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J. Cell Sci. 14, 611-63 1 (1974) 6l I Printed in Great Britain THE CYTOPROCT OF PARAMECIUM CAUDATUM: STRUCTURE AND FUNCTION, MICROTUBULES, AND FATE OF FOOD VACUOLE MEMBRANES RICHARD D. ALLEN AND R. W. WOLF The Pacific Biomedical Research Center and Department of Microbiology, University of Hawaii, Honolulu, Hazvaii 96822, U.S.A. SUMMARY The cytoproct or cell anus of Paramecium caudatum was studied, using light optics and electron microscopy, at known times before, during and following food vacuole egestion. This was accomplished by microscopically observing single cells, fixing these cells at specific times and finally serial sectioning these individually processed cells. The cytoproct, at rest, is a long narrow ridge along the posterior suture. It contains 2 uniquely positioned components which identify this structure as the cytoproct: piles of fibres along the inside surfaces of the ridge, and microtubules passing from the epiplasm at the summit of the ridge down into the cytoplasm. The plasma membrane is continuous over the top of the ridge. The cortical basal bodies adjacent to the ridge have bundles of microtubules passing into the cytoplasm from an opaque plaque at their proximal ends. These 2 sets of microtubules may function in guiding the food vacuolcs to the cytoproct. A model is presented in which motive forces generated between the microtubules and the food vacuole membrane bring the food vacuole to the cytoproct and, in addition, pull the cytoproct lips apart so that the food vacuole membrane and plasma membrane come into contact and fuse together, thus opening the food vacuole to the outside. The plasma membrane increases in area between the parting lips, possibly, as the result of intercalation of membrane vesicles into the plasma membrane at the top of the ridge. Immediately after this opening is formed the food vacuole membrane changes from a smooth topography to a highly convoluted one. The membrane is engulfed through a process of endocytosis resulting in an accumulation of membranous fragments in the cytoplasm below the cytoproct. The endocytic forces probably bring about the restitution of the cytoproct ridge by pulling the lips back together as the membrane is engulfed. A filamentous meshwork underlying the food vacuole membrane may be active in this endocytic process. INTRODUCTION The process of egestion in ciliated protozoa has received little attention in recent years, even though use of the transmission electron microscope has made it potentially possible to study this normal cell function at a level not permitted by light microscopy. The few electron micrographs of thin-sectioned cytoprocts which have appeared in the literature have usually constituted only a small part of a larger study of the general morphology of a particular ciliate or group of ciliates. This is true of the studies on Paramecium aurelia (Jurand, 1961) and Paramecium caudatum (Ehret & McArdle, 1974); the gymnostomes Alloizona and Didesmis (Grain, 1966), as well as Pseudomicro- thorax (Peck, 1971); the trichostome Paraisotricha (Grain, 1966); the scuticociliate Dexiotricha (Peck, 1971); and the thigmotrich Conchophthirus (Antipa, 1971). Two ,|O C I! I. 14

THE CYTOPROCT OF PARAMECIUM CAUDATUM ...612 R. D. Allen and R. W. Wolf other studies specifically on the cytoproct of Paramecium, that on P. aurelia (Schneider, 1964) and P. caudatum

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Page 1: THE CYTOPROCT OF PARAMECIUM CAUDATUM ...612 R. D. Allen and R. W. Wolf other studies specifically on the cytoproct of Paramecium, that on P. aurelia (Schneider, 1964) and P. caudatum

J. Cell Sci. 14, 611-63 1 (1974) 6l IPrinted in Great Britain

THE CYTOPROCT OF PARAMECIUM

CAUDATUM: STRUCTURE AND FUNCTION,

MICROTUBULES, AND FATE OF FOOD

VACUOLE MEMBRANES

RICHARD D. ALLEN AND R. W. WOLF

The Pacific Biomedical Research Center and Department of Microbiology,University of Hawaii, Honolulu, Hazvaii 96822, U.S.A.

SUMMARY

The cytoproct or cell anus of Paramecium caudatum was studied, using light optics andelectron microscopy, at known times before, during and following food vacuole egestion. Thiswas accomplished by microscopically observing single cells, fixing these cells at specific timesand finally serial sectioning these individually processed cells. The cytoproct, at rest, is a longnarrow ridge along the posterior suture. It contains 2 uniquely positioned components whichidentify this structure as the cytoproct: piles of fibres along the inside surfaces of the ridge,and microtubules passing from the epiplasm at the summit of the ridge down into the cytoplasm.The plasma membrane is continuous over the top of the ridge. The cortical basal bodiesadjacent to the ridge have bundles of microtubules passing into the cytoplasm from an opaqueplaque at their proximal ends. These 2 sets of microtubules may function in guiding the foodvacuolcs to the cytoproct. A model is presented in which motive forces generated between themicrotubules and the food vacuole membrane bring the food vacuole to the cytoproct and, inaddition, pull the cytoproct lips apart so that the food vacuole membrane and plasma membranecome into contact and fuse together, thus opening the food vacuole to the outside. The plasmamembrane increases in area between the parting lips, possibly, as the result of intercalation ofmembrane vesicles into the plasma membrane at the top of the ridge. Immediately after thisopening is formed the food vacuole membrane changes from a smooth topography to a highlyconvoluted one. The membrane is engulfed through a process of endocytosis resulting in anaccumulation of membranous fragments in the cytoplasm below the cytoproct. The endocyticforces probably bring about the restitution of the cytoproct ridge by pulling the lips backtogether as the membrane is engulfed. A filamentous meshwork underlying the food vacuolemembrane may be active in this endocytic process.

