NUTRIENT UPTAKE IN TETRAHYMENA PYRIFORMIS

by LEIF RASMUSSEN The Biological Institute of the Carlsberg Foundation 16 Tagensvej, DK-2200 Copenhagen N, Denmark

Key words:food vacuoles;pinocytic vesicles;plasma membrane

Several morphological structures have been implicated in nutrient uptake in the ciliate protozoon, Tetrahymena pyriformis: food vacuoles, various types of vesicles and the plasma membrane. It is the object of this report to dis- cuss the roles of these organelles in food uptake. Measurements of multiplication rates under conditions where food vacuole formation could be controlled experimentally suggested that the food vacuoles (about 5 rtm in diameter) were essential for rapid cell multiplication in various standard growth media. If, however, con- centrations of certain specific nutrients (different for different strains of T. pyriformis) were high, then the cells could multiply rapidly even when food vacuoles were absent. Furthermore, multiplication rates of cells supplied with particulate or dissolved egg albumin as the amino acid source, suggested that the food vacuoles took up particulate egg albumin well, but dissolved egg albumin poorly. The role in food uptake of vesicles with a diameter of less than 1 vm remains largely unknown. Our present knowledge of them is not yet sufficiently detailed to permit estimations of the rates with which they are formed or of their total number per cell. The plasma membrane has carrier-mediated uptake sites for a number of nutrients such as amino acids and nucleosides. It is likely that this type of uptake mechanism plays a quantitatively important role in T. pyriformis whenever such compounds are present in the extracellular fluid. L. RASMUSSEN;Nutrient uptake in Tetrahymena

CONTENTS

page 1. Introduction ...... 145 2. Organelles implicated in nutrient uptake ...... 146

2.1. Phagocytic vacuoles ...... 146

2.1. 1. The food vacuole ...... 146 2.1. 2. Induction of food vacuoles ...... 148 2.1. 3. Rates of food vacuole formation ...... 148 2.1. 4. Food vacuoles and lysosomes ...... 149 2.1. 5. Egestion of food vacuoles ...... 150 2.1. 6. Efficiency of particle collection ...... 150 2.1. 7. Total volume of food vacuoles formed in a generation time ...... 150 2.1. 8. Area of food vacuole membranes ...... 151 2.1. 9. Mucus ...... 151 2.1.10. Other phagocytic vocuoles ...... 151 2.1.11. Summary ...... 151

2.2 Pinocytic vacuoles and vesicles ...... 152

2.3 The plasma membrane ...... 152

3. On nutrient uptake ...... 153

3.1 Nutrient uptake by membrane invaginations ...... 153

3.1.1. The roles of the food vacuoles ...... 153 3. I. I. 1. Cell multiplication and reduced food vacuole formation ...... 153 3.1.1.2. Cell multiplication and particulate material ...... 155 3. I. 1.3. On the role of the food vacuole ...... 156 3.1.1.4. Food vacuole volumes and nutrient uptake ...... 157 3.1.1.5. Summary ...... 158

3.1.2. The roles of vesicles ...... 158

3.2. Nutrient uptake without membrane invaginations ...... 159

3.2.1. The roles of the plasma membrane ...... 159

4. Concluding remarks ...... 161

5. Summary ...... 162

6. References ...... 164

144 Carlsberg Res. Commun. Vol. 41, No 3, 1976 L. RASMUSSEN"Nutrient uptake in Tetrahymena

1. INTRODUCTION How do ciliates take up their nutrients? It is generally accepted that free-living ciliates digest particulate food in their food vacuoles prior to uptake and that parasitic, astomatous ciliates do not form food vacuoles: they take nutrients up through the entire body surface (see e.g. ref. 36). It is also accepted that these astomatous forms have developed from free-living forms (36). This gives rise to the question if free-li- ving ciliates can feed in rich nutrient media in the absence of food vacuoles. This and other problems on nutrient uptake will be discussed for a free-living ciliate, Tetrahymena pyriformis (Fig. 1). It was proposed long ago that T. pyriformis could take up all nutrients in the absence of food vacuoles. Thus LWOFF (55) reported in 1923 that ciliates, identified as T. pyriformis (56) grew and multiplied in sterile solutions of peptone without having food vacuoles. He Figure 1. Tetrahymena pyriformis. Scanning electron proposed that food uptake in T. pyriformis micrograph. The length of the cell is about 50 ~tm. could occur by an ,,ectoplasmic route~. This Reproduced by courtesy of Dr. H. E. Buhse, Jr. proposition appears to have been forgotten, but the fact remains that experimental evidence for a dual capacity for nutrient uptake in T. pyriformis was presented more than 50 years ago. acids for rapid cell multiplication; (c) include evidence for mediation of transport sites, Few investigators have compared the contribu- ,,pumps,,, in the uptake of amino acids, tion of the food vacuoles to total nutrient up- carbohydrates and nucleosides in T. pyriformis. take of T. pyriformis (LWOFF (55), SEAMAN (98, These results allow us to see under which 99), PRUETT, CONNER & PRUETT (77), NILSSON environmental conditions food vacuoles are es- (63), RASMUSSEN(79, 80), RASMUSSEN • OR1AS sential, stimulatory or inadequate for nutrient (84, 85) and ORIAS & RASMUSSEN (71, 72)). uptake required for continued growth and cell There are, therefore, no reasonably detailed multiplication. The results permit the sugges- descriptions of nutrient uptake for this cell. tion that the major part of the required Such descriptions should include accounts of nutrients when present in their free, un- the roles of food vacuoles, other vesicles and polymerized form can permeate the cell surface cell surface in the uptake processes. Ultimately, fast enough to support rapid cell multiplication the relative contributions of these organelles in in rich media. cell nutrition must be established for a variety Recently, the literature on T. pyriformis has of culture conditions. been reviewed in two books, one authored by Recent findings provide a foundation to HILL (40) and the other edited by ELLIOTT(27). evaluate the roles of some of the organelles CONNER (17) summarized our knowledge of participating in nutrient uptake in T. pyriformis. nutrient uptake in protozoa, and HOLZ (48) and They (a) confirm LWOFF's proposal that the DUNHAM& KROPP (24) surveyed nutrient up- cells can grow and multiply without food take in T. pyriformis. HUINER, BAKER, FRANK vacuoles; (b) demonstrate that particulate, but & COX (51) made a review of the field of nutri- not dissolved, egg albumin can provide amino tion in protozoa.

Carlsberg Res. Commun. Vol. 41, No 3, 1976 145 L. RASMUSSEN:Nutrient uptake in Tetrahymena

2. ORGANELLES IMPLICATED IN nutrients are internalized: these may include NUTRIENT UPTAKE food particles, compounds dissolved in the fluid Nutrient uptake in T. pyriformis is mediated by surrounding the particles, and possibly mucous vacuoles, vesicles and the plasma membrane. material with adsorbed molecules as proposed The first of these include phagocytic vacuoles by NILSSON (63). Digestion then takes place in (food vacuoles, and possibly others) containing the vacuole and digestion products penetrate particles visible in the light microscope the food vacuole membrane on their way to the (particles larger than 0.2 ~tm) and the second cytoplasm of the cell. pinocytic vesicles containing no such particles. Phagocytic vacuoles of the type known as food All contain some of the fluid in which the cells vacuoles (Fig. 2) are produced by the oral are suspended. There is a tendency to use the apparatus. The beating of the three oral collective term, endocytosis, for uptake involv- membranelles and the undulating membrane ing vacuoles and vesicles. (FUR~ASON (34)) maintains a current of extra- cellular fluid and suspended particles through 2.1. Phagocytic Vacuoles the buccal cavity. Food particles and their sur- Phagocytic vacuoles play an important role in rounding medium fill up the receiving vacuole food uptake. Each time a vacuole is formed (CORLlSS (18)), which is pinched off to form a food vacuole. The oral apparatus has been isolated in mass and subjected to microscopic and biochemical studies (WILLIAMS& ZEUTHEN (115), FORER, NILSSON & ZEUTHEN (30), ELLIOTT • KENNEDY (29), WILLIAMS, MICHELSEN & ZEUTHEN (113), WOLFE (117), RANNESTAD & WILLIAMS (78), WILLIAMS & NELSON (114), and GAVIN(35)).

2.1.1. Thefood vacuole When viewed under the phase contrast microscope food vacuoles usually appear as spherical bodies. They become clearly visible in the normal light microscope after addition of particulate material to the medium, for exam- ple bacteria (ELLIOTT & CLEMMONS (28) and RICKETTS (90)), India ink particles - Fig. 3 - (ELLIOTT & CLEMMONS (28) and ORIAS & POLLOCK (70)), carmine particles (CHAPMAN- ANDRESEN & NILSSON (13) and NILSSON (63)), yeast cells and latex particles (RICKETTS (90)), dimethylbenzanthracene particles (ROTHSTEIN & BLUM (93)) and heat-coagulated egg albumin particles (RASMUSSEN, unpublished observations). CHAPMAN-ANDRESEN & NILSSON (13) reported an average of 30 food vacuoles per cell in a pop- ulation growing exponentially in the standard medium of 2 per cent proteose peptone, liver Figure 2. Mid-longitudinal section of Tetrahymena extract and salts (PLESNER, RASMUSSEN & Abbreviations: b.c.: buccal cavity; f.v.: pyriformis. ZEUTHEN (76)). RASMUSSEN & KLUDT (82) food vacuole. Reproduced by courtesy of Drs. I. L. Cameron and D. L. Hill and Academic Press from found 26 vacuoles per cell in autoclaved 2 per ref. 40. cent proteose peptone broth.

