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Cell Motility and the Cytoskeleton 44:202–208 (1999)
Characterization of Outer Arm Dynein in Sea
Anemone, Anthopleura midori
Hideo Mohri,1* Kazuo Inaba,2 Miyoko Kubo-Irie,3 Hiroyuki Takai,4
and Yoko Yano-Toyoshima5
1National
Institute for Basic Biology, Okazaki, Aichi, Japan
Marine Biological Station, Tohoku University,
Asamushi, Aomori, Japan
3University of the Air, Chiba, Japan
4Department of Geriatric Research, National Institute of Longevity Science,
Obu, Aichi, Japan
5Department of Life Sciences, Graduate School of Arts and Sciences,
University of Tokyo, Komaba, Tokyo, Japan
2Asamushi
Outer arm dynein was purified from sperm flagella of a sea anemone, Anthopleura
midori, and its biochemical and biophysical properties were characterized. The
dynein, obtained at a 20S ATPase peak by sucrose density gradient centrifugation,
consisted of two heavy chains, three intermediate chains, and seven light chains.
The specific ATPase activity of dynein was 1.3 µmol Pi/mg/min. Four polypeptides
(296, 296, 225, and 206 kDa) were formed by UV cleavage at 365 nm of dynein in
the presence of vanadate and ATP. In addition, negatively stained images of dynein
molecules and the hook-shaped image of the outer arm of the flagella indicated that
sea anemone outer arm dynein is two-headed. In contrast to protist dyneins, which
are three-headed, outer arm dyneins of flagella and cilia in multicellular animals
are two-headed molecules corresponding to the two heavy chains. Phylogenetic
considerations were made concerning the diversity of outer arm dyneins.
Cell Motil. Cytoskeleton 44:202–208, 1999. r 1999 Wiley-Liss, Inc.
Key words: flagella; cilia; axoneme; phylogeny; metazoa; protist
INTRODUCTION
Dynein is a motor protein essential for microtubuledependent cell motility. It is classified into axonemal
dynein, which supports flagellar/ciliary movement, and
cytoplasmic dynein, which is responsible for intracellular
transport. Furthermore, in axonemes of flagella and cilia
there are several dyneins including outer arm and inner
arm dyneins. Studies on axonemal dyneins have shown
that the isolated outer arm dynein molecule is a threeheaded bouquet in protists (e.g., Tetrahymena [Johnson
and Wall, 1983; Toyoshima, 1987], Chlamydomonas
[Goodenough and Heuser, 1984; Takada et al., 1992], and
Paramecium [Larsen et al., 1991]). In contrast, the
molecule is two-headed in multicellular animals belonging to Deuterostomia (e.g., bull [Marchese-Ragona et al.,
1987], fish [Gatti et al., 1989], and sea-urchin [Sale et al.,
1985]) as well as Protostomia (e.g., oyster [Wada et al.,
r 1999 Wiley-Liss, Inc.
1992]). On the other hand, there are both two-headed and
three-headed molecules on inner arms within a single
flagellum or cilium [Piperno et al., 1990; Smith and Sale,
1992]. More complicated components of inner dynein
arms, including a single-headed molecule, have recently
been shown [Taylor et al., 1999]. The number of heads
typically corresponds to the number of heavy chains
composing a dynein molecule.
In Chlamydomonas, there is a mutant whose outer
arm dynein molecule lacks one of three heavy chains, the
␣-heavy chain [Sakakibara et al., 1991]. Electron microscopy revealed that the outer arm of wild type Chlamydo-
*Correspondence to: Dr. H. Mohri, National Institute for Basic Biology,
Myodaiji-cho, Okazaki 444–8585, Japan. E-mail mohri@nibb.ac.jp
Received 16 July 1999; accepted 9 September 1999
Sea Anemone Outer Arm Dynein
monas looks like a pistol in the cross-section of the
axoneme, whereas the mutant shows a hook- or fist-like
shape, suggesting that the number of heads of the outer
arm dynein molecule may be predicted by observing
cross-sections of flagella and cilia. In fact, the outer arm
of sea urchin sperm flagella resembled that of the
Chlamydomonas mutant. Previously, we examined the
cross-sections of sperm flagella and cilia in various
animals including mammals, tunicates, echinoderms, molluscs, annelids, arthropods, flatworms, sea anemones, and
sponges [Mohri et al., 1995]. In contrast to the pistol-like
shape in protist flagella and cilia, the outer arms in all the
multicellular animals were the hook- or first-like shape,
reflecting their two-headed molecule.
