Subpellicular Microtubtjles of Euplotes Eurystomus: Their Geometry Relative to Cell Form, Surface Contours and Ciliary Organelles

J. Cell Set. 56, 471-484 (198*) 471 Printed in Great Britain

SUBPELLICULAR MICROTUBTJLES OF EUPLOTES EURYSTOMUS: THEIR GEOMETRY RELATIVE TO CELL FORM, SURFACE CONTOURS AND CILIARY ORGANELLES

J. NORMAN GRIM Box 5640, Northern Arizona University, Flagstaff, Arizona 86011, U.S.A.

SUMMARY There is a layer of microtubulea (MT) beneath the innermost pellicular membrane of the ciliated protozoan Euplotes eurystomus. These MT have been revealed for scanning electron microscopic study by chemical dissection techniques. In much of the body of this ciliate, these MT are oriented parallel to its long axis. Those directed towards cirri, which are complex ciliary structures of the ventral surface, either abut or bend around the cirral base. MT adjacent to or closely associated with the ciliary feeding structures (membranelles of the adoral zone of membranelles or AZM) are oriented parallel to the long axis of the AZM. Some of the MT within the oral cavity have quite complex paths. The various orientations of these subpellicular MT are discussed and evaluated for hypothetical functions of cyto- skelctal support, cell shaping and organelle movement. Each of these roles is considered to be theoretically possible.

INTRODUCTION Sheets of microtubules (MT) occur beneath the pellicle of Euplotes, as was noted over 20 years ago (Roth, 1957). Subsequently, one of these sheets was described as consisting of parallel groups of three MT each (for figs, by Wise, see Pitelka, 1969). It is now known that Euplotes eurystomus has two sheets of MT below the pellicle; MT of the sheet immediately beneath the innermost pellicular membrane (inner alveolar membrane) are in groups of three and are oriented parallel to the long axis of the organism, while the second sheet is slightly deeper and has single parallel MT that are perpendicular to the long axis (Grim, 1967). The dorsal and ventral surfaces of this flattened ciliate have different MT patterns, as will be described. Microtubules of the sheet just beneath the pellicle, hence those most distal within the cytoplasm, are not parallel to the long axis of Euplotes in all regions of the body (Grim, Halcrow & Harshbarger, 1980). This report presents the geometry of this distal sheet of MT in regions of complex surface geometry, around several different ciliary structures (cirri, membranelles and dorsal bristles) and the contractile vacuole pore. The geometry of these MT suggests possible functions of cytoskeletal support, cell shaping, ciliary organelle displacement, or combinations of these. The possible relevance for these functions will be discussed. 472 J. N. Grim

MATERIALS AND METHODS This strain of Euplotes eurystomus Wrzesniowski was isolated in northern Arizona and identified after Chatton-Lwoff silver staining and comparison with the recent descriptions (and extensive arguments) of Curds (1975). It was maintained in culture as noted previously (Grim et al. 1980). The distal MT were revealed for scanning electron microscopic (SEM) study after chemical dissection techniques (Gibbons, 1965; Grim et al. 1980). One millilitre of a culture of living Euplotes was placed in 4 ml of 10 % (v/v) ethanol and o-i % (w/v) EDTA (ethylene-diaminetetraacetic acid) for 1-2 min, then 1 drop of 1 M-CaClj was added and the ciliates were immediately drawn into and out of a Pasteur pipette several times. They were next fixed in Parducz' (1967) solution for 3-5 min, freeze-dried (Small & Marzalek, 1969) and gold/palladium coated. Specimens were examined in an AMR1000 SEM.