146 Carlsberg Res. Commun. Vol. 41, No 3, 1976 L. RASMUSSEN:Nutrientuptake in Tetrahymena

Figure 3. Food vacuoles in Tetrahymenapyriformis, syngen 1, DIII, labelled with India ink particles. A 10 ml cell suspension was incubated for 30 minutes with 1 ml of a 0.1 per cent ink suspension and fixed. The India ink was obtained from ~Rotring~, W. Germany, and it was used throughout this study.

LYKKESFELDT (personal communication) and 72)). The cells were incubated with India ink RASMUSSEN & KLUDT (unpublished particles for 30 minutes or more and then fixed observations) found 15 and 17 food vacuoles (Fig. 3). Microscopy of the cells revealed per cell, respectively, in cells growing ex- vacuoles with diameters conservatively es- ponentially in the chemically defined medium timated to exceed 1 ~tm (NILSSON, personal of HOLZ, ERWIN, ROSENBAUM & AARONSON communication; ORIAS & RASMUSSEN (72)). (49). This method therefore disclosed only relatively The average diameter of newly-formed large phagocytic vacuoles, among these the vacuoles in cells from good nutrient media is 5 food vacuoles; vesicles with diameters below 1 rtm (CHAPMAN-ANDRESEN & NILSSON (13) and ~tm may have passed undetected. NILSSON(63)) or 6 ~tm (RASMUSSEN, BUHSE & Material enclosed in food vacuoles is sur- GROH (81)), RICKETTS (90) recorded average rounded by a unit membrane (ELLIOTT d~ diameters up to 7.3 ~tm. No values are available CLEMMONS (28) and NILSSON & WILLIAMS for the lower limit of newly-formed vacuoles. (67)). WEIDENBACH& THOMPSON (109) studied Food vacuoles decrease in size after their phospholipid composition in different sub-cel- formation: CHAPMAN-ANDRESEN 8s NILSSON lular fractions of starving T. pyriformis. They (13) found that ,,old, food vacuoles had an found that the vacuolar distribution of average diameter of 2 ~tm in starving cells. phospholipids resembled most closely that of In the experiments to be described in section 3 the microsome fraction and proposed that the the presence or absence of food vacuoles in T. former might arise from the latter. This conclu- pyriformis was established by phase contrast sion was supported by measurements of and ordinary light microscopy, at radioactivity of P32-phosphate in sub-cellular magnifications of 40 x 12 times (RASMUSSEN & fractions: after induction of food vacuoles for KLUDT (82), RASMUSSEN (79), RASMUSSEN & 10 minutes by addition of ferric oxide particles ORIAS (84, 85) and ORIAS ,~ RASMUSSEN (71, to starving cells, the activity was lower in the

Carlsberg Res. Commun. Vol. 41, No 3, 1976 147 L. RASMUSSEN:Nutrient uptake in Tetrahymena food-vacuolar fraction than in any other frac- NtLSSON (13) found that no food vacuoles were tion. The authors suggested on basis of these formed in cells suspended in starvation results that the lipid components of the food medium, unless inert particles such as carmine vacuoles were not made de novo at the time of were added as a stimulant. NILSSON (63) later the food vacuole formation. They also found reported that glucose and radioactive that the cells formed as many as four vacuoles thymidine and uridine induced formation of in the absence of protein synthesis and took this some vacuoles, but the addition of particles as evidence that there was an intra-cellular does not seem to have been excluded. It is very store of vacuolar membrane proteins. In agree- difficult to establish if particles are necessary to ment with this interpretation of the biochemical induce vacuole formation. In fact it may be studies ALLEN's (2) electron microscopical in- questioned whether reliable methods exist to vestigations suggested a re-cycling of food permit measurements of rates of formation of vacuole membranes in Paramecium caudatum food vacuoles in populations of T. pyriformis. (section 2.1.5). The methods used depend on adding foreign If the food-vacuolar membranes arise from the particles in numbers which nearly always cause microsome fraction in T. pyriformis this points a large increase in the concentration of to a difference between this and other cells: particles in the cell suspension. The conditions ULSAMER, SMITH & KORN (106) and ULSAMER, under which each experiment is made should WRIGHT, WETZEL & KORN (107) working with be stated: conclusions are only valid for specific amoebae and STOSSEL, MASON & VAUGHAN situations. (103) working with leukocytes proposed that vacuolar membranes in these cells arose from 2.1.3. Rates of food vacuoleformation surface membranes. The frequency with which food vacuoles are formed has been studied under many con- 2.1.2. Induction offood vacuoles ditions. CHAPMAN-ANDRESEN & NILSSON (13) Formation of food vacuoles can be induced by found that the frequency varied with the posi- particulate material. The process was induced tion of the cell in the cell cycle: no food by digestible particles: bacteria (R1CKETTS (90)), vacuoles were formed for about 20 minutes bits of spleen tissue (M~LLER & ROHLICH (61)) prior to cell division. NILSSON(63) reported that and heat-coagulated egg albumin (RASMUSSEN changes in external factors such as & MODEWEG-HANSEN(83)). Formation of food temperature, acidity and composition of the vacuoles was also induced by non-digestible ionic environment inhibited vacuole formation. particles: latex granules (MI~LLER & ROHLICH Using carmine particles to label new food (61)), carmine particles (CHAPMAN-ANDRESEN vacuoles, she found that T. pyriformis, GL, & NILSSON (13)), polystyrene particles suspended in 2 per cent proteose peptone with (RASMUSSEN & MODEWEG-HANSEN (83)) and liver extract and salts, formed an average of India ink particles (RASMUSSEN, unpublished about three vacuoles per 10 minutes at 28~ observations). NILSSON(63) stated that very few Similarly, using India ink particles to label new particles may be needed to initiate food vacuole food vacuoles, HOFFMANN, RASMUSSEN & formation. ZEUTHEN (43) found that cells in media of the It is an open question if compounds in solution same type formed from 7.2 to 9.2 vacuoles in 25 can induce formation of food vacuoles. minute periods, and LVKKESFELDT (personal RASMUSSEN & KLUDT (82) observed as few as 3- communication) obtained an average of 10 food 4 vacuoles per cell in a filter-sterilized, vacuoles per 30 minutes in a chemically defined ,,particle-free,, 2 per cent proteose peptone medium. These values all indicate a formation medium. RASMUSSEN(79) found under certain rate of about 1 vacuole per 3 minutes. strict conditions an average value of less than 1 However, carmine and India ink particles do vacuole per celt. No food vacuoles were formed not give the same results over longer incubation in Single live cells under these conditions (see periods. NILSSON (63) reported about 3 food section 3.1.1.1.). CHAPMAN-ANDRESEN & vacuoles per average cell after 10 to 15 minutes

148 Carlsberg Res. Commun. Vol. 41, No 3, 1976 L. RASMUSSEN: Nutrient uptake in Tetrahymena

1 1 I membrane and they have acid phosphatase activity (ELLIOTT(26)). NILSSON (62) observed vesicles which she assumes to be primary lysosomes. They have an electron dense con- tent and differ from those reported as primary lysosomes by ELLIOTT (26) with respect to size (0.3 rtm) and general morphology (laminated 8 content). MOLLER, BAUDHUIN & de DOVE (60) isolated lysosomes from T. pyriformis and found that they contained five hydrolases - acid phosphatase, ribonuclease, deoxyribonuclease, protease and amylase. In the cells these lysosomes fuse with newly-formed food =E vacuoles and release their enzymes into the Z I I I vacuoles thus transforming them into digestive 15 30 ~5 vacuoles, or secondary lysosomes. MINUTES T. pyriform& secrete lysosomal enzymes to the Figure 4. Number of food vacuoles per cell as a func- extracellular fluid. SMITH (101) found that tion of time. At time zero 1 ml of a 0.1 per cent India suspension medium contained four of the ink suspension was added to a 10 ml cell suspension of Tetrahymena pyriform&, strain GL, growing on 2 hydrolases mentioned above, even after brief per cent proteose peptone broth supplemented with incubation. DICKIE& LtENER (22, 23) found the 2 mM citrate and 0.036 mM ferric chloride. Cell fifth: protease. samples were fixed with 2 per cent formaldehyde There is evidence for heterogeneity of (final concentration) at pre-selected time points. lysosomes in T. pyriformis. MULLER (59) Food vacuoles were counted in 200 cells for each point. reported that the source of the hydrolases secreted into non-nutrient salt solutions was a special population of lysosomes characterized of incubation and no more than 4 after a pariod by its specific density. This secretion of of 30 minutes. Fig. 4 shows that the number of enzymes was not influenced by the induction of India ink labelled food vacuoles per average food vacuoles. Furthermore, ROTHSTEIN & cell increased steadily throughout a period of BLUM (94) observed that ingested 45 minutes at which time it amounted to 12. dimethylbenzanthracene particles did not co- The reason for this discrepancy in results sediment with either acid phosphatase or obtained with two different markers is not ribonuclease activity. They suggested that known. The media are not identical. NILSSON's either many of the particle-containing vacuoles medium (2 per cent proteose peptone, liver ex- did not fuse with lysosomes, or that those which tract and salts) contained a black precipitate did formed a class of food vacuoles which was which obscured the uptake of India ink and separable from the acid phosphatase-rich made it impossible to do the experiment in her lysosomes. BLUM & ROTHSTEIN (4) discussed medium with this marker. the idea that maturation changes subsequent to the fusion between primary lysosomes and food 2.1.4. Food vacuoles and lysosomes vacuoles may contribute to the heterogeneity Primary lysosomes are single-membraned of the lysosomal population. vesicles containing acid hydrolase which has According to RICKETTS (89) feeding causes not yet been involved in digestive events (de parallel stimulation of vacuole formation and of DUVE (25)). T. pyriform& contains numerous phosphatase activity in T. pyriformis. RICKETTS slightly dense, oval or spherical vesicles varying (92) also reported that latex particles in saline in size from 0.5 to 1 ~tm (ELLIOTT (26)). At the failed to induce endocytosis, and so did a ultrastructural level they consist of fine variety of potentially digestible solutes studied granular material enveloped in a unit singly in saline. However, some mixtures of

