These authors contributed equally to this work.
A novel neuronal calcium sensor family protein, calaxin, is a potential Ca2+-dependent regulator for the outer arm dynein of metazoan cilia and flagella
Article first published online: 3 JAN 2012
2009 Société Française des Microscopies and Société Biologie Cellulaire de France
Biology of the Cell
Volume 101, Issue 2, pages 91–103, February 2009
How to Cite
Mizuno, K., Padma, P., Konno, A., Satouh, Y., Ogawa, K. and Inaba, K. (2009), A novel neuronal calcium sensor family protein, calaxin, is a potential Ca2+-dependent regulator for the outer arm dynein of metazoan cilia and flagella. Biology of the Cell, 101: 91–103. doi: 10.1042/BC20080032
The nucleotide sequence data reported for Ciona intestinalis calaxin will appear in the DDBJ, EMBL, GenBank® and GSDB Nucleotide Sequence Databases under the accession number AB079059.
- Issue published online: 3 JAN 2012
- Article first published online: 3 JAN 2012
- Received 16 June 2008; Accepted 11 July 2008
- epithelial cilia;
- neuronal calcium sensor (NCS);
- sperm flagella
- Top of page
- Materials and methods
Background information. Spermatozoa show several changes in flagellar waveform, such as upon fertilization. Ca2+ has been shown to play critical roles in modulating the waveforms of sperm flagella. However, a Ca2+-binding protein in sperm flagella that regulates axonemal dyneins has not been fully characterized.
Results. We identified a novel neuronal calcium sensor family protein, named calaxin (Ca2+-binding axonemal protein), in sperm flagella of the ascidian Ciona intestinalis. Calaxin has three EF-hand Ca2+-binding motifs, and its orthologues are present in metazoan species, but not in yeast, green algae or plant. Immunolocalization revealed that calaxin is localized near the outer arm of the sperm flagellar axonemes. Moreover, it is distributed in adult tissues bearing epithelial cilia. An in vitro binding experiment indicated that calaxin binds to outer arm dynein. A cross-linking experiment showed that calaxin binds to β-tubulin in situ. Overlay experiments further indicated that calaxin binds the β-dynein heavy chain of outer arm dynein in the presence of Ca2+.
Conclusions. These results suggest that calaxin is a potential Ca2+-dependent modulator of outer arm dynein in metazoan cilia and flagella.
Ca2+-binding axonemal protein
Coomassie Brilliant Blue R-250
differential interference contrast
neuronal calcium sensor
simple modular architecture research tool
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- Materials and methods
The motility of eukaryotic cilia and flagella is caused by the sliding of doublet microtubules due to dyneins in the 9+2 microtubule structure called the axoneme. Axonemal dyneins are multisubunit force-generating ATPases composed of HCs (heavy chains), ICs (intermediate chains) and LCs (light chains) (King, 2000). They are classified into two molecular species based on the position of attachment on the doublet microtubule: the outer and inner arm dyneins. Both types of dyneins are regulated by phosphorylation and Ca2+. It is well known that the Tctex2-related LC of the outer arm dynein in sperm flagella is phosphorylated in a cAMP-dependent manner when sperm motility is activated. An IC of an inner arm dynein, IC138, is dephosphorylated on activation of flagellar motility in response to light in Chlamydomonas (for reviews, see Porter and Sale, 2000; Inaba, 2003). On the other hand, Ca2+ has been shown to play critical roles in modulating the waveforms of cilia and flagella. The basic regulation of flagellar waveform is performed by the specific activation of the dynein arms, resulting in changes in flagellar waveforms (Brokaw, 1979; Lindemann and Goltz, 1988). Accumulating evidence indicates that the activity of axonemal dyneins is regulated by signals from the radial spoke/central pair in a Ca2+-dependent manner (Smith, 2002). Calmodulin has been identified as a radial spoke component, designated RSP20 (Yang et al., 2001). Recent proteomic analysis has identified a new Ca2+-binding protein, RSP7, with a consensus module for binding to the regulatory subunit RIIa of cAMP-dependent protein kinase (Yang et al., 2006).