RESULTS General body form and major structures E. eurystomus is a dorso-ventrally flattened ciliated protozoan with most of its locomotor and feeding structures on the ventral surface. The ciliary feeding structures, collectively called the adoral zone of membranelles (AZM), contain many membranelles, each consisting of two to four rows of fused cilia. The AZM starts on the ventral surface just above cirrus no. 2-3 (Fig. i), traverses the anterior dorsal surface, curves over to the ventral surface and extends posteriorly about two-thirds of the body length. The cavity to the right (as viewed from inside the organism) of the ventral AZM is the 'buccal cavity*. It is relatively shallow in its anterior aspect (Fig. 1, bcB) and appreciably deeper posteriorly and to the right of that (Fig. 1, bca, see also Fig. 9). Extending from the anterior end of the deeper groove on the right is a very shallow groove (Figs. 9-10), which will be described later. Many of the cilia at the ventral surface are in clusters, with the cilia adherent. These are called cirri, and consist of up to 160 cilia each. All are given special numbers that relate to the nature of their formation prior to cytokinesis (Wallengren, 1900; and see Fig. 1). The nine most anterior are called fronto-ventral; the group of five (four of which form a diagonal row) posterior to these nine are the transverse cirri; and the most posterior four, at the rear boundary of the cell, are the caudal cirri. The contractile vacuole pore is to the right of the transverse cirri (Figs. 1, 2).

Microtubular patterns Dorsal surface. Over most of this surface the distal MT are parallel to the long axis of E. eurystomus (Figs. 4-5). They continue in this orientation to the posterior boundary of the cell (Fig. 6). At the anterior end, however, the MT bend towards the right (Fig. 7). This cannot be accomplished unless many MT either end some- where within the path of this curvature or plunge deeper into the cytoplasm. This, apparently random, MT termination (or disappearance from the surface) has been observed with the SEM (Fig. 7, inset). Cilia on the dorsal surface are in rows, are quite short, and do not beat. Each extends from a cavity or pit and is surrounded by a collar. As the MT 'approach' a collar, those near the collar edge appear to plunge slightly beneath it and emerge on the other side (Fig. 5), while others Subpellicular microtubules of Euplotes 473

Fig. i. Diagrammatic sketch of E. eurystomus, ventral view. The buccal cavity is large shaded area (be,, superficial region; bcit deep region). All numbered features are cirri showing the approximate shape of their bases. The contractile vacuole is indicated by an arrowhead, r, ridges between transverse cirri; azm, adoral zone of membranelles. Fig. 2. Sketch of E. eurystomus showing the MT geometry at the ventral surface. Not all MT are shown, only enough to show those (key) features described and discussed.

Fig. 3. Sketch of anterior right (seen from inside the organism, looking out) portion of the buccal cavity (4c). At the left some MT abut (arrowheads) thoie curving within the be.

16 CEL 56 474 jf. N. Grim Subpellicular microtubules of Euplotes 475 possibly abut the collar, or plunge into the cytoplasm. Finally, dorsal MT are also parallel to the long axis of the body at the sides of this ciliate and continue around this boundary to the ventral surface (Fig. 7). The dorsal and ventral surfaces in these preparations also contain a network of territorial fibrils, which are about 70 nm in diameter. These have been observed only recently with the transmission electron microscope (TEM) (Foissner, 1978) and the SEM (Grim et al. 1980). These fibrils are within the cytoplasm. Even so, they are very distinct after chemical dissection. Without chemical dissection, the pellicle forms a small ridge over the fibrils (Foissner, 1978); thus, their presence is often evident without chemical dissection, as noted previously (Grim et al. 1980). Many MT are in register on either side of these fibrils (Figs. 4, 5, 8, 16, 17) and thus probably pass uninterrupted beneath them.

Ventral surface General pattern and oral structures. The geometry of this surface is quite complex and varied, due especially, but not entirely, to the feeding apparatus (Fig. 9). For clarity it will be presented in several sections. Near the posterior end of E. eurystomus the MT curve around parallel to the posterior boundary (Fig. 2). From a few micrometres anterior to the caudal cirri up to the transverse cirri the MT are very rarely seen; hence, their geometry here is not known. Over most of the remainder of the ventral surface MT are parallel (or nearly so) to the long axis of the body. Very near the anterior end of this ciliate, MT bend rather acutely, but in different directions depending upon the location. On the right side (viewed from the inside) they bend to the left or towards the AZM (Fig. 2). In the anterior region of the buccal cavity (Figs. 1, 2, 3, 8) MT curve to the right and many MT in this region can be observed ending or disappearing from the surface, as noted previously for the dorsal anterior region (see Fig. 8,