Carlsberg Res. Commun. Vol. 41, No 3, 1976 149 L. RASMUSSEN:Nutrient uptake in Tetrahymena solutes and latex particles in saline did induce WOLF & ALLEN (106) saw that the food vacuole endocytosis. Protein, polypeptides and RNA membrane was re-engulfed by endocytosis. In were highly effective inducers while glutamate, agreement with these observations the fecal amino acid mixtures, polysaccharides and pellets of T. pyriformis are not surrounded by glucose were moderately effective. RaCKETTS membranes (ELLIOTT& CLEMMONS (28)). (91) also found that starved cells showed a According to BERGER (3) food vacuoles in marked periodicity in the rate of formation of Paramecium aurelia were not egested in the vacuoles when they were presented with a mix- temporal order in which they were formed. He ture of peptone and polystyrene latex particles. based his opinion on the following observation: He suggested that supply of energy and of cells incubated with radioactive bacteria lysosomes may be more important than supply formed food vacuoles whose egestion could be of new membrane material for formation of followed upon transfer of the cells to non-ra- vacuoles. NILSSON (63) also found periodicity in diactive bacteria suspensions. The number of rates of formation of endocytic vacuoles. labelled food vacuoles per cell plotted against It seems that the acidity of the food vacuole in time decreased exponentially rather than Paramecium varies in a programmed fashion linearly. This indicates randomness rather than during the life of a vacuole (MAST (57), sequential order in the expulsion of food WICHTERMAN (110), KITCH1NGS(53)). The same vacuoles. A similar conclusion for T. pyriformis may happen in T. pyriformis. An increase in was reached by ROTHSTEIN& BLUM (93). vacuolar acidity may make some compounds, ROTHSTE1N & BLUM (93) observed that otherwise unavailable to the cell, soluble as well colchicine, vinblastine and cytochalasin B, all as denature proteins and other macromolecules of which inhibited particle ingestion, did not in- to facilitate enzyme action. terfere with particle egestion. They stated that It is still unknown how the digestion products this points to a significant difference between pass through the food vacuole membrane. ingestion and egestion mechanisms. WEIDENBACH & THOMPSON (109) recently succeeded in isolating food vacuoles in vitro 2.1.6. Efficiency ofparticle collection from T. pyriformis, so the necessary ex- The undulating membrane and the three perimental material for solving this problem is membranelles of the oral apparatus beat fast now available. and incessantly in intact cells. This creates a current of fluid with suspended material 2.1.5. Egestion offood vacuoles flushing the buccal cavity, the cytopharynx and Food vacuoles void their indigestible content perhaps even the receiving vacuole. This results through the cytoproct. Using the electron in a collection of particulate material, probably microscope ALLEN (2) studied this process in assisted by mucus present in these cavities (see Paramecium caudatum. Just before the food section 2.l.9). Cox (19) and RASMUSSEN,BUHSE vacuole emptied, its membrane fused with the & GROH (81) measured the rates with which In- plasma membrane in the cytoproct region of dia ink particles disappeared from the suspen- the cell; then after the content of the food sion medium. The results agreed well. The vacuole was released into the suspension latter authors found that the volume of extra- medium, the vacuole membrane underwent a cellular fluid cleared of particles by the cells transformation to form disc-shaped vesicles; was about 500-fold larger than the volume of these became part of the membrane pool. food vacuoles formed in the same time. Thus T. Apparently the vacuole membrane, at least in pyriformis is very efficient in collecting small Paramecium caudatum, is not formed de novo at particles from large volumes of suspension the time of food vacuole formation. Although medium. the oral regions of Paramecium and Tetrahymena are different, ELLIOT]"& KENNEDY 2.1.7. Total volume of food vacuoles formed in a (29) and SATTLER (97) observed vesicles near generation time the forming food vacuoles in T. pyriformis, and RASMUSSEN (80) found that cells suspended in a

150 Carlsberg Res. Commun. Vol. 41, No 3, 1976 L. RASMUSSEN:Nutrient uptake in Tetrahymena chemically defined medium formed food 2.1.9. Mucus vacuoles which had a total volume of about 50 Mucus material is extruded from small per cent of the average cell volume in a genera- argentophilic granules situated under the cell tion time. CHAPMAN-ANDRESEN& NILSSON(13) surface. They have been called protrichocysts calculated the average volume of a newly-for- (PITELKA (74)), mucigenic bodies (PITELKA med food vacuole (0.2 per cent of the average (75)), and more recently, mucocysts (TOKuV- cell volume) in cells grown in the standard ASU & SCHERBAUM (105), N1LSSON & BEHNKE proteose peptone medium, and NILSSON (63) (65), SATIR, SCHOOLEY& SATIR (96) and SATIR reported that the cells formed about 60 food (95). These granules also appear under the cell vacuoles per cell generation under identical surface in the buccal cavity (WILLIAMS& LUFT conditions. From the two sets of values it can (ll2) and NILSSON, (63)). NILSSON & BEHNKE be calculated that the total volume of vacuoles (65) observed that T. pyriformis from the formed in a cell generation time is about 15 per stationary growth phase were coated by a cent of the average cell volume. We have no es- mucosubstance approximately 0.02 ~tm thick. timate of the volume of food vacuoles formed Cells from the exponential growth phase ex- per cell generation in cells feeding on bacteria, hibited no such surface layer. but it is quite possible that values higher than NILSSON (63) proposed that this mucus plays a those quoted above can be obtained under role in uptake processes: it may adsorb these conditions. Extreme, low values nutrients prior to being enclosed in the food (probably < 1 per cent of those given above) are vacuoles. A similar role has been established found in the so-called food-vacuole-less cells, for the hair-like extensions of the plasmalemma NP1 (see section 3.1.1.1. ). of the large amoebae (CHAPMAN-ANDRESEN& HOLTER (12)) - see section 3.1.1.4. There is no experimental evidence as yet for concentrative 2.1.8. Area of food vacuole membranes adsorption of any nutrient in the case of T. CHAPMAN-ANDRESEN & N1LSSON (13) calcu- pyriformis (compare section 3.2.1). NILSSON(63) lated that one newly-formed food vacuole observed, however, that the mucus of the ex- represented a surface area equal to 1.6 per cent truded mucocysts stained with alcian blue and of the cell surface of an average cell. In 2 per then appeared with a more intense colour than cent proteose peptone medium, enriched with the surrounding solution, indicating an adsorp- 0.4 per cent liver fraction L and salts (PLESNER, tion of the dye to the extruded mucous RASMUSSEN & ZEUTHEN (76)) they showed that material. exponentially-growing T. pyriformis, strain GL, The mucus may also play a part in retaining had an average generation time of about 2.5 food particles swept into the buccal cavity and hours, made an average of 1 food vacuole per 3 the receiving vacuole by the beating action of minutes, forming about 50 food vacuoles per the undulating membrane and the three generation time. Thus the food vacuole membranelles. membrane made available in a generation time equalled 50 x 1.6 per cent of a cell surface, or 2.1.10. Other phagocytic vacuoles about 80 per cent of the cell surface. It is still not known if T. pyriformis forms There are thirty food vacuoles per average cell phagocytic vacuoles from the cell surface other grown under the experimental conditions than the food vacuoles described above. described above. The average food vacuole has a diameter of 3.5 um (RASMUSSEN, unpublished 2. I. 11. Summary observations). Thus it can be calculated that the The oral structures of T. pyriformis efficiently membrane area available for transport of collect small particles in the food vacuoles. The nutrients from vacuoles into the cytoplasm at vacuoles fuse with lysosomes carrying digestive any time in the average cell is in the range of 20 enzymes; uptake of digestion products then per cent of the cell surface under these occurs by unknown mechanisms through the conditions. food vacuole membrane. Dissolved nutrients