INTRODUCTION

The process of egestion in ciliated protozoa has received little attention in recentyears, even though use of the transmission electron microscope has made it potentiallypossible to study this normal cell function at a level not permitted by light microscopy.The few electron micrographs of thin-sectioned cytoprocts which have appeared inthe literature have usually constituted only a small part of a larger study of the generalmorphology of a particular ciliate or group of ciliates. This is true of the studies onParamecium aurelia (Jurand, 1961) and Paramecium caudatum (Ehret & McArdle,1974); the gymnostomes Alloizona and Didesmis (Grain, 1966), as well as Pseudomicro-thorax (Peck, 1971); the trichostome Paraisotricha (Grain, 1966); the scuticociliateDexiotricha (Peck, 1971); and the thigmotrich Conchophthirus (Antipa, 1971). Two

,|O C I! I. 14

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612 R. D. Allen and R. W. Wolf

other studies specifically on the cytoproct of Paramecium, that on P. aurelia (Schneider,1964) and P. caudatum (Esteve, 1969), made no systematic attempt to follow themorphological variations which this structure exhibits during the excretory process.Foissner (1972) has recently provided an extensive light-microscope study of thecytoproct of Uronema and a review of the literature on this structure.

This situation, in which only a few randomly acquired electron micrographs ofcytoprocts in several ciliates at unknown functional stages have been published, hasled to a general confusion about the fine structure of the cytoproct. This same situationhas also helped to obscure a functional explanation at the ultrastructural level of theactivity of the cytoproct. The present work describes a study in which single Para-mecium caudatum cells were observed and fixed at known times with regard to thetime of food vacuole defaecation. These individual cells were then serially sectionedand their cytoproct regions carefully identified and studied. From this study theultrastructural morphology of both resting and active cytoprocts will be described.The method by which the food vacuole finds the cytoproct and the disposition of thefood vacuole membrane will also be discussed.

MATERIALS AND METHODS

Selection of cells to be studied

Paramecium caudatum, growing either in hay infusion or lettuce infusion medium, was usedin this study. Individual cells were isolated from the culture and observed in a wet mountpreparation, without a coverglass, under the low power of a compound microscope. A drop ofmethyl cellulose was added to the slide to retard the swimming movements of the cell. Justbefore food vacuole release, it was observed that the cell becomes distended in the region ofthe cytoproct. Some cells were fixed at this stage. This was performed by flooding the slidewith buffered fixative. The moment of food vacuole release could also be observed by thismethod; other cells were fixed immediately following the observation of the release of foodvacuole contents through the cytoproct. Cells were also fixed at intervals of 3, 10 and 30 s and1 min following the opening of the cytoproct. Only cells whose cytoprocts could be identifiedand the time of cytoproct opening clearly established were used for the last 4 fixation times.Following fixation, individual cells were passed separately through the sequence of washing,dehydration, and embedding by transferring the single cells with a small-bore pipette and byusing a dissecting microscope to locate the cell.

Preparation for electron microscopy

Cells were fixed for 15 min in either 2 or 4 % glutaraldehyde buffered with either 005 M oro-i M collidine at pH 7-4. The compactness of the cell's cytoplasm can, to some extent, bealtered by using fixatives of different osmolalities. The cells were then washed in buffer andpostfixed in 1 % or 2 % OsO4. After washing, the cells were placed for 15 min in 0-5 % aqueousuranyl acetate stain. This was followed by washing in distilled water and then dehydrating ina series of increasing concentrations of ethanol and finally 100% propylene oxide. Cells wereflat embedded in Epon 812. Following evacuation and hardening, the cells were cut out of theblock and glued to another polymerized preshaped block. These cells were serially sectionedin a longitudinal orientation and all of the sections were picked up on Formvar-supported gridshaving one large opening. A diamond knife and Sorvall MT-11 ultramicrotome were used forsectioning. Sections were then stained with uranyl acetate (Watson, 1958) and lead citrate(Reynolds, 1963) for times which varied from 15 s to 5 min in each stain. A Hitachi HU-11Aelectron microscope operated at 75 kV was used throughout the study.

During sectioning the exact number of sections on each grid was recorded and the grids

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The cytoproct of Paramecium 613

were kept in their correct sequence. This permitted the positive identification of the cytoproctin each cell by making it possible to relate it to the overall morphology of that cell and it alsopermitted the cytoproct to be studied from its anterior to its posterior extremities. Serialsectioning of preoriented cells has the advantage of allowing the observer to know the orientationand precise position of each section in the cell, even though a 3-dimensional reconstruction isnot attempted and electron micrographs of all sections are not taken.