Axonemal dyneins possess Ca2+-binding subunits and are possibly regulated directly by Ca2+. For example, the Ca2+-binding protein centrin is a subunit of a subset of inner arm dynein in Chlamydomonas (Piperno et al., 1992). The outer arm dynein in Chlamydomonas has a Ca2+-binding LC, LC4, as a subunit (King and Patel-King, 1995). One of the components of the outer arm dynein DC (docking complex) also has Ca2+-binding sites that are redox-sensitive in Chlamydomonas flagella (Casey et al., 2003; Sakato and King, 2003). On the other hand, Ca2+-binding protein that regulates axonemal dyneins has not been fully characterized in sperm flagella. Calmodulin was reported to regulate flagellar motility and could be extracted from axonemes with outer arm dynein, but it has not been clarified whether calmodulin can bind directly to outer arm dynein (Tash et al., 1988). In fact, isolated outer arm dynein does not contain calmodulin as a subunit (Inaba, 2007).
Recent proteomic study has shown that no Ca2+-binding protein is included as a subunit of outer arm dynein in the ascidian Ciona intestinalis (Hozumi et al., 2006). Ciona outer arm dynein contains two HCs, five ICs and six LCs. The ICs include two WD repeat proteins (IC1 and IC2), a protein with thioredoxin/nucleoside diphosphate kinase domains (IC3), and two coiled-coil proteins related to Chlamydomonas outer arm DC2 (IC4 and IC5). The LCs include a leucine-rich repeat protein, Tctex1- and Tctex2-related proteins, a protein similar to Drosophila roadblock, and two components related to Chlamydomonas LC8. Since the structure and function of axonemes have been conserved throughout evolution, it is possible that some unknown Ca2+-binding protein is involved in the Ca2+-dependent regulation of outer arm dynein.
We have previously identified 76 axonemal proteins in C. intestinalis by immunoscreening with antiserum against axonemal proteins (Padma et al., 2003). Among the cDNA clones for these proteins, six showed sequence similarity to calcineurin B subunit. In the present study, we analysed this protein in detail and found that it is a member of the vertebrate NCS (neuronal calcium sensor) family. Further analysis with antibody against this protein showed its localization at outer arm dynein, and biochemical analysis demonstrated that this protein binds to dynein HC in a Ca2+-dependent manner. Thus, we found a potential Ca2+-dependent regulator for the outer arm dynein of sperm flagellar axoneme, and named this protein ‘calaxin’ (for calcium-binding axonemal protein).
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- Materials and methods
Calaxin is a novel protein in the NCS family
Immunoscreening of testis cDNA library with anti-axonemal protein antibody resulted in the isolation of six clones of axonemal proteins showing sequence homology to the calcineurin B subunit (Padma et al., 2003). The clone (CiAx83) with the longest insert contained an open reading frame encoding 203 amino acids with a predicted molecular mass of 23772 Da and an isoelectric point of 4.95 (GenBank® accession number AB079059).
A BLAST search of the amino acid sequence of CiAx83 showed the highest similarity to a novel EF-hand protein in Xenopus tropicalis (E=7e−61; GenBank® accession number CAJ83954) with 65% amino acid identity and 77% homology. It also showed significant homology to the NCS family of proteins, such as NCS-1 (E=5e−15) and calsenilin (E=2e−14) of Danio rerio. A SMART (simple modular architecture research tool) search for modules detected three potential EF-hand Ca2+-binding modules (amino acids 62–90, 98–126 and 151–166) (Figure 1A). A ScanProsite search found a further motif of intermediate filament signature (amino acids 30–38) (Figure 1A). The homology search and motif scans suggest that CiAx83 encodes a novel Ca2+-binding protein related to NCS. Comparison of domain structures of other Ca2+-binding proteins clearly showed that it is a unique EF-hand axonemal protein, different from any other Ca2+-binding proteins reported to be present in the axonemes (Figure 1A).
