In the polymorphic ciliate Tetrahymena vorax, the non-selective phagocytosis seen in microstomes changes to a highly selective process in macrostomes
Department of Biology, University of Oslo, PO Box 1051 Blindern, N-0316 Oslo, Norway
* e-mail: h.k.gronlien{at}bio.uio.no
Accepted 22 April 2002
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Summary |
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After capturing a T. thermophila cell, the macrostomal cell, which normally swims in a forward direction, reverses direction and swims backwards for a short time before starting to rotate. Macrostomal cells did not change their swimming pattern after capturing a latex bead. We believe, therefore, that backward swimming is more likely to be related to signals resulting from phagocytosis than from mechanical stimulation of the pouch.
Cytochalasin B (10 µg ml-1) inhibits phagocytosis in both microstomes and macrostomes, indicating that actin filaments play an active role in phagocytosis in both cell types. The antitubulin drug nocodazole (0.3-30 µmol l-1) inhibits the formation of more than one phagosome in the macrostome, indicating that membrane transport to the oral apparatus in macrostomes is guided by microtubules. Nocodazole has no effect on the process of phagocytosis in microstomes.
Key words: Tetrahymena vorax, polymorph, microstome, macrostome, phagocytosis, phagosome, latex bead, Tetrahymena thermophila, cytochalasin B, nocodazole, microtubule, actin filament
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Introduction |
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The polymorphic ciliate Tetrahymena vorax appears in two forms,
termed microstomes and macrostomes
(Williams, 1961). When a
microstome transforms into a macrostome, its small oral apparatus is resorbed
and replaced with a larger one of different shape termed a pouch
(Buhse, 1966b
; Smith,
1982a
,
b
). Transformation occurs in
the presence of another ciliate, Tetrahymena thermophila, and permits
T. vorax to live as a carnivore preying on T. thermophila
(Buhse, 1966a
). The signal from
T. thermophila that triggers this differentiation in T.
vorax is a complex of iron and nucleic acid catabolites termed stomatin
(Smith-Somerville et al.,
2000
).
Little is known about the process of phagocytosis in macrostomes. The
transformation of microstomes results in cells that are able to capture,
phagocytose and digest T. thermophila. Here, we have investigated
whether the phagocytotic process in macrostomes is non-selective or whether it
is specifically aimed at catching T. thermophila. Phagocytosis by
microstomes seems to be non-selective. Food particles are swept into the oral
apparatus by beating of the oral ciliary membranelles. The process may be
compared with so-called macropinocytosis in macrophages, whereby relatively
large volumes of medium are internalized in large endosomes
(Swanson, 1989). If
macrostomes select a certain ciliate for feeding, the capture of these cells
may be initiated by binding of the prey to specific binding sites, similar to
the interaction between receptor and prey that occurs before phagocytosis by
macrophages and neutrophils. To obtain information about the selectivity of
phagocytosis in macrostomes, we compared phagocytotic uptake of T.
thermophila cells, microstomes and latex beads by macrostomal cells. To
further characterize phagocytosis by macrostomes, we studied the effects of
drugs that affect the cytoskeleton (microtubules, microfilament) in these
cells. In parallel control experiments, we assessed the effects of the same
drugs on phagocytosis by microstomes.
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Materials and methods |
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Animals
Tetrahymena vorax strain V2 and Tetrahymena thermophila
were kept at room temperature (21-23 °C) in standard growth medium
(Plesner et al., 1964). The
experiments were performed on cells in the mid-logarithmic phase of
exponential growth (Grelland,
1988
). Cell densities were approximately 5x105
cells ml-1 for T. vorax and 8x105 cells
ml-1 for T. thermophila. The size of the elongated
microstomal cell was approximately 60 µmx20 µm. The size of the
more spherical T. thermophila was approximately 35 µmx25
µm. The size of the T. vorax cells was approximately the same as
that of interphase T. thermophila cells immediately after
division.
