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 Tetrahymena
thermophila were used to investigate a possible
connection between acid hydrolase secretion and
the major processes through which membranes
are recycled in this ciliated protozoon. These
processes consist of food vacuole formation (endocytosis), and food vacuole egestion and mucocyst release (both exocytosis). We have found that
a mutant (MS-1, see") blocked in hydrolase secretion is not blocked in either food vacuole
formation or egestion and that it has normal
mucocyst exocytosis. Another line of experiments
with wild-type cells showed also that hydrolase
secretion and endocytosis are independent of
each other. Thus, sucrose (0-1M) did not interfere
with hydrolase secretion, but blocked food
vacuole formation. Furthermore, release of acid
hydrolases was selectively stimulated by dibucaine without any effect on food vacuole egestion.
Finally, exocytosis of mucocysts could occur
without simultaneous release of acid hydrolases,
as when cells were exposed to (M5 M-NaCl, which
evokes a massive secretory response of mucocysts. Our results demonstrate that formation and
egestion of food vacuoles and exocytosis of mucocysts are unrelated to secretion of acid hydrolases. Furthermore, they suggest that secretion of acid hydrolases is not a secondary
effect of membrane recycling through these processes.
Introduction
should fuse with the plasma membrane before release
of enzymes. Here, we investigate a possible connection
of this process with other events taking part in membrane traffic in Tetrahymena.
Release of acid hydrolases to the extracellular medium
by the ciliate Tetrahymena has been the object of many
studies (Blum & Rothstein, 1975; Muller, 1971;
Nilsson, 1979). Muller (1972) found that released acid
hydrolases originate from the denser of two populations
of lysosomes and considered this release to be secretion. We have recently substantiated this view and
shown that enzyme release is enhanced by Ca 2+ mobilizing agents and that the hydrolases are liberated
without loss of other cytoplasmic components (Tiedtke
el al. 1984).
The cellular route of the hydrolases is unknown. To
date, lysosomal enzymes are not known to pass the
membrane of the lysosome (Creek & Sly, 1984) and
therefore vesicles that contain lysosomal enzymes
Journal of Cell Science 89, 515-520 (1988)
Printed in Great Britain © The Company of Biologists Limited 1988
Key words: membrane recycling, secretion, acid
hydrolases, Tetrahymena thermophila.
Materials and methods
Strains
The wild-type strains B i l l and B VII, and the mutant MS-1
of Tetrahymena theniiophila blocked constitntively in release
of lysosomal enzymes (Hiinseler el al. 1987«), were used in
this study. The phenotype of MS-1 is caused by a recessive
single gene mutation (Tiedtke et al. 1987).
Culture conditions
The cells were grown in PPYS medium: 1% proteose
peptone (Difco), 0 1 % yeast extract (Difco), 0003 % sequestrene (Geigy). Cell cultures were inoculated at initial concentrations of 2x 104 cells ml"' and grown at 30°C in a shaker
515
1A
B
Fig. 1. Bright-field micrographs of T. themiophila
containing food vacuoles labelled with India ink. A. The
initial stage of the formation of an India ink-labelled food
vacuole; B, distribution of labelled food vacuoles after
20-30min incubation with 0-05% India ink;
C, accumulation of food vacuoles at the posterior end of
the cell as observed after a chase of 30 min.
(90 revs min~ ) up to cell concentrations around
8X 10 s cellsml~'. Cell-free supernatants were collected by
centrifugation (3 min, 600g) of the cell suspensions into a
cushion of 10% (w/v) Ficoll.
Evaluation of egestion rates
Cells in PPYS medium were labelled with India ink (005 %
final concn) for 20 min at 30°C. During this pulse the cells
ingested India ink particles and contained many black food
vacuoles. The cells were then washed twice with PPYS
medium to remove remaining India ink particles, resuspended in fresh PPYS medium and incubated at 30°C for
30min. At the end of this chase period many labelled food
vacuoles were observed in the posterior parts of the cells
ready for egestion through the cytoproct (Fig. 1A-C). In this
way, egestion could be followed from the start of the
experiment. The wild-type (B VII) and the mutant MS-1 cell
suspensions were adjusted to Sx 103 cells ml" 1 following the
pulse-labelling with India ink. At each time point indicated
four samples of cells were removed and OS ml of each sample
was fixed for food vacuole countings, the remaining cell
suspensions were centrifuged and the cell-free supernatants
were collected on ice for assays of enzyme activity. Out of
each fixed sample 60 cells were chosen randomly and the
number of labelled food vacuoles per cell was found.