Multiple alignment of EF-hand proteins from Ciona, Chlamydomonas, mouse and humans using the ClustalW program (http:clustalw.ddbj.nig.ac.jptop-j.html), followed by phylogenetic analysis, revealed that calaxin is closely related to NCS family members such as NCS-1 (or frequenin in Drosophila) but is classified into the same clade as uncharacterized EF-hand proteins in mouse (NP_080045) and human (NP_078869) (Figure 1B). A search of the C. intestinalis genome database (http:genome.jgi-psf.orgCioin2Cioin2.home.html) found a clear orthologue of calmodulin, calcineurin B and frequenin in the Ciona genome (Figure 1B). Hence, we named this novel Ca2+-binding NCS family protein ‘calaxin’, for calcium-binding axonemal protein.
A BLASTP search of Ciona CiAx83 against the Chlamydomonas database (http:genome.jgi-psf.orgChlre3Chlre3.home.html) detected two EF-hand Ca2+-binding proteins with proper molecular mass: protein IDs 195964 and 119565. Both proteins showed high similarity to calcineurin B, but not to any NCS family proteins. Homology search against databases of other organisms demonstrated that calaxin orthologues were present in Xenopus (CAJ83954), lancelet (ABP06309), sea urchin (XP_001193804), Drosophila (NP_572437) and sea anemone (ID235777; http:genome.jgi-psf.orgNemve1Nemve1.home.html), but could not be found in yeast, Volvox, Trypanosoma or Arabidopsis. Intriguingly, search against recently published database of the choanoflagellate Monosiga brevicollis (http:genome.jgi-psf.orgMonbr1Monbr1.home.html) resulted in a significant hit with an orthologue (protein ID 27286). Multiple alignment of this orthologue with those in chordates showed high identity in amino acid sequence, although there is a long insertion at EF-hand2 and both EF-hand1 and EF-hand2 were less homologous than EF-hand3 (Figure 1C).
Immunolocalization of calaxin in Ciona tissues
To localize calaxin in the axonemes, a specific antibody against this protein was prepared. The antibody recognized a single band with an apparent molecular mass of 25 kDa in the sperm flagellar axonemes, which is in agreement with the size deduced from the cDNA sequence (Figure 2A). Immunofluorescence microscopy showed that calaxin localized along the sperm flagellum (Figures 2B and 2C). Immunogold localization revealed the presence of gold particles near the outer arm dyneins (Figure 2D). To exclude the possibility that antibodies could not get access to inside the axonemes, disintegrated axonemes were used for immunogold labelling. The result clearly showed the localization of calaxin at outer arm dynein on the doublet microtubules (Figure 2E).
We performed Western blotting of proteins from several adult tissues of C. intestinalis to determine the distribution of calaxin in adult tissues (Figure 3A). Calaxin was present in a high amount in sperm and testis. Other tissues, such as endostyle and branchial basket, were shown to express calaxin, suggesting that calaxin is also present in motile cilia in adult tissues. Immunofluorescent microscopy of branchial basket clearly showed that calaxin is localized at epithelial cilia (Figure 3B).
Ca2+-dependent binding of calaxin to the outer arm dynein
Immunogold localization suggested that calaxin is structurally or functionally associated with outer arm dynein (Figure 2D). Outer arm dynein can be extracted from the axonemes by high salt treatment. Successive extraction of the Ciona axonemes and subsequent Western blotting showed that calaxin is exclusively extracted with high salt solution (Figure 4A). Binding of Ca2+ to Ca2+-binding protein results in the changes in the electrophoretic mobility. SDS/PAGE detected the change in the mobility of the calaxin band in the presence and absence of Ca2+ (Figure 4B), indicating the binding of Ca2+ to calaxin.