Transformation of microstomes into macrostomes
Transformation of microstomes into macrostomes is initiated by a
transforming substance called stomatin that is released into the medium by
T. thermophila (Smith-Somerville
et al., 2000; Buhse,
1967
). To obtain a stomatin-containing solution, 100 ml of the
T. thermophila culture was centrifuged to pellet the cells, and the
cells were subsequently suspended in 50 ml of deionized water and incubated
for 3 h at room temperature. The suspension was centrifuged to remove the
cells, and the supernatant was filtered through a 0.45 µm pore diameter
filter. The stomatin-containing solution was added to the T. vorax
culture in a ratio of 1:10 (v/v). The first macrostomes appeared after
approximately 5 h (at 23 °C), and macrostomes continued to be formed for
the next 5-7 h. The first division of a macrostome into microstome daughters
occurred after 15-17 h. In these experiments, we used macrostomal cells that
had been in the stomatin solution for 8 h; approximately 20 % of the cells had
been transformed to macrostomes at this point. The size of the macrostomal
cell was approximately 120 µmx80 µm.
Prey selection and formation of phagosomes in macrostomes
To study the process of prey selection and the formation of phagosomes in
macrostomes, the cells were incubated with latex beads or with ciliated or
deciliated T. thermophila cells. T. thermophila was
mechanically deciliated using the procedure described by Hawkins
(1975). To determine the prey
concentration that gave optimal phagocytotic uptake, dose/response curves for
both latex beads and deciliated T. thermophila were obtained (data
not shown). The optimal concentrations were 106 beads
ml-1 and 105 cells ml-1. To compare
phagocytosis of latex beads and deciliated T. thermophila by
macrostomes, the cells were exposed to the same concentrations (106
prey ml-1) of the two types of prey. The macrostomes were exposed
to latex beads and deciliated T. thermophila cells for 30 min with
manual agitation every 10 min to keep the prey in suspension. The deciliated
cells were immobile during the experiments. This is in accordance with results
reported by Rannestad (1974
),
who found that deciliated T. thermophila stay immobile for 40 min
after deciliation. At the end of the incubation period, the cells were quickly
but gently mixed with formaldehyde to a final concentration 0.4 % (the
formaldehyde was methanol-free; Code F017/3 from TAAB Laboratories, England,
UK). In one set of experiments, repeated three times, the numbers of latex
beads in the pouch and/or T. thermophila in the food vacuoles in 100
macrostomes were counted using light microscopy (magnification 20x).
In the experiments in which phagosome formation in individual macrostomes was studied, macrostomal cells were selected with a manually operated micropipette and placed in separate drops under liquid paraffin in Petri dishes. A single macrostomal cell was placed in the droplet together with either five small microstomal cells (approximately 35 µmx20 µm) or a mixture of 25 small microstomes and five. T. thermophila cells. All prey were manually collected using a suction pipette. The T. thermophila cells were marked with Texas-Red-labelled ovalbumin, and the cells were observed with both a fluorescence and a light microscope. The Texas-Red-labelled T. thermophila culture was washed three times before the cells were added to the droplets. The behaviour of the animals was recorded using a video camera recording at 25 frames s-1.
Drug treatments
To determine the effects of cytochalasin B and nocodazole on phagocytosis
in microstomes and macrostomes, cells were first preincubated with the drugs
for 8 h and 2 h, respectively. Microstomes were exposed to carmine particles
(50 µg ml-1) for 6 min, and three-quarters of the culture was
quickly but gently fixed with formaldehyde (final concentration 0.4 %). In one
set of experiments, repeated three times, labelled food vacuoles were counted
in 100 microstomes. The remaining 25 % of the cells were washed five times in
Dryl solution and allowed to stand for 30 min before being fixed in
formaldehyde (0.4 %). Macrostomes were allowed to feed on T.
thermophila cells (105 cells ml-1) for 30 min
before they were gently fixed in formaldehyde (final concentration 0.4 %). In
one set of experiments, repeated three times, food vacuoles were counted in
100 macrostomes.