Evaluation of techniques

The techniques used in handling and fixing the individual cells caused some damage anddistortion both to the cell surface and to the internal cytoplasm. However, these disadvantageswere judged to be more than compensated by the advantages of the techniques. The outer 2pellicular membranes frequently break away during the handling procedures, while thecollidine-buffered fixative causes most trichocysts to discharge internally with some consequentdistortion of large cytoplasmic organelles and some localized disarrangement of the cytoplasm.On the other hand, by handling individual cells we were able to study cells at a known stageof cytoproct function. Also, the collidine-buffered fixative preserves microtubular, fibrous andfilamentous cellular details in such a manner as to make them more easily recognizable in thin-sectioned cells (Allen, 1971).

RESULTS

The cytoproct was always found to be located, as reported in light-microscopestudies, on the ventral side of the organism and posterior to the opening to the oralregion. In longitudinal thin sections the posterior end of the cell can always be readilyidentified by observing the attachment of the kinetodesmal fibres to the basal bodies;these fibres always extend more-or-less anteriorly from the basal bodies. The ventralside of the organism can be identified by the opening to the oral region, and the dorsalside by the contractile vacuole pores. Knowing these landmarks, one can find thecytoproct in thin sections even when the cytoproct is closed, the period during theegestion cycle when it is least conspicuous.

The closed cytoproct

In thin sections the closed or resting cytoproct (Fig. 2) can be identified as a longnarrow ridge running in an anterior-posterior direction. Its width near its apex(Figs. 3, 4) is about o-2/tm and its overall length is 12/*m or more. This cytoproctridge forms a straight line along the posterior suture and is continuous with the inter-secting ridges (t) of adjacent cortical units (Fig. 2).

This ridge is, at low magnification, not easily distinguished from other thin-sectioned somatic cortical ridges. However, there are significant differences in thecytoproct ridge which, when seen, allow its immediate identification as the closedcytoproct. The presence of an underlying food vacuole is not a necessary criterion foridentifying the cytoproct, since at this resting stage a food vacuole may not be closeto the cytoproct. This ridge is devoid of such components as kinetodesmal fibres andthe 2 fibrillar systems, the infraciliary lattice and the striated bands, which are nor-mally found within or underlying other somatic ridges (Allen, 1971). Trichocysts,although usually absent, have been observed within the connecting transverse ridgesvery near to the cytoproct ridge. Alveolar sacs are found overlying and passing up the2 sides of the ridge to the summit (Figs. 3, 8, 10, a). The plasma membrane covers

40-2

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614 R. D. Allen and R. W. Wolf

these alveolar sacs and is continuous over the narrow, 20-50 nm, gap between thesacs at the peak of the ridge (Figs. 3, 10, g). A layer of epiplasm 15 nm thick covers thecytoplasmic surfaces of the alveolar sacs but does not appear to cross under this gap.

Two uniquely arranged structures are found within this cytoproct ridge. They area group of microtubules and a pair of short electron-opaque fibrous mats. These fibrescover the epiplasm from the base of the ridge to its summit on both sides of the ridgeand form a coating like the pile of a carpet (Figs. 2, 3, 4, 5, 8, 13,/). These fibrousunits are each of the order of 14 nm in diameter at their base and are at least 60 nmlong. They may fray out into smaller fibrils at their free end. In a cross-section of aridge the fibres appear tilted, so that their free ends are closer to the ridge summitthan their attached ends which lie in the epiplasm. Passing up the middle of the ridgefrom the cytoplasm to the summit are microtubules, 24 nm in diameter (Figs. 4,5, cm). These microtubules appear to insert in the epiplasm, some on each side of thegap at the very top of the ridge. Their opposite ends pass into the cytoplasm for anunknown distance (Fig. 4). These cytoproct microtubules do not appear to be asso-ciated together into bundles or sheets but apparently remain separated over theirlengths. A meshwork of very thin filaments fills the middle of the ridge between the2 piles of fibres and around the microtubules (Figs. 3, 5, 13, fm).

One other specialized feature of the cytoproct area is found in the cytoplasm adjacentto the cytoproct ridge. The basal bodies lining the 2 sides of the cytoproct ridge oftendo not bear cilia. This was previously observed by Ehret & McArdle (1974). However,these basal bodies do have bundles of up to 15 microtubules arising from their proximalends and extending into the cytoplasm (Figs. 2, 6, 8). The microtubules in this bundlemay be held together by cross bridges (Fig. 7). They begin next to the basal body inan electron-opaque plaque which lies against the outer margin of the cartwheel portionof the basal body, on its right anterior quadrant. These are probably not modifiedtransverse microtubules, which also come from this same position next to the basalbody, since both transverse microtubules and bundles of microtubules can sometimesbe observed arising from the same basal body. Their length has not been determined,although they are at times at least 2-5 /tm long; they may be much longer. When afood vacuole is located next to the cytoproct, these bundles of microtubules can beseen to course over the food vacuole membrane in very close association with thismembrane (Figs. 6, 7). Electron-opaque connexions are occasionally seen apparentlylinking these microtubules with the food vacuole membrane (Fig. 6, inset).

