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Oral cavity of Tetrahymena pyriformis
JOURNAL OF ULTRASTRUCTURE RESEARCH
66, 132-150 (1979)
Oral Cavity of Tetrahymena Pyriformis A Freeze-Fracture and High-Voltage Electron Microscopy Study of the Oral Ribs, Cytostome, and Forming Food Vacuole CAROL A. SATTLER I AND L. ANDREW STAEttELIN
Department Of Molecular, Cellular, and Developmental Biology, University of Colorado, Boulder, Colorado 80309 Received December 28, 1978, and in revised form, November 7, 1978 The architecture of the oral ribs and the cytostome region of the in situ oral cavity of Tetrahymena pyriformis has been studied. The overall organization of the ribs depends on two sets of underlying microtubules, linkers that connect individual microtubules to the plasma membrane and to the alveolar sacs and a fine filamentous reticulum that supports all of these structures and which exhibits many cross-connections to the microtubules and the alveolar sacs. Freeze-fracture replicas of the plasma membrane that covers the ribs reveal longitudinal rows of evenly spaced particles located near the crest and irregular, short rows of closely spaced particles in the troughs between the ribs. The longitudinal rows of particles could correspond to attachment sites for the linkers connecting the outermost microtubules in the rows of four to the plasma membrane. The short, irregular rows of particles might mediate plasma membrane-alveolar sac interactions. Serial thick sections indicate that microtubules from some of the oral ribs extend around the cytostome and insert into the underside of the foot of the trap door-like cytostomal lip. Cytoplasmic vesicles appear to contribute membrane material to the adjacent forming food vacuole. A model describing the functional significance of the different oral cavity components is presented.
Tetrahymena pyriformis, a pear-shaped, freshwater ciliated cell approximately 50 /~m in length and 30/~m in width, has been used as a model system in many laboratoties to study a variety of biological phenomena such as cell division, cell synchrony, structure of cilia, ciliate genetics and cortical patterns, and membrane biosynthesis. Excellent reviews of pertinent literature in Tetrahymena research are available in Hill's (1972) and Elliott's (1973) books and in several review articles (Everhart, 1973; Corliss, 1973; Nilsson, 1976). The purpose of this paper is to provide additional morphological observations on the highly differentiated oral cavity of Tetrahymena pyriformis. Initial studies on the isolated oral apparatus (Metz and Westfall, 1954) described three membranelles and an undulating membrane that are held toPresent address: McArdle Laboratory for Cancer Research, University of Wisconsin, Madison, Wis. 53706.
gether by a complex fiber system. Further studies on the ultrastructure of the anteriorly located oral cavity have included observations on thin-sectioned in situ oral cavities (Nilsson and Williams, 1966; Williams and Luft, 1968) which reveal specific information on the hexagonally packed kinetosomes in the membranelles, the staggered arrangement of the two rows of kinetosomes in the undulating membrane, the oral connectives, and the fine structure of the oral ribs that line the undulating membrane side of the cavity. The complexity of the oral connectives and the size of the oral cavity itself have made the reconstruction of the oral cavity a formidable task. Although observations from thin-sectioned oral cavities provide data on many individual components of the oral cavity, one cannot deduce the in situ shape of the cavity, the coarse of the oral connectives within the cavity, and the overall orientation of the membranelles, oral ribs, cyto132
0022-5320/79/020132-19502.00/0 Copyright © 1979 by Academic Press, Inc. All rights of reproduction in any form reserved.
ORAL CAVITY OF TETRAHYMENA PYRIFORMIS stome, and forming food vacuole. Therefore, s e v e r a l i n v e s t i g a t o r s ( N i l s s o n a n d W i l l i a m s , 1966; F o r e r et al., 1970; W o l f e , 1970) h a v e s t u d i e d w h o l e - m o u n t p r e p a r a t i o n s to obtain additional information on the complex network of oral connectives. Scanning e l e c t r o n m i c r o s c o p y s t u d i e s ( B u h s e et al., 1973) also p r o v i d e i n f o r m a t i o n o n t h e orientation of the components of the cavity. Our recent publication (Sattler and Staeh e l i n , 1976) o n r e c o n s t r u c t i o n o f t h e o r a l cavity utilizing high-voltage electron microscopy on serial sections of oral cavities in situ d e s c r i b e d t h e s h a p e o f t h e c a v i t y a n d t h e o r i e n t a t i o n of t h e m e m b r a n e l l e s , o r a l ribs, c y t o s t o m a l lip, a n d f o r m i n g f o o d v a c u o l e w i t h r e s p e c t to t h e c a v i t y . W e h a v e also r e p o r t e d o n t h e p r e s e n c e o f s t r u c t u r a l differentiations within the ciliary memb r a n e s of s o m e , b u t n o t all, o r a l cilia o f Tetrahymena pyriformis (Sattler and Staehelin, 1974). T h i s p a p e r , finally, p r e s e n t s fine-structural details of the oral ribs and other specialized regions of the oral cavity wall as determined by correlating observat i o n s f r o m t h i n s e c t i o n s , 0.5-/~m t h i c k sections examined with a high-voltage electron microscope (HVEM), and replicas obtained by freeze-fracturing. MATERIALS AND METHODS
Tetrahymenapyriformis were grown in 2% proteose peptone supplemented with 0.1% liver extract. The cells were harvested by centrifugation after 2 days growth and washed with starvation medium (Cameron and Jeter, 1970) before fixation. Thin sections. The cells were fixed in 0.1 M cacodylate-buffered 2% glutaraldehyde for 30 rain and postfixed in 0.1 M cacodylate-buffered 1% osmium tetroxide. Following dehydration in ethanol the cells were embedded in Araldite 6005. Thin sections were cut with glass knives on a Porter-Blum MT 1 ultramicrotome. Sections stained with uranyl acetate and lead citrate were examined under a Philips EM 200 operated at 60 kV. Thick sections. The fixation of cells for high-voltage electron microscopy was the same as that described for cells to be thin sectioned. The en bloc staining, embedding in Epon-Araldite, serial sectioning, and poststaining with lead citrate have been previously described (Sattler and Staehelin, 1976). Serial thick sections were examined in the JEM-100B operated at
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100 kV to determine which cells had appropriate oral regions to warrant further examination in the JEM1000 operated at 1000 kV. Freeze-fracturing. The ceils were fixed for 45 rain in 0.1 M cacodylate-buffered 4% glutaraldehyde and then slowly infiltrated with glycerol over a 2-day period until a final concentration of 30% glycerol in the medium was obtained. Specimens were placed on gold or copper grids, immersed in Freon 12, and stored in liquid nitrogen until fractured in a Balzers 360 freezeetch apparatus (Balzers, Liechtenstein). Replicas were cleaned with bleach followed by chromic acid and then picked up on 200-mesh grids coated with 1% polybutene 4000 in 1,2-dichloroethane. Replicas were examined with a Philips EM 200 or JEM 100B operated at 60 kV. The fracture faces are labeled P, representing the cytoplasmic membrane leaflet, and E, representing the extracellular membrane leaflet, according to the recently adopted freeze-etch nomenclature (Branton, et al., 1975). RESULTS The terminology used to describe the oral c a v i t y o f Tetrahyrnena p y r i f o r m i s corresponds to that employed by Nilsson and W i l l i a m s (1966). O r a l r i b s a r e l o c a t e d o n t h e r i g h t s i d e o f t h e o r a l c a v i t y o f t h e cell a n d t h u s a p p e a r o n t h e l e f t side of t h e c a v i t y as a v i e w e r e x a m i n e s a m i c r o g r a p h . The ribs extend from the top of the oral cavity adjacent to the basal bodies of the undulating membrane and descend down i n t o t h e o r a l c a v i t y t e r m i n a t i n g in t h e reg i o n o f t h e f o r m i n g f o o d v a c u o l e . T h e 17 o r a l r i b s ( N i l s s o n a n d W i l l i a m s , 1966) a p p e a r a s a s e r i e s of c r e s t s a n d t r o u g h s . T h e r e is a d i s t i n c t c u r v a t u r e in t h e o r a l ribs, simi l a r to t h e l e v e l i n g - o f f of a slide, in t h e p o s t e r i o r e n d of t h e o r a l c a v i t y c l o s e s t to the area where the forming food vacuoles p i n c h off ( S a t t l e r a n d S t a e h e l i n , 1976).
Pellicle o f O r a l R i b s T h e o r a l r i b s a r e d e l i n e a t e d on t h e i r outer surface by the plasma membrane (Figs. 1 a n d 2). E a c h r i b c o n t a i n s six m i c r o t u b u l e s a r r a n g e d in t w o rows, t w o m i c r o t u b u l e s in o n e r o w a n d f o u r m i c r o t u b u l e s in t h e o t h e r . B e t w e e n t h e r o w of f o u r m i c r o t u b u l e s o f o n e r i b a n d t h e r o w of t w o m i c r o t u b u l e s in t h e a d j a c e n t r i b t h e a l v e o l a r s a c s a r e a r r a n g e d in t h e f o r m of l o n g
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Fza. 1. Cross section of oral ribs. The top microtubule of the set of four microtubules is linked (arrows) to the adjacent plasma membrane. The filaments of the fine filamentous reticulum (FR) lie in the ribosome-free cytoplasm underlying the microtubules and alveolar sacs (AS). Note the regular densities (arrowheads) along the filament bundle that parallels the membranes of the reticulum (ER), which separates the ribosome-free from the ribosome-rich cytoplasm. Some cross filaments can be seen to anchor the microtubules and the alveolar sacs to the filamentous reticulum, x 94 500. FIo. 2. Cross section of oral ribs. The alveolar sacs (AS) are distended in this fixation. Linkers between the alveolar sacs and the adjacent microtubule are indicated with arrows. Disk-shaped membranes with a dense luminal leaflet are present in the cytoplasm in the posterior region of the oral ribs. The complexity of the layer of fine filamentous reticulum associated with the oral ribs gradually decreases in these most posteriorly oriented ribs. x 68 500. tubes which underlie the plasma membrane a n d r u n p a r a l l e l to t h e ribs. D e p e n d i n g u p o n t h e fixation, t h e a l v e o l a r sacs c a n be d i s t e n d e d (Fig. 2) or f l a t t e n e d (Fig. 1).
