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Localization of projectin in locust flight muscle
Comparative Biochemistry and Physiology Part B 136 (2003) 419–423
Localization of projectin in locust flight muscle Jinen Shimamuraa, Koscak Maruyamab, Sumiko Kimuraa,* a
Department of Biology, Faculty of Science, Chiba University, Chiba 263-8522, Japan b National Center for University Entrance Examinations, Tokyo 153-8501, Japan
Received 11 March 2003; received in revised form 18 July 2003; accepted 21 July 2003
Abstract Projectin is a giant filamentous protein of arthropod striated muscle. By using immunofluorescence microscopy, projectin was shown to span between the I band and the A band in locust (Locusta migratoria) flight muscle sarcomeres. The N- and C-terminal regions of projectin molecules were localized in the I band and A band, respectively. This observation explains the controversial reports of previous studies that projectin is localized either in the I band or in the A band of locust flight muscle sarcomeres. It is also observed that the N-terminal region of projectin is located in the I band of locust leg muscle sarcomeres. 䊚 2003 Elsevier Inc. All rights reserved. Keywords: Projectin; I-connectin; Locust; Flight muscle; Leg muscle; Mini-titin; Connectin; Titin; Insect
1. Introduction Projectin is a high molecular weight filamentous protein in arthropod striated muscle. The entire primary sequence of projectin was recently elucidated for Drosophila melanogaster (approx. 9210 residues, approx. 1 MDa) (Southgate and AymeSouthgate, 2001) and for crayfish claw closer muscle (8625 residues; 962 634 Da) (Oshino et al., 2003). The kinase domain of locust flight muscle projectin has been shown to act as a protein ¨ kinase (Weitkamp et al., 1998; Fahrmann et al., 2002). Projectin was first discovered as a C (connecting)-filament linking the Z line to the tip of the thick filament in asynchronous flight muscle of the honeybee (Saide, 1981). Since then projectin has been regarded as being localized in the I band of asynchronous flight muscle sarcomeres of D. *Corresponding author. Fax: q81-43-290-2811. E-mail address: [email protected] (S. Kimura).
melanogaster (Saide et al., 1989) and Lethocerus indicus (giant water bug) (Lakey et al., 1990). In contrast, projectin has been shown to be localized in the A band of synchronous flight, leg and larval muscles (Saide et al., 1990; Vigoreaux et al., 1991; cf. Ayme-Southgate et al., 2000). Conflicting observations have been reported for the localization of projectin in locust synchronous flight muscle. On the one hand, Nave and Weber (1990) described that mini-titin (projectin in this case) was localized in the I band using polyclonal antibodies against mini-titin. On the other hand, Weitkamp et al. (1998) reported that gold particles bound to polyclonal antibodies against locust projectin were distributed only in the A band and that they bound to thick filaments freed from locust flight muscle sarcomeres. However, Weitkamp et al. (1998) noted that projectin was localized in the I band of asynchronous flight muscle sarcomeres of the blowfly, Phormia terrae novae. To clarify these contradictory observations, we
1096-4959/03/$ - see front matter 䊚 2003 Elsevier Inc. All rights reserved. doi:10.1016/S1096-4959Ž03.00252-5
420
J. Shimamura et al. / Comparative Biochemistry and Physiology Part B 136 (2003) 419–423
