J. Cell Set. 51, 1-13 (1981)Printed in Great Britain © Company of Biologist! Limited ig8i

CILIARY SPECIALIZATIONS IN MATINGCELLS OF THE CILIATE EUPLOTESCRASSUS

PIERANGELO LUPORINI1 AND ROMANO DALLAI1

^Institute of Zoology of Camerino (MC) and^Institute of Zoology of Siena, Italy

SUMMARY

In the ciliate Euplotes crassus, mixed cells of different compatible mating types pass throughan induction period before agglutinating with each other by means of cilia in the matingreaction. We examined the ciliary membranes of cells involved in the mating reaction by thefreeze-fracture technique and detected at least 5 distinct types of specialization, each indicatedby a special congregation of intramembrane particles. Near to the necklace, at the ciliarybase, we observed a set of several parallel transverse rows of 10-15 n m particles; a longitudinalrow of 12 nm particles appeared more distally from the necklace, preferentially in replicas ofintermediate regions of the ciliary shaft. These 3 specializations were common to both matingand vegetative cells. The other 2 appeared as more dynamic specializations found exclusively,at least in their most patterned organization, in the ciliary membranes of mating cells. Takingon the aspects of rosette-like arrays and patches, respectively, the former were positionedregularly on ciliary membrane bulges (containing an electron-opaque granule) and consistedof 8—9 nm particles; the latter had an elliptic shape and contained up to 50 closely packed9-10 nm particles.

INTRODUCTION

Cilia of ciliated protozoa have a primary role in cell locomotion and feeding duringvegetative life. Another function of cilia occurs during sexual interaction when theybecome involved in processes of cellular recognition and adhesion between cells ofdifferent mating types (Sonneborn, 1939). In Paramecium, cilia detached from cellsof one mating type are able to adhere specifically to cilia detached from cells of adifferent compatible mating type (Takahashi, Takeuchi & Hiwatashi, 1974).

In this study we sought to find out, by the freeze-fracture technique, what typesof structural specialization occur in the membranes of cilia functionally transformedas belonging to sexually interacting cells of Euplotes crassus. Therefore we examinedcilia of cells isolated from mixtures during the mating (ciliary) reaction. This is thestage of sexual interaction of Euplotes that can only be performed by cells thatcompleted an induction period and it precedes the fusion of cell bodies into conjugalpairs (Heckmann & Siegel, 1964). Our attention was mainly focused on the develop-ment of membrane specialization in the cilia that are organized to form the mem-branelles that surround the anterior left margin of the cell's ventral surface. Thesecompound ciliary organelles and, to a lesser extent, the cirri are involved in theestablishment of intercellular contacts and union during the mating reaction (Luporini

2 P. Luporini and R. Dallai

& Dallai, 1980). The ciliary membrane specializations were revealed by the presenceof organized particle arrays. According to Branton (1966), the particles representintramembrane, protein-containing elements of the fluid lipid bilayer.

MATERIALS AND METHODS

The 2 mating-type complementary strains LLtD and LLtR of E. crassus were used. Thecharacteristics of these strains have been described previously (Luporini & Dallai, 1980). Theywere cultured at 22-23 CC in Erd-Schreiber sea-water medium inoculated withDunaliella salina, on a cycle of 12 h of light and 12 h of darkness. Observations wereperformed on 2- to 3-day starved cells, which should have accumulated homogeneously inthe Gx stage of the cell cycle (Dini & Luporini, 1979).

Freeze-fracturing

Cells were fixed for 20-30 min in c i M-sodium cacodylate-buffered 2-5 % glutaraldehydeand infiltrated at 30 min intervals with a graded series of glycerol solutions (10, 20 and 30 %),at 4 °C. Fixed cells were then pelleted, placed on gold support plates, rapidly frozen inFreon 22 cooled with liquid nitrogen, and fractured at — 115 °C in a Balzer BAF 301 freeze-etching device. Replicas were then cleaned with Chlorox, recovered on grids, and examinedwith a Philips EM301 electron microscope.

