- Email: [email protected]
Titin-related proteins in invertebrate muscles
Camp. Biochem. Physiol. Vol. 109A. No. 4, pp. 823-833. 1994 Elwier Science Ltd Printed in Great Britain 0300-9629/94 $26.00 + 0.00
REVIEW Titin-related
proteins in invertebrate
muscles
Clemens Ziegler Institute of Animal Physiology, Hindenburgplatz
55, D-48143 Miinster, F.R.G.
The localization of filaments connecting the Z-line and the A-band in insect flight muscles and the identification of very large proteins as their components is reviewed. The characterization of twitchin in the obliquely striated muscles of Cuenorhabditis ekgans is reported and the deductions made from its amino acid sequence are considered. The characterization of mini-titins in obliquely striated molhrscan muscles is compared, The identification of projectin in the muscles of Drosophila mefunogaster by anti-twit&in-antibodies, its sequence analysis and the characterization of mini-titins in arthropod and mollusc fast-striated muscles are summarized. The possible biological functions of the different proteins in various invertebrate muscles are discussed. Key words: Twitchin;
Titin; Mini-titin; Projectin; Connectin; Connecting filaments; Insect flight muscle; Obliquely striated muscle; Drosophila melanogaster; Caenorhabditis elegans. Comp. Biochem. Physiol. 109A, 823-833,
1994.
Introduction into a group of direct and indirect flight muscles. The indirect flight muscles may then be further subdivided into a group of non-fibrillar synchronous and a group of fibrillar, stretch-activated asynchronous flight muscles. The direct flight muscles are not considered in this review. The second main group comprises the obliquely striated muscles of worms and molluscs which-in the case of the wormscan be subdivided into the vulval, anal, body wall and the pharynx muscles, and, in the case of the molluscs, into the motor muscles (for instance byssus retractor) and the very specialized “catch” muscle which is able to develop strong tension with a minimum of ATPase activity. It was among the fibrillar, asynchronous Correspondence to: C. Ziegler, Institute of Animal flight muscles of insects where a third Physiology, Hindenburgplatz 55, D-48143 Miinfilament system was detected that was ster, F.R.G. Tel. 251833878; Fax 251838371. to constitute the morphological Received 22 January 1994; revised 4 July 1994; thought accepted 18 July 1994. basis of stretch activation (Auber and
A survey of the invertebrate titin-relatives first necessitates a general review of the different muscle constructions in the invertebrates. In this review, most of the known invertebrate muscles are therefore assigned to various groups. The first main group is that of striated muscles which may be subdivided into a group of the flight and a group of the non-flight muscles. To the latter, the claw, leg and the other striated muscles of arthropods, the supercontractile gut muscles and the fast striated muscles of molluscs will be added. The group of insect flight muscles may be further subdivided
823
824
C. Ziegler
Couteaux, 1962,1963). The filaments of this system could be seen as elastic structures of variable length, connecting the ends of the thick filaments and the Z-line (Garamviilgyi, 1965a,b). This very instructive concept led to the discovery of titin in vertebrate muscles (Wang et al., 1979, “Connectin”, Maruyama et al., 1976,1977a,b, 1981). This giant protein connects the A-band and the Z-line and reaches up to the M-line of vertebrate skeletal muscles. In these muscles, the accessory C-, X- and H-proteins and an 86 kDa protein were shown to share epitopes with titin when probed with the respective monospecific or monoclonal antibodies (Offer et al., 1973; Starr and Offer, 1983; BBhler et al., 1985a,b; Fiirst et al., 1988, 1989a; Nave et al., 1989; Vinkemeier et al., 1993). Meanwhile, attempts to characterize the proteins from invertebrate muscles that are able to maintain myofibrillar elasticity and muscle cell structure have led to different conclusions depending on the type of muscle which was studied. Genomic analysis, immunohistochemistry as well as characterization of purified proteins, led to an enhanced but often contradictory understanding of the possible functions of the previously described connecting proteins and their close relatives from other invertebrate muscles. As a result of these different approaches and the multiplicity of more or less specialized invertebrate muscles, the denomination of proteins which are thought