describe here immunofluorescence localization of projectin in locust flight muscle using two kinds of antibodies raised against the N- and C-terminal regions of projectin. In the present work, the Nterminal region of projectin was shown to be located in the I band of locust flight muscle as well as of locust leg muscle. 2. Materials and methods 2.1. SDS gel electrophoresis and immunoblot tests A piece of freshly excised locust (L. migratoria) flight muscle was dissolved in 5 volumes of an SDS solution containing 10% SDS, 40 mM dithiothreitol, 10 mM EDTA and 100 mM Tris– HCl at pH 8.0, boiled for 3 min and clarified by centrifugation at 15 000=g for 10 min. SDS gel electrophoresis was carried out according to the method of Laemmli (1970) using 2–10% polyacrylamide gradient gels. Electrically transferred nitrocellulose sheets were treated with polyclonal antibodies, PcNIG, raised against the N-terminal region (229 amino acids (1–229; Ig 1qIg 2), molecular weight: 25 595) of crayfish claw closer muscle projectin (Oshino et al., 2003) and monoclonal antibody, Mc3B9, reactive with the C-terminal region of projectin (Hu et al., 1990; Oshino et al., 2003). The secondary antibodies used were horseradish peroxidase-conjugated anti-rabbit or anti-mouse Ig (DAKO). 2.2. Immunofluorescence microscopy Freshly excised muscle fibers of locust flight muscle were fixed at a given sarcomere length by a cold relaxing solution containing 3.5% formalin and 0.5% Triton X-100. After blocking with 1% bovine serum albumin (BSA), suspended myofibrils were treated with the primary antisera for 14 h at 4 8C; PcNIG was diluted 10 times with 0.15 M NaCl and 20 mM sodium phosphate buffer at pH 7.2; Mc3B9 was used without dilution. After washing with PBS several times, myofibrils were treated with FITC-labeled anti-rabbit or anti-mouse Ig (Cappel, West Chester, PA) for 2 h at room temperature. Immunofluorescence observations were performed as described previously (Manabe et al., 1993) with a Zeiss Axioskop 2 fluorescence. The antibody epitope spacing across the M line in a sarcomere was measured and the distance
Fig. 1. SDS gel electrophoresis patterns and immunoblot tests of locust flight and leg muscles projectin. (a) SDS gel electrophoresis patterns. 1, rabbit back muscle; 2, crayfish claw closer muscle; 3, locust flight muscle; 4, locust leg muscle. (b) Immunoblot tests. 1, Amido Black stain (locust flight muscle); 2, treated with PcNIG (locust flight muscle); 3, treated with Mc3B9 (locust flight muscle); 4, Amido Black stain (locust leg muscle); 5, treated with PcNIG (locust leg muscle); 6, treated with Mc3B9 (locust leg muscle). Electrophoresed on 2–10% polyacrylamide gradient gels. Con, titinyconnectin; Neb, nebulin; MHC, myosin heavy chain; I-con, I-connectin; P, projectin.
(sarcomere lengthythe epitope spacing) was divided by two to obtain the Z line–epitope distance in a half sarcomere. The distance between the epitope and the A–I junction was calculated from the width of the A band and the epitope spacing within a sarcomere. 3. Results and discussion 3.1. Immunoblot tests The SDS gel electrophoresis pattern depicted in Fig. 1a showed two distinct bands corresponding to projectin in locust flight and leg muscles (Fig. 1a3,a4). The upper band of locust flight muscle projectin corresponded to the main band of crayfish claw closer projectin (Fig. 1a2) and to the upper band of locust leg muscle projectin (Fig. 1a4). The lower band of locust projectin was much more abundant in flight muscle than in leg muscle (Fig. 1a3,a4). Immunoblot analyses were done using PcNIG against the N-terminal region of projectin (Oshino et al., 2003) and Mc3B9 reactive with the Cterminal region of projectin (Hu et al., 1990; Oshino et al., 2003). Fig. 1b shows that PcNIG reacted only with the upper band of locust projectin, while Mc3B9 reacted with both the upper and lower bands of locust projectin (cf. Fig. 1b2 vs. b3; Fig. 1b5 vs. b6).
J. Shimamura et al. / Comparative Biochemistry and Physiology Part B 136 (2003) 419–423
Fig. 2. Immunofluorescence localization of projectin in locust flight and leg muscles sarcomeres. (a), (b), locust flight muscle; (c), (d), locust leg muscle; (a), (c), treated with PcNIG; (b), (d), treated with Mc3B9. (a), (b), (c), (d), relaxed muscle sarcomeres; (a’), (b’), (c’), (d’), stretched muscle sarcomeres. Upper, phase contrast image; lower, fluorescence image. Bar, 5 mm.
