J. Cell Set. 13, 663-675 (1973) 663

Printed in Great Britain

CYCLIC MEMBRANE FLOW IN THE INGESTIVE-

DIGESTIVE SYSTEM OF PERITRICH

PROTOZOANS

I. VESICULAR FUSION AT THE CYTOPHARYNX

J. A. McKANNA*Department of Anatomy, University of Wisconsin, Madison, WI. 53706, U.S.A.

SUMMARY

Food vacuoles in peritrichs form by pinching off the distal half of the cytopharynx; and thusthe pharyngeal membrane must be renewed during feeding. Correlation of light- and electron-microscopic observations indicates that the membranes of the ingestive—digestive systemrecycle. As the young food vacuoles enter the first stage of digestion (condensation of thevacuole), they pinch off cup-shaped coated vesicles which, in association with the microtubularpost-oral fibres, return to the pharynx. In the peripharyngeal region, the o-2$-fim diametercups flatten into o-^2-/im diameter disks, which then fuse with the pharyngeal membrane,thereby providing membrane for future food vacuoles. Ultrastructural evidence for similarpatterns of cyclic membrane flow and the functional implications of the association of thepharyngeal membrane with the microtubules of the pharyngeal ribs are considered for otherprotozoa, including suctorians.

INTRODUCTION

Peritrich ciliates like Vorticella and Epistylis are voracious feeders. In a 3-minburst of feeding, the organism may form more than twenty new food vacuoles. Eachof the membrane-bounded food vacuoles is created by endocytosis at the cytopharynx,a specialized region of the oral apparatus continuous with the cell surface. Since thesurface area of each new food vacuole is more than half the area of the cytopharyngealsurface, the membrane of the cytopharynx must be capable of turning over severaltimes per minute. This system thus suggested itself for examination of membranetransport. Light- and electron-microscopic investigation of ingestion and digestionin several species of peritrichs supports a model of cyclic membrane flow in whichfood vacuole membrane pinches off small vesicles which then return to fuse with thecytopharynx, thereby supplying membrane for new food vacuoles. The present paperis concerned with the addition of membrane at the pharynx, and discusses the func-tional implications of the basic pattern of microtubule-membrane interactions whichcharacterize ciliate oral cytostomes.

• Present address: Department of Anatomy, Upstate Medical Center, Syracuse, New York13210, U.S.A.

664 J- A- McKanna

MATERIALS AND METHODS

Vorticella, Epistylis plicatilis, and an unidentified species of Zoothamnium were maintainedin 0025 % Cerophyl infusion inoculated with Aerobacter aerogenes. A variety of fixatives weretried. Best results were achieved with aldehyde fixation (1 % glutaraldehyde + 1 % acrolein +1 % formaldehyde) for 1 h, followed by several changes of buffer wash (30 min), followed by1 % OsO4 for 2 h. All solutions were at room temperature, and buffered with 0-075 M sodiumcacodylate (pH 7-2) containing 1 rrtM CaClj. As extensively documented elsewhere (McKanna,1972), we have found that even in protozoans where penetration is not a problem, osmicationfor at least 2 h at room temperature is essential for the preservation and staining of membranecoats. The organisms were embedded in Epon 812, thin sections stained with uranyl acetate(saturated solution in 5 0 % ethanol) followed by lead citrate (50 mg in 10 ml HjO + 3 drops10 N NaOH), and examined with a Philips EM 200 electron microscope.

RESULTS

Light microscopy

Protozoa are advantageous subjects for studies correlating structure and functionbecause their functional activity may be observed at high resolution under essentiallyin vivo conditions. Epistylis plicatilis, the organism used most extensively in the presentinvestigation, exhibits the active ingestive-digestive system characteristic of peritrichs(Fig. 1). The feeding process involves the peristomal cilia which create a current ofculture fluid through the buccal cavity. By means of a poorly understood mechanism,bacteria and other particulate food are strained from the fluid and collected in thecone-shaped cytopharynx. At a rate that may exceed 6 times per minute, the distalhalf of the cytopharynx begins moving toward the base of the cell, assuming a fusiformshape as a constriction forms between it and the remainder of the cytopharynx. Assoon as its continuity with the cytopharynx is broken, the food vacuole is propelledrapidly to the base of the cell (Fig. 1). Remaining in this position for a few seconds,the new vacuole becomes spherical and begins condensing to the volume of itsparticulate food. It subsequently migrates apically to join the other food vacuoles inthe streaming cytoplasm.