Sucrose density gradient centrifugation showed that only a trace amount of calaxin sedimented in the outer arm dynein fraction in the absence of Ca2+. However, it sedimented with the outer arm dynein in the presence of Ca2+ (Figure 4C). The molar ratio of calaxin in the outer arm dynein appeared the same as that of LC1 or LC2. To estimate the quantity of calaxin in the axonemes, a quantitative analysis of protein spot on two-dimensional gel was performed (Figure 5). The spots of calaxin, LC1 (a LC with leucine-rich repeats), IC1 and IC2 were detected by Western blotting using specific antibodies (Hozumi et al., 2008). Quantification of each spot indicated that the relative ratio of the quantity of calaxin, LC1, IC1 and IC2 was 1:0.85:1.1:1.3. This suggests that calaxin is exclusively bound to the outer arm dynein in the axoneme at a 1:1 ratio.
Axonemes were treated with EDC [1-ethyl-3-(3-dimethylaminopropyl)carbodiimide], followed by Western blotting with anti-calaxin antibody, to identify the protein interacting with calaxin in situ. Western blotting with anti-calaxin antibody showed the formation of a cross-linked product of ∼80 kDa with increasing concentration of EDC (Figure 6). The cross-linked product was recognized by anti-β-tubulin antibody, but not by anti-α-tubulin antibody (Figure 6B). As the product could be discretely observed in CBB (Coomassie Brilliant Blue R-250)-stained gel, it was subjected to N-terminal amino acid sequencing. The sequence obtained was MREIVHLQAGQXGNQI, a complete match with β-tubulin. No other N-terminal sequence was obtained, indicating that the N-terminus of calaxin is blocked. Direct amino acid sequencing of purified calaxin also supported this result. It is known that the N-terminal is modified by myristoylation in many NCS family proteins (Burgoyne and Weiss, 2001), but N-terminal modification of calaxin could not be deciphered. The ∼80 kDa cross-linked product was also formed in the presence of Ca2+, indicating that calaxin interacts with β-tubulin in both the absence and presence of Ca2+ (Figure 6A).
Interactions of calaxin with the subunit of outer arm dynein were further examined by overlay experiments (Figure 7). The outer arm dynein, isolated by sucrose density gradient centrifugation, was separated by SDS/PAGE and transferred on to PVDF membrane. The fraction rich in calaxin was overlaid onto the membrane in the absence and presence of Ca2+, followed by detection with anti-calaxin antibody. In the absence of Ca2+, the most intense signal was observed on β-tubulin (Figure 7A), which is in agreement with the EDC cross-linking experiment results (Figure 6). In the presence of Ca2+, however, no signal was observed on β-tubulin and intense signal was observed on the dynein HC (Figure 7A). In both the presence and the absence of Ca2+, a weak signal was observed at the region of IC1/2. Further analysis using a low concentration of polyacrylamide gel showed the binding of calaxin to IC2 (Figure 7B). The SDS gel, which separated dynein HCs with high resolution, revealed that the binding of calaxin occurred on the β HC (Figure 7C). Partial amino acid sequences of a fragment of β HC (MHGMYSLEXPGD and TVQTAENVNPAF) showed complete matches with the amino acid sequence deduced from a dynein HC gene (Kyotograil2005.2.152.1; http:ghost.zool.kyoto-u.ac.jpindexr1.html). Analysis by MS confirmed this identification (results not shown). On the other hand, mass spectrometry of the α HC resulted in a significant match with another gene product (Kyotograil2005.431.1.1). A phylogenetic analysis of the HCs of outer arm dyneins was carried out to find out the relationship among multiple HCs of outer arm dynein from different organisms. The result indicated that the Ciona β HC is closely related to the Chlamydomonas γ HC and to sea urchin α HC (Figure 7D).
