Exocytosis, endocytosis and membrane recycling in

Tetrahymena thermophila

ARNO TIEDTKE, PETER HUNSELER

Institute of/.oology, University of Minister, Schlofipkitz 5, D-4400 Minister, FRG

JORGE ELORIN-CHRISTENSEN and MONICA FLORIN-CHRISTENSEN

Center for Parasite and Vector Research, Xational University of La Plata, Calle 2, Xo. 5S4, 1900 La Plata, Argentina

Summary

Mutant and wild-type cell lines of Tetrahymenathermophila were used to investigate a possibleconnection between acid hydrolase secretion andthe major processes through which membranesare recycled in this ciliated protozoon. Theseprocesses consist of food vacuole formation (en-docytosis), and food vacuole egestion and muco-cyst release (both exocytosis). We have found thata mutant (MS-1, see") blocked in hydrolase se-cretion is not blocked in either food vacuoleformation or egestion and that it has normalmucocyst exocytosis. Another line of experimentswith wild-type cells showed also that hydrolasesecretion and endocytosis are independent ofeach other. Thus, sucrose (0-1M) did not interferewith hydrolase secretion, but blocked food

vacuole formation. Furthermore, release of acidhydrolases was selectively stimulated by dibu-caine without any effect on food vacuole egestion.Finally, exocytosis of mucocysts could occurwithout simultaneous release of acid hydrolases,as when cells were exposed to (M5 M-NaCl, whichevokes a massive secretory response of muco-cysts. Our results demonstrate that formation andegestion of food vacuoles and exocytosis of muco-cysts are unrelated to secretion of acid hy-drolases. Furthermore, they suggest that se-cretion of acid hydrolases is not a secondaryeffect of membrane recycling through these pro-cesses.

Key words: membrane recycling, secretion, acidhydrolases, Tetrahymena thermophila.

Introduction

Release of acid hydrolases to the extracellular mediumby the ciliate Tetrahymena has been the object of manystudies (Blum & Rothstein, 1975; Muller, 1971;Nilsson, 1979). Muller (1972) found that released acidhydrolases originate from the denser of two populationsof lysosomes and considered this release to be se-cretion. We have recently substantiated this view andshown that enzyme release is enhanced by Ca2+-mobilizing agents and that the hydrolases are liberatedwithout loss of other cytoplasmic components (Tiedtkeel al. 1984).

The cellular route of the hydrolases is unknown. Todate, lysosomal enzymes are not known to pass themembrane of the lysosome (Creek & Sly, 1984) andtherefore vesicles that contain lysosomal enzymes

Journal of Cell Science 89, 515-520 (1988)Printed in Great Britain © The Company of Biologists Limited 1988

should fuse with the plasma membrane before releaseof enzymes. Here, we investigate a possible connectionof this process with other events taking part in mem-brane traffic in Tetrahymena.

Materials and methods

StrainsThe wild-type strains B i l l and B VII, and the mutant MS-1of Tetrahymena theniiophila blocked constitntively in releaseof lysosomal enzymes (Hiinseler el al. 1987«), were used inthis study. The phenotype of MS-1 is caused by a recessivesingle gene mutation (Tiedtke et al. 1987).

Culture conditionsThe cells were grown in PPYS medium: 1% proteosepeptone (Difco), 0 1 % yeast extract (Difco), 0003 % seques-trene (Geigy). Cell cultures were inoculated at initial concen-trations of 2x 104 cells ml" ' and grown at 30°C in a shaker

515

1A B

Fig. 1. Bright-field micrographs of T. themiophilacontaining food vacuoles labelled with India ink. A. Theinitial stage of the formation of an India ink-labelled foodvacuole; B, distribution of labelled food vacuoles after20-30min incubation with 0-05% India ink;C, accumulation of food vacuoles at the posterior end ofthe cell as observed after a chase of 30 min.

(90 revs min~ ) up to cell concentrations around8X 10scellsml~'. Cell-free supernatants were collected bycentrifugation (3 min, 600g) of the cell suspensions into acushion of 10% (w/v) Ficoll.

