Connectin, an Elastic Filamentous Protein of Striated Muscle

INTERNATIONAL REVIEW OF CYTOLOGY. VOL. 104

Connectin, an Elastic Filamentous Protein of Striated Muscle KOSCAK MARUYAMA Department of Biology, Faculty of Science, Chiba University, Chibn 260, Japan

I. Introduction Connectin, also called titin, is a very long, flexible filamentous protein of striated muscle that links thick myosin filaments to Z disks in a sarcomere. Despite its presence in rather high amount (about 10%) in myofibrils next to myosin (43%) and actin (22%), as listed in Table I, connectin has only recently been characterized. This was because physicochemical investigations have been very difficult due to its huge molecular weight of a few million. At present, a proteolytic product of the mother molecule (a-connectin, T,), p-connectin or T, has been purified as native form, but a-connectin has not yet been isolated. Connectin superthin filaments are shown to serve as an elastic component of striated muscle. Therefore, it is regarded as the fourth type of cytoskeletal structure following microtubules, intermediate filaments, and actin filaments. The problem is whether connectin-like filaments are present in nonmuscle cells or not. The present review deals with this problem after a full description of muscle connectin (cf. Wang, 1984, 1985; Locker, 1984a; Maruyama and Kimura, 1985; Ohtsuki et af., 1986).

11. The Third Filament? Over a century ago, a great German physiologist Johannes Miiller indicated in his famous textbook on human physiology that skeletal muscle could be considered to be elastic bodies (Miiller, 1840). Since then, it has long been assumed that muscle consists of two components, contractile and elastic. The elastic property of muscle was ascribed to the function of extracellular collagen fibers attached to muscle cell membranes (sarcolemma). In 1954, Natori first demonstrated that an elastic component exists in muscle cells using his famous demembraned fibers of the Natori type (Natori, 1954). He postulated the presence of an “internal elastic structure.” In the same year, H. E. Huxley and Hanson, together with A. F. Huxley and Niedergerke (1954), proposed the sliding theory based on the movement of the thin (actin) filaments relative to the thick (myosin) 81 Copyrighl 0 1986 by Academic Press. Inc. All rights or reprduclion in any form reserved.

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KOSCAK MARUYAMA TABLE I MYOFIBRILLAR PROTEIN OF VERTEBRATE SKELETAL MUSCLE“

Protein Contractile Myosin Actin Regulatory Troponin Tropomyosin M-Protein C-F’rotein a-Actinin Cytoskeletal Connectin Nebulin

Molecular weight (x 103)

Content wt%

Localization (band)

520 42

43 22

A I I I M line A Z line

70

33 x 2 165 135 95 x 2 2800 750

10 5

A-I I

OMinor structural proteins less than 1% of the total proteins are omitted. For a complete list, see Ohtsuki er al. (1986).

filaments in a sarcomere (H. E. Huxley and Hanson, 1954). In order to explain the continuity of myosin-removed myofibrils, they assumed the presence of an elastic filament called an S-filament linking the free ends of actin filaments in a sarcomere. However, this elastic filament model was not subsequently mentioned in the development of the sliding theory. In 1962, Sjostrand observed very thin filaments at the gap region between myosin and actin filaments when muscle was extremely stretched beyond the overlap of the two sets of filaments. Sjostrand (1962) called these filaments that were thinner than actin filaments “gap filaments,” and assumed that they were continuous with the tapered ends of myosin filaments. A year earlier, A. F. Huxley and Peachy (1961) mentioned the possible presence of “fine filaments” connecting the ends of both myosin and actin filaments from their observations of highly stretched muscle fibers. Graham Hoyle and his associates examined fine structures of a variety of striated muscles both in invertebrates and vertebrates and reached the conclusion that there were “superthin” or T-filaments (3 nm in width) connecting adjacent Z lines in a sarcomere (McNeill and Hoyle, 1967; Hoyle et af., 1968). Guba et al. (1968) reported that there were residual filaments after the extraction of myosin and actin. They claimed that those superthin filaments consisted of a protein designated fibrillen. dos Remedios (1969) observed that there was a third filament in addition to the myosin and actin filaments in a sarcomere that was resistant to salt extraction (dos Remedios and Gilmour, 1978). Revival of Sjostrand’s gap filament was brought about by Locker in 1975.

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TABLE II CHRONOLOGICAL LIST OF THE CONCEFT OF SUPERTHIN FILAMENT IN STRIATED MUSCLE Reference Inner elastic structure S filament connecting actin filaments Fine filament connecting myosin and actin filaments Gap filaments connecting myosin and actin filaments Connecting filaments connecting myosin and Z disk in insect muscle Superthin T filament running from Z to Z disks Fibrillen Third thin filament running from Z to Z disks G-filament connecting myosin and Z disks Connectin 10-nm filament connecting Z to Z disks Titin Projectin in insect muscle Cross-linked matrix

Natori (1954) H. E. Huxley and Hanson (1954) A. F. Huxley and Peachy (1961) Sjostrand (1962) Auber and Couteaux (1963) McNeill and Hoyle (1967) Guba er al. (1968) dos Remedios (1969); dos Remedios and Gilmour (1978) Locker and Leet (1975) Maruyama et al. (1976, 1977a) Price and Sanger (1977) Wang et al. (1979) Saide (1981) Lowey et al. (1983)

Locker and Leet (1975) first observed that bovine neck muscle (sternomandibularis) can be stretched to five times its rest length, up to 12 pm between the neighboring Z disks, spanned by very thin gap filaments, continuous with the thick filaments (Locker and h a t , 1976a,b). Locker called them G-filaments and assumed that they formed cores of myosin filaments, emerging at one end only, and arriving at the Z disk (cf. Locker, 1984a). Thus, as listed in Table 11, morphological and physiological observations strongly suggested the presence of the third superthin filament in addition to the thick and thin filaments in a sarcomere of vertebrate striated muscles. However, this was not widely accepted. One reason was, and still is, the lack of a clear-cut image of the compatibility of the third filament model toward the sliding theory of muscle contraction. Finally, it is to be noted that there are third filaments connecting the edges of myosin filaments to Z disks in fibrillar flight muscles of some insects, e.g., bee, fly, waterbug, etc. that rhythmically and quickly repeat contraction and relaxation (Auber and Couteaux, 1963; reviewed by Pringle, 1978). Projectin, the connecting filament protein, was isolated from honeybee thoracic muscle (Saide, 1981). 111. Connectin versus Titin

Stimulated by Natori’s pioneering work (Natori, 1954), the present writer began to identify the chemical entity of the elastic filament connecting Z disks in myofibrils of vertebrate skeletal muscle after removal of myosin and actin (rab-

