Localization of connectin-like proteins in leg and flight muscles of insects

Tissue & Cell, 1996 28 (1) 1-8 © 1996 Pearson Professional Ltd.

Localization of connectin-like proteins in leg and flight muscles of insects Y. Ohtani, S. Maki, S. Kimura, K. Maruyama

Abstract. In leg muscle sarcomeres of a beetle, approximately 6 ~tm sarcomere length at rest, projectin (~1200 kDa) was located on the myosin filament up to 2 ~tm from the both ends of the filament, using immunofluorescence and immunoelectron microscopy. On the other hand, projectin linked the Z line to the myosin filament and bound on the myosin filament in beetle flight muscle, approximately 3-4 pm sarcomere length at rest. Connectin-like protein ( ~ 3000 kDa) was detected by immunoblot tests in beetle, bumblebee and waterbug leg muscles. Immunofluorescence and immunoelectron microscopic observations revealed that the connectin-like protein linked the myosin filament to the Z line in beetle leg muscle. Keywords: Connectin, projectin, leg muscle, flight muscle, insect

Introduction Connectin, also called titin, is the largest polypeptide, MW, ~ 3 million in vertebrate striated muscle. It largely consists of repeated motifs I and II, fibronectin Type III and immunoglobulin C2 domains. Connectin links the Z line to the myosin filament running up to the M line in a sarcomere. The portion of connectin mainly consisting of the motif II in tandem is responsible for the muscle elasticity that positions the myosin filament at the center of a sarcomere and generates tension upon stretch (for reviews, see Trinick, 1992; Maruyama, 1994). In all the vertebrate skeletal muscles examined, connectin is present although the size differences exist (Hu et al., 1986). In invertebrates, several kinds of connectin-related proteins have been characterized: 753 kDa twitchin in Caenor habditis elegans ((2. elegans) bodywall muscle (Benian et al., 1989; 1993); 1200 kDa projectin in arthropod muscles (Saide, 1981; Saide et al., 1989; Nave and Weber, 1990; Hu et al., 1990; Lakey et al., 1990; Vigoreaux et al., 1991; Maki et al., 1994) and 3000-5000 kDa connectin-like protein in annelid bodywall muscle (Kawamura et al., 1994) and arthropod muscles (Manabe et al., 1993; Maki et al., 1994). Department of Biology, Faculty of Science, Chiba University, Chiba 263, Japan Received 27 March 1995 Accepted 31 August 1995 Correspondence to K, Maruyama, Tel: 81-43-290-2811; Fax: 81-43-290-2812,

The localization of projectin is complicated. In regular sizes of sarcomeres (3-4 gm sarcomere lengths at rest), projectin links the Z line to the myosin filament (honeybee flight muscle (Saide, 1981; Saide et al., 1990), locust flight muscle (Nave and Weber 1990), waterbug flight muscle (Lakey et al., 1990), and crayfish tail flexor muscle (Manabe et al., 1993). On the other hand, in giant sarcomeres of the crustacean muscles (8-t0 gm sarcomere length at rest), projectin is localized on the myosin filament (crayfish claw opener and closer muscles (Manabe et al., 1993) and barnacle adductor muscles (Maki et al., 1994)). What is responsible for linking the Z line to the myosin filament in such giant sarcomeres? Manabe et al. (1993) showed that ~ 3 0 0 0 k D a connectin-like protein links the Z line to the myosin filament, but not up to the M line in the giant sarcomeres of crayfish claw muscle. This was also the case in barnacle adductor muscle (Maki et al., 1994). The present study is concerned with the localization of projectin and connectin-like protein in insect leg muscles, 5-8 ~tm sarcomere length at rest (Tiegs, 1955). The results are in good agreement with those observed with the giant sarcomeres of crayfish claw and barnacle adductor muscles.

Materials and methods Materials Horned beetle Allomyrina dichotomus and waterbug Lethocerus were purchased from a local pet shop.

2

O H T A N I ET AL.

Bumblebee Bombus terrestris was a gift of Dr M. Ono, Honeybee Science Research Center, Tamagawa University, Tokyo. Chicken breast muscle and crayfish closer muscle were used for comparison. SDS gel electrophoresis Bundles of muscle fibers were dissolved in 2 vol. of 10% (w/v) SDS, 40 mM dithiothreitol, 10 mM EDTA and 0.1 M Tris-HC1 buffer, pH 8.0, boiled for 2 rain and clarified by centrifugation for 20 min at 15 000 g. SDS gel electrophoresis was carried out according to Laemmli (1970) using 2.3-4% polyacrylamide gels.