Binding of calaxin to the outer arm dynein was examined in the presence of different concentrations of Ca2+. At 10−6 M Ca2+, almost all the calaxin was bound to the outer arm dynein (Figure 8). The binding appeared quite strong and the unbound calaxin was detected only in trace amounts even at 10−8 M Ca2+ (Figure 8). This agrees with the Ca2+-binding property of the NCS family of proteins: Ca2+ affinities of NCS proteins lie within 0.1–0.75 μM (Burgoyne and Weiss, 2001).
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- Materials and methods
We characterized a novel Ca2+-binding protein of the NCS family, calaxin, as an axonemal protein localized at outer arm dynein. NCS family proteins are encoded by 14 genes in the human genome (Burgoyne et al., 2004). Most of the proteins in the NCS family are reported to be expressed predominantly in neurons. NCS-1 was first found in Drosophila and named frequenin, and is involved in frequency-dependent synaptic facilitation. NCS-1 in Caenorhabditis elegans was reported to participate in learning and memory. However, studies have shown the diverse functions of NCS family members, including gene expression, ion channel regulation, membrane trafficking, and controlling apoptosis (Burgoyne et al., 2004). The N-termini of most NCS proteins are myristoylated, and becomes exposed outside the protein molecules, allowing them to associate with membranes. The Ciona calaxin appears to be modified at the N-terminal region, but the modification has not been elucidated. In this study, calaxin was shown to bind both β-tubulin (possibly to microtubules) and dynein β HC. It is therefore less likely that the N-terminal modification enables it to associate with membranes. The consensus sequence for myristoylation is N-terminal GXXXSX (where X is any amino acid) (Towler et al., 1988), which is observed in mammalian NCS-1 and calcineurin B, but not in calaxin and its mammalian orthologues. This also supports the idea that the N-terminal modification is not myristoylation. Considering the binding of calaxin to dyneins in the presence of Ca2+ (Figure 7), it is most likely that the N-terminal portion of calaxin is involved in the interaction with the dynein HC.
NCS proteins undergo large conformational changes upon binding to Ca2+. In this study, calaxin bound to β tubulin and possibly to IC2. IC2 is a WD-repeat protein and is thought to be involved in the assembly of outer arm dynein and binding to the doublet microtubules (King, 2000). The overlay experiment showed that the binding of calaxin to β tubulin was weak in the presence of Ca2+ (Figure 7). However, the EDC cross-linking experiment indicated the clear interaction of calaxin with β tubulin in the presence of Ca2+ (Figure 6). Therefore, IC2 may complement the anchoring of calaxin to the microtubule (Figure 9). It is worth noting that IC2 is dephosphorylated upon activation of sperm motility (Hozumi et al., 2008), implying the regulatory mechanism for the function of calaxin.
In the presence of Ca2+, calaxin binds to the β dynein HC. Although it has been shown that Chlamydomonas LC4 binds to the stem region of the γ HC through two IQ motifs (Sakato and King, 2003), it is not clear whether calaxin interacts with the stem or the head of the dynein HC, or whether calaxin associates with an adjacent dynein molecule (Figure 9). A search of the amino acid sequence of the β HC of Ciona outer arm dynein failed to find an IQ motif. Another search of calmodulin-binding sites (http:calcium.uhnres.utoronto.cactdbctdbhome.html) could not find any calmodulin-binding sites in the Ciona β HC, suggesting no binding of calmodulin-like Ca2+-binding proteins.