Carlsberg Res. Commun. Vol. 41, No 3, 1976 151 L. RASMUSSEN"Nutrient uptake in Tetrahyrnena may be present in the mucus of the food surface pits and associated membrane-bound vacuoles in higher concentrations than in the cisternae with a diameter of about 0.1 ~tm suspension medium. throughout the oral and somatic regions. They The food vacuoles may be considered cellular noted that the parasomal sacs were somewhat compartments in which the chemical environ- similar in morphology to the coated vesicles ment can be changed to promote release of known from certain metazoan cells in which compounds from previous physical and they transport absorbed protein into epidermal chemical combinations, serving to facilitate cells (FRIEND St, FARQUHAR (33)). WILLIAMS 8s their uptake. The same purpose is likely to be LUFT (112) suggested that the pits and cisternae served by water uptake from the vacuole might function in T. pyriformis in a similar way. leading to its shrinkage and to increase in con- We do not know how often these vesicles are centrations of enclosed dissolved compounds. formed, and we do not know their number per The phagocytic vacuoles enlarge the area of the cell. Hence we cannot assess their quantitative transporting surfaces of the cell. role for nutrient uptake. It is not known if other types of vesicles parti- 2.2. Pinocytic Vacuoles and Vesicles cipate in nutrient uptake. Several authors reported cytoplasmic vacuoles It is a far cry from the static electron or vesicles, to which they have attributed micrographs of the small vesicles in 7". functions in nutrient uptake in T. pyriformis. pyriformis to a demonstration of their role in ELLIOTT & CLEMMONS (28) found clear vacu- food uptake. It will be shown in section 3.1.1.1. oles near the plasma membrane which they in- that cells without food vacuoles suspended in a terpreted to be pinocytic vacuoles. These rich, standard proteose peptone broth are in- vacuoles appear to be about 2 om in diameter. capable of sustained growth and division. Thus Their origin is unknown, but it is stated that these small vesicles cannot fulfill the nutrient they are often seen in the vicinity of breaks in requirement for rapid multiplication under the plasma membrane described as regions these experimental conditions. through which the content of mucocysts has It is regrettable that our knowledge of food up- been discharged (ELLIOT'r & KENNEDY(29)). take at levels different from those of the ALLEN (1) saw cisternae with smooth mem- phagocytic vesicles in T. pyriformis is so scanty, branes in the subpellicular cytoplasm. These when compared to our knowledge of pino- smooth-membraned cisternae frequently had at cytosis in the large amoebae. Here extensive their margins enlargements coated on their and fruitful studies have revealed the im- cytoplasmic surfaces with bristles similar in portance of adsorption of charged molecules, appearance to those coating the parasomal the existence of inducers of pinocytosis, and the sacs, and indeed a direct connection between timing and the extent of the uptake of both the two systems is possible. The parasomal sacs high- and low- molecular weight compounds: are indentations of the outer membrane found for reviews see HOLTER (44, 45, 46) and CHAP- just anterior to each kinetosome or basal body MAN-ANDRESEN(7, 8, 9) and for original reports of the cilium (PITELKA (75)). Thus the mem- see HOLTER & MARSHALL(47), CHAPMAN-AN- brane of the cisternae might be continuous with DRESEN & HOLTER (11, 12), CHAPMAN-ANDRE- the outer pellicular membrane. ALLEN (1) SEN & PRESCOTT (14), CHAPMAN-ANDRESEN(6), proposed that the parasomal sacs are sites of CHAPMAN-ANDRESEN & CHRISTENSEN (10), selective uptake of certain large molecules, HENDIL(39) and BRANDT & HENDIL (5). such as proteins. According to him the outer membrane could give rise to the smooth-mem- 2.3. The Plasma Membrane braned cisternae, and these could pinch off The surface of T. pyriformis encompasses a vesicles carrying loads of externally adsorbed variety of structures: cilia, fibers, mucocysts molecules into the cell by a process similar to etc. (see ELLIOTT& KENNEDY (29) for a recent pinocytosis. review). The cell is enclosed in a system of WILLIAMS • LUFT (112) observed a system of membranes which all show the unit membrane

152 Carlsberg Res. Commun. Vol. 41, No 3, 1976 L. RASMUSSEN:Nutrient uptake in Tetrahymena structure (PITELKA (75) and TOKUYASU & 3.1.1. The roles of thefood vacuoles SCHERBAUM (105)). The outer membrane, the T. pyriformis grows well both in complex plasma membrane, covers the entire surface of nutrient media (proteose peptone broth with or the cell, also the cilia; immediately beneath the without supplements) and in chemically defined plasma membrane is a mosaic of flat, solutions. Many nutrients are required in- membrane-bounded ~alveoli,,; cilia and muco- cluding amino acids, vitamins, nucleosides and cysts emerge from the underlying ectoplasm salts (KIDDER & DEWEY (52)). The minimum between adjacent alveoli (PITELKA (75)). generation times under optimum conditions are Because it has been suggested that the outer less than 2 hours for T. pyriformis, syngen 1, surface of T. pyriformis is extended by ,,pits,, (optimum temperature: about 37~ (RASMUS- ,,sacs,,, or by ,,cisternae,, - stretching deep into SEN, unpublished observations)) and 2.5 hours the cell - in periodic or permanent open con- for T. pyriformis, strain GL (optimum tempe- nection with the surroundings, it is difficult to rature: 28~ THORMAR (104)). define what distinguishes ,,the outer cell sur- T. pyriformis grows and forms many food face,, from ,,the inner cell surface,(. vacuoles in the nutrient media traditionally Operationally, these inner surfaces could be ex- used (section 2.1.1), and cells which form few or tensions of the outer surface and for my no food vacuoles do not grow (RASMUSSEN & purpose they will be treated as such. Thus all KLUDT (82), RASMUSSEN & MODEWEG-HANSEN nutrients which are taken up by the outer cell (83), HOFFMANN, RASMUSSEN & ZEUTHEN (43), surface pass the plasma membrane. ORIAS • POLLOCK (70) and RASMUSSEN R, It will be shown later that the plasma mem- ORIAS (84)). However, it has been found that brane seems to be permeable to the majority of the cells can grow without forming food vacu- the nutrients which T. pyriformis requires for oles, provided that the media are enriched in rapid growth and multiplication and it will be certain defined ways (RASMUSSEN ~/; KLUDT shown that the cell seems to have specific up- (82), RASMUSSEN (79), HOFFMANN, RASMUSSEN take sites for a number of compounds, such as & ZEUTHEN(43) and RASMUSSEN & ORIAS (84)). carbohydrates, amino acids etc. Nothing is These observations have formed the basis for known about the structure of the single experiments carried out in order to charac- transport sites, nor about their distribution on terize the roles of the food vacuole in nutrient the celt surface. uptake. The following pages present results of experiments designed to show whether or not all compounds required for continued cell mul- tiplication could be taken up by the cells under 3. ON NUTRIENT UPTAKE different sets of conditions. All compounds Detailed information on food uptake i T. were judged to be taken up, if the cells kept pyriformis is scarce. One reason is that several multiplying with normal generation times under types of organelles participate in the uptake the new experimental conditions. On the other processes. Another, that the relative contri- hand, if the ceils grew very slowly or ceased to butions of these organelles may vary in media multiply, it was concluded that they had failed of different composition. The possible roles of in taking up one or more of the essential nutri- food vacuoles, vesicles and the cell surface will ents. be discussed below. 3.1.1.1. Cell multiplication and reduced food 3.1. Nutrient Uptake by Membrane Invaginations vacuoleformation This section deals with the role of food vacuoles RASMUSSEN & KLUDT (82) reported that T. and vesicles in food uptake. The significance of pyriformis, strain GL, suspended in autoclaved these structures is evaluated from the aspect of and filter-sterilized proteose peptone broth how well they contribute towards uptake of all multiplied with generation times of 6 and > 40 nutrients required for reasonably fast cell hours, respectively. The cells in the autoclaved growth and multiplication. proteose peptone medium contained many

Carlsberg Res. Commun. Vol. 41, No 3, 1976 153 L. RASMUSSEN"Nutrient uptake in Tetrahymena

Table I

Multiplication of two lines of Tetrahymena pyriformis, syngen 1, in the chemically defined medium with various sup- plementations.

Cell concentrations per ml x 10-3.

NPI cells forming no Dill cells forming food vacuoles food vacuoles Medium Oh 18h 43h Oh 18h I 43h a. DM 4 5 300 550 b. DM + Fe&Cu 10 c. DM + folinic acid 4 d. DM + Fe&Cu+ folinic acid 36 300 300 500

DM: chemically defined medium (HoLz et al. (49)); Fe: ferric chloride (900 ~tM); Cu: copper sulphate (30 ~tM); folinic acid: 2 ~tM; time zero: cell concentration at inoculation. General conditions: source of inocula: cells subjected to two transfers in unsupplemented DM; temperature 37~ Unpublished data, ORIAS & RASMUSSEN. food vacuoles (see section 2.1.1.) and obser- that cytochalasin B reduced formation of food vations of live ceils from this nutrient medium vacuoles in T. pyriformis. HOFFMANN, RASMUS- showed frequent formation of food vacuoles. SEN 8s ZEUTHEN (43) found that the inhibitor On the other hand, cells from the filtered also decreased multiplication rates of cells in medium showed no food vacuole formation: 20 the nutrient medium, and observed that glucose single cells from the filter-sterilized medium and nucleosides relieved this effect, while hav- were kept under continuous observation each ing no effect on vacuole formation. for a period of 10 to 15 minutes, and no These studies have been extended to other cells vacuoles were seen to be formed during the without food vacuole formation. OR1AS 8s total observation period of 4 hours (RASMUS- POLLOCK (69) isolated a cell line, NPI, in which SEN, unpublished). food vacuole formation could be controlled. The slow cell multiplication in the filter-ste- The cell line was derived from T. pyriformis, rilized broth was probably a result of lack of syngen l, mating type III, inbred family D. The food-vacuole-inducing particles, since addition NPI cells did not form food vacuoles at 37~ as of particulate materials such as clay, iron, observed by optical microscopy (ORIAS & aluminium hydroxide particles (RASMUSSEN & POLLOCK (70)) - see Fig. 5 - and by transmis- KLUDT (82)), polystyrene particles, heat-co- sion electron microscopy (ORIAS, CHARVAT, agulated egg albumin (RASMUSSEN & LING & POLLOCK(68); NILSSON, personal com- MODEWEG-HANSEN (83)) and India ink munication). They formed, however, normal particles (RASMUSSEN, unpublished obser- food vacuoles at 28~ NPI cells without food vations) stimulated cell growth back to control vacuoles ceased to multiply in standard values. proteose peptone media at 37~ (ORIAS & Multiplication of T. pyriformis, strain GL, with POLLOCK (69); RASMUSSEN, unpublished ob- reduced food vacuole formation could also be servation). However, the cells could be grown stimulated by addition of specific nutrients in for hundreds of generations, if the nutrient high concentrations. RASMUSSEN (79) found media were enriched with high concentrations that addition of glucose and nucleosides in of both trace metal salts and of vitamins rather high concentrations promoted mul- (RASMUSSEN & ORIAS (84)). The cell generation tiplication substantially; again direct observa- times of the NPI cells were about twice as long tion of live cells in the microscope failed to as those of the DIII wild-type cells under these reveal any formation of food vacuoles. conditions (RASMUSSEN • OR1AS (84, 85) and NILSSON, RJCKETTS & ZEUTHEN (66) showed ORIAS & RASMUSSEN(71, 72)). Further studies