The homology of the Ig 1qIg 2 region (229 amino acid residues; antigen for PcNIG) of crayfish projectin has high identity of 62.6% to the same region of D. melanogaster (insect) projectin. In addition, there are four segments consisting of the same sequence of over ten amino acids in both the N-terminal regions. In contrast, the homology of the Ig 1qIg 2 region of crayfish projectin has identity of only 25.4% at the best to the C-terminal region of Drosophila projectin (6658 amino-acid residues). The PcNIG is regarded to recognize the N-terminal region of both locust flight and leg muscle projectins. Therefore, it is suggested that the lower band of locust projectin corresponds to an N-terminal region-deficient projectin. The lower band could be an isoform of projectin lacking the N-terminal region as described for D. melanogaster (Southgate and Ayme-Southgate, 2001). 3.2. Immunofluorescence microscopy of locust flight muscle Immunofluorescence microscopy using the polyclonal antibody (PcNIG) showed that the N-ter-
421
minal region of projectin was clearly localized in the I band of both relaxed and stretched flight muscle sarcomeres (Fig. 2a,a’). The Z line was not fluorescent. However, the epitope recognized by the monoclonal antibody (Mc3B9) in the Cterminal region of projectin was localized in the A band near the A–I junction area (Fig. 2b,b’). Faint fluorescent areas were also seen in the A band, except in the center of the A band. The distance from the center of the Z line to the center of the main fluorescent band due to PcNIG or to Mc3B9 binding was plotted at various I band lengths (Fig. 3). The distance from the A–I junction to the Mc3B9 epitope was approximately 0.05 mm within the A band indicating that the Cterminal region of projectin was certainly localized in the A band (Fig. 3). Although the band positions were considerably scattered, no systematic change occurred due to sarcomere stretch. However, the distance from the A–I junction to the PcNIG epitope was approximately 0.23 mm within the I band and thus the N-terminal region of projectin was evidently localized in the I band. A single projectin molecule, approximately 0.3 mm long (Hu et al., 1990; Nave and Weber, 1990), may span the I band to the A band in locust flight muscle. It is suggested that projectin molecules may be associated with each other side by side, but in a staggered pattern (Ayme-Southgate et al., 2000). Now the discrepancy between previous studies for the localization of projectin in locust flight muscle may be explained. Nave and Weber (1990) might have used antibodies against the N-
Fig. 3. Distances between the Z line and the epitopes for antiprojectin antibodies in locust flight muscle sarcomeres at various sarcomere lengths. d, epitope for polyclonal antibody against the N-terminal region of projectin (PcNIG); m, epitope for monoclonal antibody against the C-terminal region of projectin (Mc3B9). Solid line, distance between the A–I junction and the Z line.
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terminal regions of projectin and, therefore, these authors localized them in the I band. In contrast, Weitkamp et al. (1998) showed projectin’s localization in the A band because antibodies against the C-terminal regions of projectin might have been employed. A question arises whether the N-terminal region of projectin is somehow positioned or is free in the I band of locust flight muscle sarcomeres. The N-terminus of projectin filament does not extend to the Z line. One possibility is that the N-terminus binds to the elastic filament, invertebrate connectin (I-connectin), linking the Z line and the thick (myosin) filament in the A band as observed with crayfish closer sarcomeres (Fukuzawa et al., 2001). Although this 2 MDa I-connectin was not found by the SDS PAGE (Fig. 1a3), a faint band was observed in a large dose-loaded gel (data not shown). Note that a distinct band of I-connectin, lower than the crayfish band, was recognized in the