Thin sectionsCells were fixed in o-i M-sodium cacodylate-buffered 2-5% glutaraldehyde for 20 min at

4 °C and postfixed in o-1 % osmium tetroxide. Thin sections were cut with an LKB ultratome,stained with uranyl acetate and lead citrate and examined under a Philips EM301 operatedat 60 kV.

Specimens observed with the scanning electron microscope were prepared as reported pre-viously (Luporini & Dallai, 1980).

All the figures shown in the text refer to mating cells with the exception of Fig. 14, whichrefers to a freeze-fracture replica of a vegetative cell.

RESULTS

The mating reaction of E. crassus is not a highly synchronous event. Thus mixedcells are not able to start the mating reaction simultaneously, even if the extremelymating-reactive cells, such as those of the LLXD and LLXR strains that we usedin this study, are mixed. Therefore, we isolated mating cells from the mixtures bysorting them by hand with a micropipette. Isolated and fixed cells were then con-centrated by sedimentation into blind capillaries. By this method we also isolatedand concentrated vegetative cells that we examined as controls.

Particles were unevenly distributed between the 2 fracture faces of the ciliarymembranes. Most of the particles remained constantly attached to the cytoplasmicleaflet (or P-face) as was found in the cilia of Tetrahymena (Sattler & Staehelin, 1974)and flagella of Chlamydomonas (Snell, 1976).

In addition to particles randomly dispersed in the smooth areas of the membranelipid phase, we observed particles aggregated to constitute at least 5 distinct types ofspecialization of the ciliary membranes: (i) necklace, (ii) parallel transverse rows,(iii) longitudinal row, (iv) rosette-like arrays and (v) patches. The first 3 appearedas stable structures in the ciliary membrane and had an organization fundamentally

Cilia of Euplotes 3

identical to that of both vegetative and mating cells. The last 2 specializations appearedas more dynamic membranal structures able to take on more than a single aspect;their most patterned organization having been detected in mating cells only.

(i) Necklace. This appears typically just above the site where cilia emerge fromthe cell body and is composed of 2 strands of closely associated 12-13 nm particles(Fig. 1). Strands appear scalloped such as those seen in the gill cilia of Elliptio(Gilula & Satir, 1972), rather than being straight or slightly wavy as seen in Para-mecium (Plattner, Miller & Bachmann, 1973) and Tetrahymena (Sattler & Staehelin,1974; Satir, Sale & Satir, 1976).

Fig. 1. The 2 strands of particles forming the necklace are indicated by lines; E and Pindicate the external and the protoplasmic fracture faces, respectively; the asteriskmarks the ridge that occurs to separate the cilia of a membranelle from those of anadjacent membranelle. x 82000. In all freeze-fracture figures an encircled arrow-head indicates direction of shadow.

(ii) Parallel transverse rows. Three to 7 largely incomplete rows are usuallyorganized around the cilium, just above the necklace, at an angle of 70-750 relativeto the ciliary axis (Fig. 3). The rows are spaced about 30 nm from one another and,in the ciliary portion exposed to the observer, each of them may contain up to10 particles. These have diameters ranging from 10 to 15 nm and are arranged at awider and less regular distance (which varies from 8 to 20 nm) with respect to thenecklace particles.

(iii) Longitudinal row. No more than 1 longitudinal row was detected for each

4 P. Luporini and R. Dallai

cilium (Figs. 4, 5). It appears perfectly aligned with the ciliary axis and consistsof closely positioned 12 nm particles. Although the longitudinal row was locatedmore frequently in intermediate regions of the ciliary shaft, its distance from thenecklace varied strikingly, fromo-2 to 2-2 /im. In one instance (not illustrated) individualrows appeared on adjacent ciliary membranes (of the same cirrus); each row beganat an increasing distance from the necklace, in a staircase-shaped aspect. The length

Fig. 2. Longitudinal thin section of membranelle cilia. Arrows indicate electron-opaque granules in the space between the ciliary membrane and the axonemaldoublets. Note the core granules inside the kinetosomes (see Ruffolo, 1976). x 55000.Fig. 3. Parallel transverse rows of particles appearing on the P-face of fracturedcilia are indicated by lines. Rosette-like arrays, consisting of a few particles, areindicated by arrows; each array appears on a small bulge of the ciliary membrane,x 82000.

of the row was also found to be different; e.g. one as long as 1-4 fim is visible inFig. 4. Our impression was that longer longitudinal rows are more common in thecilia of mating cells.