Evaluation of egestion ratesCells in PPYS medium were labelled with India ink (005 %final concn) for 20 min at 30°C. During this pulse the cellsingested India ink particles and contained many black foodvacuoles. The cells were then washed twice with PPYSmedium to remove remaining India ink particles, resus-pended in fresh PPYS medium and incubated at 30°C for30min. At the end of this chase period many labelled foodvacuoles were observed in the posterior parts of the cellsready for egestion through the cytoproct (Fig. 1A-C). In thisway, egestion could be followed from the start of theexperiment. The wild-type (B VII) and the mutant MS-1 cellsuspensions were adjusted to Sx 103 cells ml"1 following thepulse-labelling with India ink. At each time point indicatedfour samples of cells were removed and OS ml of each samplewas fixed for food vacuole countings, the remaining cellsuspensions were centrifuged and the cell-free supernatantswere collected on ice for assays of enzyme activity. Out ofeach fixed sample 60 cells were chosen randomly and thenumber of labelled food vacuoles per cell was found.

To measure the action of dibucaine on the egestion oflabelled food vacuoles, wild-type cells (Bi l l ) were washedafter a chase of 30 min in PPY'S medium into lOniM-Tris-HCl, pi I 6-0. The cells were exposed to the drug at25°C for 15 min and then processed for food vacuole countingand enzyme assays as described above for control cells nottreated with the drug.

Dibucaine treatmentSolutions of 6mmoll~' of dibucaine (Sigma) in lOmM-Tris- IICI, pi I 6 0 , were freshly prepared before each exper-iment. At pi I 6-0 but not at higher pH values dibucaine wasreadily soluble. No meaningful pH variations were observedduring the experiments in spite of the reduced buffer capacityof Tris- IICI at pi I 6 0 . Cultures of the wild type (Bi l l ) were

adjusted to 1-5x10''cells ml ' after the end of the 30-minchase period and samples of one ml (containing cells withlabelled food vacuoles) were added to 15-ml centrifugationtubes containing 2ml of the corresponding drug solution.The drug concentrations before addition of the cells were I -5times the final concentration indicated in Results. Controlexperiments were performed identically using the corre-sponding buffer without drugs. The tubes with the cells wereplaced in a tilted rack to ensure aeration of the cell suspen-sion. Immediately before the end of the incubation periodssmall samples were withdrawn and the status and behaviourof the drug-treated cells were controlled under a microscope.Samples for food vacuole countings and enzyme assays werewithdrawn as described above. The data were statisticallyanalysed with the help of Student's /-test.

Alcian Blue-induced mucocyst exocytosisThe method is based on previous observations of the sccreta-gogue activity of this dye (Tiedtkc, 1976). It consisted ofexposing cell suspensions starved for 2 h to 0-1 % Alcian Blueand observing in the light microscope the blue-stainedmucocyst aggregates around the cells and in the medium.The question whether the Alcian Blue treatment also causedan enhanced secretion of acid hydrolases was not investi-gated: the Alcian Blue remaining in the supernatant interactsstrongly with proteins and prevents meaningful enzymedeterminations.

Exposure to 0-1 M-sucroseWild-type cells (Bi l l ) were washed twice in lOmM-Tris H O , pH7-4, and the washed cell suspension adjustedto 106cellsml~' was divided into two portions. One wasmixed with an equal volume of 02M-sucrose in 10niM-Tris- HC1, pH 7-4, while the other received the same volumeof buffer without sucrose. Each of these suspensions wasimmediately divided in two. One was used to measurereleased enzyme activities and the other to follow foodvacuole formation after addition of India ink (0-05%, finalconcn). The cells were incubated at 25°C and samples werewithdrawn at 30 and 60 min for food vacuole countings andenzyme assays.

Exposure to 015 M-NaClIn this experiment washed Bi l l cells in 10 mM-Tris'1IC1,pH 7-4, were either mixed with an equal volume of the samebuffer or the same buffer supplemented with 03M-NaCl,under continuous stirring. Suspensions were allowed to standat 25°C for 2min and centrifuged. Supernatants were col-lected for enzyme assays and mucocyst inspection.

Inspection of mucocystsSamples of the supernatants of both control and NaCl-treatedcells were exposed to cationized ferritin after addition of anequal volume of 2mM-MgCl2. Observation of mucocystsstained in this way was performed using a Zeiss Photomicro-scope. For electron microscopy (EM), the samples werespread on carbon-coated grids, negatively stained with a 1 %aqueous solution of uranyl acetate and observed with aSiemens Elmiskop at 60 kV.

