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Original

Distribution of Actin-like Proteins in the Ciliate Spirostomum ambiguum
Hideki ISHIDA1,*, Toshinobu SUZAKI2, Chiharu KURIBAYASHI1,
Etsuko MASUYAMA3 and Osamu NUMATA4
1Department of Biological Science, Faculty of Life and Environmental Science, Shimane University, Matsue 690-8504, 2Department of Biology, Faculty of Sciences, Kobe University, Kobe 6578501, 3Department of Health Science, Hiroshima Prefectural Women’s University, Minami-ku,
Hiroshima 734-8558, and 4Institute of Biological Sciences, University of Tsukuba, Tsukuba, Ibaraki 305-8572, JapanSUMMARY
Previous studies on cell contraction in theciliate Spirostomum have suggested that actin may
not be present, in spite of its ubiquity in otherciliates including Paramecium and Tetrahymena.
In this study, different anti-actin antibodies wereused to re-examine whether actin occurs in Spirostomum. Western blotting analysis with theseanti-actin antibodies showed that actin-like proteins
may exist in Spirostomum. Intracellular localization of actin-like proteins was examined by immunofluorescence microscopy with the sameantibodies. Fluorescent signals were seen at thecleavage furrow, around the macronucleus, and at
the ciliary bases including those of the membranelle. These results suggest that actin may exist inSpirostomum, but it is different from skeletal mus-*Corresponding author
Tel/Fax: +81-852-32-6449
E-mail: h-ishida@life.shimane-u.ac.jp
Received: 25 July 2003; Accepted: 11 Aug 2003.

cle actin and Tetrahymena actin in antigenic characteristics.

INTRODUCTION
The large heterotrichous ciliate Spirostomum isknown for its remarkable rapid cell contraction thatis triggered by external stimuli, and the cytoskeleton-based motile systems of contraction and subsequent re-elongation have been studied in order tounderstand its underlying molecular machineries(Yogosawa-Ohara et al., 1985; Ishida et al., 1988).
The possible involvement of actin in cell contraction was investigated by Valeri et al. (1983), whodemonstrated by transmission electron microscopythat myosin S-1 from rabbit skeletal muscle did notform its characteristic “arrowhead” complex in thecortex of Spirostomum. They further showed noDNase-I inhibition activity by Spirostomum cellhomogenate, and concluded that actin may notoccur in Spirostomum. Based on these results, it
has been surmised that contraction in Spirostomum
does not involve actin.
In Tetrahymena, Yasuda et al. (1980) and142

Actin-like Proteins in Spirostomum

Jerka-Dziadose (1981) showed that dividing cells
have actin filaments as a component of the contractile ring structure. Tetrahymena actin sharesfundamental characteristics with other actin proteins, including polymerization activity and interaction with myosin, but it does not bind to phalloidin, DNase-I and α-actinin (Hirono et al. 1989),
indicating that protozoan actin may be different
from vertebrate actins. In view of these facts, wehave re-examined the possible existence of actin inSpirostomum using biochemical and immunohistological techniques.

MATERIALS AND METHODS

ing gels. Gels were stained with Coomassie brilliant blue.
Immunoblotting
The specimens of Spirostomum, Tetrahymena
and chicken skeletal muscle were electrophoresedand blotted onto a polyvinylidene fluoride (PVDF)
membrane. The blotted PVDF membranes were
blocked with 5% skim milk in TBS (140 mM NaCl,
50 mM Tris HCl, pH 7.4) for 60 min at room
temperature (RT) and incubated with ×100 diluted
primary antibodies for 1 h at RT. After the treatedmembranes were washed with TBS and TBSTween, they were reacted with ×1,000 diluted
secondary antibodies for 1 h at RT. After being
washed, they were stained with diaminobenzidine.