Beginning at the point illustrated in Fig. 1, we should like to work back in theingestive sequence at the level of fine structure. We shall be asking several questionsraised by the in vivo observations. How is membrane added to the pharynx? How isthe membrane transported to the pharynx, and where is it recruited? The paper whichfollows this one (McKanna, 1973) is concerned with the source of supply forrecruitment.

Fine structure

Electron microscopy of peritrichs reveals the cytopharynx lying in the cytoplasmnear the lateral surface of the cell (Fig. 2). The pharyngeal wall is a complex structure,composed of a surface membrane with underlying microtubules and stacks of diskoidalvesicles. In contrast to the cell surface, which is covered by pellicle composed of theplasmalemma plus 2 membranes of a subsurface cistern (pellicular alveolus), thecytopharynx is lined by a single membrane. This feature serves to define the outerboundary of the pharynx. The cytopharyngeal membrane is continuous with the

Vesicular fusion at the cytopharynx 665

plasmalemma; but the subsurface cistern of the pellicle, which extends into the buccalcavity, terminates at the outer edge of the cytopharynx.

Some of the microtubule bundles which form the post-oral fibres are also shownin Figs. 2 and 3. These fibres run from the cytopharynx toward the base of the cell,terminating at approximately the level reached by the fusiform food vacuole in Fig. 1.

The cytopharyngeal membrane is thrown into longitudinal plicae, the pharyngealribs, which give the pharynx a serrated appearance in transverse section (Fig. 2).The wide end of the conical pharynx has more plicae than the narrow end; and weassume that Y-shaped branching occurs at points as indicated in Fig. 3. The plicaeare 100 nm high and 40 nm wide. A 6-o-nm layer of dense material, which may becomposed of longitudinal filaments, is situated 3-0 nm beneath the membrane oneach side of a plica (Fig. 3, inset). In addition, a fascicle of 22-nm diameter micro-tubules is associated with the base of each pharyngeal rib. In regions of apparentbranching, the fascicles merge. Assuming that the branching hypothesis is correct,some of the rib microtubules must terminate at a branch, since the number of micro-tubules in a fascicle is usually 5 + 1. Bridges or arms connecting the microtubules toeach other or to the membrane are observed only infrequently; and the lib micro-tubules do not appear to be in paracrystaUine array. Punctate elements, 5 nm indiameter, which are interpreted to be transverse sections of longitudinal filaments,are present in the vicinity of the rib microtubules.

Although the dynamic process of vesicular fusion cannot be proven by static electronmicrographs, that process is strongly suggested by the images recorded in Figs. 5 and6. Thus, in the feeding organism, fixation preserves diskoidal vesicles as well assmaller spherical vesicles which appear to insert between the rib microtubule fasciclesand finally fuse with the cytopharyngeal membrane. The point of fusion consistentlyappears to be the trough between 2 plicae. In sufficiently thin sections, one may detectfusion of the 2 cytoplasmic dense laminae (vesicular and pharyngeal membranes) intoa single thick dense line.

The steps preceding vesicular fusion in the peritrich membrane flow cycle are,first, the recruitment and transport of cup-shaped coated vesicles (CSCVs) to thevicinity of the pharynx and, secondly, the transformation of the CSCVs into diskoidalvesicles. With regard to the recruitment and transport of CSCVs to the pharynx, wemay benefit from further description of the process in vivo. The cytoplasm, includingthe food vacuoles, in the living cell is generally in a state of constant cyclosis, streamingin counterclockwise direction when viewed from the perspective in Fig. 1. Thus thecyclosis is in the same direction as the path of the new fusiform food vacuole as itproceeds to the base of the cell, but the fusiform food vacuole travels much morerapidly than the cytoplasmic streaming. It has been noted previously that the bundlesof microtubules composing the post-oral fibres run from the pharynx toward thebase of the cell; and it is possible that the post-oral fibres are involved in the rapidprogress of the fusiform food vacuoles. At the same time as the fusiform food vacuoleis rushing to the base of the cell and the cytoplasm is streaming counterclockwise,however, there may be observed in vivo a counter current of cytoplasmic granules atthe side of the cell (left border in Fig. 1). These granules proceed along the path of