In the phylogenic tree of the animal kingdom,
Coelenterata (sea anemone) is located just prior to the
divergence of the two main branches, Protostomia and
Deuterostonia. The reduction in the number of heads of
outer arm dynein has so far been observed between
protists (Protozoa) and Metazoa. Therefore, it is significant to ascertain whether the outer arm dynein in sea
anemone is really a two-headed molecule, as suggested
by electron microscopy [Mohri et al., 1995]. In the
present study, we examined outer arm dynein isolated
from sperm flagella of a sea anemone both biochemically
and biophysically.
203
was loaded on a 5–20% sucrose density gradient and
separated by ultracentrifugation as described previously
[Inaba and Mohri, 1989]. Fractions were collected from
the bottom of the tube. Protein concentration or ATPase
activity in each fraction was determined according to
Bradford [1976] or Tausskey and Shorr [1953), respectively. Sedimentation coefficients were estimated with
thyroglobulin (19 S), catalase (11.3 S), and bovine serum
albumin (4.75 S) as markers.
Photocleavage
Photosensitized cleavage was performed in the
presence of 1 mM ATP and 50 mM Vi according to the
method of Gibbons et al. [1987].
SDS-PAGE
Proteins were analyzed by SDS-PAGE according to
the method of Laemmli [1970]. Molecular mass markers
were myosin heavy chain (205 kDa), ␤-galactosidase
(116 kDa), phosphorylase b (97 kDa), bovine serum
albumin (66 kDa), ovalbumin (45 kDa), carbonic anhydrase (31 kDa), soybean trypsin inhibitor (21.5 kDa),
lysozyme (14.4 kDa), and aprotinin (6.5 kDa). Dynein
heavy chains (␣ and ␤) were separated by SDS-PAGE
using the system reported by King et al. [1986].
Electron Microscopy
MATERIALS AND METHODS
Collection of Gametes
Gonads were excised from mature males of the sea
anemone, Anthopleura midori, washed once and minced
in filtered sea water. The suspension, usually prepared
from three to five mature sea anemones, was filtered
through 60 µm nylon mesh and centrifuged at 8,000g for
10 min at 4°C to collect sperm.
High Salt Extraction and Sucrose Density
Gradient Centrifugation
All procedures were performed at 4°C. Sperm were
demembranated with a demembranation buffer (0.15 M
KCl, 1 mM MgCl2, 1 mM EGTA, 0.1 mM DTT, 0.1%
Triton X-100, 20 mM Tris-HCl, pH 8.0, 0.1 mM chymostatin, 1 mM PMSF, 0.1 mM leupeptin) and centrifuged at 10,000g for 10 min. The pellet was suspended in
a wash buffer (0.15 M KCl, 1 mM MgCl2, 1 mM EGTA,
0.1 mM DTT, 20 mM Tris-HCl, pH 8.0, 0.1 mM
chymostatin, 1 mM PMSF, 0.1 mM leupeptin) and
centrifuged at 10,000g for 10 min. After a second wash,
the pellet was treated with a high salt buffer (0.6 M KCl, 1
mM MgCl2, 1 mM EGTA, 0.1 mM DTT, 20 mM
Tris-HCl, pH 8.0, 0.1 mM chymostatin, 1 mM PMSF, 0.1
mM leupeptin) on ice for 30 min. The suspension was
then centrifuged at 12,000g for 10 min. The supernatant
Demembranated axonemes of sea anemone sperm
and Chlamydomonas flagella were fixed with a mixture of
2.5% glutaraldehyde and 1% tannic acid in 0.2 M sodium
cacodylate, followed by post-fixation with 1% OsO4 and
block-staining with uranyl acetate. Specimens were dehydrated and embedded in Quetol 812 (Epon 812 in the case
of Chlamydomonas flagella). Thin sections were made
with a Sorvall ultra-microtome MT-2 and observed under
a JEOL 1200A electron microscope at a 80 kV accelerating voltage.