Longitudinal rows were also observed in replicas of the cell's dorsal, immotilebristle cilia like those shown in Fig. 6. These rows (which usually appeared notsingly but arranged in couples as shown in Fig. 7) run parallel to the bristle axisand consist of 12 nm particles as is found in the longitudinal rows of the membranellecilia.

Cilia of Euplotes

Fig. 4. Longitudinal rows of particles 0-33 /im distant from the necklace are indi-cated by arrows on the P-face of 2 fractured cilia, x 43 500.Fig. 5. Longitudinal rows of particles (arrows) are shown at higher magnification onintermediate ciliary regions, x 74000.Fig. 6. Scanning electron micrograph of a portion of the cell's dorsal surface. Threebristle cilia emerging from pits in the cellular surface are evident, x 10 000.Fig. 7. The white arrowhead indicates a pair of longitudinal rows of particles presenton the P-face of a dorsal bristle cilium. An asterisk marks an ampoule (see Ruffolo,1976) facing the ciliary pocket, x 74000.

P. Luporini and R. Dallai

Fig. 8. Poorly organized rosette-like arrays are visible. They appear to be arrangedin lines parallel to the ciliary axis, x iooooo.Fig. 9. A rosette-like array is shown that consists of a few particles clustered arounda central one. x 125000.Fig. 10. Lateral view of 3 bulges of the ciliary membrane each of which shows acrater-like depression; an arrow indicates particles on the slopes of a bulge, x 92000.Fig. 11. Frontal view of 2 bulges with crater-like depressions; a few particles (arrow)decorate the ridge of a depression, x 99000.

Cilia of Euplotes 7

(iv) Rosette-like arrays. These are regularly positioned, each on a bulge of theciliary membrane, and appear more or less uniformly disposed to form more than1 line running parallel to the ciliary axis. Their distribution appears limited to theproximal half of the ciliary membrane starting about 0-45 /u.m from the necklace(Fig. 3). Thin sections of this same ciliary region (see Fig. 2) revealed roundishelectron-opaque granules, about 20 nm in diameter, arranged with a centre-to-centrespace ranging around 80 nm. These granules occupy the space between the micro-tubule doublets and the ciliary membrane, and apparently cause the surroundingmembrane to bulge out. In their most patterned organization, the rosette-like arrayseach consist of 5 or 6 8-nm particles (the smallest ones we ever observed in thereplicas of cilia of Euplotes) clustered around a central particle (Fig. 9). However,they also appeared to be composed either of 2-3 particles only (Fig. 3), or of a greaternumber of poorly organized particles (Fig. 8), the latter being an aspect very similarto that of the scattered rosette-like arrays we observed in the vegetative cells. Excep-tionally, and only in the ciliary membranes of mating cells, we observed what webelieve to represent a further aspect of the rosette-like arrays, that is, particlesarranged in a rather disorderly fashion along both the sides of the bulges and theridge of a 70 nm crater-like depression formed in the top of the bulge (Figs. 10, 11).Usually, only one type of rosette-like array was ever found on the same ciliarymembrane.

(v) Patches. These usually show an elliptic shape and average dimensions of0-15 fim x 01 fan. Well-defined patches, like those detected in replicas of membranellecilia interconnecting 2 mating cells (Fig. 12), each consist of as many as 50 closelypacked 9-10 nm particles (Fig. 13). They appear particularly concentrated all overthe distal portions of the ciliary shaft, where 2 or even more patches were also seenmerging with each other to form larger congregations. Conversely, patches thatoccur over intermediate ciliary regions as a rule appear distinctly separated fromone another.

In the absence of patches we observed, especially in replicas of intermediate anddistal ciliary portions of vegetative cells, congregations of particles that appearedwithout order, except 1 type consisting of a doublet of unequal rows. These rowshave an average length of 0-14 /im and are arranged regularly at a slight angle withthe ciliary axis (Fig. 14). The 9-10 nm diameter of the component particles (identicalto that of the patch particles), as well as their absence from the basal ciliary portions,suggests that the doublets of rows might represent initial stages of patch formation.