The distended cytoproct prior to egestion

In the period just before food vacuole defaecation, the pellicular region around thecytoproct can be seen, in the light microscope, to bulge out from the cell surface. Cellsfixed at this stage always have a food vacuole in close proximity to the cytoproct. Thisfood vacuole is frequently elongated, with one or two annular constrictions, suggestingthat it is the product of a fusion of two or three food vacuoles; Foissner (1972) reportsa similar observation in a light-microscope study of Uronema. The food vacuole inFig. 9 which appears to be a product of the fusion of 3 smaller food vacuoles is 13 /*mlong and 4-4 /«m in diameter. The membrane-lined margins of these intact food

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The cytoproct of Paramecium 615

vacuoles are relatively smooth, with little sign of convolution except for the marginclosest to the cytoproct. Here the food-vacuole membrane may be drawn out intolong tubular evaginations which are often associated with microtubules (Fig. 8, arrows).This whole complex composed of microtubules and food vacuoles always projectsaway from the cytoproct region in an anterior direction.

The food vacuole margin closest to the cytoproct comes to lie very near to theplasma membrane which, in turn, covers the top of the cytoproct ridge. In order forthis to happen, the cytoproct ridge appears to widen and flatten (Fig. 10). The twohalves of the ridge, which can still be identified by their unique fibres (/), spreadapart (Figs. 11, 12) and the plasma membrane surface area increases between theselips of the cytoproct ridge. The food vacuole membrane and plasma membrane arenow separated by only a narrow gap (Figs. 11, 12, arrowheads). This close approachoccurs first along only a small fraction of the length of the cytoproct but, beforeegestion occurs, much of its length lies next to the food vacuole membrane. In theone cell in which the cytoproct ridge was just beginning to flatten and spread (Figs.10-13), this portion was found to be close to the middle of the long axis of thecytoproct.

Within the cytoplasm, adjacent to the region of the flattening cytoproct ridge,membrane-limited vesicles may be found (Fig. 13, v). At times these vesicles (v)appear to be drawn up into the partly flattened ridge (Fig. 10). Microtubules, probablyfrom the ridge summit, can usually be found close to these vesicles. These vesiclesvary in size and outline but always appear flat and relatively empty of electron-opaquematerial.

The cytoproct during egestion

Egestion occurs over a short period of time and can be observed either as the releaseof a ball of undigested material or as a streaming of this material from the cytoproct.Prior to egestion the cytoproct ridge has parted along the greater portion of its length.The edges of this ridge are always recognizable by their pile of fibres. The plasmamembrane (pm) becomes continuous with the membrane of the evacuating foodvacuole (fvm) (Figs. 14, 15). No breaks in the covering membrane system weredetected which could allow the underlying cytoplasm to come into direct contactwith the outside environment. The food vacuole appears to be deflated and its marginscome close together so as to resemble an inelastic bag which has been flattened (Fig.14). However, the membrane of the food vacuole (fvm) is no longer smooth but hasassumed a highly irregular surface, with many short invaginations into the cytoplasm(Figs. 14, 15). These invaginations are surrounded by a highly filamentous cytoplasm(Figs. 15, 16, fc). The cytoproct microtubules which are still found at the ridge lipspass along the inward-directed margins of these filament-enclosed invaginations ofthe food vacuole membrane (Fig. 16). The whole complex continues to maintain itsanteriorly-directed orientation.

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616 R. D. Allen and R. W. Wolf

Three seconds after egestion

At this time the deep invagination of the food vacuole is still evident but the outlineof the food vacuole membrane appears to have a greater irregularity (Fig. 17) than atthe moment of egestion. Also an increased number of flattened vesicles and tubules(arrows) are present in the cytoplasm surrounding the evacuated food vacuole.

Ten to thirty seconds after egestion

The time of recovery of the cytoproct seems to vary from organism to organismand may vary from evacuation to evacuation within a single cell as well. Thus, theobserved state of recovery of the cytoproct of one cell does not necessarily resemblethat of other cells following identical periods of post-egestion time. At some point intime, between 10 and 30 s after its opening, the cytoproct no longer displays a deepinvagination into the cell but the opening is covered over with a single membranewhich stretches between the 2 edges (cl) of the cytoproct ridge (Fig. 18). This mem-brane is not smooth but has an irregular surface with many short invaginations whichare surrounded by a microfilamentous material (fc) that excludes other structuresfrom the region next to the membrane (Fig. 20). Occupying the cytoplasmic spaceimmediately under this membrane are a number of both flattened and tubular vesiclesinterspersed with microtubules (Figs. 18, 19). This mass of microtubules and vesiclesprojects anteriorly. In some cells at this stage the 2 edges of the cytoproct appear,along most of their lengths, to be closely appressed together and are, in all respects,identical to the resting cytoproct ridge. In one cell which was fixed 10 s after egestion,the cytoproct was closed over its posterior half and open for only a short distance inits anterior part. The closed portion of the cytoproct contained the fibrous mats andthe microtubules passing up to its summit. The bundles of microtubules which arisefrom the basal bodies adjacent to the cytoproct were also evident.

Another food vacuole may already be positioned below the cytoproct region at thisstage. It has not been determined if the cytoproct must be completely closed beforeanother round of egestion can occur, although this does not seem likely, since foodvacuole release has been observed in the light microscope within 15 s of the precedingegestion.

One minute after egestion

By 60 s the 2 cytoproct lips are back together along their full length. The cytoproct,at the ultrastructural level, can no longer be distinguished from any other non-func-tioning cytoproct. Its morphology is identical to that described under a previousheading, The closed cytoproct.