For observation of isolated dynein molecules, a
dynein solution of 0.02 mg/ml was deposited onto a
carbon-coated grid and negatively stained with 1% uranyl
acetate. The specimens were examined under a Hitachi
HF 2000 electron microscope at a 200 kV accelerating
voltage and the images were recorded using a Gatan slow
scan CCD camera.
RESULTS
Morphological Observation of Outer Arm
Images of outer arms appearing in the crosssections of sperm flagella from sea anemone and flagella
from both wild type and mutant (oda11) Chlamydomonas
were compared. As seen in Figure 1, the outer arm of sea
anemone sperm was hook- or fist-like-shaped and indistinguishable from that of the mutant Chlamydomonas. In
204
Mohri et al.
Fig. 1. Cross-sections of axonemes. a: Flagellum of wild-type Chlamydomonas; b: Flagellum of a mutant
(oda11) Chlamydomonas; c: Sperm flagellum of sea anemone Anthopleura midori. Arrows indicate outer
arms. Bar ⫽ 50 nm.
contrast, the outer arm of the wild type Chlamydomonas
exhibited a pistol-like shape. These results suggest that
outer arm dynein in sea anemone consists of two heavy
chains and should be a two-headed bouquet.
Biochemical Characterization of Sea Anemone
Outer Arm Dynein
The outer arm dynein extracted from axonemes by a
high salt solution sedimented through a 5–20% sucrose
density gradient around 20 S (Fig. 2). The disappearance
of outer arms from the axonemes was confirmed by
electron microscopy after the high salt extraction. The
specific ATPase activity of the dynein was 1.3 µmol
Pi/mg/min. SDS-PAGE analysis of the fraction from the
sucrose density gradient revealed that the dynein was
composed of two heavy chains (␣ and ␤), three intermediate chains (78, 76, and 68 kDa), and seven light chains
(26, 24, 14, 12, 11, 10, and 8 kDa) (Fig. 3). All these
polypeptides cosedimented at the 20S ATPase peak
through sucrose density gradient (data not shown), indicating that they were subunits of the outer arm dynein.
The heavy chains of the outer arm dynein were
cleaved into four polypeptides (two HUVs and two
LUVs) by irradiation at 365 nm in the presence of ATP
and Vi (Fig. 4). The HUVs showed one band, but the
intensity was approximately twofold that of each LUV,
indicating that the band was composed of two HUVs. The
LUVs with higher or lower electrophoretic mobilities
appear to be derived from ␣ or ␤ heavy chain, respectively. The dynein heavy chain is thought to be photocleaved at one of the P-loops, called the V1 site. Based on
the amino acid sequence of the sea urchin outer arm
dynein ␤ heavy chain [Ogawa, 1991], the molecular
masses of the HUV and the LUV from the sea urchin ␤
Fig. 2. Sedimentation profile of proteins and ATPase activity of a 0.6
M KCl extract from sea anemone sperm through a 5–20% sucrose
density gradient. The ATPase activity of outer arm dynein appears at 20
S (arrow). Arrowheads indicate the sedimentation of marker proteins;
thyroglobulin (19 S), catalase (11.3 S), and bovine serum albumin (4.75 S).
Sea Anemone Outer Arm Dynein
Fig. 3. Analysis of the components of sea anemone outer arm dynein
by SDS-PAGE. The 3–5% gradient gel (A), 12% gel (B), or 16.5% gel
(C) shows two heavy chains, three intermediate chains (ICs), or seven
light chains (LCs), respectively. Proteins were stained by Coomassie
brilliant blue (A,B) or silver (C).
heavy chain were calculated as 296 and 206 kDa,
respectively. Using these fragments as molecular mass
markers, the molecular masses of HUVs, LUV␣ and
LUV ␤ from sea anemone dynein heavy chains were
calculated as 296, 225, and 206 kDa, respectively (Fig.
4). Thus, the molecular masses of the ␣ and ␤ heavy
chains of sea anemone outer arm dynein would be 521and
512 kDa, respectively.