to play a role in the formation of connecting filaments and related structures is manifold. “Projectin” and “mini-titin” (Saide, 1981; Nave and Weber, 1990; Ziegler et al., 1990) have been identified in fibrillar and non-fibrillar insect flight muscles (classification according to Pringle, 1977). The very closely related proteins from the invertebrate non-fibrillar, nonflight muscles which rarely exhibit connecting filaments, and from the obliquely straited muscles, which, until recently, were shown to lack connecting filaments, are also called “projectin” and “mini-titin” as well as “connectin” or “twitchin” (Benian et al., 1989; Hu et al., 1990; Saide et al., 1989, 1990; Vigoreaux et al., 1991; Nave et al., 1991; Manabe et al., 1993; Vibert et al., 1993). When sequences of these proteins, as well as those from vertebrate M-protein,
skelemin, myosin light chain kinase, myomesin and nematode twitchin, are compared, it is possible to assign all of them to a large group of very similar proteins which are apparently responsible for the development of cellular shape and are related to cell adhesion and recognition proteins. the proteins of this Characteristically, group are characterized by a pattern of two repeating motifs (Benian et al., 1989; Einheber and Fischman, 1990, 1991; Okagaki et al., 1993; Tan et al., 1993; Price, 1987; Price et al., 1990; Price and Gomer, 1993; Olson et al., 1990; Shoemaker et al., 1990; Lakey et al., 1993; Hill et al., 1986; Fi_irst et al., 1989b; Schultheiss et al., 1990). Past studies on the identification and characterization of various invertebrate proteins from this group are reviewed, and their significance in the functioning of invertebrate muscles is considered.
Fibrillar Insect Flight Muscles as Objects of Identification and Localization of Connecting Filament Proteins Since their detection in the asynchronous flight muscles of the fly Calliphora erythrocephala (Auber and Couteaux, 1962, 1963), biochemical studies on the proteins from invertebrate muscles have been typically aggravated by limited amounts of muscle tissue. As a result, the molecular basis of connecting filaments in such muscles was primarily studied in the water-bug (Lethocerus maximus), the dung beetle (Heliocopris japetus) and the honey-bee (Apis mellijica). These species are particularly useful because of their size and the predominance of the flight muscles which permit isolation of reasonable amounts of homogeneous myofibers. Dung beetle fibrillar flight muscles, which were used for paramyosin purification and then stained with anti-paramyosin antibodies, exhibited epitopes at the M-line and at the ends of the thick filaments. Only staining in the centre of the sarcomeres was thought to show epitopes for paramyosin. This led to the conclusion that paramyosin, which constitutes a considerable proportion of the filament mass, should be localized within the core of the thick filaments. The staining at the ends was thought to be
Titin-related proteins in invertebrate muscles
caused by an additional protein that was identified as an impurity of the paramyosin preparation (Bullard et al., 1977). Parallel with the characterization of purified proteins from various different muscles, another line of investigation used purified Z-discs from the asynchronous fibrillar flight muscles of the honey-bee. These structures proved to be a valuable tool for making antibodies. As a result, a protein that projects from the periphery of the Z-lines into the (short) I-band was identified. This so-called “projectin” is arranged in the region which was earlier thought to be the binding site for connecting filaments in the flight muscles of the fly and for the protein associated with the paramyosin from the flight muscles of the dung beetle (Auber and Coutaux, 1962, 1963; Bullard et al., 1977; Saide, 1981). This protein is larger than myosin and is related to a set of very large proteins which are components of almost all invertebrate muscles. In SDS-PAGE it could be seen that the mobilities of these types of proteins vary signi~cantly among a wide variety of invertebrate muscles (Locker and Wild, 1986; Hu et al., 1986). Thus, the study of relationship between connecting the filament proteins, the huge proteins from other invertebrate muscles and vertebrate titin was a distinct goal of research in this area. Since then, many proteins have been identified in various invertebrate muscles that exhibit a very close relationship to projectin and to the vertebrate titin.
