Jpn. J. Protozool. Vol. 43, No. 2. (2010)101

Short Communication㻌

Changes in cell-cell adhesion ability of artificiallyinduced autogamous cells in Paramecium caudatum
Satoshi OHTA and Akira YANAGI*
Department of Biotechnology, Senshu University of Ishinomaki, Ishinomaki 986-8580, JapanSUMMARY
To analyze cell-cell adhesion in Paramecium caudatum, we examined methyl cellulose-induced
autogamous cells for their ability to adhere. It was found that the autogamous cells have cell-celladhesion ability. In addition, we examined the proportion of adhering (conjugating) pairs derived fromautogamous cells at various stages (0.5, 1, 2, 4, 6, 9 and 12 h after the onset of induction of autogamy).
The proportion of adhering cells reached a maximum about 2 h after the onset of induction of autogamy,
and thereafter, rapidly decreased by 4 h after the onset, eventually reaching almost zero 6 h after theonset. These results suggest that there is a transient expression or activation of molecule(s) involved incell-cell adhesion around 2 h after the onset of induction of autogamy.
Key words: Autogamy, Cell-cell adhesion, Cell adhesion molecule, Methyl cellulose, Paramecium
caudatum

INTRODUCTION
In vertebrates, the mechanism of cell-celladhesion is well documented and many kinds ofcell adhesion molecules such as cadherins havebeen identified and characterized (Alberts et al.,2002). On the other hand, in the ciliate Paramecium caudatum, a unicellular eukaryote, no celladhesion molecules have been identified and there*Corresponding author
Tel: 㸩81-225-22-7716
Fax: 㸩81-225-22-7746㻌
E-mail: yanagi@isenshu-u.ac.jp㻌㻌㻌
Received: 30 November 2009; Accepted: 19 April2010.

have only been a few studies investigating themechanism of adhesion between cell bodies without ventral cilia (Miyake, 1969), though there aremany reports about mating reaction, in which cells
adhere to each other on the surface of the ciliary
membrane (Hiwatashi, 1981). In the presentstudy, we refer to the adhesion between cell bodies
without ventral cilia in P. caudatum as cell-celladhesion. Cells of P. caudatum undergo cell-celladhesion during their sexual reproduction process,also known as conjugation, which is one of examples of cell-cell adhesion in eukaryotic organisms.
The ciliate P. caudatum occupies a key position ina major branch of eukaryotic evolution, the Alveolata. Therefore, an investigation of cell-cell adhesion in P. caudatum contributes to the understand-102

Cell adhesion in Paramecium

ing of the evolution of cell-cell adhesion.
To examine cell-cell adhesion in P. caudatum, we used artificially induced autogamous cells.
Autogamy is a sexual reproduction process occurring in single cells without the direct association ofcells, and in P. caudatum autogamy can be artificially induced by treatment with chemical agents
such as trypsin (Mikami and Koizumi, 1979), KCland papain (Tsukii and Hiwatashi, 1979) andmethyl cellulose (Yanagi and Haga, 1996). Under
artificially induced autogamy, cells have the samecharacteristic as conjugating cells, in which thecilia on the ventral surface degenerate (Watanabe,1978; Yanagi and Haga, 1996). But, in natural
autogamy occurring in P. tetraurelia, ventral cilia
do not degenerate (Watanabe, 1978). Thus, wepresumed that methyl cellulose-induced autogamous cells in P. caudatum could adhere to eachother on the ventral surface without cilia. In thepresent study, we examined this possibility andfound that methyl cellulose-induced autogamous
cells have the ability to conduct cell-cell adhesion.
This is the first report showing changes in cell-celladhesion ability during sexual reproduction process in Paramecium.
MATERIALS AND METHODS
Strains and culture conditions
Cells of a stock KNZ2 (mating type VI) in
syngen 3 of Paramecium caudatum were mainly
used. We also used stocks KoscA4 (V), 27aG3(V) and KoscA3 (VI) in syngen 3 of P. caudatum.
The culture medium was 1.25% (w/v) fresh lettuce
juice diluted with modified Dryl's solution, pH 7.0,
and inoculated with Klebsiella pneumoniae one
day before use (Hiwatashi, 1968). The modified
Dryl's solution (Dryl, 1959) (K-DS) substituted
KH2PO4 for NaH2PO4. Cells were cultured at 2425℃.
Induction of autogamy
Methyl cellulose (400 centipoises) (SIGMA)