Ca2+-dependent binding of calaxin occurred on the β HC but not on the α HC (Figure 7). The β HC of Ciona outer arm dynein is related to the α HC of sea urchin and the γ HC of Chlamydomonas. The microtubule sliding assay with subparticles of outer arm dynein showed that the α HC of sea urchin outer arm dynein mediates structural and rigor binding to microtubules; the β HC does not participate in such binding but is more involved in microtubule sliding (Moss et al., 1992). In Chlamydomonas, the γ HC of outer arm dynein is bound to the calmodulin-type LC4 and exhibits Ca2+-regulated and ATP-sensitive interaction with microtubules (Sakato and King, 2003). The γ HC shows no microtubule translocation activity under conventional conditions because of ATP-insensitive binding to microtubules; however, when preincubated with tubulins, the γ HC efficiently translocates microtubules (Sakakibara and Nakayama, 1998). Thus it is possible that calaxin is involved in Ca2+-dependent regulation of outer arm dynein by direct interaction with the Ciona β HC, which may modify ATP-insensitive interactions with microtubules.
Reactivated axonemes lacking the radial spokes still display waveform conversion in response to changes in Ca2+ (Wakabayashi et al., 1997). The outer arm dynein in Chlamydomonas has a Ca2+-binding protein, LC4, as a subunit. Chlamydomonas mutants lacking outer arm dynein show abnormalities in the photophobic response, and generate no symmetric waveform (Kamiya and Okamoto, 1985) or show a decrease in bend angle (Mitchell and Rosenbaum, 1985). One of the subunits of the outer arm dynein docking complex also has Ca2+-binding sites that are redox-sensitive (Casey et al., 2003). The DC3-null strain rescued with its mutant form lacking Ca2+-binding property showed normal swimming behavior (Casey et al., 2003). These observations suggest that the outer dynein arm contains a Ca2+ sensor that is at least partly responsible for flagellar waveform conversion during the photophobic response. In many animals, sperm chemotaxis to the egg is Ca2+-dependent (Cosson et al., 1984; Yoshida et al., 1994; Bohmer et al., 2005; Wood et al., 2005). However, no direct data has been reported on the Ca2+-binding proteins in the outer arm dynein of sperm flagella. Considering the Ca2+-dependent interaction of calaxin to the dynein HC, it is possible that calaxin is responsible for Ca2+-dependent changes in flagellar waveform in the process of chemotaxis in Ciona sperm.
The functions of Chlamydomonas LC4 and Ciona calaxin appear similar in terms of Ca2+-dependent regulation of outer arm dynein by binding to a similar type of dynein HC. Recently, Ca2+/LC4-dependent conformational changes of outer arm dynein have been reported (Sakato et al., 2007), in which the similarity of Chlamydomonas LC4 to Ciona calaxin has also been shown (Figure 9). However, a search of the database found no homologue of the calaxin gene in the Chlamydomonas genome. Searches in the genomes of several other organisms also suggest that the calaxin gene is not present in multicellular green algae, plant or fungi. Therefore it is most likely that its gene had been acquired during the onset of metazoa. It is not known but intriguing if the acquirement of calaxin may have lead to the diverse function of cilia and flagella in the neuronal or sensory system or in the establishment of body plan in metazoa.
Western blot analysis in Ciona showed that calaxin is abundantly expressed in mature sperm, but it is also widely distributed in adult tissues that apparently possess epithelial cilia (Figure 3; Burighel and Cloney, 1997). This suggests that calaxin functions not only in sperm flagella but also commonly in cilia in several tissues as a Ca2+-sensor. Recent progress in research on cilia has revealed the wide distribution of this organelle in a variety of tissues or organs in multicellular organisms and their close involvement in human diseases (Wheatley et al., 1996; Ibanez-Tallon et al., 2003). The role of Ca2+ in the control of ciliary functions is quite essential in many tissues (Gao et al., 2003; Marshall and Nonaka, 2006; Singla and Reiter, 2006). Further studies on the structure and function of calaxin should shed light on the general mechanism and functions of the Ca2+-dependent regulation of ciliary movement in a variety of tissues in metazoa.