825
tangular direction (see Heumann and Zebe, 1967; Francis and Waterston, 1985). The quickly developing nematode Caenorhabditis elegans provides a powerful tool for using a genetic approach to investigate these muscles (Epstein et al., 1974). Therefore, this species is frequently used for the molecular characterization of proteins when the development of contractile structures and of muscle attachment is studied (Moerman et al., 1986, 1988; Mori et af., 1988; Francis and Waterston, 1985, 1991; Epstein et al., 1993; Williams and Waterston, 1994; Hresko ef al., 1994). A useful tool involves the disruption of normal larval development of the nematode by application of mutagenic agents. Thus, the genes which are responsible for biosynthesis of contractile proteins and for muscle development and assembly are well known (see Hresko et al., 1994; Williams and Waterston, 1994). One of the mutations, unc-22, has been shown to be responsible for a constant twitching of the body wall muscles in C. elegans (Moerman et al., 1988) and thus the product of this gene-a very large protein-was called “twitchin”. The myofibrillar lattice is disrupted in such zdnc-22-muscles, but the numbers of thick filaments are nearly normal. Provided that certain mutations within the myosin head are present, even the assembly of these filaments into the sarcomere is almost the same as in the unaffected muscle. The mutant phenotype is relatively normal, probably because of the redundancy of motifs within the gene which might be able to mask the absence of several units. As a result, this gene (more than 20 kb) is able to Characterization of Various Titin- tolerate mutations. Furthermore, a 4.3 kb Related Proteins as Components of open reading frame exhibits several sites for transposon insertions which may result in a Numerous Invertebrate Muscles distinct plasticity of the gene (Kiff et al., Titin -like proteins in obliquely striated 1988; Mori et al., 1988). Consequently, muscles however, the molecular weight of the (a) Twitchin in the muscles of nematodes. product is not always well defined: two The obliquely striated muscles of worms possible starting sites for its m-RNA have been identified, and a molecular weight of and molluscs are characteristically different from striated muscles. The thin filaments about 668 or 753 kDa may be calculated. are attached to conical dense bodies which Twitchin was the first example of an intraare arranged in oblique strings within the cellular protein whose complete sequence muscle cell and are attached to the cuticle. was demonstrated to be composed of a regular repeating pattern of two motifs. Therefore, the muscle appears to be smooth when viewed from one direction but Each motif has a length of about 100 amino obliquely striated when viewed from a rec- acids, and the sequences reveal similarity
826
C. Ziegler
not only to the C2-subset of immunoglobulins (the class of IgC2 domains), but also to the type III-repeats of fibronectin (the class of Fn III-domains). A single kinase domain which is encoded within its sequence may act as a novel regulating factor in muscle contraction of the nematode (Benian et al., 1989, 1993). Monospecific antibodies against an in vitro-expressed peptide that represented part of its sequence, identified the product of the genes as a component of the A-band. The antibodies stained the body wall, anal and vulva1 muscles and the pharynx of wild-type animals but only the pharynx in unc-22-mutants (Moerman et al., 1988). Therefore, the epitopes in the pharynx are thought to be the result of a similar protein from a different gene. Staining with monoclonal antibodies against p-connectin, a proteolytic product of a-connectin (Maruyama et al., 1981) with a sequence very similar to that of twitchin, labelled not only the A-band of the nematode muscles but the dense bodies as well (Matsuno et al., 1989). Therefore, a very closely related modular protein is most probably present in these structures. Probing the epitopes in the body wall muscles of C. elegans with antibodies against a mini-titin from insect striated muscles showed a pattern of cross-reactions quite similar to the