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bit, chicken, and frog) in 1975. The residue was completely insoluble in salt solution, e.g., 1 M K1, LiBr, KSCN, etc. Dilute acid (1 N acetic acid, 0.1 N HCl, etc.) or alkali (0.01 and 0.1 N NaOH) also failed to solubilize. Most of the residue was resistant to even 0.1% SDS or 8 M urea. Alkali-treated ghost skinned fibers of frog muscle behaved just like rubber (Maruyama et al., 1976). When the insoluble residue, after removal of myosin, actin, and regulatory proteins and also connective tissue, was washed with water and then solubilized in I % SDS solution, the SDS-gel electrophoresis pattern showed that the main components were a high-molecular-weight (HMW) component which hardly moved and a 42kDa band (Maruyama et al., 1977a). The HMW component was cut out of the gel and used as an antigen. The FITC-labeled antiserum stained the filamentous material in the muscle residues (Maruyama et al. 1977a). Immunofluorescence observation showed that the A-I junction area of a sarcomere was most intensely stained (Maruyama et al., 1980). The 42-kDa component was nothing but denatured actin (Maruyama et al., 1983). King and Kurth (1980) isolated HMW connectin by chromatography on DEAE-Sepharose CL-6B in the presence of guanidine-HC1 and urea. Locker and Daines (1980) separated maleylated protein by DEAE-cellulose chromatography. The relationship between ‘‘gap filaments” and salt-insoluble connectin was discussed by Locker and Daines (1980). It is worth mentioning the peculiar solubility behavior of muscle structural proteins. Myosin can be easily extracted with a large volume of HasselbachSchneider solution: the remaining myosin would be very small in amount, if extraction is repeated several times. Actin extraction with 0.6 M KI is always incomplete. Some actin is left behind. If myofibrils are treated with 0.6 M KI from the beginning, a large amount of actin remains unextracted together with myosin. The proteins aggregate around deteriorated Z disks as first pointed out by Granger and Lazarides (1978). The most likely situation is that actin, myosin, and other proteins are bound to free connectin filaments extending from the Z disk and the whole mass aggregates near the Z disk. These become completely insoluble in salt solutions and some of them are not soluble even in a SDS solution. This explains the formation of the “elastic matrix” mentioned by Lowey et al. (1983). Quite independent of the connectin work aimed at the elastic filament of skeletal muscle, Kuan Wang accidentally discovered a giant protein by SDS-gel electrophoresis of total SDS e.xtract of whole muscle (Wang et al., 1979). Originally, he intended to find smooth muscle actin-binding protein (filamin according to Wang’) in skeletal muscle without success. Instead, he found at least three

’

Actin-binding protein (ABP) was first purified from rabbit lung macrophages (Hartwig and Stossel, 1975; Stossel and Hartwig, 1975). Chicken gizzard ABP was isolated by Wang (1977; cf. Wang er al., 1975) and named filamin.

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1

2

3

4

a

b

c

d

FIG. 1. SDS-gel electrophoresis patterns of a direct SDS extract of chicken breast muscle myofibrils. (a) 2.8% polydcrylamide gels, (b) 2.5%. (c) 2.3%,(d) 2.0%.( I ) a-Connectin (titin I ) , (2) p-connectin (titin 2), (3) nebulin, (4) myosin heavy chain. D. H. Hu (unpublished).

high-molecular-weight protein bands (bands 1, 2, and 3). Figure 1 shows SDSgel electrophoresis pakerns of a SDS extract of chicken breast muscle. By gel filtration in the presence of 0.1% SDS, Wang was able to isolate bands 1 and 2 in a denatured state and to raise antibodies against them. Bands 1 and 2 were named titin because of their huge molecular weight of one million. In myofibrils, they were located in the A - I junction area and also in Z disks as revealed by an immunofluorescence study. The content of titin was as large as 10% of the total myofibrillar proteins, and therefore Wang called it the third major structural protein of muscle. We immediately confirmed that Wang’s titin was identical to connectin (Maruyama et al., 1981a). Although King and Kurth (1980) separated connectin by gel filtration from guanidine- and HCI-solubilized muscle residues, Wang’s procedure was simpler and more reproducible. Then, our efforts were concentrated on the isolation of connectin in a native form. We noticed that some amount of connectin is soluble in a salt solution, coextractable with myosin in Guba-Straub solution (Maruyama et al., 1981a). It turned out that this is p-connectin (T,), derived from a-connectin (T2) by endog-

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enous protease. a-Connectin is not soluble. We have selected the conditions in which myosin is not solubilized and isolated native p-connectin (Kimura et al., 1982; Kimura and Maruyama, 1983a; Kimura et al., 1984b). Meanwhile, Wang’s group in the United States and Trinick’s group in the United Kingdom were successful in isolating connectin by different means (Wang et al., 1984; Trinick et al., 1984).

IV. Native Connectin A. PREPARATION With our observations that some connectin is solubilized from chicken breast muscle together with myosin in Guba-Straub or Hasselbach-Schneider solution (Maruyama et al. 1981a), we sought the conditions in which connectin alone is soluble without myosin. First, it was ascertained that connectin is soluble in the presence of 0.2 M NaCl (pH 7.0) or 0.075 M phosphate buffer (pH 6.5). With 0.1 M sodium phosphate buffer, connectin was solubilized above pH 6.5. At pH 7.0, both connectin and myosin were soluble, but the latter was not soluble at pH 6.5. However, a-actinin, actin, and other proteins were also extracted by 0.1 M phosphate buffer (pH 6.5). Therefore, myofibrils were first washed well with 5 mM NaHCO, followed by extraction with 0.1 M phosphate buffer at pH 5.6. These procedures removed a-actinin and other proteins. The precipitate was briefly washed with water and then extracted with 0.1 M phosphate buffer, pH 6.6. The filtrate consisted largely of connectin (Kimura and Maruyama, 1983a). For further purification, hydroxyapatite chromatography is highly recommended. However, it was noticed that fresh myofibrillar preparations resulted in a low yield of connectin. Therefore, myofibrils were prepared from muscle strips stored overnight at 0°C (Kimura et al., 1984b). This was due to proteolysis of aconnectin to p-connectin (see Section IV). The native connectin we obtained was p-connectin. By this procedure the yield was as high as 400 mg starting from 100 g of muscle. This is approximately 40% of the original content of connectin. Trinick and his associates (1984) have separated connectin from myosin in a salt extract of rabbit psoas myofibrils by sedimenting myosin in 0.2 M KCI. Contaminated C-protein and other proteins can be removed by DEAE-cellulose column chromatography. The eluted connectin is precipitated by (NH,),SO, at 35% saturation and it was dissolved in 0.5 M KCI containing 50 mM Tris-HC1 buffer, pH 7.9. This procedure is very convenient for concentrating connectin. Pure connectin can be obtained by gel filtration. The yield is approximately 200 mg from 100 g of muscle. Wang’s procedure (Wang er al., 1984) was similar to the procedures of C-protein purification (Reinach et al., 1982) and the yield was low: 100 mg from 100 g of muscle. Crude myosin preparations were subjected to

CONNECTIN, AN ELASTIC FILAMENTOUS PROTEIN

87

DEAE-Sephadex chromatography to remove myosin. The unabsorbed material containing C-protein and connectin was then subjected to hydroxyapatite chromatography. Connectin tends to degrade during this preparation procedure, unless the procedure is performed quickly in the cold. B. SIZEAND SHAPE The mobility of connectin in SDS-gel electrophoresis is very slow: in a 510% polyacrylamide gel, connectin hardly moves and remains at the top of the gel. In 2-3% polyacrylamide gels it moves slowly (cf. Fig. 1). Since the apparent molecular weight (MW) of connectin is very large, appropriate markers for MW determination are not available. Therefore, artificial markers were prepared by cross-linking myosin heavy chains (MHC) with a maleimide derivative (Knight and Offer, 1978). Myosin heavy chains were rapidly dimerized by the cross-linking reaction of their SH groups with the maleimide reagent and within a few minutes of incubation monomers completely disappeared. Dimers were further cross-linked with each other forming 2 X dimers, 4 X dimers, etc. A linear relationship between logarithms of MWs and electrophoretic mobilities was observed up to 3200 kDa (8 X dimers) using 2.0% polyacrylamide gels, as seen in Fig. 2. From the mobility of isolated native connectin, its MW was roughly estimated to be 2100 kDa between 5 X dimers (2000 kDa) and 6 X dimers (2400 kDa) of MHC. This native connectin corresponds to the lower band (p-connectin) of direct SDS extract of intact myofibrils. The upper band (a-connectin) had an apparent MW of 2800 kDa. Wang (1982) claimed the MWs of 1800 and 1200 kDa for T , and T, (a-and p-connectins) by a similiar procedure, but he assumed that dimers, trimers, etc. had been formed by cross-linking reaction of MHC. Our sedimentation equilibrium measurements of MW of p-connectin in 0.5 M KCl and 0.1 M phosphate buffer (pH 7.0) also showed a value of 2700 kDa (Maruyama et al., 1984a). The sedimentation pattern was a single hypersharp peak having a sedimentation coefficient of 17 S in 0.1 M phosphate buffer, pH 7.0, in agreement with the value of 13.4 S in 0.5 A4 KCI and 0.05 M phosphate buffer, pH 7.5 (Trinick et al.. 1984). The shape of the sedimentation pattern showed that the connectin molecule is highly asymmetric. At the same time, the presence of a smaller peak of approximately 30-40 S suggested formation of side-by-side aggregates. An interesting observation was made in the presence of 1% SDS: the sedimentation coefficient of 15 S in the native state changed to 11 S in the denatured state. Since the peak became more hypersharp, it is likely that the change in the sedimentation coefficient was not due to subunit dissociation, but to a shape change in the presence of SDS. King (1984) estimated the MW of connectin in SDS to be lo6 by lightscattering technique. Electron microscopic images by a low angle rotary shadowing procedure showed that there were more straight rods in the presence of SDS

