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Comparative studies on troponin, a Ca2+-dependent regulator of muscle contraction, in striated and smooth muscles of protochordates
Methods 56 (2012) 3–10
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Comparative studies on troponin, a Ca2+-dependent regulator of muscle contraction, in striated and smooth muscles of protochordates Takashi Obinata a,⇑,1, Naruki Sato b,1 a b
Department of Biology, Faculty of Science, Chiba University, Yayoi-cho, Inage-ku, Chiba 263-8522, Japan Department of Nanobiology, Graduate School of Advanced Integration Science, Chiba University, Yayoi-cho, Inage-ku, Chiba 263-8522, Japan
a r t i c l e
i n f o
Article history: Available online 18 October 2011 Keywords: Troponin Calcium Muscle contraction Protochordate Ascidian Amphioxus
a b s t r a c t Troponin is well known as a Ca2+-dependent regulator of striated muscle contraction and it has been generally accepted that troponin functions as an inhibitor of muscle contraction or actin–myosin interaction at low Ca2+ concentrations, and Ca2+ at higher concentrations removes the inhibitory action of troponin. Recently, however, troponin became detectable in non-striated muscles of several invertebrates and in addition, unique troponin that functions as a Ca2+-dependent activator of muscle contraction has been detected in protochordate animals, although troponin in vertebrate striated muscle is known as an inhibitor of the contraction in the absence of a Ca2+. Further studies on troponin in invertebrate muscle, especially in non-striated muscle, would provide new insight into the evolution of regulatory systems for muscle contraction and diverse function of troponin and related proteins. The methodology used for preparation and characterization of functional properties of protochordate striated and smooth muscles will be helpful for further studies of troponin in other invertebrate animals. Ó 2011 Elsevier Inc. All rights reserved.
1. Introduction Troponin is a mediator of Ca2+-dependent regulation of striated muscle contraction [1]. It is linked to actin filaments and functions in combination with tropomyosin. Troponin is a complex of three components: troponin T (TnT), a tropomyosin-binding component that stabilizes the troponin complex along actin filaments, troponin I (TnI), an inhibitory component of the actin–myosin interaction and troponin C (TnC), a Ca2+-binding component [1]. Troponin of vertebrate striated muscle is known to function as an inhibitor (or a brake) of the actin–myosin interaction (actomyosin Mg2+-ATPase activity) at low Ca2+ concentrations. Binding of Ca2+ to TnC induces a structural change of the troponin complex, specifically in TnI within the complex. Subsequently, the inhibitory action of troponin is removed, enabling myosin to interact with actin [2]. This troponin–tropomyosin regulatory system also exists in a variety of invertebrate striated and obliquely striated muscles [3,4]. In most cases, troponin of invertebrate muscle also inhibits the actin–myosin interaction at low Ca2+ concentrations in a similar manner to vertebrate striated muscle troponin. Although troponin is predominantly expressed in striated muscles in both vertebrate and invertebrate animals, there are several instances for the presence of troponin in non-striated muscles. Troponin has been detected in multinucleated smooth muscle cells ⇑ Corresponding author. Fax: +81 43 290 2804. 1
E-mail address: [email protected] (T. Obinata). These authors contributed equally to this work.
1046-2023/$ - see front matter Ó 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.ymeth.2011.09.026
of ascidian body wall [5,6] and adductor smooth muscle of Akazara scallop [7]. In these muscles, troponin functions as a Ca2+-dependent activator of actin–myosin interaction. Recently, expression of troponin was detected in mammalian vascular smooth muscle, although the physiological roles in the contraction of vertebrate smooth muscle are not yet to be established [8]. More recently, troponin was detected in myoepithelial cells, primitive non-striated muscle-like cells, which are present in the somatic gonad of the nematode Caenorhabditis elegans. In these cells, actin and myosin are organized in non-striated filament networks, and TnC and TnI are associated with actin filaments [9,10]. Their contraction is tightly coupled with oocyte maturation, and troponin plays an essential role for the contractile regulation on the occasion of ovulation [9–11]. Furthermore, TnI is involved in the regulation of chromosomal stability and cell polarity in early Drosophila embryos [12]. A TnI-like protein that is associated with actin filaments has been detected in smooth muscle-like cells in tardigrades (water bears) [13]. These observations suggest that troponin may play important roles not only in striated muscles but also in non-striated muscle in invertebrate animals. Therefore, it will be interesting to investigate how the actin-linked troponin regulatory system is distributed among non-striated muscles of a variety of invertebrate animals and how it functions in the contractile regulation of invertebrate muscles, especially whether it exhibits a brake-type property or an accelerator-type property. Purification procedures of vertebrate striated muscle troponin and its components have been established [14,15]. However, extraction and purification of troponin from various invertebrate
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muscles may not be achieved by simply applying the method that has been established for vertebrate striated muscle troponin for several reasons: (1) amount of muscle tissues is occasionally limited. (2) If a mixture of heterogeneous tissues or whole animal body is used as a starting material, purification of troponin from the crude protein extracts is not easy and in addition, proteolytic enzymes that are contaminated in the extract tend to degrade troponin that is sensitive to proteases during preparation. Nevertheless, troponin has been purified from striated and smooth muscles of protochordate animals, namely ascidian and amphioxus. This experience may be helpful for the preparation of troponin from other invertebrate animals and the analysis of its functional characteristics. Therefore, the preparation procedure for protochordate troponin and analysis of its function are described in the following. Protochordata, comprising subphyla Cephalochordata and Urochordata, is the closest to Vertebrata. A recent study on chordate evolution has shown that Urochordata and Vertebrata diverged from an ancestor of subphylum Cephalochordata [16]. Phylogenic analysis of troponin components based on their amino acid sequences across Chordata have demonstrated that chordate troponin can be classified into three different monophyletic groups, cephalochordate (amphioxus) troponin, urochordate (ascidian) troponin and vertebrate troponin [6,17–19] as summarized in Fig. 1A. Cephalochordate (amphioxus) troponin seems to have diverged first from a chordate troponin ancestor. Functional analysis of troponin showed that troponin in amphioxus striated muscle inhibits the actin–myosin interaction in the absence of Ca2+ in a similar manner to vertebrate striated muscle troponin [19]. Interestingly, however, troponin isolated from giant multi-nucleated smooth muscle that constitutes the body wall of a urochordate ascidian (Halocynthia roretzi) (Fig. 2) [20] scarcely inhibits the actin–myosin interaction in the absence of Ca2+, but enhances it remarkably in the presence of Ca2+ [5]. This unique property of ascidian troponin has been attributed to TnI and TnT;
Fig. 2. Electron micrograph showing fine structure of multinucleated smooth muscle in body wall of ascidian, H. roretzi. Thin filaments (marked by white triangles) and thick filaments (marked by black triangles) are running along the longitudinal axis of the cell. No striations are visible. The photograph was taken by Dr. K. Terakado (see Ref. [20]). Bar: 1 lm.
that is, isolated ascidian TnT alone remarkably promotes actomyosin Mg2+-ATPase activity, regardless of Ca2+ ion concentration, while isolated ascidian TnI alone scarcely inhibits this activity [5]. Based on these functional characteristics, ascidian (H. roretzi) smooth muscle troponin has been regarded as ‘‘accelerator-type’’ troponin, a Ca2+-dependent accelerator, compared with vertebrate and amphioxus striated muscle troponin that has the property of a ‘‘brake-type’’ troponin: a Ca2+-sensitive inhibitor [6,19]. The ascidian smooth muscle troponin might have acquired the unique property of a Ca2+-dependent accelerator during evolution, while amphioxus and vertebrate striated muscle troponin may have succeeded the brake property from the ancestor troponin. Another ascidian species, Ciona intestinalis, belongs to Urochordata as H. roretzi, but they are phylogenetically categorized in different orders, Enterogona and Pleurogona, respectively. Between
Fig. 1. Phylogenetic relationships and functional variation of troponin during chordate evolution. (A) Neighbor-Joining tree showing relationships among cephalochordate (amphioxus), urochordate (ascidian) and vertebrate TnT amino acid sequences. Numbers at the forks indicate the percentage of 1000 bootstrap resamplings that support these topological elements. The scale bar indicates an evolutionary distance of 0.05 amino acid substitutions per position in the sequence. The Accession Numbers for TnT of Danio rerio fast (Danio F), slow (Danio S), and cardiac (Danio C) muscle and TnT of Xenopus laevis fast (Xenopus F), slow (Xenopus S) and cardiac (Xenopus C) muscle are NP_857636, AAI62595, NP_690853, AAM55471, NP_001086207 and NP_001082509, respectively. The accession numbers for TnT of Homo sapiens fast (Homo F), slow (Homo S), and cardiac (Homo C) muscle and TnT of Gallus gallus fast (Gallus F), slow (Gallus S) and cardiac (Gallus C) muscle are P45378, P13805, P45379, NP_990253, NP_990445 and NP_990780, respectively. The accession numbers for TnT of H. roretzi smooth (Halo Sm), larval striated muscle (Halo St), Branchiostoma floridae (Branchiostoma f) and Branchiostoma belcheri (Branchiostoma b) are BAA09463, BAA12720, EEA60346 and BAI67730, respectively. The accession numbers for TnT of C. intestinalis cardiac and smooth and larval striated muscle (Ciona Card/St/Sm) is KH.C4.57.v1.A.SL1-1 in the Ghost database. The C. elegans (Caenorhabditis) sequence, used as an outgroup, is from Q27371. (B) Functional variation of troponin during chordate evolution. Based on the evolutionary relationship of TnT amino acid sequences of various chordate species and the functional characteristics of troponin, vertebrate striated muscle troponin appears to have inherited the properties of ‘‘brake-type’’ troponin from the ancestral protein of amphioxus troponin. Ascidian (H. roretzi and C. intestinalis) striated and smooth muscle troponin evolved to acquire the unique property of ‘‘accelerator-type’’ troponin, characterized as a Ca2+-dependent activator of actin–myosin interaction during chordate evolution.
T. Obinata, N. Sato / Methods 56 (2012) 3–10 Table 1 Number of the genes encoding troponin components and the troponin isoforms generated in two ascidian species, H. roretzi and C. intestinalis. (A) Number of genes and protein isoforms. (B) The isoforms in C. intestinalis, their sizes (amino acid numbers), and the tissue expression. H. roretzi (A) TnT TnI TnC (B) CiTnT
CiTnI
CiTnC
C. intestinalis
2 Genes 4 Genes 1 Gene
2 isoforms 5 isoforms 2 isoform
1Gene 1 Gene 3 Genes
3 Isoforms 2 Isoforms 4 Isoforms
Isoforms
a.a.