anti-twitchin-antibodies (Nave et al., 1991). Research with antibodies against this mini-titin had demonstrated earlier the presence of closely related, if not identical, proteins in a wide variety of different invertebrate muscles (Nave and Weber, 1990; compare Locker and Wild, 1986; Hu et al., 1986). Mini-titins from nematode and molluscan muscles, as well as mini-titins and projectins from crayfish and insect muscles, are flexible, rod-like molecules, about 260 nm long and about 5 nm wide. Characteristically, the proteins from all these muscles are composed of a large number of P-sheets which makes them closely related to vertebrate C-, X- and H-protein and to titin (Starr and Offer, 1983; Hu et al., 1990; Nave and Weber, 1990; Ziegler et al., 1990; Nave et al., 1991; Vibert et al., 1993; Fiirst et al., 1992). (b) Mini-titins in mollusc obliquely striated muscles. Molluscs make use of striated
as well as obliquely striated muscles, and in
both types of muscles, epitopes for insect mini-titins have been found exclusively in the A-band and most prominently in the A-I-junction. Because the specialization of their obliquely striated muscles is significantly different from the muscles of worms, the relationship between the molluscan mini-titins and twitchin was carefully studied. Comparing reactions of the antibodies against mollusc-mini-titin in the muscles of scallop and mussel with their cross-reaction in the myofibrils of crayfish, Limulus and the giant water-bug, demonstrated that the analogous proteins of arthropods seem to be slightly larger than the molluscan minititins. In addition, the molecular weights of the mini-titins from molluscs differed slightly from those of nematode muscles. Antibodies against twitchin’s repetitive epitopes stain molluscan mini-titins in a similar pattern as was found with twitchin in nematode muscles. Also, use of monoclonal antibodies localized the kinase domain of twitchin near the M-line of the striated muscles of the muscular system of molluscs (Vibert et al., 1993). Additionally, the molecular shape of molluscan mini-titins proved to be identical to that from nematode or arthropod muscles and evidence for an ability to bind to the myosin rods has been found (Vibert et al., 1993; Nave et al., 1991). This behaviour is reminiscent of projectin in arthropod non-flight muscles where even the whole A-band may be stained after suitable preparation of the myofibrils (Saide et al., 1990; Vigoreaux et al., 1991; Fig. lb, b’). The slight differences among twitchins, projectins and relatives in many invertebrate muscles, however, seem to indicate a distinct functional diversity (Table 1). Titin-like proteins muscles
in arthropod striated
For several of its contractile proteins, melunoguster makes use of single copy genes (Bernstein et al., 1983; Rozek and Davidson, 1983; Becker et al., 1992); one of these genes (sited 102 C/D) encodes a protein which has been detected by probes for the twitchin gene (Moerman et al., 1988; Benian et al., 1989; AymeSouthgate et al., 1991). This protein is very similar and closely related to twitchin and to vertebrate titin. It is expressed in every Drosophila
1200
900
360 600
800
Projectin
Locust-HMWP
Projectin
P800
500 700
668 753
600800
> 500/700
600-900
Kettin
Twitchin
Mini-titin
Twitchin/ mini-titin
Projectin
nd.
700
Mini-titin
Twitchin/projection
3000
(E)
Connectin
Term
Mollusc Annelid
Mollusc (Scallop, mussell) (Nematode (C. elegans)
Nematode (C. elegans)
Arthropod (Water-bug) Insect (D. ~e~ano~~ter) Arthropod (Water-bug, Fruitfly)
Arthropod Annelid Nematode Arthropod (Crayfish, Honey-bee) Arthropod (Locust, Crayfish, FleshBy) Arthropod (Honey-bee, Fruitfly)
Crayfish claw
Animals
Immunostaining Molecular Shape Immunostaining/ Immunostaining Molecular shape Immunostaining
Sequence Immunostaining
Partial Sequence in situ hybridization Sequence
Immunostaining
Immunostaining Molecular shape Immunostaining Physical data Molecular shape Immunostaining Molecular shape Immunostaining Molecular shape Immunostaining
Relation to titin as revealed by
+
4 +
non-flight
non-flight
+
non-flight
non-flight
+
+ + flight non-flight flight non-flight flight non-flight
+ + Z-d. n.d.