DISCUSSION

Congregations of intramembrane particles that indicate functionally specialized areasof the ciliary membranes (portions of the plasma membrane surrounding the cellbody) appeared more numerous and, in general, more organized in the cilia of matingcells than in those of vegetative cells of E. crassus.

The necklace is a specialization commonly found at the base of cilia of differentorganisms. It has been shown to consist of particles connected to the peripheral

P. Luporini and R. Dallai

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14

Cilia of Euplotes 9

doublets of the axoneme and may be involved in the control of local membranepermeability (Gilula & Satir, 1972).

The localization of the parallel transverse rows just above the necklace suggeststhat they correspond to the 'ciliary granule plaques' described in Paramecium(Plattner et al. 1973; Plattner, 1975; Beisson et al. 1976) and in Tetrahymena (Wunder-lich & Speth, 1972; Sattler & Staehelin, 1974; Satir et al. 1976). Plattner (1975)suggests that these plaques could be involved in the ciliary activity of Paramaceium(including mating agglutination), acting as binding sites for calcium ions. However,the parallel transverse rows of Euplotes consist of more heterogeneous particles witha larger average size and, more importantly, form a ciliary specialization with acompletely different geometrical organization. This lacks, above all, any kind ofresemblance to the 9 rectangular arrays that reflect the 9-fold symmetry of theaxonemal structure. Rather, the resemblance appears closer to an array detected onthe E-face of the tip region of some of the oral cilia of Tetrahymena, the particles ofwhich could serve as points of attachment for the globular units of ' bristles' arisingfrom the ciliary membrane (Sattler & Staehelin, 1974). But we were unable to observesuch ciliary structures associated with the cilia of Euplotes.

Particles aligned in rows parallel to the main ciliary axis are frequently foundin replicas of ciliary (Gilula & Satir, 1972; Sattler & Staehelin, 1974) and flagellarmembranes (Bergstrom & Henley, 1973; Friend & Fawcett, 1974; Bergman et al.1975; Snell, 1976; De Souza, Martinez-Palomo & Gonzales-Robles, 1978). Theseparticles have been found to provide attachment sites for crosslinking elements frommicrotubular doublets in Tetrahymena (Sattler & Staehelin, 1974). Connexionsbetween peripheral doublets and the ciliary membrane were also recognized in sometransversely cleaved and etched cilia of Euplotes (our unpublished micrographs).The ciliary beat may be modified as a consequence of the restriction in sliding of thelinked doublets (Sattler & Staehelin, 1974). If this were particularly true for thecilia of interacting Euplotes, one can imagine that a reduced ciliary beat facilitateseither more stable contacts between specialized regions of the ciliary membranesof complementary cells or, like the immotile cilia of mechanoreceptors, the trans-mission of perturbations generated by contact with the cell body. The fact thatparticles arranged in longitudinal rows were also detected in replicas of the cell'sdorsal bristle cilia needs to be considered in the light of the supposed relation betweenreduced ciliary mobility and promotion of signalling transduction. In fact, theseorganelles of Euplotes, as well as of other hypotrichous ciliates, are typically non-motile and usually considered to be sensory in function (Corliss, 1979).

The rosette-like arrays bear a resemblance to the rosettes of particles detected

Fig. 12. Membranelle cilia interconnecting 2 mating cells are visible, x 13000.Fig. 13. Detail of Fig. 12 showing a cilium heavily decorated with patches ofparticles, x 74000.Fig. 14. A doublet of unequal rows of particles arranged obliquely to the ciliaryaxis is indicated by an arrow, x 47500.

