J. Cell Set. i3> 677-686 (1973) 677Printed in Great Britain

CYCLIC MEMBRANE FLOW IN THE INGESTIVE-

DIGESTIVE SYSTEM OF PERITRICH

PROTOZOANS

II. CUP-SHAPED COATED VESICLES

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

SUMMARY

As peritrich food vacuoles condense during the initial stage of digestion, excess membranepinches off as cup-shaped vesicles which exhibit a structured coat on the non-cytoplasmicsurface of the membrane. As the membrane cycles from cup-shaped vesicles to diskoidalvesicles to cytopharynx to food vacuoles, the coat undergoes structural transformations fromthe condensed form (5x16 nm peg-shaped elements) to an extended form (long thin filaments).Review of the literature reveals morphologically similar coats which undergo similar trans-formations in the digestive organelles of flagellate protozoa, Hydra absorptive cells, insectpericardial cells, ileal absorptive cells of suckling rats, cells of the guinea-pig placenta, mam-malian Langerhans cells, and macrophages. The similar functional situation in which thesecoated membranes occur suggests that the coat is important to the recognition and binding ofmacromolecules.

INTRODUCTION

Electron microscopy of peritrichs has revealed that the membranes of organellesinvolved in food uptake and digestion have unique structural features which dis-tinguish them from other membranes of the cell. The trilaminar membranes of thecytopharynx, food vacuoles, and vesicles are asymmetric; and, in addition, anorganized coat is apparent on the non-cytoplasmic surface of the membranes incertain situations. This type of membrane coat has been implicated in the recognitionand binding of macromolecules for pinocytotic uptake in Hydra (Slautterback, 1967);and has been morphologically demonstrated on membranes in metazoan phagocytesand absorptive cells. The present paper documents the involvement of cup-shapedcoated vesicles (CSCVs) in peritrich intracellular digestion, and reviews the functionalimplications of comparative ultrastructural data on similarly coated membranes.

MATERIALS AND METHODSPeritrichs were prepared for electron microscopy as described previously (McKanna, 1973).

In tracer experiments, ferritin and Thorotrast (Dextrin-stabilized colloidal thorium dioxide)were added to the normal culture medium.

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

678 J. A. McKanna

RESULTS

In addition to their association with the post-oral fibres and the cytopharynx asdocumented in the preceding paper, the cup-shaped coated vesicles are found in theimmediate vicinity of food vacuoles at certain stages of the digestive cycle (Fig. 1);and, in fact, their membrane is continuous with that of the food vacuoles in somecases (Figs. 2-4). Although many of the vesicles free in the cytoplasm appear as 2concentric circles of membrane, consideration of the geometry of the vesicles hasconvinced us that these profiles all represent various planes of section through cup-shaped vesicles. The membrane forming the outer surface of the vesicle doubles backupon itself at the lip of the cup's narrow mouth. The lumen of the cup is open to thecytoplasm, and contains cytoplasmic matrix, ribosomes, glycogen, etc. The presenceof similar cytoplasmic material in most of the vesicles for which the section plane hasnot passed through the mouth (Fig. 6) constitutes further evidence that all of these'double-membrane' vesicles are cup-shaped.

The outside diameter of the CSCVs is 0-23—0-28 fim, and the lumen of the cup is~ 0-16 fim in diameter. The diameter of the mouth varies, being narrow (50 nm) inthe immediate vicinity of the food vacuoles (Figs. 2, 4), somewhat wider in the cyto-plasm and associated with the post-oral fibres (Fig. 6), and completely non-existentwhen the cup-shaped vesicle flattens at the pharynx as demonstrated in the precedingpaper (McKanna, 1973). As is apparent in Figs. 3 and 4, the membrane of the CSCVsexhibits the asymmetry characteristic of other membranes of the peritrich ingestive-digestive system. The cytoplasmic dense lamina is 4-0 nm thick, while the electron-lucent lamina and non-cytoplasmic dense lamina are each 2-5 nm thick. We havechosen to refer to cytoplasmic and non-cytoplasmic laminae in order to maintainconsistency for both intracellular and surface membranes.

