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Cell Motility and the Cytoskeleton 33:175-182 (1996)
Improved Preparation and Swimming
Behavior of Triton-Extracted Models of
Didinium nasutum
Yoshiaki lwadate and Hiroshi Asai
Department of Physics, Waseda University, Okubo, Shinjuku-ku, Tokyo, Japan
An improved method for the preparation of Triton X-100 extracted models of
Didiniim nasutum was established. Didinium models prepared by treatment with
a Triton X- 100 solution, containing specific proteolysis inhibitors and dimethyl
sulfoxide, maintained an intact shape at 25°C for a longer time than models
prepared by treatment with a Triton X-100 solution not containing the proteolysis
inhibitors and dimethyl sulfoxide.
The improved Didinium models were reactivated so as to swim forward in the
presence of Mg2+ and ATP or ADP. They did not swim backward in response to
C a 2 + , in contrast to well-known Paramecium models. However, the improved
Didiniiim models showed circular swimming and spinning in response to
M
or higher concentrations of C a 2 + . This observation suggests that the quick directional change as well as the spinning, which are characteristic of live Didinium,
are due to an increase in the endoplasmic Ca2+ concentration around the ciliary
system. The response of the Didinium ciliary system to Ca2+ may differ from the
response of the Paramecium ciliary system to Ca2'.
0 1996 Wiley-Liss, Inc.
Key words: ciliary girdles, circular movement, Didinium, Didinium models, spinning, Triton models
INTRODUCTION
Triton X-100 extracted models of Paramecium
caudatum have been reported [Naitoh and Kaneko,
1972, 1973; Nakaoka and Toyotama, 19791. Naitoh and
Kaneko prepared Paramecium models in an extracting
solution not containing Mg2+. The swimming velocity
of the Paramecium models depended on the concentrations of Mg2+ and ATP as well as the pH [Naitoh and
Kaneko, 19731. The swimming direction of the models
depended on the Ca2+ concentration [Naitoh and
Kaneko, 19721. Nakaoka and Toyotama [ 19791 reported
an extraction method utilizing a Triton X-100 extracting
solution containing Mg2+, suggesting the importance of
this ion in the process of preparing of Triton X-100 Paramecium models. The Paramecium models extracted by
the method of Nakaoka and Toyotama changed swimming velocity in the presence of Ca2+, and this response
was very sensitive to the Ca2+ concentration.
Didinium nasutum, a Gymnostomatida ciliate, has
two ciliary girdles. It was reported that Didinium swam
both forward and backward along a right-handed helical
0 1996 Wiley-Liss, Inc.
path [Machemer, 19741. Furthermore, we often observed
that Didinium showed a characteristic swimming behavior, quick directional change and spinning, caused by the
beating of two ciliary girdles. In the case of live didinia,
little study has been devoted t o this characteristic swimming behvior. Moreover, no studies on the characteristic
swimming behavior using Didinium models have yet
been conducted. We previously found that Didinium
models were obtained by treatment with a Triton X-100
extracting solution containing Mg2+ [Iwadate and Asai,
19951, using a preparation method similar to that used
for Paramecium models by Nakaoka and Toyotama
[ 19791. It was also found that Mg2+ is indispensable for
the preparation of Triton X-100 extracted models of Didinium nasutum [Iwadate and Asai, 19951.
Received September 12, 1995; accepted November 20, 1995
Address reprint requests to Yoshiaki Iwadate, Asai Laboratory (Room
51-706), Department of Physics, Waseda University, 3-4-1 Okubo,
Shinjuku-ku, Tokyo 169, Japan.
176
Iwadate and Asai
However, even with the method of Nakaoka and
Toyotama [ 19791, most of these Didinium models maintained an intact shape for less than 5 rnin at 25°C. It is
thus important to investigate the preparation method used
to extract Didinium models. In order to prepare longer
lasting reactivated Didinium models, we investigated an
improved method for the preparation of Triton X-100
extracted models of Didinium nasutum. The Didinium
models, prepared by treatment with a Triton X- 100 extracting solution containing 1 mM p-APMSF, 0.1 mM
leupeptin, 0.1 mM pepstatin, and 1% (vh) dimethyl sulfoxide (DMSO), remained intact morphologically at
25°C for a longer time.