Tissue expression
TnT-card TnT-st TnT-sm TnI-l TnI-s
304 275 253 229 182
TnC-3 TnC-1a TnC-1b
162 158 158
TnC-2
164
Cardiac muscle Larval striated muscle Adult smooth muscle Cardiac muscle Larval striated muscle Adult smooth muscle Larval striated muscle Larval striated muscle Adult smooth muscle Cardiac muscle Larval striated muscle
the two species, distinct differences are present in the number of genes encoding troponin components and the troponin isoforms generated (Table 1). Namely, in the case of H. roretzi, two genes and two protein isoforms for TnT [21], four genes and five protein isoforms for TnI [22] and one gene and two protein isoforms for TnC [23], while in the case of C. intestinalis, there is one gene and three protein isoforms for TnT [24], one gene and two protein isoforms for TnI [17,25] and three genes and four protein isoforms for TnC [26]. Adult smooth muscle and larval striated muscles of C. intestinalis use the same TnI isoform but different TnT and TnC isoforms, while smooth and striated muscles of H. roretzi use different TnT, TnC, and TnI isoforms. Characterization of the functional properties of these troponin components and their complex, has been achieved by preparing recombinant proteins based on the genome information in addition to preparation of native proteins from tissues. Interestingly, it was revealed that troponin of ascidian striated muscle functions as a Ca2+-dependent accelerator of actin–myosin interaction just as troponin of ascidian smooth muscle [6]. Functional variation of troponin during chordate evolution is summarized in Fig. 1B.
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be obtained from the body wall. Another ascidian, C. intestinalis, can also be used but the amount of muscle tissue is much smaller in this animal. The outline of the procedure is illustrated in Fig. 3. First, dissect out fresh smooth muscle tissue from the body wall, and rinse the isolated tissue with a KMP solution (50 mM KCl, 10 mM phosphate buffer (pH 7.0), 1 mM MgCl2) containing 1 mM ethylenediamine tetra acetic acid (EDTA) and 1 mM 2-mercaptoethanol, several times. Since troponin is sensitive to heavy metal ions, the EDTA-containing solution is helpful for protecting troponin from inactivation by removing heavy metal ions that may be found in seawater. If necessary, the isolated muscle tissue can be stored in the same solution containing 50% glycerol at 20 °C for at least several months. The isolated tissue is homogenized in the KMP solution containing 1 mM EDTA with a tissue grinder (blender) or a Dounce homogenizer. The homogenate is centrifuged for 10 min at 14,000g. The supernatant that is to be discarded will be turbid because of the presence of polysaccharides. Take the pellet, suspend it in the same solution and centrifuge the mixture again. These procedures are repeated until the supernatant becomes transparent. The final pellet is suspended in the 3 volume of the KMP solution containing 6 mM ATP and 0.2 mM ethyleneglycolbis (aminoethyl ether)–N-N0 -tetra acetic acid (EGTA) and incubated with stirring for about 10 min at 4 °C. A KMT solution where 10 mM Tris(hydroxymethyl)aminomethane (Tris)–HCl buffer (pH 7.4 at 4 °C) is used in place of phosphate buffer is preferable at this step, if isolated thin filaments are used for examining their interaction with myosin by ATPase assay, to avoid contamination of inorganic phosphate in the thin filament fraction. Through this process, troponin-containing actin filaments are released from myosin filaments in the contractile apparatus. The suspension is centrifuged for 1 h at 70,000g to precipitate myosin filaments and the residue of contractile apparatus leaving actin filaments in the supernatant. The resultant supernatant is taken and subjected to centrifugation at 100,000g for 3 h. The resultant precipitate contains actin thin filaments that have tropomyosin and
2. Isolation of troponin from tissues The procedure for preparing troponin from vertebrate striated muscles has been established [14,15]. This procedure, however, may not be directly applicable for preparing troponin from invertebrate muscles. In the case of non-striated muscles such as smooth muscle, troponin-containing actin filaments are easily released from contractile structures under the conditions that makes muscle relaxed, namely by incubating tissue homogenates in the solution containing EGTA, a Ca2+-chelater, and ATP, because actin filaments are not tightly anchored to Z-disks as in striated muscles. Troponin-containing actin thin filaments can be isolated as follows and the isolated thin filaments are used as a good source for obtaining troponin. 2.1. Isolation of actin thin filaments Isolation of actin thin filaments from ascidian smooth muscle can be achieved as first described by Toyota et al. [27], with a slight modification of the method that was originally devised for molluscan smooth muscle [28]. Ascidian, H. roretzi, is an excellent material for this purpose, because a large amount of smooth muscle can
Fig. 3. Schematic representation of the procedure for isolation of native thin filaments and extraction of troponin and tropomyosin from the thin filaments.
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troponin in a considerably high purity (see Fig. 4). By applying slight modification of these procedures, native actin filaments have also been successfully isolated from the muscles of other invertebrate animals such as sea urchin [29], nematodes (Ascaris) [30], sea cucumber [31], earthworm (unpublished data), and tardigrade (water bear) [13]. 2.2. Extraction of troponin from the actin filaments Troponin and tropomyosin can be dissociated from the isolated actin filaments by incubating the actin filaments in 0.6 M KCl, 1 mM MgCl2 and 10 mM Tris–HCl buffer, pH 7.5 at 4 °C for 1–2 h at 25 °C (Figs. 3 and 5A). This method is based on the report by Spudich and Watt [32] that was devised for preparing tropomyo-
sin-free F-actin. Troponin–tropomyosin complex can be obtained in the supernatant leaving actin in the pellet by spinning the mixture at 100,000g for 3 h and then, salted out by the addition of saturated ammonium sulfate (pH 7.0) to 30–75% saturation. Troponin can be separated from tropomyosin by isoelectric precipitation of tropomyosin at pH 4.5 in the presence of 0.4 M LiCl. Namely, 1.2 M LiCl is added to the solution of troponin–tropomyosin complex to a final concentration of 0.4 M and then, the pH of the solution is adjusted to 4.5 with 0.2 N HCl. After 30 min, the solution is centrifuged for 15 min at 16,000g. The supernatant containing troponin is neutralized by gradual addition of diluted NaOH solution, and then, troponin can be precipitated by the addition of solid ammonium sulfate to 70% saturation (Fig. 5B, the lane marked TN). The precipitate, a complex of three troponin components, is dissolved and dialyzed against in 1 mM NaHCO3. Troponin complex is soluble in a low salt buffer solution at neutral pH such as 1 mM NaHCO3, or 10 mM Tris–HCl buffer (pH 7.4 at 4 °C).
2.3. Purification of ascidian troponin components
Fig. 4. Electron micrograph of native thin filaments that were isolated from ascidian (H. roretzi) body wall smooth muscle. The filaments were negatively stained with 2% uranyl acetate. Bar: 0.5 lm.
Ascidian three troponin components can be purified by applying the method described for vertebrate striated muscle troponin components [14] with a slight modification [5] (Fig. 5B). The complex of ascidian three troponin components is dialyzed twice or more against the solution containing 6 M urea, 50 mM glycine– NaOH buffer (pH 9.0) and 0.5 mM CaCl2 (termed Buffer A). This dialyzed sample is then centrifuged at 20,000g for 20 min to remove insoluble aggregate in the solution. Proteins in the supernatant are applied to an SP-Sephadex C-50 (Sigma) column equilibrated with Buffer A. Proteins are eluted by a linear gradient of NaCl. TnI–TnC complex is eluted at void volume and TnT at about 0.17 M NaCl. The TnI–TnC complex is dialyzed against 6 M urea, 50 mM Tris–HCl buffer (pH 8.0 at 4 °C) containing 2 mM EGTA (Buffer B) and separated into each component by SP-Sephadex C50 column chromatography. Under this condition, TnC becomes free from TnI because of the absence of Ca2+. TnC is eluted at void volume and TnI at about 0.15 M NaCl of the linear NaCl gradient. TnC is further purified by chromatography on a DEAE–cellulose column equilibrated with Buffer B. TnC is eluted by a linear NaCl gradient as a single peak at about 0.18 M NaCl. The purified TnC is dialyzed against 1 mM NaHCO3 (or 10 mM Tris–HCl, pH 7.4), while TnT and TnI are against 0.4–0.5 M NaCl containing 1 mM NaHCO3 (or 10 mM Tris–HCl, pH 7.4) because they are insoluble in a solution of low salt concentration. The advantage of using NaHCO3 is that this chemical makes the solution slightly alkaline pH and does not disturb assays of protein concentration. The purified troponin components can be simply frozen and stored at 20 °C to 80 °C without loosing activity.
3. Preparation of recombinant troponin components
Fig. 5. SDS–PAGE patterns of each fraction that was obtained during the isolation process of ascidian (H. roretzi) regulatory proteins. (A) Extraction of troponin– tropomyosin from isolated native thin filaments. (a): native thin filaments, (b) and (c): 100,000g supernatant and precipitate from 0.6 M KCl-treated thin filaments (see detail in the text), respectively. (B) Isolated troponin complex (TN) and three troponin components, TnT, TnI and TnC. A, actin; TM, tropomyosin; T, TnT; I, TnI; C, TnC. The protein marked by asterisk is HR-29 [44] that is always contaminating the thin filament fraction from ascidian, H. roretzi. This protein is not detectable in the thin filament fraction of another ascidian, C. intestinalis.
In most of invertebrate animals, it is not easy to obtain sufficient amounts of muscle tissue to prepare troponin as for the ascidian H. roretzi. On the other hand, nowadays genomic and/or cDNA sequence information is available for troponin components in a variety of invertebrate animals. Multiple isoforms of troponin components have been detected in many invertebrate animals. These isoforms can be prepared only as recombinant proteins in E. coli or other expression systems. In our experience with ascidian and amphioxus, recombinant troponin components show the same functional properties as native proteins that have been isolated from muscle tissue. In the following, the preparation procedures that we used to obtain recombinant ascidian and amphioxus troponin components are described.