In addition to the characteristic trilaminar asymmetry, however, the CSCV mem-brane exhibits further modification of the non-cytoplasmic dense lamina. This modi-fication, customarily called a membrane coat, consists of electron-dense elementsapposed or attached to the non-cytoplasmic dense lamina. In micrographs of ourmost favourably fixed and stained tissues, the electron-dense elements of the coatappear to be 5-0 nm in diameter and 16 nm long. They may be associated into pairsseparated by a centre-to-centre distance of 10 nm (Fig. 5). The coat in this state isreferred to as being in the condensed configuration on the basis of its similarity tothe coat demonstrated in Hydra absorptive cells as discussed below (Slautterback,1967).

The observation of CSCVs free in the cytoplasm and in continuity with the foodvacuole membrane leads to the question of whether they are fusing with or pinchingoff from the vacuoles. The answer is not easily learned. Previous authors havereported that the cup-shaped vesicles fuse with the food vacuoles (Goldfischer,Carasso & Favard, 1963); and, as considered in the Discussion, we feel that suchfusion is certainly possible. Our own observations, however, strongly suggest that,especially in the early stages of the digestive cycle, the predominant direction in theCSCV—food vacuole association is for the vesicles to pinch off from the vacuoles.

Cup-shaped coated vesicles in peritrichs 679

As related in the preceding paper (McKanna, 1973), it appears in vivo that when thefusiform food vacuole reaches the base of the cell, it becomes spherical and begins tocondense to the volume of its particulate food. It subsequently enters the more centralstreaming cytoplasm where further condensation leading to the first (acid) stage ofdigestion occurs. When observed in the living cell, the condensing vacuole appears togenerate a halo or cloud around its periphery. The individual components of thecloud, however, cannot be resolved with the light microscope.

In the interest of examining very young food vacuoles with the electron microscope,we fed ferritin to the peritrichs for short periods. The food vacuole shown in Fig. 1is less than 1 min old, since the organism had been placed in a ferritin solution for1 min preceding fixation. The ingested bacterium is still intact. As noted above, cup-shaped coated vesicles are present in the cytoplasm adjacent to the vacuole. Thesevesicles appear to be the only candidates for the cloud components; and their size isappropriate to our inability to resolve the individual cloud components in vivo.

Further suggestion as to the composition of the cloud derives from a section passingadjacent to the surface of a young vacuole (Fig. 6). The CSCVs fill the region. Inaddition, Fig. 6 demonstrates the association of the CSCVs with the post-oral fibresas discussed in the preceding paper (McKanna, 1973).

DISCUSSION

At various times in the digestive cycle, the membrane limiting a food vacuole willbe in excess of that necessary to bound the vacuolar contents. In the proposed modelof peritrich cyclic membrane flow, cup-shaped coated vesicles remove excess food-vacuole membrane for transport back to the cytopharynx. The first instance in whichexcess membrane might be expected arises with the transition in vacuolar shape fromfusiform to spherical as observed in vivo. For vacuoles bounding identical volumes,the surface area is reduced by more than 16%. Next, as the vacuole loses water andcondenses to approximately the volume of its contained particulate material, itssurface undergoes a proportionate reduction, usually greater than 50%. The shapetransition and condensation occur during the period when a cloud appears to emanatefrom the vacuole.