Arrangement of Microtubules in Oral Ribs W i l l i a m s a n d L u f t (1968) h a v e r e p o r t e d t h a t t h e g r o u p of four m i c r o t u b u l e s occupies a r e g i o n i n t h e c r e s t of t h e rib w h i c h is l o c a t e d a n t e r i o r l y i n t h e cell, w h e r e a s t h e
g r o u p of two m i c r o t u b u l e s i n l o c a t e d posteriorly. W e d i s a g r e e w i t h t h o s e results. A l l e n (1967) h a s s h o w n t h a t t h e k i n e t o d e s m a l fibers of t h e s o m a t i c cilia e x t e n d tow a r d t h e a n t e r i o r e n d of t h e cell. S i n c e t h e k i n e t o d e s m a l fibers i n Fig. 3 e x t e n d t o w a r d t h e a n t e r i o r e n d of t h e cell, we c o n c l u d e t h a t t h e m e m b r a n e l l a r cilia a r e p r o t r u d i n g f r o m t h e a n t e r i o r e n d of t h e oral c a v i t y i n t o t h e p o s t e r i o r p a r t of t h e cavity. F i g u r e 4 is
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Fro. 3. Longitudinal thick section (0.5 #m) through the three membranelles in the oral region. Since the kinetodesmal fiber (k) extends toward the anterior end of the cell, the cilia from the first, second, and thh'd membranelles extend from the anterior side of the oral cavity toward the posterior side. The oral ribs (or) line the undulating membrane side of the cavity and the ribosome-free cytoplasm of the cytostomal lip (cl) is visible in the posterior portion of the cavity. × 6000. Fro. 4. Longitudinal thick section (0.5 #m) with the three membranelles extending from the anterior side of the oral cavity. The arrangement of microtubules in the oral ribs (see insert) is a repeating 2 + 4 pattern with the group of two microtubules being anterior to the group of four microtubules in each oral rib. x 7500; inset, x 17 000. another thick section through a similarly o r i e n t e d o r a l c a v i t y in w h i c h t h e m i c r o t u b u l e s in t h e o r a l r i b s a r e n i c e l y c r o s s - s e c t i o n e d in t h e p o s t e r i o r e n d o f t h e r i b b e d wall. I n t h e i n s e t to Fig. 4 it is q u i t e c l e a r t h a t t h e r o w of t w o m i c r o t u b u l e s lies a n t e r i o r to t h e r o w of f o u r m i c r o t u b u l e s in t h e s a m e rib. W e h a v e d e t e c t e d t h e p r e s e n c e o f a fine f i b e r (Fig. 1, s m a l l a r r o w s ) l i n k i n g t h e t o p m i c r o t u b u l e o f t h e r o w of f o u r m i c r o t u b u l e s to t h e a d j a c e n t p l a s m a m e m b r a n e c o v e r i n g t h e o r a l ribs. O c c a s i o n a l l i n k e r s a r e also d e t e c t e d b e t w e e n t h e a l v e o l a r sac m e m -
b r a h e a n d a m i c r o t u b u l e l o c a t e d in t h e a d j a c e n t r o w of t w o or f o u r m i c r o t u b u l e s (Fig. 2, a r r o w s ) . Fine Filamentous Reticulum I n c r o s s s e c t i o n s of t h e o r a l r i b s (Fig. 1) ribosomes are conspicuously absent from t h e c o r t i c a l r e g i o n c o n t a i n i n g t h e r o w s of m i c r o t u b u l e s , t h e a l v e o l a r sacs, a n d t h e filament-rich band of cytoplasm directly b e n e a t h t h e m i c r o t u b u l e s . L a r g e s h e e t s of e n d o p l a s m i c r e t i c u l u m (Fig. 1) s e p a r a t e this layer of ribosome-free cytoplasm from the ribosome-rich cytoplasm. The filaments
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in the ribosome-free cytoplasm of the ribs (Fig. 1) have a diameter of approximately 6 nm.~Filaments that run parallel to the cell surface and filaments that seem to anchor the microtubules and the alveolar sacs to the filamentous reticulum as a whole can be distinguished (Fig. 1). Williams and Luft (1968) introduced two names for this cortical layer: fine filamentous reticulum and ectoplasm depending on whether or not they were able to resolve filaments in the layer. Since all of our high-resolution micrographs reveal the filamentous nature of this layer, and since we have found no distinctive local variations in its structure, we will use only the term fine filamentous reticulum. However, specific regions of the oral ribs differ in the size and complexity of the associated fine filamentous reticulum. The oral ribs in Fig. I have a large, complex fine filamentous reticulum while those shown in Fig. 2 (located in the posterior portion of the oral cavity close to the forming food vacuole) demonstrate the gradual termination of the fine filamentous reticulum in the ribs located closest to the forming food vacuole. In oblique sections (Fig. 5) the fine filamentous reticulum is seen to underlie all the oral ribs. In the posterior part of the oral cavity the fine filamentous reticulum borders the microtubules of the undulating membrane network (Fig. 5) while in the anterior part of the cavity the fine filamentous reticulum curves away from the undulating membrane network and remains associated with the oral ribs. In tangential sections (Fig. 6, arrows) and occasionally in cross sections (Fig. 1, arrowheads) the fine filamentous reticulum exhibits a distinctive regular pattern of dense bands oriented at right angles to the filaments. The dense bands in Fig. 6 (arrows) are spaced at intervals of 82.5 to 105 nm and they make an angle of between 48 and 54 ° with the microtubules of the ribs. It is not clear whether individual fine filaments (Fig. 6, arrowheads) interconnect only adjacent
dense bands or whether they are sufficiently long to interlink several.
H V E M of Oral Ribs In 0.5-ttm sections the microtubules of the oral ribs remain in the plane of section over a considerable distance and thus they are ideal Structures to use in the alignment of serial sections through the oral region. Figures 7 and 8 are consecutive 0.5-/~m cross sections through the oral region directly beneath the cytostome [Fig. 9 in Sattler and Staehelin (1976) is serial section No. 11, and these micrographs are section Nos. 12 and 13 of the same oral cavity]. In Fig. 7 it appears that microtubules from several of the anteriorly located oral ribs (large arrow) extend under the cytostomal lip and reappear (large arrowheads) on the cytostomal lip side of the forming food vacuole. In Fig. 8, a 0.5 #m below Fig. 7, microtubules from the oral ribs are clearly seen extending into the forming food vacuole. Exactly where the microtubules of the oral ribs terminate is a difficult question to answer. Alignment of enlarged transparencies of serial thick sections suggests that some of the oral rib microtubules crossover below the forming food vacuole and then turn upward on the cytostomal lip side of the forming food vacuole. Figure 9, a thin section through a different oral cavity just below the plane of the cytostome, also suggests that microtubules which are continuous with the oral ribs (arrows) crossover to the cytostomal lip side of the food vacuole. In Fig. 10 the sets of two or three microtubules (small arrowheads) observed on the food vacuole side of the cytostomal lip may represent the ends of the rows of oral rib microtubules that have inserted into the cytostomal lip. If the oral rib microtubules are terminating in the lip, the 2 + 4 microtubular arrangement characteristic of oral ribs could be incomplete which would explain the variation in number of microtubules in other thin sections through similar regions.
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FIG. 5. Thin section through lower region of oral cavity. Cilia from the third membranelle (3) extend into the cavity. The cytostomal lip (cl) and the oral ribs (or) border the cytostome (large arrow). A fine filamentous reticulum (FR) consisting of 6-nm filaments in a ribosome-free cytoplasm underlies all the oral ribs. Microtubules from the undulating membrane network (umn) separate the fine filamentous reticulum from the basal bodies of the undulating membrane, x 23 000. FIG. 6. High magnification of the fine filamentous reticulum. The fine filaments (arrowheads) are oriented at right angles to the dense bands (arrows) which are spaced at intervals of 82.5 to 105 nm. x 105 500.
