate-buffered saline (PBS) containing 0.3% Tween 20 and 3% bovine serum albumin (BSA), cells were incubated for 1-2 hours in the primary antibody diluted in the same washing solution. Then they were washed several times and incubated for 1 hour in the secondary antibody diluted 1:200 in the same buffer. After washing, cells were mounted in glycerol containing 2% N-propyl gallate and observed under a Zeiss III epifluorescent microscope and photographed with Kodak Tri-X film. The same results were obtained using the same method without fixation. In this case, PHEM buffer was used throughout the process. Antibodies The primary antibodies used in this study, as well as the experimental conditions, sources and specificities, are shown in Table 1. The second antibodies were FITC-labelled goat anti-rabbit antibody, FITC-labelled anti-mouse Ig (Pasteur Production, Paris) or biotinylated sheep anti-mouse Ig, detected by Texas Red-labelled streptavidin (Amersham). RESULTS Basal bodies The monoclonal antibody 1A2, anti-tyrosylated α-tubulin was used as the main marker of basal bodies. In Parame - cium visualized by immunofluorescence, this antibody only decorates basal bodies, part of the contractile vacuole rootlets and part of the postoral fibres. In interphase cells, immunofluorescence with anti-tyr reveals exactly the same pattern of basal body distribution as that deduced by Iftode et al. (1989) from silver-stained images (Fig. 1). Our results show that the interphase pattern of basal bodies is altered during conjugation by resorption of one basal body in every unit of the 2-bb field. Furthermore, the right side of the oral vestibulum is disorganized because of the cell fusion and its basal bodies are later rearranged into rows in the exconjugant (Figs 3, 4A, 5A). In pairs at an advanced phase of conjugation, as well as in early exconjugants, every unit in the 2-bb field of the cortex has lost one basal body and so displays only a single basal body (Figs 2, 3, 38B). Basal body resorption continues in the exconjugant following an anteroposterior sequence on both the ventral and dorsal surfaces. This resorption wave spreads over the mixed field as well, where normally 1-bb and 2-bb units coexist. Most of the 2-bb units lose one of their basal bodies, so, as the resorption wave progresses, the mixed fie l d is reduced and the zone with single basal bodies is increased. The dorsal surface (Figs 6-8) is the last region invaded by the resorption wave. At the end of this process, resorption of one basal body has occurred in every 2bb unit. Resorbed basal bodies are replaced by new ones that reconstruct the normal pattern of basal body distribution. When the resorption wave reaches the dorsal surface of the cell, basal body duplication has already begun on the ventral surface (Figs 4A, 7, 38B). Basal body duplication begins at the basal body rows closest to the buccal opening. This duplication process rapidly propagates to the adjacent rows on both the right and left. Duplication starts from the anterior suture line and progresses posteriorly along each row of basal bodies (Fig. 9), adding one basal body in the units that normally display two basal bodies in a mature vegetative cell (Fig. 5A,B). During basal body duplication in exconjugant Parame - cium, the anterior and the posterior basal bodies of a cortical unit show different reactivities towards some antitubulin antibodies, the anterior one being the most strongly decorated by universal anti-tubulin antibodies such as antiβ-tubulin (data not shown) and by the monoclonal antibody 1A2 against α-tyr tubulin (Fig. 4B). However, the posterior basal body, faintly decorated by these antibodies, is M. R. Romero and A. Torres Table 1. Antibodies used in this study Antibody Specificity References Dilution Buffer Anti-kd Ciliary rootlet Sperling, 1991 1/500 PBS I4B7Epiplasm Adoutte, Clerot 1/5 PBS I3D3Outer lattice Adoutte, Clerot 1/5 PBS 1A2Tyrosinylated α-tubulin Kreis, 1987 1/400 PBS 6-11B-1 Acetylated α-tubulin Piperno, 1985 1/5 TBS, PBS MPM-2 Mitotic phosphoproteins Davis, 1983 1/50 PBS Anti-β-tubulin β-tubulin Amersham 1/100 TBS, PBS 1101Cortical development in conjugant Paramecium more strongly stained with anti-PA tubulin than the anterior one (data not shown). These basal body staining patterns observed in exconjugants during basal body duplication exactly coincide with those seen during basal body proliferation in division (Adoutte et al., 1991; our unpublished observations). Cytospindle The cytospindle is a transitory cortical structure consisting of bundles of 4 to 20 longitudinal microtubules that run from pole to pole on the right side of the ciliary rows, at the top of the longitudinal ridges of the cortex (Kaneda and Hanson, 1974; Sundararaman and Hanson, 1976; Cohen et al., 1982). This microtubular array appears on the cell cortex during division and sexual processes. Cytospindle assembly in exconjugant cells (Fig. 38A) begins about 5 minutes after pair separation. At