To measure the action of dibucaine on the egestion of
labelled food vacuoles, wild-type cells ( B i l l ) were washed
after a chase of 30 min in PPY'S medium into lOniMTris-HCl, pi I 6-0. The cells were exposed to the drug at
25°C for 15 min and then processed for food vacuole counting
and enzyme assays as described above for control cells not
treated with the drug.
Dibucaine treatment
Solutions of 6mmoll~' of dibucaine (Sigma) in lOmMTris- IICI, pi I 6 0 , were freshly prepared before each experiment. At pi I 6-0 but not at higher pH values dibucaine was
readily soluble. No meaningful pH variations were observed
during the experiments in spite of the reduced buffer capacity
of Tris- IICI at pi I 6 0 . Cultures of the wild type ( B i l l ) were
516
A. Tiedtke et al.
adjusted to 1-5x10''cells ml ' after the end of the 30-min
chase period and samples of one ml (containing cells with
labelled food vacuoles) were added to 15-ml centrifugation
tubes containing 2ml of the corresponding drug solution.
The drug concentrations before addition of the cells were I -5
times the final concentration indicated in Results. Control
experiments were performed identically using the corresponding buffer without drugs. The tubes with the cells were
placed in a tilted rack to ensure aeration of the cell suspension. Immediately before the end of the incubation periods
small samples were withdrawn and the status and behaviour
of the drug-treated cells were controlled under a microscope.
Samples for food vacuole countings and enzyme assays were
withdrawn as described above. The data were statistically
analysed with the help of Student's /-test.
Alcian Blue-induced mucocyst exocytosis
The method is based on previous observations of the sccretagogue activity of this dye (Tiedtkc, 1976). It consisted of
exposing cell suspensions starved for 2 h to 0-1 % Alcian Blue
and observing in the light microscope the blue-stained
mucocyst aggregates around the cells and in the medium.
The question whether the Alcian Blue treatment also caused
an enhanced secretion of acid hydrolases was not investigated: the Alcian Blue remaining in the supernatant interacts
strongly with proteins and prevents meaningful enzyme
determinations.
Exposure to 0-1 M-sucrose
Wild-type cells ( B i l l ) were washed twice in lOmMTris H O , pH7-4, and the washed cell suspension adjusted
to 10 6 cellsml~' was divided into two portions. One was
mixed with an equal volume of 02M-sucrose in 10niMTris- HC1, pH 7-4, while the other received the same volume
of buffer without sucrose. Each of these suspensions was
immediately divided in two. One was used to measure
released enzyme activities and the other to follow food
vacuole formation after addition of India ink (0-05%, final
concn). The cells were incubated at 25°C and samples were
withdrawn at 30 and 60 min for food vacuole countings and
enzyme assays.
Exposure to 015 M-NaCl
In this experiment washed B i l l cells in 10 mM-Tris'1IC1,
pH 7-4, were either mixed with an equal volume of the same
buffer or the same buffer supplemented with 03M-NaCl,
under continuous stirring. Suspensions were allowed to stand
at 25°C for 2min and centrifuged. Supernatants were collected for enzyme assays and mucocyst inspection.
Inspection of mucocysts
Samples of the supernatants of both control and NaCl-treated
cells were exposed to cationized ferritin after addition of an
equal volume of 2mM-MgCl2. Observation of mucocysts
stained in this way was performed using a Zeiss Photomicroscope. For electron microscopy (EM), the samples were
spread on carbon-coated grids, negatively stained with a 1 %
aqueous solution of uranyl acetate and observed with a
Siemens Elmiskop at 60 kV.