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Results |
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We next compared the capture by macrostomes of latex beads with the capture of T. thermophila. Latex beads with a diameter of 30 µm were chosen since this size is nearest that of T. thermophila. We used deciliated T. thermophila since swimming T. thermophila cells may conceivably be easier to capture than immobilised cells. The rate of phagocytosis, given as the number of digestive vacuoles formed in 30 min, is more than twice as high for ciliated (2.6±0.1; mean ± S.E.M., N=4) as for deciliated (0.93±0.3; N=6) prey.
Deciliated T. thermophila or latex beads (30.0 µm indiameter) were added to T. vorax cultures 8 h after exposure to stomatin-containing solution. Prey concentrations were 106 ml-1. The control results presented in Fig. 2 show that the percentage of macrostomes capturing deciliated T. thermophila when exposed for 30 min to cells only was 37.7±0.9 % (mean ± S.E.M.) (Fig. 2) and that the percentage of macrostomes capturing latex beads when exposed for 30 min to beads only was 30.7±3.5 % (Fig. 2). If the medium contained both deciliated cells and latex beads (final concentration 106 prey ml-1), the macrostomes consistently selected cells rather than beads (Fig. 2). The percentage of macrostomes capturing deciliated T. thermophila in this medium during the 30 min exposure was 32.7±6.1 %, and the percentage capturing latex beads was 4.0±5.1 %.
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In an immature macrostome, the prey attaches to the mouth for 2-5 s, but the macrostomal cell is unable to engulf it since the mouth is not yet fully developed (data not shown). We did not observe this abortive interaction between macrostomes and prey when immature macrostomes were exposed to latex beads.
To determine whether the macrostome recognizes a signal that is specific for T. thermophila, a macrostomal cell was placed in a droplet of medium (under liquid paraffin) together with either a mixture of small microstomes and T. thermophila or only small microstomes. If the droplet contained only microstomes, the macrostomal cell did phagocytose microstomal cells. In another set of experiments, six droplets contained a mixture of 25 microstomes and five T. thermophila each, in addition to the macrostome. In this experiment, the effect of the size difference between a normal microstome and T. thermophila cells was reduced by manually collecting microstomal cells of a size close to that of a T. thermophila cell (approximately 35 µmx20 µm) using a suction pipette. In all six droplets, the macrostome selected exclusively T. thermophila.
The macrostome does not phagocytose latex beads
We do not know the mechanisms whereby phagocytosis is initiated in ciliates
(Allen and Fok, 2000). In the
presence of only latex beads in the medium, macrostomes captured but did not
phagocytose the beads (Fig.
3A). When both latex beads and T. thermophila were
present in the medium, a bead was occasionally phagocytosed together with the
cell (Fig. 3B). This was,
however, a rare event. At least 300 macrostomal cells were observed during
these experiments, and concurrent uptake of beads and cells was observed only
four times. Since the latex beads were of the same size as T.
thermophila, the results indicate that the signal for phagocytosis in
macrostomes is not merely mechanical but probably due to signal molecules
associated with or released from T. thermophila.
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Behavioural response to phagocytosis in macrostomes
When T. thermophila has been captured in the pouch, the
macrostomal cell continues to swim in the same direction before starting a
short backward swim that is followed by cell rotation
(Fig. 4). The time from capture
to the reverse reaction was found to be 1.7±0.6 s (N=8). The
total duration of the chain of events depicted was 28.3±5.2 s (means
± S.E.M., N=8). The macrostomal cells did not change their
swimming pattern after capturing a latex bead. At the same time as the
macrostome swims backwards in response to phagosome formation, the anterior
part of the cell contracts. We believe, therefore, that backward swimming is
related to the process of phagocytosis.
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Effects of cytoskeletal inhibitors on phagocytosis in macrostomes and
microstomes
To further characterize phagocytosis in macrostomes, we studied the effects
of cytochalasin B, an inhibitor of microfilament polymerization, and
nocodazole, an inhibitor of microtubules, on phagocytosis by macrostomes. The
drugs were dissolved in DMSO, and the final DMSO concentration in the cell
suspensions was far below the critical concentration (2.5% v/v) for
suppressing phagocytosis in Tetrahymena pyriformis
(Nilsson, 1974). The effects
of these drugs on phagocytosis in microstomes were studied in parallel
experiments.