Hu et al. (1990)
+ + +
flight non-flight tlight non-thght flight non-flight
Matsuno et al. (1989) Nave et al. (1991) Hu et al. (1990)
d.b. -
-
Moerman et al. (1988) Benian et al. (1989) Benian et al. (1993) Vibert et al. (1993)
-
Ayme-Southgate et al. (1991) Lakey et al. (1993)
Ziegler et al. (1990) Kolsch et al. (subm.) Saide (1981) Saide et al. (1989) Saide et a/. (1990) Lakey et al. (1990)
Nave and Weber (1990) Nave et al. (1991)
flight non-flight
-
References Manabe et al. (1993)
-
Localization A-b. A-I-Z +
non-flight
Muscle
Table 1. Titin-like proteins as identified in various invertebrate muscles. The designations are those, used by the different authors for the identified proteins. nd. = not determined, MW = molecular weight. d.b. = dense bodies, A-b. = A-band, Z-d. = Z-disc
828
C. Ziegler
stage of development, except those stages and tissues lacking muscles, yet it is considerably similar to projectin from the muscles of the honey-bee (Saide et al., 1990; Ayme-Southgate et al., 1991; Fryberg et al., 1992). Its biological activity seems to stabilize muscle function: a mutant phenotype in the larval stage results in animals that suffer from a characteristic weakness of the body muscles (Fryberg et al., 1992). The fibrillar flight muscles of Drosophila exhibit epitopes for the monospecific antibodies against projectin and for the antibodies against twitchin’s peptides. These epitopes represent connections between the A-band and the Z-line at the same position as in the previously mentioned flight muscles of the honey-bee and the waterbug. The non-fibrillar muscles of these insects, the striated muscles of other arthropods and the fast striated muscles of molluscs, however, display the targetprotein predominantly in the A-band without any visible connection to the Z-line (Hu et al., 1990; Lakey et al., 1990; Saide et al., 1989, 1990; Vigoreaux et al., 1991; Vibert et al., 1993). The only exception up to now is the flight muscle of locusts, Though these muscles are non-fibrillar, their mini-titins bind to the Z-lines; in isolated thick filaments, monospecific antibodies against this mini-titin stained the protein in that region where the additional protein in dung beetle flight muscles had been previously localized (Bullard et al., 1977; Ziegler et al., 1990; Nave and Weber, 1990; Fig. la). In some other non-fibrillar muscles, a connection to the Z-line is apparently accomplished via a much larger protein, called “connectin”, which has most recently been identified in the giant sarcomeres of crayfish claw muscles. Projectin, on the other hand, is bound within the A-band predominantly in the A-I-junction in these muscles (Manabe et al., 1993; Hu et al., 1990; Ziegler et al., 1990; Table 1). The functional role of projectin in the non-flight muscles of the arthropods, as well as in the flight muscles of insects, may be outlined as follows: in the asynchronous fibrillar as well as the synchronous nonfibrillar flight muscles, projectin is part of the structures connecting the A-band and the Z-line. No regulatory activities have been described for these structures. In the
striated non-flight muscles, projectin is thought to act as a regulatory component of contraction quite similar to twitchin in the obliquely striated muscles (Saide et al., 1989, 1990; Vigoreaux et al., 1991; Benian et al., 1989, 1993). As a hint of function, Drosophila homogenates projectin in becomes labelled when incubated in viz,0 with [“PI. Also, projectin from the myofibers of crayfish muscles performs autophosphorylation which is thought to be catalysed by a kinase domain within the This activity is Mg’+same sequence. and/or Mn2+ -dependent and phosphorylates serine and threonine residues (Maroto et al., 1992). Crayfish muscles are exclusively non-fibrillar, and the kinase activity confirms the functional predictions. The small size of the fruitflies, however, makes it usually very difficult to separate the fibrillar flight muscles from the non-fibrillar non-flight muscles. Therefore, discrimination between possible fibrillar and nonfibrillar projectin (iso)forms cannot yet be made definitively. Although the target of these kinase activities is not yet established, a solely autophosphorylating activity, as also observed for titin, seems unlikely (Takano-Ohmuro et al., 1992; Somerville and Wang, 1987; Benian et al., 1989). Therefore, twitchin, projectin and related mini-titins may have many diverse roles, e.g. in anchoring thick filaments to the Z-discs, in filament formation and in regulation of contraction (Fig. 1).