SDS PAGE pattern of locust leg muscle (Fig. 1a2,a4). Another possibility is that the projectin binds to the thin (actin) filament in the I band. The interaction of projectin with the thin filaments has been reported (Weitkamp et al., 1998). We made a preliminary experiment using the Far Western method with anti-actin antibodies that showed a weak binding of recombinant NIG peptide to F-actin (data not shown), and, therefore, the N-terminal end of the projectin filament may weakly attach to the actin filament in the I band. In contrast, it is reported that projectin definitely interacts with myosin (Weitkamp et al., 1998; Ayme-Southgate et al., 2000). Thus, projectin bound to myosin in the thick filament is regarded as a regulator of the function of myosin. 3.3. Immunofluorescence microscopy of locust leg muscle In locust leg muscle sarcomeres, PcNIG binding resulted in a fluorescent band in the I band (Fig. 2c) and moved away from the Z line when stretched (Fig. 2c’). This observation suggests that the N-terminal region of projectin is bound to some elastic filament, possibly I-connectin, in the I band of leg muscle sarcomeres. However, the Mc3B9 stained the both half regions of the A band except for the center region (Fig. 2d) and did not appreciably change upon stretch of sarcomeres (Fig. 2d’). The present work clearly indicates that the N-terminal region of projectin is also extended
into the I band in leg muscle sarcomeres as seen in flight muscle. Acknowledgments We thank Dr S. Tanaka of the Institute of Insect and Animal Sciences, Tsukuba for his kind supply of live locusts. This work was supported by a Grant-in-Aid for Scientific Research (B) of the Ministry of Education, Culture, Sports, Science and Technology of Japan. References Ayme-Southgate, A., Southgate, R., McEliece, M.K., 2000. Drosophila projectin: a look at protein structure and sarcomeric assembly. Adv. Exp. Med. Biol. 481, 251–264. ¨ Fahrmann, M., Fonk, I., Beinbrech, G., 2002. The kinase activity of the giant protein projectin of the flight muscle of Locusta migratoria. Insect Biochem. Mol. Biol. 32, 1401–1407. Fukuzawa, A., Shimamura, J., Takemori, S., Kanzawa, N., Yamaguchi, M., Sun, P., et al., 2001. Invertebrate connectin spans as much as 3.5 mm in the giant sarcomeres of crayfish claw muscle. EMBO J. 20, 4826–4835. Hu, D.H., Matsuno, A., Terakado, K., Matsuura, T., Kimura, S., Maruyama, K., 1990. Projectin is an invertebrate connectin (titin): isolation from crayfish claw muscle and localization in crayfish claw muscle and insect flight muscle. J. Muscle Res. Cell Motil. 11, 497–511. Lakey, A., Ferguson, C., Labeit, S., Reedy, M., Larkins, A., Butcher, G., et al., 1990. Identification and localization of high molecular weight proteins in insect flight and leg muscle. EMBO J. 9, 3459–3467. Manabe, T., Kawamura, Y., Higuchi, H., Kimura, S., Maruyama, K., 1993. Connectin, giant elastic protein, in giant sarcomeres of crayfish claw muscle. J. Muscle Res. Cell Motil. 14, 654–665. Nave, R., Weber, K., 1990. A myofibrillar protein of insect muscle related to vertebrate titin connects Z band and A band: purification and molecular characterization of invertebrate mini-titin. J. Cell Sci. 95, 535–544. Oshino, T., Shimamura, J., Fukuzawa, A., Maruyama, K., Kimura, S., 2003. The entire cDNA sequences of projectin isoforms of crayfish claw closer and flexor muscles and their localization. J. Muscle Res. Cell Motil. in press. Saide, J.D., 1981. Identification of a connecting filament protein in insect fibrillar flight muscle. J. Mol. Biol. 153, 661–679. Saide, J.D., Chin-Bow, S., Hogan-Sheldon, J., Busquets-Turner, L., Vigoreaux, J.O., Valgeirsdottir, L., et al., 1989. Characterization of components of Z-bands in the fibrillar flight muscle of Drosophila melanogaster. J. Cell Biol. 109, 2157–2167. Saide, J.D., Chin-Bow, S., Hogan-Sheldon, J., Busquets-Turner, L., 1990. Z-band proteins in the flight muscle and leg muscle of the honeybee. J. Muscle Res. Cell Motil. 11, 125–136.