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3.1. Vector construction of amphioxus troponin Amphioxus TnI (BbTnI, AB035664 for GenBank) and the TnC (BbTnC, AB175946) cDNAs were cloned from Branchiostoma belcheri (B. belcheri) adult whole body RNA by reverse transcription (RT)-PCR. B. belcheri TnT (BbTnT, AB543715) cDNA was obtained from a cDNA library that was produced from the nerve cord and muscle tissues [33]. The coding regions of BbTnC, BbTnI and BbTnT cDNA were obtained by PCR using specific primer sets (See reference [19]). The cDNA fragment of BbTnC was cloned into pBluescript SK(-) (Stratagene, Cedar Creek, TX), containing synthesized nucleotides that encode 8 histidine residues, and then the DNA fragment encoding BbTnC containing a His-tag at the C-terminus was subcloned into the expression vector pGEX4T-1 that carries an N-terminal glutathione S-transferase (GST)-tag coding sequence followed by a thrombin cleavage recognition site (GE Healthcare UK Ltd., UK). The cDNA fragment encoding BbTnI was cloned into pQE-30 (Qiagen, Valencia, CA). The cDNA fragment encoding BbTnT was cloned into the expression vector pGEX6P-1 that carries an N-terminal GST-tag coding sequence followed by a PreScission cleavage recognition site (GE Healthcare). B. belcheri troponin expression vectors were transformed to E. coli strain BL21 (DE3) pLysS (Novagen, Darmstadt, Germany). Expression of amphioxus troponin components was induced in E. coli with 1 mM IPTG (isopropyl b-D-thiogalactopyranoside). 3.2. Purification of recombinant BbTnI, BbTnC and BbTnT For purification of recombinant BbTnI with a His-tag at the Nterminus, the insoluble fraction of E. coli lysates was dissolved in 6 M urea solution containing 10 mM imidazole, 0.5 M NaCl, 0.5 mM DTT, and 20 mM phosphate buffer (pH 7.4), and then dialyzed against the same buffer without urea. The solubilized protein was applied to a HisTrap affinity column (GE Healthcare Ltd., UK) in a His-buffer (10 mM imidazole, 20 mM phosphate buffer, and 0.5 M NaCl, pH 7.4). Purified BbTnI was eluted using a gradient of 10–500 mM imidazole in 20 mM phosphate buffer (pH 7.4) containing 0.5 M NaCl. The protein was dialyzed against 0.5 M KCl containing 10 mM sodium bicarbonate and concentrated with Aquacide II (Calbiochem, La Jolla, CA) for analysis. For purification of recombinant BbTnC, the supernatant of E. coli lysates that contained BbTnC with a GST-tag at the N-terminus and a His-tag at the C-terminus (GCH protein) was applied to a glutathione Sepharose 4B (GS) column (GE Healthcare) that had been pre-washed with the His-Buffer. GCH protein was allowed to bind
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to the GS column. After that, the protein was incubated with thrombin (GE Healthcare) at a 1:100 dilution (equivalent to 50 U) overnight at 4 °C. BbTnC was eluted and immediately applied to a HisTrap affinity column in the same buffer conditions for further purification. Purified BbTnC (with a His-tag) was eluted from the column and prepared for analysis in the same manner as BbTnI. One typical example of expression and purification of recombinant amphioxus TnC is shown in Fig 6. The purification procedure for recombinant BbTnT was almost the same as that for BbTnC, except for the following changes. For the GS column, the protein was incubated with a 1:100 dilution (50 U) of PreScission (GE Healthcare) overnight at 4 °C. BbTnT was then eluted with 10 mM phosphate buffer (pH 7.4) containing 0.15 M NaCl and directly absorbed on an ion exchange HiTrap column (GE Healthcare) for further purification. BbTnT was eluted from the column with a gradient of 0.15–0.5 M NaCl in 10 mM phosphate buffer (pH 7.4). 3.3. Vector construction of ascidian troponin C. intestinalis TnI cDNA (TnI-s; U55261 for GenBank, KH.C11.673.v1 for Ghost Database) and TnC cDNA (TnC-1b; AK112490, KH.C8.251.v2) were provided by C. intestinalis genomic and cDNA resources (Kyoto University, Japan). The coding regions of TnI and TnC cDNA were obtained by PCR using specific primer sets (see Ref. [6]). The cDNA fragments of the TnI and TnC were inserted into pBluescript SK(-) containing synthesized nucleotides encoding eight histidine residues following a termination codon. The cDNA fragments of TnI and TnC carrying a C-terminal His-tag coding sequence were then subcloned into the expression vector pET3a (Novagen, Darmstadt, Germany). In C. intestinalis, different TnT isoforms are expressed in larval striated muscle and adult smooth muscle; they were termed TnT-st (striated muscle type) and TnT-sm (smooth muscle type), respectively, in this paper. To obtain these isoforms, C. intestinalis TnT cDNA (TnT-st; KH.C4.57.v3 and TnT-sm; KH.C4.57.v4) were cloned from C. intestinalis body-wall muscle RNA by RT-PCR. The coding regions of TnT-st and TnT-sm were obtained by PCR using the primer sets (see Ref. [6]). The PCR products were then inserted into pBluescript KS(-), containing synthesized nucleotides encoding eight histidine residues. These cDNA fragments carrying an N-terminal His-tag coding sequence were subcloned into the expression vector pET3a. All C. intestinalis troponin expression vectors were transformed to E. coli strain BL21(DE3) pLysS. Expression of ascidian troponin components was induced in E. coli with 1 mM IPTG. 3.4. Purification of recombinant TnI, TnC and TnT of C. intestinalis
Fig. 6. Expression and purification of recombinant amphioxus TnC. Expression of amphioxus TnC (BbTnC) was induced in E. coli with IPTG. Whole extract (WE; lane 1) were centrifuged to obtain the supernatant (S; lane 2) and precipitate (P; lane 3). The initial translated BbTnC containing both a GST-tag and His-tag (GCH) in the supernatant was bound to glutathione beads and then cleaved with thrombin. BbTnC (CH) in the eluate (G; lane 4) was further purified with a HisTrap affinity column (H; lane 5). The molecular weight markers are shown on the left side.
For the purification of recombinant C. intestinalis TnI and TnC, the soluble fractions of bacterial lysates were applied to a HisTrap affinity column in the His-buffer. Proteins were eluted using a gradient of 50 to 500 mM imidazole in 20 mM phosphate buffer (pH 7.4) containing 500 mM NaCl. TnC was further dialyzed against Q-buffer (20 mM NaCl, 25 mM Tris–HCl, 1 mM DTT, pH 8.0) and then applied to an anion exchange Econo-Pac Q cartridge (BioRad Laboratories). The purified TnC was obtained by a gradient of 40–500 mM NaCl. For the purification of recombinant TnT-st and TnT-sm, the insoluble fraction of bacterial lysates were dissolved in the Hisbuffer containing 6 M urea, and then dialyzed against the same buffer without urea. The solubilized proteins were subjected to HisTrap affinity chromatography in His-buffer. Purified proteins were eluted using a gradient of 10 to 500 mM imidazole in 20 mM phosphate buffer (pH 7.4) containing 0.5 M NaCl.
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4. Functional assay of troponin components 4.1. Interaction of troponin components and specific binding to other myofibrillar proteins 4.1.1. Ca2+-responsiveness of TnC and Ca2+-dependent interaction of TnC and TnI To clarify whether troponin components function in a Ca2+dependent manner, an essential property of troponin, Ca2+-responsiveness of TnC and Ca2+-dependent TnI–TnC interaction can be examined by electrophoresis. The Ca2+-dependent mobility shift on SDS–PAGE gels is a pronounced property of TnC [34]. SDS–PAGE is carried out using a 13.5% polyacrylamide gel and a discontinuous Tris–glycine buffer system. To examine the Ca2+-dependent mobility shift of TnC on SDS–PAGE, 2 mM EGTA or 2 mM CaCl2 is added to the TnC sample at the final concentration. After SDS-treatment, the specimens are subjected to SDS–PAGE. In the presence of either EGTA or CaCl2, purified TnC shows a single band on an SDS–PAGE gel. However, TnC in the presence of CaCl2 migrates faster on the gel than in the presence of EGTA (or in the absence of Ca2+). The results obtained with ascidian (H. roretzi) TnC are shown in Fig. 7A. This phenomenon has been observed for TnC in another ascidian (C. intestinalis) [6], amphioxus [19] and other animals [34] and confirms that the purified ascidian or amphioxus recombinant TnC has Ca2+-binding ability. This Ca2+-binding mobility shift is attributable to the conformational change of TnC following the binding of Ca2+. To examine the Ca2+-dependent interaction of TnI and TnC, alkaline-urea PAGE is carried out in the presence or absence of Ca2+ [35]. In this electrophoresis, 8% polyacrylamide gels containing 8 M urea, 25 mM Tris and 80 mM glycine, pH 8.3 are used. Either 10 mM EGTA or 2 mM CaCl2 at the final concentration is added to the mixture of TnI and TnC or troponin complex that is dissolved in a Tris–glycine buffer containing 8 M urea as in the electrophoresis gel. Urea concentration can be decreased to 6 M. The results obtained with ascidian (H. roretzi) TnI and TnC are shown in Fig. 7B. As shown in this figure, in the presence of Ca2+, a slower migrating band (marked I–C) is observed (the right lane, Ca2+) and in the absence of Ca2+, a faster migrating band (marked
Fig. 7. Ca2+-responsiveness of TnC and Ca2+-dependent interaction of TnC and TnI. (A). Ca2+-dependent mobility shift of TnC on SDS–PAGE. Difference in mobility of TnC is observed in the presence of Ca2+ and in the presence of EGTA on SDS–PAGE. Purified ascidian (H. roretzi) TnC in an SDS buffer solution with 2 mM CaCl2 (right lane) showed a single band with faster mobility than the same purified TnC in an SDS buffer solution with 2 mM EGTA (left lane) (T. Nogami and T. Obinata, unpublished data). (B). Ca2+-dependent interaction of TnI and TnC in alkaline urea-PAGE analysis. A binding assay of ascidian (H. roretzi) TnI (TnI) with ascidian (H. roretzi) TnC (TnC) was carried out in the presence of Ca2+ (2 mM CaCl2, Ca2+) or in the absence of Ca2+ (10 mM EGTA, EGTA). The upper band (I–C) corresponds to the TnI–TnC complex in the presence of Ca2+ and the lower band (C) to TnC (according to Endo and Obinata [5]).
C) is observed (the left lane, EGTA). The slower migrating band corresponds to the TnI–TnC complex that is formed in a Ca2+-dependent manner and the faster migrating band is TnC alone. Under these electrophoresis conditions, TnI alone does not move in the gel regardless of the presence or absence of Ca2+, because it is a basic protein. These behaviors of TnC and TnI have been observed in vertebrate [35], ascidian (C. intestinalis) [6] and amphioxus proteins [19]. Thus, we can confirm that purified recombinant TnI and TnC of ascidian [6] and amphioxus [19] can bind to each other in the characteristic manner of chordate troponin components. 4.1.2. Binding of TnT to tropomyosin TnT is the tropomyosin-binding component of the troponin complex. Therefore, whether an isolated protein has this expected property of TnT can be tested by examining its binding to tropomyosin. The TnT–tropomyosin binding can be examined by different ways. First, binding of TnT to tropomyosin that is associated with F-actin can be examined by a co-sedimentation assay. Purified tropomyosin and F-actin are pre-incubated in a solution containing 0.1 M KCl and 1 mM MgCl2, then ‘‘TnT’’ is added to the actin–tropomyosin complex and incubated for 1 h or so. The mixture is then centrifuged at 350,000g for 30 min and the precipitate is subjected to SDS–PAGE to check whether ‘‘TnT’’ is co-precipitated with the F-actin–tropomyosin complex. Secondly, binding of TnT to tropomyosin can be examined by electron microscopy. In this analysis, ascidian or rabbit tropomyosin with or without ascidian TnT is dialyzed against 1 M KCl in 50 mM Tris–HCl buffer, pH 8.0 at 4 °C and then, dialyzed against 50 mM MgCl2 in 50 mM Tris–HCl buffer, pH 8.0 at 4 °C to form paracrystals of tropomyosin. In the absence of TnT, the electron microscopic image of tropomyosin paracrystals consists of light staining bands (26 nm in width) and dark staining bands (14 nm in width) that alternate to give a periodicity of 40 nm as described by Cohen and Longly [36]. When the tropomyosin paracrystals were formed in the presence of ascidian TnT, however, the band patterns of the paracrystals were markedly transformed as the result of TnT-binding to tropomyosin (Fig. 8). Namely, the presence of TnT resulted in the appearance of lighter staining zone (marked by triangles in the figure) in the center of the light staining bands of the paracrystals with the 40 nm periodicity. This remarkable phenomenon was first demonstrated with rabbit TnT and rabbit tropomyosin by Nonomura et al. [37].