Didinium models are useful for studying characteristic swimming behavior. Utilizing our improved Didinium models, we were able to confirm characteristic
swimming behavior. This report describes an improved
preparation method of preparing Didinium models and
the swimming behavior of these models.
MATERIALS AND METHODS
Cell Culture
The ciliate used in this report was a clone harvested
from a local pond and identified as Didinium nasutum.
The stock cultures of the encysted didinia were excysted
in a hay infusion. They were then fed Paramecium caudatum, cultured in a bacterized hay infusion at room
temperature. Didinia were washed in standard saline solution containing 0.25 mM CaCI,, 7.5 mM KCl, 2 mM
Tris-HC1 at pH 7.2 for 30 rnin at 25°C before all experiments.
Previous Method of Didinium nasutum Extraction
The didinia were gently centrifuged to make a
loose pellet. The pellet was suspended in an ice-cold
(0-1°C) extracting solution which contained 10 mM KCl,
10 mM MgCI,, 5 mM ethylene glycol bis(P-aminoethyl
ether)-N,N,N’,N’-tetraacetic acid (EGTA), 0.008% (v/v)
Triton X-100, and 10 mM Tris-HC1, at pH 7.2, for 60
min. The extracted Didinium cells were washed in an
ice-cold (0-1°C) solution containing 20 mM KCl, 5 mM
MgCI,, 3 mM EGTA, and 10 mM Tris-HC1, at pH 7.2,
for 15 min to remove the Triton X-100. The Didinium
cells were then further washed in an ice-cold (0-1°C)
washing solution of 20 mM KC1 and 10 mM Tris-HC1, at
pH 7.2, for 15 rnin to remove the EGTA. The models
prepared utilizing this procedure are termed type A.
Improved Method of Didinium nasutum Extraction
The didinia were gently centrifuged to make a
loose pellet. The pellet was suspended in an ice-cold
(0-1°C) extracting solution which contained 20 mM KC1,
5 mM MgCI,, 5 mM EGTA, 0.01% (v/v) Triton X-100,
1 mM p-APMSF, 0.1 mM leupeptin, 0.1 mM pepstatin,
1% (v/v) DMSO, and 10 mM Tris-HC1, at pH 7.2, for 45
min. The extracted Didinium cells were washed in an
ice-cold (0-1°C) solution containing 20 mM KC1, 3 mM
EDTA, and 10 mM Tris-HC1, at pH 7.2, for 15 rnin to
remove the Triton X- 100 and Mg2 . The Didinium cells
were then washed again in an ice-cold (0-1°C) washing
solution containing 20 mM KCl and 10 mM Tris-HC1, at
pH 7.2, for 15 rnin to remove the EDTA. The models
prepared utilizing this procedure are termed type B.
+
Comparison of Durability Between Type A and
Type B Models
Models kept in an ice bath during 15 min were used
for the experiment. Twenty microliters of model suspension, containing 50-100 models, was pipetted onto a
depression slide. The model suspension on the depression slide was kept at 25°C. Those models which maintained two intact ciliary girdles and showed a clear proboscis outline were defined as morphologically intact.
The numbers of such intact models were counted every
other minute for 20 min. The percentage of intact models
remaining at each time point was calculated. Separate
measurements of type A and B models were repeated
three times.
Swimming Behavior of Type B Models
A 20 ~1 type B model suspension, containing 50100 models, was mixed with 20 pl of each reactivation
solution at 25°C. The Mg2+ reactivation solutions contained various concentrations of MgCI,, 8 mM ATP, 6
mM EDTA, 20 mM KCI, and 10 mM Tris-HC1, at pH
7.2. The ATP reactivation solution contained various
concentrations of ATP, 16 mM MgCl,, 6 mM EGTA, 20
mM KCl, and 10 mM Tris-HC1, at pH 7.2. The ADP
reactivation solution contained various concentrations of
ADP, 16 mM MgCl,, 6 mM EGTA, 20 mM KC1, and 10
mM Tris-HC1, at pH 7.2. The Ca2+ reactivation solution
contained various concentrations of CaCl,, adjusted with
2 mM EGTA, 8 mM ATP, 16 mM MgCI,, 20 mM KCI ,
and 10 mM Tris-HC1, at pH 7.2.
Swimming behavior was observed under dark-field
microscopy and recorded on videotapes. The video image was photographed for a pre-determined exposure
time. The average swimming velocity was calculated
from 10-50 traces of the swimming models obtained
with each measurement.