Figs. 4-16. SEM micrographs of chemically dissected E. eurystomus. Various magnifications. Fig. 4. Dorsal surface showing pit from which single cilium normally extends (c), 70 nm territorial fibrils {tr) and many parallel MT. x 10 000. (Fig. by permission from J. Protozool., see Grim et al. 1980.) Fig. 5. Dorsal surface showing MT triad running beneath (arrows) a dorsal cilium collar (c). MT are in register on either side of territorial fibrils (arrowheads), x 24000. Fig. 6. Dorsal surface-posterior boundary (pb). MT here are still parallel to the long axis of the body, x 20000. Fig. 7. Right anterior edge («) between dorsal (d) and ventral (i>) surfaces. MT curving prominently to the right at d. Arrowhead shows termination of MT. x 10000. Inset: MT termination, x 30000. Fig. 8. MT curving to the right (from inside) in anterior be,. Termination of MT is apparent (arrowhead), x 19500. Inset: MT termination, x 39000. Fig. 9. Low magnification of the anterior third of E. eurystomus showing much of the AZM and buccal cavity. The arrow indicates the most anterior extremity of the AZM. x 1000. 16-3 476 J. N. Grim Subpellicular microtubules of Euplotes 477 arrow). The cessation of MT is not along a line and appears to be random in this region. At the right-anterior aspect of the buccal cavity there is the beginning of a very shallow groove that arches to the right as it passes anteriorly (Fig. 10). Posterior to this groove the cavity is, abruptly, much deeper (Figs. 9, 10). MT appear to originate in the shallow groove (Fig. 10), extend anteriorly, then curve to the right, along with all other MT in this locality. MT near the anterior aspect of cirri nos. 1-1 and 2-3 extend toward these curving MT of the buccal cavity and groove (Fig. 2). Just to the right of the groove (Fig. 10) the curving MT appear to be abutted by the former MT (Figs. 3, 10). There are MT also within the deepest portion of the buccal cavity (bca) and those seen thus far are parallel to the long axis of the body (Fig. 2). MT outside and close to the AZM on the left side are parallel to the AZM, and thus take on a shallow S-shaped curve (compare Figs. 1, 2). The posterior extremity of the buccal cavity is V-shaped and MT bend away from this feature (Figs. 2, 11). As these MT continue towards the posterior of the cell they bend gradually to the right. There is a collar along each side of the AZM, formed by an evagination of the pellicle (Fig. 12). MT occur on the inside of each collar, and are oriented parallel to the long axis of the collar (thus also to the AZM) (Fig. 12). The 70 nm territorial fibrils are found inside the two collars and one branch extends towards the end, parallel to the long axis of each membranelle (Fig. 12, arrowhead). As noted previously, the AZM curves around on the dorsal surface and begins on the ventral surface, very near cirrus no. 2-3 (Figs. 1, 9). MT inside the outer collar, which is contiguous with the dorsal surface, curve counter-clockwise around the posterior boundary of the first membranelle, at the starting end of the AZM. The general features, but not the MT, can be seen in Fig. 9. Ruffolo (1980) has recently described, in the anterior superficial buccal cavity, a 'buccal flap' that appears to lift out and (his speculation) interfere with food organisms attempting to swim out of the buccal cavity. This flap, in his SEM micrographs, appears rather complex in form. The distal MT sheets of the present study do not seem to have any specializations corresponding to the (apparent) surface complexities of the flap.

Fig. 10. Groove (g) and deeper portion of the be. Some MT at the left (arrowhead) abut other curving MT (see also Fig. 3). x 10000. Fig. 11. Posterior (surface) extremity of the buccal activity (be); MT separate and bend around this feature, x 20000. Fig. 12. Portion of AZM at dorsal surface showing collar (c), three membranelles (m), and a territorial fibril directed towards a membranelle (arrowhead). The long arrow indicates the long axis of the AZM. x 10000. Fig. 13. Cirrus no. 4-3, right-anterior in the figure is anterior in the organism and many MT appear to abut here. At the posterior end most MT bend around. Also present are a collar (c) and a connecting territorial fibril (tr). x 15000. Fig. 14. Cirrus no. 6-2, anterior end to the right. Most MT bend around the posterior end. Some granular material is present (g). x 15000. Fig. 15. Region of transverse cirri showing cirrus no. 2-1, ridges (r), and apparent termination of MT (t) approaching from the anterior, x 9600. 478 J. N. Grim Cirri. Many but not all cirri have been observed with a distinct collar around them (Figs. 13, 14, 16). This feature is unlike the AZM collar and appears as a small cylindrical structure larger in diameter than the territorial fibrils; however, some of these latter fibrils connect to the collar (Fig. 13). Outside the collar in somewhat anterior and posterior locations there are small spaces (possibly artifacts), often containing granular material (Fig. 14). In other cirri the collar is much smaller and may be the 70 nm territorial fibril. It would seem, a priori, that MT that