this time micronuclei are in metaphase and fragments of the old macronucleus are observed in the cytoplasm (Fig. 13). The first microtubular bundles of the cytospindle appear around the oral cavity, initially on the right side (Fig. 10) and then on the left (Fig. 11). The cytospindle bundles gradually increase their length and thickness. Longitudinal growth occurs at both ends of the bundles, until they reach the anterior and posterior suture lines (Figs 11, 12, 16). At the same time, new microtubular bundles appear, adjacent to the preexisting ones, following a transverse sequence which starts from the oral opening and spreads to the right and left towards the dorsal surface of the cell (Figs 14, 15). In this manner, every longitudinal ridge running between rows becomes occupied by a microtubular bundle (Figs 16, 17). Cytospindle assembly is completed by bidirectional elongation of all the bundles, which first reach the anterior pole and then the posterior pole of the cell. The process of cytospindle formation takes about 10-15 minutes. During this time, micronuclear mitosis continues and, when the cytospindle is completed, micronuclei are in telophase. Resorption of old macronuclear fragments progresses during this time as well. At late phases of cytospindle assembly most of the fragments of the old macronucleus have been resorbed. The cytoplasmic microtubular network at this phase has fewer microtubules than normal (Fig. 18). When the cytospindle reaches the posterior end of the cell it immediately begins to disassemble (Figs 19-23, 38A). The regression of each microtubular bundle starts from the anterior and posterior suture lines, i.e. bundles shorten from both ends until they disappear. The bundles closest to the oral apparatus are the first ones to be disassembled (Fig. 19). The cytospindle disassembly wave progresses from the buccal opening to the right and left sides of the cell, this progression being faster on the left side. Moreover, the shrinkage of the microtubular bundles is faster at the posterior extreme than at the anterior one. The left ventral surface is the first zone to lose the cytospindle (Fig. 20), followed by the right ventral surface. At later stages of the disassembly process, short bundles of microtubules, shrunk at their extremes, remain on the dorsal anterior surface of the exconjugant cell (Fig. 21). In exconjugant paramecia, cytospindle assembly and development of the new oral apparatus overlap. As conjuFig. 1. Immunofluorescence image of an interphase Paramecium (ventral surface) labelled with the antibody 1A2. This antibody reveals the pattern of basal body distribution over the cell surface. Black lines delimit the three different fields existing in interphase: the 1-bb field, in which every cortical unit contains a single basal body, the 2-bb field, where every unit contains two basal bodies and the mixed field, where 1-bb and 2-bb units coexist. R, right side of the cell; L, left side. ×650. Fig. 2. Immunofluorescence image of a late conjugating pair decorated by the antibody 1A2. One basal body of each cortical unit in the 2-bb field has been resorbed (arrowheads). ×800. 1102 gating cells separate, the new oral apparatus is poorly developed. It is small and C-shaped (stage 4, Ng and Newman, 1984; Figs 3, 10). When the cytospindle has formed, lying along the entire cell, the oral apparatus has attained its mature pattern (stage 6, Ng and Newman, 1984), but it still lacks the postoral fibres that were also disassembled during conjugation (Fig. 18). The postoral fibres grow while the cytospindle regresses. Thus, at the end of the disassembly process the postoral fibres have attained their normal length (Fig. 24). At this stage (stage 8, Ng and Newman, 1984) exconjugating cells recover their ability to feed. Ciliary rootlets Ciliary rootlets (kinetodesmal fibres) are striated fibrils arisM. R. Romero and A. Torres 1103Cortical development in conjugant Paramecium ing from the basal bodies (in cortical units with two basal bodies it arises from the posterior one). They run straight anteriorly, close to the right side of the basal body row. They extend for a distance of several cortical units, overlapping others in the same longitudinal row. The overlapping fibres constitute longitudinal bundles running the full length of the cell (Dippell, 1964). Ciliary rootlets have been visualized by means of immunofluorescence with the anti-kd antiserum. In conjugation of Paramecium, the first modification of the ciliary rootlet pattern is observed when cells separate. As mentioned above, the right vestibulum is disorganized in the new exconjugants, with disorganized basal bodies, which lack ciliary rootlets (Fig. 25A,B, 38C). At this stage, ciliary rootlets on the rest of the cortex have not been modified yet. They are still long fibres constituting longitudinal bundles (Fig. 25A). As the basal bodies on the right vestibulum are being arranged in rows, small fibres start growing from them (Figs 