Subpellicular Microtubtjles of Euplotes Eurystomus: Their Geometry Relative to Cell Form, Surface Contours and Ciliary Organelles
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J. Cell Set. 56, 471-484 (198*) 471 Printed in Great Britain <Q Company of Biologists Limited 1982 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 mem- branelles, 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.