Cytochalasin B, which is a potent inhibitor of actin polymerization
(Wessels et al., 1971), has been shown to inhibit the separation of the
nascent phagosome from the oral apparatus in ciliates
(Nilsson et al., 1973;
Allen and Fok, 1985
;
Fok et al., 1985
;
Leakey et al., 1994
). Actin
polymerization may be required just before the nascent phagosome starts to
move along the microtubular bundle (Cohen et al.,
1984a
,b
;
Allen and Fok, 2000
). To test
the effects of cytochalasin B on phagocytosis in macrostomes, the drug (final
concentration 10 µg ml-1) was added to the culture together with
stomatin-containing solution 8 h before phagocytosis was initiated. The long
preincubation period was chosen because it has been shown that, to achieve a
maximal effect, the cells need to be exposed to the drug for at least 8 h
(Leakey et al., 1994
). As a
control, we tested the effect of the drug on the microstomes in the same
culture. Cytochalasin B was found to inhibit phagosome formation in both
macrostomes and microstomes (Table
1). In cytochalasin-B-treated macrostomal cells, a T.
thermophila cell was often captured but the prey continued to swim in the
pouch (Fig. 5) it was also
frequently able to escape. These results indicate that the formation of the
phagosome requires actin assembly in the macrostomal form of T.
vorax.
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In Paramecium spp., phagosomes arise in the lower part of the oral
apparatus and grow as discoidal vesicles fuse with the membrane of the oral
apparatus (Allen, 1974). The
discoidal vesicles are transported to the oral apparatus along microtubular
ribbons (Schroeder et al.,
1990
) anchored to the membrane of the oral apparatus
(Allen, 1974
). In
Tetrahymena spp., these ribbons are less well developed; they are
shorter and the number of microtubules per ribbon is smaller than in
Paramecium (R. D. Allen, personal communication).
To determine whether phagocytosis in the two forms of T. vorax is
dependent on microtubules, we used nocodazole, which has been shown to
depolymerize microtubules (Adelman et al.,
1968). As depicted in Fig.
6, phagocytosis in microstomal cells was not significantly
(t-test, P>0.05) inhibited by nocodazole (final
concentration 5 µmol l-1). However, the movement of the
phagosomes was prevented by the drug, and the phagosomes stayed near the oral
apparatus. To count the number of phagosomes, the cells were washed five times
in Dryl solution after the 6 min incubation period with carmine particles and
allowed to stand for 30 min before being fixed in formaldehyde (0.4 %). In
macrostomes, the concentrations of nocodazole used were 0.3, 3.0 and 30.0
µmol l-1. The nocodazole-treated macrostomes rarely formed more
than one phagosome (Fig. 7),
possibly as a result of the inhibition of membrane transport (in the form of
discoidal vesicles transported along microtubules) to the pouch. Membrane
transport in microstomal cells did not seem to be affected by nocodazole since
the drug did not inhibit phagocytosis in these cells.
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Discussion |
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The percentage of macrostomes in the process of capturing prey during 30 min of incubation with either deciliated T. thermophila or latex beads was approximately 30 % (Fig. 2), which may seem low. It should be noted, however, that all cells with a large pouch were counted. Thus, both immature and dividing macrostomes, which do not phagocytose, were counted. In immature macrostomal cells, which are incapable of engulfing prey since the pouch is not completely developed, the T. thermophila remained bound for 2-5 s to the anterior surface of the cell. There were no signs of any interaction between the anterior part of an immature macrostome cell and a latex bead, supporting the idea that macrostomes recognize T. thermophila selectively.
Phagocytosis in macrostomes is not triggered by mechanical
stimulation
The mechanism whereby phagosomes are pinched off from the oral apparatus in
ciliates is unknown. It has been suggested that the formation of food vacuoles
is induced by the mechanical action of captured particles
(Allen and Fok, 2000).