io P. Luporini and R. Dallai

(Satir, Schooley & Satir, 1973; Planner, 1974; Beisson etal. 1976; Allen & Hausmann,1976; Dallai & Luporini, 1980) in the ciliate plasma membrane at the sites whereextrusomes discharge, as well as in neurosecretory cells in which the rosettes mostlikely represent sites of endocytic activity (Theodosis, Dreifuss & Orci, 1978). Yet,in analogy, the rosette-like arrays could mark sites of exo-endocytosis in the ciliarymembranes of mating Euplotes. Two observations support this view: (1) a denselypacked electron-opaque granule is present (in thinly-sectioned cilia) inside themembrane pouches, which are identifiable (because of the close correspondence inboth disposition and size) with the bulges exposing the rosette-like arrays on theirtops; and (2) a depression may appear inside the protrusion. The formation of sucha depression might represent a transient stage of tight connexion between the ciliarymembrane and the underlying granule. However, the fractured necks could beregions where the fracture plane skips out of the membrane, since the 2 membraneleaflets are tightly bound at these foci and since the membrane bulges out at thesesites.

Light could be thrown on the function of the rosette-like array by the knowledgeof the composition of the electron-opaque granules of the membrane pouches. Theyseem comparable, at the first glance, to the granules enriched in calcium detectedin the ciliary bases of Paramecium fixed in the presence of CaCl2 (Plattner, 1975;Plattner & Fuchs, 1975; Fischer, Kaneshiro & Peters, 1976; Tsuchiya, 1976; Tsuchiya& Takahashi, 1976).

The function and origin of the patches is difficult to understand since nothingsimilar to this has, to our knowledge, been observed in the ciliary membranes ofother ciliates. The fact that they did not appear (or appeared much less organized)in cilia of vegetative cells suggests that they are dynamic structures possibly organizedduring the induction period of the interaction. It might be that the complete organi-zation of the patches is the result of a morphogenetic event that causes analogousparticles (either already present although dispersed in the lipid phase of the mem-branes of vegetative cells, or newly synthesized by the cells after mixing) to movewithin the lipid bilayer and to produce the orderly aggregation required for function.

Particles organized in very extensive ' plates' have been described in Paramecium(Allen, 1978), in the plasma membrane of the anterior ventral surface, which is theregion of the cell directly involved in the formation of the conjugal union. Allen(1978) proposed, among other explanations, a possible involvement of the plates inthe conjugal interaction. Similarly, one might hypothesize that the patches of thecilia connecting interacting Euplotes have a role in intercellular contact and/orcommunication since they might represent elements of some type of junction. Severalconsiderations seem to support this intriguing, yet speculative, hypothesis:(1) Euplotes interacting with each other during the mating reaction do not appearsubstantially different from differentiating metazoan cells in reciprocal contact.Specifically, they exchange polypeptides (Luporini, Bracchi & Esposito, 1979;Luporini & Gabrielli, unpublished data) and are able to synchronize and coordinatetheir nuclear maturation (Dini & Luporini, 1979; Luporini & Giachetti, 1980).Likewise, embryonic heart myocytes having specialized junctions can adjust their

Cilia of Euplotes 11

rhythms of contraction within a few minutes of mutual contact (De Haan & Hirakow,1972). (2) Protozoa have been found that are able to develop extensive membrane-to-membrane junctions between structures of the same cytoplasm (Planner et al. 1973;Smith, Njogu, Cayer & Jarlfors, 1974; Plattner, Wolfram, Bachmann & Watcher,1975; Hogan & Patton, 1976; Martinez-Palomo, De Souza & Gonzales-Robles, 1976;De Souza et al. 1978; Sattler & Staehelin, 1979). These 'pericellular' junctions appearcomparable to the 'reflexive' junctions developed by metazoan cells and detected,for instance, in the ovarian decidual cells by Herr (1976). (3) The simplest forms ofmulticellular organization show junctions that appear structurally well defined(Filshie & Flower, 1977). Such junctions might have evolved from more primitivetypes developed by unicellular organisms that undergo interaction by intimate contactduring some stages of their life-cycle.

Certainly, in order to prove that the patches in cilia of Euplotes actually representintramembrane structures possibly capable of uniting with respective counterpartsacross the intercellular space, one would have to demonstrate 2 patches coincidentin both the P- and E-fracture faces. One would also have to find close approximationof membranes at the sites where patches appear. Such studies could perhaps befacilitated by freeze-fracturing only agglutinated cilia detached from cells engagedin the mating reaction.