Fig. 16. Caudal cirrus no. C-i; the anterior end is at the upper-right corner of the figure. Prominent here are a collar (c), a network of territorial fibrils (tr) and unique side-abutting MT (arrowhead), x 11000. Fig. 17. Lower portion of Fig. 16, at higher magnification, to show MT better in register beneath a territorial fibril (tr). x 22000.

'approach' a cirrus have several 'options': (1) to abut the collar, (2) to separate and bend around, (3) to plunge into the cytoplasm, or (4) to exist as partial combinations of the above three. The SEM views best support the first two of these possibilities, but do not fully exclude (3). Of the fronto-ventral cirri, there are always some MT that bend around and others that appear to abut. For most of these fronto-ventral cirri, a high percentage of MT seem to abut their anterior side; however, the possibility of their abruptly bending into the deeper cytoplasm cannot be excluded. Most MT appear to bend around the posterior side (Figs. 13, 14). This results in a polarity along the anterior-posterior axis of a cirrus. As can be seen by comparing Figs. 13 and 14, it may be difficult to tell if MT are indeed abutting a cirrus or perhaps plunging slightly deeper into the cytoplasm to pass around the side. Rigorous study of cleaner SEM preparations as well as TEM studies may resolve this quandary. At present, it appears that some cirri have little polarity with respect to MT, due to a smaller percentage abutting the anterior side. All five transverse cirri (nos. 2-1 to 6-1) have MT that approach their anterior side (Fig. 15) but 'stop' somewhat short where they seem to terminate at what Subpellicular microtubules of Euplotes 479 may be a territorial fibril. A similar MT-cirrus relationship has been observed for the posterior side of transverse cirrus no. 3-1. No MT have been observed in the region immediately posterior to the other transverse cirri; this does not imply that MT do not exist there, as the membrane may be relatively resistant to chemical dissection in this region. MT both abut and bend around each of the caudal cirri, although not in the same ratio for each. The left caudal cirri (C-i, C-2) have an unusual polarity in their association with MT. Several MT posterior to these cirri bend sharply, somewhat towards the anterior, and appear to abut them from the side (Fig. 16). MT are probably continuous beneath the territorial fibril (Figs. 16, 17).

Other features Six ridges exist in association with the transverse cirri (Figs. 1, 15). The leftmost ridge extends up to the vicinity of fronto-ventral cirrus no. 2-2 and is about two or more times the length of the other ridges. MT bend up to ' cover' the ridges from the adjacent flat parts of the ventral surface so that ridges, too, contain MT parallel to their long axes (Fig. 15). It appears from some micrographs that additional MT occur or originate in the ridges, as an apparent adjustment to the increased surface area. The contractile vacuole pore seems to be quite labile to the chemical dissection procedures, because this region generally appears fragmented and amorphic. How- ever, in some cells it remains intact. MT approach this pore and coil helically clockwise (looking at the ventral surface) down into the pore. Territorial fibrils also are found here.

Dividing cell A few cells have been seen that were in an intermediate stage of division; in this stage new cirri for both 'daughter' cells have formed in five rows, each with anterior-posterior orientation (Wallengren, 1900; Wise, 1965). These primordial cirri are quite close together and contain many cilia in a normal hexagonal packing. In many organisms, the distal MT layer can be seen adjacent to these cirri. MT that approach the anterior side of the cirrus, which is itself at the anterior of each developing row, bend around the cirrus. The next cirrus to the posterior is, at this stage, very close to the first one and MT have not been observed between these two. The last cirrus of each row (second or third cirrus, depending upon the row; see Fig. 1) has MT that bend around their posterior side. Other details have not been seen, partly because there is an unusual amount of membrane debris over these cirral rows.