Immunoblot tests The protein bands separated by SDS gel electrophoresis were electrically transferred onto a nitrocellulose sheet. The sheet was treated with monoclonal antibodies, SM1 and 3B9 to vertebrate skeletal muscle connectin (Itoh et al., 1988) and the antiserum against crayfish closer muscle connectin (Manabe et al., 1993), and the antiserum against crayfish claw muscle projectin (Hu et al., 1990). The detection of the antibody binding was carried out using horseradish peroxidase-conjugated anti-mouse IgG (DAKOPATTS, Copenhagen) or anti-rabbit IgG (Bio-Rad). Immunofluorescence microscopy Chemically skinned fibers with 0.5% Triton X-100 in relaxing buffer (80 mM KC1, 5 mM MgCI2, 4 mM ATP, 4 mM EGTA and 10 mM phosphate buffer, pH 7.0) of beetle and bumblebee leg and flight muscles were fixed for 15 min at room temperature with 3.5% formaldehyde in relaxing buffer. The fibers were treated by 1% bovine serum albumin in phosphate-buffered saline (pH 7.2) and then with the primary antibodies followed by the treatment with secondary antibody (FITC-labeled antirabbit IgG or anti-mouse IgG (Cappel, West Chester, PA) as described before (Manabe et al., 1993). The specimen were observed under Leitz Ortholax-2 fluorescence microscope. Immunoelectron microscopy Chemically skinned fibers of beetle and bumblebee leg and flight muscles were fixed for 10 min at room temperature with 2% paraformaldhehyde in relaxing buffer, followed by the antibody treatments as described (Itoh et al., 1988). The fibers were embedded by a standard procedure. Thin sections stained with lead citrate and uranyl acetate were observed under a JEOL 100S electron microscope.

Results SDS gel electrophoresis patterns Figure 1 shows SDS gel electrophoresis patterns of leg and flight muscles of beetle, bumblebee and waterbug. Projectin (1200kDa) was common to all the insect

muscles examined, but not present in chicken breast muscle. It is of interest to note that the amount of projectin relative to that of myosin heavy chain was much more in leg muscles than in fight muscles (Fig. la, lanes 2 and 3). In the SDS gel electrophoresis patterns shown in Figure 1, connectin-like protein was present in all the leg muscles but not in flight muscles. However, this apparent absence might have been due to a very trace amount of the connectin-like protein in flight muscle, because Lakey et al. (1993) clearly demonstrated its presence in waterbug flight muscle. In fact, immunoblot tests using antiserum to crayfish claw muscle 3000 kDa protein revealed the presence of ~ 3000 kDa protein in waterbug flight muscle also in the present study (see below). There were two or three bands below the projectin band. One of them might be kettin first described in waterbug flight muscle (700kDa) and leg (500kDa) muscles (Lakey et al., 1993).

Immunoblot tests The antiserum to crayfish connectin-like protein reacted ~3000 kDa proteins in leg muscles (Fig. 2Aa, lane 3). The proteins also reacted with SM1, a monoclonal antibody to vertebrate striated muscle connectin (Fig. 2Aa, lane 2). On the other hand, the antiserum to crayfish connectin-like protein did detect ~3000 kDa protein in waterbug fight muscle (Fig. 2Cb, lane 3) but not in beetle (Fig. 2Ab, lane 3) and bumblebee fight muscles (Fig. 2Bb, lane 3). Projectin was detected in each muscle by the antiserum to crayfish projectin and 3B9, a monoclonal antibody to vertebrate skeletal muscle connectin (Fig. 2A, 2B and 2C, lanes 4 and 5). 3B9 also reacted with a band below projectin. There were two bands reactive with 3B9 and the antiserum to crayfish claw muscle projectin in waterbug flight muscle (Fig. 2Ca, lanes 4 and 5). Localization of connectin-like protein in leg muscle Immunofluorescence microscopy revealed that connectin-like protein was localized from the side of the Z line up to the center region of the myosin filament in beetle leg muscle. It is shown that the antiserum to crayfish connectin-like protein stained both sides of the Z line of beetle leg muscle sarcomeres (Fig. 3a) and the staining patterns remained unchanged upon stretch to a sarcomere length of 11.1 pm from 5.2 ~tm (Fig. 3c). On the other hand, SM1 bound both I and A bands except for the Z line and the mid portion of the A band (Fig. 3b). Upon stretch the staining of the periphery of the Z line did not change, but the staining of the I band became faint and that of the A band became stronger (Fig. 3d). These changes strongly suggest that an elongatable filament-like connectin links the Z line to the myosin filament. Figure 4 shows immunoelectron micrographs of beetle leg muscle sarcomeres, using the antiserum to crayfish