205
Fig. 4. Photosensitized cleavage of dynein heavy chains. The sperm
outer arm dyneins from sea anemone (A) and sea urchin (B) were
subjected to photosensitized cleavage at 365 nm in the presence of ATP
and Vi and analyzed by SDS-PAGE (5% gel). Irradiation time is
indicated at the top of each gel. HUV ␣, ␤ or LUV ␣, ␤ shows heavy
fragments from ␣, ␤ dynein heavy chain or light fragments from ␣, ␤
dynein heavy chain, respectively.
Fig. 5. Electron micrographs of 20S dynein particles prepared from
sperm flagella of the sea anemone. Bar ⫽ 50 nm.
Image of Isolated Dynein Molecule
Based on the negatively stained images of outer arm
dynein molecules obtained as the 20S peak on a sucrose
density gradient (Fig. 5), it is evident that sea anemone
outer arm dynein is a two-headed bouquet type similar to
other metazoans including mammals [Marchese-Ragona
et al., 1987], fish [Gatti et al., 1989], sea urchin [Sale et
al., 1985], and oyster [Wada et al., 1992].
DISCUSSION
Morphological Aspects
Since Afzelius [1959] presented the first, schematic
cross-section drawing of sea urchin sperm flagellum and
named the ultrastructures seen ‘‘arms’’ and ‘‘spokes,’’
many such representations have been made for flagella
and cilia [Allen, 1968; Warner and Satir, 1974; Mohri,
1976; Sugrue et al., 1991; Kamiya, 1992]. Until the
discovery that Tetrahymena cilia outer arm dynein molecules were three-headed bouquets [Johnson and Wall,
1983], and sea urchin sperm flagella dyneins were
two-headed [Sale et al., 1985], no special attention has
been paid to the shape of outer arms on cross-sections of
flagella or cilia. People have continued drawing hook- or
fist-like images of the outer arms for both metazoan
flagella/cilia and protozoan flagella/cilia. In fact, immediately after the description of Tetrahymena outer arm
dynein as a three-headed molecule, it was thought that
206
Mohri et al.
each part of a hook corresponded to each of the three
heads. Recently, however, the schematic drawing of the
outer arms in protist flagella/cilia is drawn as either a
three-headed bouquet [Sugrue et al., 1991] or an extended, pistol-like shape [Kamiya, 1992]. Since all metazoan outer arm dyneins studied to date have been
identified as two-headed molecules either electron microscopically or biochemically [Sale et al., 1985; MarchaseRagona et al., 1987; Gatti et al., 1989; Wada et al., 1992;
Mohri et al., 1995; Lupetti et al., 1998 and the present
data] and protists have three-headed outer arm dyneins
[Johnson and Wall, 1983; Goodenough and Heuser, 1984;
Toyoshima, 1987; Larsen et al., 1991; Takada et al.,
1992], we present here schematic cross-section drawings
of both metazoan and protist flagella and cilia with
special reference to the outer arms (Fig. 6).
Molecular Aspects
Molecular phylogenetic analyses of amino acid
sequences of dynein heavy chains [Gibbons, 1995] indicate that sea urchin outer arm dynein heavy chains ␣ and
␤ constitute different subfamilies. From the phylogenetic
tree, sea urchin ␣ and ␤ chains are shown to be related to
Chlamydomonas ␥ and ␤ chains, respectively. The Chlamydomonas ␣ and ␤ chains are likely to have arisen from
gene duplication before the diversification between metazoan animals and green plants including Chlamydomonas. Thus, outer arm dynein is considered to originally be
a two-headed molecule in ancient times and three-headed
molecules such as those seen in Chlamydomonas, are
thought to have acquired a distinctive function and
topology during evolution. The outer arms in the flagellar
cross sections of Bryopsis, a green alga, also resembled a
pistol, suggesting that its outer arm dyneins are threeheaded molecules (unpublished results).
The ␣ and ␤ heavy chains in sea urchin sperm
flagella are known to have different functions. The ␣
chain mediates structural and rigor binding to microtubules, whereas the ␤ chain is involved in force generation
necessary for microtubule sliding [Moss et al., 1992a, b].