DISCUSSION

Comparative morphology

It is evident from this study that the morphology of the cytoproct varies with itsfunctional state. The failure to recognize this fact has led to some confusion in the

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The cytoproct of Paramecium 617

literature, some of which, and in particular that involving Paramecium, can now beresolved. In most cases the previously published pictures of structures reported to becytoprocts oi Paramecium have been of active stages of this structure. Schneider (1964)shows cytoprocts which are at an early stage of opening, at which the lips of thecytoproct have separated prior to egestion. Ehret & McArdle (1974) show later stagesfollowing egestion in which the food vacuole membrane is highly convoluted. Theirscanning electron micrograph shows a resting or closed cytoproct. The pictures ofEsteve (1969), which are reported to be from the same organism as used in the presentstudy, do not resemble the cytoproct described here and are certainly pictures ofcontractile vacuole pores. The one picture reported to be of a cytoproct published byJurand (1961) may indeed be of the cytoproct but, if so, the cell is damaged and themembranes in this region have broken. Our study shows that the cytoproct opens asthe result of the fusion of the plasma membrane with the food vacuole membrane andnot by tearing of one or both membranes.

Whether the cytoprocts of other ciliates resemble those of Paramecium remains tobe studied. However, the available electron micrographs of other ciliate cytoproctssuggest that at least some do. In particular, the micrograph of the cytoproct of thegymnostome Pseudomicrothorax (Peck, 1971) looks very much like the closed cyto-proct of Paramecium. However, the cytoprocts of the two gymnostomes and thetrichostome described by Grain (1966) and the thigmotrich of Antipa (1971) appearto resemble each other and to be unlike Paramecium, since they have microtubulesunderlying the plasma membrane of the cytoproct in a fashion more like that of thecontractile vacuole pore of Paramecium than of its cytoproct.

The function of the cytoproct

By using the information obtained from the electron micrographs of organismswhich are in known states of cytoproct activity, a correlated functional-structuralsequence can be deduced. As the food vacuole approaches the cytoproct region, thefirst change in the resting cytoproct (Fig. 1 A) involves a flattening of the ridge and aparting of the lips of the cytoproct (Fig. 1 B). The plasma membrane remains intactand continuous between the separating lips. Thus either new plasma membrane isformed by the intercalation of membrane vesicles or membrane subunits into themembrane covering the cytoproct ridge or, alternatively, there is a flow of plasmamembrane from adjacent pellicular regions into this area of the opening cytoproct.The presence of membrane-limited vesicles very close to the opening-cytoproct regionleads us to favour the notion of membrane growth by intercalation of vesicles.

The topography of the plasma membrane and food vacuole membrane are bothrelatively smooth at this time and they come to lie very close to each other. In thelight microscope one can see an overall bulging of the pellicle surrounding, andincluding, the opening cytoproct. At the actual moment of egestion the 2 apposedmembranes must fuse (Fig. 1 c) and the contents are then released either as a non-membrane-limited faecal ball or as a stream of undigested debris. The compactnessof the faecal material is dependent on the kinds of materials on which the organismwas feeding at the time of food vacuole formation. The characteristics of this faecal

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618 R. D. Allen and R. W. Wolf

material can be altered experimentally by changing the environment of the organism.The forces involved in causing the parting of the cytoproct lips, the fusion of themembranes and the expulsion of the faecal material can only be hypothesized. How-ever, it seems reasonable that the internal cell pressure exerted on the cytoplasmicsurface of the food vacuole membrane, once the opening to the outside is formed,causes the flattening of the food vacuole and subsequent expulsion of the contents.

w.i-,.-.-'/m

Fig. i A-D. Three-dimensional drawings each depicting a short segment of the cyto-proct at a different moment in its functional cycle. A, a resting cytoproct with a foodvacuole close by. B, the stage just before egestion in which the cytoproct ridge has beenpulled out and flattened and the food vacuole membrane and plasma membrane lieclose together, c depicts the opened cytoproct and shows the food vacuole membrane,which is now continuous with the plasma membrane, undergoing endocytosis.Finally, in D, most of the food vacuole membrane has been engulfed by the cell andthe cytoproct opening is covered by a single convoluted membrane. Subsequent tothis state the cycle will be completed and the cytoproct will return to its resting shapeas in the first drawing. For further explanation see text, a, alveolus; 66, basal body;bm, microtubular bundle; cm, cytoproct microtubules; / , cytoproct fibres; /c, fila-mentous cytoplasm; fv, food vacuole; pm, plasma membrane; v, vesicle.

This would be comparable to the previously proposed mechanism for the expulsionof water from water-expulsion vesicles (contractile vacuoles) of ciliates (Organ, Bovee,Jahn, Wigg & Fonseca, 1968; Organ, Bovee & Jahn, 1972).