Fig. 8. Electron micrographs of magnesium paracrystals of rabbit tropomyosin with (B) or without (A) ascidan (H. roretzi) smooth muscle TnT. The paracrystals were negatively stained with 2% uranyl acetate. TnT bound to the center of the light band (marked by triangles) of the tropomyosin paracrystals, just as observed with rabbit TnT and rabbit tropomyosin paracrystals [37]. The photographs were taken by Dr. T. Endo [5]. Bar: 0.1 lm.
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Troponin is a Ca2+-dependent regulator of muscle contraction or actin–myosin interaction. To assess this fundamental property of troponin, troponin is combined with tropomyosin, and the effects of troponin–tropomyosin complex on actomyosin-Mg2+-ATPase activity are measured in the presence of either CaCl2 or EGTA. Rabbit or chick reconstituted actomyosin, a mixture of purified actin and myosin, can be used for the assay. Myosin, actin and tropomyosin are prepared as described [38–40]. Actin and tropomyosin can also be prepared from body wall muscle of ascidian H. roretzi, as from vertebrate muscle tissue. To obtain ascidian myosin, an improved method as described [41] is required, because ascidian myosin tends to become insoluble and subsequently the ATPase activity is decreased, when the precipitation and solubilization of myosin are repeated by changing ionic strength during preparation. In the improved method, myosin is extracted from the homogenate of ascidian body wall muscle for 1 h at 0 °C with a pyrophosphate solution containing 40 mM Na-pyrophosphate, 2 mM MgCl2, 2 mM EGTA, 5 mM ATP, 2 mM 2-mercaptoethanol, 10% glycerol and 0.1 M sucrose, pH 7.0. The mixture is centrifuged for 3 h at 72,000g. Myosin is, then, purified from the supernatant by DEAE cellulose column chromatography. Myosin is eluted with a linear NaCl gradient from 0 to 0.2 M NaCl in the presence of pyrophosphate (See detail in reference [41]). In a standard assay to examine the effects of troponin on actin– myosin interaction, myosin (0.4 lM), actin (1.6 lM), tropomyosin (0.23 lM), and the troponin components (TnT: 0.23 lM, TnI: 0.23 lM, TnC: 0.23 lM) are mixed in a solution containing 40– 50 mM KCl, 1 mM MgCl2, 20 mM Tris–HCl, pH 7.5 with 0.1 mM CaCl2 or 1 mM EGTA. In other words, the actin:tropomyosin:troponin molar ratio in the mixtures is 7:1:1. Relative proportion of troponin in the mixture can be increased to see the effects of troponin more obviously: After pre-incubation for 5 min at 25 °C, ATP (1 mM at final concentration) is added to the mixture to initiate ATPase reaction and allowed to further incubate for about 10 min. The reaction is stopped by adding 5% trichloroacetic acid (TCA) at final concentration. Inorganic phosphate (Pi) liberated as the result of ATP hydrolysis by actomyosin ATPase is measured according to Taussky and Shorr [42]. In this assay method, 1/4 volume of 4% sodium molybdate in 2 N H2SO4 is added to the test
(%)
+Ca2+ 0.5 0.4
+Ca2+
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+EGTA
0.1 0
0
200 150 100 50
M+A+TM (Control)
M+A+TM +Tst +I +C
M+A+TM +Tsm +I +C
10 15 Time (min)
20
(B) Amphioxus
(%) 200 150 100 50 0
0
5
Fig. 10. Effects of ascidian (H. roretzi) troponin on the superprecipitation of reconstituted rabbit actomyosin. Reaction mixtures contain 80 mM KCl, 1 mM MgCl2, 20 mM Tris–HCl buffer (pH 7.5 at 4 °C), 0.2 mM ATP, 0.1 mM EGTA (open circle and open triangle) or 0.1 mM CaCl2 (closed circle and closed triangle), 300 lg/ ml of rabbit myosin, 100 lg/ml of rabbit actin, 40 lg/ml of ascidian tropomyosin and 40 lg/ml of ascidian troponin, when added. Reaction temperature is 20 °C. Superprecipitation was initiated by adding ATP at 0 time point. Closed and open triangle: actin + myosin + tropomyosin; Closed and open circle: actin + myosin + tropomyosin + troponin.
Actomyosin-ATPase Activity (relative value)
250
+EGTA
0.2
1 mM EGTA
Ascidian Actomyosin-ATPase Activity (relative value)
0.6
0.1 mM CaCl2
(A)
300
sample solution. After quick mixing, 1/16 volume of 40% FeSO4 in 1 N H2SO4 is added. After another quick mixing, the color of the solution is measured with a spectrophotometer at 660 nm and then, Pi (lmoles) in the solution is calculated. The ATPase activity is shown as lmoles Pi liberated/min/mg myosin. The value for the activity of actin–myosin without troponin under this condition is usually 0.2–0.3. Typical examples of the results showing the effects of troponin (or a complex of TnT, TnI and TnC) on actomyosin ATPase activity in the presence or absence of Ca2+ are shown in Fig. 9. Ascidian troponin enhances the ATPase activity in a Ca2+-dependent manner (Fig. 9A) (See detail in Ref. [6]), while amphioxus troponin suppresses the ATPase activity in the absence of Ca2+ (Fig. 9B) (See detail in Ref. [19]).
A660
4.2. Ca2+-dependent regulation of actin–myosin interaction
M+A+TM (Control)
M+A+TM +native TN
M+A+TM +T +I +C
Fig. 9. Effects of ascidian (C. intestinalis) and amphioxus troponin on the Mg2+-ATPase activity of reconstituted rabbit actomyosin. (A): The effects of the ascidian recombinant troponin complex, combinations of striated muscle-type (Tst, I and C) and smooth muscle-type (Tsm, I and C) forms, on actomyosin ATPase. (B): The effects of the amphioxus native troponin (native TN) and recombinant troponin complex (TnT, TnI and TnC) on actomyosin ATPase. The actin, tropomyosin, TnT, TnI and TnC were incubated roughly in a molar ratio of 4:1:1:1:1 and then mixed with myosin. In the control mixture (control), actin, myosin and tropomyosin were combined without any troponin components. The gray and white bars represent the reaction mixture with 0.1 mM CaCl2 or 1 mM EGTA, respectively. M, myosin; A, actin; TM, tropomyosin; Tst, ascidian striated muscletype TnT; Tsm, ascidian smooth muscle-type TnT; T, amphioxus TnT; I, TnI; C, TnC. The salts concentrations of the reaction mixtures are 50 mM KCl, 1 mM MgCl2, 20 mM Tris– HCl buffer (pH 7.5 at 4 °C), 1 mM ATP, 0.1 mM CaCl2 or 1 mM EGTA.
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Acknowledgments We thank Dr T. Endo in Chiba University for creating the electron micrographs used in this paper. They also thank the Ministry of Education, Science and Culture, Japan for financial support. References
Fig. 11. Detection of TnC along isolated ascidian native thin filaments by immuno electron microscopy. Isolated ascidian (H. roretzi) thin filaments as shown in Fig. 4 were incubated with the antibody raised against ascidian TnC in 0.1 M KCl, 1 mM MgCl2, 10 mM phosphate buffer, pH 7.0 for 30 min at 20 °C. The aggregates formed were collected by centrifugation, washed with 0.1 M KCl. 10 mM Tris–HCl buffer, pH 7.5 at 4 °C, followed by staining with 2% uranylacetate, and then observed under an electron microscope. Cross striations with about 38 nm intervals were formed along the thin filaments, indicating location of TnC along actin filaments. The photographs were taken by Dr. T. Endo. Bar: 0.2 lm.
[1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11]
Another way of examining the effect of troponin on actin–myosin interaction is monitoring of superprecipitation. Actomyosin dissolved in dilute KCl concentration shows a marked flocculent precipitation when ATP is added. This phenomenon, so-called superprecipitation, is coupled with ATP hydrolysis by actomyosin ATPase. Occurrence of superprecipitation can be monitored as the change in turbidity (absorbance at 660 nm) of an actomyosin solution. The reaction mixture for assay including actin, myosin, tropomyosin and troponin (or its component) is the same as in the ATPase assay described above, except that the KCl concentration is slightly higher (0.08–0.1 M) and ATP concentration is lower (0.2 mM) than in the ATPase assay. Immediately after addition of ATP, monitoring of the absorbance at 660 nm is started. A typical example of the assay is shown in Fig 10. Troponin causes a remarkable difference in time course of the turbidity change of the actomyosin solution between the presence and absence of Ca2+. An advantage of this assay is that smaller amounts of proteins are needed for this assay in comparison with the ATPase assay.
[12] [13] [14] [15] [16]
5. Intracellular localization of troponin
[25]
Troponin is involved in actin-linked regulation of muscle contraction or actin–myosin interaction. Therefore, it is important to confirm the location of troponin or each troponin component along actin filaments in cells. In order to achieve this, antibodies that specifically recognize troponin components are indispensable. If purified troponin components are obtained, polyclonal or monoclonal antibodies can be prepared by standard procedures. Specificity of antibodies needs to be carefully checked by Western blotting. Sometimes, antibodies recognize the counterparts of the animal species other than the animals that were used for preparing the immunogens, but we must always keep in mind that they may interact with other proteins non-specifically. Co-localization of actin and troponin components can be visualized by dual staining of muscle tissues with rhodamine–phalloidin and anti-troponin antibodies [10,13]. Location of troponin along actin filaments can be directly confirmed by immunoelectron microscopy by staining isolated native actin filaments with anti-troponin antibody as described by Ohtsuki [43] and Endo and Obinata [5] (Fig. 11).