We would like to thank Mr P. Salvatici and Mr P. Bracchi for skilful technical assistance.Financial support was provided by Italian C.N.R.; grants to P. Luporini and to the Instituteof Zoology of Siena for the project ' Biology of reproduction'. We also thank the Referee forvaluable suggestions.

REFERENCESALLEN, R. D. (1978). Particle arrays in the surface membrane of Paramecium: junctional and

possible sensory sites. J. Ultrastruct. Res. 63, 64-78.ALLEN, R. D. & HAUSMANN, K. (1976). Membrane behavior of exocytic vesicles. I. The ultra-

structure of Paramecium trichocysts in freeze-fracture preparations. J. Ultrastruct. Res. 54,224-234.

BEISSON, J., LEFORT-TRAN, M., POUPHILE, M., ROSSIGNOL, M. & SATIR, B. (1976). Geneticanalysis of membrane differentiation in Paramecium. Freeze-fracture study of trichocystcycle in wild-type and mutant strains. J. Cell Biol. 69, 126-143.

BERGMAN, K., GOODENOUGH, U. W., GOODENOUGH, D. A., JAWITZ, J. & MARTIN, H. (1975).Gametic differentiation in Chlamydomonas reinhardi. II. Flagellar membranes and theagglutination reaction. J. Cell Biol. 67, 606-622.

BERGSTROM, B. H. & HENLEY, C. (1973). Flagellar necklaces: freeze-etch observations.J. Ultrastruct. Res. 42, 551-553-

BRANTON, D. (1966). Fracture faces of frozen membranes. Proc. natn. Acad. Sri. U.S.A. 55,1048-1056.

CORLISS, J. (1979). The riliated Protozoa. Oxford: Pergamon.DALLAI, R. & LUPORINI, P. (1980). Membrane specializations in the ciliate Euplotes crassus

at the sites of interaction of the ampules with the plasma membrane. Eur. J. Cell Biol. 23,286-294.

DE HAAN, R. L. & HIRAKOW, R. (1972). Synchronization of pulsation rates in isolated cardiacmyocytes. Expl Cell Res. 70, 214-220.

DE SOUZA, W., MARTINEZ-PALOMO, A. & GONZALES-ROBLES, A. (1978). The cell surface ofTrypanosoma aruzi: cytochemistry and freeze-fracture. J. Cell Sri. 33, 285-299.

12 P. Luporini and R. Dallai

DINI, F. & LUPORINI, P. (1979). Preconjugant cell interaction and cell cycle in the ciliateEuplotes crassus: switching from the vegetative to the sexual stage. Devi Biol. 69, 506-516.

FILSHIE, B. K. & FLOWER, N. E. (1977). Junctional structures in Hydra. J. Cell Sci. 23,151-172-

FISHER, G., KANESHIRO, E. S. & PETERS, P. D. (1976). Divalent cation affinity sites in Para-mecium aurelia. J. Cell Biol. 69, 429-442.

FRIEND, D. S. & FAWCETT, D. W. (1974). Membrane differentiations in freeze-fracturedmammalian sperm. J. Cell Biol. 63, 641—664.

GILULA, N. B. & SATIR, P. (1972). The ciliary necklace. A ciliary membrane specialization.J. Cell Biol. S3, 494-509.

HECKMANN, K. & SIEGEL, R. W. (1964). Evidence for the induction of mating-type substancesby cell to cell contacts. Expl Cell Res. 36, 688-691.

HERR, J. C. (1976). Reflexive gap junctions. J. Cell Biol. 69, 495-501.HOGAN, J. C. & PATTON, C. L. (1976). Variation in intramembrane components of Trypanosoma

brucei from intact and X-irradiated rats: a freeze-fracture study. J. Protozool. 23, 205-215-

LUPORINI, P., BRACCHI, P. & ESPOSITO, F. (1979). Specific contact-dependent cell-to-cellcommunication during preconjugant interaction of the ciliate Euplotes crassus. J. Cell Sci.39, 201-213.

LUPORINI, P. & DALLAI, R. (1980). Sexual interaction of Euplotes crassus: differentiation ofcellular surfaces in cell-to-cell union. Devi Biol. 77, 167-177.

