J. Cell Set. i 3 > 677-686 (1973)
Printed in Great Britain
677
CYCLIC MEMBRANE FLOW IN THE INGESTIVEDIGESTIVE 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 membrane
pinches off as cup-shaped vesicles which exhibit a structured coat on the non-cytoplasmic
surface of the membrane. As the membrane cycles from cup-shaped vesicles to diskoidal
vesicles to cytopharynx to food vacuoles, the coat undergoes structural transformations from
the 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 transformations in the digestive organelles of flagellate protozoa, Hydra absorptive cells, insect
pericardial cells, ileal absorptive cells of suckling rats, cells of the guinea-pig placenta, mammalian Langerhans cells, and macrophages. The similar functional situation in which these
coated membranes occur suggests that the coat is important to the recognition and binding of
macromolecules.
INTRODUCTION
Electron microscopy of peritrichs has revealed that the membranes of organelles
involved in food uptake and digestion have unique structural features which distinguish them from other membranes of the cell. The trilaminar membranes of the
cytopharynx, food vacuoles, and vesicles are asymmetric; and, in addition, an
organized coat is apparent on the non-cytoplasmic surface of the membranes in
certain situations. This type of membrane coat has been implicated in the recognition
and binding of macromolecules for pinocytotic uptake in Hydra (Slautterback, 1967);
and has been morphologically demonstrated on membranes in metazoan phagocytes
and absorptive cells. The present paper documents the involvement of cup-shaped
coated vesicles (CSCVs) in peritrich intracellular digestion, and reviews the functional
implications of comparative ultrastructural data on similarly coated membranes.
MATERIALS AND METHODS
Peritrichs 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 York
13210, U.S.A.
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J. A. McKanna
RESULTS
In addition to their association with the post-oral fibres and the cytopharynx as
documented in the preceding paper, the cup-shaped coated vesicles are found in the
immediate 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 some
cases (Figs. 2-4). Although many of the vesicles free in the cytoplasm appear as 2
concentric circles of membrane, consideration of the geometry of the vesicles has
convinced us that these profiles all represent various planes of section through cupshaped vesicles. The membrane forming the outer surface of the vesicle doubles back
upon itself at the lip of the cup's narrow mouth. The lumen of the cup is open to the
cytoplasm, and contains cytoplasmic matrix, ribosomes, glycogen, etc. The presence
of similar cytoplasmic material in most of the vesicles for which the section plane has
not 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) in
the immediate vicinity of the food vacuoles (Figs. 2, 4), somewhat wider in the cytoplasm and associated with the post-oral fibres (Fig. 6), and completely non-existent
when the cup-shaped vesicle flattens at the pharynx as demonstrated in the preceding
paper (McKanna, 1973). As is apparent in Figs. 3 and 4, the membrane of the CSCVs
exhibits the asymmetry characteristic of other membranes of the peritrich ingestivedigestive system. The cytoplasmic dense lamina is 4-0 nm thick, while the electronlucent lamina and non-cytoplasmic dense lamina are each 2-5 nm thick. We have
chosen to refer to cytoplasmic and non-cytoplasmic laminae in order to maintain
consistency for both intracellular and surface membranes.
In addition to the characteristic trilaminar asymmetry, however, the CSCV membrane exhibits further modification of the non-cytoplasmic dense lamina. This modification, customarily called a membrane coat, consists of electron-dense elements
apposed or attached to the non-cytoplasmic dense lamina. In micrographs of our
most favourably fixed and stained tissues, the electron-dense elements of the coat
appear to be 5-0 nm in diameter and 16 nm long. They may be associated into pairs
separated by a centre-to-centre distance of 10 nm (Fig. 5). The coat in this state is
referred to as being in the condensed configuration on the basis of its similarity to
the coat demonstrated in Hydra absorptive cells as discussed below (Slautterback,
1967).