Freeze-Fracture of Plasma Membrane of Oral Ribs I n f r e e z e - f r a c t u r e r e p l i c a s of Tetrahymena cells in w h i c h t h e o r a l c a v i t y is re-
v e a l e d , t h e s h e e r n e s s of t h e oral rib side of t h e c a v i t y is v e r y s t r i k i n g (Fig. 11). N e a r t h e t o p of t h e o r a l c a v i t y (Figs. 11 a n d 12) the oral ribs display a corrugated appearance. E x a m i n a t i o n o f m a n y r e p l i c a s h a s
Fro. 7. Serial section [No. 12 of a series, part of which is published in Sattler and Staehelin (1976)] 0.5 t~m thick through oral region taken at 100 kV. The section is located just below the cytostome. Posterior oral ribs are protruding into the forming food vacuole (FV). Part of the cytostomal lip (cl) is also present. Four or five of the anteriorly located oral ribs (large arrow) extend around the cytostome (see also Fig. 8). After passing under the cytostomal lip, the oral ribs curve upward and into the plane of the section again (arrowheads). x 10 000. FIo. 8. Serial section [No. 13 of a series, part of which is published in Sattler and Staehelin (1976)] 0.5 ttm thick just below section shown in Fig. 7 taken at 1000 kV. {Note the improved resolution when compared with 138
ORAL CAVITY OF T E T R A H Y M E N A P Y R I F O R M I S and 9) the forming food vacuole is a t t a c h e d to the posterior end of the oral cavity. T h e P face of the forming food vacuole (Figs. 16 and 17) contains relatively few r a n d o m l y distributed particles. T h e p l a s m a mereb r a n e covering the r a t h e r flattened crests and troughs of the posterior portion of the oral ribs is continuous with the p l a s m a revealed t h a t the distribution of particles on the fracture faces of the oral ribs varies depending u p o n w h e t h e r the oral ribs fract u r e d are located n e a r the top of the oral cavity or in the b o t t o m close to the forming food vacuole. N e a r the top of the cavity the P face of the p l a s m a m e m b r a n e covering each rib (Figs. 11 and 12) h a s a distinct longitudinal row of particles regularly spaced at intervals of 33.5 nm. In order to specify m o r e precisly the location of the longitudinal row of particles with respect to o t h e r rib c o m p o n e n t s , we designate the highest point in the P face covering the rib, the crest. T h e p l a s m a m e m b r a n e descends on b o t h sides of the crest down into the adjacent troughs. In Fig. 11 we h a v e design a t e d the m e m b r a n e t h a t extends f r o m the crest down into the t r o u g h located m o r e anteriorly in the oral cavity, the anterior side of the rib. I n similar fashion, the m e m b r a n e t h a t extends f r o m the crest down into the t r o u g h located m o r e posteriorly in the cavity is designated the posterior side of the rib. T h e r e f o r e in Fig. 11 the longitudinal rows of particles on the P face of the p l a s m a m e m b r a n e are located on the posterior sides of the rib as are the rows of four m i c r o t u b u l e s underlying the ribs in Figs. 1, 2, and 4. In the s a m e area of the oral ribs there is a second t y p e of m e m b r a n e differentiation within the troughs. T h i s differ-
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entiation consists of short, irregular lines of particles spaced a p p r o x i m a t e l y 13.5 n m a p a r t (Fig. 12, arrowheads). As one descends down the oral cavity, the contour of the ribs b e c o m e s less p r o n o u n c e d and the longitudinal row of evenly spaced particles disappears. In contrast, the short rows of closely p a c k e d particles in the troughs persist until the ribs flatten out in the vicinity of the c y t o s t o m a l lip (Fig. 13). T h e replica in Fig. 13 is difficult to i n t e r p r e t because its location with respect to the c y t o s t o m e is not obvious. P e r h a p s Fig. 13 reveals an area well below the c y t o s t o m e c o m p a r a b l e to Fig. 8 or 9. If t h a t is the case, the ribs t h a t descend straight into the forming food vacuole (ribs on viewer's lower left side in Fig. 13) do not flatten out as m u c h as the ribs located in the middle of Fig. 13 t h a t extend t h r o u g h the c y t o s t o m e and p e r h a p s insert into the c y t o s t o m a l lip side of the forming food vacuole as discussed earlier. T h e flat fracture face on the right in Fig. 13 m a y be p a r t of the c y t o s t o m a l lip. T h e p l a s m a m e m b r a n e in the transition region b e t w e e n the oral ribs and the merebranellar areas (Fig. 14) is characterized b y distinctive m e m b r a n e specializations. For example, in Fig. 14 (a roughly longitudinal fracture of the oral cavity at the juncture of the oral rib and the m e m b r a n e l l a r sides of the cavity) the E f a c e of the p l a s m a m e m b r a n e exhibits a single row of closely p a c k e d particles in the junctional area bet w e e n ribs and m e m b r a n e l l e s . In some areas this row displays a zig-zag configuration (Fig. 14B). Since Fig. 14 exhibits the E face of the m e m b r a n e , the structures relating to the longitudinal rows of particles located n e a r the crest of the ribs on the P
Fig. 7). A parallel set of oral ribs (or) protrude into the forming food vacuole (FV). The tips of the long membranellar cilia (c) also reach into the food vacuole. × 7500. FIG. 9. Thin section through oral cavity (OC) and food vacuole (FV) in the vicinity of the cytostome. Microtubules (arrows) which appear to be continuous with those of the oral ribs (or) cross over to the cytostomal lip side (CLS) of the food vacuole. × 46 500. FIG. 10. Thin section of cytostome bordered by the cytostomal lip (cl) and the oral ribs (or). On the underside of the cytostomal lip there are short rows of microtubules (arrowheads) containing two or three microtubules (in other sections sometimes four microtubules) directly beneath the plasma membrane of the forming food vacuole. Flattened vesicles with a dense luminal leaflet are present in the cytoplasm on the oral rib side of the food vacuole (FV). × 89 500.
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face in Fig. 12 are found in the troughs in Figs. 14 and 14A. When the oral cavity is roughly crossfractured in its posterior portion (i.e., just above the region where the forming food vacuole is attached similar to Fig. 5) as illustrated in Fig. 15, a distinct band of particles approximately four particles wide (Fig. 15, arrow) is observed on the cytoplasmic membrane leaflet (P face) of the plasma membrane extending from the membranellar side of the oral cavity to the cytostomal lip side. Since the complete pellicle structure (i.e., plasma membrane overlying alveolar sac membranes) is characteristic of the membranellar and cytostomal lip sides of the cavity, we suggest that the band of particles located on the plasma membrane in Fig. 15 may indicate where the alveolar sac membranes terminate and are attached to the plasma membrane.
Forming Food Vacuole At the level of the cytostome (Figs. 5, 7, membrane forming the food vacuole. The plasma membrane of the forming food vacuole is also continuous with the plasma membrane of the cytostomal lip (see Fig. 10). Figure 16 illustrates the wavy contour of the plasma membrane of the food vacuole on the cytostomal lip side. The abundant small, flattened vesicles located in the cytoplasm surrounding the food vacuole plasma membrane (Figs. 16 and 17) probably represent reserve membrane material that becomes incorporated into the membrane of the forming food vacuole (Figs. 17 and 18, arrows). Both the P and the E faces of the flattened vesicles (Figs. 16 and 17) as well as those of the forming food vacuoles (Figs. 16 and 17; only E faces shown) are covered with very few particles, the E faces carrying slightly fewer particles than the P faces. The plasma membrane of the small vesicle abutting the food vacuole in Fig. 17 (arrow) appears to be fusing with the membrane of the food vacuole. In thin-sectioned
cells (Figs. 2, 5, and 10), membranes in the shape of disks are commonly found in the cytoplasm adjacent to the forming food vacuole. These flattened vesicles have a dense luminal leaflet which can be easily seen in Fig. 10. In thin-sectioned food vacuoles (Fig. 18, arrows) the plasma membrane contains many V-shaped regions where new membrane material is probably being inserted. The plasma membrane of the food vacuole (Figs. 10 and 18), including the V-shaped areas, has a dense external membrane leaflet. Therefore, if the flattened vesicles represent membrane precursor elements for the plasma membrane of the food vacuole then these vesicles must open up as they are inserted into the membrane of the food vacuole. DISCUSSION In our studies of the relationship between the structure and function of the oral cavity of Tetrahymena pyriformis (Sattler and Staehelin, 1974, 1976) we have examined the in situ oral cavity rather than isolated oral cavities to avoid changes in the architecture of the cavity during the isolation procedure. Reconstruction of the oral cavity (Sattler and Staehelin, 1976) from serial thick (0.5/~m) sections examined under the HVEM has demonstrated that the anteriorly located oral cavity is approximately 9.5/~m deep with an opening width of approximately 7 × 8 /tm at the top of the cavity. Our HVEM micrographs have also proven most valuable for interpreting location and orientation or oral cavity components observed in thin sections of the cavity. For example, Williams and Luft (1968) have reported that the group of four microtubules is located more anteriorly in the oral rib than the group of two microtubules. Figure 4 clearly shows that this is not the case since the group of four microtubules lies on the posterior side of the oral rib crest. In thin-sectioned oral ribs (Fig. 1) we have observed a fine filament linking
ORAL CAVITY OF T E T R A H Y M E N A PYRIFORMIS
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FIG. 11. Replica of the upper level of the oral cavity. The first~ second, and third membranelles are located in the anterior side of the oral cavity. Cilia from the second membranelle extend down into the cavity and, like all other membranellar cilia, are in a position to sweep down along the oral ribs. The plasma membrane (PM) covering the membraneUes is continuous with the plasma membrane covering the oral ribs (or) which descend into the cavity. A single longitudinal row of particles (arrow, see also Fig. 12) is found on the P face of the plasma membrane on the posterior side of the crest of the oral ribs. The closely spaced row of cross-fractured cilia located close to the top of the oral ribs is from the undulating membrane (UM). × 18 000.
the top m i c r o t u b u l e s of the group of four m i c r o t u b u l e s to the adjacent p l a s m a m e m b r a n e on the posterior side of the crest of the rib. We suggest, therefore, t h a t these fine filament linkers in Fig. 1 m a y be asso-
ciated with the longitudinal row of the particles observed on the P face of the posterior side of the oral rib crest (Figs. 11 and 12). B o t h the longitudinal rows of particles and the corresponding linkers s e e m to be lim-
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ited to the upper regions of the cavity where the contours of the ribs are greatest. In contrast, the short, irregular rows of particles observed in the grooves of the ribs (Figs. 12 and 13) are found over the entire length of the oral ribs. Their location suggests that they could be involved in maintaining a close relationship between the oral rib membrane and the underlying alveolar sacs. Chart 1 illustrates the morphology of the oral ribs located in the top of the oral cavity. An additional feature of the cytoplasm immediately underlying the oral ribs is the banded, fine filamentous reticulum. Our micrographs indicate that this reticulum, which can most readily be demonstrated in oblique and tangential sections (Figs. 5 and 6), underlies all the oral ribs. Its position and structure suggest two possible functions: (1) It could increase the tensile strength of the oral rib region and help prevent the flattening out of the oral ribs; or (2) because of its gross similarity to muscle (i.e., dense cross bands spaced at regular intervals of 82.5 to 105 nm are lo-
cated at right angles to fine filaments whose diameter of 6 nm is the same as the diameter of actin in muscle), it could possibly allow the oral rib region to contract and possibly even carry out peristaltic-like movements. The cytostome joining the oral cavity and the forming food vacuole near the bottom of the oral cavity is the least wellcharacterized area of the oral cavity. Originally Nilsson and Williams (1966) in a study of whole-mount preparations concluded that the microtubules of the oral ribs extended toward the cytostome where they meet with the "membranellar deep fiber" originating from the third membranelle. A more recent study (Forer et al., 1970) using a polarization microscope on whole-mount preparations concluded that the oral ribs terminate at the cytopharyngeal region and do not intertwine with the membranellar deep fiber. In contrast this study has produced results indicating that some of the oral rib microtubules extend below the level of the cytostome into the forming food vacuole region (Figs. 7 and 8).