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Comparative studies on troponin, a Ca2+-dependent regulator of muscle contraction, in striated and smooth muscles of protochordates Takashi Obinata a,⇑,1, Naruki Sato b,1 a b
Department of Biology, Faculty of Science, Chiba University, Yayoi-cho, Inage-ku, Chiba 263-8522, Japan Department of Nanobiology, Graduate School of Advanced Integration Science, Chiba University, Yayoi-cho, Inage-ku, Chiba 263-8522, Japan
a r t i c l e
i n f o
Article history: Available online 18 October 2011 Keywords: Troponin Calcium Muscle contraction Protochordate Ascidian Amphioxus
a b s t r a c t Troponin is well known as a Ca2+-dependent regulator of striated muscle contraction and it has been generally accepted that troponin functions as an inhibitor of muscle contraction or actin–myosin interaction at low Ca2+ concentrations, and Ca2+ at higher concentrations removes the inhibitory action of troponin. Recently, however, troponin became detectable in non-striated muscles of several invertebrates and in addition, unique troponin that functions as a Ca2+-dependent activator of muscle contraction has been detected in protochordate animals, although troponin in vertebrate striated muscle is known as an inhibitor of the contraction in the absence of a Ca2+. Further studies on troponin in invertebrate muscle, especially in non-striated muscle, would provide new insight into the evolution of regulatory systems for muscle contraction and diverse function of troponin and related proteins. The methodology used for preparation and characterization of functional properties of protochordate striated and smooth muscles will be helpful for further studies of troponin in other invertebrate animals. Ó 2011 Elsevier Inc. All rights reserved.
1. Introduction Troponin is a mediator of Ca2+-dependent regulation of striated muscle contraction [1]. It is linked to actin filaments and functions in combination with tropomyosin. Troponin is a complex of three components: troponin T (TnT), a tropomyosin-binding component that stabilizes the troponin complex along actin filaments, troponin I (TnI), an inhibitory component of the actin–myosin interaction and troponin C (TnC), a Ca2+-binding component [1]. Troponin of vertebrate striated muscle is known to function as an inhibitor (or a brake) of the actin–myosin interaction (actomyosin Mg2+-ATPase activity) at low Ca2+ concentrations. Binding of Ca2+ to TnC induces a structural change of the troponin complex, specifically in TnI within the complex. Subsequently, the inhibitory action of troponin is removed, enabling myosin to interact with actin [2]. This troponin–tropomyosin regulatory system also exists in a variety of invertebrate striated and obliquely striated muscles [3,4]. In most cases, troponin of invertebrate muscle also inhibits the actin–myosin interaction at low Ca2+ concentrations in a similar manner to vertebrate striated muscle troponin. Although troponin is predominantly expressed in striated muscles in both vertebrate and invertebrate animals, there are several instances for the presence of troponin in non-striated muscles. Troponin has been detected in multinucleated smooth muscle cells ⇑ Corresponding author. Fax: +81 43 290 2804. 1
E-mail address: [email protected] (T. Obinata). These authors contributed equally to this work.
1046-2023/$ - see front matter Ó 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.ymeth.2011.09.026
of ascidian body wall [5,6] and adductor smooth muscle of Akazara scallop [7]. In these muscles, troponin functions as a Ca2+-dependent activator of actin–myosin interaction. Recently, expression of troponin was detected in mammalian vascular smooth muscle, although the physiological roles in the contraction of vertebrate smooth muscle are not yet to be established [8]. More recently, troponin was detected in myoepithelial cells, primitive non-striated muscle-like cells, which are present in the somatic gonad of the nematode Caenorhabditis elegans. In these cells, actin and myosin are organized in non-striated filament networks, and TnC and TnI are associated with actin filaments [9,10]. Their contraction is tightly coupled with oocyte maturation, and troponin plays an essential role for the contractile regulation on the occasion of ovulation [9–11]. Furthermore, TnI is involved in the regulation of chromosomal stability and cell polarity in early Drosophila embryos [12]. A TnI-like protein that is associated with actin filaments has been detected in smooth muscle-like cells in tardigrades (water bears) [13]. These observations suggest that troponin may play important roles not only in striated muscles but also in non-striated muscle in invertebrate animals. Therefore, it will be interesting to investigate how the actin-linked troponin regulatory system is distributed among non-striated muscles of a variety of invertebrate animals and how it functions in the contractile regulation of invertebrate muscles, especially whether it exhibits a brake-type property or an accelerator-type property. Purification procedures of vertebrate striated muscle troponin and its components have been established [14,15]. However, extraction and purification of troponin from various invertebrate
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muscles may not be achieved by simply applying the method that has been established for vertebrate striated muscle troponin for several reasons: (1) amount of muscle tissues is occasionally limited. (2) If a mixture of heterogeneous tissues or whole animal body is used as a starting material, purification of troponin from the crude protein extracts is not easy and in addition, proteolytic enzymes that are contaminated in the extract tend to degrade troponin that is sensitive to proteases during preparation. Nevertheless, troponin has been purified from striated and smooth muscles of protochordate animals, namely ascidian and amphioxus. This experience may be helpful for the preparation of troponin from other invertebrate animals and the analysis of its functional characteristics. Therefore, the preparation procedure for protochordate troponin and analysis of its function are described in the following. Protochordata, comprising subphyla Cephalochordata and Urochordata, is the closest to Vertebrata. A recent study on chordate evolution has shown that Urochordata and Vertebrata diverged from an ancestor of subphylum Cephalochordata [16]. Phylogenic analysis of troponin components based on their amino acid sequences across Chordata have demonstrated that chordate troponin can be classified into three different monophyletic groups, cephalochordate (amphioxus) troponin, urochordate (ascidian) troponin and vertebrate troponin [6,17–19] as summarized in Fig. 1A. Cephalochordate (amphioxus) troponin seems to have diverged first from a chordate troponin ancestor. Functional analysis of troponin showed that troponin in amphioxus striated muscle inhibits the actin–myosin interaction in the absence of Ca2+ in a similar manner to vertebrate striated muscle troponin [19]. Interestingly, however, troponin isolated from giant multi-nucleated smooth muscle that constitutes the body wall of a urochordate ascidian (Halocynthia roretzi) (Fig. 2) [20] scarcely inhibits the actin–myosin interaction in the absence of Ca2+, but enhances it remarkably in the presence of Ca2+ [5]. This unique property of ascidian troponin has been attributed to TnI and TnT;
Fig. 2. Electron micrograph showing fine structure of multinucleated smooth muscle in body wall of ascidian, H. roretzi. Thin filaments (marked by white triangles) and thick filaments (marked by black triangles) are running along the longitudinal axis of the cell. No striations are visible. The photograph was taken by Dr. K. Terakado (see Ref. [20]). Bar: 1 lm.
that is, isolated ascidian TnT alone remarkably promotes actomyosin Mg2+-ATPase activity, regardless of Ca2+ ion concentration, while isolated ascidian TnI alone scarcely inhibits this activity [5]. Based on these functional characteristics, ascidian (H. roretzi) smooth muscle troponin has been regarded as ‘‘accelerator-type’’ troponin, a Ca2+-dependent accelerator, compared with vertebrate and amphioxus striated muscle troponin that has the property of a ‘‘brake-type’’ troponin: a Ca2+-sensitive inhibitor [6,19]. The ascidian smooth muscle troponin might have acquired the unique property of a Ca2+-dependent accelerator during evolution, while amphioxus and vertebrate striated muscle troponin may have succeeded the brake property from the ancestor troponin. Another ascidian species, Ciona intestinalis, belongs to Urochordata as H. roretzi, but they are phylogenetically categorized in different orders, Enterogona and Pleurogona, respectively. Between
Fig. 1. Phylogenetic relationships and functional variation of troponin during chordate evolution. (A) Neighbor-Joining tree showing relationships among cephalochordate (amphioxus), urochordate (ascidian) and vertebrate TnT amino acid sequences. Numbers at the forks indicate the percentage of 1000 bootstrap resamplings that support these topological elements. The scale bar indicates an evolutionary distance of 0.05 amino acid substitutions per position in the sequence. The Accession Numbers for TnT of Danio rerio fast (Danio F), slow (Danio S), and cardiac (Danio C) muscle and TnT of Xenopus laevis fast (Xenopus F), slow (Xenopus S) and cardiac (Xenopus C) muscle are NP_857636, AAI62595, NP_690853, AAM55471, NP_001086207 and NP_001082509, respectively. The accession numbers for TnT of Homo sapiens fast (Homo F), slow (Homo S), and cardiac (Homo C) muscle and TnT of Gallus gallus fast (Gallus F), slow (Gallus S) and cardiac (Gallus C) muscle are P45378, P13805, P45379, NP_990253, NP_990445 and NP_990780, respectively. The accession numbers for TnT of H. roretzi smooth (Halo Sm), larval striated muscle (Halo St), Branchiostoma floridae (Branchiostoma f) and Branchiostoma belcheri (Branchiostoma b) are BAA09463, BAA12720, EEA60346 and BAI67730, respectively. The accession numbers for TnT of C. intestinalis cardiac and smooth and larval striated muscle (Ciona Card/St/Sm) is KH.C4.57.v1.A.SL1-1 in the Ghost database. The C. elegans (Caenorhabditis) sequence, used as an outgroup, is from Q27371. (B) Functional variation of troponin during chordate evolution. Based on the evolutionary relationship of TnT amino acid sequences of various chordate species and the functional characteristics of troponin, vertebrate striated muscle troponin appears to have inherited the properties of ‘‘brake-type’’ troponin from the ancestral protein of amphioxus troponin. Ascidian (H. roretzi and C. intestinalis) striated and smooth muscle troponin evolved to acquire the unique property of ‘‘accelerator-type’’ troponin, characterized as a Ca2+-dependent activator of actin–myosin interaction during chordate evolution.
T. Obinata, N. Sato / Methods 56 (2012) 3–10 Table 1 Number of the genes encoding troponin components and the troponin isoforms generated in two ascidian species, H. roretzi and C. intestinalis. (A) Number of genes and protein isoforms. (B) The isoforms in C. intestinalis, their sizes (amino acid numbers), and the tissue expression. H. roretzi (A) TnT TnI TnC (B) CiTnT
CiTnI
CiTnC
C. intestinalis
2 Genes 4 Genes 1 Gene
2 isoforms 5 isoforms 2 isoform
1Gene 1 Gene 3 Genes
3 Isoforms 2 Isoforms 4 Isoforms
Isoforms
a.a.