CultureSpirostomum ambiguum was used in this study.
Spirostomum cells were cultured at 22°C in ×100
diluted Knop’s solution (0.24 mM Ca(NO3)2, 0.14
mM KNO3, 0.06 mM MgSO4 and 0.1 mM KH2PO4)
supplemented with boiled wheat grains. Freshculture solution and boiled wheat were added to themedium every two weeks. Tetrahymena thermophila was cultured according to the method ofHirono et al. (1987a). Cells were harvested at thelogarithmic phase of growth.

Antibodies
Primary antibodies used in this study were: arabbit polyclonal IgG against 11 amino acid residues at the C-terminal of vertebrate skeletal muscle
actin (antibody-I, Sigma A2066), a rabbit antichicken skeletal muscle actin antiserum (antibodyII, Sigma A2668), and a rabbit polyclonal antibody
raised against the N-terminal portion of Tetrahymena actin (Hirono et al. 1987a; antibody-III).
Immunofluorescence stainingElectrophoresis
After the cells were washed thoroughly, they
were collected into a small test tube and homogenized. Proteins were solubilized with 8 M urea andcentrifuged at 4,000 × g for 5 min, and the supernatant was dialyzed against 10 mM Tris-HCl buffer(pH 7.2) to remove urea. The dialyzed supernatant
was used as the specimen for electrophoresis. Ascontrol samples for comparison, Tetrahymena
proteins and crude extract of chicken skeletal
muscle were prepared by the same procedure. SDS
polyacrylamide-gel electrophoresis (SDS-PAGE)
was performed following Laemmli's method(Laemmli 1970) with 3% stacking and 8% separat-Dividing cells were chosen from culture dishes
in which cells were in the logarithmic growthphase, and were washed with fresh culture medium.
The cells were then treated for immunofluorescencestaining by the method of Hirono et al. (1987b).
The primary antibodies were the same as in theimmunoblotting. FITC-labeled anti-rabbit goat IgGwas used as a secondary antibody.
Phalloidin staining
Air-dried cells prepared as for the immunostaining were fixed in Carnoy's fixative (ethanol:chloroform:acetic acid = 6:3:1) for 20 min. Afterbeing washed with ethanol and PBS, the cells wereJpn. J. Protozool. Vol. 36, No. 2.
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processed for membrane permeabilization with 1%
Triton-X100 in PBS, and then washed with PBS.
They were reacted with rhodamine-tagged phalloidin for 20 min, then washed, and examined undera fluorescence microscope.

RESULTS AND DISCUSSION
Actin plays an essential part in cell movement
in a wide range of species, from protozoans tohigher animals and plants. It is a highly conserved
protein and its primary structure shows remarkably
high homology among different species. However,
it is reported that actins in some ciliates like Tetrahymena and Paramecium show low homology toother actins in their amino acid sequence (Hirono etal., 1987a, 1990; Diaz-Ramos et al., 1998). To
detect actin in Spirostomum, Valeri et at. (1983)
applied biochemical procedures that were developed for detecting vertebrate actins. Such procedures may not be appropriate for protozoan species,
and this might be the reason they failed to detect
actin in Spirostomum.

Fig. 1. SDS-PAGE of the extract of Spirostomum cells
and immunoblots with antibody-I and antibody-III. M;
standard molecular markers (116, 84, 58, 45 and 26.5kDa), S; Spirostomum cell extract, T; Tetrahymena cell
extract, C; crude extract of chicken skeletal muscle.
Antibody-I; immunoblots showing the distribution ofactin-like proteins detected by antibody-I, Antibody-III;
immunoblots detected by antibody-III.