FIG. 12. P-Face image of the plasma membrane that covers the oral ribs near the top of the cavity. Each rib displays near its crest a longitudinal row of evenly spaced particles (arrows) with a periodicity of 33.5 nm. In the troughs between adjacent ribs there are short, irregular lines of particles (small arrowheads) spaced approximately 13.5 nm apart. At the top of the ribs, the plasma membrane forms a lip (L). x 77 500. FIG. 13. P face of the plasma membrane covering the ribs in the lowest region of the oral cavity near the cytostome. Short, irregular rows of particles (arrowheads) may be discerned in the troughs between the ribs on the left side of the micrograph. Where the ribs flatten out (center) the particle rows disappear. The plasma membrane on the right side of the micrograph is extremely flat compared to that covering the ribs on the left. This flattened membrane area may be part of the cytostomal lip. x 74 500. FIG. 14. Replica of oral ribs with adjacent membranelles (1, 2, 3). This E face of the plasma membrane of the ribs is complementary to the P face. Thus the single, longitudinal rows of particles seen in the troughs (arrowheads) correspond in their position to the rows of particles located near the crests of the rib in the P-face images (Fig. 12). Inset A: High magnification of particles in trough. Inset B. Membrane region at the edge of the ribs near the bases of the second membranelle exhibiting meandering rows of closely spaced particles (small arrow). Note how the ribs become both narrower and shallower toward the bottom of the cavity, x 29 000; inset a; x 43 000; inset b, x 41 500. Fro. 15. Replica illustrating the posterior portion of the oral cavity. Cilia from the third membranelle (3) lie within the cavity. The oral ribs (or) appear to descend below the fracture plane and then turn upward where they appear as rather flattened oral ribs on the cytostomal lip side (CLS) of the cavity. The P face of the plasma membrane (PM) covering the cytostomal lip side and the membranellar side of the cavity possesses a narrow band of particles (arrow). x 28 000. FIG. 16. E face of the plasma membrane of forming food vacuole (FV) containing relatively few particles. The membrane of the anterior end of the food vacuole is continuous with the plasma membrane covering the rather flattened oral ribs (or) (also notice where they terminate abruptly, arrows) and with the wavy plasma membrane of the cytostomal lip (cl). Small vesicles (v) are present in the cytoplasm surrounding both sides of the food vacuole, x 22 500.
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CHART 1. Oral ribs near top of oral cavity. The contour, i.e., the high crests and deep troughs, is most pronounced near the top of the oral cavity. In cytoplasm underlying the plasma membrane each rib contains six microtubules with the row of two microtubules located anterior to the row of four microtubules. Alveolar sacs underlie the plasma membrane and run parallel to the crests and troughs. Replicas indicate the presence of a longitudinal row of particles on the posterior side of the rib and closely spaced particles in the troughs.
Furthermore, we have found some of the more anteriorly located oral ribs to extend into the forming food vacuole and then turn upward on the cytostomal lip side of the food vacuole (Fig. 7, arrowheads). Superimposed negatives of serial thick sections (Figs. 7 and 8) as well as thin sections such as Fig. 9 suggest that some of the oral rib microtubules are indeed continuous with microtubules on the cytostomal lip side of the forming food vacuole. Nilsson (1976) in describing a thin section through the same region as in Fig. 10 reports that it is unclear whether the rows of two or three microtubules located on the underside of the foot of the cytostomal lip represent the merebranellar deep fiber. We propose that these rows of microtubules arise from oral rib microtubules that have dipped below the level of the cytostome opening, crossing the
region in which food vacuoles are formed and then turning upward to insert into the underside of the cytostomal lip where they terminate. However, more detailed studies of the cytostome region are needed to clarify this point. Chart 2 illustrates our interpretation of the ultrastructure of the cytostome region of the oral cavity. Replicas of the posterior portion of the oral cavity (Figs. 16 and 17) show that the plasma membrane of the forming food vacuole is continuous with the plasma membrane covering the oral ribs and the cytostomal lip. In the cytoplasm adjacent to the cytostomal lip (Figs. 16 and 17) and adjacent to the oral ribs in the cytostome region (Fig. 2, 5, 10, 16 and 18) there are many small vesicles, some of which are flattened and disk-shaped. Many studies on oral cavities of other ciliates (Miller and Stone,
Fro. 17. E face of forming food vacuole (FV). Cilia from the third membranelle with membrane specializations appear in the cavity. Cross-fractured oral ribs (or) line one side of the cavity while the cytostomal lip (cl) lines the opposite side. Vesicles (v), some of which appear disk-shaped, are present in the cytoplasm beneath the plasma membrane of the cytostomal lip. The arrow points to a vesicle that appears to be in the process of fusing with the food vacuole. × 33 500. Fro. 18. Plasma membrane of forming food vacuole (FV). Vesicles (v) are located in the cytoplasm adjacent to the plasma membrane of the food vacuole. V-Shaped areas (large arrows) which probably represent insertion points of the vesicles into the food vacuole are numerous. × 88 500.
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SATTLER AND STAEHELIN
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CHART 2. Cytostome region. At the base of the oral cavity the plasma membrane of the forming food vacuole is continuous with the plasma membrane from the cytostomal lip side of the cavity and the oral rib side. The contour of the oral ribs decreases the deeper they are located in the cavity. The most posteriorly located oral ribs extend into the forming food vacuole, dip below the level of the cytostome, and then curve upward and insert into the foot of the cytostomal lip. Cytoplasmic vesicles surrounding the food vacuole fuse with the vacuole, thus increasing the size of the vacuole.
1963; Kloetzel, 1970; McKanna, 1973; Bradbury, 1973) report that small vesicles are located in the cytoplasm in close proximity to the forming food vacuole. Allen (1974) has provided conclusive evidence that the disk-shaped vesicles which accumulate next to the cytopharynx in Paramecium caw datum fuse with the food vacuole membrane. Since thin sections of the disks (Fig. 10) reveal a dense luminal leaflet similar to the dense luminal leaflet of the food vacuole membrane as in Allen's (1974) study, we suggest that these disk-shaped vesicles contribute membrane material to the food vacuole. This notion is further supported by the V-shaped profiles in the plasma membrane of the forming vacuoles (Fig. 18) and the similar freeze-fracture morphology of the two types of membranes (Batz and Wunderlich, 1976). We have not attempted to study the origin of the disk-shaped vesicles, but it is highly probable that they are also involved in the recycling of membrane material in the digestive process. However,
structural studies of Tetrahymena pyriformis have so far failed to reveal a specific microtubule system involved in the recycling of the digestive vacuole membrane as described by Allen (1974) for Paramecium.
Functional Interpretation of the Architecture of the Oral Cavity By correlating results obtained from freeze-fracturing, scanning electron microscopy, high-voltage electron microscopy (HVEM), and conventional transmission electron microscopy as reported in this paper and our other studies (Sattler and Staehelin, 1974, 1976), we have obtained a better understanding of how the various components of the oral cavity interact and function in the movement of food particles into the oral cavity. As Tetrahymena pyriformis swims, it rotates relative to its longitudinal axis and a large volume of fluid containing food particles comes in contact with the undulating membrane cilia and the membranellar cilia located in the anterior part
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CHART 3. Oral cavity. Only a few cilia from the first, second, and third membranelles and from the undulating membrane are shown so that the other components of the cavity can be seen. Cilia from the membranelles extend from the anterior end of the oral cavity toward the posterior end. Cilia from the undulating membrane extend from the undulating membrane side to the cytostomal lip side of the cavity. Note the curvature of the oral ribs and the decreasing size of the oral cavity in the deeper parts of the cavity. The forming food vacuole, located at the bottom of the oral cavity, is not visible from the top of the cavity due to the curvature of the cavity.
of the cell. In this context the long cilia of the undulating membrane serve as a dynamic rake that can propel trapped food particles into the anterior opening of the oral cavity which is diagramed in Chart 3. Membranellar cilia, some of which have distinct membrane specializations (Sattler and Staehelin, 1974), beat peripendicular to the undulating membrane cilia (Sattler and Staehelin, 1976) and thus propel fluid containing food particles downward toward the cytostome. The membranellar cilia create a sweeping motion against and parallel to the oral ribs which line one side of the cavity. The oral ribs serve as mechanical guides for directing fluids and food particles toward the cytostome and thus the food vacuole. As such the oral ribs resemble a microslide. The microtubule-reinforced cytostomal lip resembles a trap door in that it easily allows food particles to enter the food vacuole, but prevents them from escaping into the oral cavity. The food vac-
uole appears to enlarge by fusion with the small cytoplasmic vesicles. Recycling of the food vacuole membrane, as has been demonstrated in Paramecium (Allen, 1974), most likely occurs in Tetrahymena. We thankfully acknowledge the assistance of Mr. George Wray in the operation of the JEM-1000, highvoltage electron microscope. Thanks to Mr. Gerald L. Sattler for technical assistance in the printing of micrographs and to Ms. Catherine Verhulst for making the illustrations. This work was supported by Grant GM 18639,to L. A. S. and by National Institutes of Health Grant RR-00592, Biotechnology Resources Branch, Division of Research Resources, NIH, to K. R. Porter, M. Fotino, and L. D. Peaehey. REFERENCES ALLEN, R. D. (1967) J. Protozool. 14, 553-565. ALLEN, R. D. (1974) J. Cell Biol. 63, 904-922. BATZ, W., AND WUNDERL~CH, F. (1976) Arch. Mikrobiol. 109, 215-220. BRADBURY, P. C. (1973) J. Protozool. 20, 405-414. BRANTON, D., BULLIVANT, S., GILULA, N. B., KARNOVSKY, M. J., MOOR, H., M/JHLETHALER, K., NORTHCOTE, D. H., PACKER, L., SATIR, B., SATIR, P., SPETH, V., STAEHELIN, L. A., STEERE, R. L., AND
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WEINSTEIN, R. S. (1975) Science 190, 54-56. BUHSE, H. E., STAMLER, S. J., AND CORLISS, J. O. (1973) Trans. Amer. Microsc. Soc. 92, 95-105. CAMERON, I. L., AND JETER, J. R., JR. (1970) J. "Protozool. 17, 429-431. CORLISS, J. O. (1973) Trans. Amer. Microsc. Soc. 92, 468-491. ELLIOTT, A. M. (1973) Biology of Tetrahymena, DoTden, Hutchinson and Ross, Stroudsburg, Pa. EVERHART, J. L. P. (1973) in PRESCOTT, D. M. (Ed.), in Methods in Cell Physiology, Vol., V, pp. 219-288, Acadmic Press, New York. FORER, A., NILSSON, J. R., AND ZEUTHEN, E. (1970) Compt. Rend. Tray. Lab. Carlsberg 38, 67-86. HILL, D. L. (1972) The Biochemistry and Physiology of Tetrahymena, Academic Press, New York and London.