154 Carlsberg Res. Commun. Vol. 41, No 3, 1976 L. RASMUSSEN:Nutrient uptake in Tetrahymena

Figure 5. Appearance of Tetrahymenapyriformis, syngen i, Dill (left) and NPI (right) grown at 37~ in enriched proteose peptone and exposed to India ink. Late exponential phase cultures were exposed to 0.01 per cent India ink (final concentration) at 37~ for approximately 60 minutes, fixed with 2 per cent formaldehyde (final con- centration), concentrated by low speed centrifugation and observed under the microscope.

showed that the mixture of trace metal salts formed no food vacuoles (NPI) starved on the could be replaced with iron and copper salts same diet (Table II, line c). Furthermore, alone, and the vitamins could be replaced with CURDS & COCKBURN (21) showed that T. folinic acid alone (ORIAS & RASMUSSEN (71) pyriformis could grow and divide with short and RASMUSSEN& ORIAS (85)). These additions doubling times in bacteria suspensions mul- also promoted growth in the defined medium tiplying in solutions of sucrose and salts. (Table I), and the growth rates shown in line d Apparently, the food vacuoles could provide could be maintained for hundreds of most, if not all, of the nutrients the cells re- generations (ORIAS• RASMUSSEN (72) and un- quired for rapid multiplication when the food published observations). was present in the form of particles. Thus food In conclusion, T. pyriformis could grow and vacuoles seem to be indispensable for the up- multiply in enriched media in spite of strongly take of particulate nutrients. reduced food vacuole formation. Their growth The effect on cell multiplication of particulate rates were about half of those of the food-va- material without apparent nutritional value has cuole-forming cells under the same conditions. been studied in filter-sterilized proteose Different strains of T. pyriformis required peptone broth. As already mentioned T. supplements of different nutrients. pyriformis, strain GL, grew poorly in this medium. RASMUSSEN (unpublished) observed 3.1.1.2. Cell multiplication and particulate that India ink particles stimulated growth well material in this medium. RASMUSSEN 86 MODEWEG- RASMUSSEN & ORIAS (unpublished observa- HANSEN (83) found that addition of polystyrene tions) showed that heat-coagulated, particulate particles resulted in improved growth. Both of egg albumin could supply cells which formed these types of particles induced food vacuole food vacuoles (D III) with all of the amino acids formation (see section 2.1.2) and, in turn, required for growth, whereas cells which apparently stimulation of nutrient uptake. Cell

Carlsberg Res. Commun. Vol. 41, No 3, 1976 155 L. RASMUSSEN:Nutrient uptake in Tetrahymena multiplication rates, therefore, appear to be that uptake is facilitated by shrinkage of the stimulated also by particles which are not food vacuole. Lastly, it is possible, but again not nutritious themselves. demonstrated, that the food vacuole membrane has transport abilities which are different from 3.1.1.3. On the role of thefood vacuole those of the plasma membrane. In this context The normal, wild-type, syngen 1 cells formed it is of interest that CHAPMAN-ANDRESEN pc food vacuoles and grew in media whose con- HOLTER (11) working with amoebae found centrations of Fe, Cu and folinic acid were glucose, to which the plasmalemma is im- much lower than those required by the food-va- permeable, in the cytoplasm after induction of cuole-less NPI cells (Table I, line a). What do pinocytosis by addition of protein. So far the these results mean? Fe, Cu and folinic acid may reason for this change in transport capacity is be taken up in NPI cells by the cell surface or by unknown. vesicles other than the food vacuoles. This The reason why different strains of T. pyriformis seems to require higher than normal extra-cel- need different nutrients in excess concen- lular nutrient concentrations, reflecting poor trations is not understood. surface penetration, small vesicular volumes, or ORIAS & RASMUSSEN (unpublished observa- both. In any case, as judged by the growth rates, tions) attempted to evaluate the role of the food the uptake of these compounds was greatly vacuole in uptake of dissolved protein. They facilitated when food vacuoles were present cultivated normal, food-vacuole-forming wild- (Table I, lines a to c). Chemical features of type cells in a chemically defined medium in food-vacuolar uptake of Fe, Cu and folinic acid which either the whole amino acid comple- are largely unknown. We do not know why the ment, or leucine, or phenylalanine was replaced food vacuole was more efficient than the cell by dissolved egg albumin (Table III, lines e, f surface in the uptake of certain nutrients. and g). This tested if the protein in solution ORIAS & RASMUSSEN (71) pointed out features could supply all the amino acids required, or which may or may not be important. Firstly, the leucine, or phenylalanine fast enough to allow oral structures are well suited to collect the cells to grow and multiply with generation particles (see section 2.1.6.). Secondly, the cell times which could be considered reasonably might be able to bring insoluble salts (of iron, short for T. pyriformis, namely generation times for example) in solution by lowering of the pH of a few hours. In no case where the cells had to value of the food vacuole (see section 2.1.4). rely on soluble egg albumin for a supply of Thirdly, it is possible, but not yet demonstrated amino acids was a full doubling observed during

Table II

Food vacuoles are required fur utilization of particulate egg albumin as an amino acid source; dissolved egg albumin cannot serve as an amino acid source.

Cell concentrations per I x 10-3.

NPI cells forming no DIII cells forming food vacuoles food vacuoles Medium Oh 18h 43h Oh 18h 43h a. EDM 40 190 305 300 b. EDM- a.a. 2 1 6 5 c. EDM - a.a. + EAh 2 1 307 395 d. EDM - a.a. + EAs 2 5 9 8

EDM: enriched chemically defined medium (Table I, line d); a.a.: amino acid complement of the defined medium; EAhand EAs:heat-coagulated and dissolved egg albumin, 0.1%; time zero: cell concentrations at inoculation. General conditions: source of inocula: amino acid depleted cells; temperature: 37~ pH: 7. Unpublished data, ORIAS& RASMUSSEN.

156 Carlsberg Res. Commun. Vol. 41, No 3, 1976 L. RASMUSSEN:Nutrientuptake in Tetrahymena

Table III

Egg albumin replaces neither the whole amino acid complement, nor leueine, nor phenylalanine in the chemically defined medium for growth of Tetrahymena pyriformis, syngen 1, strain Dill.

Cell concentrations per ml x 10 -3. Cell doubling Medium Oh 18h 45h times a. DM 305 300 ca. 3h b. DM-a.a. 6 5 >45h c. DM-leu 6 6 > 45h d. DM- phe 5 6 > 45h e. DM-a.a. + EAs 9 8 > 45h f. DM-leu + EAs 6 8 > 45h g. DM-phe + EAs 8 8 > 45h h. DM - a.a. + EAh 194 340 ca. 3h i. DM + EA s 307 395 ca. 3h

DM: the chemically defined medium of Hoez et al. (49); a.a.: amino acids; EAs and EAh : dissolved and heat- coagulated egg albumin, 0.1%; time zero: cell concentrations at inoculation. General conditions: source of in- ocula: amino acids depleted cells; temperature: 37~ Unpublished data, ORIAS & RASMUSSEN. a period of 45 hours. Tests were included to promotes its adsorption to the mucous layer of show that dissolved egg albumin did not inhibit the cell surface (CHAPMAN-ANDRESEN (6); cell multiplication in the complete medium CHAPMAN-ANDRESEN & CHR1STENSEN (10)). (line i) and that heat-coagulated, particulate The nutrient medium in the experiments egg albumin indeed supplied all the essential mentioned above was maintained at pH 7.0 and amins acids for T. pyriformis (line h). Two series the isoelectric point of egg albumin is around a of experiments have been made: in one (not pH-value of 4.8. Thus the egg albumin had a shown) separate addition of insoluble iron and negative net-charge in these experiments. It will Sephadex particles ensured that the cells be of interest to see if T. pyriformis can utilize formed food vacuoles; this did not affect cell dissolved proteins with a positive charge fast multiplication. enough for rapid cell multiplication. These results showed that the cells could utilize particulate egg albumin much better than the 3.1.1.4. Food vacuole volumes and nutrient uptake same protein in solution, and cells which were LWOFF (55) stated that T. pyriformis could grow incubated with particulate egg albumin had in- and multiply without food vacuoles. His state- deed their food vacuoles packed with lumps of ment would imply that there is not necessarily coagulated protein not seen in the cells fed dis- any relation between food vacuole volumes and solved albumin. Thus the difference in mul- nutrient uptake, but it has so far had no impact tiplication rates may be explained by the diffe- on the discussion on food uptake in Tetra- rent concentrations of nutrients in the food hymena. Since then, several authors have shown vacuoles. Other interpretations are possible: that the volume of food vacuoles made per unit VISWANATHA & LIENER (108) suggested that time, multiplied by the concentration of a similar results could be explained if the diges- certain compound in the suspension medium, tive enzymes of the food vacuoles were less could not account for the amount of that efficient in attacking native, than denatured nutrient taken up by the cells. Thus SEAMAN protein. Although this is probably true its (98, 99) calculated that only 2 per cent of con- significance in nutrient uptake is difficult to sumed acetate entered T. pyriformis through assess. food vacuoles. PRUETT, CONNER & PRUETT (77) In the large amoebae it has been established showed that accumulation of orthophosphate that a positive electric net-charge of a protein far exceeded the capacity of the food vacuole