Tetrahymena pyriformis incubated with polystyrene latex particles and
natural food particles (bacteria) accepted the polystyrene latex particles as
readily as the bacteria, and acid phosphatase appeared normally in the
vacuoles (Muller et al.,
1965
). Microstomal cells cultured in Dryl formed phagosomes when
they were exposed to latex beads with a diameter of 3.0 µm (data not
shown). Phagocytosis of particles in microstomes therefore seems to be a
rather non-selective process. Food particles are swept into the oral apparatus
by the beating of the oral ciliary membranelles, and mechanical stimulation by
the prey may subsequently trigger phagocytosis.
The non-selective phagocytosis seen in microstomes changes to a highly
selective process during the transformation to macrostomes. In macrostomal
cells, as in microstomal cells, latex beads were captured in the oral
apparatus but whereas the oral apparatus containing beads separates to form a
closed vacuole in microstomes, this step does not take place in macrostomes
(Fig. 3A), indicating that
mechanical stimulation by the prey does not in itself trigger phagocytosis in
the macrostomal form of T. vorax. Although macrostomes captured
T. thermophila in preference to microstomes, phagocytosis of
microstomes started immediately following capture, indicating that the
substance/molecule that triggers the formation of the phagosome is not
specific for T. thermophila cells. However, T. thermophila
seem to contain `signal' molecules with higher affinity or in greater amounts
than those in the microstomes. The macrostomes may conceivably contain
receptors in their pouch that need to be activated by signal molecules
associated with or released from the prey in order to stimulate phagocytosis.
The actual phagocytosis of the prey may be initiated by binding to receptor
sites similar to those that mediate phagocytosis in macrophages and
neutrophils (Aderem and Underhill,
1999). Receptor-mediated phagocytosis in macrophages is a
well-documented process initiated by signals that originate from the receptors
involved in binding the particle (Aderem
and Underhill, 1999
). Two-dimensional electrophoresis of the
macrostome oral apparatus of T. vorax has revealed approximately 55
polypeptides in addition to the set of approximately 145 polypeptides also
found in the microstome oral apparatus
(Gulliksen et al., 1984
). Some
of these additional peptides in macrostomes may correspond to receptor
proteins and signal proteins.
The closing of the food vacuole is dependent on actin filaments
Little is known about the molecular mechanisms that lead to the closing of
a phagosome in ciliates. In `higher' Eukaryota, receptors (e.g. Fc receptors)
mediating phagocytosis are expressed on macrophages and neutrophils. Receptor
clustering occurs upon particle binding, and this generates a phagocytotic
signal that leads to the activation of kinases (including scr and syk and
phosphatidylinositol 3-kinase) (Ninomiya
et al., 1994). Monomeric GTPases, in cooperation with
phosphatidylinositol 3-kinase, can modulate the assembly of the submembranous
actin filament system, leading to particle internalization
(Araki et al., 1996
).
Cytochalasin B, a potent inhibitor of actin polymerization, inhibits the
detachment of the nascent phagosome from the oral apparatus in both
microstomes and macrostomes. This is in accordance with earlier observations
of cytochalasin-B-treated Paramecium spp. and T. thermophila
(Nilsson et al., 1973
;
Allen and Fok, 1985
;
Fok et al., 1985
). It has been
suggested that actin polymerization may be required just before the
commencement of the movement of the nascent phagosome along the postoral
microtubular bundle (Schroeder et al.,
1990
). Antibodies against heavy meromyosin (which decorates actin)
label the periphery of phagosomes during and shortly after phagosome movement
(Cohen et al.,
1984a
,b
),
indicating that the pinching off of the nascent phagosome is dependent on
actin microfilaments.
Membrane transport to the pouch in macrostomes occurs along
microtubules
Microtubules are an important constituent of the architecture of the oral
apparatus in Tetrahymena spp. The microtubules form a deep fibre
bundle that extends far into the cytoplasm, and the food vacuoles are guided
along these tubules away from the oral apparatus
(Nilsson and Williams, 1966).