The observation of CSCVs free in the cytoplasm and in continuity with the food
vacuole membrane leads to the question of whether they are fusing with or pinching
off from the vacuoles. The answer is not easily learned. Previous authors have
reported that the cup-shaped vesicles fuse with the food vacuoles (Goldfischer,
Carasso & Favard, 1963); and, as considered in the Discussion, we feel that such
fusion is certainly possible. Our own observations, however, strongly suggest that,
especially in the early stages of the digestive cycle, the predominant direction in the
CSCV—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 the
fusiform food vacuole reaches the base of the cell, it becomes spherical and begins to
condense to the volume of its particulate food. It subsequently enters the more central
streaming cytoplasm where further condensation leading to the first (acid) stage of
digestion occurs. When observed in the living cell, the condensing vacuole appears to
generate a halo or cloud around its periphery. The individual components of the
cloud, 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. 1
is less than 1 min old, since the organism had been placed in a ferritin solution for
1 min preceding fixation. The ingested bacterium is still intact. As noted above, cupshaped coated vesicles are present in the cytoplasm adjacent to the vacuole. These
vesicles appear to be the only candidates for the cloud components; and their size is
appropriate to our inability to resolve the individual cloud components in vivo.
Further suggestion as to the composition of the cloud derives from a section passing
adjacent to the surface of a young vacuole (Fig. 6). The CSCVs fill the region. In
addition, Fig. 6 demonstrates the association of the CSCVs with the post-oral fibres
as discussed in the preceding paper (McKanna, 1973).
DISCUSSION
At various times in the digestive cycle, the membrane limiting a food vacuole will
be in excess of that necessary to bound the vacuolar contents. In the proposed model
of peritrich cyclic membrane flow, cup-shaped coated vesicles remove excess foodvacuole membrane for transport back to the cytopharynx. The first instance in which
excess membrane might be expected arises with the transition in vacuolar shape from
fusiform 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 and
condenses to approximately the volume of its contained particulate material, its
surface undergoes a proportionate reduction, usually greater than 50%. The shape
transition and condensation occur during the period when a cloud appears to emanate
from the vacuole.
It has been recognized for some time that discrete stages of the digestive cycle in
ciliates involve further alteration of the volume and surface area of the vacuoles
(Hyman, 1940). By the end of the first stage (condensation) described above, the
vacuole contents have become acidic, but the particles (bacteria) retain their discrete
form. In the second stage, the vacuole grows to slightly less than its original size, it
becomes alkaline, and the bacteria disintegrate. In the third stage, the vacuolar
volume is reduced again and its pH returns to neutrality. At the conclusion of this
final condensation, the residual vacuolar contents are defaecated by exocytosis at the
cytopyge. During the second stage, vacuolar expansion and pH rise are accompanied
by fusion of primary lysosomes with the vacuole (Miiller, Rohlich, Toth & Toro,
1963). Although conclusive evidence is not available on this point, it seems possible
that the fusing lysosomal membrane adds to the surface membrane of the food vacuole.
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J. A. McKanna
Indeed, since lysosomal enzymes are transported within endoplasmic reticulum or
Golgi membranes (Miiller et al. 1963; Goldfischer et al. 1963), it is possible that this
is the point of addition of membrane to an otherwise closed cycle.
In the final condensing phase, a reduction in vacuolar volume results from the
pinching off of small vesicles filled with digested material (Favard & Carasso, 1964).
According to these investigators, the molecular contents of the vesicles pass out
through 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 cannot
add substantially to the evidence for this hypothesis, it is consistent with the model
of coat transformations (condensed/extended) as discussed below.
Since current methods are incapable of denning absolutely the direction of net
flow, even less certainty may be ascribed to the direction of an isolated individual
event. Thus it is possible that any micrograph presented to demonstrate the evolution
of a small vesicle from a food vacuole (Favard & Carasso, 1964) may have recorded
an unlikely instance in which the vesicle was fusing with the vacuole. Similarly, an
individual CSCV shown in membranous continuity with a food vacuole may actually
have been in the process of fusion, as reported by Goldfischer et al. (1963). Knowledge
of the forces involved in membrane fusion is insufficient to eliminate the possibility
that a CSCV could collide with a food vacuole and fuse with it. Our data strongly
suggest, however, that the net flow of CSCVs is from the food vacuoles toward the
cytopharynx.
Two features of the cup-shaped coated vesicles seem especially appropriate to
their functional role in membrane transport. The first arises from consideration of
the shape of a vesicle relative to its movement through the cytoplasm. Although the
amount of membrane surface in a CSCV 0-25 fim in diameter is identical to that
in a disk of 0-42 /an diameter, the frictional coefficient of the CSCV would be less
than half that of the disk (Bull, 1964). This physical consequence of a change in shape
may be important to the flow of cup-shaped vesicles toward the cytopharynx and
the accumulation of diskoidal vesicles in the peripharyngeal cytoplasm.