DISCUSSION Cytoskeletal function The pellicles of Euplotes species are remarkably rigid compared to most members of the order (Hypotrichida) to which they belong. All euplotids studied thus far 480 J. N. Grim (Faure'-Fremiet & Andre, 1968; Tuffrau, Payne & de Haller, 1968; Ruffolo, 1976^; Foissner, 1978) have subpellicular MT similar to those presented here and originally described earlier (Grim, 1967). It is axiomatic that the shape of an aquatic organism, as well as the distribution of locomotor and feeding structures, is important for efficient locomotion and feeding. The surface geometry of E. eurystomus is very complex, and if the pellicle were appreciably flexible this geometry would not be constant. For Euplotes at least (but not all ciliates) it seems, a priori, that a relatively constant body form is important. I propose that the MT are acting, at least in part, as a cytoskeleton to help maintain this surface form. The surface geometry of Euplotes could be altered by several different stresses to which this protist is exposed. These stresses and possible MT buttressing will be discussed individually. The major ciliary structures involved in feeding are the numerous membranelles (at least 50) that make up the AZM. Larger membranelles such as those on the dorsal surface may contain up to 80 cilia each in the strain used in this study. There are, then, several thousand cilia beating in the oral apparatus alone. A study on another hypotrich ciliate (Machemer, 1966) has shown that the power stroke for these AZM cilia is parallel to the long axis of the AZM. One would expect this to place the pellicle under considerable stress and potential deformation. The major stress should be parallel to the AZM and in a plane parallel to the body surface beneath each membranelle. Buttressing against these stresses would be provided, in theory, by long rigid and anchored structures that are parallel to these stress orientations. The MT around the periphery and within the collar of the AZM are so oriented. In some TEM micrographs, linkers connect these MT to the inner alveolar membrane (C. A. Stroup, unpublished). Thus their orientation fits a reasonable model for these MT being cytoskeletal. The need for support structures around the mouth has been proposed previously (Pitelka, 1969). Also, other fibrous components beneath the pellicular (or outer limiting) membranes may be cytoskeletal, as suggested by several other workers (Goldman, 1971; Williams, Vaudaux & Skriver,