Immunoblotting
Antibody-I specifically stained a single band of
40.5 kDa in an extract of Spirostomum (Fig 1). This
antibody scarcely stained Tetrahymena actin.
Antibody-II did not react with protozoan cell
extracts, but exclusively reacted with chickenskeletal muscle. Antibody-III stained a band of
40.5 kDa in Spirostomum as with antibody-I.
However, antibody-III gave weaker staining thanantibody-I, and an additional 27-kDa band was also
stained. Antibody-III did not detect chicken skeletal muscle actin. These findings show that antigens
for anti-actin antibodies exist in Spirostomum cell
extract. In particular, the fact that antibody-I
detected a single band with high specificity strongly
suggests the existence of an actin-like protein withmolecular weight of about 40.5 kDa in Spirosto-mum. This protein cross-reacted with an antiTetrahymena actin antibody, indicating that theprotein has epitopes binding to both anti-vertebrate
actin and anti-Tetrahymena actin antibodies. Sinceskeletal muscle actin does not cross-react with theanti-Tetrahymena actin antibody (antibody III), theactin-like protein in Spirostomum may have aunique amino acid sequence that is different fromboth skeletal muscle and Tetrahymena actins.
Phalloidin staining
Fluorescence microscopy after rhodaminephalloidin staining failed to show any fluorescentstructure in Spirostomum (data not shown here). It
has been reported that Tetrahymena actin cannot bestained with phalloidin because Tetrahymena actin

144

P.Actin-liketetraureliaProteins
Autogamy-relatedin Spirostomum
Genes

Fig. 2. Immunofluorescence micrographs of Spirostomum ambiguum stained with antibody-I (a-c) or antibody-III (d).
In the middle part of a dividing cell (a), a line of bright fluorescence is present at the division furrow (shown by anarrow). At the cell surface, fluorescence signals are localized as small dots aligned along the ciliary lines, which areclearly seen in an enlarged picture (b) of the area shown by a rectangle in (a). Materials around the macronucleus (N ina and d) and along the membranelle (arrow in c and M in d) were also stained. Scale bars: 20 µm (a), 5 µm (b), 20 µm(c) and 50 µm (d).

lacks the binding site for phalloidin (Hirono et al.,1989). The Spirostomum actin-like protein may
also lack the phalloidin binding site.
Immunofluorescence microscopy
When Spirostomum was stained with antibodyI, fluorescent signals were detected at the peripheryof the macronucleus, at the cell surface as dots
distributed along the ciliary line, and along themembranelle (Fig. 2). In cells undergoing division,
ring-shaped fluorescence was observed at thedivision furrow (arrow in Fig. 2a). The same
patterns of antibody staining were obtained whencells were stained with antibody-III, althoughstaining was weaker than with antibody-I (Fig. 2d).
As in the immunoblotting experiment, almost nofluorescence was found in Spirostomum when it
was stained with antibody-II.
In Tetrahymena, antibody-III has been reported
to recognize cortical rows of basal bodies, division
furrows, intracellular filaments, a part of the oralstructure, food vacuoles and contractile vacuole
pores (Hirono et al., 1987a, b). The specific staining at the division furrow in Spirostomum suggests
the possibility that actin may be involved in cytokinesis. It may also correspond to the filamentous

Jpn. J. Protozool. Vol. 36, No. 2.
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ring-like structure observed under the division
furrow of Stentor coeruleus, a heterotrichous ciliate
closely related to Spirostomum (Diener et al.,1983). The spotty localization of antibody-binding
structures on the cell surface along ciliary lines andthe membranelle suggests that at least some, if notall, of the fluorescent dots may correspond to basal
bodies. In Tetrahymena, the ciliary basal body is
known to contain actin as one of its constituentproteins (Hirono, 1987b; Hoey and Gavin, 1992).
In contrast to Tetrahymena, the dot-like structureson the cell surface of Spirostomum are not aligned
in a straight line, but are located in bands that lie
along ciliary lines. They are apparently more
numerous than the basal bodies, indicating that only
some of the dots can be attributed to basal bodies.
Further studies with electron microscopy areneeded to understand the precise localization of theactin-like proteins in Spirostomum.
In summary, this study demonstrated actin-likeproteins in Spirostomum which had previously beensurmised to contain no actin. These proteins may
be different from actins in higher animals andTetrahymena in both molecular weight and antigenicity.
ACKNOWLEDGMENTS
We thank Cathy Busby for her critical readingof the manuscript. This work was partly supportedby the River Environment Fund (REF) in charge of
the Foundation of River and Watershed Environment Management (FOREM) to TS.

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