CONNECTIN IN INSECT LEG MUSCLE

1

2

3

4

1

2

3

1

2

3 ~C

N

MHC

a

b

c

Fig. 1 SDS gel electrophoresis patterns of insect flight and leg muscles, a, beetle; b, bumblebee; c, waterbug. 1, chicken breast muscle; 2, flight muscle; 3, leg muscle; 4, crayfish closer muscle, ct, ct-connectin; [3, ~-connectin; N, nebulin; MHC, myosin heavy chain; P, projectin; C, connectin. o Laemmli's system using 2.3-4V0 polyacrylamide gels.

A

B

C

1 2345

1345

12345

1345

a

b

a

b

1 2345

1345

C P

MHC

a

b

Fig. 2 Immunoblot tests of insect leg and flight muscles. A, beetle; B, bumblebee; C, waterbug, a, leg muscle; b, flight muscle. 1, Amido Black stain; 2, treated with SM1; 3, treated with the antiserum to crayfish claw muscle connectin; 4, treated with 3B9; 5, treated with the antiserum to crayfish claw muscle projectin. Laemmli's system using 2.3 4% polyacrylamide gels.

3

4

OHTANI ET AL.

a

c

b

d

Fig. 3 Immimofluorescence location of connectin-like protein in beetle leg muscle, a, c, treated with antiserum to crayfish claw muscle connectin; b, d, treated with SM1. Sarcomere length: 5.2 ~tm (a); 7.8 ~tm (b); 11.1 gm (c); 11.7 gm (d). Arrowhead indicates Z line. Upper photograph, phase-contrast image; lower photograph, fluorescence image. Bar, 10 pan.

connectin-like protein and monoclonal antibody, SM1. Deposits of anti-connectin antibodies were found at the regions in the I band 200-400 nm apart from the Z line, when muscle was somewhat contracted (Fig. 4b). In stretched muscle, the antibodies were widely distributed in the I band, although more deposits were observed near the Z line (Fig. 4c). On the contrary, SM1 widely bound in the I band as well as in the A band (Fig.4d). In stretched muscle, the deposits were abundant in the I band region, 2-3 gm from the edge of the A band (Fig. 4e). Similar localization of connectin-like protein was also observed With bumblebee leg muscle (data not shown).

Localization of projectin in leg and flight muscles In beetle leg muscle, 3B9 reactive with projectin, stained the A band except for the mid portion regardless of sarcomere length (6.1 ~tm or ll.3gm), as shown in Fig. 5a, c). The Z line and I band were not fluorescent at all. This was also the case with bumblebee leg muscle (Fig. 5b, d). On the other hand, in beetle flight muscle, 3B9 stained the both sides of the Z line (Fig. 6a). It appeared that the antiserum to crayfish projectin stained the Z line (Fig. 6d) and the separation of adjacent two bands at the Z line became recognizable upon stretch (data not shown). This was also the case with bumblebee flight muscle (Fig. 6b). Immunoelectron microscopy revealed that 3B9 anti-

bodies were deposited solely in the A band except for the mid portion approximately 600 nm wide in beetle leg muscle (Fig. 7b, c). Projectin was present on the myosin filament up to about 2 pm from the both ends of the filament. On the other hand, in beetle flight muscle at rest, there were two stripes at the edge of the A band and at 180 nm inside from the edge of the A band (Fig. 8b). When stretched, the two bands fused into a diffuse band but did not move into the I band (Fig. 8c). When the antiserum to crayfish projectin was used, there was one stripe formation at the edge of the A band at rest length (Fig. 8d). Upon stretch, the stripe disappeared and random deposits of the antibodies were recognized between the edge of the A band and the Z line (Fig. Be).

Discussion In vertebrate striated muscle, only a single giant molecule (connectin/titin; approximately 3000kDa) connects the myosin filament to the Z line as an elastic spring (see Trinick, 1992; Maruyama, 1994). Biodiversity exists in invertebrate muscle. At least three kinds of connectin-related proteins are known at present: connectin-like protein, projectin and twitchin. Twitchin is located on the myosin filament (C. elegans (Benian et al., 1989); annelid bodywall muscle (Kawamura et al., 1994); scallop adductor muscle (Vibert et al., 1993)).