KLOETZEL, J. A. (1970) J. Cell Biol. 47,108a. MCKANNA, J. A. (1973) J. Cell Sci. 13, 663-686. METZ, C. B., AND WESTFALL, J. A. (1954) Biol. Bull. 107, 106-122. MILLER, O. L., AND STONE, G. E. (1963) J. Protozool. 10, 280-288. NILSSON, J. R. (1976) Compt. Rend. Tray. Lab. Carlsberg 49, 215-355. NILSSON, J. R., AND WILLIAMS. N. E. (1966) Compt. Rend. Tray. Lab. Carlsberg 35, 119-141. SATTLER, C. A., AND STAEHELIN, L. A. (1974) J. Cell Biol. 62, 473-490. SATTLER, C. A., AND STAEHELIN, L. A. (1976) Tissue Cell 8, 1-18. WILLIAMS, N. E., AND LUFT, J. H. (1968) J. Ultrastruct. Res. 25, 271-292. WOLFE, J. (1970) J. Cell Sci. 6, 679-700.
66, 132-150 (1979)
Oral Cavity of Tetrahymena Pyriformis A Freeze-Fracture and High-Voltage Electron Microscopy Study of the Oral Ribs, Cytostome, and Forming Food Vacuole CAROL A. SATTLER I AND L. ANDREW STAEttELIN
Department Of Molecular, Cellular, and Developmental Biology, University of Colorado, Boulder, Colorado 80309 Received December 28, 1978, and in revised form, November 7, 1978 The architecture of the oral ribs and the cytostome region of the in situ oral cavity of Tetrahymena pyriformis has been studied. The overall organization of the ribs depends on two sets of underlying microtubules, linkers that connect individual microtubules to the plasma membrane and to the alveolar sacs and a fine filamentous reticulum that supports all of these structures and which exhibits many cross-connections to the microtubules and the alveolar sacs. Freeze-fracture replicas of the plasma membrane that covers the ribs reveal longitudinal rows of evenly spaced particles located near the crest and irregular, short rows of closely spaced particles in the troughs between the ribs. The longitudinal rows of particles could correspond to attachment sites for the linkers connecting the outermost microtubules in the rows of four to the plasma membrane. The short, irregular rows of particles might mediate plasma membrane-alveolar sac interactions. Serial thick sections indicate that microtubules from some of the oral ribs extend around the cytostome and insert into the underside of the foot of the trap door-like cytostomal lip. Cytoplasmic vesicles appear to contribute membrane material to the adjacent forming food vacuole. A model describing the functional significance of the different oral cavity components is presented.
Tetrahymena pyriformis, a pear-shaped, freshwater ciliated cell approximately 50 /~m in length and 30/~m in width, has been used as a model system in many laboratoties to study a variety of biological phenomena such as cell division, cell synchrony, structure of cilia, ciliate genetics and cortical patterns, and membrane biosynthesis. Excellent reviews of pertinent literature in Tetrahymena research are available in Hill's (1972) and Elliott's (1973) books and in several review articles (Everhart, 1973; Corliss, 1973; Nilsson, 1976). The purpose of this paper is to provide additional morphological observations on the highly differentiated oral cavity of Tetrahymena pyriformis. Initial studies on the isolated oral apparatus (Metz and Westfall, 1954) described three membranelles and an undulating membrane that are held toPresent address: McArdle Laboratory for Cancer Research, University of Wisconsin, Madison, Wis. 53706.
gether by a complex fiber system. Further studies on the ultrastructure of the anteriorly located oral cavity have included observations on thin-sectioned in situ oral cavities (Nilsson and Williams, 1966; Williams and Luft, 1968) which reveal specific information on the hexagonally packed kinetosomes in the membranelles, the staggered arrangement of the two rows of kinetosomes in the undulating membrane, the oral connectives, and the fine structure of the oral ribs that line the undulating membrane side of the cavity. The complexity of the oral connectives and the size of the oral cavity itself have made the reconstruction of the oral cavity a formidable task. Although observations from thin-sectioned oral cavities provide data on many individual components of the oral cavity, one cannot deduce the in situ shape of the cavity, the coarse of the oral connectives within the cavity, and the overall orientation of the membranelles, oral ribs, cyto132
0022-5320/79/020132-19502.00/0 Copyright © 1979 by Academic Press, Inc. All rights of reproduction in any form reserved.
ORAL CAVITY OF TETRAHYMENA PYRIFORMIS stome, and forming food vacuole. Therefore, s e v e r a l i n v e s t i g a t o r s ( N i l s s o n a n d W i l l i a m s , 1966; F o r e r et al., 1970; W o l f e , 1970) h a v e s t u d i e d w h o l e - m o u n t p r e p a r a t i o n s to obtain additional information on the complex network of oral connectives. Scanning e l e c t r o n m i c r o s c o p y s t u d i e s ( B u h s e et al., 1973) also p r o v i d e i n f o r m a t i o n o n t h e orientation of the components of the cavity. Our recent publication (Sattler and Staeh e l i n , 1976) o n r e c o n s t r u c t i o n o f t h e o r a l cavity utilizing high-voltage electron microscopy on serial sections of oral cavities in situ d e s c r i b e d t h e s h a p e o f t h e c a v i t y a n d t h e o r i e n t a t i o n of t h e m e m b r a n e l l e s , o r a l ribs, c y t o s t o m a l lip, a n d f o r m i n g f o o d v a c u o l e w i t h r e s p e c t to t h e c a v i t y . W e h a v e also r e p o r t e d o n t h e p r e s e n c e o f s t r u c t u r a l differentiations within the ciliary memb r a n e s of s o m e , b u t n o t all, o r a l cilia o f Tetrahymena pyriformis (Sattler and Staehelin, 1974). T h i s p a p e r , finally, p r e s e n t s fine-structural details of the oral ribs and other specialized regions of the oral cavity wall as determined by correlating observat i o n s f r o m t h i n s e c t i o n s , 0.5-/~m t h i c k sections examined with a high-voltage electron microscope (HVEM), and replicas obtained by freeze-fracturing. MATERIALS AND METHODS
Tetrahymenapyriformis were grown in 2% proteose peptone supplemented with 0.1% liver extract. The cells were harvested by centrifugation after 2 days growth and washed with starvation medium (Cameron and Jeter, 1970) before fixation. Thin sections. The cells were fixed in 0.1 M cacodylate-buffered 2% glutaraldehyde for 30 rain and postfixed in 0.1 M cacodylate-buffered 1% osmium tetroxide. Following dehydration in ethanol the cells were embedded in Araldite 6005. Thin sections were cut with glass knives on a Porter-Blum MT 1 ultramicrotome. Sections stained with uranyl acetate and lead citrate were examined under a Philips EM 200 operated at 60 kV. Thick sections. The fixation of cells for high-voltage electron microscopy was the same as that described for cells to be thin sectioned. The en bloc staining, embedding in Epon-Araldite, serial sectioning, and poststaining with lead citrate have been previously described (Sattler and Staehelin, 1976). Serial thick sections were examined in the JEM-100B operated at
133
100 kV to determine which cells had appropriate oral regions to warrant further examination in the JEM1000 operated at 1000 kV. Freeze-fracturing. The ceils were fixed for 45 rain in 0.1 M cacodylate-buffered 4% glutaraldehyde and then slowly infiltrated with glycerol over a 2-day period until a final concentration of 30% glycerol in the medium was obtained. Specimens were placed on gold or copper grids, immersed in Freon 12, and stored in liquid nitrogen until fractured in a Balzers 360 freezeetch apparatus (Balzers, Liechtenstein). Replicas were cleaned with bleach followed by chromic acid and then picked up on 200-mesh grids coated with 1% polybutene 4000 in 1,2-dichloroethane. Replicas were examined with a Philips EM 200 or JEM 100B operated at 60 kV. The fracture faces are labeled P, representing the cytoplasmic membrane leaflet, and E, representing the extracellular membrane leaflet, according to the recently adopted freeze-etch nomenclature (Branton, et al., 1975). RESULTS The terminology used to describe the oral c a v i t y o f Tetrahyrnena p y r i f o r m i s corresponds to that employed by Nilsson and W i l l i a m s (1966). O r a l r i b s a r e l o c a t e d o n t h e r i g h t s i d e o f t h e o r a l c a v i t y o f t h e cell a n d t h u s a p p e a r o n t h e l e f t side of t h e c a v i t y as a v i e w e r e x a m i n e s a m i c r o g r a p h . The ribs extend from the top of the oral cavity adjacent to the basal bodies of the undulating membrane and descend down i n t o t h e o r a l c a v i t y t e r m i n a t i n g in t h e reg i o n o f t h e f o r m i n g f o o d v a c u o l e . T h e 17 o r a l r i b s ( N i l s s o n a n d W i l l i a m s , 1966) a p p e a r a s a s e r i e s of c r e s t s a n d t r o u g h s . T h e r e is a d i s t i n c t c u r v a t u r e in t h e o r a l ribs, simi l a r to t h e l e v e l i n g - o f f of a slide, in t h e p o s t e r i o r e n d of t h e o r a l c a v i t y c l o s e s t to the area where the forming food vacuoles p i n c h off ( S a t t l e r a n d S t a e h e l i n , 1976).
Pellicle o f O r a l R i b s T h e o r a l r i b s a r e d e l i n e a t e d on t h e i r outer surface by the plasma membrane (Figs. 1 a n d 2). E a c h r i b c o n t a i n s six m i c r o t u b u l e s a r r a n g e d in t w o rows, t w o m i c r o t u b u l e s in o n e r o w a n d f o u r m i c r o t u b u l e s in t h e o t h e r . B e t w e e n t h e r o w of f o u r m i c r o t u b u l e s o f o n e r i b a n d t h e r o w of t w o m i c r o t u b u l e s in t h e a d j a c e n t r i b t h e a l v e o l a r s a c s a r e a r r a n g e d in t h e f o r m of l o n g
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Fza. 1. Cross section of oral ribs. The top microtubule of the set of four microtubules is linked (arrows) to the adjacent plasma membrane. The filaments of the fine filamentous reticulum (FR) lie in the ribosome-free cytoplasm underlying the microtubules and alveolar sacs (AS). Note the regular densities (arrowheads) along the filament bundle that parallels the membranes of the reticulum (ER), which separates the ribosome-free from the ribosome-rich cytoplasm. Some cross filaments can be seen to anchor the microtubules and the alveolar sacs to the filamentous reticulum, x 94 500. FIo. 2. Cross section of oral ribs. The alveolar sacs (AS) are distended in this fixation. Linkers between the alveolar sacs and the adjacent microtubule are indicated with arrows. Disk-shaped membranes with a dense luminal leaflet are present in the cytoplasm in the posterior region of the oral ribs. The complexity of the layer of fine filamentous reticulum associated with the oral ribs gradually decreases in these most posteriorly oriented ribs. x 68 500. tubes which underlie the plasma membrane a n d r u n p a r a l l e l to t h e ribs. D e p e n d i n g u p o n t h e fixation, t h e a l v e o l a r sacs c a n be d i s t e n d e d (Fig. 2) or f l a t t e n e d (Fig. 1).