Materials and methods
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- Materials and methods
Molecular cloning of calaxin
We previously isolated six cDNAs by immunoscreening with antisera against axonemal proteins of Ciona spermatozoa. The clone with the longest insert, CiAx83, was analysed. The insert of the cDNA in λZAP II vector was subcloned into pBluescript by in vivo excision and sequenced using a BigDye terminator sequencing kit with an ABI310 DNA sequencer. Translation of DNA sequences into amino acid sequences, calculation of molecular masses, and estimation of isoelectric points were conducted using GENETYX-Mac software. Multiple sequence alignment was performed with ClustalW. SMART, PROSITE and ProfileScan were used for the prediction of functional sites and domains in amino acid sequences.
Antibodies and immunohistochemistry
PCR primers used for the amplification of the open reading frame of calaxin (CiAx83) were 5′-CGCGGGATCCATGAGCAAAAAGAATCA-3′ (sense) and 5′-CGCGGAATTCATCAAAACATTTCATCCC-3′ (antisense). The PCR product was subcloned into pET32a vector and transfected into Escherichia coli AD494. Protein expression was induced by 1 mM IPTG (isopropyl β-D-thiogalactoside). Thioredoxin—calaxin fusion protein was purified by His-Bond metal chelation resin (Novagen). Production of the antibody against thioredoxin—calaxin fusion protein with a purity more than 95% was performed as described (Padma et al., 2003). Immunofluorescence microscopy and immunogold electron microscopy were conducted according to methods described previously (Padma et al., 2003). For immunostaining of branchial basket, a small piece of the tissue was excised and washed with filtered seawater and then fixed with 10% formalin in filtered seawater. After fixation overnight at 4°C, the sample was washed five times with TPBS (PBS containing 0.1% Tween 20) and sequentially washed with 25%, 50% and 70% ethanol. Samples were stored at −30°C until used. After washing with TPBS, the sample was incubated in a blocking buffer containing 10% goat serum in TPBS at 4°C for 3 h. Primary antibody (anti-calaxin antiserum or non-immune serum, 1:1000) was added to the blocking buffer and incubated at 4°C overnight. The sample was washed five times with TPBS and incubated with secondary antibody (Alexa Fluor® 555-conjugated anti-mouse IgG, 1:1000) in blocking buffer for 8 h at 4°C. After washing with TPBS, the sample was mounted with vectashield mounting medium (Vector Laboratories) with 1 μM DAPI (4′,6-diamidino-phenylindole).
Isolation of axonemes and outer arm dynein
Sperm flagella and axonemes were prepared from C. intestinalis sperm pellets as described previously (Inaba et al., 1998; Padma et al., 2001). Selective extraction of the axonemes was performed according to Satouh et al. (2005). For isolation of outer arm dyneins, the axonemes were extracted with a high salt buffer containing 0.6 M KCl, 20 mM Tris/HCl, pH 8.0, 1 mM MgSO4, 0.5 mM EGTA and 0.2 mM dithiothreitol. The extract was further centrifuged at 100000 g for 30 min. The clear supernatant was layered on a 5–20% sucrose gradient made with a buffer containing 0.15 M KCl, 20 mM Tris/HCl, pH 8.0, 1 mM MgSO4, 0.5 mM EGTA and 0.2 mM dithiothreitol, and centrifuged with a Hitachi Himac CP65β centrifuge with a Hitachi P28S2 rotor at 26000 rev./min for 21 h at 4°C. Fractions of 500 μl were collected. The protein concentration was determined by the method of Bradford (1976). The fraction of outer arm dynein was determined by measuring ATPase activity in each fraction by the method of Taussky and Schorr (1953). The fraction of calaxin was determined by Western blotting using anti-calaxin antibody. SDS/PAGE was performed as described by Laemmli (1970). The percentage of acrylamide in the separating gel was selected according to the protein to be analysed. To separate the dynein HC, a gradient gel from 2.8% polyacrylamide/0 M urea to 5% polyacrylamide/4 M urea was used as the separating gel. Two-dimensional gel electrophoresis was carried out according to Hozumi et al. (2008). Quantification of protein spot was performed using the software PDQuest (Bio Rad Laboratories, Tokyo, Japan). Western blotting was performed on PVDF membrane with the ECL® (enhanced chemiluminescence)-plus detection system (Amersham Biosciences).