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KOSCAK MARUYAMA

a

C

e

rno bil it y FIG. 2. Molecular weight determinations of a- and p-connectins by SDS-gel electrophoresis: 2% polyacrylamide gels. The two arrows indicate a- and p-connectins. (a) Cross-linked myosin heavy chains, (b) isolated native connectin, (c) a + b, (d) direct SDS extract of chicken breast muscle. Myosin oligomers, (0)albumin oligomers. Modified (e) cross-linked bovine serum albumin. (0) from Maruyama ei al. (1984a).

than in its absence (H. Sawada and S. Kimura, unpublished). From the asymmetric nature of connectin, it was expected that the viscosity value was large. In a conventional Ostwald type viscometer, the value of intrinsic viscosity was less than 2 (g/dl) suggesting an axial ratio of about 50. However, it turned out that the viscosity of connectin greatly depended on the velocity gradient in the measurements. Thus at a very low velocity gradient of 0.0007 second-', a connectin solution of 0.3 mg/ml had a viscosity value of as high as 17,000 CPand the value dropped to 230 CP at 0.08 second-'. This is a thyxotropic nature owing to an entanglement of very thin filaments that can be easily disentangled by weak force. Electron microscopic observations have revealed that connectin is a very long,

CONNECTIN, AN ELASTIC FILAMENTOUS PROTEIN

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flexible filament (Maruyama et al., 1984a; Wang et al., 1984; Trinick ef al., 1984). A low angle rotary shadowing method gave various kinds of images: from straight filament to entangled knitting wool (Fig. 3). The length distribution was heterogeneous ranging from 0.2 to 1 pm. Flow birefringence measurements

FIG. 3. Low angle shadowing images of connectin filaments. Note that myosin molecule (160 nm long) is included. Bar, 0.2 Fm. From H . Sawada and S. Kimura (unpublished; cf. Maruyarna er a / . , 1984a).

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suggested an approximate length of 0.4 p n in solution. Electron microscopic pictures showed that there were some beaded structures in the filaments. Trinick et al. (1984) observed that the width of the connectin filament was 4-5 nm in negatively stained samples. C. OTHERPROPERTIES Connectin is soluble as filaments in KCl concentrations higher than 0.2 M at pH 7-8 and aggregates in lower concentrations of KCl. At 0.05 M KCl it precipitates. Even in 0.2 M KCl, shaking or agitation results in fiber-like aggregate formation. On concentration by a rotary evaporator, lateral association occurs leading to formation of an elastic rubber-like bundle. UV absorption spectra are of a protein nature with a maximum at 280 nm. The value of A,, at 1 mg/ml was approximately 1.2 (light path, 1 cm). A slight shoulder around 290 nm is always seen, but its origin is unknown. Amino acid composition shows that connectin is an acidic protein (Table 111). On the whole, connectin is similar to actin in amino acid composition: proline, TABLE 111 AMINOACIDCOMPOSITION OF CONNECTIN FROM CHICKEN BREASTMUSCLP

Native connectin Asx Thr Ser Glx Pro GlY Ala Cysl2

Val Met Ile Leu TYr Phe LYS

His Arg

96 76 60 111 74 74 65 2 87 12 60

66 31 26 86 15 59

Denatured connectinb 1

2

95 75 69 116 74 71 62 II 85 10 59 67 30 27 82 15 55

93 66 67 I I8 67 76 75 6 78 16 56 76 30 29 79

uNumber of residues per lo00 residues. bPrepared according to Wang et al. (1979).

18

50

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91

TABLE IV A M I N OACID COMFQSITIONS OF CONNECTIN, C-PROTEIN, AND PROJECT IN",^

Asx Thr Ser Glx Pro GlY Ala Cysl2 Val Met Ile Leu TYr Phe LYS His Arg

T

co

CI

c2

c3

P

95 75 69 I16 74 71 62 I1 85 10 59 67 30 27 82 15 55

93 66 67 118 67 76 75 6 78 16 56 76 30 29 79 18 50

96 59 57 I I7 71 71 66 14 I 04 17 51 69 29 37 86 14 44

99 58 62 I24 69 85 74 ND 85 13 43 68 27 35 89 17 52

I07 69 58 I I9 70 70 80 ND 68 18 62

I09 66 74 I25 76 93 68 8 53 12 36 69 27 32 87 23 51

64 35

32 93 15 48

UNumber of residues per 1000 residues. "T, Denatured connectin (chicken breast) (Maruyama c/ a/.. 1981); CO, native connectin (chicken breast) (Maruyama CI a / ., 1981 a); C I, C-protein (rabbit skeletal) (Offer cr a/.. 1973); C2, C-protein (rabbit white) (Callaway and Bechtel, 1981); C3, C-protein (rabbit red) (Callaway and Bechtel, 1981); P, projectin (Honeybee flight) (Saide, 1981); ND, not determined.

valine, and lysine are abundant and alanine, methionine, isoleucine, and tyrosine occur less in connectin than in actin. Methylhistidine is not present in connectin. A rather high content of proline (-9%) is in parallel with the lack in the a-helix portion (see below). The fact that connectin is rich in nonpolar amino acids may be related to its tendency to form salt-insoluble aggregates. A striking fact in the amino acid composition is that connectin is almost identical with C-protein (Table IV; Fig. 4). Although immunological crossreactivity was not detected using both antisera against connectin and C-protein, this fact is of some interest in view of their localizations in myofibrils (see Section VI). The amino acid composition of connectin is also very similar to projectin, an elastic protein of insect flight muscle (Table 1V; Fig. 4), whose MW is 360 kDa (Saide, 1981). This is of special interest in that both proteins serve as the elastic component of muscle. It is generally thought that a polypeptide of such a huge MW as connectin does not consist of a single peptide. It is reasonable to assume that several peptides are cross-linked as in collagen or elastin. The presence of hydroxylysinonorleucine,

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KOSCAK MARUYAMA

FIG. 4. Star diagrams of the amino acid compositions of connectin, C-protein, and projectin. Relative contents of the amino acids listed in Table IV are presented in star diagrams. ( I ) Denatured connectin (a + p). (2) native p-connectin, (3) projectin, (4) C-protein (rabbit skeletal), (5) C-protein (rabbit white), and (6) C-protein (rabbit red).

a cross-linker in collagen and elastin, was suggested by tritium incorporation experiments (Fujii and Maruyama, 1982). However, the amino acid analyses showed that the presence of the diamino acid was negligible (Maruyama et al., 1983). Gruen et al. (1982) also denied its presence. They could not detect glutamyllysine either. The carbohydrate content is very small and less than 1% by weight (Table V). Recently, Gassner et al. (1985) have emphasized the possibility of connectin as a glycoprotein. It is not clear whether the small amount of carbohydrates is covalently bonded to connectin or not (cf. Gassner, 1986). Circular dichroism measurements led Trinick and his associate (1984) to the conclusion that connectin completely consists of a random coil. This was an important finding. However, when we tried to measure circular dichroism in

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TABLE V CARBOHYDRATE CONTENTSI N CONNECTIN FROM CHICKEN BREASTMUSCLE"

Fucose Mannose Galactose Glucose GluNAc NeuNAc

Native connectin

Denatured connectinb

0.38 0.48 0.81 5.77 0.34 0

0.68 0.95 0.91 9.41

0.12 0

<'Dataare given as nrnol/rng dry weight. bPrepared according to Wang el al. (1979) (S Handa and S. Kimura, unpublished).