666 J. A. McKanna

the post-oral fibres from the base of the cell up to the pharynx. The granules are verytiny, and they are no longer visible in the region of the cytopharynx due to the thick-ness of the cell and the presence of other organdies. Ultrastructural examination ofthe post-oral fibres in the region of the pharynx (Fig. 2) and in the basal region of thecell (Fig. 7), however, indicates that the o-z^-fim diameter cup-shaped coated vesiclesare likely candidates for the granules observed in vivo.

Ultrastructural data indicate that the transformation of CSCVs into diskoidalvesicles is a 2-stage process. The actual transformation of the coat from its condensedconformation in the cup-shaped coated vesicles to its extended conformation in thediskoidal vesicles (Figs. 5, 6) will be considered in the following paper; however,since the transition from cups to disks is integral to membrane transport, the dataare presented here. In peritrichs fixed during feeding, it is not uncommon to observeCSCVs with very wide mouths (partially flattened cups) adjacent to the pharynx(Fig. 3). In addition, fully flattened diskoidal vesicles with the condensed form ofthe coat are sometimes present (Fig. 8). The flattened vesicles with the condensedcoat are not present in other regions of the cell; and cup-shaped vesicles with theextended coat have not been detected. These data suggest that the CSCVs, followingtransport to the pharynx, transform into disks; and subsequently, the coat assumesthe extended conformation. The fact that the membrane surface area of a CSCV0-25 /im in diameter is identical to that of a diskoidal vesicle 0-42 /tm in diameter isconsistent with the hypothesis that the cups transform into disks.

DISCUSSION

The ultrastructural data on the cytopharyngeal membranes and microtubules inperitrichs complement data from other protozoans to support hypotheses of membranetransport, membrane stability, and microtubule function. Although these correlationswill be made for data from protozoan systems only, we assume that the models devel-oped are generally applicable to other organisms.

In addition to peritrichs, supplementation of the pharyngeal membrane by meansof vesicles has been reported previously for Paramecium, Euplotes, and suctorians.Schneider (1964) demonstrated that peripharyngeal diskoidal vesicles in Parameciumjoined with the pharyngeal membrane; and Yagui & Shigenaka (1966) recognizedthe role of this addition in providing membrane for food vacuole formation. Improvedresolution in recent studies has shown a thickened membrane or coat on the non-cytoplasmic (luminal) surface of these diskoidal vesicles (Pitelka, 1969; Jurand &Selman, 1969). In addition, Pitelka (1969) reported the association of diskoidalvesicles with the post-oral microtubules, much like the cup-shaped vesicles describedhere.

Kloetzel (1970) suggested a membrane-transport role for rod-shaped bodies whichhe observed in the peripharyngeal vicinity in Euplotes. These bodies were seen incontinuity with the pharynx; and their numbers were depleted after a burst of feeding.We have confirmed Kloetzel's observations, although the membranous bodies werediskoidal or ellipsoid in the specimens which we examined (McKanna, 1972).

Vesicular fusion at the cytopharynx 667

The process of feeding in suctorians is of special interest in this Discussion becauseof its relevance to questions of membrane transport and microtubule function. Suc-torians are predators, equipped with tentacles which capture prey protozoa andpenetrate their cell bodies. In feeding, the prey cytoplasm passes down through thetentacle into the suctorian cell body where food vacuoles form at the base of, thetentacle. Thus the suctorian feeding apparatus is superficially quite different fromthe cytopharynx of other ciliates; but, as has been recognized recently, its functionalanatomy is very similar to other ciliate oral cytostomes (Rudzinska, 1965; Bardele &Grell, 1967).

The defining features of an oral cytostome are: (1) its external boundary is thepoint at which the pellicle ends and the plasmalemma continues as the single cyto-stomal membrane; (2) the cytostomal membrane is subtended by bands or fasciclesof microtubules; and (3) vesicles, which provide membrane for the formation of newfood vacuoles by fusing with the cytostomal membrane, are present in the surroundingcytoplasm.