Tissue expression
TnT-card TnT-st TnT-sm TnI-l TnI-s
304 275 253 229 182
TnC-3 TnC-1a TnC-1b
162 158 158
TnC-2
164
Cardiac muscle Larval striated muscle Adult smooth muscle Cardiac muscle Larval striated muscle Adult smooth muscle Larval striated muscle Larval striated muscle Adult smooth muscle Cardiac muscle Larval striated muscle
the two species, distinct differences are present in the number of genes encoding troponin components and the troponin isoforms generated (Table 1). Namely, in the case of H. roretzi, two genes and two protein isoforms for TnT [21], four genes and five protein isoforms for TnI [22] and one gene and two protein isoforms for TnC [23], while in the case of C. intestinalis, there is one gene and three protein isoforms for TnT [24], one gene and two protein isoforms for TnI [17,25] and three genes and four protein isoforms for TnC [26]. Adult smooth muscle and larval striated muscles of C. intestinalis use the same TnI isoform but different TnT and TnC isoforms, while smooth and striated muscles of H. roretzi use different TnT, TnC, and TnI isoforms. Characterization of the functional properties of these troponin components and their complex, has been achieved by preparing recombinant proteins based on the genome information in addition to preparation of native proteins from tissues. Interestingly, it was revealed that troponin of ascidian striated muscle functions as a Ca2+-dependent accelerator of actin–myosin interaction just as troponin of ascidian smooth muscle [6]. Functional variation of troponin during chordate evolution is summarized in Fig. 1B.
5
be obtained from the body wall. Another ascidian, C. intestinalis, can also be used but the amount of muscle tissue is much smaller in this animal. The outline of the procedure is illustrated in Fig. 3. First, dissect out fresh smooth muscle tissue from the body wall, and rinse the isolated tissue with a KMP solution (50 mM KCl, 10 mM phosphate buffer (pH 7.0), 1 mM MgCl2) containing 1 mM ethylenediamine tetra acetic acid (EDTA) and 1 mM 2-mercaptoethanol, several times. Since troponin is sensitive to heavy metal ions, the EDTA-containing solution is helpful for protecting troponin from inactivation by removing heavy metal ions that may be found in seawater. If necessary, the isolated muscle tissue can be stored in the same solution containing 50% glycerol at 20 °C for at least several months. The isolated tissue is homogenized in the KMP solution containing 1 mM EDTA with a tissue grinder (blender) or a Dounce homogenizer. The homogenate is centrifuged for 10 min at 14,000g. The supernatant that is to be discarded will be turbid because of the presence of polysaccharides. Take the pellet, suspend it in the same solution and centrifuge the mixture again. These procedures are repeated until the supernatant becomes transparent. The final pellet is suspended in the 3 volume of the KMP solution containing 6 mM ATP and 0.2 mM ethyleneglycolbis (aminoethyl ether)–N-N0 -tetra acetic acid (EGTA) and incubated with stirring for about 10 min at 4 °C. A KMT solution where 10 mM Tris(hydroxymethyl)aminomethane (Tris)–HCl buffer (pH 7.4 at 4 °C) is used in place of phosphate buffer is preferable at this step, if isolated thin filaments are used for examining their interaction with myosin by ATPase assay, to avoid contamination of inorganic phosphate in the thin filament fraction. Through this process, troponin-containing actin filaments are released from myosin filaments in the contractile apparatus. The suspension is centrifuged for 1 h at 70,000g to precipitate myosin filaments and the residue of contractile apparatus leaving actin filaments in the supernatant. The resultant supernatant is taken and subjected to centrifugation at 100,000g for 3 h. The resultant precipitate contains actin thin filaments that have tropomyosin and
2. Isolation of troponin from tissues The procedure for preparing troponin from vertebrate striated muscles has been established [14,15]. This procedure, however, may not be directly applicable for preparing troponin from invertebrate muscles. In the case of non-striated muscles such as smooth muscle, troponin-containing actin filaments are easily released from contractile structures under the conditions that makes muscle relaxed, namely by incubating tissue homogenates in the solution containing EGTA, a Ca2+-chelater, and ATP, because actin filaments are not tightly anchored to Z-disks as in striated muscles. Troponin-containing actin thin filaments can be isolated as follows and the isolated thin filaments are used as a good source for obtaining troponin. 2.1. Isolation of actin thin filaments Isolation of actin thin filaments from ascidian smooth muscle can be achieved as first described by Toyota et al. [27], with a slight modification of the method that was originally devised for molluscan smooth muscle [28]. Ascidian, H. roretzi, is an excellent material for this purpose, because a large amount of smooth muscle can
Fig. 3. Schematic representation of the procedure for isolation of native thin filaments and extraction of troponin and tropomyosin from the thin filaments.
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troponin in a considerably high purity (see Fig. 4). By applying slight modification of these procedures, native actin filaments have also been successfully isolated from the muscles of other invertebrate animals such as sea urchin [29], nematodes (Ascaris) [30], sea cucumber [31], earthworm (unpublished data), and tardigrade (water bear) [13]. 2.2. Extraction of troponin from the actin filaments Troponin and tropomyosin can be dissociated from the isolated actin filaments by incubating the actin filaments in 0.6 M KCl, 1 mM MgCl2 and 10 mM Tris–HCl buffer, pH 7.5 at 4 °C for 1–2 h at 25 °C (Figs. 3 and 5A). This method is based on the report by Spudich and Watt [32] that was devised for preparing tropomyo-
sin-free F-actin. Troponin–tropomyosin complex can be obtained in the supernatant leaving actin in the pellet by spinning the mixture at 100,000g for 3 h and then, salted out by the addition of saturated ammonium sulfate (pH 7.0) to 30–75% saturation. Troponin can be separated from tropomyosin by isoelectric precipitation of tropomyosin at pH 4.5 in the presence of 0.4 M LiCl. Namely, 1.2 M LiCl is added to the solution of troponin–tropomyosin complex to a final concentration of 0.4 M and then, the pH of the solution is adjusted to 4.5 with 0.2 N HCl. After 30 min, the solution is centrifuged for 15 min at 16,000g. The supernatant containing troponin is neutralized by gradual addition of diluted NaOH solution, and then, troponin can be precipitated by the addition of solid ammonium sulfate to 70% saturation (Fig. 5B, the lane marked TN). The precipitate, a complex of three troponin components, is dissolved and dialyzed against in 1 mM NaHCO3. Troponin complex is soluble in a low salt buffer solution at neutral pH such as 1 mM NaHCO3, or 10 mM Tris–HCl buffer (pH 7.4 at 4 °C).
2.3. Purification of ascidian troponin components
Fig. 4. Electron micrograph of native thin filaments that were isolated from ascidian (H. roretzi) body wall smooth muscle. The filaments were negatively stained with 2% uranyl acetate. Bar: 0.5 lm.
Ascidian three troponin components can be purified by applying the method described for vertebrate striated muscle troponin components [14] with a slight modification [5] (Fig. 5B). The complex of ascidian three troponin components is dialyzed twice or more against the solution containing 6 M urea, 50 mM glycine– NaOH buffer (pH 9.0) and 0.5 mM CaCl2 (termed Buffer A). This dialyzed sample is then centrifuged at 20,000g for 20 min to remove insoluble aggregate in the solution. Proteins in the supernatant are applied to an SP-Sephadex C-50 (Sigma) column equilibrated with Buffer A. Proteins are eluted by a linear gradient of NaCl. TnI–TnC complex is eluted at void volume and TnT at about 0.17 M NaCl. The TnI–TnC complex is dialyzed against 6 M urea, 50 mM Tris–HCl buffer (pH 8.0 at 4 °C) containing 2 mM EGTA (Buffer B) and separated into each component by SP-Sephadex C50 column chromatography. Under this condition, TnC becomes free from TnI because of the absence of Ca2+. TnC is eluted at void volume and TnI at about 0.15 M NaCl of the linear NaCl gradient. TnC is further purified by chromatography on a DEAE–cellulose column equilibrated with Buffer B. TnC is eluted by a linear NaCl gradient as a single peak at about 0.18 M NaCl. The purified TnC is dialyzed against 1 mM NaHCO3 (or 10 mM Tris–HCl, pH 7.4), while TnT and TnI are against 0.4–0.5 M NaCl containing 1 mM NaHCO3 (or 10 mM Tris–HCl, pH 7.4) because they are insoluble in a solution of low salt concentration. The advantage of using NaHCO3 is that this chemical makes the solution slightly alkaline pH and does not disturb assays of protein concentration. The purified troponin components can be simply frozen and stored at 20 °C to 80 °C without loosing activity.
3. Preparation of recombinant troponin components
Fig. 5. SDS–PAGE patterns of each fraction that was obtained during the isolation process of ascidian (H. roretzi) regulatory proteins. (A) Extraction of troponin– tropomyosin from isolated native thin filaments. (a): native thin filaments, (b) and (c): 100,000g supernatant and precipitate from 0.6 M KCl-treated thin filaments (see detail in the text), respectively. (B) Isolated troponin complex (TN) and three troponin components, TnT, TnI and TnC. A, actin; TM, tropomyosin; T, TnT; I, TnI; C, TnC. The protein marked by asterisk is HR-29 [44] that is always contaminating the thin filament fraction from ascidian, H. roretzi. This protein is not detectable in the thin filament fraction of another ascidian, C. intestinalis.
In most of invertebrate animals, it is not easy to obtain sufficient amounts of muscle tissue to prepare troponin as for the ascidian H. roretzi. On the other hand, nowadays genomic and/or cDNA sequence information is available for troponin components in a variety of invertebrate animals. Multiple isoforms of troponin components have been detected in many invertebrate animals. These isoforms can be prepared only as recombinant proteins in E. coli or other expression systems. In our experience with ascidian and amphioxus, recombinant troponin components show the same functional properties as native proteins that have been isolated from muscle tissue. In the following, the preparation procedures that we used to obtain recombinant ascidian and amphioxus troponin components are described.