Carlsberg Res. Commun. Vol. 41, No 3, 1976 157 L. RASMUSSEN:Nutrient uptake in Tetrahymena volumes. NILSSON (63) found that the food 3.1.1.5. Summary vacuole volume could not account for bringing T. pyriformis could grow and multiply in in nutrients in sufficient amounts to allow the absence of food vacuole formation, provided cells to double their dry weight in a generation the nutrient media were strongly supplemented. time. Finally, RASMUSSEN (80) calculated that Different strains were not identical with respect food vacuole volumes were insufficient for the to their requirements for excess concentrations uptake of nucleosides from a chemically of nutrients. The biochemical foundation for defined medium. promotion of food uptake by the food vacuole These observations were interpreted in diffe- is not known. rent ways. SEAMAN (98, 99) proposed that acet- The food vacuoles were very effective in col- ate could be taken up through the cell surface; lecting and processing particulate food, such as PRUETT, CONNER & PRUETT (77) were inclined bacteria and heat-coagulated egg albumin. In to assume that a highly selective binding site in contrast, food vacuoles could not supply the the oral region allowed initial concentration of cells with amino acids from dissolved egg albu- phosphate; NILSSON (63) proposed that min fast enough to sustain rapid cell multiplica- nutrients were concentrated in the mucous sub- tion. stance of the food vacuole prior to uptake. This latter proposal was made in analogy with CHAPMAN-ANDRESEN & HOLTER's (12) results 3.1.2. The roles of vesicles on Amoeba proteus which showed that serum Morphological investigations have shown that albumin was concentrated about 36-fold in the vesicles are formed from the surface of the oral mucus of the amoebae prior to uptake. region and possibly also from the cell surface RASMUSSEN (80) proposed that uptake of (section 2.2). These vesicles tend to be small. nutrients could occur through the cyto- WILLIAMS & LUFT (112) showed the presence pharyngeal membrane before the food vacuole of vesicles with a diameter of about 0.1 ~tm, or is formed. Thus all authors agreed that about 2 per cent of the diameter of a newly-for- mechanisms exist which either concentrate med food vacuole. This means that the volume nutrients prior to, or concomitant with, their of one food vacuole is about 100,000-fold larger uptake through vacuoles, or which transport than that of one vesicle. No studies have yet nutrients into the cells through parts of the sur- shown how many types of vesicles are formed, face which may or may not be associated with or with what frequency they appear. the feeding apparatus. No direct measurements of the contribution

Table IV

Growth of three lines of Tetrahymena pyriformis in filter-sterilized and heat-sterilized proteose peptone broth.

Cell concentrations per ml x 10-3.

PPf PPh

Oh 18h 43h*) Oh 18h 43h a. Syngen t, strain DIII 1 43 210 b. Syngen 1, strain NPI 1 40 230 c. Strain GL 1 39 220

PPf and PPh: 2% proteose peptone broth, sterilized by filtration and by heating, respectively; time zero: cell con- centrations at inoculation. General conditions: cells were starved for 24h and then transferred to medium of the above description; 24 hours later the cells were inoculated at 0 hours; temperature 28~ Unpublished data, ORIAS • RASMUSSEN. *) No increase in size is seen in these cells.

158 Carlsberg Res. Commun. Vol. 41, No 3, 1976 L. RASMUSSEN:Nutrient uptake in Tetrahymena towards nutrient uptake by these vesicles have they may have significant qualitative roles in been made. We may infer that they cannot the uptake of specific nutrients. bring into the cell all the required nutrients at a fast rate, since three cell lines of T. pyriformis 3.2. Nutrient Uptake without Membrane grew very poorly in filter-sterilized media com- lnvagination pared to the cells having food vacuoles in the This section deals mainly with uptake through autoclaved proteose peptone broth (RASMUS- the plasma membrane, that is, uptake without SEN & KLUDT (82) and Table IV). The same participitation of membrane invagination as argument can be applied to NPI cells without typified by food vacuole formation, and without food vacuoles suspended in 2 per cent proteose quantitatively significant formation of other peptone broth or in the chemically defined vesicles. medium, both of which support fast growth of the wild type cells: the mutant cells do not mul- 3.2.1. The roles of the plasma membrane tiply in these media (RASMUSSEN & ORIAS(84)). It is important to decide if carrier-mediated up- Therefore it can be concluded that the summed take of nutrients occurs in T. pyriformis. In the activity of all non-food-vacuolar vesicles is in following discussion some results which throw no way sufficient to supply all the nutrients in light on this problem are presented: amounts required for reasonably fast continued (i) The oral apparatus partially dedifferentiated growth and multiplication under these con- (WILLIAMS • ZEUTHEN (115)) and food vacuole ditions. Such vesicles also failed to take up formation ceased for a period of about 20 either heat-denatured or dissolved egg albumin minutes before cell division (CHAPMAN- at a rate fast enough to support continued cell ANDRESEN & NILSSON (13)). CROCKETT, multiplication (Table II, lines c and d, DUNHAM & RASMUSSEN(20) found that the in- respectively). However, it is possible that the corporation of radioactive phenylalanine and vesicles may take up specific nutrients present histidine administered separately in short pulses in high concentrations in the medium in which occurred at nearly constant rates in heat-syn- the NPI cells grow. Enriched media which chronized cells (SCHERBAUM gr ZEUTHEN (100)) support growth of these cells contain Fe, Cu studied in an otherwise inorganic salt solution and folinic acid in much higher concentrations (HAMBURGER & ZEUTHEN (38)). This showed than those required for growth of normal cells. that these amino acids could be taken up during The whole question of small vesicles is unclear, the period in which no food vacuoles were but a good survey of them is given by NILSSON formed. (64, see Fig. 7 in this reference). Some of the (ii) HOFFMANN & RASMUSSEN (41) measured small vesicles may be membrane recycling the initial rates of methionine uptake in the vesicles, others membrane renewal vesicles, i.e. presence and absence of food-vacuole-in- adding rather than removing membranes at the ducing particles. These rates were independent cytopharynx, while still others may be of food vacuole formation. LING ~; ORIAS micropinocytic, in any case their contribution (personal communication) measured the rates to uptake is unknown, and until satisfactory of uptake of L-phenylalanine using wild type electron microscope tracer experiments are cells in the absence and presence of food-va- made, no clarification of this problem can be cuole-inducing particles, and in the food-va- expected. Such experiments have been in cuole-less mutant, NP1. They found no signifi- progress for a year in collaboration between cant differences. Overall uptake rates are, Drs. J. NILSSON, E. ORIASand the author. therefore, not measurably influenced by the In conclusion, T. pyriformis contain many activity of the food vacuoles. vesicles which are smaller than the food (iii) ORIAS & RASMUSSEN (71) showed that the vacuoles some of which may be pinched off requirements for each of the four vitamins from the plasma membrane by pinocytosis. nicotinic acid, pantothenic acid, pyridoxal, and These vesicles are not likely to make a major riboflavin-5'-P were similar in food-vacuole- contribution to the ceils' overall nutrition, but producing wild type and in food-vacuole-less

Carlsberg Res. Commun. Voi. 41, No 3, 1976 159 L. RASMUSSEN"Nutrient uptake in Tetrahymena