Examination of the membranes and microtubules of the suctorian feeding apparatusreveals that it is a typical oral cytostome. The plasmalemma of the cell soma continuesup the shaft of the tentacle. At the tip, this membrane invaginates into the lumen ofa hollow cylinder denned by bands of microtubules, much like the fascicles of micro-tubules associated with the pharyngeal ribs in peritrichs. The microtubule cylinderopens into the cytoplasm at the base of the tentacle. At the time of feeding, thismembranous tube, containing prey cytoplasm, runs down the length of the tentacleto the basal end of the microtubule cylinder, where food vacuoles are formed(Bardele & Grell, 1967).

The suctorian feeding apparatus may be considered in relation to the featurescharacteristic of oral cytostomes. The subsurface cisternae of the pellicle are foundbeneath the plasmalemma of the tentacle shaft; but they are not present at the verytip where the membrane invaginates into the microtubule cylinder. Thus the pellicleends and the cytostome begins at the tentacle tip. The bands of microtubules subtendthe cytostomal membrane as mentioned above. The third feature, the addition ofmembranous vesicles to the pharyngeal membrane, is especially interesting in thesuctorians because the small diameter of the tentacles has allowed high-resolytionobservation in vivo. Simultaneous bidirectional streaming in the tentacle is apparentin the living feeding organism. Organelles of the prey cytoplasm stream down theaxis of the tentacle within the cytostomal membrane, but, in addition, granules areobserved peripheral to the axial stream, moving out toward the tip of the tentacle.With the electron microscope, these granules have been shown to be osmiophilic,with lamellar organization of the luminal contents in Acineta tuberosa (Bardele &Grell, 1967). The lamellae exhibit a periodicity similar to that of the diskoidal bodiesin Euplotes (Kloetzel, 1970; McKanna, 1972). Bardele & Grell (1967) considered thepossibility that the osmiophilic granules play a role in the generation of cytostomaland food vacuole membrane; and, more recently, Hauser (1970) demonstrated thatsimilar granules fuse with the cytostomal membrane at the tip of the tentacle inanother suctorian, Paracineta limbata. In addition to the osmiophilic granules, vesicles

668 J. A. McKanna

with a thickened (possibly coated) membrane and an electron-lucent lumen have beendemonstrated in recent studies of ingestion and digestion in another suctorian,Tokophrya infusionum (Rudzinska, 1970). These vesicles have been shown pinchingoff from new food vacuoles, piled as disks at the base of the tentacle, and at the tipof the tentacle (figs. 15, 17; i, 10; and 6, respectively, in Rudzinska, 1970). Thesedata suggest not only vesicular membrane addition, but also membrane recycling insuctorians as seen in other protozoa. Such recycling would be especially importantfor new food vacuoles in suctorians because the area of membrane needed to containa certain volume of food in the narrow cylinder of the tentacle would be far greaterthan the membrane surface bounding that volume as a spherical food vacuole.

The basic pattern of microtubules in these various situations, along with the high-resolution observations of the in vivo activity, suggest that the microtubules play arole in ciliate ingestion. As developed extensively in a previous communication(McKanna, 1972), we feel that sufficient evidence exists to allow the assumption thatforces develop between microtubules and other cellular elements in their immediatevicinity. The directions of the forces are parallel to the long axis of the microtubules;and the forces may result in movement of the cellular elements relative to the micro-tubule, or in stabilization of these elements relative to external forces. Included amongthe cellular elements which interact with microtubules are other microtubules, mem-branes, and cytoplasmic matrix.

With specific reference to ciliate oral cytostomes, we suggest that forces developbetween the rib microtubules, serving to stabilize the cytopharyngeal structure. Otherforces developed between the rib microtubules and the pharyngeal membrane areresponsible for pinching off the new food vacuole, creating stresses resulting in itsfusiform shape, and propelling it toward the end of the post-oral fibres. As suggestedby Bardele (1972), similar forces would result in the movement of the cytopharyngealmembrane and its contents down the suctorian tentacle. We would caution, however,that we are aware of no evidence that microtubules ever shorten or ' contract'; andthat linear displacement of the microtubules seems unlikely.