J. Shimamura et al. / Comparative Biochemistry and Physiology Part B 136 (2003) 419–423 Southgate, R., Ayme-Southgate, A., 2001. Alternative splicing of an amino-terminal PEVK-like region generates multiple isoforms of Drosophila projectin. J. Mol. Biol. 313, 1035–1043. Vigoreaux, J.O., Saide, J.D., Pardue, M.L., 1991. Structurally different Drosophila striated muscles utilize distinct variants
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of Z-band-associated proteins. J. Muscle Res. Cell Motil. 12, 340–354. Weitkamp, B., Jurk, K., Beinbrech, G., 1998. Projectin–thin filament interactions and modulation of the sensitivity of the actomyosin ATPase to calcium by projectin kinase. J. Biol. Chem. 273, 19 802–19 808.
Localization of projectin in locust flight muscle Jinen Shimamuraa, Koscak Maruyamab, Sumiko Kimuraa,* a
Department of Biology, Faculty of Science, Chiba University, Chiba 263-8522, Japan b National Center for University Entrance Examinations, Tokyo 153-8501, Japan
Received 11 March 2003; received in revised form 18 July 2003; accepted 21 July 2003
Abstract Projectin is a giant filamentous protein of arthropod striated muscle. By using immunofluorescence microscopy, projectin was shown to span between the I band and the A band in locust (Locusta migratoria) flight muscle sarcomeres. The N- and C-terminal regions of projectin molecules were localized in the I band and A band, respectively. This observation explains the controversial reports of previous studies that projectin is localized either in the I band or in the A band of locust flight muscle sarcomeres. It is also observed that the N-terminal region of projectin is located in the I band of locust leg muscle sarcomeres. 䊚 2003 Elsevier Inc. All rights reserved. Keywords: Projectin; I-connectin; Locust; Flight muscle; Leg muscle; Mini-titin; Connectin; Titin; Insect
1. Introduction Projectin is a high molecular weight filamentous protein in arthropod striated muscle. The entire primary sequence of projectin was recently elucidated for Drosophila melanogaster (approx. 9210 residues, approx. 1 MDa) (Southgate and AymeSouthgate, 2001) and for crayfish claw closer muscle (8625 residues; 962 634 Da) (Oshino et al., 2003). The kinase domain of locust flight muscle projectin has been shown to act as a protein ¨ kinase (Weitkamp et al., 1998; Fahrmann et al., 2002). Projectin was first discovered as a C (connecting)-filament linking the Z line to the tip of the thick filament in asynchronous flight muscle of the honeybee (Saide, 1981). Since then projectin has been regarded as being localized in the I band of asynchronous flight muscle sarcomeres of D. *Corresponding author. Fax: q81-43-290-2811. E-mail address: [email protected] (S. Kimura).
melanogaster (Saide et al., 1989) and Lethocerus indicus (giant water bug) (Lakey et al., 1990). In contrast, projectin has been shown to be localized in the A band of synchronous flight, leg and larval muscles (Saide et al., 1990; Vigoreaux et al., 1991; cf. Ayme-Southgate et al., 2000). Conflicting observations have been reported for the localization of projectin in locust synchronous flight muscle. On the one hand, Nave and Weber (1990) described that mini-titin (projectin in this case) was localized in the I band using polyclonal antibodies against mini-titin. On the other hand, Weitkamp et al. (1998) reported that gold particles bound to polyclonal antibodies against locust projectin were distributed only in the A band and that they bound to thick filaments freed from locust flight muscle sarcomeres. However, Weitkamp et al. (1998) noted that projectin was localized in the I band of asynchronous flight muscle sarcomeres of the blowfly, Phormia terrae novae. To clarify these contradictory observations, we
1096-4959/03/$ - see front matter 䊚 2003 Elsevier Inc. All rights reserved. doi:10.1016/S1096-4959Ž03.00252-5
420
J. Shimamura et al. / Comparative Biochemistry and Physiology Part B 136 (2003) 419–423