1983, it was not possible due to a strong orientation effect. Recently, we have obtained data by decreasing the protein concentration to 0.1 mg/ml in 0.5 M KCl. Trinick et al. (1984) used a connectin solution of 1 mg/ml for their study. The curve we obtained is quite different from that of Trinick et al. (1984), although the presence of a-helix is very small, if any (Fig. 5 ) . Although an accurate analysis is difficult, our curve suggests abundance of p-structure in addition to a smaller content of random coil (Maruyama et al., 1986). D. PROTEOLYSIS 1. Spontaneous Breakdown

Connectin is very easily degraded into smaller but still large fragments both in vivo and in vitro. As was first demonstrated by Wang et al. (1979), direct SDS extract of fresh whole muscle contains a- and p-connectins in an approximate ratio of 5: 1. In addition, a faint band just below p-connectin is always seen. This is tentatively called p'-connectin (Yoshidomi et a l . , 1985), but it has not yet been characterized (cf. Fig. 13). Isolated native p-connectin is mainly derived from a-connectin hydrolyzed in situ. Therefore, the question arises of whether p-connectin present in a SDS extract of whole muscle fibers is a real component of myofibrillar structural proteins or not. a-connectin might be partly degraded by intrinsic protease before its inactivation with SDS. This possibility cannot be ruled out, although we cannot readily agree with this view, because the ratio of p-connectin to aconnectin did not change by procedures of SDS extraction: muscle was first homogenized in water followed by addition of a SDS solution or directly homogenized in a SDS solution. Connectin was degraded into a form corresponding to

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FIG. 5 . Circular dichroism spectra of native connectin solution: 0.5 M KCI, 5 mM phosphate buffer, pH 7.5. From H. Yoshidomi (unpublished; cf. Maruyama et a / . . 1986).

the smear without the two distinct bands in the SDS-gel electrophoresis pattern, when stored in a SDS solution for more than a few hours at room temperature. It must be kept in the semisolid condition at 0°C. When chicken breast muscle was placed in the ice, degradation of a-connectin to p-connectin completed within a day, and only p-connectin was present after 8

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hours at 30°C (D. H. Hu, unpublished). This observation led us to prepare native (3-connectin effectively using muscle strips kept overnight at 0°C (Kimura et al., 1984b). On the other hand, it took 4-5 days at 0°C in well-washed myofibrils before a-connectin disappeared. Protease inhibitors, phenylmethylsulfonate (0.5 mM), p-chloromercuribenzoate (2 mM), leupeptin (10 pg/ml), and pepstatin (10 kg/ml) did not prevent the degradation at all. On the other hand, 1 mM CaCI, promoted it, although 10 mM EGTA only slightly delayed hydrolysis. Since calcium-activated neutral protease did not effectively hydrolyze a-connectin, it is still unknown what type of protease is responsible for in vivo hydrolysis of connectin. Seki and Watanabe (1984) observed that in carp muscle stored at 0°C aconnectin changed to (3-connectin within 10 hours, whereas it took 72 hours in rabbit skeletal muscle. a-Connectin was detected in a small amount in myofibrils prepared from bovine skeletal (longissimus) muscle stored for 3 days at 2°C and only a trace of a-connectin was present in myofibrils prepared from the muscle kept for 24 hours at 25°C (Lusby et al., 1983). Both a- and (3-connectins were largely broken down by heating bovine longissimus dorsi muscle for 40 minutes at 60 or 80°C (King et al., 1981; King, 1984; Locker and Wild, 1984). Isolated native connectin in solution is slowly degraded even at 0°C. It took 2 weeks at 0°C for (3-connectin to be split into 1900- and 400-kDa fragments that were similar to tryptic or chymotryptic products (see Section IV,D,2). 2. Effects of Proteases Various kinds of proteolytic enzymes, trypsin, chymotrypsin, papain, serine protease, and nagarse rapidly digested connectin in myofibrils. Interestingly, calcium-activated protease was not effective (Maruyama et al., 198Ib). Here, effects of trypsin and chymotrypsin will be described in detail. Both are very effective even in the weight ratio of 1:lOOO to chicken skeletal myofibrils. Trypsin ( I : 1000) digested a-connectin to (3-connectin within 1 minute at 25"C, and slowly split (3-connectin to 1900- and 400-kDa fragments. Chymotrypsin also acted in a similar way. In the weight ratio of 1:250, tryptic action produced I900-, 1700-, I400-, I 300-, I050-, 800-, 600-, and 400-kDa fragments, respectively. In the chymotryptic peptides, 1700- and 1300-kDa fragments were not present, and 1200-kDa fragments were produced. In both cases, the 1900- and 400-kDa fragments remained after prolonged hydrolysis, but this tendency was more evident in the chymotryptic action. Isolated native connectin was also very sensitive to trypsin and chymotrypsin. As seen in Fig. 6 , trypsin, in the weight ratio of 1:lOO or 1500, was more effective in splitting connectin than chymotrypsin. However, chymotrypsin in these weight ratios did not hydrolyze the 400-kDa fragment. Therefore, chymotryptic digestion has been used for the preparation of this fragment (Kimura et al., 1984b).

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FIG.6. Digestion of native p-connectin by trypsin and chymotrypsin. Connectin, 0.5 mg/ml, was incubated in 0.1 M sodium phosphate buffer, pH 7.0 at 25°C. The reaction was stopped by the addition of trypsin inhibitor, I mg/ml or 1 mM PMSF; 2% polyacrylamide gels were used. (a) Trypsin, 1/100 or 11500 by weight ratio; (b) chymotrypsin, 1/100 or 1/500 by weight ratio. Incubation time (minutes) is given under each lane. Lane C, intact sample. From H. Yoshidomi (unpublished).

V. Interaction with Myosin and Actin A. BINDING TO MYOSIN It was observed that connectin causes aggregation of myosin filaments around 50-120 mM KCI at pH 7.0. Turbidity increased and then gradually percipitation of aggregates occurred as seen in Fig. 7 (Kimura and Maruyama, 1983b; Kimura

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et al., 1984a). The turbidity measurements revealed that the action of connectin is effective up to 0.15 M KCl, and the KCl dependence is reversible: upon dilution to 75 mM KCl aggregation took place, and on elevation to 0.15 M KCl the suspension became clear again. The aggregate formation of myosin filaments was ascertained by flow birefringence measurements and directly visualized under an electron microscope (Fig. 7). Myosin filaments were randomly aggregated in the presence of connectin. The myosin aggregates are easily sedimented in the presence of connectin under conditions in which myosin filaments or connectin alone remained in the supernatant. Thus the extent of binding of connectin to myosin filaments was

Fic. 7 . Effects of native connectin on aggregation of myosin and actin filaments at 80 mM KCI. (a) 1, Myosin; 2, connectin: 3, 4, myosin and connectin; (b) I , actin; 2, connectin; 3, 4, actin and connectin; (c) myosin + connectin; (d) actin + connectin. Modified from Kimura and Maruyama (1983b) and Kimura e r a / . (1984a).