In order for the cytoproct to close, it appears that the food vacuole membrane and'excess' plasma membrane must first be removed. This takes place through an endo-

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The cytoproct of Paramecium 619

cytic engulfment of the membranes (Fig. 1 c). Immediately following food vacuoleegestion the once relatively smooth membrane of the undischarged food vacuolebecomes highly convoluted and numerous tubular profiles can be seen along its surfaceextending into the adjacent cytoplasm. A fine filamentous meshwork surrounds thecytoplasmic surface of these tubular invaginations. This meshwork resembles thethin filamentous material found in the vicinity of pinocytic channels of the giantamoeba Chaos chaos (Nachmias, 1964; Marshall & Nachmias, 1965). Within a periodof 10-30 s all of the food vacuole membrane has been taken into the cytoplasm in theform of elongated tubules and flattened vesicles and the cytoproct opening is nowcovered with a highly convoluted plasma membrane stretching from one cytoproctlip to the opposite lip (Fig. 1 D). AS time goes on this covering membrane is alsoengulfed in apparently the same manner as the food vacuole membrane.

It seems likely that the endocytic forces bringing about the engulfment of thesemembranes and the consequent removal of the membranes cause the 2 lips of thecytoproct to come back together. The final step of the reformation of the closed ridgemay result from an interaction between the 2 piles of fibres covering the internal sidesof the ridge or to some other obscure force. The whole process of opening and closingof the cytoproct reminds one of the action of a zipper which can be opened or closed,starting at any point or points along its long axis.

Role of microtubules in cytoproct function: a model

There are at least 2 sets of microtubules which are unique to the cytoproct. Theseare distinguishable by their points of attachment to the pellicular region. One settakes the form of bundles arising from near the proximal region of basal bodies. Thesebasal bodies, which are otherwise similar to somatic basal bodies, are found adjacentto, and on either side of, the cytoproct ridge. These microtubular bundles pass intothe cytoplasm slanting in an anterior direction. The second set of microtubules, whichare not found associated together, pass directly from the cytoplasm into the closedcytoproct ridge where they end in the epiplasm on either side of the narrow alveolar-sac gap at the summit of the ridge.

The bundles of microtubules arising from the basal bodies may be involved intrapping the circulating food vacuoles and guiding them to the cytoproct. Foodvacuoles in the vicinity of the cytoproct have microtubules lying along their surfaces,either singly or in groups, which occasionally appear to be linked to the membraneby cross bridges. These microtubules can sometimes be traced to the basal bodiesnext to the cytoproct. We feel that it is reasonable to postulate, in light of studies onthe role of microtubules in many other organisms (e.g. Holmes & Choppin, 1968;Freed & Lebowitz, 1970; Hepler, Mclntosh & Cleland, 1970; Smith, Jarlfors &Beranek, 1970; Smith, 1971; Bardele, 1972; Tucker, 1972) that these microtubulesare able to make contact with old food vacuoles and direct them to the site of thecytoproct.

The motive forces for the final movement of the food vacuole are not known but amodel can be postulated. Our model would require the application of a shearing forceat the sites of contact between relatively rigid microtubules, either of cytoproct ridge

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or basal body origin, and the food vacuole membrane. The shearing or sliding of themicrotubules relative to the food vacuole surface would displace the food vacuoletowards the cytoproct, since the microtubules themselves are anchored next to orwithin the cytoproct. If this is true, this would also explain how the food vacuole isable to apply force on the pellicle which, in turn, causes the pellicle to bulge out andalso the lips of the cytoproct to part. If such unidirectional microtubule-to-membraneshearing forces were sufficient and could persist long enough, the microtubules wouldconceivably be able to bring the food vacuole and cytoproct together and, in addition,pull the cytoproct ridge down against the food vacuole, flattening the ridge andeventually separating the lips of the cytoproct as the plasma membrane area enlargesbetween these lips. A sustained shearing force of the microtubules over this hemi-sphere-shaped part of the food vacuole would eventually bring the plasma membraneinto contact with the food vacuole membrane as the cytoproct lips are moved fartherapart. Fusion of these membranes would then be possible. This would be the laststep in the process of cytoproct opening. Egestion could then occur, as suggestedabove, as a result of the pressure exerted by the cytoplasm against the food vacuolemembrane.

The technical contributions of Vernon Azuma and Marilynn Ueno to this study are grate-fully acknowledged. The work was supported by a U.S. Public Health Service grant numberGM-17991.

REFERENCESALLEN, RICHARD D. (1971). Fine structure of membranous and microfibrillar systems in the

cortex of Paramecium caudatum. J. Cell Biol. 49, 1—20.ANTIPA, G. A. (1971). Structural differentiation in the somatic cortex of a ciliated protozoan,

Conchophthirus curtus Engelmann 1862. Protistologica 7, 471—501.BARDELE, C. F. (1972). A microtubule model for ingestion and transport in the suctorian

tentacle. Z. Zellforsch. mikrosk. Anat. 126, 116-134.EHRET, C. F. & MCARDLE, E. W. (1974). The structure of Paramecium as viewed from its

constituent levels of organization. In Biology of Paramecium (ed. W. van Wagtendonk), inPress. New York: Elsevier.

ESTEVE, J.-C. (1969). Observation ultrastructurale du cytopyge de Paramecium caudatum.C. r. hebd. Se'anc. Acad. Sci., Paris 268, 1508-1510.

FOISSNER, W. (1972). The cytopyge of Ciliata. I. Its function, regeneration and morphogenesisin Uronema parduczi. Ada biol. hung. 23, 161-174.

FREED, J. J. & LEBOWITZ, M. M. (1970). The association of a class of saltatory movements withmicrotubules in cultured cells. J. Cell Biol. 45, 334-354.