describe here immunofluorescence localization of projectin in locust flight muscle using two kinds of antibodies raised against the N- and C-terminal regions of projectin. In the present work, the Nterminal region of projectin was shown to be located in the I band of locust flight muscle as well as of locust leg muscle. 2. Materials and methods 2.1. SDS gel electrophoresis and immunoblot tests A piece of freshly excised locust (L. migratoria) flight muscle was dissolved in 5 volumes of an SDS solution containing 10% SDS, 40 mM dithiothreitol, 10 mM EDTA and 100 mM Tris– HCl at pH 8.0, boiled for 3 min and clarified by centrifugation at 15 000=g for 10 min. SDS gel electrophoresis was carried out according to the method of Laemmli (1970) using 2–10% polyacrylamide gradient gels. Electrically transferred nitrocellulose sheets were treated with polyclonal antibodies, PcNIG, raised against the N-terminal region (229 amino acids (1–229; Ig 1qIg 2), molecular weight: 25 595) of crayfish claw closer muscle projectin (Oshino et al., 2003) and monoclonal antibody, Mc3B9, reactive with the C-terminal region of projectin (Hu et al., 1990; Oshino et al., 2003). The secondary antibodies used were horseradish peroxidase-conjugated anti-rabbit or anti-mouse Ig (DAKO). 2.2. Immunofluorescence microscopy Freshly excised muscle fibers of locust flight muscle were fixed at a given sarcomere length by a cold relaxing solution containing 3.5% formalin and 0.5% Triton X-100. After blocking with 1% bovine serum albumin (BSA), suspended myofibrils were treated with the primary antisera for 14 h at 4 8C; PcNIG was diluted 10 times with 0.15 M NaCl and 20 mM sodium phosphate buffer at pH 7.2; Mc3B9 was used without dilution. After washing with PBS several times, myofibrils were treated with FITC-labeled anti-rabbit or anti-mouse Ig (Cappel, West Chester, PA) for 2 h at room temperature. Immunofluorescence observations were performed as described previously (Manabe et al., 1993) with a Zeiss Axioskop 2 fluorescence. The antibody epitope spacing across the M line in a sarcomere was measured and the distance
Fig. 1. SDS gel electrophoresis patterns and immunoblot tests of locust flight and leg muscles projectin. (a) SDS gel electrophoresis patterns. 1, rabbit back muscle; 2, crayfish claw closer muscle; 3, locust flight muscle; 4, locust leg muscle. (b) Immunoblot tests. 1, Amido Black stain (locust flight muscle); 2, treated with PcNIG (locust flight muscle); 3, treated with Mc3B9 (locust flight muscle); 4, Amido Black stain (locust leg muscle); 5, treated with PcNIG (locust leg muscle); 6, treated with Mc3B9 (locust leg muscle). Electrophoresed on 2–10% polyacrylamide gradient gels. Con, titinyconnectin; Neb, nebulin; MHC, myosin heavy chain; I-con, I-connectin; P, projectin.
(sarcomere lengthythe epitope spacing) was divided by two to obtain the Z line–epitope distance in a half sarcomere. The distance between the epitope and the A–I junction was calculated from the width of the A band and the epitope spacing within a sarcomere. 3. Results and discussion 3.1. Immunoblot tests The SDS gel electrophoresis pattern depicted in Fig. 1a showed two distinct bands corresponding to projectin in locust flight and leg muscles (Fig. 1a3,a4). The upper band of locust flight muscle projectin corresponded to the main band of crayfish claw closer projectin (Fig. 1a2) and to the upper band of locust leg muscle projectin (Fig. 1a4). The lower band of locust projectin was much more abundant in flight muscle than in leg muscle (Fig. 1a3,a4). Immunoblot analyses were done using PcNIG against the N-terminal region of projectin (Oshino et al., 2003) and Mc3B9 reactive with the Cterminal region of projectin (Hu et al., 1990; Oshino et al., 2003). Fig. 1b shows that PcNIG reacted only with the upper band of locust projectin, while Mc3B9 reacted with both the upper and lower bands of locust projectin (cf. Fig. 1b2 vs. b3; Fig. 1b5 vs. b6).
J. Shimamura et al. / Comparative Biochemistry and Physiology Part B 136 (2003) 419–423
Fig. 2. Immunofluorescence localization of projectin in locust flight and leg muscles sarcomeres. (a), (b), locust flight muscle; (c), (d), locust leg muscle; (a), (c), treated with PcNIG; (b), (d), treated with Mc3B9. (a), (b), (c), (d), relaxed muscle sarcomeres; (a’), (b’), (c’), (d’), stretched muscle sarcomeres. Upper, phase contrast image; lower, fluorescence image. Bar, 5 mm.