Sliding disintegration of the axonemes
Axonemes were suspended in a buffer containing 0.15 M KCl, 20 mM Tris/HCl, pH 8.0, 1 mM MgSO4, 0.5 mM EGTA, and 0.2 mM dithiothreitol. They were treated with 0.4 μg/ml trypsin for 5 min at room temperature. After termination of digestion by soybean trypsin inhibitor (1 mg/ml), ATP was added to 1 mM. Disintegrated axonemes were centrifuged at 12000 g for 20 min and the pellet was processed for immunogold labelling according to Padma et al. (2003).
The axonemes were washed with a buffer containing 0.15 M KCl, 20 mM HEPES/NaOH, pH 8.0, 1 mM MgSO4 and 0.5 mM EGTA. EDC of various concentrations was added to aliquots of the samples, and the mixtures were incubated at 20°C for 1 h. The cross-linking reactions were stopped by addition of 1/4 vol. of 5×sample buffer for SDS/PAGE.
The fraction of outer arm dynein from the sucrose density gradient was separated by SDS/PAGE and transferred onto PVDF membrane. The membrane was incubated with a blocking buffer containing 10 mg/ml BSA in 20 mM Tris/HCl (pH 8.0) for 1 h at room temperature (25°C). The membrane was then incubated in calaxin solution in the blocking buffer in the presence of 10 mM EGTA or 10 mM CaCl2 for 1 h at room temperature. The membrane was washed thrice with a blocking buffer and then once with 0.1 M NaCl and 10 mM triethanolamine, pH 8.0, in the presence of 10 mM EGTA or 10 mM CaCl2. Protein—calaxin complex was cross-linked by incubation with 2.5 mM disuccinimidyl suberate in 0.1 M NaCl and 10 mM triethanolamine, pH 8.0, in the presence of 10 mM EGTA or 10 mM CaCl2 for 1 h at room temperature. The membrane was washed once with 0.1 M NaCl and 10 mM triethanolamine, pH 8.0, three times with distilled water, once with 100 mM glycine/HCl, pH 2.5, for 10 min, and finally for three times with distilled water. The membrane was subjected to Western blotting with anti-calaxin antibody.
Binding assay by immunoprecipitation
The fraction of outer arm dynein and calaxin were mixed at 1:1 and dialysed against a buffer containing 20 mM Tris/HCl, pH 8.0, 1 mM MgCl2, 0.15 M KCl, 10 mM EGTA and various concentrations of CaCl2 at 4°C for 4 h. The concentration of Ca2+ was calculated by the program Calcon (Goldstein, 1979). The mixture (400 μl) was immunoprecipitated with anti-IC2 antibody immobilized to 50 μl of Protein A—Sepharose (Amersham Biosciences) by disuccimidyl suberate. After incubation with the resin at 4°C for 4 h, the mixture was centrifuged and the supernatant was analysed by Western blotting with anti-calaxin antibody.
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We thank the members of the Education and Research Center of Marine Bio-Resources, Tohoku University, Department of Zoology, Kyoto University (National Bio-Resource Project; NBRP) and Otsuchi Marine Research Center, University of Tokyo, for supplying C. intestinalis; Ms Yumiko Makino, National Institute for Basic Biology, for protein sequencing (NIBB Cooperative Research Program No. 3-133). The manuscript was edited by Crimson Interactive Pvt. Ltd.
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This work was supported in part by a grant from MEXT (Ministry of Education, Culture, Sports, Science and Technology), Japan and by JST-BIRD (Japan Science and Technology Agency-Institute for Bioinformatics Research and Development), Japan to K.I.
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- Materials and methods
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