RESULTS
Difference in Durability Between Type A and
Type B Models
The time courses of the percentages of intact type
A and type B models remaining are shown in Figure 1.
Swimming Behavior of Didinium Models
0 '
0
5
10
15
177
20
Time [min 1
Fig. 1. The time courses of the percentages of type A and B models remaining intact at 25°C. The values
are averages of 50-100 models from three independent measurements. Models prepared by extraction
without proteolysis inhibitors are termed type A , and those with proteolysis inhibitors are termed type B.
0:type A. 0:
type B.
It is clear that, by 3 min, more than half of the type A
models had collapsed, whereas more than 80% of type B
models were still intact. At 20 minutes, most of the type
A models were no longer intact, whereas about 30% of
type B models were still intact.
Figure 2 shows the results typically observed in this
experiment. As shown in Figure 2A, after 6 min, the
type A model began to collapse around its cell surface.
Whereas, as shown in Figure 2B, the type B model maintained an intact shape for more than 12 min despite gradually becoming more spherical and showing contraction
of the ciliary girdle near the proboscis.
Effect of Mg2+ Concentration on
Swimming Behavior
Swimming velocity, as a function of the Mg2+
concentration, is shown in Figure 3. The swimming velocity was measured in each Mg2 t reactivation solution,
at various Mg2+ concentrations (0-8 mM), in the presence of 4 mM ATP. The swimming velocity of the models increased with increasing Mg2+ concentration, up to
8 mM. It took about 1 min to reach maximum velocity,
after the model suspension had been mixed with each of
the Mg2+ reactivation solutions.
Effect of ATP Concentration on
Swimming Behavior
Swimming velocity as a function of the ATP concentration is illustrated in Figure 4. The swimming velocity was measured in each ATP reactivation solution,
at various ATP concentrations (0-8 mM). The higher the
ATP content, the greater the swimming velocity of the
models, up to 4 mM. It took about 1 min to reach the
maximum velocity after the model suspension had been
mixed with each ATP reactivation solution.
Reactivation was also obtained with ADP and
Mg2+, without ATP, but 2-3 min was required to reach
maximal velocity.
Effect of Ca2+ Concentration on
Swimming Behavior
Swimming traces of the models at pCa of 8.0 and
6.0, are shown in Figure 5. Swimming velocities as a
function of the Ca2+ concentration are shown in Figure
6. The swimming velocity was measured in each Ca2+
reactivation solution, at various Ca2 concentrations
( 10-9- 10-4.5 MI.
At pCa 6.5 and lower Ca2+ concentrations, the
reactivated Didinium models swam forward. The models
+
178
Iwadate and Asai
Fig. 2. Series of photomicrographs of typical type A and B models at 25°C. A: Type A, prepared by
extraction without proteolysis inhibitors. B: Type B, prepared by extraction with proteolysis inhibitors.
Bar = 20 p,m.
I
i
-
-
1
100
i
I
-
50 C
0
L_-__
0
1
L
L
L
L
I
_
_
2
3
5
4
Mg2' concentrat I on
6
7
L
8
[mMl
Fig. 3. Effect of Mg2+ concentration on swimming velocity of type B models. Final concentrations of
ATP (4 mM), EGTA (3 mM), KCl(20 mM), and Tris-HCl(l0 mM) were kept constant throughout. Data
represent means and standard errors for measurements on 50-100 models.
shown in Figure 5A swam along a right-handed helical
path, as illustrated in Figure 5C. The spots in Figure 5A
are non-reactivated models. As shown in Figure 6, the
swimming velocities were essentially constant, regardless of the Ca2+ concentration (10-9-10-6.5 M).
At pCa 6.0 and higher Ca2+ concentrations, reactivated models showed circular swimming not along a
right-handed helical path, as illustrated in Figure 5D, or
spinning, as in Figure 5E. The reactivated models did
not swim backward. As shown in Figure 5B, the swimming courses at pCa 6.0 were not straight. The spots in
Figure 5B, which are indicative of no migration, are
non-reactivated models as well as reactivated spinning
models.