Arrangement of Microtubules in Oral Ribs W i l l i a m s a n d L u f t (1968) h a v e r e p o r t e d t h a t t h e g r o u p of four m i c r o t u b u l e s occupies a r e g i o n i n t h e c r e s t of t h e rib w h i c h is l o c a t e d a n t e r i o r l y i n t h e cell, w h e r e a s t h e
g r o u p of two m i c r o t u b u l e s i n l o c a t e d posteriorly. W e d i s a g r e e w i t h t h o s e results. A l l e n (1967) h a s s h o w n t h a t t h e k i n e t o d e s m a l fibers of t h e s o m a t i c cilia e x t e n d tow a r d t h e a n t e r i o r e n d of t h e cell. S i n c e t h e k i n e t o d e s m a l fibers i n Fig. 3 e x t e n d t o w a r d t h e a n t e r i o r e n d of t h e cell, we c o n c l u d e t h a t t h e m e m b r a n e l l a r cilia a r e p r o t r u d i n g f r o m t h e a n t e r i o r e n d of t h e oral c a v i t y i n t o t h e p o s t e r i o r p a r t of t h e cavity. F i g u r e 4 is
ORAL CAVITY OF TETRAHYMENA PYRIFORMIS
135
Fro. 3. Longitudinal thick section (0.5 #m) through the three membranelles in the oral region. Since the kinetodesmal fiber (k) extends toward the anterior end of the cell, the cilia from the first, second, and thh'd membranelles extend from the anterior side of the oral cavity toward the posterior side. The oral ribs (or) line the undulating membrane side of the cavity and the ribosome-free cytoplasm of the cytostomal lip (cl) is visible in the posterior portion of the cavity. × 6000. Fro. 4. Longitudinal thick section (0.5 #m) with the three membranelles extending from the anterior side of the oral cavity. The arrangement of microtubules in the oral ribs (see insert) is a repeating 2 + 4 pattern with the group of two microtubules being anterior to the group of four microtubules in each oral rib. x 7500; inset, x 17 000. another thick section through a similarly o r i e n t e d o r a l c a v i t y in w h i c h t h e m i c r o t u b u l e s in t h e o r a l r i b s a r e n i c e l y c r o s s - s e c t i o n e d in t h e p o s t e r i o r e n d o f t h e r i b b e d wall. I n t h e i n s e t to Fig. 4 it is q u i t e c l e a r t h a t t h e r o w of t w o m i c r o t u b u l e s lies a n t e r i o r to t h e r o w of f o u r m i c r o t u b u l e s in t h e s a m e rib. W e h a v e d e t e c t e d t h e p r e s e n c e o f a fine f i b e r (Fig. 1, s m a l l a r r o w s ) l i n k i n g t h e t o p m i c r o t u b u l e o f t h e r o w of f o u r m i c r o t u b u l e s to t h e a d j a c e n t p l a s m a m e m b r a n e c o v e r i n g t h e o r a l ribs. O c c a s i o n a l l i n k e r s a r e also d e t e c t e d b e t w e e n t h e a l v e o l a r sac m e m -
b r a h e a n d a m i c r o t u b u l e l o c a t e d in t h e a d j a c e n t r o w of t w o or f o u r m i c r o t u b u l e s (Fig. 2, a r r o w s ) . Fine Filamentous Reticulum I n c r o s s s e c t i o n s of t h e o r a l r i b s (Fig. 1) ribosomes are conspicuously absent from t h e c o r t i c a l r e g i o n c o n t a i n i n g t h e r o w s of m i c r o t u b u l e s , t h e a l v e o l a r sacs, a n d t h e filament-rich band of cytoplasm directly b e n e a t h t h e m i c r o t u b u l e s . L a r g e s h e e t s of e n d o p l a s m i c r e t i c u l u m (Fig. 1) s e p a r a t e this layer of ribosome-free cytoplasm from the ribosome-rich cytoplasm. The filaments
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SATTLER AND STAEHELIN
in the ribosome-free cytoplasm of the ribs (Fig. 1) have a diameter of approximately 6 nm.~Filaments that run parallel to the cell surface and filaments that seem to anchor the microtubules and the alveolar sacs to the filamentous reticulum as a whole can be distinguished (Fig. 1). Williams and Luft (1968) introduced two names for this cortical layer: fine filamentous reticulum and ectoplasm depending on whether or not they were able to resolve filaments in the layer. Since all of our high-resolution micrographs reveal the filamentous nature of this layer, and since we have found no distinctive local variations in its structure, we will use only the term fine filamentous reticulum. However, specific regions of the oral ribs differ in the size and complexity of the associated fine filamentous reticulum. The oral ribs in Fig. I have a large, complex fine filamentous reticulum while those shown in Fig. 2 (located in the posterior portion of the oral cavity close to the forming food vacuole) demonstrate the gradual termination of the fine filamentous reticulum in the ribs located closest to the forming food vacuole. In oblique sections (Fig. 5) the fine filamentous reticulum is seen to underlie all the oral ribs. In the posterior part of the oral cavity the fine filamentous reticulum borders the microtubules of the undulating membrane network (Fig. 5) while in the anterior part of the cavity the fine filamentous reticulum curves away from the undulating membrane network and remains associated with the oral ribs. In tangential sections (Fig. 6, arrows) and occasionally in cross sections (Fig. 1, arrowheads) the fine filamentous reticulum exhibits a distinctive regular pattern of dense bands oriented at right angles to the filaments. The dense bands in Fig. 6 (arrows) are spaced at intervals of 82.5 to 105 nm and they make an angle of between 48 and 54 ° with the microtubules of the ribs. It is not clear whether individual fine filaments (Fig. 6, arrowheads) interconnect only adjacent
dense bands or whether they are sufficiently long to interlink several.
H V E M of Oral Ribs In 0.5-ttm sections the microtubules of the oral ribs remain in the plane of section over a considerable distance and thus they are ideal Structures to use in the alignment of serial sections through the oral region. Figures 7 and 8 are consecutive 0.5-/~m cross sections through the oral region directly beneath the cytostome [Fig. 9 in Sattler and Staehelin (1976) is serial section No. 11, and these micrographs are section Nos. 12 and 13 of the same oral cavity]. In Fig. 7 it appears that microtubules from several of the anteriorly located oral ribs (large arrow) extend under the cytostomal lip and reappear (large arrowheads) on the cytostomal lip side of the forming food vacuole. In Fig. 8, a 0.5 #m below Fig. 7, microtubules from the oral ribs are clearly seen extending into the forming food vacuole. Exactly where the microtubules of the oral ribs terminate is a difficult question to answer. Alignment of enlarged transparencies of serial thick sections suggests that some of the oral rib microtubules crossover below the forming food vacuole and then turn upward on the cytostomal lip side of the forming food vacuole. Figure 9, a thin section through a different oral cavity just below the plane of the cytostome, also suggests that microtubules which are continuous with the oral ribs (arrows) crossover to the cytostomal lip side of the food vacuole. In Fig. 10 the sets of two or three microtubules (small arrowheads) observed on the food vacuole side of the cytostomal lip may represent the ends of the rows of oral rib microtubules that have inserted into the cytostomal lip. If the oral rib microtubules are terminating in the lip, the 2 + 4 microtubular arrangement characteristic of oral ribs could be incomplete which would explain the variation in number of microtubules in other thin sections through similar regions.
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FIG. 5. Thin section through lower region of oral cavity. Cilia from the third membranelle (3) extend into the cavity. The cytostomal lip (cl) and the oral ribs (or) border the cytostome (large arrow). A fine filamentous reticulum (FR) consisting of 6-nm filaments in a ribosome-free cytoplasm underlies all the oral ribs. Microtubules from the undulating membrane network (umn) separate the fine filamentous reticulum from the basal bodies of the undulating membrane, x 23 000. FIG. 6. High magnification of the fine filamentous reticulum. The fine filaments (arrowheads) are oriented at right angles to the dense bands (arrows) which are spaced at intervals of 82.5 to 105 nm. x 105 500.