To the extent of our knowledge, convincing demonstration of vesicular additionat the cytopharynx is limited to the papers cited above. Numerous other papers,however, have documented the presence of vesicles in the peripharyngeal vicinity inother organisms; and it may be assumed that these organisms, as well, utilize vesiculartransport and addition. We interpret the fact that vesicular fusion is only infrequentlypreserved to indicate that this step in membrane recycling proceeds rapidly.

Experiments which diminish the demand for cytopharyngeal membrane by inter-ruption of the feeding process suggest that the molecular constituents of this membranesystem do not turn over very rapidly. During mating in Tetrahymena, a processinvolving the oral region and thus preventing feeding, the vesicles have been shownto accumulate in stacks adjacent to the pharyngeal microtubules (Elliott & Zieg,1968). Peripharyngeal vesicles are present in increased numbers in Tetrahymena afterstarvation (Levy & Elliott, 1968); and we have also demonstrated the accumulationof stacks of diskoidal vesicles in the vicinity of the pharynx in starvation (Fig. 4).Especially in these starvation experiments, where the vesicles were shown to persist

Vesicular fusion at the cytopharynx 669

while significant numbers of other organelles were being incorporated into auto-lysomes, it seems clear that the membrane is a stable entity. We reason that were themembrane a labile association of molecules or micelles, with membrane subunitsequilibrating with free cytoplasmic species, the vesicles would disappear as the dis-sociated proteins and phospholipids were catabolized in starvation.

An additional feature of membrane transport is apparent in the presence of vesiclesat the oral cytostome of slow eaters like the flagellates (Mignot, 1966; Leedale, 1967;Schuster, 1968). One might expect in these organisms, where the demand for mem-brane is not nearly so great as in the ciliates, that molecular or micellar transport andaddition (if such occurs) would be sufficient. The observation of vesicles, however,indicates that vesicular transport is important in this situation as well, and suggeststhat smaller subunits do not play a significant role.

These data, while not obviously related to the question of membrane synthesis,suggest for the membranes of protozoan ingestive-digestive systems that the mem-brane is a stable association which does not dissociate into molecular subunits duringits normal functional cycling. They also suggest that the normal transport and additionof membrane in these systems occurs by means of membranous vesicles rather thansmaller subunits.

Supported by NIH Training Grant AS Toi GM00723-10.

REFERENCES

BARDELE, C. F. (1972). A microtubule model for ingestion and transport in the suctoriantentacle. Z. Zellforsch. mikrosk. Anat. 126, 116—134.

BARDELE, C. F. & GRELL, K. G. (1967). Elektronenmikroskopische Beobachtungen zurNahrungsaufnahme bei dem Suktor Acineta tuberosa Ehrenberg. Z. Zellforsch. mikrosk.Anat. 80, 108-123.

ELLIOTT, A. M. & ZrEG, R. G. (1968). A Golgi apparatus associated with mating in Tetra-hymena pyriformis. J. Cell Biol. 36, 391-398.

HAUSER, M. (1970). Elektronenmikroskopische Untersuchung an dem Suktor Paracinetalimbata Maupas. Z. Zellforsch. mikrosk. Anat. 106, 584-614.

JURAND, A. & SELMAN, G. G. (1969). The Anatomy of Paramecium aurelia, pp. 29-36. London:Macmillan.

KLOETZEL, J. A. (1970). The role of 'cytoplasmic rods' in food vacuole formation in Euplotes.J. Cell Biol. 47, 108 a.

LEEDALE, G. F. (1967). Euglenoid Flagellates, pp. 190-195. Englewood Cliffs, N.J.: Prentice-Hall.

LEVY, M. R. & ELLIOTT, A. M. (1968). Biochemical and ultrastructural changes in Tetrahymenapyriformis during starvation. J. Protozool. 15, 208-222.

MCKANNA, J. A. (1972). Contractile Vacuoles in Protozoans and Sponges: Comparative Studiesof Fine Structure and Function in Relation to the Physical Properties of Membranes and Waterin Biological Systems. Ph.D. Dissertation, The University of Wisconsin.