The discharge of pexicysts and toxicysts in response to Ca2+ was not observable in this experiment,
because of the movement of the models in the presence
of ATP and Mg2+. This issue is beyond the scope of the
present report.
dinium in response to Ca2+ has not previously been studied. In this study, owing to an improved preparation of
Didinium models, the swimming behavior of Didinium
models in response to Ca2+ was observable. We demonstrated circular swimming and spinning of Didinium
models in response to
M or higher concentrations
of Ca2+ (see Fig. 5 ) . We speculate that the circular
swimming and the spinning of the Didinium models,
respectively, correspond to the quick directional change
and the spinning of live Didinium. The characteristic
swimming behavior of live Didinium, quick directional
change and spinning, may thus be caused by an increase
in the endoplasmic Ca2+ concentration to lop6 M or
more around the ciliary system.
Paramecium extracted models have been reactivated to swim forward at Ca2+ concentrations below
lop6 M, and to swim backward at Ca2+ concentrations
higher than lop6 M [Naitoh and Kaneko, 1972, 19731.
These results indicate that the ciliary beat direction in
Paramecium is regulated by the intracellular Ca2 concentration. In this investigation, however, we did not
observe backward swimming of Didinium type B models
M or higher concentrations of Ca2+.
in response to
In the case of Paramecium, an increase in the endoplasmic Ca2+ concentration around the ciliary system simply
causes ciliary reversal and backward swimming. How+
DISCUSSION
In studying the ciliary beat of live didinia in response to Ca2+, each Didinium cell had to be fixed for
the purpose of Ca2+ injection [Hara and Asai, 1980;
Hara et al., 19851. Thus, the swimming behavior of Di-
180
Iwadate and Asai
0
U
U
1
2
3
-
1
5
4
ATP concentrat ion
-
-
L
6
7
[mMl
Fig 4 Effect of ATP concentration on swimming velocity of the type B models Final concentrations of
MgCI, (8 mM), EGTA (3 mM), KCI (20 mM), and Tns-HC1 (10 mM) were kept constant throughout
Data represent means and standard errors for measurements on 50-100 models
ever, the beat of the two ciliary girdles of Didinium is suggested that the ciliary beat of Paramecium requires
apparently more complicated. The two ciliary girdles, Mg2 as co-factors for ATP-energized reactivation [Naipeculiar to Didinium nasutum, may cause the quick di- toh and Kaneko, 19731. In the present Didinium type B
rectional change and the spinning, rather than the back- models as well, there was no reactivation swimming in
ward swimming, in response to a
M or higher con- solutions containing ATP but no Mg2+. We thus propose
centration of Ca2 .
that, as with Paramecium, the cyclic ciliary beating of
Presumably, the two types of ciliary girdles have Didinium requires Mg2+. In other words, the substrate
different roles in the characteristic swimming behavior of of dynein ATPase in cilia of Paramecium and also DiDidinium, quick directional change and spinning. The dinium is Mg2+-ATP rather than free ATP.
difference between, and character of these roles of the
In this experiment, we observed the reactivation of
two ciliary girdles have yet to be clarified. What change ciliary beating of Didinium models in the presence of
in the ciliary beat pattern of our Didinium models is ADP and a delay of about 1 min for reactivation with
responsible for the circular swimming and what is re- addition of ADP instead of ATP. This delay may responsible for the spinning are questions that require fur- flect the conversion of ADP to ATP by an adenylate
ther investigation.
kinase.
The ATP, ADP, and Mg2+ concentrations all afWe previously found that the preparation of Didifected the swimming velocity. However, when the Ca2+ nium models was possible in Triton X-100 solution conconcentration was lower than lop6 M, no effect on taining Mg2+ [Iwadate and Asai, 19951. Mg2+ was inswimming velocity was observed (see Fig. 5). These dispensable for the preparation of Didinium models. It
results suggest that an increase in the endoplasmic Ca2+ was previously impossible to prepare Didinium models
concentration around the ciliary system, within physio- in the absence of Mg2+, in contrast to models of Paralogical range, does not affect the beat frequency.
mecium and other ciliates. Paramecium has two fibrous
In the case of Paramecium models, it was reported networks spanning the entire cell surface, an outer lattice
by Naitoh and Kaneko [1973] that the reactivation of and an infraciliary lattice, serving as a cortical cytoskelciliary beating by ATP required Mg2+ or Mn2+. They eton [Garreau de Loubresse et a]. , 19881. Didinium does
+
+
Swimming Behavior of Didinium Models
181
C
Fig. 5 . Swimming traces of the models at pCa 8.0 and 6.0. A: Exposure time 2 sec. At pCa 8.0, the
Didinium models swam along a right-handed helical path, as illustrated in C. B: Exposure time 4 sec. At
pCa 6.0, some of the models showed circular swimming, as in D, and others showed spinning in one spot,
as in E. The spots in A are non-reactivated models. The spots in B, which are indicative of no migration,
represent non-reactivated models and spinning models. Both circular swimming and spinning, which are
characteristic of the Gymnostomatida ciliate Didinium nasurum, were restored by utilizing our improved
Didinium models. Bar = 200 pm.