26A,B, 27A,B). Our observations of immunofluorescence with the anti-kd antiserum indicate that ciliary rootlets are renewed over the entire cortex (Fig. 38C): the old fibres regress and new ones replace them. First, old fibres undergo a partial regression, becoming shorter and thinner. Soon they detach from their basal bodies, to be completely disassembled in the internal cytoplasm. A new ciliary rootlet then develops in every unit to replace the old ones (Figs 26B, 27B). Detached old fib r e s accumulate in the cytoplasm and are clearly visualized by i m m u n o fluorescence with the anti-kd antibody. Anti-kd decoration inside the cell changes throughout the ciliary rootlet remodelling process. At an early stage, as in interphase cells, this antibody does not recognize any structure within the cytoplasm (Fig. 28). However, when some of the cortical units have lost their old ciliary rootlets, many scattered fibrils are seen in the cytoplasm (Fig. 29). Later, as new ciliary rootlets grow, the number of inner fibrils decreases until eventually they disappear. As mentioned above, the new exconjugant cells keep their old ciliary rootlets over the entire cortex, except on the right side of the oral apparatus. At this time, a wave of regression and posterior detachment of the old fibres begins. This wave commences in the basal body rows closest to the oral opening (Fig. 25B), from where it extends to the adjacent rows on the right and left, until it reaches the dorsal surface. Within each row, it starts from the anterior suture line and progress posteriorly. Thus, the first fibres to shorten are the ones closest to the preoral suture, which no longer extend across it (Fig. 26B). Then, the remodelling wave extends posteriorly along the rows, more quickly on the left side than on the right (Fig. 27A). At the end of this process all ciliary rootlets of the exconjugant will have been replaced (Figs 30, 31). Sperling et al. (1991) reported that the decoration of ciliary rootlets with the monoclonal antibody MPM-2, specific for phosphoproteins, just precedes fibre disassembly. They also observed MPM-2 staining in exconjugant cells. In order to find out whether this staining is correlated with ciliary rootlet reorganization (as it occurs during division), immunofluorescence with MPM-2 was performed on exconjugant cells (Figs 32, 33). We observed that MPM-2 staining in exconjugant Paramecium starts in the basal body rows closest to the oral apparatus and proceeds in an anteroposterior wave originating from the anterior suture line within each row. It precedes fibre disassembly and follows exactly the spatial sequence observed for ciliary rootlet remodelling. Outer lattice and epiplasm Cortical units in Paramecium are delineated by meshes that constitute a regular cortical network, the outer lattice (Von Gelei, 1937; Parducz, 1962; Ehret and MacArdle, 1974; Cohen el al., 1987). Underlying the cortical membranes there is a fibrous layer, the epiplasm. The epiplasm is organized as individual scales within each cortical unit, i.e. within each mesh of the outer lattice. Epiplasm and outer lattice development are therefore strictly coordinated (Fig. 38D). When cells separate following conjugation, the basal body rows surrounding the oral cavity are disorganized and cortical units in this region are not well defined, especially on the right side of the oral apparatus. I3D3 and I4B7 decoration in this area of the cortex is very faint in early exconjugants (Figs 34, 35). During conjugation the cells become shorter, so that the outer lattice meshes in the exconjugant are smaller than in interphase cells. Figs 3-9. Immunofluorescence images of exconjugants labelled by the antibody 1A2, showing the reorganization of the basal body pattern. Fig. 3. At an early stage, the anterior region of the cell (outlined zone) displays a single basal body in each cortical unit. The 2-bb field and part of the mixed field have been affected by the basal body resorption wave. The pattern of basal bodies to the right of the oral apparatus is disorganized (arrow). as, anterior suture; ps, posterior suture. ×875. Fig. 4. A later stage, when basal body duplication is taking place. (A) View of the entire cell. Basal bodies to the right of the oral apparatus are still disorganized (arrows). ×875. (B) Enlargement of a portion of the anterior region of this cell. Basal bodies have just been duplicated. The anterior basal body of each 2-bb unit appears more strongly stained by the antibody 1A2 than the posterior one. ×1600. Fig. 5. A later stage of basal body duplication. (A) View of the entire cell. Basal bodies on the right of the oral apparatus have been arranged in rows. The ventral surface of the exconjugant has recovered the normal pattern of basal bodies. ×875. (B) Enlargement of the anterior region of the cell in A. The two basal bodies of each unit show the same reactivity towards the antibody. ×1600. Figs 6-8. Dorsal views of the cells in Figs 3, 4 and 