NPI cells. This suggested that uptake of these centration range studied, he found that this vitamins by the food vacuole could not play any sugar was not concentrated by the cells. The essential role. Uptake must occur across the further addition of glucose to a culture plasma membrane or by micropinocytosis, if equilibrated with arabinose resulted in a loss of the latter has any quantitative significance. arabinose from the cells against a concentration (iv) Several groups of investigators studied up- gradient. Thus he found exchange of in- take of phenylalanine in T. pyriformis. STEPHENS tracellular arabinose with extra-cellular & KERR (102), HOFFMANN & RASMUSSEN (41) glucose. According to WILBRANDT 8s and LING & ORIAS (personal communication ROSENBERG (111) exchange kinetics, such as found that the uptake showed saturation those observed with the amino acids and kinetics, and STEPHENS & KERR (102) and LING monosaccharides, can be explained by the & ORIAS (54) found that it was stereo-specific, assumption of the existence of binding sites temperature-dependent and inhibitable with which can transport compounds both into and 2,4-dinitrophenol, which is apparantly an un- out of the cells. Such demonstrations of ex- coupler of oxidative phosphorylations in T. change kinetics call for specific, binding sites pyriformis (HAMBURGER & ZEUTHEN (38)). Up- on the plasma membrane, rather than for take of methionine also showed saturation vesicle-bound transport. kinetics and was stereo-specific (HOFFMANN & These results (i)-(iv) considered together RASMUSSEN (41)). suggest that the plasma membrane of T. Uptake of nucleosides has also been studied. pyriformis possesses carrier-mediated uptake FREEMAN & MONER (31, 32) and WOLFE (118) mechanisms. Such mechanisms are well-known found that uptake of uridine by T. pyriformis from bacteria and higher cells (see e.g. showed saturation kinetics. FREEMAN & CHRISTENSEN (15)). Experimental evidence for MONER (32) reported that thymidine, deoxy- them is available, however, only in the case of guanosine, deoxycytidine, deoxyadenosine, 6- few of the required nutrients. It is likely that mercaptopurine riboside, and 5-fluoro- this type of uptake plays an important role, deoxyuridine inhibited uptake of uridine com- both in food-vacuole-less NPI mutant cells and petitively. Ribose and deoxyribose did not in wild-type cells of T. pyriformis suspended in affect uridine transport, thus providing nutrient medium containing amino acids, evidence for the view that the specificity of the nucleosides, vitamins and other compounds transport mechanism resided in the purine or which can be utilized by the cells without prior pyrimidine portion of the nucleoside. digestion. FREEMAN & MONER'S (32) results supported ZEUTHEN'S (119) proposal that several NILSSON(63) proposed that mucus - ultimately to be nucleosides, particularly uridine and deoxy- seen in the buccal cavity and in the food vacuole - uridine, interfered with mechanisms which might concentrate nutrients prior to its appearance in transport thymidine into the cells. the food vacuole in analogy with what CHAPMAN- RASMUSSEN & ZEUTHEN's (86), HOLZ, ANDRESEN & HOLTER(12) observed in studies on the relation between the glycocalyx coat and pinocytosis RASMUSSEN & ZEUTHEN'S(50), and RASMUSSEN in amoebae. Single nutrients or many may be in- & ZEUTHEN's (87, 88) studies on amino acid volved. However, if such a concentration mechanism antagonisms have been continued by is to play a significant role in general food uptake in HOFFMANN, RASMUSSEN& ZEUTHEN (42). They T. pyriformis it must operate with a wide variety of compounds required by the cells: at least 10 essential found that certain, but not all, amino acids in- amino acids, 2 essential nucleosides, 7 vitamins, and terfered with the uptake of extra-celllalar L- with a number of inorganic ions (KIDDER • DEWEY phenylalanine, and with the exchange of intra- (52)). Furthermore, the adsorption must show stereo- cellular L-phenylalanine with extra-cellular specificity in relation to at least two amino acids: amino acids (Fig. 6). Before this CIRILLO (16) HOFFMANN & RASMUSSEN(41) showed that the initial uptake rate of D-methionine was about I l per cent of studied uptake kinetics of arabinose in T. the uptake rate of L-methionine at the same con- pyriformis. Arabinose is a pentose which is not centration, and LING& OinkS (54) showed that the in- metabolized by these cells. Within the con- itial uptake rate of D-phenylalanine was less than 20

160 Carlsberg Res. Commun. Vol. 41, No 3, 1976 L. RASMUSSEN; Nutrient uptake in Tetrahymena

1 I I I I .... i' I I ! cpm

2.000 // addition of either L-phe(a) L- teu (b) L-met (c) .//" 1.500 L-try (d) or L-pro (e) ./

1.000

500 L,0 I I I 1 I I I I 0 30 60 90 120 150 180 210 240 MINUTES AFTER WASH Figure 6. The effect of various amino acids (added singly to different subcultures at the point indicated by the arrow) on the radioactivity of the extra-cellular fluid of a culture in which the cells had been pre-incubated with C~'-L-phenylalanine for one hour and then transferred by three washings into fresh, non-radioactive inorganic medium (at time zero). Curve f shows the level of extra-cellular radioactivity if no amino acid is added. Reproduced from ref. 42 with permission from the Danish Science Press.

per cent of that of L-phenylalanine. In view of this the part of the cell's nutrients under different sets present author would prefer to suggest that specific, of conditions. In most cases, however, both rather than general adsorption mechanisms function organdies most likely contribute to nutrient up- in nutrient uptake in T. pyriform&. take at the same time. Even though WEIDENSACH & THOMPSON (109) found differences in the lipid composition of 4. CONCLUDING REMARKS the plasma and vacuolar membranes, it is possi- It has been shown that both the plasma ble that these membranes are similar with membrane and the food vacuoles are active in respect to transport abilities. If we accept this food uptake in T. pyriform&. Separately, each of idea for the moment, then it follows that low- them seems able to supply at least the major molecular weight compounds like amino acids

Carlsberg Res. Commun. Vol. 41, No 3, 1976 161 L. RASMUSSEN:Nutrient uptake in Tetrahymena

and glucose pass both membranes equally well, it seems possible that natural habitats offer both whereas macromolecules would not be ex- particulate and dissolved food. This prompts pected to pass any of them at a fast rate. the question: Are dissolved compounds like Similarly, insoluble or complex-bound heavy amino acids, vitamins and carbohydrates taken metal salts would be expected not to be taken up through the cell surface at rates which con- up by any of the two routes or to be taken up tribute significantly to the cells' need? In the poorly. According to such views the food few cases where Km-values (half-saturation vacuoles might serve to bring nutrients into a concentrations) have been determined, they chemical form which can pass the food-va- seem to be fairly low. For strain GL-cells we cuolar membrane. Digestive enzymes hydrolyse have the following values (in /aM con- organic macromolecules, and possible changes centrations): for L-phenylalanine 15 (SrEPHENS in the vacuolar acidity might bring otherwise in- & KERR (102)) and 94 (HOFFMANN 8s soluble salts or their complexes in solution. This RASMUSSEN (41)); for L-methionine 7.8 is a broad formulation of the generally accepted (HOFFMANN & RASMUSSEN (41)); for uridine view of the function of the food vacuole. 2.3 (MONER, personal communication) and 2 If contrary to what was suggested above the (WOLFE (117)). For syngen 1 cells L1NG & transport abilities of the plasma membrane and OR1AS (personal communication) found two up- the food vacuole membrane are different, then take systems for L-phenylalanine: system l is the latter membrane would appear to be more half saturated at 20 /aM (same for NPI in the permeable to iron and copper compounds than food-vacuole-less state) and system 2 at a con- the former. This might reflect the demonstrated centration of about 5 /aM in both cell types. difference in composition of the two types of These concentrations were not influenced by membranes, or it might reflect that addition of induction of new food vacuoles. These values lysosomal membrane - in the process of fusion will now be compared with those determined of lysosomes with food vacuoles - endows the for amino acid uptake in bacteria. PIPERNO & food vacuole membrane with new OXENDER (73) found Km-values of 1.1, 1.2 and specifications. 8/aM for L-leucine, L-isoleucine and L-valine, Because the plasma membrane is equipped respectively, in Escherichia coil; in Streptococcus with chemical pumps, the cells can grow on a faecalis MORA & SNELL (58) found values of 80 balanced diet of low-molecular weight com- to 10t) and 50 /aM for glycine and L-alanine, pounds, represented by the chemically defined respectively; and in Comamonas sp. GUROFF & medium. Because of the cell's ability to create BROMWELL (37) found 20 and 60 uM for L- changes in the micro-environment in the food phenylalanine and L-tyrosine, respectively. vacuole the cell can feed on bacteria, organic Thus Kin-values which relate to uptake of particles and macromolecules. This dual amino acids seem to be of the same order of capacity for uptake makes it possible for T. magnitude in bacteria and in T. pyriformis. pyrifo~'mis to grow and multiply in a variety of The natural habitat of T. pyriformis is abundant media, ranging from suspensions of bacteria to in bacteria, micro-organisms and detritus chemically defined solutions with exclusively particles. The results presented here show that low-molecular weight solutes like amino acids, this cell is well equipped for a dual uptake of glucose, vitamins etc. nutrients: by means of the food vacuoles it may There is no doubt that T. pyriformis in nature - collect and digest bacteria and micro-or- in lakes etc. - feeds on micro-organisms smaller ganisms, and by means of uptake mechanisms than itself, such as bacteria and diatoms, and in the plasma membrane it may compete with laboratory experiments have shown that a sin- the micro-organisms for dissolved low-mole- gle species of bacteria can serve as the sole food cular weight nutrients. source (CURDS & COCKBURN (21)). On the other hand, laboratory experiments have also 5. SUMMARY shown that T. pyriformis can feed exclusively on Quantitative aspects of food uptake in the dissolved low-molecular compounds. Presently ciliate protozoon, Tetrahymena pyriformis,