The beat patterns for cirri in Euplotes have not been fully analysed, to my know- ledge, but power and recovery strokes are, generally, anterior-posterior (Epstein & Eckert, 1972); and the power stroke frequently changes by 1800, as an individual often darts back and forth. Again, the MT studied here are oriented 'properly' to provide possible support to the pellicle against deformation. Those that seem to abut a cirrus at the anterior or posterior will be under one period of compression and one period of tension stress during each beat cycle. Also, each beat should place stresses on the cirral base, which would tend to rotate it out of the plane of the body surface; for example, a power stroke towards the posterior would tend to lift the posterior end of a cirral base away from the surface. In this case, adjacent MT will have shear stresses placed upon them and, again, their observed orientation could reasonably provide mechanical support to retard related surface deformations. Those cirri with more MT abutting anteriorly than posteriorly may be provided with maximum support for an 'unusual' beat pattern, but without further information Subpellicular microtubules of Euplotes 481 this relationship is enigmatic. Additionally, there are distinct rootlet MT bundles that may anchor the cirri within the organism. Comparing the MT sheets of this study with the rootlets stained for light microscopy (Hammond & Kofoid, 1937) shows that they are different MT. Their orientations differ greatly. Moving euplotids often make abrupt changes in direction by rotating on their long axes. The cirri at either the anterior or posterior extremity would be most effective in this change of orientation, acting like a dynamic rudder. Interestingly, two of the most posterior cirri (caudals) have a unique pattern of side-abutting MT. Their orientation (almost perpendicular to the long axis) could provide some of the support expected of a cytoskeletal component for laterally directed power-and- recovery strokes. It is of further interest that during cell division these two cirri develop close together on the ventral surface and far from the other two caudal cirri, which form on the dorsal surface (Hammond, 1937; Hufnagel & Torch, 1967). The other MT associated with C-2, C-3 and C-4 (Fig. 2) could provide mechanical support for lateral power strokes as well. Both swimming and bumping into the substrate should cause some compressive forces parallel to the long axis of the body. Perhaps the many anterior-posterior MT help to provide the necessary mechanical stability. The deeper layer of subpellicular MT, which is perpendicular to the MT of this study (Grim, 1967), is not visible after chemical dissection. Their orientation does not 'argue against' the cytoskeletal arguments presented for the distal MT, since they may provide support in other directions. Pitelka (1969) considered the possibility that many MT of protozoa, especially those organized into linked arrays, serve the function of mechanical support. This idea is reasonable for certain structures in at least some flagellates (Bouck & Brown, 1973; Brown & Bouck, 1973), amoeba (Kitching & Craggs, 1965; Tilney & Porter, 1965; Cachon & Cachon, 1971), ciliates (Tucker, 1968; Sattler & Staehelin, 1979; others), and cells of higher organisms (see a review by Dustin, 1978). As Dustin has summarized, there is a growing controversy over MT as cytoskeletal components. In this paper, the possibility of MT providing cytoskeletal support has been recog- nized and assumed for the sake of the preceding discussion. Cell-shaping function. MT functioning to help shape a cell was strongly suggested in an early elegant study on sperm by Mclntosh & Porter (1967). However, nuclear shaping in some sperm occurs without associated MT (Fawcett, Anderson & Phillips, 1971). There are numerous studies that argue for or against shaping as a real function of MT in sperm and other cells (see Dustin, 1978, for a recent review). The shape of a mature (feeding) Euplotes is remarkable in complexity for a single cell. Ions, molecules and complex assemblages of macromolecules may interact to shape the precise, ornate curves in its surface. The molecular events that control shape as well as organelle location are unknown, as summarized in a recent review by Aufderheide, Frankel & Williams (1980), although some provocative models have been proposed (Sonneborn, 1975; Roth & Pihlaja, 1977). A reasonable model would propose interaction(s) between cytoskeleton and outer limiting membranes. In E. eurystomus, the membrane adjacent to the MT layer described here is the inner 482 J. N. Grim alveolar and appears, after freeze-fracture, to contain a very high concentration of intramembrane particles (Grim, unpublished; also see Allen, 1978). If these MT were linked extensively to this membrane, they might somehow cause, or be part of, bend-inducing stresses. Also, the space between the two alveolar membranes contains a fine filamentous mesh work (Ruffolo, 1976 a), which could conceivably hold these membranes together. Recent studies on the 'ground substance' of certain cells (Ellisman & Porter, 1980) suggest that a variety of cytoplasmic components such as MT, microfilaments, large organelles and microtrabeculae may be inter- connected into a complex network. Theoretically, several of these structures could interact to induce genetically controlled bends in the surface. High-resolution and high-voltage TEM, further freeze-fracture studies, or special experimental techniques will be important in testing these or other possible models.

Morphogenesis and ciliary organelle placement It is conceivable that the loci of ciliary organelles and other structures within or very near the surface of a ciliate could be controlled, at least in part, by MT. In general, this might be similar to the movement and positioning of chromosomes accomplished during anaphase by MT of the spindle apparatus. The positioning of cortical or pellicular structures during the morphogenesis of ciliated protozoa has been of interest for many years. This is especially so because this phenomenon does not always seem to be under immediate genetic control (Sonneborn, 1963, 1970; Grimes & L'Hernault, 1979; Aufderheide et al. 1980), but is also under the influence of existing structures, such as ciliary organelles. As for a role for MT in this phenomenon, it is possible that MT could move a cirrus to its 'final' location in Euplotes by one of at least three mechanisms: (1) pull it there by depolymerization, (2) push it there by polymerization, or (3) push or pull it by MT sliding on one another or some other structure. During cell division in euplotids, new cirri for both daughter cells form from apparently disorganized streaks of kinetosomes. During one period of this morphogenesis the kinetosomes aggregate into clusters, each cluster being a cirral primordium. Perhaps one function for the distal MT sheets is to move (or have some role in the movement of) kinetosomes together and/or to separate others during this sequestering event. A similar model could apply to the organization of ' disorganized' kinetosomes within the oral anlagen into membranelles. These techniques for visualizing MT and some of the results stimulate new possibilities and questions regarding microtubular-organizing centres (MTOC) for the complex array of MT shown in this study. Since not all MT seem to be associated with kinetosomes or other 'obvious' nucleating structures, there is probably a good likelihood of identifying and characterizing new MTOCs. This possibility is certainly relevant to future studies of this nature. Also of interest is the length of certain MT, such as those running parallel to the long axis on both dorsal and ventral surfaces. I have taken a lengthy contiguous series of SEM micrographs, at suitable magnification; however, it has not been Subpellicular microtubules of Euplotes 483 possible to follow MT on either surface for a sufficient distance to evaluate this. This is due, in part, to occasional debris and regions of incomplete chemical dissection. Future modifications of the techniques may resolve this problem. A second question is whether or not the MT that appear to abut other structures (or other MT) stop at these locations or, instead, plunge deeper into the cytoplasm. Very precise cell orientation and serial thin-sectioning will be required to answer this question. The author wishes to thank Kenneth Halcrow of the same university for critical reading of the manuscript and helpful suggestions.