It has been recognized for some time that discrete stages of the digestive cycle inciliates involve further alteration of the volume and surface area of the vacuoles(Hyman, 1940). By the end of the first stage (condensation) described above, thevacuole contents have become acidic, but the particles (bacteria) retain their discreteform. In the second stage, the vacuole grows to slightly less than its original size, itbecomes alkaline, and the bacteria disintegrate. In the third stage, the vacuolarvolume is reduced again and its pH returns to neutrality. At the conclusion of thisfinal condensation, the residual vacuolar contents are defaecated by exocytosis at thecytopyge. During the second stage, vacuolar expansion and pH rise are accompaniedby fusion of primary lysosomes with the vacuole (Miiller, Rohlich, Toth & Toro,1963). Although conclusive evidence is not available on this point, it seems possiblethat the fusing lysosomal membrane adds to the surface membrane of the food vacuole.

680 J. A. McKanna

Indeed, since lysosomal enzymes are transported within endoplasmic reticulum orGolgi membranes (Miiller et al. 1963; Goldfischer et al. 1963), it is possible that thisis the point of addition of membrane to an otherwise closed cycle.

In the final condensing phase, a reduction in vacuolar volume results from thepinching off of small vesicles filled with digested material (Favard & Carasso, 1964).According to these investigators, the molecular contents of the vesicles pass outthrough the membrane into the cytoplasm for utilization in the cell's metabolism.As the vesicles empty, they collapse to form cup-shaped vesicles. Although we cannotadd substantially to the evidence for this hypothesis, it is consistent with the modelof coat transformations (condensed/extended) as discussed below.

Since current methods are incapable of denning absolutely the direction of netflow, even less certainty may be ascribed to the direction of an isolated individualevent. Thus it is possible that any micrograph presented to demonstrate the evolutionof a small vesicle from a food vacuole (Favard & Carasso, 1964) may have recordedan unlikely instance in which the vesicle was fusing with the vacuole. Similarly, anindividual CSCV shown in membranous continuity with a food vacuole may actuallyhave been in the process of fusion, as reported by Goldfischer et al. (1963). Knowledgeof the forces involved in membrane fusion is insufficient to eliminate the possibilitythat a CSCV could collide with a food vacuole and fuse with it. Our data stronglysuggest, however, that the net flow of CSCVs is from the food vacuoles toward thecytopharynx.

Two features of the cup-shaped coated vesicles seem especially appropriate totheir functional role in membrane transport. The first arises from consideration ofthe shape of a vesicle relative to its movement through the cytoplasm. Although theamount of membrane surface in a CSCV 0-25 fim in diameter is identical to thatin a disk of 0-42 /an diameter, the frictional coefficient of the CSCV would be lessthan half that of the disk (Bull, 1964). This physical consequence of a change in shapemay be important to the flow of cup-shaped vesicles toward the cytopharynx andthe accumulation of diskoidal vesicles in the peripharyngeal cytoplasm.

The second feature is that the cupped-shape allows reduction of the vesicle lumento a volume just adequate to accommodate the coat. The vesicle is, otherwise, effec-tively empty. Although Favard & Carasso (1964) reported the presence of Thorotrastin some CSCVs, the amount of label in the CSCVs was miniscule relative to theconcentration in the food vacuoles; and we have been unable to demonstrate traceruptake into CSCVs in any of our peritrichs fed ferritin or Thorotrast. This obser-vation is also compatible with the model of coat function as discussed below.

Data on the modified non-cytoplasmic dense lamina of the peritrich ingestive-digestive system membranes are complementary to reports on other systems in otherorganisms. The highly ordered coat found on the cup-shaped vesicles is similar tomembrane coats observed in a variety of cells with active protein uptake systems;and we feel that these comparative data are especially interesting regarding the basicmechanisms of macromolecular recognition by cells.