The second feature is that the cupped-shape allows reduction of the vesicle lumen
to a volume just adequate to accommodate the coat. The vesicle is, otherwise, effectively empty. Although Favard & Carasso (1964) reported the presence of Thorotrast
in some CSCVs, the amount of label in the CSCVs was miniscule relative to the
concentration in the food vacuoles; and we have been unable to demonstrate tracer
uptake into CSCVs in any of our peritrichs fed ferritin or Thorotrast. This observation is also compatible with the model of coat function as discussed below.
Data on the modified non-cytoplasmic dense lamina of the peritrich ingestivedigestive system membranes are complementary to reports on other systems in other
organisms. The highly ordered coat found on the cup-shaped vesicles is similar to
membrane coats observed in a variety of cells with active protein uptake systems;
and we feel that these comparative data are especially interesting regarding the basic
mechanisms of macromolecular recognition by cells.
The best preservation and most detailed examination of this type coat has been
in 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 in
the cytoplasm and at the luminal surface. The basic subunit of the coat has dimensions
of 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 vesicles
are involved in the pinocytotic uptake of macromolecules from the digested food in the
gastrocoel; and as the vesicle membrane follows its functional path, the configuration
of the coat undergoes changes similar to those observed in peritrichs. In the first
stage of the functional sequence, the empty vesicles in the apical cytoplasm exhibit
the condensed form of the coat as described above. Then, as digestion proceeds in
the gastrocoel, these vesicles fuse with the apical plasmalemma and the coat assumes
the appearance of extended filaments similar to the glycocalyx demonstrated on the
microvilli of intestinal absorptive cells (Ito, 1965). In this form, the coat appears to
bind specific food molecules (as demonstrated by the binding of ferritin). The membrane with bound macromolecules subsequently pinches off back into the cytoplasm;
and in this position the coat appears to resume its condensed conformation and
release the food into the lumen of the vesicle.
The condensed form of the coat in the peritrich CSCVs and that in the Hydra
diskoidal vesicles are structurally similar; and we feel that certain aspects of the coat
transformations may show functional similarity. Favard & Carasso (1964) demonstrated the extended form of the coat on the membrane of the food vacuoles and
micropinocytotic vesicles observed in the third stage of digestion. In terms of the
model of coat function, the extended form of the coat serves to select macromolecules
from the digested brei in the vacuole. Once the pinocytotic vesicles pinch off from
the food vacuole, the coat reverts to the condensed form, releasing the molecules to
diffuse into the cytoplasm. The other situation in which the importance of coat
conformation may be appreciated involves the CSCVs pinching off from the food
vacuoles. The condensed coat prevents macromolecular binding and thereby allows
generation of a ' clean' membrane for return to the cytopharynx.
Studies in flagellate protozoa have suggested that a similar coat may be involved
in the uptake of nutrients by these organisms. Investigation of intracellular digestion
in the Euglenida led to the discovery of vesicles composed of membrane with the
condensed form of the coat associated with the digestive vacuoles in Entosiphon
sulcatwn (Mignot, 1966). Later, in the dinoflagellate, Gymnodinium, Mignot (1970)
described the pusule, a large fluid-filled vacuole in which digestion occurs. He
demonstrated membranous tubules confluent with the pusule; and showed that the
membrane of the tubules possesses a non-cytoplasmic coat similar to that on the
peritrich CSCVs. We believe that this coat may be involved in the selection of nutrient
molecules from the pusule fluid. Tubules of similar dimensions, but which appear
to have a coat in the extended conformation, were reported open to the digestive
vacuoles in another flagellate, Ochromonas (Schuster, Hershenov & Aaronson, 1968).
Experimental studies in higher organisms also have suggested that the membrane
coat is involved in recognition and uptake of macromolecules. A similar coat is present
on 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; and
Sanger & 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; and
the functional correlations in these systems are most intriguing because of their
implications for immunology. The coat is prominent on membranes forming the
apical endocytic complex (tubular imaginations of the apical plasmalemma) of
absorptive 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 jejunum
in these animals (Rodewald, 1970; J. W. Anderson, personal communication). At
this stage of development, 2 functions of the alimentary canal are separated along its
length. Proximally, in the duodenum and jejunum, maternal antibodies are absorbed
from the milk and transferred intact into the pup's circulation (Brambell, 1970). By
means of ferritin-labelled antibody, it has been demonstrated that antibody is bound
to the extended coat on the apical plasmalemma (Rodewald, 1970). The antibody
subsequently is taken into the cell by pinocytosis, and finally released into the extracellular space. In Rodewald's micrographs, it appears that the antibody does not bind
to regions of the plasmalemma where the coat is in the condensed configuration.