Enzyme assays
Enzyme activities of the cell-free supernatants were assayed
at 37CC, using /)-nitrophenyl-conjugated substrates for acid
phosphatase (EC 3.1.3.2), A'-acetyl-j8-D-hexosaminidase (EC
3.2.1.52), cr-mannosidase (EC 3.2.1.24) and <r-glucosidase
(EC 3.2.1.20) at final concentrations of lOmmoll" 1 , according to Tiedtke (1983«). One milliunit (1 ml)) is defined as the
amount of enzyme that releases 1 nmol of />-nitrophenol per
min at 37°C. Isocitrate dehydrogenase (IDH, EC 1.1.1.42)
was assayed colorimetrically according to the supplier's
information (Biochemica Information II, Boehringer-Mannheim). Isocitrate dehydrogenase, a sensitive cytosolic marker
enzyme of Telrahyniena (Borden el al. 197S), was measured
during the drug experiments to exclude cell lysis as a source
of released lysosomal enzymes.
Results
Acid hydrolase secretion (AHS) and egestion of
marked food vacuoles in the wild-type and a mutant
blocked in AJiS
Food vacuoles of wild-type (B VII) and mutant (MS-1)
cell lines were pulse-labelled with India ink. After the
30-min chase period that allows the food vacuoles to
collect at the posterior end, the cells were incubated for
3h at 30°C in nutrient medium. Samples were
removed every 30 min for determinations of enzyme
activity and every 60 min for estimation of number of
food vacuoles per cell. The results are shown in Fig. 2.
It can be observed that the mean number of food
vacuoles decreased gradually in the wild type and in the
mutant MS-1 blocked in AHS. Although the initial
mean number of food vacuoles was slightly higher in
the wild type (10 per cell) than in the mutant (9 per
cell) both strains released a similar number of marked
food vacuoles per unit of time, indicating that MS-1
was not impaired in egestion of food vacuoles. In spite
of this the mutant did not release /3-hexosaminidase and
acid phosphatase (and other lysosomal enzymes, data
not shown) above the background levels characteristic
for this mutant.
Mucocyst exocytosis in wild-type and mutant cells
Mucocyst secretion was induced in wild-type ( B V I I )
and mutant (MS-1) cells by exposure to Alcian Blue.
T h e cell lines were alike in their release of mucocysts,
showing that MS-1 has normal mucocyst exocytosis.
Influence of dibucaine on release of acid hydrolases
and egestion of marked food vacuoles
Table 1 shows the results of exposure of wild-type cells
(B III) to 100 or 200/iM-dibucaine on release of four
lysosomal enzymes and egestion of labelled food
vacuoles. The drug considerably stimulated the release
of acid phosphatase, /3-hexosaminidase, cv-glucosidase
and O'-mannosidase. The mean number of remaining
/3-Hexosaminidasc
200
BVII
10
100
200
X.
Acid phosphatase
10
>B VII
100
Ms-1
Fig. 2. Egestion of food vacuoles and kinetics of release of
acid hydrolases in the wild type (B VII) and the mutant
MS-1, blocked in release of acid hydrolases during 3 h in
nutrient medium. The mean number of food
vacuoles ± standard deviation from four independent
experiments are shown. Open columns, wild type (B VII);
hatched columns, MS-1. The standard deviation of each
point in the kinetics of release of /3-hexosaminidasc and
acid phosphatase was below 10%.
food vacuoles per cell was, however, similar in control
and drug-treated cells showing that food vacuole egestion was not increased by dibucaine. These results
point to an uncoupling of AHS and food vacuole
egestion.