516 A. Tiedtke et al.

Enzyme assaysEnzyme activities of the cell-free supernatants were assayedat 37CC, using /)-nitrophenyl-conjugated substrates for acidphosphatase (EC 3.1.3.2), A'-acetyl-j8-D-hexosaminidase (EC3.2.1.52), cr-mannosidase (EC 3.2.1.24) and <r-glucosidase(EC 3.2.1.20) at final concentrations of lOmmoll"1, accord-ing to Tiedtke (1983«). One milliunit (1 ml)) is defined as theamount of enzyme that releases 1 nmol of />-nitrophenol permin at 37°C. Isocitrate dehydrogenase (IDH, EC 1.1.1.42)was assayed colorimetrically according to the supplier'sinformation (Biochemica Information II, Boehringer-Mann-heim). Isocitrate dehydrogenase, a sensitive cytosolic markerenzyme of Telrahyniena (Borden el al. 197S), was measuredduring the drug experiments to exclude cell lysis as a sourceof released lysosomal enzymes.

Results

Acid hydrolase secretion (AHS) and egestion ofmarked food vacuoles in the wild-type and a mutantblocked in AJiS

Food vacuoles of wild-type (B VII) and mutant (MS-1)cell lines were pulse-labelled with India ink. After the30-min chase period that allows the food vacuoles tocollect at the posterior end, the cells were incubated for3h at 30°C in nutrient medium. Samples wereremoved every 30 min for determinations of enzymeactivity and every 60 min for estimation of number offood vacuoles per cell. The results are shown in Fig. 2.It can be observed that the mean number of foodvacuoles decreased gradually in the wild type and in themutant MS-1 blocked in AHS. Although the initialmean number of food vacuoles was slightly higher inthe wild type (10 per cell) than in the mutant (9 percell) both strains released a similar number of markedfood vacuoles per unit of time, indicating that MS-1was not impaired in egestion of food vacuoles. In spiteof this the mutant did not release /3-hexosaminidase andacid phosphatase (and other lysosomal enzymes, datanot shown) above the background levels characteristicfor this mutant.

Mucocyst exocytosis in wild-type and mutant cellsMucocyst secretion was induced in wild-type (BVII )and mutant (MS-1) cells by exposure to Alcian Blue.The cell lines were alike in their release of mucocysts,showing that MS-1 has normal mucocyst exocytosis.

Influence of dibucaine on release of acid hydrolasesand egestion of marked food vacuolesTable 1 shows the results of exposure of wild-type cells(B III) to 100 or 200/iM-dibucaine on release of fourlysosomal enzymes and egestion of labelled foodvacuoles. The drug considerably stimulated the releaseof acid phosphatase, /3-hexosaminidase, cv-glucosidaseand O'-mannosidase. The mean number of remaining

200

100

200

100

/3-HexosaminidascBVII

X. Acid phosphatase

>B VII

Ms-1

10

10

Fig. 2. Egestion of food vacuoles and kinetics of release ofacid hydrolases in the wild type (B VII) and the mutantMS-1, blocked in release of acid hydrolases during 3 h innutrient medium. The mean number of foodvacuoles ± standard deviation from four independentexperiments are shown. Open columns, wild type (B VII);hatched columns, MS-1. The standard deviation of eachpoint in the kinetics of release of /3-hexosaminidasc andacid phosphatase was below 10%.

food vacuoles per cell was, however, similar in controland drug-treated cells showing that food vacuole eges-tion was not increased by dibucaine. These resultspoint to an uncoupling of AHS and food vacuoleegestion.