LUPORINI, P. & GIACHETTI, C. (1980). Multiconjugant complexes of Euplotes crassus: aninstance of coordination of nuclear events. J. Protozool. 27, 108-112.

MARTINEZ-PALOMO, A., DE SOUZA, W. & GONZALES-ROBLES, A. (1976). Topographicaldifferences in the distribution of surface coat components and intramembrane particles.J. Cell Biol. 69, 507-513.

PLATTNER, H. (1974). Intramembranous changes on cationophore-triggered exocytosis inParamecium. Nature, Lond. 252, 722-724.

PLATTNER, H. (1975). Ciliary granules plaques: membrane-intercalated particle aggregatesassociated with Cat+-binding sites in Paramecium. J. Cell Sci. 18, 257-269.

PLATTNER, H. & FUCHS, S. (1975). X-ray microanalysis of calcium binding sites in Paramecium.Histochemistry 45, 23-47.

PLATTNER, H., MILLER, F. & BACHMANN, L. (1973). Membrane specializations in the formof regular membrane-to-membrane attachment sites in Paramecium. A correlated freeze-etching and ultrathin-sectioning analysis. J. Cell Sci. 13, 687-719.

PLATTNER, H., WOLFRAM, D., BACHMANN, L. & WATCHER, E. (1975). Tracer and freeze-etchinganalysis of infra-cellular membrane-junctions in Paramecium. Histochemistry 45, 1-21.

RUFFOLO, J. J. JR (1976). Fine structure of the dorsal bristle complex and pellicle of Euplotes.J. Morph. 148, 469-488.

SATIR, B., SALE, W. S. & SATIR, P. (1976). Membrane renewal after dibucaine deciliation ofTetrahymena. Expl Cell Res. 97, 83-91.

SATIR, B., SCHOOLEY, C. & SATIR, P. (1973). Membrane fusion in a model system. Mucocystsecretion in Tetrahymena. J. Cell Biol. 56, 153-176.

SATTLER, C. A. & STAEHELIN, L. A. (1974). Ciliary membrane differentiations in Tetrahymenapyriformis. Tetrahymena has four types of cilia. J. Cell Biol. 62, 473-490.

SATTLER, C. A. & STAEHELIN, L. A. (1979). Oral cavity of Tetrahymena pyriformis. A freeze-fracture and high voltage electron microscopy study of the oral ribs, cytostome, and formingfood vacuole. J. Ultrastruct. Res. 66, 132-150.

SMITH, D. S., NJOCU, A. R., CAYER, M. & JARLFORS, U. (1974). Observations of freeze-fractured membranes of a trypanosome. Tiss. Cell 6, 223-241.

SNELL, W. J. (1976). Mating in Chlamydomcnas: a system for the study of specific cell adhesion.I. Ultrastructural and electrophoretic analysis of flagellar surface components involvedin adhesion. J. Cell Biol. 68, 48-69.

SONNEBORN, T. M. (1939). Paramecium aurelia: mating types and groups; lethal interactions;determination and inheritance. Am. Nat. 73, 390-413.

TAKAHASHI, M., TAKEUCHI, N. & HIWATASHI, K. (1974). Mating agglutination of cilia detachedfrom complementary mating types of Paramecium. Expl Cell Res. 87, 415-417.

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THEODOSIS, D. T., DREIFUSS, J. J. & ORCI, L. (1978). A freeze-fracture study of membraneevents during neurohypophysial secretion. J. Cell Biol. 78, 542-553.

TSUCHIYA, T. (1976). Electron microscopy and electron probe analysis of the Ca-bindingsites in the cilia of Paramecium caudalum. Experieniia 32, 1176-1177.

TSUCHIYA, T. & TAKAHASHI, K. (1976). Localization of possible calcium-binding sites in thecilia of Paramecium caudatum. J. Protozool. 23, 523-526.

WUNDERLICH, F. & SPETH, V. (1972). Membranes in Tetrahymena. I. Cortical pattern. J.Ultrastnict. Res. 41, 258-269.

{Received 7 January 1981)