Dibucaine also stimulated AHS in the mutant MS-1
in about the same proportions as it did in wild-type
cells. Mutant cells treated for 15 min with dibucaine
1
(IOOJUM) released 0-51 ± 0-03 mU ml" of acid phosphatase and 0-47 ± 0-027 mU ml"' of j3-hcxosaminidase. The activities released by the cells not treated
with dibucaine were 0-16 ± 0"01 mil ml""' and
0-18 ± 0-005 mU ml"', respectively. This represents a
3-2-fold stimulation of acid phosphatase and a 2-6-fold
stimulation of /3-hexosaminidase release. The corresponding increases in wild-type cells were 2-97-fold for
Membrane recycling in Tetrahymcna
517
Table 1. Influence of dibucaine on the egestion offood vacuoles and the release of acid hydrolases in the wild
type (Bill) ofT. thermophila
Acid phosphatase
/3-Hexosaminidase
cv-Glucosidase
cv-Mannosidase
Mean
number of
food vacuoles
per cell*
1-07 ±0-045
318 ± 0-27
5-33 ± 0 1 1
1-12 ±0-07
2-5 ±0-03
3-1610-13
1-33 ±0-09
4-36 ±0-35
7-12±0-64
1-83 + 0-10
3-52 + 0-14
5-0±0-19
8-3 ±0-8
8-9±0-4
8-98 ±0-02
Released enzyme activities (mil ml ') and mean numbers
of labelled food vacuoles per cell
Control
100/iM-dibucaine
200;iM-dibucaine
• T h e mean number of labelled food vacuoles at the beginning of the drug treatment was 11-3 ± 0-3. Release of acid hydrolases by the
drug-treated cell suspensions is significantly higher (I3<0-005) than release of acid hydrolases in the untreated control suspension. The
mean numbers of food vacuoles per cell are not significantly different from control samples in dibucaine-treated cells. Data were analysed
with the help of Student's /-test.
acid phosphatase and 2-23-fold for /3-hexosaminidase
(Table 1).
Addition of sucrose and effects on food vacuole
formation and AIIS
Incubation of wild-type cells (B III) in buffer contain
ing O'lM-sucrose completely prevented food vacuol
formation during a 60-min period. Control cells formei
10-6 ± 0-8 vacuoles per cell within the same period
Release of acid phosphatase and /?-hexosaminidase wa
identical in treated and non-treated cells.
high rates of endocytosis and exocytosis. We have dealt
here with a Tetrahymena mutant that is blocked in acid
hydrolase secretion (AHS), which can grow and multiply normally (Hiinseler el al. 1987c;). This mutant
•r
NaCl induced inucocyst exocytosis
Massive exocytosis of mucocysts can be induced by
brief (2min) exposure of the cells to 150mM-NaCl
Discharged mucocysts stayed in the supernant afte
centrifugation. They were detected in the light micro
scope as rod-like structures after negative staining witl
cationized ferritin (Fig. 3A). The identity of these rod
as decondensed mucocysts matrices was verified b
EM. Samples negatively stained with uranyl acetat
exhibited the regular fibrillar pattern and uniform siz
characteristic of decondensed mucocysts matrices (Fig
3B). Supernatants of control preparations only oc
casionally contained mucocysts. As summarized ii
Table 2, the NaCl treatment induced massive exocy
tosis of mucocysts but did not stimulate AHS. Treat
ment with 150mM-NaCl rapidly immobilized the cell
and caused osmotic shrinkage. However, the cell
recovered from this treatment when diluted 10-foli
with 10mM-Tris- HC1, pH 7-4, at the end of the 2-mii
incubation. Cells resumed normal shape and motilit
during the following 2h.
Discussion
Membrane recycling is the means by which a cell
maintains its plasma membrane surface constant in
spite of processes that continuously add and withdraw
portions of it. This is an obvious need for cells with
518
A. Tiedtke et al.
Fig. 3. Mucocysts in the supernatant of NaCl-trcatecl
Tetrahvmena. The cells were incubated for 2min in lOniMT r i s H C l , pH7-4, plus ISOmM-NaCI and then
sedimented. A sample of the supernatant was mixed with
an equal volume of 2mM-MgCl2 and negatively stained
with cationized ferritin (A) or uranyl acetate (B). Bars: A,
11 flm; B, 1 fim.
Table 2. Secretion of acid hydrolases by
Tetrahymena after a 2-min incubation in 10mMTrisHCl, pill-4, with and without 150mM-NaCl
Released enzyme activities
(mUml" 1 )
Control
NaCl
Acid phosphatasc
/3-Hexosaminidase
0-83 ±0-07
0-55 ±0-06
0-80 ±0-09
0-43 + 004
Secretion of acid hydrolases is significantly lower (P< 0-005) in
NaCl-treated cells.
has membrane recycling as shown by its ability to form
and egest food vacuoles, and to discharge mucocysts.