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studied quantitatively. There was a saturation around 0.12 mg connectin per 0. I mg myosin. This observation indicates a stoichiometric ratio of 0.3 mol of connectin per mol of myosin (Yoshidomi and Maruyama, 1985). Assuming that a myosin filament consists of 300 myosin monomers, as much as 90 connectin filaments can bind to one myosin filament. This value is much greater than the content of connectin relative to myosin in myofibrils (12 connectin filaments per myosin filament). Thick filaments in sarcomere are associated with several regulatory proteins of which C-protein is the most abundant (Offer et a l . , 1973). It is of interest whether C-protein affects the binding of connectin to a myosin filament. So far, there was no difference in the binding of connectin to C-protein-free and Cprotein-bound myosin filaments. Although binding of C-protein to connectin was reported (Koretz and Wang, 1984), we could not confirm this (Yoshidomi and Maruyama, 1985). The interaction of connectin with myosin appeared to be electrostatic. Using various neutral salt ions, such as K + , Na+, Li+, C1-, Br-, I - , SO:-, and citrate it was observed that the interaction depends on ionic strength but not on molar concentrations (Y. P. Huang, unpublished). It is well known that a myosin molecule consists of head S , , neck S , , and rods. We have examined the binding of connectin to these subfragments (Maruyama et al., 1985a). The head portion did not interact with connectin at all, whereas L-meromyosin and the rod portion effectively caused aggregation of myosin filaments. The neck S , fragments formed some complex with connectin at 0.04-0.06 M KCl. Heavy meromyosin formed a complex with connectin in the same range. This was likely due to contaminated light meromyosin that interacted with connectin in the presence of KCI lower than 0.15 M. Thus connectin binds to the tail portion of myosin that forms a filamentous structure. So far p-connectin (2100 kDa) is used in experiments for the interaction with myosin. Does a smaller fragment of connectin filament bind to myosin filament? The answer is yes. The 400-kDa fragment formed by chymotryptic action has been isolated by gel filtration (Kimura et al., 1984b). The one-fifth fragments reacted with myosin filaments to form sediment. However, smaller fragments of chain weights of 100 kDa or less did not cause aggregation of myosin. Finally, we should mention that a crude myosin preparation is easily precipitable upon dilution to an ionic strength of less than 0.05, whereas purified myosin is not. These familiar phenomena may be explained by myosin-aggregating action of connectin.

B. BINDINGTO ACTIN Actin filaments are also affected by connectin filaments in the presence of KCI up to 0.15 M (Kimura and Maruyama, 1983b; Kimura et al., 1984a). The turbidity increased and flocculent precipitates were formed when kept standing

CONNECTIN, AN ELASTIC FILAMENTOUS PROTEIN

99

-0-0-

I

5

10

Time(min)

15

20

FIG. 8. Effects of native connectin on the degree of flow birefringence of an F-actin solution. Factin. 0.25 mglml, 40 mM KCI, 10 mM phosphate buffer. At the first arrow connectin, 0.1 mglml, was added. At the second arrow KCI was added to give a final concentration of 150 mM. Velocity gradient, 100 seconds - I . From K . Maruyama (unpublished).

for a few hours. As shown in Fig. 8, degree of flow birefringence of an F-actin solution decreased by addition of connectin at a low velocity gradient just as by a-actinin (Maruyama and Ebashi, 1965). In fact, bundles of actin filaments are formed (Fig. 7). The effects of connectin on F-actin were also dependent on KCl concentrations as on myosin. The bundling action of connectin disappeared by raising the KCl concentration to 0.15 M (Fig. 8). Distinct from the action of aactinin (Drabikowski et al., 1968), the bundle formation by connectin was not affected by tropomyosin at all (H. Yoshidomi, unpublished). On the other hand, 400-kDa fragments of connectin did not exert any action on actin filaments at 50-120 mM KCl (Kimura er al., 1984b). Therefore, it is believed that flexible connectin filaments enhance an intrinsic tendency of bundle formation of actin filaments. The effect of connectin filaments on actin filaments may be attributed to topological restrictions. It may not be physiologically significant. Kimura et al. (1984a) reported that connectin enhances the onset of superprecipitation of actomyosin by MgATP resulting in an increase in the ATPase activity. The extent of this elevation was to a much smaller extent than a-actinin (Maruyama, 1966). Therefore, this effect of connectin appears to be an artifact in vitro.

VI. Location in Myofibrils A. IMMUNOFLUORESCENCE

STUDY

A simple method to locate a given protein in situ is an immunofluorescence observation using fluorescein isothiocyanate (F1TC)-labeled antibodies against

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KOSCAK MARUYAMA

the protein. Anti-connectin was first prepared using cut top gels of salt-extracted muscle residues after electrophoresis in the presence of SDS (Maruyama et al., 1977a, 1980). Wang et al. (1979) used denatured connectin isolated by gel filtration in the presence of SDS. The two groups showed that connectin is most concentrated at the A-I junstion area in a sarcomere. It is also present in the A band except for the center region. The 1 band is only faintly fluorescent. We have observed that antiserum against chicken skeletal muscle connectin reacted with frog skeletal muscle connectin. When skinned muscle fibers of the frog were highly stretched so that gaps were formed between the A and I bands (sarcomere length, 3.5 pm), treatment with antibodies resulted in an elongation of an A band from 1.6 to 2.8 pm, as shown in Fig. 9 (Maruyama et al., 1984b). This clearly indicates that connectin filaments are present in the gap region. In fact, as presented in Fig. 9, very thin filaments are seen in this area in confirmation with Sjostrand ( 1962). More direct evidence that anti-connectin antibodies were deposited on the gap filament was reported (La Salle et al., 1983; Wang, 1985).

FIG. 9. “Gap filaments” in stretched myofibrils of frog skeletal muscle and immunofluorescence location of connectin in stretched myofibrils. ( I ) Thin section. Bar, 0.5 pm. (2) a, phase contrast image of a myofibril; b, phase contrast image of anti-connectin-treated myofibril; c , fluorescent image ofanti-connectin-treated myofibril. Bar, 10 pm. Modified from Maruyama ei a/. (1984b).

CONNECTIN, AN ELASTIC FILAMENTOUS PROTEIN

101

When myosin and actin are thoroughly extracted away from myofibrils, the strongest fluorescence is observed at both sides of the Z lines (Maruyama et af., 1977a, 1984b; Wang et af.,1979). This turned out to be due to accumulation of residual myosin, actin, and other proteins together with connectin (cf. Granger and Lazarides, 1978; Hattori and Takahashi, 1979). Fluorescent fiber-like structures are seen between Z lines. On the contrary, when myosin was removed, connectin was shown to be translocated to the M band region (Wang, 1985).