The other process, which occurs predominantly in the ileum, involves uptake and
intracellular digestion of proteins from the milk. The pattern of structure and function
in the ileal cells is very similar to Hydra, since protein from the intestinal lumen is
taken up by pinocytosis into coated vesicles. The protein then is transferred to a
large central vacuole which has been shown to contain active hydrolytic enzymes
(Cornell & Padykula, 1969). Similar extended and condensed forms of the coat are
apparently involved in protein absorption and intracellular digestion in the intestinal
absorptive cells of such diverse species as the goldfish (Gauthier & Landis, 1972) and
the neonatal calf (Staley, Corley, Bush & Jones, 1972).
A non-cytoplasmic coat exhibiting condensed and extended conformations also is
present on the membranes of vesicles active in protein absorption and transport in
cells of the guinea-pig yolk sac placenta (King & Enders, 1970). These authors also
demonstrated that the coat binds peroxidase and ferritin; but, like Hydra, does not
bind a saccharide like Thorotrast.
A peg-like condensed coat has been observed on surface membranes and diskoidal
vesicles (' 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 ultrastructural characteristics suggest that they may be a specialized type of macrophage.
In preliminary studies, we have found that condensed and extended forms of the
coat are present on vesicles and surface membranes of lymph node macrophages in
the squirrel monkey following antigenic insult (McKanna, unpublished).
The similarity of ultrastructural data on membrane coats from such diverse
organisms supports the hypothesis that these coats are involved in basic biological
phenomena 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 investigation of the components and properties of membrane coats.
The author is grateful to Dr David B. Slautterback for his valuable advice and encouragement. 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 the
suckling rat. Am. J. Anat. 125, 291-315.
FAVARD, P. & CARASSO, N. (1964). Etude de la pinocytose au niveau des vacuoles digestives de
cili£s P6ritriches. J. Microscopie 3, 671-696.
GAUTHIER, G. F. & LANDIS, S. C. (1972). The relationship of ultrastructural and cytochemical
features to absorptive activity in the goldfish intestine. Anat. Rec. 17a, 675-702.
GOLDFISCHER, S., CARASSO, N. & FAVARD, P. (1963). The demonstration of acid phosphatase
activity 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 York
and 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. Protistologica 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 the
neonatal rat. J. Cell Biol. 45, 635-640.
SAGEBIEL, R. W. & REED, T . H. (1968). Serial reconstruction of the characteristic granule of
the Langerhans cell. J. Cell Biol. 36, 595-602.
SANGER, J. W. & MCCANN, F. V. (1968). Fine structure of the pericardial cells of the moth
Hyalophora cecropia, and their role in protein uptake. J. Insect Physiol. 14, 1839-1845.
SCHUSTER, F. L., HERSHENOV, B. & AARONSON, S. (1968). Ultrastructural observations on
aging 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,
563-572STALEY, T . E., CORLEY, L. D., BUSH, L. J. & JONES, E. W. (1972). T h e 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 les
inclusions des cellules de Langerhans. J'. Microscopie 8, 113-122.
WISSIG, S. & GRANEY, D. O. (1968). Membrane modifications in the apical endocytic complex
of ileal epithelial cells. J. Cell Biol. 39, 564-579.
WOLFF, K. (1967). The fine structure of the Langerhans cell granule. J. Cell Biol. 35, 468-473.
(Received 9 April 1973)
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Figs. 1-6. Electron micrographs of E. plicatilis. Details noted are identical to those
found in Vorticella and Zoothamnium.
Fig. i. Cup-shaped coated vesicles are situated adjacent to this young food vacuole
containing ferritin and an intact bacterium, x 32000.
Fig. 2. As CSCV pinches off from food vacuole; the last point of attachment is near
the lip of the cup (arrow). The mouth of the cup is < 50 nm diameter, and thus one
edge is contained within the 50-nm thin section, x 58000.
Figs. 3, 4. Continuity of the CSCV with a food vacuole (Jv), and membrane
asymmetry 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
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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.