GRAIN, J. (1966). Etude cytologique de quelques cilies holotriches endocommensaux desruminants et des equides. Protistologica 2, 5-51.

HEPLER, P. K., MCINTOSH, J. R. & CLELAND, S. (1970). Intermicrotubule bridges in mitoticspindle apparatus. J. Cell Biol. 45, 438—444.

HOLMES, K. V. & CHOPPIN, P. W. (1968). On the role of microtubules in movement andalignment of nuclei in virus-induced syncytia. J. Cell Biol. 39, 526—543.

JURAND, A. (1961). An electron microscope study of food vacuoles in Paramecium aurelia.jf. Protozool. 8, 125-130.

MARSHALL, J. M. & NACHMIAS, V. T. (1965). Cell surface and pinocytosis. J. Histochem.Cytochem. 13, 92-104.

NACHMIAS, V. T. (1964). Fibrillar structures in the cytoplasm of Chaos chaos. J. Cell Biol. 23,183-188.

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ORGAN, A. E., BOVEE, E. C. & JAHN, T. L. (1972). The mechanism of the water expulsionvesicle of the ciliate Tetrahymena pyriformis. J. Cell Biol. 55, 644-652.

ORGAN, A. E., BOVEE, E. C, JAHN, T. L., WIGG, D. & FONSECA, J. R. (1968). The mechanismof the nephridial apparatus of Paramecium multimicronucleatum. I. Expulsion of water fromthe vesicle. J. Cell Biol. 37, 139-145.

PECK, R. K. (1971). Fine Structure, Morphogenesis and Interrelationships Within Representativesof Three Ciliated Protozoan Genera. PhD. Thesis, University of Illinois, Urbana, Illinois.

REYNOLDS, E. S. (1963). The use of lead citrate at high pH as an electron-opaque stain inelectron microscopy. J. Cell Biol. 17, 208-212.

SCHNEIDER, L. (1964). Elektronenmikroskopische Untersuchungen an den Ernahrungs-organellen von Paramecium. II. Die Nahrungsvakuolen und die Cytopyge. Z. Zellforsch.mikrosk. Anat. 62, 225-245.

SMITH, D. S. (1971). On the significance of cross-bridges between microtubules and synapticvesicles. Phil. Trans. R. Soc. Ser. B 261, 395-405.

SMITH, D. S., JARLFORS, U. & BERANEK, R. (1970). The organization of synaptic axoplasm inthe lamprey (Petromyson marinus) central nervous system. J. Cell Biol. 46, 199-219.

TUCKER, J. B. (1972). Microtubule-arms and propulsion of food particles inside a large feedingorganelle in the ciliate Phascolodon vorticella. jf. Cell Sci. 10, 883-903.

WATSON, M. L. (1958). Staining of tissue sections for electron microscopy with heavy metals.J. biophys. biochem. Cytol. 4, 475-478.

(Received 6 July 1973)

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Fig. 2. Closed cytoproct ridge sectioned along its longitudinal axis. The ridge seemsalmost removed from the cell due to the section's oblique orientation through the cellsurface at this point. This ridge is continuous with intersecting transverse pellicularridges (t). Within the cytoproct ridge are microtubules (cm) and short fibres (/). Basalbodies lying along the side of the cytoproct ridge have bundles of microtubules(arrows) connected to dense plaques next to their proximal ends. The more anteriorend of the figure is at the bottom, x 35 000.Fig. 3. Cytoproct in cross-section. The cytoproct ridge is closed and contains micro-tubules as well as fibrous mats or piles along both of its internal sides. An incon-spicuous filamentous meshwork fills the remainder of the ridge (Jm). Alveolar sacs(a) cover both sides of the ridge but are not continuous over the ridge summit. Herea gap (g) of about 50 nm separates the 2 alveolar sacs along with their underlyingepiplasm (e). The plasma membrane (pm) is continuous over the top of the ridge andover the alveolar sacs. / , cytoproct fibres, x 75000.

Fig. 4. Cytoproct in cross-section. The same features are present as described inFig. 3. In addition the cytoproct microtubules (cm) can be seen to arise from theepiplasm (e) at the summit of the ridge and pass from there into the cytoplasm. Thefibres (/) within the ridge arise all along the epiplasm coating the cytoplasmic surfaceof the inner alveolar membrane and at such an angle that their distal ends lie closestto the summit of the ridge, x 50000.Fig. 5. The cytoproct ridge sectioned approximately along its mid-longitudinal axisand perpendicular to the cell surface. Microtubules (cm) pass up to the ridge summit.Some cytoproct fibres (/) appear to be cut in cross-section. The meshwork of filaments(fm) filling the middle of the ridge is particularly evident in this micrograph, x 55 000.

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Fig. 6. A basal body (bb) lying adjacent to the cytoproct (which is out of view in thisfigure) has a bundle of microtubules arising from an electron-opaque plaque at itsproximal end. These microtubules come into close association with the membrane ofa food vacuole (fv), which is shown here in grazing section. Other microtubules canbe found at various points around the food vacuole (arrows). Also a finely filamentouscytoplasm (curved brackets) is found lining the surface of the food vacuole membranein several places, x 30000. Inset: higher magnification of the bordered area at the upperleft of Fig. 6, showing what may be bridges (above bars) linking the microtubule withthe food vacuole membrane, x 100000.