B. ELECTRONMICROSCOPY Trinick ( I 98 1) described “end-filament” attached to isolated thick filaments, 5 nm wide and 85 nm long. Later, Trinick and his associates (1984) identified the end-filaments as connectin filaments by comparison with isolated connectin under an electron microscope using negatively stained samples. Three to four connectin filaments are extended from each end of a thick filament of rabbit psoas myofibrils. These observations are in good agreement with our observations on thin sections of stretched fibers of frog muscle (Maruyama et af., 1984b). The question every investigator in this field has raised is where connectin filaments starting from thick filaments are terminated. Locker (cf. 1984a) and Maruyama and Kimura (1985) assumed that connectin filaments are directly linked to Z disks. On the other hand, Wang (1984, 1985) has claimed that connectin is attached to nebulin meshwork and eventually linked to the Z disk. Moreover, Wang ( I 985) assumes that a connectin filament spans from an N, line to other N, lines in a sarcomere. The difficulty is that connectin filaments cannot be identified in intact 1 bands because of the presence of many actin filaments. Also its exact location on a myosin filament remains obscure. We have prepared thin sections of anti-connectin-treated muscle fibers of frog skeletal muscle. First there was no sign of antibody staining in a sarcomere (Maruyama et al., 1984b). However, it was noticed that antibody deposits were only restricted to a few layers of peripheral myofibrils (Maruyama et af., 1985b), as seen in Fig. 10. Evidently, penetration of antibodies into myofibrillar bundles was very slow. Several distinct stripes were symmetrically present in each half of the A band of anti-connectin-treatedmuscle fiber at rest length (Fig. 10).The first stripe was seen approximately 0.15 pm away from the center of the A band. Thus the two stripes are at the edges of a pseudo H zone. The positions of other stripes from the center of the A band were approximately 0.3, 0.6, 0.7, 0.8, 0.85, and 0.9 pm, respectively. The latter two were outside of the A band. These bands were more markedly recognized in antiserum-treated myofibrils swollen at a low ionic strength (Fig. lo), where diffusion of antibodies was more rapid than in intact fibers.

FIG. 10. Immunoelectron micrographs of anti-connectin-treated myofibrils of frog skeletal muscle. (a) Rest length myofibrils; (b) stretched myofibrils; (c) low ionic strength extracted myofibrils. Bar, 1 pm. From T. Yoshioka (unpublished; cf. Maruyama et al., 1985b).

CONNECTIN, AN ELASTIC FILAMENTOUS PROTEIN

103

More important were the deposits of antibodies in I bands and Z lines (Fig. lo), clearly indicating that antigenic sites distribute all the way through Z lines. Stretched fibers did not show regular stripes at all. Antibody labeling was randomized because myosin filaments were pulled randomly to either side of Z disks (Fig. 10). These observations with polyclonal antibodies against connectin show that connectin filaments directly link each end of a myosin filament to its neighboring Z disk as an elastic component.

VII. Connectin as an Elastic Component As Natori (1954) first showed, there is an elastic component in myofibrils that results in passive tension generation upon extreme stretch and also in the return of separated thick and thin filaments to the original state on release (cf. Rb. Natori et al., 1980; Yoshioka et a/., 1981). First we postulated that an elastic filament like connectin directly links neighboring Z disks in a sarcomere (Maruyama et al., 1976). Electron microscopic observations suggest the presence of relatively few filaments running through Z disks in myosin- and actin-extracted myofibrils (cf. Wang and Ramirez-Mitchell, 1983). Wang and his associate (1983) regarded the remaining filaments as intermediate filaments (cf. Price and Sanger, 1979). However, intermediate filaments are known to connect adjacent Z disks at the periphery of a myofibril in cardiac muscles (Tokuyasu, 1983), but not in skeletal muscle except in early stages of embryonic development (Tokuyasu er d., 1984). Therefore, Wang’s view is not valid. Although the span of a few connectin filaments between adjacent Z disks is not definitely denied, the remaining filaments are likely to be associated connectin filaments freed from thick filaments. Some myosin or actin might be trapped in them. It is true that those remaining filaments connect KI-extracted Z disks in ghost myofibrils (Wang and Ramirez-Mitchell, 1983; Maruyama et a/., 1984b). Our work using polyclonal antibodies against connectin has definitely shown that connectin filaments directly link myosin filaments to Z lines starting from 0. I5 k m away from the center of the A band in a symmetrical way (Maruyama et al., 1985b). The presence of distinct stripes in each half of a sarcomere indicates that connectin filaments are in register along myosin filaments at rest length (see Fig. 10). A tentative model of connectin filaments in a sarcomere is depicted in Fig. 1 1 . However, we cannot exclude the possibility that a connectin filament

c--

FIG I I

I A model of connectin filaments

in

a sarcomere

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KOSCAK MARUYAMA

spans a whole sarcomere by way of its association with thick filament between adjacent Z disks. If so, a connectin filament must consist of an end-to-end (or head-to-head) dimer as judged by symmetrical distributions of epitopes (Wang and Ramirez-Mitchell, 1984; Maruyama et al., 1985b). Locker (1984a) and Magid et al. (1984) have postulated that connectin is present in the core of a thick filament. This possibility is not excluded, but the presence of connectin filaments has been observed alongside the end region of a thick filament (Trinick et al., 1984; Maruyama et al., 1984b). Therefore, at least several connectin filaments attach to the surface of a thick filament starting from the edge of the center bare zone (Maruyama et al., 1985b). Taking the MW values of connectin and myosin as 3 X lo6 and 5 X lo5,their contents of 10 and 44% in the total myofibrillar proteins, and 300 myosin monomers to form a thick filament, the presence of 12 connectin filaments per thick filaments is calculated. It is expected that six connectin filaments are present in each half of the thick filament. In the above calculation, it is assumed that a connectin filament consists of a single chain of molecule. Recent work using monoclonal antibodies has showed the specific location of each species as a pair of symmetrical stripes at the I band, A-I junction, and A band (Wang and Ramirez-Mitchell, 1984). This stimulating observation strongly suggests that a long peptide of connectin with one epitope for one species of monoclonal antibody extends from the myosin filament to the Z disk. Since a connectin molecule does not contain any a-helical structure but consists of random coil (Trinick et al., 1984) and p structure (Maruyama et a l . , 1986), a single peptide of 3 X lo6 MW could have a length of up to 7 pm. At rest length, half sarcomere length is about 1.2 pm and at extreme stretch it is 3.5 pm. Therefore, connectin filaments must be compactly folded in a sarcomere. Trinick (1981) pointed out that “end-filament” has beaded structure, and this has been confirmed in isolated connectin filaments (Trinick et al., 1984; Wang er al., 1984). In this connection, it is of special interest to determine whether the pspiral structure in elastin fibers is present in connectin filament or not (cf. uny, 1984). Higuchi and Umazume (1985) made an important observation on the passive tension generation by stretch of skinned muscle fibers of the bullfrog. A brief extraction of myosin with a salt solution resulted in a decrease in tension development in proportion to the amount of myosin extracted. Only 10% of tension generation remained after almost complete removal of myosin. The decrease in tension generation can easily be explained by the release of connectin filaments at the attachment site on the myosin filament. It has already been indicated that some connectin is solubilized together with myosin. Another significant aspect of passive tension generation of skinned muscle fibers is the effect of a brief trypsin digestion. Mild tryptic treatment (0.2 pg/ml) led to the decrease in passive tension generation (Fig. 12). At the same time, a-connectin was de-

105

CONNECTIN, AN ELASTIC FILAMENTOUS PROTEIN

1.o

0

I

1

3

5

7

(min)

C

.-0 v)

C

al I-

0-l

I

1

1

r

5 Trypsin Treatment

I

10 ( m i d

FIG.12. Decrease in generation of passive tension and hydrolysis of connectin during trypsin treatment of skinned fibers of frog skeletal muscle. SDS-gel electrophoresis patterns show progressive breakdown of a- and p-connectins. From H. Higuchi and S . Kimura (unpublished).

graded to p-connectin. Under this mild tryptic digestion, any other proteins including nebulin were not hydrolyzed. This fact strongly suggests that a-connectin filaments serve as an elastic component in a sarcomere. It is to be noted that a string of glycerinated myofibril from rabbit psoas muscle contracted into a small mass on addition of MgATP, whereas after a mild treatment with trypsin it fell into a number of small pieces in the presence of MgATP (Maruyama and Yamamoto, 1979). This observation again suggests that a trypsin-sensitive structure is involved in the elastic continuity of a myofibril. The change in length of connectin filament during contraction and relaxation has been shown in cardiac myofibrils using a fluorescent stripe stained with monoclonal antibodies as marker (S. M. Wang and Greaser, 1986).