The best preservation and most detailed examination of this type coat has beenin the gastrodermal absorptive cells in Hydra, where Slautterback (1967) described

Cup-shaped coated vesicles in peritrichs 681

an ordered array of coat elements attached to the membrane of diskoidal vesicles inthe cytoplasm and at the luminal surface. The basic subunit of the coat has dimensionsof approximately 5 x 20 nm, and looks like a peg with a globule near the distal end.These subunits usually appear in a complex composed of a pair of pegs side by side,with a third globule situated between them. The Hydra diskoidal coated vesiclesare involved in the pinocytotic uptake of macromolecules from the digested food in thegastrocoel; and as the vesicle membrane follows its functional path, the configurationof the coat undergoes changes similar to those observed in peritrichs. In the firststage of the functional sequence, the empty vesicles in the apical cytoplasm exhibitthe condensed form of the coat as described above. Then, as digestion proceeds inthe gastrocoel, these vesicles fuse with the apical plasmalemma and the coat assumesthe appearance of extended filaments similar to the glycocalyx demonstrated on themicrovilli of intestinal absorptive cells (Ito, 1965). In this form, the coat appears tobind specific food molecules (as demonstrated by the binding of ferritin). The mem-brane with bound macromolecules subsequently pinches off back into the cytoplasm;and in this position the coat appears to resume its condensed conformation andrelease the food into the lumen of the vesicle.

The condensed form of the coat in the peritrich CSCVs and that in the Hydradiskoidal vesicles are structurally similar; and we feel that certain aspects of the coattransformations may show functional similarity. Favard & Carasso (1964) demon-strated the extended form of the coat on the membrane of the food vacuoles andmicropinocytotic vesicles observed in the third stage of digestion. In terms of themodel of coat function, the extended form of the coat serves to select macromoleculesfrom the digested brei in the vacuole. Once the pinocytotic vesicles pinch off fromthe food vacuole, the coat reverts to the condensed form, releasing the molecules todiffuse into the cytoplasm. The other situation in which the importance of coatconformation may be appreciated involves the CSCVs pinching off from the foodvacuoles. The condensed coat prevents macromolecular binding and thereby allowsgeneration of a ' clean' membrane for return to the cytopharynx.

Studies in flagellate protozoa have suggested that a similar coat may be involvedin the uptake of nutrients by these organisms. Investigation of intracellular digestionin the Euglenida led to the discovery of vesicles composed of membrane with thecondensed form of the coat associated with the digestive vacuoles in Entosiphonsulcatwn (Mignot, 1966). Later, in the dinoflagellate, Gymnodinium, Mignot (1970)described the pusule, a large fluid-filled vacuole in which digestion occurs. Hedemonstrated membranous tubules confluent with the pusule; and showed that themembrane of the tubules possesses a non-cytoplasmic coat similar to that on theperitrich CSCVs. We believe that this coat may be involved in the selection of nutrientmolecules from the pusule fluid. Tubules of similar dimensions, but which appearto have a coat in the extended conformation, were reported open to the digestivevacuoles in another flagellate, Ochromonas (Schuster, Hershenov & Aaronson, 1968).

Experimental studies in higher organisms also have suggested that the membranecoat is involved in recognition and uptake of macromolecules. A similar coat is presenton the non-cytoplasmic surface of the membrane which forms vesicles, tubules, and

682 J. A. McKanna

surface imaginations in the pericardial cells of the moth, Hyalophora cecropia (Sanger& McCann, 1968). These cells are considered to be stationary phagocytes; andSanger & McCann demonstrated that they take up ferritin by means of coated vesicles.In this process, the ferritin binds to the coat in its extended form.

A morphologically similar coat has been demonstrated in mammalian cells; andthe functional correlations in these systems are most intriguing because of theirimplications for immunology. The coat is prominent on membranes forming theapical endocytic complex (tubular imaginations of the apical plasmalemma) ofabsorptive cells in the ileum of suckling rats (Porter, Kenyon & Badenhausen, 1967;Wissig & Graney, 1968); and it is also present on cells of the duodenum and jejunumin these animals (Rodewald, 1970; J. W. Anderson, personal communication). Atthis stage of development, 2 functions of the alimentary canal are separated along itslength. Proximally, in the duodenum and jejunum, maternal antibodies are absorbedfrom the milk and transferred intact into the pup's circulation (Brambell, 1970). Bymeans of ferritin-labelled antibody, it has been demonstrated that antibody is boundto the extended coat on the apical plasmalemma (Rodewald, 1970). The antibodysubsequently is taken into the cell by pinocytosis, and finally released into the extra-cellular space. In Rodewald's micrographs, it appears that the antibody does not bindto regions of the plasmalemma where the coat is in the condensed configuration.