REFERENCES ALIEN, R. D. (1978). Membranes of ciliates: infrastructure, biochemistry and fusion. In Membrane Fusion (ed. G. Poste & G. L. Nicolson), pp. 657-763. Amsterdam: Elsevier/ North-Holland Biochemical. AUFDERHEIDE, K. J., FRANKEL, J. & WILLIAMS, N. E. (1980). Formation and positioning of surface-related structures in protozoa. Microbiol. Rev. 44, 252-302. BOUCK, G. B. & BROWN, D. L. (1973). Microtubule biogenesis and cell shape in Ochromonas. I. The distribution of cytoplasmic and mitotic microtubules. Jf. Cell Biol. 56, 340-359. BROWN, D. L. & BOUCK, G. B. (1973). Microtubule biogenesis and cell shape in Ochromonas. II. The role of nucleating sites in shape development. J. Cell Biol. 56, 360-378. CACHON, P. J. & CACHON, M. (1971). Le Systeme axopodial des Radiolaires Nassellaires. Arch. Protistenk. 113, 80-97. CURDS, C. R. (1975). A guide to the species of the genus Euplotes (Hypotrichida, Ciliatea). Bull. Br. Mus. ttatl. Hist. 28(1), 1-61. DUSTIN, P. (1978). Microtubules. Berlin: Springer-Verlag. ELLISMAN, M. H. & PORTER, K. R. (1980). Microtrabecular structure of the axoplasmic matrix: visualization of cross linking structures and their distribution. J. Cell Biol. 87, 464-479. EPSTEIN, M. & ECHERT, R. (1972). Membrane control of ciliary activity in the protozoan Euplotes. J. exp. Biol. 58, 437-462. FAURE-FREMIET, E. & ANDRE, J. (1968). Structure fine de YEuplotes eurystomus (WRZ.). Arch. Anat. Microsc. 57, 53-78. FAWCETT, D. W., ANDERSON, W. A. & PHILLIPS, D. M. (1971). Morphogenetic factors influencing the shape of the sperm head. Devi Biol. a6, 220-251. FOISSNER, W. (1978). Euplotes moebiusi f. quadricirattus (Ciliophora, Hypotrichida). I. Die Feinstruktur des Cortex und der argyrophilen Strukturen. Arch. Protistenk. iao, 86—117. GIBBONS, I. R. (1965). Chemical dissection of cilia. Archs Biol., Liege 76, 317-352. GOLDMAN, R. D. (1971). The role of three cytoplasmic fibers in BHK-21 cell motility. I. Microtubules and the effects of colchicine. Jf. Cell Biol. 51, 752-762. GRIM, J. N. (1967). Ultrastructure of pellicular and ciliary structures of Euplotes eurystomus. Jf. Protozool. 14, 625-634. GRIM, J. N., HALCROW, K. R. & HARSHBARGER, R. (1980). Microtubules beneath the pellicles of two ciliate protozoa as seen with the SEM. J. Protozool. 27, 308-310. GRIMES, G. W. & L'HERNAULT, S. W. (1979). Cytogeometrical determination of ciliary pattern formation in the hypotrich Stylonychia mytilus. Devi Biol. 70, 372-395. HAMMOND, D. M. (1937). The neuromotor system of Euplotes patella during binary fission and conjugation. Q. J. microsc. Sci. 79, 507-557. HAMMOND, D. M. & KOFOID, C. A. (1937). The continuity of structure and function in the neuromotor system of Euplotes patella during its life cycle. Proc. Am. phil. Soc. 77, 207- 218. HUFNAGEL, L. A. & TORCH, R. (1967). Intraclonal dimorphism of caudal cirri in Euplotes vamtus: cortical determination. J. Protozool. 14, 429-438. KITCHING, J. A. & CRAGGS, S. (1965). The axopodial filamentso f the heliozoan Actinosphaerium nucleofilum. Expl Cell Res. 40, 658-660. 