T. Obinata, N. Sato / Methods 56 (2012) 3–10
3.1. Vector construction of amphioxus troponin Amphioxus TnI (BbTnI, AB035664 for GenBank) and the TnC (BbTnC, AB175946) cDNAs were cloned from Branchiostoma belcheri (B. belcheri) adult whole body RNA by reverse transcription (RT)-PCR. B. belcheri TnT (BbTnT, AB543715) cDNA was obtained from a cDNA library that was produced from the nerve cord and muscle tissues [33]. The coding regions of BbTnC, BbTnI and BbTnT cDNA were obtained by PCR using specific primer sets (See reference [19]). The cDNA fragment of BbTnC was cloned into pBluescript SK(-) (Stratagene, Cedar Creek, TX), containing synthesized nucleotides that encode 8 histidine residues, and then the DNA fragment encoding BbTnC containing a His-tag at the C-terminus was subcloned into the expression vector pGEX4T-1 that carries an N-terminal glutathione S-transferase (GST)-tag coding sequence followed by a thrombin cleavage recognition site (GE Healthcare UK Ltd., UK). The cDNA fragment encoding BbTnI was cloned into pQE-30 (Qiagen, Valencia, CA). The cDNA fragment encoding BbTnT was cloned into the expression vector pGEX6P-1 that carries an N-terminal GST-tag coding sequence followed by a PreScission cleavage recognition site (GE Healthcare). B. belcheri troponin expression vectors were transformed to E. coli strain BL21 (DE3) pLysS (Novagen, Darmstadt, Germany). Expression of amphioxus troponin components was induced in E. coli with 1 mM IPTG (isopropyl b-D-thiogalactopyranoside). 3.2. Purification of recombinant BbTnI, BbTnC and BbTnT For purification of recombinant BbTnI with a His-tag at the Nterminus, the insoluble fraction of E. coli lysates was dissolved in 6 M urea solution containing 10 mM imidazole, 0.5 M NaCl, 0.5 mM DTT, and 20 mM phosphate buffer (pH 7.4), and then dialyzed against the same buffer without urea. The solubilized protein was applied to a HisTrap affinity column (GE Healthcare Ltd., UK) in a His-buffer (10 mM imidazole, 20 mM phosphate buffer, and 0.5 M NaCl, pH 7.4). Purified BbTnI was eluted using a gradient of 10–500 mM imidazole in 20 mM phosphate buffer (pH 7.4) containing 0.5 M NaCl. The protein was dialyzed against 0.5 M KCl containing 10 mM sodium bicarbonate and concentrated with Aquacide II (Calbiochem, La Jolla, CA) for analysis. For purification of recombinant BbTnC, the supernatant of E. coli lysates that contained BbTnC with a GST-tag at the N-terminus and a His-tag at the C-terminus (GCH protein) was applied to a glutathione Sepharose 4B (GS) column (GE Healthcare) that had been pre-washed with the His-Buffer. GCH protein was allowed to bind
7
to the GS column. After that, the protein was incubated with thrombin (GE Healthcare) at a 1:100 dilution (equivalent to 50 U) overnight at 4 °C. BbTnC was eluted and immediately applied to a HisTrap affinity column in the same buffer conditions for further purification. Purified BbTnC (with a His-tag) was eluted from the column and prepared for analysis in the same manner as BbTnI. One typical example of expression and purification of recombinant amphioxus TnC is shown in Fig 6. The purification procedure for recombinant BbTnT was almost the same as that for BbTnC, except for the following changes. For the GS column, the protein was incubated with a 1:100 dilution (50 U) of PreScission (GE Healthcare) overnight at 4 °C. BbTnT was then eluted with 10 mM phosphate buffer (pH 7.4) containing 0.15 M NaCl and directly absorbed on an ion exchange HiTrap column (GE Healthcare) for further purification. BbTnT was eluted from the column with a gradient of 0.15–0.5 M NaCl in 10 mM phosphate buffer (pH 7.4). 3.3. Vector construction of ascidian troponin C. intestinalis TnI cDNA (TnI-s; U55261 for GenBank, KH.C11.673.v1 for Ghost Database) and TnC cDNA (TnC-1b; AK112490, KH.C8.251.v2) were provided by C. intestinalis genomic and cDNA resources (Kyoto University, Japan). The coding regions of TnI and TnC cDNA were obtained by PCR using specific primer sets (see Ref. [6]). The cDNA fragments of the TnI and TnC were inserted into pBluescript SK(-) containing synthesized nucleotides encoding eight histidine residues following a termination codon. The cDNA fragments of TnI and TnC carrying a C-terminal His-tag coding sequence were then subcloned into the expression vector pET3a (Novagen, Darmstadt, Germany). In C. intestinalis, different TnT isoforms are expressed in larval striated muscle and adult smooth muscle; they were termed TnT-st (striated muscle type) and TnT-sm (smooth muscle type), respectively, in this paper. To obtain these isoforms, C. intestinalis TnT cDNA (TnT-st; KH.C4.57.v3 and TnT-sm; KH.C4.57.v4) were cloned from C. intestinalis body-wall muscle RNA by RT-PCR. The coding regions of TnT-st and TnT-sm were obtained by PCR using the primer sets (see Ref. [6]). The PCR products were then inserted into pBluescript KS(-), containing synthesized nucleotides encoding eight histidine residues. These cDNA fragments carrying an N-terminal His-tag coding sequence were subcloned into the expression vector pET3a. All C. intestinalis troponin expression vectors were transformed to E. coli strain BL21(DE3) pLysS. Expression of ascidian troponin components was induced in E. coli with 1 mM IPTG. 3.4. Purification of recombinant TnI, TnC and TnT of C. intestinalis
Fig. 6. Expression and purification of recombinant amphioxus TnC. Expression of amphioxus TnC (BbTnC) was induced in E. coli with IPTG. Whole extract (WE; lane 1) were centrifuged to obtain the supernatant (S; lane 2) and precipitate (P; lane 3). The initial translated BbTnC containing both a GST-tag and His-tag (GCH) in the supernatant was bound to glutathione beads and then cleaved with thrombin. BbTnC (CH) in the eluate (G; lane 4) was further purified with a HisTrap affinity column (H; lane 5). The molecular weight markers are shown on the left side.
For the purification of recombinant C. intestinalis TnI and TnC, the soluble fractions of bacterial lysates were applied to a HisTrap affinity column in the His-buffer. Proteins were eluted using a gradient of 50 to 500 mM imidazole in 20 mM phosphate buffer (pH 7.4) containing 500 mM NaCl. TnC was further dialyzed against Q-buffer (20 mM NaCl, 25 mM Tris–HCl, 1 mM DTT, pH 8.0) and then applied to an anion exchange Econo-Pac Q cartridge (BioRad Laboratories). The purified TnC was obtained by a gradient of 40–500 mM NaCl. For the purification of recombinant TnT-st and TnT-sm, the insoluble fraction of bacterial lysates were dissolved in the Hisbuffer containing 6 M urea, and then dialyzed against the same buffer without urea. The solubilized proteins were subjected to HisTrap affinity chromatography in His-buffer. Purified proteins were eluted using a gradient of 10 to 500 mM imidazole in 20 mM phosphate buffer (pH 7.4) containing 0.5 M NaCl.
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4. Functional assay of troponin components 4.1. Interaction of troponin components and specific binding to other myofibrillar proteins 4.1.1. Ca2+-responsiveness of TnC and Ca2+-dependent interaction of TnC and TnI To clarify whether troponin components function in a Ca2+dependent manner, an essential property of troponin, Ca2+-responsiveness of TnC and Ca2+-dependent TnI–TnC interaction can be examined by electrophoresis. The Ca2+-dependent mobility shift on SDS–PAGE gels is a pronounced property of TnC [34]. SDS–PAGE is carried out using a 13.5% polyacrylamide gel and a discontinuous Tris–glycine buffer system. To examine the Ca2+-dependent mobility shift of TnC on SDS–PAGE, 2 mM EGTA or 2 mM CaCl2 is added to the TnC sample at the final concentration. After SDS-treatment, the specimens are subjected to SDS–PAGE. In the presence of either EGTA or CaCl2, purified TnC shows a single band on an SDS–PAGE gel. However, TnC in the presence of CaCl2 migrates faster on the gel than in the presence of EGTA (or in the absence of Ca2+). The results obtained with ascidian (H. roretzi) TnC are shown in Fig. 7A. This phenomenon has been observed for TnC in another ascidian (C. intestinalis) [6], amphioxus [19] and other animals [34] and confirms that the purified ascidian or amphioxus recombinant TnC has Ca2+-binding ability. This Ca2+-binding mobility shift is attributable to the conformational change of TnC following the binding of Ca2+. To examine the Ca2+-dependent interaction of TnI and TnC, alkaline-urea PAGE is carried out in the presence or absence of Ca2+ [35]. In this electrophoresis, 8% polyacrylamide gels containing 8 M urea, 25 mM Tris and 80 mM glycine, pH 8.3 are used. Either 10 mM EGTA or 2 mM CaCl2 at the final concentration is added to the mixture of TnI and TnC or troponin complex that is dissolved in a Tris–glycine buffer containing 8 M urea as in the electrophoresis gel. Urea concentration can be decreased to 6 M. The results obtained with ascidian (H. roretzi) TnI and TnC are shown in Fig. 7B. As shown in this figure, in the presence of Ca2+, a slower migrating band (marked I–C) is observed (the right lane, Ca2+) and in the absence of Ca2+, a faster migrating band (marked
Fig. 7. Ca2+-responsiveness of TnC and Ca2+-dependent interaction of TnC and TnI. (A). Ca2+-dependent mobility shift of TnC on SDS–PAGE. Difference in mobility of TnC is observed in the presence of Ca2+ and in the presence of EGTA on SDS–PAGE. Purified ascidian (H. roretzi) TnC in an SDS buffer solution with 2 mM CaCl2 (right lane) showed a single band with faster mobility than the same purified TnC in an SDS buffer solution with 2 mM EGTA (left lane) (T. Nogami and T. Obinata, unpublished data). (B). Ca2+-dependent interaction of TnI and TnC in alkaline urea-PAGE analysis. A binding assay of ascidian (H. roretzi) TnI (TnI) with ascidian (H. roretzi) TnC (TnC) was carried out in the presence of Ca2+ (2 mM CaCl2, Ca2+) or in the absence of Ca2+ (10 mM EGTA, EGTA). The upper band (I–C) corresponds to the TnI–TnC complex in the presence of Ca2+ and the lower band (C) to TnC (according to Endo and Obinata [5]).
C) is observed (the left lane, EGTA). The slower migrating band corresponds to the TnI–TnC complex that is formed in a Ca2+-dependent manner and the faster migrating band is TnC alone. Under these electrophoresis conditions, TnI alone does not move in the gel regardless of the presence or absence of Ca2+, because it is a basic protein. These behaviors of TnC and TnI have been observed in vertebrate [35], ascidian (C. intestinalis) [6] and amphioxus proteins [19]. Thus, we can confirm that purified recombinant TnI and TnC of ascidian [6] and amphioxus [19] can bind to each other in the characteristic manner of chordate troponin components. 4.1.2. Binding of TnT to tropomyosin TnT is the tropomyosin-binding component of the troponin complex. Therefore, whether an isolated protein has this expected property of TnT can be tested by examining its binding to tropomyosin. The TnT–tropomyosin binding can be examined by different ways. First, binding of TnT to tropomyosin that is associated with F-actin can be examined by a co-sedimentation assay. Purified tropomyosin and F-actin are pre-incubated in a solution containing 0.1 M KCl and 1 mM MgCl2, then ‘‘TnT’’ is added to the actin–tropomyosin complex and incubated for 1 h or so. The mixture is then centrifuged at 350,000g for 30 min and the precipitate is subjected to SDS–PAGE to check whether ‘‘TnT’’ is co-precipitated with the F-actin–tropomyosin complex. Secondly, binding of TnT to tropomyosin can be examined by electron microscopy. In this analysis, ascidian or rabbit tropomyosin with or without ascidian TnT is dialyzed against 1 M KCl in 50 mM Tris–HCl buffer, pH 8.0 at 4 °C and then, dialyzed against 50 mM MgCl2 in 50 mM Tris–HCl buffer, pH 8.0 at 4 °C to form paracrystals of tropomyosin. In the absence of TnT, the electron microscopic image of tropomyosin paracrystals consists of light staining bands (26 nm in width) and dark staining bands (14 nm in width) that alternate to give a periodicity of 40 nm as described by Cohen and Longly [36]. When the tropomyosin paracrystals were formed in the presence of ascidian TnT, however, the band patterns of the paracrystals were markedly transformed as the result of TnT-binding to tropomyosin (Fig. 8). Namely, the presence of TnT resulted in the appearance of lighter staining zone (marked by triangles in the figure) in the center of the light staining bands of the paracrystals with the 40 nm periodicity. This remarkable phenomenon was first demonstrated with rabbit TnT and rabbit tropomyosin by Nonomura et al. [37].