The other process, which occurs predominantly in the ileum, involves uptake andintracellular digestion of proteins from the milk. The pattern of structure and functionin the ileal cells is very similar to Hydra, since protein from the intestinal lumen istaken up by pinocytosis into coated vesicles. The protein then is transferred to alarge central vacuole which has been shown to contain active hydrolytic enzymes(Cornell & Padykula, 1969). Similar extended and condensed forms of the coat areapparently involved in protein absorption and intracellular digestion in the intestinalabsorptive cells of such diverse species as the goldfish (Gauthier & Landis, 1972) andthe neonatal calf (Staley, Corley, Bush & Jones, 1972).

A non-cytoplasmic coat exhibiting condensed and extended conformations also ispresent on the membranes of vesicles active in protein absorption and transport incells of the guinea-pig yolk sac placenta (King & Enders, 1970). These authors alsodemonstrated that the coat binds peroxidase and ferritin; but, like Hydra, does notbind a saccharide like Thorotrast.

A peg-like condensed coat has been observed on surface membranes and diskoidalvesicles (' granules') of Langerhans cells found in mammalian epidermis, lymph nodes,and other tissues (Wolff, 1967; Sagebiel & Reed, 1968; Tusques & Pradal, 1969).The function of these cells is not known; however, their distribution and ultra-structural characteristics suggest that they may be a specialized type of macrophage.In preliminary studies, we have found that condensed and extended forms of thecoat are present on vesicles and surface membranes of lymph node macrophages inthe squirrel monkey following antigenic insult (McKanna, unpublished).

The similarity of ultrastructural data on membrane coats from such diverseorganisms supports the hypothesis that these coats are involved in basic biologicalphenomena like selective binding and recognition; and we feel that the peritrich

Cup-shaped coated vesicles in peritrichs 683

ingestive-digestive system is an accessible system for isolation and further investi-gation of the components and properties of membrane coats.

The author is grateful to Dr David B. Slautterback for his valuable advice and encourage-ment. Supported by NIH Training Grant AS TOI 'GMOO723-IO.

REFERENCES

BRAMBELL, F. W. R. (1970). The Transmission of Passive Immunity from Mother to Young,pp. 102-141. Amsterdam and London: North-Holland Publishing.

BULL, H. B. (1964). An Introduction to Physical Biochemistry, pp. 263-266. Philadelphia:F. A. Davis.

CORNELL, R. & PADYKULA, H. A. (1969). A cytological study of intestinal absorption in thesuckling rat. Am. J. Anat. 125, 291-315.

FAVARD, P. & CARASSO, N. (1964). Etude de la pinocytose au niveau des vacuoles digestives decili£s P6ritriches. J. Microscopie 3, 671-696.

GAUTHIER, G. F. & LANDIS, S. C. (1972). The relationship of ultrastructural and cytochemicalfeatures to absorptive activity in the goldfish intestine. Anat. Rec. 17a, 675-702.

GOLDFISCHER, S., CARASSO, N. & FAVARD, P. (1963). The demonstration of acid phosphataseactivity by electron microscopy in the ergastoplasm of the ciliate Campanella umbellaria L.J. Microscopie 2, 621-628.

HYMAN, L. H. (1940). The Invertebrates: Protozoa through Ctenophora, pp. 58-62. New Yorkand London: McGraw-Hill.