We can therefore conclude that membrane recycling
can proceed without acid hydrolase release (AHS) in
Tetrahymena. In other words, membrane recycling is
not a sufficient condition for acid hydrolase release.
We have also tested the possibility that blockage of
release of hydrolases in this mutant could be due to
deficient dissociation of the enzymes from receptors at
the plasma membrane after exocytosis. In such a case,
it should be expected that hydrolases accumulate on the
cell surface. We used the method described by Gottlieb
& Dwyer (1981) for determination of membrane surface enzymes and found that MS-1 has a significantly
lower content of acid phosphatase and /3-hexosaminidase activities on the cell surface than wild-type cells
(unpublished data). Since biosynthesis of lysosomal
enzymes is not impaired in this mutant (Hiinseler et al.
19876), the mutation probably affects the behaviour of
the vesicles containing acid hydrolases, preventing
their fusion with the plasma membrane. This emphasizes the view that AHS is separate from the mainstream of membrane recycling in Tetrahymena.
Dissociation of food vacuole formation from AHS in
wild-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 in
food vacuole formation but secretes essentially normal
amounts of acid hydrolases. It also confirms the report
by Miiller (1975) of the independence of AHS from the
contractile vacuole activity, which, unlike AHS, is
inhibited in media with high osmolarity.
It was proposed that the acid hydrolases left the cells
via the egested food vacuoles. Rothstein & Blum
(1974) based this view on a simultaneous stimulation of
AHS and food vacuole egestion mediated by catecholamine antagonists. Our results, however, both with the
mutant MS-1 and with the wild-type cells exposed to
dibucaine, which may enhance enzyme release by an
increase in cytoplasmic free Ca 2+ (Tiedtke et al. 1984),
demonstrate that egestion of vacuoles is unrelated to
AHS. This seems to be the case also for both
Dictyostelium (Dimonde/c//. 1984) and Acanthamoeba
(Hohman & Bowers, 1984). We must point out that the
secretory mutant MS-1 can grow on bacteria as sole
food source as fast as the wild-type cells (Hiinseler el
al. 1987a). It has, therefore, a normal intracellular
digestion process, i.e. fusion of lysosomes with food
vacuoles. Such fusion has recently been demonstrated
by Fok & Schokley (1985) in Tetrahymena growing in
PPY medium. As a consequence, we can conclude that
retrieval or inactivation of lysosomal enzymes must
take place prior to egestion, since MS-1 normally egests
food vacuoles but does not release enzymes. Evidence
for retrieval of lysosomal enzymes has been obtained
for another ciliate, Paramecium caudatum (Allen &
Fok, 1984).
A possible role for mucocyst exocytosis in AHS was
suggested by the finding of acid phosphatase activity
associated with these organelles (Tiedtke & Gortz,
1983). The method employed, however, permitted no
quantification of the amounts of enzyme released in this
way. Our present experiments, in which exposure to
0-15M-NaCl results in massive discharge of mucocysts
without enhancement in AHS, show that these two
processes are essentially independent. This agrees well
with our previous observation that another secreted
hydrolase, /3-hexosaminidase, is not associated with
discharging mucocysts, as shown by immunostaining
with specific antibodies against this enzyme (Tiedtke,
19836). As to the biological role of the secreted
hydrolases, their exploitation in extracellular digestion
has already been shown for Tetrahymena (Rasmusscn
et al. 1986). The observations presented here suggest
that AHS, at least in Tetrahymena, should be regarded
as an independent cellular event and not just as an
incidental consequence of membrane recycling, as
Hohman & Bowers (1984) proposed for Acanthamoeba
castellani.
We thank Dr L. Rasmussen for critical reading of and for
many helpful comments on the manuscript. Support from
the Deutsche Forschungsgemeinschaft to A.T. is gratefully
acknowledged, M.F.-C. was the recipient of a HcinrichHertz-Stiftung fellowship, and J.F.-C. of an EM BO shortterm fellowship.
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