The homology of the Ig 1qIg 2 region (229 amino acid residues; antigen for PcNIG) of crayfish projectin has high identity of 62.6% to the same region of D. melanogaster (insect) projectin. In addition, there are four segments consisting of the same sequence of over ten amino acids in both the N-terminal regions. In contrast, the homology of the Ig 1qIg 2 region of crayfish projectin has identity of only 25.4% at the best to the C-terminal region of Drosophila projectin (6658 amino-acid residues). The PcNIG is regarded to recognize the N-terminal region of both locust flight and leg muscle projectins. Therefore, it is suggested that the lower band of locust projectin corresponds to an N-terminal region-deficient projectin. The lower band could be an isoform of projectin lacking the N-terminal region as described for D. melanogaster (Southgate and Ayme-Southgate, 2001). 3.2. Immunofluorescence microscopy of locust flight muscle Immunofluorescence microscopy using the polyclonal antibody (PcNIG) showed that the N-ter-
421
minal region of projectin was clearly localized in the I band of both relaxed and stretched flight muscle sarcomeres (Fig. 2a,a’). The Z line was not fluorescent. However, the epitope recognized by the monoclonal antibody (Mc3B9) in the Cterminal region of projectin was localized in the A band near the A–I junction area (Fig. 2b,b’). Faint fluorescent areas were also seen in the A band, except in the center of the A band. The distance from the center of the Z line to the center of the main fluorescent band due to PcNIG or to Mc3B9 binding was plotted at various I band lengths (Fig. 3). The distance from the A–I junction to the Mc3B9 epitope was approximately 0.05 mm within the A band indicating that the Cterminal region of projectin was certainly localized in the A band (Fig. 3). Although the band positions were considerably scattered, no systematic change occurred due to sarcomere stretch. However, the distance from the A–I junction to the PcNIG epitope was approximately 0.23 mm within the I band and thus the N-terminal region of projectin was evidently localized in the I band. A single projectin molecule, approximately 0.3 mm long (Hu et al., 1990; Nave and Weber, 1990), may span the I band to the A band in locust flight muscle. It is suggested that projectin molecules may be associated with each other side by side, but in a staggered pattern (Ayme-Southgate et al., 2000). Now the discrepancy between previous studies for the localization of projectin in locust flight muscle may be explained. Nave and Weber (1990) might have used antibodies against the N-
Fig. 3. Distances between the Z line and the epitopes for antiprojectin antibodies in locust flight muscle sarcomeres at various sarcomere lengths. d, epitope for polyclonal antibody against the N-terminal region of projectin (PcNIG); m, epitope for monoclonal antibody against the C-terminal region of projectin (Mc3B9). Solid line, distance between the A–I junction and the Z line.