C O N N E C T I N IN INSECT L E G M U S C L E

a

b

C

cl

e

Fig. 4 Immunoelectron microscopic location of connectin-like protein in beetle leg muscle, a, control; b, c, treated with the antiserum to crayfish claw muscle connectin-like protein; d, e, treated with SM 1. a, c, e, highly stretched muscle. Z, Z line; M, middle of the A band. Bar, 1 gin.

a

b

C

d

Fig. 5 Immunofluorescence location of projectin in beetle and bumblebee leg muscles. Treated with 3B9. a, c, beetle; b, d, bumblebee. Sarcomere length: 6.1 gm (a); 4.0 gm (b); 11.3 gm (c); 6.7 gm (d). Arrowhead indicates Z line. Upper photograph, phase-contrast image; lower photograph, fluorescence image. Bar, 10 ~un. L o c a l i z a t i o n o f p r o j e c t i n present in a r t h r o p o d muscle is in two ways: in the g i a n t s a r c o m e r e s (10 ~tm at rest) p r o j e c t i n is on the m y o s i n filament ( H u et al., 1990). I n

the r e g u l a r sizes o f s a r c o m e r e s (3 4 g m at rest), p r o jectin links the Z line to the m y o s i n filament ( N a v e a n d Weber, 1990; M a u a b e et al., 1993). C o n n e c t i n - l i k e

6

OHTANI ET AL.

b

e

C

Fig. 6 Immunofluorescence observations on beetle, bumblebee and waterbug flight muscles, a, d, beetle; b, e, bumblebee; c, waterbug, a, b, c, treated with 3B9; d, e, treated with the antiserum to crayfish claw muscle projectin. Arrowhead indicates Z line. Bar, 10 gm.

El

b

C

Fig. 7 Immunoelectron microscopic location of projectin in beetle leg muscle, a, control; b, c, treated with 3B9. c, highly stretched muscle. Z, Z line. Bar, 1 gm.

proteins always link the Z line to the myosin filament (Manabe et al.,1993; Maki et al.,1994). What is the situation with projectin in intermediate size sarcomeres of insect leg muscle (sarcomere lengths, 5-8 ~tm at rest) ? The present work gives a definite answer. Projectin is on the myosin filament as in giant

sarcomeres of crayfish claw and barnacle adductor muscles (sarcomere length, 10 gm at rest). We have confirmed this result in beetle and bumblebee leg muscles. This localization of projectin was first reported by Saide et al. (1989) in Drosophila larval gut muscle using immunofluorescence microscopy. Certainly

CONNECTIN IN INSECT LEG MUSCLE

Z

"]

Z

a Z

b C

ct

e

Fig. 8 Immunoelectron microscopic location of projectin in beetle flight muscle, a, control; b, c, treated with 3B9; d, e, treated with the antiserum to crayfish claw muscle projectin. Z, Z line. Bar, 1 gm. Right sets of photographs are enlarged. Bar, 0.5 gm.

260 nm projectin filament would not be able to cover a long distance such as the wide I band (up to 2-4 gin) in the giant sarcomeres. In the present study, it was shown that approximately 2 gm portion of the both sides of the myosin filament of beetle leg muscle was bound with projectin (Fig. 7). It is not possible that a single projectin molecule covers this long distance. Therefore, it is likely that at least several projectin filaments run end-to-end as well as side-by-side (cf. Vibert et al., 1993). The presence of connectin-like protein was already reported in annelid obliquely striated muscle (Kawamura et al., 1994), crayfish claw muscle (Manabe et al., 1993) and barnacle adductor muscle (Maki et al., 1994). It was shown that it connects the Z line to the

myosin filament but to what portion of the myosin filament was uncertain. The present immunoelectron microscopy clearly revealed that it reaches the center region of the myosin filament. From the changes in the antibodies binding upon stretch, it is regarded that the connectin-like filament is elastic. Reactivity with SM1, a monoclonal antibody reactive with chicken skeletal muscle connectin (motif II, immunoglobulin C2 domains (Maruyama et al., 1993) suggests that insect connectinlike protein has a structure homologous to connectin. It is, however, noted that the connectin-like protein is also present in some sarcomeres of regular size in insect flight muscles (honeybee, (Hu et al., 1990); waterbug, (Lakey et al., 1993; this paper)).