Dibucaine also stimulated AHS in the mutant MS-1in about the same proportions as it did in wild-typecells. Mutant cells treated for 15 min with dibucaine(IOOJUM) released 0-51 ± 0-03 mU ml"1 of acid phos-phatase and 0-47 ± 0-027 mU ml"' of j3-hcxosamini-dase. The activities released by the cells not treatedwith dibucaine were 0-16 ± 0"01 mil ml""' and0-18 ± 0-005 mU ml" ' , respectively. This represents a3-2-fold stimulation of acid phosphatase and a 2-6-foldstimulation of /3-hexosaminidase release. The corre-sponding increases in wild-type cells were 2-97-fold for

Membrane recycling in Tetrahymcna 517

Table 1. Influence of dibucaine on the egestion of food vacuoles and the release of acid hydrolases in the wildtype (Bill) ofT. thermophila

Released enzyme activities (mil ml ') and mean numbersof labelled food vacuoles per cell

Acid phosphatase /3-Hexosaminidase cv-Glucosidase cv-Mannosidase

Meannumber of

food vacuolesper cell*

Control100/iM-dibucaine200;iM-dibucaine

1-07 ±0-045318 ± 0-275-33 ± 0 1 1

1-12 ±0-072-5 ±0-03

3-1610-13

1-33 ±0-094-36 ±0-357-12±0-64

1-83 + 0-103-52 + 0-145-0±0-19

8-3 ±0-88-9±0-4

8-98 ±0-02

•The mean number of labelled food vacuoles at the beginning of the drug treatment was 11-3 ± 0-3. Release of acid hydrolases by thedrug-treated cell suspensions is significantly higher (I3<0-005) than release of acid hydrolases in the untreated control suspension. Themean numbers of food vacuoles per cell are not significantly different from control samples in dibucaine-treated cells. Data were analysedwith the help of Student's /-test.

acid phosphatase and 2-23-fold for /3-hexosaminidase(Table 1).

Addition of sucrose and effects on food vacuoleformation and AIIS

Incubation of wild-type cells (B III) in buffer containing O'lM-sucrose completely prevented food vacuolformation during a 60-min period. Control cells formei10-6 ± 0-8 vacuoles per cell within the same periodRelease of acid phosphatase and /?-hexosaminidase waidentical in treated and non-treated cells.

NaCl induced inucocyst exocytosisMassive exocytosis of mucocysts can be induced bybrief (2min) exposure of the cells to 150mM-NaClDischarged mucocysts stayed in the supernant aftecentrifugation. They were detected in the light microscope as rod-like structures after negative staining witlcationized ferritin (Fig. 3A). The identity of these rodas decondensed mucocysts matrices was verified bEM. Samples negatively stained with uranyl acetatexhibited the regular fibrillar pattern and uniform sizcharacteristic of decondensed mucocysts matrices (Fig3B). Supernatants of control preparations only occasionally contained mucocysts. As summarized iiTable 2, the NaCl treatment induced massive exocytosis of mucocysts but did not stimulate AHS. Treatment with 150mM-NaCl rapidly immobilized the celland caused osmotic shrinkage. However, the cellrecovered from this treatment when diluted 10-foliwith 10mM-Tris- HC1, pH 7-4, at the end of the 2-miiincubation. Cells resumed normal shape and motilitduring the following 2h.

high rates of endocytosis and exocytosis. We have dealthere with a Tetrahymena mutant that is blocked in acidhydrolase secretion (AHS), which can grow and mul-tiply normally (Hiinseler el al. 1987c;). This mutant

•r

Discussion

Membrane recycling is the means by which a cellmaintains its plasma membrane surface constant inspite of processes that continuously add and withdrawportions of it. This is an obvious need for cells with

Fig. 3. Mucocysts in the supernatant of NaCl-trcateclTetrahvmena. The cells were incubated for 2min in lOniM-Tr isHCl , pH7-4, plus ISOmM-NaCI and thensedimented. A sample of the supernatant was mixed withan equal volume of 2mM-MgCl2 and negatively stainedwith cationized ferritin (A) or uranyl acetate (B). Bars: A,11 flm; B, 1 fim.

518 A. Tiedtke et al.

Table 2. Secretion of acid hydrolases byTetrahymena after a 2-min incubation in 10mM-TrisHCl, pill-4, with and without 150mM-NaCl

Released enzyme activities(mUml"1)

Acid phosphatasc /3-Hexosaminidase

ControlNaCl

0-83 ±0-070-55 ±0-06

0-80 ±0-090-43 + 004

Secretion of acid hydrolases is significantly lower (P< 0-005) inNaCl-treated cells.

has membrane recycling as shown by its ability to formand egest food vacuoles, and to discharge mucocysts.We can therefore conclude that membrane recyclingcan proceed without acid hydrolase release (AHS) inTetrahymena. In other words, membrane recycling isnot a sufficient condition for acid hydrolase release.