VIII. Connectin Transformation during Differentiation A. CHANGES DURING DEVELOPMENT OF THE CHICK It has been well established that a number of muscle structural proteins undergo characteristic changes in isoform expression during embryonic and neonatal development (cf. Obinata et al., 1984). We have investigated changes in connectin isoforms during embryonic and

106

KOSCAK MARUYAMA

a

b

C

Fic. 13. Connectin isoforms of embryonic, neonatal, and adult skeletal muscles. (a) Just hatched chick. Note that the uppermost band is embryonic cx-connectin and the band just below is neonatal aconnectin. Two faint bands, embryonic P- and P'-connectins are seen. (b) a + c. ( c ) Adult connectin. Note that there is adult P'-connectin just below p-connectin. Modified from Yoshidomi c'r a/. ( 1985).

neonatal development of the chick using SDS-gel electrophoresis checked by an immunoblot technique (Yoshidomi et al., 1985). Although the band was very faint, connectin-like high-molecular-weight protein was detected in breast muscles of 7-day incubated chick embryo. Cruen e?al. (1982) reported that connectin is expressed in 7.5-week-old sheep fetus much later than the appearance of myosin. The 13-day chick embryo has bands which reacted with antiserum against adult breast muscle connectin. The embryonic a-connectin showed slower mobility in SDS-gel electrophoresis than the adult one, as clearly seen in Fig. 13. Its apparent MW was estimated to be as high as 3.4 million (Table VI). This embryonic a-connectin is present up to 3 days posthatch. It is of some interest to note that the high-MW band of embryonic a-connectin corresponds to that of slow muscle (Section IX,A). In developing chick fast muscle, the first cardiac TABLE VI APPARENT MOLECULAR WEIGHTSOF CONNECTIN ISOFORMSI X J R I N G DEVELOPMENT OF THE CHICK"

Embryonic Neonatal Adult

a

P

P'

3.4 3.1 2.8

2.4 2.4 2. I

2. I 2.1 2.0

G i v e n as million (lo6).

CONNECTIN, AN ELASTIC FILAMENTOUS PROTEIN

107

type of C-protein is expressed, followed by the slow muscle type; finally the fast muscle type is expressed (Obinata et a/., 1984). Unfortunately, our polyclonal antibodies are not tissue and species specific and, therefore, tissue specificity cannot be distinguished. Neonatal a-connectin appears just after hatch and is present up to 7 days posthatch (Fig. 13). Adult type a-connectin, its MW being slightly smaller than the neonatal one, is expressed at 5 days posthatch. Changes in p-connectin are more complicated. First, on close examination, the connectin bands are not doublet bands as claimed before (Wang et al., 1979). There is another faint band just below the p band (Fig. 13). This p’ band appears not to be a proteolytic product of a-connectin, because the amount does not change when a-connectin is degraded (H. Yoshidomi, unpublished). Adult pconnectin appears at 5 days posthatch. In 17-day embryo, there are two p bands: the mobility of the lower band is the same as that of adult p-connectin. Although this band is not distinguishable from adult p, it is tentatively called embryonic p’, and the upper embryo-specific p band is termed embryonic p, which is present up to 3 days posthatch. The changes in the types of connectin during development of the chick are summarized in Table VI. The relative amount of connectin markedly increased after hatch reaching a value of adult connectin (10% of the total myofibrillar protein) at 7-10 days after hatch.

B . CHANGES DURING MYOFIBRILLOGENESIS Recently, Greaser and his associates (S. M. Wang et al., 1984) have studied the fate of connectin during myofibrillar differentiation in cultured rat leg muscle cells, using fluorescent monoclonal antibodies against bovine cardiac connectin (S. M. Wang and Greaser, 1985). Newly synthesized connectin filaments are scattered randomly in the cytoplasm, and then these filaments gradually associate with actin and myosin to form longitudinal bundles. As myofibrils are formed, the banded patterns of connectin at the A-I junction area are observed. Sometimes, the appearance of connectin periodicity preceded the band formations of myosin and actin. This suggests an important role of connectin in sarcomere assembly. Immunoelectron microscopic examinations will be of great help in understanding the situation.

IX. Comparative Biochemistry A. GENERAL SURVEY There are two ways to detect connectin in various types of tissues or organisms: detection of high-molecular-weight protein bands in 2-3% polyacrylamide gels in the presence of SDS preferably with confirmation by an

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KOSCAK MARUYAMA

immunoblot and also by immunofluorescenceexaminations of fixed cells. Antibodies raised against chicken breast muscle connectin cross-reacted with frog leg muscle connectin (Maruyama et al., 1984b). Ikeya et al. (1983) investigated cryostat sections of chicken tissues using an immunofluorescence technique: connectin was detected only in striated muscles, breast, anterior latissimus dorsi (ALD), and cardiac muscles. There was no sign of connectin in tendon, blood vessels, gizzard, nerves, and liver. A SDS-gel electrophoresis survey confirmed these observations (Hu et al., 1986a). It is rather surprising that connectin is specific for striated muscle just like troponin. S. M. Wang et al. (1984) also noticed that connectin is not found in cultured nonmuscle cells. It is noteworthy to mention “high-molecular-weight (HMW)” proteins in chicken gizzard. As already reported by Wang et al. (1979), total SDS extract of fresh gizzard does not contain any HMW bands corresponding to a MW of 1 million in SDS-gel electrophoresis. However, when gizzard was thoroughly extracted with Hasselbach-Schneider solution and 0.6 M KI to extract myosin and actin followed by 1 N acetic acid (to remove desmin), connectin-like HMW band appeared at the top of the gel. The fraction was separated by gel filtration in the presence of SDS. Amino acid analyses revealed that it contained hydroxyproline by as much as lo%, suggesting that the HMW protein in question was crosslinked collagen (Y. Kuwano and K. Maruyama, unpublished). Our earlier observation that salt- and acid-extracted residue of gizzard contained connectin (Maruyama et al., 1977b) is no longer valid. SDS-electrophoresis examinations showed that slow muscle (ALD) a-connectin has a larger MW than fast breast muscle a-connectin. Chicken cardiac aand p-connectins have the same molecular weights as breast muscle ones, but there is no p’-connectin. In all the vertebrate skeletal muscles examined, connectin has been detected in SDS-gel electrophoresis: rabbit (Wang et al., 1979; Trinick et al., 1984), bovine (Locker and Daines, 1980; Lusby et al., 1983; King, 1984), rat (Gruen et al., 1982), sheep (King and Kurth, 1980; King etal., 1981), chicken (Maruyama et al., 1981a, 1984a), snake (Hu et al., 1986a), frog (Maruyama et al., 1984b), and carp (Seki and Watanabe, 1984). In invertebrates, detailed studies have not yet been made except for a brief note: insect (Wang et al., 1979; Locker and Wild, 1986; Hu et al., 1986a); crayfish (Hu et al., 1986a); C . elegans (Hu et al., 1986a), and chordate, Amphioxus (Hu et al., 1986a).

B. NATIVECONNECTIN FROM CARDIAC MUSCLE Native connectin has been purified from pig heart (Ito et al., 1986). The method used was the same as that for the preparation from chicken breast muscle (Kimura et al., 1984b) except that it was not necessary to keep excised muscle overnight at 0°C. It appears that a-connectin is rapidly degraded to p-connectin

CONNECTIN, AN ELASTIC FILAMENTOUS PROTEIN

109

in cardiac muscle. Myofibrils were prepared from pig heart immediately after delivery from a local slaughter house. The yield was approximately 80 mg starting from 100 g of muscle. The SDS-gel electrophoresis pattern showed that cardiac native connectin moved at the same mobility as chicken breast muscle p-connectin. A negatively stained sample showed under an electron microscope that there were entanglements of very thin filaments as elegantly presented by Trinick et a f . (1984). Surprisingly, the amino acid composition was almost identical with that of chicken native connectin (Table IV). Cardiac connectin also interacted with both myosin and actin filaments in the presence of 50-150 mM KCl at pH 7.0 and 25°C.