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terminal regions of projectin and, therefore, these authors localized them in the I band. In contrast, Weitkamp et al. (1998) showed projectin’s localization in the A band because antibodies against the C-terminal regions of projectin might have been employed. A question arises whether the N-terminal region of projectin is somehow positioned or is free in the I band of locust flight muscle sarcomeres. The N-terminus of projectin filament does not extend to the Z line. One possibility is that the N-terminus binds to the elastic filament, invertebrate connectin (I-connectin), linking the Z line and the thick (myosin) filament in the A band as observed with crayfish closer sarcomeres (Fukuzawa et al., 2001). Although this 2 MDa I-connectin was not found by the SDS PAGE (Fig. 1a3), a faint band was observed in a large dose-loaded gel (data not shown). Note that a distinct band of I-connectin, lower than the crayfish band, was recognized in the SDS PAGE pattern of locust leg muscle (Fig. 1a2,a4). Another possibility is that the projectin binds to the thin (actin) filament in the I band. The interaction of projectin with the thin filaments has been reported (Weitkamp et al., 1998). We made a preliminary experiment using the Far Western method with anti-actin antibodies that showed a weak binding of recombinant NIG peptide to F-actin (data not shown), and, therefore, the N-terminal end of the projectin filament may weakly attach to the actin filament in the I band. In contrast, it is reported that projectin definitely interacts with myosin (Weitkamp et al., 1998; Ayme-Southgate et al., 2000). Thus, projectin bound to myosin in the thick filament is regarded as a regulator of the function of myosin. 3.3. Immunofluorescence microscopy of locust leg muscle In locust leg muscle sarcomeres, PcNIG binding resulted in a fluorescent band in the I band (Fig. 2c) and moved away from the Z line when stretched (Fig. 2c’). This observation suggests that the N-terminal region of projectin is bound to some elastic filament, possibly I-connectin, in the I band of leg muscle sarcomeres. However, the Mc3B9 stained the both half regions of the A band except for the center region (Fig. 2d) and did not appreciably change upon stretch of sarcomeres (Fig. 2d’). The present work clearly indicates that the N-terminal region of projectin is also extended
into the I band in leg muscle sarcomeres as seen in flight muscle. Acknowledgments We thank Dr S. Tanaka of the Institute of Insect and Animal Sciences, Tsukuba for his kind supply of live locusts. This work was supported by a Grant-in-Aid for Scientific Research (B) of the Ministry of Education, Culture, Sports, Science and Technology of Japan. References Ayme-Southgate, A., Southgate, R., McEliece, M.K., 2000. Drosophila projectin: a look at protein structure and sarcomeric assembly. Adv. Exp. Med. Biol. 481, 251–264. ¨ Fahrmann, M., Fonk, I., Beinbrech, G., 2002. The kinase activity of the giant protein projectin of the flight muscle of Locusta migratoria. Insect Biochem. Mol. Biol. 32, 1401–1407. Fukuzawa, A., Shimamura, J., Takemori, S., Kanzawa, N., Yamaguchi, M., Sun, P., et al., 2001. Invertebrate connectin spans as much as 3.5 mm in the giant sarcomeres of crayfish claw muscle. EMBO J. 20, 4826–4835. Hu, D.H., Matsuno, A., Terakado, K., Matsuura, T., Kimura, S., Maruyama, K., 1990. Projectin is an invertebrate connectin (titin): isolation from crayfish claw muscle and localization in crayfish claw muscle and insect flight muscle. J. Muscle Res. Cell Motil. 11, 497–511. Lakey, A., Ferguson, C., Labeit, S., Reedy, M., Larkins, A., Butcher, G., et al., 1990. Identification and localization of high molecular weight proteins in insect flight and leg muscle. EMBO J. 9, 3459–3467. Manabe, T., Kawamura, Y., Higuchi, H., Kimura, S., Maruyama, K., 1993. Connectin, giant elastic protein, in giant sarcomeres of crayfish claw muscle. J. Muscle Res. Cell Motil. 14, 654–665. Nave, R., Weber, K., 1990. A myofibrillar protein of insect muscle related to vertebrate titin connects Z band and A band: purification and molecular characterization of invertebrate mini-titin. J. Cell Sci. 95, 535–544. Oshino, T., Shimamura, J., Fukuzawa, A., Maruyama, K., Kimura, S., 2003. The entire cDNA sequences of projectin isoforms of crayfish claw closer and flexor muscles and their localization. J. Muscle Res. Cell Motil. in press. Saide, J.D., 1981. Identification of a connecting filament protein in insect fibrillar flight muscle. J. Mol. Biol. 153, 661–679. Saide, J.D., Chin-Bow, S., Hogan-Sheldon, J., Busquets-Turner, L., Vigoreaux, J.O., Valgeirsdottir, L., et al., 1989. Characterization of components of Z-bands in the fibrillar flight muscle of Drosophila melanogaster. J. Cell Biol. 109, 2157–2167. Saide, J.D., Chin-Bow, S., Hogan-Sheldon, J., Busquets-Turner, L., 1990. Z-band proteins in the flight muscle and leg muscle of the honeybee. J. Muscle Res. Cell Motil. 11, 125–136.
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