Freeze-Fracture of Plasma Membrane of Oral Ribs I n f r e e z e - f r a c t u r e r e p l i c a s of Tetrahymena cells in w h i c h t h e o r a l c a v i t y is re-
v e a l e d , t h e s h e e r n e s s of t h e oral rib side of t h e c a v i t y is v e r y s t r i k i n g (Fig. 11). N e a r t h e t o p of t h e o r a l c a v i t y (Figs. 11 a n d 12) the oral ribs display a corrugated appearance. E x a m i n a t i o n o f m a n y r e p l i c a s h a s
Fro. 7. Serial section [No. 12 of a series, part of which is published in Sattler and Staehelin (1976)] 0.5 t~m thick through oral region taken at 100 kV. The section is located just below the cytostome. Posterior oral ribs are protruding into the forming food vacuole (FV). Part of the cytostomal lip (cl) is also present. Four or five of the anteriorly located oral ribs (large arrow) extend around the cytostome (see also Fig. 8). After passing under the cytostomal lip, the oral ribs curve upward and into the plane of the section again (arrowheads). x 10 000. FIo. 8. Serial section [No. 13 of a series, part of which is published in Sattler and Staehelin (1976)] 0.5 ttm thick just below section shown in Fig. 7 taken at 1000 kV. {Note the improved resolution when compared with 138
ORAL CAVITY OF T E T R A H Y M E N A P Y R I F O R M I S and 9) the forming food vacuole is a t t a c h e d to the posterior end of the oral cavity. T h e P face of the forming food vacuole (Figs. 16 and 17) contains relatively few r a n d o m l y distributed particles. T h e p l a s m a mereb r a n e covering the r a t h e r flattened crests and troughs of the posterior portion of the oral ribs is continuous with the p l a s m a revealed t h a t the distribution of particles on the fracture faces of the oral ribs varies depending u p o n w h e t h e r the oral ribs fract u r e d are located n e a r the top of the oral cavity or in the b o t t o m close to the forming food vacuole. N e a r the top of the cavity the P face of the p l a s m a m e m b r a n e covering each rib (Figs. 11 and 12) h a s a distinct longitudinal row of particles regularly spaced at intervals of 33.5 nm. In order to specify m o r e precisly the location of the longitudinal row of particles with respect to o t h e r rib c o m p o n e n t s , we designate the highest point in the P face covering the rib, the crest. T h e p l a s m a m e m b r a n e descends on b o t h sides of the crest down into the adjacent troughs. In Fig. 11 we h a v e design a t e d the m e m b r a n e t h a t extends f r o m the crest down into the t r o u g h located m o r e anteriorly in the oral cavity, the anterior side of the rib. I n similar fashion, the m e m b r a n e t h a t extends f r o m the crest down into the t r o u g h located m o r e posteriorly in the cavity is designated the posterior side of the rib. T h e r e f o r e in Fig. 11 the longitudinal rows of particles on the P face of the p l a s m a m e m b r a n e are located on the posterior sides of the rib as are the rows of four m i c r o t u b u l e s underlying the ribs in Figs. 1, 2, and 4. In the s a m e area of the oral ribs there is a second t y p e of m e m b r a n e differentiation within the troughs. T h i s differ-
139
entiation consists of short, irregular lines of particles spaced a p p r o x i m a t e l y 13.5 n m a p a r t (Fig. 12, arrowheads). As one descends down the oral cavity, the contour of the ribs b e c o m e s less p r o n o u n c e d and the longitudinal row of evenly spaced particles disappears. In contrast, the short rows of closely p a c k e d particles in the troughs persist until the ribs flatten out in the vicinity of the c y t o s t o m a l lip (Fig. 13). T h e replica in Fig. 13 is difficult to i n t e r p r e t because its location with respect to the c y t o s t o m e is not obvious. P e r h a p s Fig. 13 reveals an area well below the c y t o s t o m e c o m p a r a b l e to Fig. 8 or 9. If t h a t is the case, the ribs t h a t descend straight into the forming food vacuole (ribs on viewer's lower left side in Fig. 13) do not flatten out as m u c h as the ribs located in the middle of Fig. 13 t h a t extend t h r o u g h the c y t o s t o m e and p e r h a p s insert into the c y t o s t o m a l lip side of the forming food vacuole as discussed earlier. T h e flat fracture face on the right in Fig. 13 m a y be p a r t of the c y t o s t o m a l lip. T h e p l a s m a m e m b r a n e in the transition region b e t w e e n the oral ribs and the merebranellar areas (Fig. 14) is characterized b y distinctive m e m b r a n e specializations. For example, in Fig. 14 (a roughly longitudinal fracture of the oral cavity at the juncture of the oral rib and the m e m b r a n e l l a r sides of the cavity) the E f a c e of the p l a s m a m e m b r a n e exhibits a single row of closely p a c k e d particles in the junctional area bet w e e n ribs and m e m b r a n e l l e s . In some areas this row displays a zig-zag configuration (Fig. 14B). Since Fig. 14 exhibits the E face of the m e m b r a n e , the structures relating to the longitudinal rows of particles located n e a r the crest of the ribs on the P
Fig. 7). A parallel set of oral ribs (or) protrude into the forming food vacuole (FV). The tips of the long membranellar cilia (c) also reach into the food vacuole. × 7500. FIG. 9. Thin section through oral cavity (OC) and food vacuole (FV) in the vicinity of the cytostome. Microtubules (arrows) which appear to be continuous with those of the oral ribs (or) cross over to the cytostomal lip side (CLS) of the food vacuole. × 46 500. FIG. 10. Thin section of cytostome bordered by the cytostomal lip (cl) and the oral ribs (or). On the underside of the cytostomal lip there are short rows of microtubules (arrowheads) containing two or three microtubules (in other sections sometimes four microtubules) directly beneath the plasma membrane of the forming food vacuole. Flattened vesicles with a dense luminal leaflet are present in the cytoplasm on the oral rib side of the food vacuole (FV). × 89 500.
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face in Fig. 12 are found in the troughs in Figs. 14 and 14A. When the oral cavity is roughly crossfractured in its posterior portion (i.e., just above the region where the forming food vacuole is attached similar to Fig. 5) as illustrated in Fig. 15, a distinct band of particles approximately four particles wide (Fig. 15, arrow) is observed on the cytoplasmic membrane leaflet (P face) of the plasma membrane extending from the membranellar side of the oral cavity to the cytostomal lip side. Since the complete pellicle structure (i.e., plasma membrane overlying alveolar sac membranes) is characteristic of the membranellar and cytostomal lip sides of the cavity, we suggest that the band of particles located on the plasma membrane in Fig. 15 may indicate where the alveolar sac membranes terminate and are attached to the plasma membrane.
Forming Food Vacuole At the level of the cytostome (Figs. 5, 7, membrane forming the food vacuole. The plasma membrane of the forming food vacuole is also continuous with the plasma membrane of the cytostomal lip (see Fig. 10). Figure 16 illustrates the wavy contour of the plasma membrane of the food vacuole on the cytostomal lip side. The abundant small, flattened vesicles located in the cytoplasm surrounding the food vacuole plasma membrane (Figs. 16 and 17) probably represent reserve membrane material that becomes incorporated into the membrane of the forming food vacuole (Figs. 17 and 18, arrows). Both the P and the E faces of the flattened vesicles (Figs. 16 and 17) as well as those of the forming food vacuoles (Figs. 16 and 17; only E faces shown) are covered with very few particles, the E faces carrying slightly fewer particles than the P faces. The plasma membrane of the small vesicle abutting the food vacuole in Fig. 17 (arrow) appears to be fusing with the membrane of the food vacuole. In thin-sectioned
cells (Figs. 2, 5, and 10), membranes in the shape of disks are commonly found in the cytoplasm adjacent to the forming food vacuole. These flattened vesicles have a dense luminal leaflet which can be easily seen in Fig. 10. In thin-sectioned food vacuoles (Fig. 18, arrows) the plasma membrane contains many V-shaped regions where new membrane material is probably being inserted. The plasma membrane of the food vacuole (Figs. 10 and 18), including the V-shaped areas, has a dense external membrane leaflet. Therefore, if the flattened vesicles represent membrane precursor elements for the plasma membrane of the food vacuole then these vesicles must open up as they are inserted into the membrane of the food vacuole. DISCUSSION In our studies of the relationship between the structure and function of the oral cavity of Tetrahymena pyriformis (Sattler and Staehelin, 1974, 1976) we have examined the in situ oral cavity rather than isolated oral cavities to avoid changes in the architecture of the cavity during the isolation procedure. Reconstruction of the oral cavity (Sattler and Staehelin, 1976) from serial thick (0.5/~m) sections examined under the HVEM has demonstrated that the anteriorly located oral cavity is approximately 9.5/~m deep with an opening width of approximately 7 × 8 /tm at the top of the cavity. Our HVEM micrographs have also proven most valuable for interpreting location and orientation or oral cavity components observed in thin sections of the cavity. For example, Williams and Luft (1968) have reported that the group of four microtubules is located more anteriorly in the oral rib than the group of two microtubules. Figure 4 clearly shows that this is not the case since the group of four microtubules lies on the posterior side of the oral rib crest. In thin-sectioned oral ribs (Fig. 1) we have observed a fine filament linking
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FIG. 11. Replica of the upper level of the oral cavity. The first~ second, and third membranelles are located in the anterior side of the oral cavity. Cilia from the second membranelle extend down into the cavity and, like all other membranellar cilia, are in a position to sweep down along the oral ribs. The plasma membrane (PM) covering the membraneUes is continuous with the plasma membrane covering the oral ribs (or) which descend into the cavity. A single longitudinal row of particles (arrow, see also Fig. 12) is found on the P face of the plasma membrane on the posterior side of the crest of the oral ribs. The closely spaced row of cross-fractured cilia located close to the top of the oral ribs is from the undulating membrane (UM). × 18 000.
the top m i c r o t u b u l e s of the group of four m i c r o t u b u l e s to the adjacent p l a s m a m e m b r a n e on the posterior side of the crest of the rib. We suggest, therefore, t h a t these fine filament linkers in Fig. 1 m a y be asso-
ciated with the longitudinal row of the particles observed on the P face of the posterior side of the oral rib crest (Figs. 11 and 12). B o t h the longitudinal rows of particles and the corresponding linkers s e e m to be lim-
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ited to the upper regions of the cavity where the contours of the ribs are greatest. In contrast, the short, irregular rows of particles observed in the grooves of the ribs (Figs. 12 and 13) are found over the entire length of the oral ribs. Their location suggests that they could be involved in maintaining a close relationship between the oral rib membrane and the underlying alveolar sacs. Chart 1 illustrates the morphology of the oral ribs located in the top of the oral cavity. An additional feature of the cytoplasm immediately underlying the oral ribs is the banded, fine filamentous reticulum. Our micrographs indicate that this reticulum, which can most readily be demonstrated in oblique and tangential sections (Figs. 5 and 6), underlies all the oral ribs. Its position and structure suggest two possible functions: (1) It could increase the tensile strength of the oral rib region and help prevent the flattening out of the oral ribs; or (2) because of its gross similarity to muscle (i.e., dense cross bands spaced at regular intervals of 82.5 to 105 nm are lo-
cated at right angles to fine filaments whose diameter of 6 nm is the same as the diameter of actin in muscle), it could possibly allow the oral rib region to contract and possibly even carry out peristaltic-like movements. The cytostome joining the oral cavity and the forming food vacuole near the bottom of the oral cavity is the least wellcharacterized area of the oral cavity. Originally Nilsson and Williams (1966) in a study of whole-mount preparations concluded that the microtubules of the oral ribs extended toward the cytostome where they meet with the "membranellar deep fiber" originating from the third membranelle. A more recent study (Forer et al., 1970) using a polarization microscope on whole-mount preparations concluded that the oral ribs terminate at the cytopharyngeal region and do not intertwine with the membranellar deep fiber. In contrast this study has produced results indicating that some of the oral rib microtubules extend below the level of the cytostome into the forming food vacuole region (Figs. 7 and 8).
