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 firstname.lastname@example.org 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  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. . SDS-PAGE Proteins were analyzed by SDS-PAGE according to the method of Laemmli . 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. . 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  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. 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