Chlamydomonas ␥ and ␤ chains appear to have similar
respective functions. On the other hand, the Chlamydomonas ␣ chain is classified in the same subfamily as sea
urchin and Chlamydomonas ␤ chains and is likely to
induce microtubule sliding. However, it has been shown
that the Chlamydomonas ␣ chain is not essential for
flagellar motility [Sakakibara et al., 1991], whereas the ␤
chain is necessary for the function of outer arm dynein
[Sakakibara et al., 1993].
In Chlamydomonas axonemal cross-sections,
␣-heavy chains form the outermost appendage, ␤-heavy
chains make the mid-portion, and ␥-heavy chains corre-
spond to the innermost lobe of the outer arm [Sakakibara
et al., 1991, 1993]. In Metazoa, one of the heavy chains
topologically corresponding to the Chlamydomonas ␣
chain is lacking. Consequently metazoan ␤-heavy chains
seem to represent the outer part and ␣-heavy chains
correspond to the inner lobe of the outer arm in cross
sections of their flagella or cilia (see Fig. 6).
Phylogenetic Aspects
As described above, the outer arm dynein in flagella
of Coelenterata is confirmed to be a two-headed molecule. Together with our previous observations [Mohri et
al., 1995] as well as biochemical or ultrastructural
analyses by other investigators [Sale et al., 1985; Marchese-Ragona et al., 1987; Gatti et al., 1989; Lupetti et
al., 1998; and also Wada et al., 1992], this finding
indicates that the two-headed molecule is common among
outer arm dyneins in flagella and cilia of all multicellular
animals including Mesozoa [see Mohri et al., 1995]. In
contrast, outer arm dyneins in flagella and cilia of the
protists investigated thus far are three-headed molecules
[Johnson and Wall, 1983; Goodenough and Heuser, 1984;
Toyoshima, 1987; Larsen et al., 1991; Takada et al.,
1992].
It is plausible that the outer arm dynein in protist
flagella and cilia was rather specialized, such that the
addition of an extra head facilitates their complex movements. The outer arm dynein in metazoan flagella and
cilia has kept the original two-headed structure that could
also be found in the common ancestors of protists and
animals. According to recent phylogenetic trees based on
the small subunit rRNA sequences [Wainright et al.,
1993; Ragan et al., 1996], however, this possibility is
unlikely. The protist world is polyphyletic and is likely to
cluster more basally than metazoans in the eukaryotic
lineage. Metazoan animals are likely to have evolved
from the ancestor shared with choanoflagellates. Both
animals and fungi then diverged from the ancestor of
green plants including Chlamydomonas. Furthermore,
ciliates such as Tetrahymena belong to a different cluster
from the one containing green plants and fungi and
animals.
Based on the phylogenetic tree of dynein genes
[Gibbons, 1995], Chlamydomonas ␣ and ␤ chains are
produced by gene duplication. The sea urchin ␤ chain
gene and the Chlamydomonas ␣ chain gene form a clade
with which the Chlamydomonas ␤ gene forms a sister
group, suggesting that a sea urchin ortholog of the
Chlamydomonas ␤ chain gene has been lost in the
metazoan lineage. The number of heavy chains of outer
arm dynein was reduced during evolution of metazoans
from their ancestoral unicellular organisms.
Sea Anemone Outer Arm Dynein
207
Fig. 6. Schematic cross-sections of flagella or cilia in protists and lower plants (left) and in animals
(right). Positions of each heavy chain subunit (head) are also shown.
The same phylogenetic tree showed that the ␤ chain
gene of a ciliate, Paramecium, is sister to the sea urchin
␤-Chlamydomonas ␣-Chlamydomonas ␤ clade, indicating that Chlamydomonas ␣ and ␤ chain genes were
duplicated after the divergence of the ciliates and the
common ancestor of metazoan animals and green plants.
Although more sequence data of heavy chains in protists
are needed, the phylogenetic analysis suggests that threeheaded outer arm dynein of Chlamydomonas and ciliates
are not homologous. The three-headed feature of outer
dynein arms in Chlamydomonas and ciliates appears to be
the result of parallel evolution. As the next step, it is
worth examining flagella of choanoflagellates and protists, which are more ancient than Chlamydomonas and
ciliates.
ACKNOWLEDGMENTS
The authors thank Dr. C. Morrey for his kind help in
preparing the manuscript. They also thank Drs. K. Ogawa
and M. Hasebe for their helpful discussion.
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