was dissolved in K-DS. Cells at a density of 1,500
cells/ml were treated with 1.25% methyl cellulose
in a plastic Petri dish (6 cm in diameter, Corning)
to induce autogamy according to the methods described by Yanagi and Haga (1996). Experiments
were carried out at 25-26°C.
Examination of changes in cell-cell adhesion
ability during autogamy
We examined whether methyl celluloseinduced autogamous cells can adhere to form pairs
and whether cell-cell adhesion ability varies during
autogamy by the following protocol.
1. Prepare cells (12,000 cells/8 ml) 0.5, 1, 2, 4, 6,
9 and 12 h after the treatment with methyl cellulose. Examine the percentage of conjugating
pairs in the population of methyl cellulosetreated cells just after the treatment. This percentage is referred to as [control]. The population of methyl cellulose-treated cells contains
not only autogamous cells but also conjugating
pairs and vegetative cells, because methyl cellulose induces autogamy (Yanagi and Haga,1996) and conjugation (Yanagi and Haga,1998) in P. caudatum.
2. Wash methyl cellulose-treated cells three times
with K-DS using a hand-operated centrifuge toremove methyl cellulose just after the treatment
with methyl cellulose.
3. Suspend methyl cellulose-treated cells in 1 mlof K-DS to adjust the cell density to 12,000
cells/ml in order to increase the cell density andenhance the probability of collisions between
the cells. The percentage of pairs formed after
this was calculated and referred to as[experiment 1].
4. Incubate methyl cellulose-treated cells in K-DS
for 1 h to allow them to form pairs. The percentage of pairs formed was calculated andreferred to as [experiment 2].
5. Calculate the percentage of pairs derived fromautogamous cells by subtracting [control] from[experiment 1] or [experiment 2].

Jpn. J. Protozool. Vol. 43, No. 2. (2010)

RESULTS AND DISCUSSION
Changes in cell-cell adhesion ability during
autogamy
To ascertain whether methyl celluloseinduced autogamous cells can adhere to form pairs
and whether cell-cell adhesion ability varies during
autogamy, the percentage of pairs was examined in
autogamous cells derived at various stages (0.5, 1,2, 4, 6, 9 and 12 h after the treatment with methyl
cellulose) (Table 1). As a result, {[experiment 1] –[control]} values varied in a similar manner tothose of {[experiment 2] – [control]} during
autogamy (Table 1 and Fig. 1). This indicates that103

most adhesion-capable autogamous cells have already paired just after the adjustment of cell density to 12,000 cells/ml and continue to pair overthe next 1 h.
The percentage of adhering pairs derived
from autogamous cells varied during autogamy(Table 1 and Fig. 1). In the early stages ofautogamy (0.5 and 1 h after treatment with methyl
cellulose), the percentage of pairs formed was very
low. This appears to be due to incomplete degeneration of the ventral cilia in autogamous cells, as
the degeneration of cilia begins at the anterior tipsof cells 0.5 h after methyl cellulose treatment andcontinues toward the posterior regions of the cells

Table 1. The percentage of pairs in methyl cellulose-treated cells
Time of methyl cellulose treatment (h)

0.5

12

469

12[control]* (%)

0(143#)
0㻌(41)
1.9(212)1.7(115)
4.4(135)
4.5(219)4.0(100)
22.7(220)24.0(225)

26.1(46)
12.8(219)
16.8(226)