Fig. 7. The serial section of the upper left part of Fig. 6 at higher magnification. Themicrotubules arising from the basal body appear to be linked together by bridges(above bracket). Bridges may also occur between the microtubule to the right of thefigure and the food vacuole membrane, x 100000.Fig. 8. The cytoproct ridge in a cell fixed before cytoproct opening while the pelliclearound the cytoproct was distended. The food vacuole (fv), seen close to the cytoproct,has finger-like evaginations (arrows) extending toward the cytoproct. Basal bodies (bb)on both sides of the cytoproct have bundles of microtubules directed toward the foodvacuole. The infraciliary lattice (il) does not pass under the cytoproct ridge, a, alveolarsacs;/ , cytoproct fibres, x 30000.

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Fig. 9. A food vacuole lying near the cytoproct (c). The food vacuole membrane has arelatively smooth profile, and it may be the result of fusion of 3 smaller ones; this issuggested by the annular constrictions (arrows) in its surface, x 8500.

Fig. 10. Figs. 10-13 a r e from a cell in which the cytoproct lips are just starting toseparate. The cell surface is indented along this region. In Fig. 10 the sides of theridge are partially separated and flattened. This has begun at their proximal ends.The fibrous mats (brackets) can be seen to be pulled apart in this zone. A membrane-limited vesicle (v) is located between these separated fibrous mats. This view of theridge is 9 sections away from Fig. 11 and is of a portion of the ridge closest to the leftarrowhead in Fig. 11. a, alveolar sacs, x 50000.

Figs. 11, 12. Two views of the same cytoproct fixed during the process of opening.The figures are 9 sections apart. The cytoproct is lying within an indented groove inthe cell pellicle. Arrowheads indicate regions in which the lips of the cytoproct haveseparated and in which the cytoplasm is covered only by a plasma membrane. Theseareas contain an electron-opaque precipitate in this cell as do other parts of this cell'ssurface. The cytoproct lips, identifiable by the fibrous mats (/), are separated andflattened along that part of their length seen in these figures. The surface indentationdips down into the food vacuole (Jv), which thus appears in Fig. 12, to surround asmall portion of the indented pellicle. The food vacuole membrane lies close to theplasma membrane (pvi) at the right of this figure. Corner marks in Fig. 12 are ex-plained in Fig. 13. Figs. 11, 12, x 19000.

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CE L 14

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Fig. 13. The cytoproct 2 sections from that of Fig. 12 and 11 sections from that ofFig. 11. This is in the transition zone between its being closed (to the right) and open(to the left of the figure). This is adjacent to the region in Fig. 12 enclosed by cornermarks. The food vacuole (fv) membrane lies close to a small indentation of the plasmamembrane (pm). Fibrous material (/) lies against the indented segments of the alveolarsacs (a) which pass over the flattened cytoproct lips. In this transition area membrane-limited flattened vesicles (v) interspersed with microtubules may be seen, frn, fila-mentous meshwork. x 38000.Fig. 14. The cytoproct of a cell fixed immediately after the contents could be seenleaving the cytoproct. The plasma membrane (pm) is continuous with the food vacuolemembrane (fvm) allowing the internal portion of the food vacuole to be open to thecell's exterior. The food vacuole is flattened and undigested material can no longerbe seen within its lumen. The profile of the food vacuole membrane is highly con-voluted and invaginated, forming many tubular channels, x 20000.

Fig. 15. An opened cytoproct at higher magnification. One lip of the cytoproct is seenat the left with its characteristic fibres (/). The plasma membrane (pm) covering thislip is seen to be continuous with the convoluted food vacuole membrane (fvm). Afilamentous cytoplasm (fc) is found against the internal surface of the food vacuolemembrane. The outside surface appears also to have a thin coating of amorphousmaterial, x 50000.Fig. 16. Another view of the same cell as seen in Figs. 14 and 15. This section showsthe cytoproct microtubules (cm) passing from the cytoproct lip at the left along theinvaginating food vacuole membrane (fvm). The filamentous cytoplasm (fc) lining themembrane is also recognizable, x 30000.

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Fig. 17. By 3 s after egestion the deflated food vacuole membrane shows a still greaterirregularity in its profile. In addition flattened vesicles (arrows) begin to appear in theadjacent cytoplasm, x 20000.Fig. 18. By 10-30 s after egestion the deep indentation formed by the deflated foodvacuole is no longer present and the gap between the cytoproct lips {cl) is closed by asingle unit membrane which is continuous and is coated with filamentous cytoplasm.An accumulation of flattened vesicles and elongated membrane-bound tubules inter-spersed with microtubules occupies the cytoplasm just under this single membrane.This mass of membranes and microtubules passes in a generally anterior direction(toward the right) away from the cytoproct. x 21000.Fig. 19. Membranous and microtubular (m) fragments found in the region formerlyoccupied by the deflated food vacuole. x 75000.Fig. 20. A single membrane covers the region between the cytoproct lips within10-30 s following cytoproct egestion. The filamentous cytoplasm (fc) underlying thismembrane is particularly prominent in this enlarged view, x 50000.

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