New membrane is incorporated into the membrane of the oral apparatus during
phagocytosis. Small vesicles are present in the oral region of
Tetrahymena spp. (Nilsson and
Williams, 1966
; Nilsson,
1976
), and their membranes resemble those of the food vacuoles
(Nilsson, 1979
). In
Paramecium spp., the membrane of the nascent phagosome is provided by
discoidal vesicles that fuse with the membrane of the oral apparatus
(Allen, 1974
). The discoidal
vesicles are transported to the oral apparatus along microtubular ribbons
(Schroeder et al., 1990
)
anchored to the membrane of the oral apparatus
(Allen, 1974
). It has been
suggested that vesicles retrieved from food vacuoles during processing as well
as after fusion with the cytoproct may be reused for vacuole formation
(Allen and Fok, 1980
).
Nocodazole, an inhibitor of microtubule formation, did not affect phagocytosis
in microstomes in our studies (Fig.
6). It is possible, however, that nocodazole does not depolymerize
microtubules in Tetrahymena spp. The microtubular ribbons in the oral
region of Paramecium spp. are unaffected by nocodazole (R. D. Allen,
personal communication). The absence of effect could also be due to a large
membrane pool already docked to the oral apparatus.
Surprisingly, nocodazole greatly inhibited the formation of more than one phagosome in macrostomes (Fig. 7); this could be due to inhibition of membrane transport along microtubules to the pouch. However, the need for membrane during phagocytosis to form the pouch may require the retrieval and recycling of membrane from previously formed phagosomes before the cell can capture another prey.
Backward swimming after capture of a T. thermophila cell is probably
related to the process of phagocytosis
When a T. thermophila has been engulfed in the pouch, the
macrostomal cell, which normally swims in a forward direction, reverses
direction and swims backwards for a short time before starting to rotate
(Fig. 4). This backward
swimming could be due to the avoidance reaction normally seen after mechanical
stimulation of the anterior part of the cell. This behaviour has been studied
in detail in Paramecium spp.
(Naitoh and Eckert, 1969).
Touching the front end of the animal opens stretch-sensitive Ca2+
channels and generates a depolarizing receptor potential that leads to a
reversal of the ciliary beat and backward swimming. T. vorax, in both
its microstome and macrostome forms, display a similar avoidance reaction to
mechanical obstacles (H. K. Grønlien, unpublished data). If the
observed backward swimming were related to the stimulation of mechanosensitive
channels in the pouch by the prey, we might expect backward swimming to occur
after capture of a latex bead. However, since the macrostomal cells did not
change their swimming pattern after capturing a latex bead, we believe that
backward swimming is more likely to be related to signals resulting from
phagocytosis. At the same time as the macrostome swims backward in response to
phagosome formation, the anterior part of the cell contracts.
Backward swimming may be the result of an increased internal
Ca2+ concentration, which will cause reversal of the cilia
(Naitoh and Eckert, 1969). It
has been shown that Ca2+ plays a role in the initiation of pouch
closure to form a closed vacuole in the macrostomal form of T. vorax
(Sherman et al., 1982
). This
observation supports the idea that influx of Ca2+ to the cytosol is
one step in the signal-transduction process during phagocytosis in
macrostomes. Contractile proteins have also been reported to be associated
with the oral region of Pseudomicrothorax dupius
(Hauser et al., 1980
). In
Tetrahymena spp., three different calmodulin proteins have been found
(Takemasa et al., 1989, 1990), and the cytosol of Tetrahymena spp.
contains Ca2+-activated ATPases
(Chua et al., 1977
). The
increased cytosolic Ca2+ concentration may be a result of influx of
Ca2+ from the environment and/or from intracellular organelles.
Abundant alveoli, the main storage organelles of Ca2+
(Stelly et al., 1991
), are
associated with the oral apparatus in Tetrahymena pyriformis
(Nilsson and Williams, 1966
).
Ca2+ chelators (EDTA and EGTA) are known to cause collapse and
resorption of the pouch in the macrostomal form of T. vorax
(Sherman et al., 1982
).
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Acknowledgments |
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