ITO, S. (1965). The enteric surface coat on cat intestinal microvilli. J. Cell Biol. 27, 475-491.KING, B. F. & ENDERS, A. C. (1970). Protein absorption and transport by the guinea pig

visceral yolk sac placenta. Am. J. Anat. 129, 261-287.MCKANNA, J. A. (1973). Cyclic membrane flow in the ingestive-digestive system of peritrich

protozoans. I. Vesicular fusion at the cytopharynx. J. Cell Sci. 13, 663—675.MIGNOT, J.-P. (1966). Structure et ultrastructure de quelques Eugl^nomonadines. Protisto-

logica 2, 51-118.MIGNOT, J.-P. (1970). Remarques sur le deVeloppement du reticulum endoplasmique et du

systeme vacuolaire chez les Gymnodiniens. Protistologica 6, 267—281.MULLER, M., ROHLICH, P., TOTH, J. & TORO, I. (1963). Fine structure and enzymic activity

of protozoan food vacuoles. In Ciba Fdn Symp. on Lysozomes (ed. A. V. S. de Reuck &M. P. Cameron), pp. 201-216. London: Churchill.

PORTER, K. R., KENYON, K. & BADENHAUSEN, S. (1967). Specializations of the unit membrane.Protoplasma 63, 262-274.

RODEWALD, R. (1970). Selective antibody transport in the proximal small intestine of theneonatal rat. J. Cell Biol. 45, 635-640.

SAGEBIEL, R. W. & REED, T . H. (1968). Serial reconstruction of the characteristic granule ofthe Langerhans cell. J. Cell Biol. 36, 595-602.

SANGER, J. W. & MCCANN, F. V. (1968). Fine structure of the pericardial cells of the mothHyalophora cecropia, and their role in protein uptake. J. Insect Physiol. 14, 1839-1845.

SCHUSTER, F. L., HERSHENOV, B. & AARONSON, S. (1968). Ultrastructural observations onaging of stationary cultures and feeding in Ochromonas. J. Protozool. 15, 335-346.

SLAUTTERBACK, D. B. (1967). Coated vesicles in absorptive cells of Hydra. J. Cell Sci. 2,563-572-

STALEY, T. E., CORLEY, L. D., BUSH, L. J. & JONES, E. W. (1972). The ultrastructure of

neonatal calf intestine and absorption of heterologous proteins. Anat. Rec. 172, 559-580.TUSQUES, J. & PRADAL, G. (1969). Analyse tridimensionnelle des inclusions rencontr^es dans

les histiocytes de l'histiocytose ' x ' , en microscopie dlectronique. Comparaison avec lesinclusions des cellules de Langerhans. J'. Microscopie 8, 113-122.

WISSIG, S. & GRANEY, D. O. (1968). Membrane modifications in the apical endocytic complexof ileal epithelial cells. J. Cell Biol. 39, 564-579.

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(Received 9 April 1973)

684 J. A. McKanna

Figs. 1-6. Electron micrographs of E. plicatilis. Details noted are identical to thosefound in Vorticella and Zoothamnium.

Fig. i. Cup-shaped coated vesicles are situated adjacent to this young food vacuolecontaining ferritin and an intact bacterium, x 32000.

Fig. 2. As CSCV pinches off from food vacuole; the last point of attachment is nearthe lip of the cup (arrow). The mouth of the cup is < 50 nm diameter, and thus oneedge is contained within the 50-nm thin section, x 58000.

Figs. 3, 4. Continuity of the CSCV with a food vacuole (Jv), and membraneasymmetry are demonstrated, x 125000.

Fig. 5. Curvature of the vesicle obscures the details of coat structure in most places,but the peg-shaped elements and their association into pairs are apparent (arrow),x 125000.

Cup-shaped coated vesicles in peritrichs 685

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686 J. A. McKanna

Fig. 6. Section adjacent to a young food vacuole showing the tightly packed CSCVs,suggestive of the cloud obsen'ed in vivo. CSCVs are associated with the post-oral fibres(arrows), x 68000.