5, respectively. ×875. Fig. 6. The basal body resorption wave has only affected the anterior pole, which contains 1-bb per unit. The equatorial region still displays 1-bb and 2-bb units randomly distributed. Fig. 7. Most of the 2-bb units on the dorsal surface have resorbed one basal body. Fig. 8. Basal bodies in the anterior pole have been duplicated, but on the rest of the dorsal surface most of the units still contain one single basal body. Fig. 9. A portion of the anterior region of an exconjugant cell showing the beginning of the basal body duplication wave. The units closest to the anterior suture line (as) in every basal body row already contain two basal bodies (arrows), whereas the more posterior units of each row still display one single basal body (asterisks). oa, oral apparatus. ×1600. 1104 Our results with the monoclonal anti-outer lattice antibody, I3D3, indicate that, after conjugation, this structure is at least partially reorganized. A progressive appearance of new transverse partitions within some of the preexisting cortical units is observed in exconjugants (Figs 36, 37). These partitions always appear close to the anterior boundary of M. R. Romero and A. Torres Figs 10-15. Immunofluorescence images of three exconjugants at sucessive stages of cytospindle formation. Figs 10, 11, 12, 14 and 15 show 6-11B-1 decoration. oa, oral apparatus. ×980. Fig. 10. Initial stage of cytospindle assembly. The first microtubular bundles appear on the right side of the oral apparatus (arrowhead). Fig. 11. A later stage. Cytospindle bundles can be seen on the right and left (arrowheads). They are growing in thickness and length. The microtubular bundles closest to the oral apparatus are thicker and they have reached the anterior suture line (arrows). Fig. 12. A more advanced stage of cytospindle formation. New microtubule bundles have been formed and the preexisting ones have grown (arrowheads). Many of them have reached the anterior suture, but still not the posterior one. Fig. 13. Interior view of the cell in Fig. 10, decorated by anti-β-tubulin antibody. Fragments of the old macronucleus remain in the cytoplasm (ma). The micronuclei (mi) are in the 2nd postzygotic metaphase. Many cytoplasmic microtubules (cm) are observed into the cell. Fig. 14. Dorsal surface of the cell in Fig. 11. Cytospindle assembly wave has not invaded the dorsal surface yet. Fig. 15. Dorsal surface of the cell in Fig. 12. Microtubular bundles have appeared on the dorsal surface of the exconjugant (arrowheads). 1105Cortical development in conjugant Paramecium the unit. Formation of transverse partitions first occurs in the rows closest to the oral apparatus, and then extends to the dorsal surface. As newly formed transverse septa appear, degradation of some of the old ones takes place (Fig. 37), so that the number of cortical units is maintained. Thus, the position of transverse partitions of the outer lattice changes and they now define different units, mixing material coming from old adjacent units. Likewise, new septa separate the material of an old unit into two new ones. This phenomenon of cortical unit reorganization is only observed in the mixed field of the cortex. Outer lattice meshes in 2-bb and 1-bb fields remain unaltered. DISCUSSION In this paper we describe the morphogenetic events occurring in the cortex of Paramecium during conjugation. The major cytoskeletal structures have been studied by means of immunological probes. The morphogenetic strategy used by Paramecium to reproduce its oral and somatic basal body arrays during cell division is significantly different from other evolutionally distant ciliates. Whereas some ciliates such as Oxytrichids resorb and reorganize most of their basal bodies at every division, Paramecium conserves all basal bodies, and the new ones are arranged following the preexisting pattern (Sonneborn, 1963; Beisson and Sonneborn, 1965). Likewise, while most other ciliates develop their new oral apparatus independently of the old one and can regenerate it, Paramecium develops its new oral apparatus close to the old one during division and normally cannot regenerate it (Tartar, 1954; Sonneborn, 1963). The sexual processes of autogamy and conjugation therefore are the only occasions in the cell cycle when Paramecium renews its oral system. Our observations indicate that conjugation is also the morphogenetic process by which other cortical elements, conserved during division, undergo a remodelling process. The development of the basal body pattern in conjugating cells has been followed mostly by using the monoclonal antibody 1A2 as a basal body marker. The results obtained with this antibody were confirmed by using other anti-tubulin antibodies, such as anti-αand anti-β-tubulin, 6-11B-1 and anti-axonemal tubulin of Paramecium (Cohen et al., 1982). Basal body resorption occurs in cortical units containing two basal bodies. The question arises as to whether the anterior or the posterior basal body is the