FIG. 12. P-Face image of the plasma membrane that covers the oral ribs near the top of the cavity. Each rib displays near its crest a longitudinal row of evenly spaced particles (arrows) with a periodicity of 33.5 nm. In the troughs between adjacent ribs there are short, irregular lines of particles (small arrowheads) spaced approximately 13.5 nm apart. At the top of the ribs, the plasma membrane forms a lip (L). x 77 500. FIG. 13. P face of the plasma membrane covering the ribs in the lowest region of the oral cavity near the cytostome. Short, irregular rows of particles (arrowheads) may be discerned in the troughs between the ribs on the left side of the micrograph. Where the ribs flatten out (center) the particle rows disappear. The plasma membrane on the right side of the micrograph is extremely flat compared to that covering the ribs on the left. This flattened membrane area may be part of the cytostomal lip. x 74 500. FIG. 14. Replica of oral ribs with adjacent membranelles (1, 2, 3). This E face of the plasma membrane of the ribs is complementary to the P face. Thus the single, longitudinal rows of particles seen in the troughs (arrowheads) correspond in their position to the rows of particles located near the crests of the rib in the P-face images (Fig. 12). Inset A: High magnification of particles in trough. Inset B. Membrane region at the edge of the ribs near the bases of the second membranelle exhibiting meandering rows of closely spaced particles (small arrow). Note how the ribs become both narrower and shallower toward the bottom of the cavity, x 29 000; inset a; x 43 000; inset b, x 41 500. Fro. 15. Replica illustrating the posterior portion of the oral cavity. Cilia from the third membranelle (3) lie within the cavity. The oral ribs (or) appear to descend below the fracture plane and then turn upward where they appear as rather flattened oral ribs on the cytostomal lip side (CLS) of the cavity. The P face of the plasma membrane (PM) covering the cytostomal lip side and the membranellar side of the cavity possesses a narrow band of particles (arrow). x 28 000. FIG. 16. E face of the plasma membrane of forming food vacuole (FV) containing relatively few particles. The membrane of the anterior end of the food vacuole is continuous with the plasma membrane covering the rather flattened oral ribs (or) (also notice where they terminate abruptly, arrows) and with the wavy plasma membrane of the cytostomal lip (cl). Small vesicles (v) are present in the cytoplasm surrounding both sides of the food vacuole, x 22 500.
ORAL CAVITY OF T E T R A H Y M E N A
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CHART 1. Oral ribs near top of oral cavity. The contour, i.e., the high crests and deep troughs, is most pronounced near the top of the oral cavity. In cytoplasm underlying the plasma membrane each rib contains six microtubules with the row of two microtubules located anterior to the row of four microtubules. Alveolar sacs underlie the plasma membrane and run parallel to the crests and troughs. Replicas indicate the presence of a longitudinal row of particles on the posterior side of the rib and closely spaced particles in the troughs.
Furthermore, we have found some of the more anteriorly located oral ribs to extend into the forming food vacuole and then turn upward on the cytostomal lip side of the food vacuole (Fig. 7, arrowheads). Superimposed negatives of serial thick sections (Figs. 7 and 8) as well as thin sections such as Fig. 9 suggest that some of the oral rib microtubules are indeed continuous with microtubules on the cytostomal lip side of the forming food vacuole. Nilsson (1976) in describing a thin section through the same region as in Fig. 10 reports that it is unclear whether the rows of two or three microtubules located on the underside of the foot of the cytostomal lip represent the merebranellar deep fiber. We propose that these rows of microtubules arise from oral rib microtubules that have dipped below the level of the cytostome opening, crossing the
region in which food vacuoles are formed and then turning upward to insert into the underside of the cytostomal lip where they terminate. However, more detailed studies of the cytostome region are needed to clarify this point. Chart 2 illustrates our interpretation of the ultrastructure of the cytostome region of the oral cavity. Replicas of the posterior portion of the oral cavity (Figs. 16 and 17) show that the plasma membrane of the forming food vacuole is continuous with the plasma membrane covering the oral ribs and the cytostomal lip. In the cytoplasm adjacent to the cytostomal lip (Figs. 16 and 17) and adjacent to the oral ribs in the cytostome region (Fig. 2, 5, 10, 16 and 18) there are many small vesicles, some of which are flattened and disk-shaped. Many studies on oral cavities of other ciliates (Miller and Stone,
Fro. 17. E face of forming food vacuole (FV). Cilia from the third membranelle with membrane specializations appear in the cavity. Cross-fractured oral ribs (or) line one side of the cavity while the cytostomal lip (cl) lines the opposite side. Vesicles (v), some of which appear disk-shaped, are present in the cytoplasm beneath the plasma membrane of the cytostomal lip. The arrow points to a vesicle that appears to be in the process of fusing with the food vacuole. × 33 500. Fro. 18. Plasma membrane of forming food vacuole (FV). Vesicles (v) are located in the cytoplasm adjacent to the plasma membrane of the food vacuole. V-Shaped areas (large arrows) which probably represent insertion points of the vesicles into the food vacuole are numerous. × 88 500.
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SATTLER AND STAEHELIN
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CHART 2. Cytostome region. At the base of the oral cavity the plasma membrane of the forming food vacuole is continuous with the plasma membrane from the cytostomal lip side of the cavity and the oral rib side. The contour of the oral ribs decreases the deeper they are located in the cavity. The most posteriorly located oral ribs extend into the forming food vacuole, dip below the level of the cytostome, and then curve upward and insert into the foot of the cytostomal lip. Cytoplasmic vesicles surrounding the food vacuole fuse with the vacuole, thus increasing the size of the vacuole.
1963; Kloetzel, 1970; McKanna, 1973; Bradbury, 1973) report that small vesicles are located in the cytoplasm in close proximity to the forming food vacuole. Allen (1974) has provided conclusive evidence that the disk-shaped vesicles which accumulate next to the cytopharynx in Paramecium caw datum fuse with the food vacuole membrane. Since thin sections of the disks (Fig. 10) reveal a dense luminal leaflet similar to the dense luminal leaflet of the food vacuole membrane as in Allen's (1974) study, we suggest that these disk-shaped vesicles contribute membrane material to the food vacuole. This notion is further supported by the V-shaped profiles in the plasma membrane of the forming vacuoles (Fig. 18) and the similar freeze-fracture morphology of the two types of membranes (Batz and Wunderlich, 1976). We have not attempted to study the origin of the disk-shaped vesicles, but it is highly probable that they are also involved in the recycling of membrane material in the digestive process. However,
structural studies of Tetrahymena pyriformis have so far failed to reveal a specific microtubule system involved in the recycling of the digestive vacuole membrane as described by Allen (1974) for Paramecium.
Functional Interpretation of the Architecture of the Oral Cavity By correlating results obtained from freeze-fracturing, scanning electron microscopy, high-voltage electron microscopy (HVEM), and conventional transmission electron microscopy as reported in this paper and our other studies (Sattler and Staehelin, 1974, 1976), we have obtained a better understanding of how the various components of the oral cavity interact and function in the movement of food particles into the oral cavity. As Tetrahymena pyriformis swims, it rotates relative to its longitudinal axis and a large volume of fluid containing food particles comes in contact with the undulating membrane cilia and the membranellar cilia located in the anterior part
ORAL CAVITY OF T E T R A H Y M E N A
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PYRIFORMIS
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CHART 3. Oral cavity. Only a few cilia from the first, second, and third membranelles and from the undulating membrane are shown so that the other components of the cavity can be seen. Cilia from the membranelles extend from the anterior end of the oral cavity toward the posterior end. Cilia from the undulating membrane extend from the undulating membrane side to the cytostomal lip side of the cavity. Note the curvature of the oral ribs and the decreasing size of the oral cavity in the deeper parts of the cavity. The forming food vacuole, located at the bottom of the oral cavity, is not visible from the top of the cavity due to the curvature of the cavity.
of the cell. In this context the long cilia of the undulating membrane serve as a dynamic rake that can propel trapped food particles into the anterior opening of the oral cavity which is diagramed in Chart 3. Membranellar cilia, some of which have distinct membrane specializations (Sattler and Staehelin, 1974), beat peripendicular to the undulating membrane cilia (Sattler and Staehelin, 1976) and thus propel fluid containing food particles downward toward the cytostome. The membranellar cilia create a sweeping motion against and parallel to the oral ribs which line one side of the cavity. The oral ribs serve as mechanical guides for directing fluids and food particles toward the cytostome and thus the food vacuole. As such the oral ribs resemble a microslide. The microtubule-reinforced cytostomal lip resembles a trap door in that it easily allows food particles to enter the food vacuole, but prevents them from escaping into the oral cavity. The food vac-
uole appears to enlarge by fusion with the small cytoplasmic vesicles. Recycling of the food vacuole membrane, as has been demonstrated in Paramecium (Allen, 1974), most likely occurs in Tetrahymena. We thankfully acknowledge the assistance of Mr. George Wray in the operation of the JEM-1000, highvoltage electron microscope. Thanks to Mr. Gerald L. Sattler for technical assistance in the printing of micrographs and to Ms. Catherine Verhulst for making the illustrations. This work was supported by Grant GM 18639,to L. A. S. and by National Institutes of Health Grant RR-00592, Biotechnology Resources Branch, Division of Research Resources, NIH, to K. R. Porter, M. Fotino, and L. D. Peaehey. REFERENCES ALLEN, R. D. (1967) J. Protozool. 14, 553-565. ALLEN, R. D. (1974) J. Cell Biol. 63, 904-922. BATZ, W., AND WUNDERL~CH, F. (1976) Arch. Mikrobiol. 109, 215-220. BRADBURY, P. C. (1973) J. Protozool. 20, 405-414. BRANTON, D., BULLIVANT, S., GILULA, N. B., KARNOVSKY, M. J., MOOR, H., M/JHLETHALER, K., NORTHCOTE, D. H., PACKER, L., SATIR, B., SATIR, P., SPETH, V., STAEHELIN, L. A., STEERE, R. L., AND
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