Fig. 8. Electron micrographs of magnesium paracrystals of rabbit tropomyosin with (B) or without (A) ascidan (H. roretzi) smooth muscle TnT. The paracrystals were negatively stained with 2% uranyl acetate. TnT bound to the center of the light band (marked by triangles) of the tropomyosin paracrystals, just as observed with rabbit TnT and rabbit tropomyosin paracrystals [37]. The photographs were taken by Dr. T. Endo [5]. Bar: 0.1 lm.
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Troponin is a Ca2+-dependent regulator of muscle contraction or actin–myosin interaction. To assess this fundamental property of troponin, troponin is combined with tropomyosin, and the effects of troponin–tropomyosin complex on actomyosin-Mg2+-ATPase activity are measured in the presence of either CaCl2 or EGTA. Rabbit or chick reconstituted actomyosin, a mixture of purified actin and myosin, can be used for the assay. Myosin, actin and tropomyosin are prepared as described [38–40]. Actin and tropomyosin can also be prepared from body wall muscle of ascidian H. roretzi, as from vertebrate muscle tissue. To obtain ascidian myosin, an improved method as described [41] is required, because ascidian myosin tends to become insoluble and subsequently the ATPase activity is decreased, when the precipitation and solubilization of myosin are repeated by changing ionic strength during preparation. In the improved method, myosin is extracted from the homogenate of ascidian body wall muscle for 1 h at 0 °C with a pyrophosphate solution containing 40 mM Na-pyrophosphate, 2 mM MgCl2, 2 mM EGTA, 5 mM ATP, 2 mM 2-mercaptoethanol, 10% glycerol and 0.1 M sucrose, pH 7.0. The mixture is centrifuged for 3 h at 72,000g. Myosin is, then, purified from the supernatant by DEAE cellulose column chromatography. Myosin is eluted with a linear NaCl gradient from 0 to 0.2 M NaCl in the presence of pyrophosphate (See detail in reference [41]). In a standard assay to examine the effects of troponin on actin– myosin interaction, myosin (0.4 lM), actin (1.6 lM), tropomyosin (0.23 lM), and the troponin components (TnT: 0.23 lM, TnI: 0.23 lM, TnC: 0.23 lM) are mixed in a solution containing 40– 50 mM KCl, 1 mM MgCl2, 20 mM Tris–HCl, pH 7.5 with 0.1 mM CaCl2 or 1 mM EGTA. In other words, the actin:tropomyosin:troponin molar ratio in the mixtures is 7:1:1. Relative proportion of troponin in the mixture can be increased to see the effects of troponin more obviously: After pre-incubation for 5 min at 25 °C, ATP (1 mM at final concentration) is added to the mixture to initiate ATPase reaction and allowed to further incubate for about 10 min. The reaction is stopped by adding 5% trichloroacetic acid (TCA) at final concentration. Inorganic phosphate (Pi) liberated as the result of ATP hydrolysis by actomyosin ATPase is measured according to Taussky and Shorr [42]. In this assay method, 1/4 volume of 4% sodium molybdate in 2 N H2SO4 is added to the test
(%)
+Ca2+ 0.5 0.4
+Ca2+
0.3
+EGTA
0.1 0
0
200 150 100 50
M+A+TM (Control)
M+A+TM +Tst +I +C
M+A+TM +Tsm +I +C
10 15 Time (min)
20
(B) Amphioxus
(%) 200 150 100 50 0
0
5
Fig. 10. Effects of ascidian (H. roretzi) troponin on the superprecipitation of reconstituted rabbit actomyosin. Reaction mixtures contain 80 mM KCl, 1 mM MgCl2, 20 mM Tris–HCl buffer (pH 7.5 at 4 °C), 0.2 mM ATP, 0.1 mM EGTA (open circle and open triangle) or 0.1 mM CaCl2 (closed circle and closed triangle), 300 lg/ ml of rabbit myosin, 100 lg/ml of rabbit actin, 40 lg/ml of ascidian tropomyosin and 40 lg/ml of ascidian troponin, when added. Reaction temperature is 20 °C. Superprecipitation was initiated by adding ATP at 0 time point. Closed and open triangle: actin + myosin + tropomyosin; Closed and open circle: actin + myosin + tropomyosin + troponin.
Actomyosin-ATPase Activity (relative value)
250
+EGTA
0.2
1 mM EGTA
Ascidian Actomyosin-ATPase Activity (relative value)
0.6
0.1 mM CaCl2
(A)
300
sample solution. After quick mixing, 1/16 volume of 40% FeSO4 in 1 N H2SO4 is added. After another quick mixing, the color of the solution is measured with a spectrophotometer at 660 nm and then, Pi (lmoles) in the solution is calculated. The ATPase activity is shown as lmoles Pi liberated/min/mg myosin. The value for the activity of actin–myosin without troponin under this condition is usually 0.2–0.3. Typical examples of the results showing the effects of troponin (or a complex of TnT, TnI and TnC) on actomyosin ATPase activity in the presence or absence of Ca2+ are shown in Fig. 9. Ascidian troponin enhances the ATPase activity in a Ca2+-dependent manner (Fig. 9A) (See detail in Ref. [6]), while amphioxus troponin suppresses the ATPase activity in the absence of Ca2+ (Fig. 9B) (See detail in Ref. [19]).
A660
4.2. Ca2+-dependent regulation of actin–myosin interaction
M+A+TM (Control)
M+A+TM +native TN
M+A+TM +T +I +C
Fig. 9. Effects of ascidian (C. intestinalis) and amphioxus troponin on the Mg2+-ATPase activity of reconstituted rabbit actomyosin. (A): The effects of the ascidian recombinant troponin complex, combinations of striated muscle-type (Tst, I and C) and smooth muscle-type (Tsm, I and C) forms, on actomyosin ATPase. (B): The effects of the amphioxus native troponin (native TN) and recombinant troponin complex (TnT, TnI and TnC) on actomyosin ATPase. The actin, tropomyosin, TnT, TnI and TnC were incubated roughly in a molar ratio of 4:1:1:1:1 and then mixed with myosin. In the control mixture (control), actin, myosin and tropomyosin were combined without any troponin components. The gray and white bars represent the reaction mixture with 0.1 mM CaCl2 or 1 mM EGTA, respectively. M, myosin; A, actin; TM, tropomyosin; Tst, ascidian striated muscletype TnT; Tsm, ascidian smooth muscle-type TnT; T, amphioxus TnT; I, TnI; C, TnC. The salts concentrations of the reaction mixtures are 50 mM KCl, 1 mM MgCl2, 20 mM Tris– HCl buffer (pH 7.5 at 4 °C), 1 mM ATP, 0.1 mM CaCl2 or 1 mM EGTA.
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Acknowledgments We thank Dr T. Endo in Chiba University for creating the electron micrographs used in this paper. They also thank the Ministry of Education, Science and Culture, Japan for financial support. References
Fig. 11. Detection of TnC along isolated ascidian native thin filaments by immuno electron microscopy. Isolated ascidian (H. roretzi) thin filaments as shown in Fig. 4 were incubated with the antibody raised against ascidian TnC in 0.1 M KCl, 1 mM MgCl2, 10 mM phosphate buffer, pH 7.0 for 30 min at 20 °C. The aggregates formed were collected by centrifugation, washed with 0.1 M KCl. 10 mM Tris–HCl buffer, pH 7.5 at 4 °C, followed by staining with 2% uranylacetate, and then observed under an electron microscope. Cross striations with about 38 nm intervals were formed along the thin filaments, indicating location of TnC along actin filaments. The photographs were taken by Dr. T. Endo. Bar: 0.2 lm.
[1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11]
Another way of examining the effect of troponin on actin–myosin interaction is monitoring of superprecipitation. Actomyosin dissolved in dilute KCl concentration shows a marked flocculent precipitation when ATP is added. This phenomenon, so-called superprecipitation, is coupled with ATP hydrolysis by actomyosin ATPase. Occurrence of superprecipitation can be monitored as the change in turbidity (absorbance at 660 nm) of an actomyosin solution. The reaction mixture for assay including actin, myosin, tropomyosin and troponin (or its component) is the same as in the ATPase assay described above, except that the KCl concentration is slightly higher (0.08–0.1 M) and ATP concentration is lower (0.2 mM) than in the ATPase assay. Immediately after addition of ATP, monitoring of the absorbance at 660 nm is started. A typical example of the assay is shown in Fig 10. Troponin causes a remarkable difference in time course of the turbidity change of the actomyosin solution between the presence and absence of Ca2+. An advantage of this assay is that smaller amounts of proteins are needed for this assay in comparison with the ATPase assay.
[12] [13] [14] [15] [16]
5. Intracellular localization of troponin
[25]
Troponin is involved in actin-linked regulation of muscle contraction or actin–myosin interaction. Therefore, it is important to confirm the location of troponin or each troponin component along actin filaments in cells. In order to achieve this, antibodies that specifically recognize troponin components are indispensable. If purified troponin components are obtained, polyclonal or monoclonal antibodies can be prepared by standard procedures. Specificity of antibodies needs to be carefully checked by Western blotting. Sometimes, antibodies recognize the counterparts of the animal species other than the animals that were used for preparing the immunogens, but we must always keep in mind that they may interact with other proteins non-specifically. Co-localization of actin and troponin components can be visualized by dual staining of muscle tissues with rhodamine–phalloidin and anti-troponin antibodies [10,13]. Location of troponin along actin filaments can be directly confirmed by immunoelectron microscopy by staining isolated native actin filaments with anti-troponin antibody as described by Ohtsuki [43] and Endo and Obinata [5] (Fig. 11).
[17] [18] [19] [20] [21] [22] [23] [24]
[26] [27] [28] [29] [30] [31] [32] [33] [34] [35] [36] [37] [38] [39] [40] [41] [42] [43] [44]
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