one disappearing. Immunofluorescence images lead us to think that the anterior basal body is the one resorbed while the posterior one remains. When stained with anti-tubulin antibodies, early exconjugant cells display, in every unit, only the fibrils normally associated with the posterior basal body, i.e. transverse and postciliary ribbons and ciliary rootlets. During basal body duplication, the anterior and posterior basal bodies show different reactivities towards anti-tubulin antibodies. Since duplicating basal bodies in exconjugant cells show the same staining pattern seen in dividing cells (see results), it may be concluded that new basal bodies in exconjugant Paramecium, as in dividers (Dippel, 1968), appear anterior to the old ones. Remodelling of transverse partitions of the outer lattice implies a modification of the preexisting cortical units. New transverse segments are formed and the epiplasmic material underlying each unit is redistributed into new units. Changes in the outer lattice meshes only occurs in the mixed field of the cortex, where 1-bb and 2-bb units appear randomly distributed. Moreover, new transverse partitions Figs 16-18. Exconjugant cell with a complete cytospindle, decorated by anti-β-tubulin antibody. ×875. Fig. 16. Ventral surface. The cytospindle bundles run all along the basal body rows from the anterior to the posterior sutures. Fig. 17. Dorsal surface. Every longitudinal ridge at this stage is occupied by a microtubular bundle running from pole to pole of the cell. Fig. 18. Interior view of the cell. The oral apparatus (oa) has attained its mature pattern, but the postoral fibres has not been formed yet. At this stage, the cell has fewer cytoplasmic microtubules (cm). Micronuclei are in telophase. Long micronuclear mitotic spindles are observed (mi). 1106 M. R. Romero and A. Torres Figs 19-24. Exconjugant cells at different stages of cytospindle disassembly. All images are from 6-11B-1 decoration, except Fig. 24 which shows anti-β-tubulin staining. Fig. 19. An early stage. Ventral surface. Microtubular bundles have begun to shorten from the suture lines (arrows), those closest to the oral apparatus being the ones more disassembled. ×900. Fig. 20. A later stage. Ventral surface. The wave of disassembly progresses (arrows). Microtubular bundles on the right side of the cell have already been resorbed. ×900. Fig. 21. At the last stages of cytospindle disassembly short fragments of microtubule bundles remain on the anterior dorsal surface (arrowhead). ×875. Fig. 22. Dorsal surface of the cell in Fig. 19. The wave of disassembly is beginning to invade the dorsal surface from both poles of the cell (arrows). ×900. Fig. 23. Dorsal view of the cell in Fig. 20. Microtubular bundles disassembly progresses from the anterior and posterior poles (arrows). ×900. Fig. 24. Interior view of a cell at a stage corresponding to that of Fig. 21, decorated by anti-β-tubulin antibody. The postoral fibres (pf) have grown and the cytoplasmic network is recovering its normal number of microtubules (cm). ×825. 1107Cortical development in conjugant Paramecium Figs 25-29. Immunofluorescence images of exconjugants decorated by the anti-kd antiserum. Fig. 25. Early exconjugant. The zone on the right of the oral cavity (arrowhead) lacks ciliary rootlets, while the rest of the cortex keeps the old fibres. (A) View of the whole cell. ×765. (B) Enlargement of a region of the cell showing the oral cavity and part of the anterior suture. The most anterior ciliary rootlets of the basal body rows closest to the oral apparatus have begun to shorten (arrow). ×1500. Fig. 26. (A) The wave of ciliary rootlet remodelling extends to the adjacent basal body rows on the right and left, starting from the anterior suture line (arrows). As the basal bodies on the right of the oral opening are being arranged in rows, small ciliary rootlets start growing from them (arrowhead). ×875. (B) Enlargement of part of the cell in Fig. 26A. The large arrows mark partially disassembled old fibres, detached from their anchoring points. The small arrows mark small new fibres which will replace the old ones. ×1870. Fig. 27. A later stage of the ciliary rootlet remodelling. The remodelling wave has spread posteriorly from the anterior suture line over the zone delimited by the arrows. On the rest of the cortex the old fibres still remain. (A) ×1000. (B) Enlargement of the central region of the same cell. Large arrows mark old detaching ciliary rootlets; small arrows mark new developing fibres. ×1375. Fig. 28. Interior view of the early exconjugant of Fig. 25. The anti-kd antiserum does not recognize any fibrilar structure in the internal cytoplasm. Fig. 29. Interior view of an exconjugant at an intermediate stage of the ciliary rootlet reorganization. Many scattered fibrils are recognized by the antiserum in the internal cytoplasm (arrows). They presumably correspond to old fibres detached from their basal bodies (compare to the image of Fig. 28).