We have also tested the possibility that blockage ofrelease of hydrolases in this mutant could be due todeficient dissociation of the enzymes from receptors atthe plasma membrane after exocytosis. In such a case,it should be expected that hydrolases accumulate on thecell surface. We used the method described by Gottlieb& Dwyer (1981) for determination of membrane sur-face enzymes and found that MS-1 has a significantlylower content of acid phosphatase and /3-hexosamini-dase activities on the cell surface than wild-type cells(unpublished data). Since biosynthesis of lysosomalenzymes is not impaired in this mutant (Hiinseler et al.19876), the mutation probably affects the behaviour ofthe vesicles containing acid hydrolases, preventingtheir fusion with the plasma membrane. This empha-sizes the view that AHS is separate from the main-stream of membrane recycling in Tetrahymena.

Dissociation of food vacuole formation from AHS inwild-type cells was obtained by exposure to ( H M -sucrose, which blocks the former but not the latter.This result agrees with the study by Silberstein (1979)on a Tetrahymena mutant cell line that is blocked infood vacuole formation but secretes essentially normalamounts of acid hydrolases. It also confirms the reportby Miiller (1975) of the independence of AHS from thecontractile vacuole activity, which, unlike AHS, isinhibited in media with high osmolarity.

It was proposed that the acid hydrolases left the cellsvia the egested food vacuoles. Rothstein & Blum(1974) based this view on a simultaneous stimulation ofAHS and food vacuole egestion mediated by catechol-amine antagonists. Our results, however, both with themutant MS-1 and with the wild-type cells exposed todibucaine, which may enhance enzyme release by anincrease in cytoplasmic free Ca2+ (Tiedtke et al. 1984),demonstrate that egestion of vacuoles is unrelated to

AHS. This seems to be the case also for bothDictyostelium (Dimonde/c//. 1984) and Acanthamoeba(Hohman & Bowers, 1984). We must point out that thesecretory mutant MS-1 can grow on bacteria as solefood source as fast as the wild-type cells (Hiinseler elal. 1987a). It has, therefore, a normal intracellulardigestion process, i.e. fusion of lysosomes with foodvacuoles. Such fusion has recently been demonstratedby Fok & Schokley (1985) in Tetrahymena growing inPPY medium. As a consequence, we can conclude thatretrieval or inactivation of lysosomal enzymes musttake place prior to egestion, since MS-1 normally egestsfood vacuoles but does not release enzymes. Evidencefor retrieval of lysosomal enzymes has been obtainedfor another ciliate, Paramecium caudatum (Allen &Fok, 1984).

A possible role for mucocyst exocytosis in AHS wassuggested by the finding of acid phosphatase activityassociated with these organelles (Tiedtke & Gortz,1983). The method employed, however, permitted noquantification of the amounts of enzyme released in thisway. Our present experiments, in which exposure to0-15M-NaCl results in massive discharge of mucocystswithout enhancement in AHS, show that these twoprocesses are essentially independent. This agrees wellwith our previous observation that another secretedhydrolase, /3-hexosaminidase, is not associated withdischarging mucocysts, as shown by immunostainingwith specific antibodies against this enzyme (Tiedtke,19836). As to the biological role of the secretedhydrolases, their exploitation in extracellular digestionhas already been shown for Tetrahymena (Rasmusscnet al. 1986). The observations presented here suggestthat AHS, at least in Tetrahymena, should be regardedas an independent cellular event and not just as anincidental consequence of membrane recycling, asHohman & Bowers (1984) proposed for Acanthamoebacastellani.

We thank Dr L. Rasmussen for critical reading of and formany helpful comments on the manuscript. Support fromthe Deutsche Forschungsgemeinschaft to A.T. is gratefullyacknowledged, M.F.-C. was the recipient of a Hcinrich-Hertz-Stiftung fellowship, and J.F.-C. of an EM BO short-term fellowship.

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(Received 24 September 1987 -Accepted, in revised fonn,16 December 19S7)

520 A. Tiedtke el al.