REFERENCES Benian, G. M., Kiff, J. E., Neckelmann, N., Moerman, D. G. and Waterston, R. H. 1989. Sequence of an unusually large protein implicated in regulation of myosin activity in C. elegans. Nature, 342, 45 50. Benian, G. M., L'Hernault, S. W. and Morris, M. E. 1993. Additional sequence complexity in the muscle gene, Unc-22, and its encoded protein, twitchin, of Caenorhabditis elegans. Genetics, 134, 1097-1104. Hu, D. H., Kimura, S. and Maruyama, K. 1986. Sodium dodecyl sulfate gel electrophoresis studies of connectin-like high molecular weight proteins of various types of vertebrate and invertebrate muscles. J. Biochem., 99, 1485-1492. Hu, D. H., Matsuno, A., Terakado, K., Matsuura, T., Kimura, S. and 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. Itoh, Y., Suzuki, T., Kimura, S., Ohashi, K., Higuchi, H., Sawada, H., Shimizu, T., Shibata, M. and Maruyama, K. 1988. Extensible and less-extensible domains of connectin filaments in stretched vertebrate skeletal muscle sarcomeres as detected by immunofluorescence and immunoelectron microscopy using monoclonal antibodies. J. Biochem., 104, 504 508. Kawamura, Y., Kimura, S. and Maruyama, K. 1994. Characterization of connectin-like proteins of obliquely striated muscle of a polychaete (annelida). J. Muscle Res. Cell Motil., 15, 623-632. Laemmli, U. K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, 227, 680-685. Lakey, A., Labeit, S.Gautel, M., Ferguson, C., Barlow, D. P.,

8

OHTANI ET AL.

Leonard, K. and Bullard, B. 1993. Kettin, a large modular protein in the Z-disc of insect muscles. EMBO J., 12, 2863-2871. Lakey, A., Ferguson, C., Labeit, S., Reedy, M., Larkins, A., Butcher, G., Leonard, K. and Bullard, B. 1990. Identification and localization of high molecular weight proteins in insect flight and leg muscle. EMBO J., 9, 3459-3467. Maki, S., Kimura, S. and Maruyama, K. 1994. Localization of connectin-like protein in the giant sarcomeres of barnacle muscle. Zool. Sci., 11, 821-824. Manabe, T., Kawamura, Y., Higuchi, H., Kimura, S. and Maruyama, K. 1993. Connectin, giant elastic protein, in giant sarcomeres of crayfish claw muscle. J. Muscle Res. Cell Motil., 14, 654-665. Maruyama, K.1994. Connectin, an elastic protein of striated muscle. Biophys. Chem., 50, 73 85. Maruyama, K., Endo, T., Kume, H., Kawamura, Y., Kanzawa, N., Nakauchi, Y., Kimura, S., Kawashima, S. and Maruyama, K. 1993. A novel domain sequence of connectin localized at the I band of skeletal muscle sarcomeres-homology to neurofilament subunits. Biochem. Biophys. Res. Commun., 194, 1288-1291. Nave, R. and 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.

Saide, J. D. 1981. Identification of a connecting filament protein in insect flbrillar flight muscle. J. Mol. Biol., 153, 661-679. Saide, J. D., Chin-Bow, S., Hogan-Sheldon, J. and 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. Saide, J. D., Chin-Bow, S., Hogan-Sheldon, J., Busquets-Turner, L., Vigoreaux, J. O., Valgeirsdottir, K. and Pardue, M. L. 1989. Characterization of components of Z-bands in the fibrillar flight muscle of Drosophila melanogaster. J. Cell Biol., 109, 2157-2167. Tiegs, O. W. 1955. The flight muscles of insects their anatomy and histology; with some observations on the structure of striated muscle in general. Phil. Trans. Roy. Soc. London, 238B, 221 348. Trinick, J. 1992. Understanding the functions of titin and nebulin. FEBS Lett., 307, 4448. Vibert, P., Edelstein, S. M., Castellani, L. and Elliott, B. W. 1993. Mini-titin in striated and smooth molluscan muscles: structure, location and immunological crossreactivity. J. Muscle Res. Cell Motil., 14, 598-607. Vigoreaux, J. O., Saide, J. D. and Pardue, M. L. 1991. Structurally different Drosophila striated muscles utilize distinct variants of Z-band-associated proteins. J. Muscle Res. Cell Motil., 12, 340-354.