162 Carlsberg Res. Commun. Vol. 41, No 3, 1976 L. RASMUSSEN:Nutrient uptake in Tetrahymena represent a series of unsolved problems. The specially supplemented media without forming cells form food vacuoles which are readily seen food vacuoles, provided the concentrations of at low magnification in the light microscope. specific nutrients, different for different strains The view has been held that the major part of of 7". pyriform& are increased very much. The the nutrients are taken up by the food vacuoles. significance of the food vacuoles thus depends The first observation to contradict this view was on the composition of the nutrient medium. published more than 50 years ago, but it seems The role of other vesicles in nutrient uptake is to have been forgotten. To-day, many cases not known. The significance of the cell surface have been reported where the food vacuoles is exemplified by measurements of uptake rates seem to be insufficient to cover the cells' actual of some amino acids and monosaccharides. It is uptake. Therefore uptake mechanisms and likely that uptake by the cell surface plays an their roles in food uptake in T. pyriformis under important rote for T. pyriformis, whenever it is various growth conditions were discussed. This suspended in nutrient media which contain subject was introduced in the first section. compounds which can be taken up without Section 2 reviews morphological studies prior digestion. pertaining to structures which may take part in Section 4 presents some ecological con- nutrient uptake: food vacuoles, other vesicles siderations. The few Kin-values (half-sa- and the cell surface. The best known of these turation values) known for uptake of amino structures are the food vacuoles: features of acids in T. pyriformis are compared with similar their development from formation to expulsion values for amino acid uptake in bacteria. Since are described; lysosomes which contain the values seem to be in the same low con- hydrolytic enzymes fuse with the food vacuoles centration range (1 to 100 ~M), it is proposed and bring about digestion of macromolecules that although the ciliate in its natural habitats present here; and later, unknown mechanisms feeds mainly on bacteria, other micro-or- take up digestion products across the food ganisms and detritus taken up by means of the vacuole membrane. Reports of vesicles food vacuoles, it is possible that under these (diameter about 0.btm)which may take part in conditions it can also compete effectively with food uptake are mentioned. So far, it is not other micro-organisms for dissolved, low-mo- known how many types of these vesicles exist in lecular weight organic nutrients. T. pyriformis and how rapidly they are formed. Section 3 deals with the roles of food vacuoles, other vesicles and the cell surface in nutrient ACKNOWLEDGEMENTS uptake. The conclusions on the role of the food It gives me much pleasure to thank everybody vacuole build on experiments in which the mul- who in some way has helped towards the com- tiplication rates of the cells are determined un- pletion of the work reported here. First and der conditions where food vacuole formation is foremost I thank Professor, dr. phil. E. controlled. The major part of the experiments ZEUTHEN, head of the Biological Institute of the were carried out on a mutant unable to form Carlsberg Foundation, for inspiring collabora- food vacuoles at 37~ but wild-type cells have tion and innumerable discussions over many been used, in which food vacuole formation years. Then I express sincere gratitude to my was regulated by manipulations with the co-authors, cand. scient. T. A. KLUDT, cand. physical and chemical characters of the growth scient. E. K. HOFFMANN, cand. med. vet. L. medium. Food vacuoles are required for rapid MODEWEG-HANSEN, Dr. H. E. BUHSE, JR. and and sustained multiplication in standard growth Dr. E. ORIASfor their contributions to the pro- media. These include solutions of unknown ject. I am grateful also to dr. phil. C. CHAPMAN- chemical composition (proteose peptone ANDRESEN, dr. phil. J. R. NILSSON and media) and chemically defined substrates con- Professor G. G. HOLZ, JR. for discussions on sisting exclusively of low-molecular weight nutrient uptake. I wish to thank Drs. G. A. compounds like amino acids, glucose, vitamins THOMPSON, R. D. ALLEN and J. G. MONER for etc. It is also shown that the cells can grow in access to unpublished material.

Carlsberg Res. Commun. Vol. 41, No 3, 1976 163 L. RASMUSSEN:Nutrient uptake in Tetrahymena

Furthermore I am pleased to be able to 12. CHAPMAN-ANDRESEN, C. & H. HOLTER: acknowledge the excellent technical assistance Differential uptake of protein and glucose by pinocytosis in Amoeba proteus. Compt. Rend. of Miss D. MOENBO and Mrs. B. DOHN, the Tray. Lab. Carlsberg 34:211-226 (1964) competent secretarial help of Mrs. K. 13. CHAPMAN-ANDRESEN,C. • J. R. NILSSON; On ANDERSEN in the preparation of the vacuole formation in Tetrahymena pyriformis manuscript, and the unfailing co-operation of GL. Compt. Rend. Trav. Lab. Carlsberg 36: 405- Mr. G. KNUDSEN in solving practical problems 432 (1968) in the workshop. Mr. J. S. WATSON and Dr. R. 14. CHAPMAN-ANDRESEN, C. 8r D. M. PRESCOTT: Studies on pinocytosis in the amoebae Chaos E. PEARLMANkindly read the manuscript and I chaos and Amoeba proteus. Compt. Rend. Trav. am grateful to them for their comments. Lab. Carlsberg 30" 57-78 (1956) 15. CHI~ISTENSEN, H. N.: Biological Transport, 2nd edit. W. A. Benjamin Inc. (Reading, Mass.) 1975. REFERENCES 16. CmtLLO, V. P.: Mechanism of arabinose 1. ALLEN, R. D.: Fine structure, reconstruction transport in Tetrahymena pyriformis. J. Bact. 84: and possible functions of components of the 754-758 (1962) cortex of Tetrahymena pyriformis. J. Protozool. 17. CONNER, R. L.: Transport phenomena in 14:553-565 (1967) protozoa. In: G. W. Kidder, ed., Chemical 2. ALLEN, R. D.: Food vacuole membrane growth Zoology 1: pp. 309-350. Academic Press (New with microtubule-associated membrane trans- York and London) 1967. port in Paramecium. J. Cell Biol. 63:904-922 18. CORLISS;J. O.: An illustrated key to the higher (1974) groups of the ciliated protozoa, with definitions 3. BEGGER, J. D.: Kinetics of incorporation of of terms. J. Protozool. 6:265-281 (1959) DNA precursors from ingested bacteria into 19. Cox, F. E. G.: A quantitative method for macromolecular DNA of Paramecia aurelia. J. measuring the uptake of carbon particles by Protozool. 18:419-429 (1971) Tetrahymena pyriformis. Trans. Am. Microsc. 4. BLUM, J. J. & T. L. ROTHSTEIN: Lysosomes in Soc. 86:261-267 (1967) Tetrahymena. In: J. T. Dingle and R. T. Dean, 20. CROCKE'Iq', R. L., P. B. DUNHAM 8s L. eds., Lysosomes in Biology and Pathology 4, pp. RASMUSSEN: Protein metabolism in Tetrahymena 33-45. North-Holland Publishing Company pyriformis cells dividing synchronously under (Amsterdam and New York) 1975. starvation conditions. Compt. Rend. Trav. Lab. 5. BRANDT, P. W. & K. B. HENDIL: Plasma mem- Carlsberg 34:451-486 (1965) brane permeability and pinocytosis in Chaos 21. CURDS, C. R. & A. COCKBURN: Continuous chaos. Compt. Rend. Trav. Lab. Carlsberg 38: monoxenic culture of Tetrahymena pyriformis. J. 423-443 (1972) Gen. Microbiol. 66; 95-108 (1971) 6. CHAPMAN-ANDRESEN,C.: Studies on pinocytosis 22. DICKIE, N. & I. E. LIENER: A study of the in amoebae. Compt. Rend. Trav. Lab. Carlsberg proteolytic system of Tetrahymena pyriformis W. 33:73-264 (1962) I. Purification and partial characterization of 7. CHAPMAN-ANDRESEN, C.: Measurement of the constituent proteinases. Biochim. Biophys. material uptake by cells: pinocytosis. In: D. M. Acta 64:41-51 (1962) Prescott, ed., Methods in Cell Physiology 1: pp. 23. DICKIE, N. &: I. E. LIENER; A study of the 277-304. Academic Press (New York and proteolytic system of Tetrahymenapyriformis W. London) 1964. II. Substrate specificity of the constituent 8. CHAPMAN-ANDRESEN,C.: Biology of the large proteinases. Biochim. Biophys. Acta 64:52-59 amoebae. Ann. Rev. Microbiol. 25:27-48 (1971) (1962) 9. CHAPMAN-ANDRESEN,C.; Endocytic processes. 24. DUNHAM, P. B. & D. L. KROPP: Regulation of In: K. W. Jeon, ed., The Biology of Amoeba, pp. solutes and water in Tetrahymena. In: A. M. 319-348. Academic Press (New York and Elliott, ed., Biology of Tetrahymena pp. 165- London) 1971. 198. Dowden, Hutchinson and Ross, Inc. 10. CHAPMAN-ANDRESEN, C. 8s S. CHRISTENSEN" (Stroudsburg, Pa.) i973. Pinocytic uptake of ferritin by the amoeba 25. de DUVE, C: From cytases to lysosomes. Fed. Chaos chaos measured by atomic absorption of Proc. 23:1045-1049 (1964) iron. Compt. Rend. Trav. Lab. Carlsberg 38; 19- 26. ELLIOTT A. M.: Primary lysosomes in Tetra- 58 (1970) hymenapyriformis. Science 149:640-641 (1965) 11. CHAPMAN-ANDRESEN,C. & H. HOLTER: Studies 27. ELLIOrr, A. M., ed., Biology of Tetrahymena. on the ingestion of 14C-glucose by pinocytosis in Dowden, Hutchinson & Ross, Inc. (Strouds- the amoeba Chaos chaos. Exptl. Cell Res. suppl. burg, Pa.) 1973. 3:52-63 (1955) 28. ELLIOTT, A. M. & G. L. CLEMMONS: An ul-

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