MCKANNA, J. A. (1973). Cyclic membrane flow in the ingestive-digestive system of peritrichprotozoans. II . Cup-shaped coated vesicles. J. Cell Sci. 13, 677-686.

MIGNOT, J.-P. (1966). Structure et ultrastructure de quelques EugleYiomonadines. Protisto-logica 2, 51-117.

PITELKA, D. R. (1969). Fibrillar systems in protozoa. In Research in Protozoology, vol. 3 (ed.T. Chen), pp. 279—388. Oxford: Pergamon.

RUDZINSKA, M. A. (1965). The fine structure and function of the tentacle in Tokophryainfusionum. J. Cell Biol. 25, 459-477.

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RUDZINSKA, M. A. (1970). The mechanism of food intake in Tokophrya infusionum and ultra-structural changes in food vacuoles during digestion. J. Protozool. 17, 626-641.

SCHNEIDER, L. (1964). Elektronenmikroskopische Untersuchungen an den Ernahrungs-organellen von Paramecium. I. Der Cytopharynx. Z. Zellforsch. mikrosk. Anat. 62, 198-224.

SCHUSTER, F. L. (1968). The gullet and trichocysts of Cyathomonas truncata. Expl Cell Res. 49,277-284.

YAGUI, R. & SHIGENAKA, Y. (1966). The food vacuole formation and the process of digestionand absorption in Paramecium caudatum. In Electron Microscopy 1966, vol. 2 (ed. R. Uyeda),pp. 237-238. Tokyo: Maruzen.

{Received 9 April 1973)

Fig. 1 A, B. Phase-contrast micrograph of E. plicatilis, and corresponding line drawingshowing a fusiform food vacuole (Jfv), which was at the tip of the cytopharynx (cp)less than 1 s previously, on its path toward the base of the cell. When viewed fromthis perspective, the cytoplasm containing other food vacuoles (fv) streams in acounterclockwise direction, b, buccal cavity; pc, peristomal cilia, x 650.Figs. 2-8. Electron micrographs of E. plicatilis. Details noted are identical to thosefound in Vorticella and Zoothamnium.

Fig. 2. Transverse section through the cytopharynx of an organism which had justbegun feeding. The ribbed pharyngeal wall is limited by a single membrane, while thecell surface is covered by pellicle (p). Diskoidal vesicles are situated in the peri-pharyngeal cytoplasm; and the post-oral fibres (pof) with associated cup-shapedvesicles are also present. Holes in the section are caused by poorly embedded calcium-and phosphate-containing granules, x 25000.

Vesicular fusion at the cytopharynx 671

.pc.

43-2

672 J. A. McKanna

Fig. 3. Cytopharynx following an extended feeding period. A number of smallspherical vesicles, but few diskoidal vesicles, are present. Two CSCVs (c) with widemouths are observed. This section is more basal than Fig. 2, and there are fewerpharyngeal plicae at this level. Arrows indicate points of branching, x 50000. Insetshows punctate images which may represent filaments underlying the membrane ofthe plicae, x 90000.

Fig. 4. In an organism which has been starved, the peripharyngeal cytoplasmcontains numerous stacks of diskoidal vesicles, cp, cytopharynx. x 19000.

Vesicular fusion at the cytopharymx 673

674 J- A. McKanna

Figs. 5, 6. Higher magnification of the cytopharynx shown in Fig. 2 reveals thediskoidal vesicles inserting between the rib microtubule fascicles to reach the pharyn-geal membrane at the trough between plicae. The trilaminar membranes of thepharynx and diskoidal vesicles are asymmetric; and in some places (arrows) thecytoplasmic dense laminae of the vesicular and pharyngeal membranes appear tohave fused. Although it is not heavily stained, a suggestion of the presence of themembrane coat in its extended conformation is apparent in the lumen of the vesiclesand on the pharyngeal surface, x 95000.

Fig. 7. Cup-shaped coated vesicles are observed in association with the micro-tubules of the post-oral fibres in the basal region of the cell, x 45000.

Fig. 8. Diskoidal vesicle with the coat in its condensed conformation (arrow)observed adjacent to the pharynx, x 45 000.

Vesicular fusion at the cytopharynx 675