484 J. N. Grim MACHEMER, H. (1966). Zur koordination und Wirkungsweise der Membranellen von Stylonyckia mylitis. Arch. Protistenk. 109, 257-277. MCINTOSH, J. R. & PORTER, K. R. (1967). Microtubules in the spermatids of the domestic fowl. J. CellBiol. 35, 153-173. PARDUCZ, B. (1967). Ciliary movement and coordination in ciliates. Int. Rev. Cytol. ai, 91- 128. PITELKA, D. R. (1969). Fibrillar systems in protozoa. In Research in Protozoology, vol. 3 (ed. T. Chen), pp. 279-388. New York: Pergamon. ROTH, L. E. (1957). An electron microscope study of the cytology of the protozoan, Euplotes patella. J. biophys. biochem. Cytol. 3, 985-1000. ROTH, L. E. & PIHLAJA, D. J. (1977). Gradionation: hypothesis for positioning and patterning. J. Protozool. 24, 2-9. RUFFOLO, J. J. (1976a). Fine structure of the dorsal bristle complex and pellicle of Euplotes. J. Morph. 148, 469-488. RUFFOLO, J. J. (19766). Cortical morphogenesis during the cell division cycle in Euplotes: an integrated study using light optical, scanning electron, and transmission electron micro scopy. J. Morph. 148, 489-528. RUFFOLO, J. J. (1980). Feeding apparatus of the ciliate protozoan, Euplotes eurystomus. Scan. elect. Microsc. 3, 533-536. SATTLER, C. A. & STAEHELIN, L. A. (1979). Oral cavity of Tetrahymena pyriformis. A freeze- fracture and high-voltage study of the oral ribs, cytostome, and forming food vacuole. J. Ultrastruct. Res. 66, 132-150. SMALL, E. B. & MARSZALEK, D. S. (1969). Scanning electron microscopy of fixed, frozen, and dried protozoa. Science, N.Y. 163, 1964-1965. SONKEBORN, T. M. (1963). Does preformed cell structure play an essential role in heredity? In The Nature of Biological Diversity (ed. J. M. Allen), pp. 165-221. New York: McGraw- Hill. SONNEBORN, T. M. (1970). Gene action in development. Proc. R. Soc. Land. B 176, 347- 366. SONNEBORN, T. M. (1975). Positional information and nearest neighbor interactions in relation to spatial patterns in ciliates. Aim. Biol. 14, 565-584. TILNEY, L. G. & PORTER, K. R. (1965). Studies on the microtubules of Heliozoa. I. Fine structure of Actmosphaerium with particular reference to axial rod structure. Protoplasma 50, 317-344- TUCKER, J. B. (1968). Fine structure and function of the cytopharyngeal basket in the ciliate Nassula.J. Cell Sci. 3, 493-514. TUFFRAU, M., PAYNE, C. K. & DE HALLER, G. (1968). Organisation de l'infraciliature chez quelques cilies hypotriches. Protistologica 4, 289-301. WALLENGREN, H. (1900). Zur Kenntnis der vergleichenden Morphologie der Hypotrichen. Bih. K. svenska VetenskAkad. Handl. 36, 1-31. WILLIAMS, N. E., VAUDAUX, P. E. & SKRIVER, L. (1979). Cytoskeletal proteins of the cell surface in Tetrahymena. I. Identification and localization of major proteins. Expl Cell Res. 123, 311-320. WISE, B. N. (1965). The morphogenetic cycle in Euplotes eurystomus and its bearing on problems of ciliate morphogenesis. J. Protozool. ia, 626-648.

(Received 11 August 1981 -Revised 20 January 1982)