10.3(78)
8.5(164)13.5(223)

2.0(99)3.1(131)7.0(228)

5.9(102)1.8(111)
6.0(232)[experiment 1]-[control]

0

2.7

18.7

-13.3

-1.8

1.1-4.1[experiment 2]-[control]

1.9

2.8

20.0

-9.3

3.2

5.0

0.1

0(167)0㻌(73)
0(149)

1.5(133)
10.1(179)
12.0(217)

2.8(143)38.1(126)
41.3(213)

16.4(159)
37.3(177)
32.9(225)

21.4(168)27.0(111)29.7(229)

21.0(124)
16.7(192)
19.9(261)

15.3(183)
15.4(169)
10.2(235)[experiment 1]-[control]

08.635.3

20.9

5.6

-4.3

0.1

[experiment 2]-[control]

0

10.5

38.5

16.5

8.3

-1.1

-5.1

0(144)
0(136)
0(150)

0(130)
0(147)
2.6(152)0(130)
28.4(148)
34.8(207)20.0(50)
24.7(146)
35.6(208)

12.2(148)
14.3(140)
6.1(163)

14.3(98)
7.0(142)9.6(188)0㻌(94)
5.1(118)
6.8(146)[experiment 1]-[control]

00

28.4

4.7

2.1

-7.3

5.1

[experiment 2]-[control]

0

2.6

34.8

15.6

-6.1

-4.7

6.8

[experiment 1]* (%)
1stexperiment*㻌 [experiment 2] (%)[control] (%)[experiment 1] (%)
2ndexperiment㻌 [experiment 2] (%)[control] (%)[experiment 1] (%)
3rdexperiment*㻌 [experiment 2] (%)

See text for an explanation of [control], [experiment 1] and [experiment 2].
#Figures in parentheses show number of cells examined.

104

Cell adhesion in Paramecium

1 h after treatment (Watanabe, 1978). The percentage of paired cells reached a peak 2 h after
methyl cellulose treatment. The percentage rapidly dropped by 4 h after the treatment and gradually decreased as autogamy progressed until 6 hafter the treatment. Paired autogamous cells werehardly observed 6, 9 and 12 h after the treatment.
These results show that methyl celluloseinduced autogamous cells have the ability to conduct cell-cell adhesion, which reaches a maximum

around 2 h after the induction of autogamy. Thissuggests that autogamous cells transiently express
or activate molecule(s) involved in cell-cell adhesion around 2 h after the induction of autogamy.
This also suggests that we must harvest autogamous cells 2 h after induction in order to identify
the molecule(s) responsible for cell-cell adhesion.
Furthermore, the cell adhesion molecule(s) may
correspond to hypothetical holdfast substances
appearing in the early stage of conjugation inParamecium (Miyake, 1969).
The present study demonstrates that the
methyl cellulose-induced autogamous cells provide a useful model system to study the cell adhesion molecule(s) and mechanism(s) controlling cell-cell adhesion in P. caudatum.
REFERENCES

Fig. 1. Changes in the percentage of pairs derived fromautogamous cells. The abscissa shows time of methyl
cellulose treatment (h) and the ordinate shows values of{[experiment 1] – [control]} (A) or {[experiment 2] –[control]} (B). The mean values of {[experiment 1] –[control]} (A) or {[experiment 2] – [control]} (B) of three
independent experiments (,  and ) in Table 1 areused to draw the lines. In A, the mean value of{[experiment 1] – [control]} at 2 h is significantly different at the P < 0.01 level from that at 9 h, and at the P <0.05 level from that at 0.5, 1, 4, 6 and 12 h. In B, themean value of {[experiment 2] – [control]} at 2 h is significantly different at the P < 0.01 level from that at 0.5, 6,
9 and 12 h, and at the P < 0.05 level from that at 1 and 4 h.
All significance tests were performed using Tukey's multiple comparison test. See text for an explanation of[control], [experiment 1] and [experiment 2].

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