C. Physarum PLASMODIAL PROTEIN Ozaki and Maruyama ( 1980) first indicated that salt-extracted residues of plasmodium of the slime mold Physarum polycephalum contained a SDS-insoluble material from which acidic polysaccharides could be separated by centrifugation as transparent gels. These SDS-insoluble residues could be partly dispersed in 6 M guanidine-HCI. Under an electron microscope, entanglements of very thin filaments were observed. Its amino acid composition was very similar to that of muscle connectin (Table VII). Therefore, it is assumed that there are connectin-like filaments in Physarum plasmodium. Recently, Wohlfarth-Bottermann’s school has been able to isolate connectinlike protein from cytoplasmic droplets of plasmodium in the presence of SDS (Gassner et a f . , 1985; Gassner, 1986). The HMW protein isolated from a SDS extract of Physarum plasmodium freed of extracellular slime formed huge, elastic, gel-like aggregate after removal of SDS by dialysis. Electron microscopy by negative staining and by rotary shadowing revealed the presence of superthin filaments 2-3 nm wide. Gassner et a f . (1985) emphasized that the isolated superthin filaments closely resemble those observed in plasmodia1 endoplasmic droplets and also in their SDS-treated ghost cytoskeletal matrix. It is suggested that Physarum connectin-like filament is a major component in the elastic cytoskeletal matrix. We have confirmed the above result (Hu et a f . , 1986b) using whole plasmodium. A homogenate in 1 mM NaHCO, was briefly centrifuged and the precipitate was used for the starting material. To the precipitate SDS was added, and the solution was centrifuged for 2 hours at 200,000 g to sediment the polysaccharides. The supernatant was subjected to gel filtration to isolate connectin-like protein. The amino acid composition was similar to that reported by Gassner et a f . ( 1985) and also to that by Ozaki and Maruyama (1980) (Table VII). Negatively stained sample showed a thin filamentous structure under an electron microscope. Native HMW protein has been obtained from direct gel filtration of a bicarbo-

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KOSCAK MARUYAMA

AMINOACIDCOMPoSlTlON

Asx Thr Ser Glx Pro GIY

Ala Val Met Ile

Leu

TYr Phe LYS His A%

TABLE VII Physarum

OF

SDS insoluble('

Isolatedh

100 64 67 116 62 70 84 60 18 49 88 29 42 74 24 49

loo 55 68 126 63 73 84 58 17 48 86 29 39 77 26 50

CONNECTIN-LIKE PROTEIN

SDS insoluble matrices" 101

53 70 I28 61 80 96 52 14 37 83 25 34 71 34 60

Isolated'' 102 58 69 I28 60 72 90 58 20 45 86 27 38 71 38 60

OOzaki and Maruyama (1980). bD.H. Hu and S . Kimuras (unpublished). G a m e r et al. (1985).

nate extract of plasmodium (Hu et al., 1986b). This fraction caused aggregation of rabbit skeletal muscle myosin as muscle connectin. Thus, it is very likely that connectin-like protein is present in the slime mold. However, judging from the mobility in SDS-gel electrophoresis and the presence of an SDS-insoluble form, Physarum connectin-like proteins are covalently cross-linked.

X. Perspectives Connectin (also called titin) has a long history of doubt, neglect, and revival. Some of the pioneering researchers include Natori (Japan), Sjostrand (United States and Sweden), Hoyle (United States), Guba (Hungary), dos Remedios (Australia), and Locker (New Zealand). Even after the present writer took up this elastic protein in 1976, followed by Wang from a different point of view in 1979, most scientists were and still are reluctant to accept it. The present writer feels that this reluctance is rather natural because the protein molecule in question is very large and quite outside the reasonable range of sizes of hitherto known proteins and, in addition, its width (5 nm) is near the limit of resolution under an

CONNECTIN, AN ELASTIC FILAMENTOUS PROTEIN

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electron microscope. It may be added that all the workers who followed our and Wang’s research so far have belonged to meat science laboratories: Takahashi (Japan), King (Australia), Locker (New Zealand), Greaser (United States), Robson (United States), and Trinick (United Kingdom). Is there really a single peptide of three million MW consisting of more than 20,000 amino acids? Wang’s elegant work showing the epitope distribution corresponding to each of monoclonal antibodies (Wang and Ramirez-Mitchell, 1984) strongly suggests so. From a protein chemical view it is extremely difficult to test this problem simply because of the size and tendency of degradation. Despite difficulty, gene cloning will give a final answer. At present we have not yet been successful in the isolation of the mother molecule, a-connectin. What has been isolated independently in Japan, the United States, and the United Kingdom is its proteolytic product, p-connectin (Kimura and Maruyama, 1983a; Wang et al., 1984; Trinick et al., 1984). aConnectin is not easily soluble in salt solution, but a small amount can be solubilized together with its degradation peptides (S. Kimura, unpublished). Therefore, isolation of a-connectin does not seem impossible. Returning to the connectin filaments in a sarcomere of striated muscle, details of the structure have remained obscure. First, are they present as a singlestranded filament consisting of random coil and p-structure? In the rest state, the length may be in the order of 1 pm and it may be extended to longer than 3 pm when stretched. To generate tension, an elastic structure should be present in connectin filaments. One of these candidates is P-spiral structure in elastin fibers (Urry, 1984), since connectin is rich in P-sheet structure (Maruyama et al., 1986). Connectin filaments directly link myosin filaments to Z disks in a sarcomere. How does this attach to the Z disk? Is there any cementing substance? Connectin filaments bind to the rod portion of myosin filaments, but details of the manner of binding are not known, It is also possible that a few connectin filaments exist in a core of myosin filament (Locker, 1984a; Magid et al., 1984). It is known that there are high-density areas called N , and N, lines in the 1 band (cf. Locker, 1984b; Cooke, 1985). Nebulin constitutes an N, line (Wang and Willaimson, 1980). Wang (1984, 1985) assumes that connectin filaments are linked to nebulin meshwork. At present the nature of native nebulin is completely unknown. Furthermore, as pointed out by Locker (1984b), there is no nebulin in cardiac myofibrils. We have also observed that the content of nebulin in cardiac myofibrils is very small, if any (Hu et a/., 1986a). It is possible that connectin attaches to both N , and N, lines in skeletal muscle and this is a problem that needs clarification. The mechanism of myofibrillar formation during differentiation of muscle cells is of vital importance from a biological point of view. Greaser’s school has presented very suggestive evidence that connectin filaments are deeply con-

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KOSCAK MARUYAMA

cerned in the sarcomeric assembly of myosin and actin filaments (S. M. Wang el al., 1984). More detailed work is due along this line. Finally, from the comparative view of cell motility, the presence of connectinlike superthin filaments in the slime mold is of special interest. A thorough investigation is desirable. Are there similar type of filaments in the amoeba and other motile cells?

ACKNQWLE~GMENTS

I express my cordial thanks to Dr. S. Kimura for painful joint work and also to other members of our laboratory for their cooperation. I am much indebted to Professors R. Natori of Jikei Medical School, S. Ebashi of the National Institute for Physiological Sciences, and Sir Andrew Huxley of Trinity College, Cambridge, for their constant encouragement. I am deeply indebted to Dr. H. Sawada of the University of Tokyo for his constant help in the present work. Finally, acknowledgments are due to Professor H. Noda of the University of the Air for his critical reading of the manuscript.

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