not appear to have such a network, making the prepara- Tris inevitably as a pH buffer at pH 7.2 in this experition of Didinium models relatively difficult. In this in- ment.
The interesting cell motilities of Didinium, the
vestigation, proteolysis inhibitors and DMSO were
found effective for preparing the better Didinium models characteristic swimming behavior [Wessenberg and An(see Fig. 1). None of the Didinium models could be tipa, 19701, the recognition of Paramecium as prey
prepared when we tried to use piperazine-N,N’ bis-(2- [Harumoto and Miyake, 1992; Miyake and Harumoto,
ethanesulphonic acid) (Pipes) (pKa = 6.8 at 20°C) or 19931, and the discharge of extrusomes [Hara and Asai,
3-(N-morpholino)propanesulfonic acid (Mops) (pKa = 1980; Hara et a]., 19851, have been investigated utilizing
7.2 at 20°C) or N-2-hydroxyethylpiperazine-N’-2-live didinia. The application of our improved Didinium
ethane-sulphic acid (Hepes) (pKa = 7.6 at 20°C) as a pH models was found to be of great value for investigating
buffer instead of Tris (pKa = 8.3 at 20°C). So, we used cell motilities on a molecular basis.
182
Iwadate and Asai
600
-
9
8.5
8
7.5
I
6.5
6
5.5
5
4.5
4
pCa
Fig. 6. Effect of Ca2+ concentration on swimming velocity of the type B models. Final concentrations
of ATP (4 mM), MgCI, (8 mM), KCI (20 mM), and Tris-HC1 (10 mM) were kept constant throughout.
The Ca2+ concentration was adjusted with Ca-EGTA buffer. Data represent means and standard errors
for measurements on 50-100 models.
REFERENCES
Garreau, de Loubresse N., Keryer, G., Viguts, B., and Beisson, J.
( I 988): A contractile cytoskeletal network of Paramecium: The
infraciliary lattice. J. Cell Sci. Y0:35 1-364.
Hara, R., and Asai, H. (1980): Electrophysiological response of Didinium nasutum to Paramecium capture and mechanical stimulation. Nature 283:869-870.
Hara, R.,Asai, H., and Naitoh, Y . (1985): Electrical responses of the
carnivorous ciliate Didinium nasutum in relation to discharge of
the extrusive organelles. J. Exp. Biol. 119:211-224.
Harumoto, T., and Miyake, A. (1992): Possible participation of surface antigens of Paramecium in predator-prey interaction. J.
Protozool. 39:47A.
Iwadate, Y., and Asai, H. (1995): Success in preparing detergentextracted models of Didinium nasutum and extrusive organelle
discharge in response to calcium ions. Cytobios 82:29-37.
Machemer, H. (1974): Ciliary activity and metachronism in Protozoa.
In Sleigh, M.A. (ed.): “Cilia and Flagella.” London: Academic Press, pp. 199-286.
Miyake, A,, and Harumoto, T. (1993): Possible participation of surface antigens in the predator-prey interaction between Didinium
and Paramecium. J. Euk. Microbiol. 40:27a.
Naitoh, Y., and Kaneko, H. (1972): Reactivated Triton-extracted
models of Paramecium: Modification of ciliary movement by
calcium ions. Science 176:523-524.
Naitoh, Y., and Kaneko, H. (1973): Control of ciliary activities by
adenosine-triphosphate and divalent cations in Triton-extracted
models of Paramecium caudatum. J. Exp. Biol. 58:657-676.
Nakaoka, Y . , and Toyotama, H. (1979): Directional change of ciliary
beat effected with Mg2+ in Paramecium. J. Cell Sci. 40:207214.
Wessenberg, H., and Antipa, G . (1970): Capture and ingestion of
Paramecium by Didinium nasutum. J . Protozool. 17:250-270.
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