Conclusions In invertebrate muscles, proteins with a similar size as titin have only been detected in several cases until recently (“Connectin”, about 3000 kDa, Manabe et al., 1993). Thus, the different invertebrate mini-titins may reflect the diversity of molecular and functional variations in invertebrate muscles. As a result, the nomenclature also varies considerably, and the wide range of molecular weights from 500-l 200 kDa possibly reflects the variability of invertebrate sarcomeric muscles (Table 1). This might be a result of distinct genetic differences, a variable splicing pattern or possibly a result of post-translational modifications, as presumed earlier for some other insect proteins (Honda and Epstein,
Titin-related
proteins
1990; “Flightin”, Vigoreaux et al., 1993). The latter modifications might lead to differential binding of the titin-related invertebrate proteins to other muscular structures. On the other hand, it has also been speculated that this behaviour might be the result of a kinase domain alternately encoded within the sequences of projectin from fibrillar and non-fibrillar muscles (Vigoreaux et al., 1991). The in vitro binding of mini-titins from molluscan muscles to myosin rods reveals
a
a’
b
b'
C
C' Fig. 1. Myofibrils from various muscles of the locust labelled with anti-mini-titin-antibodies. a, b, c: phase contrast and a’, b’, c’: immunofluorescent micrographs of myofibrils from the respective muscle. a and a’: stretched myofibrils from locust flight muscles. b, b’: myofibrils from locust leg muscles presoaked (24 hr) in a buffer of low NaCl-concentration (50 mM) in an ice bath. c, c’: myofibrils from locust leg muscles immediately stained after preparation. Staining was done according to established procedures. The arrows indicate the Z-lines; bar = 10pm.
in invertebrate
muscles
829
similarity to bacterially expressed motifs of vertebrate titin (Labeit et al., 1992; Vibert et al., 1993). This might indicate a distinct interaction between these proteins and other contractile proteins. Accordingly, purified mini-titins from insect flight muscles seem to co-operate with paramyosin in determining the lengths of synthetic thick filaments (Kolsch et al., 1992, submitted). In contrast, the role of twitchin in the assembly of invertebrate thick filaments is assumed to be negligible. This would be quite similar to the effect of vertebrate C-protein on the in vitro-formation of thick filaments from vertebrate myosin (Benian et al., 1989; Goldfine et al., 1991). Thus, in invertebrate muscles, the co-operation is most likely more complex. Paramyosin is a protein characteristically found in invertebrate muscles and is localized within the core of invertebrate thick filaments (Bullard et al., 1977). It might therefore be an essential part in their construction which is probably supported by mini-titins. A brief review of the literature on the inner structure of the thick filaments from the flight muscles of various insects is therefore of interest. Superimposed image analysis of crosssections from insect flight muscles suggests that myosin molecules are arranged at the surface of the thick filaments (Ashton et al., 1987; Beinbrech et al., 1988). The core, however, is very different (Beinbrech et al., 1992), and mini-titins have not yet been detected here, possibly because they bind exclusively to the ends of thick filaments in insect flight muscles (Bullard et al., 1977; Ziegler et al., 1990). This behaviour might be restricted to this type of muscle where no regulatory functions of mini-titins are presumed (Vigoreaux et al., 1991). The binding of the mini-titins, however, is not only a function of their primary structure or the experimental conditions used during investigations. It must be remembered that contractile and cytoskeletal proteins vary their binding according to even slight differences in dielectric properties of the surrounding solvent (Ziegler et al., 1990; Ziegler, unpublished). Generally, however, both paramyosin and insect mini-titin are capable of promoting the binding of myosin to a protein matrix in vitro (Ziegler et al., 1994). It is therefore
830
C. Zie:gler
reasonable that both proteins are essential in the construction of a framework of invertebrate muscle cells. This stroma might constitute a nucleation center for the formation of thick filaments, in a similar manner as the cytoskeleton attachment structures, which connect the plasma membrane with the cytoskeleton. These latter structures are directly involved in myofibrillar assembly (Pardo et al., 1983; Dlugosz et al., 1984; Francis and Waterston, 1991; Epstein et al., 1993; Hresko et al., 1994) and provide a basis for anchoring the contractile and regulatory proteins and for filament nucleation (Epstein and Fischman, 1991). This relation of titin to the cytoskeleton can most easily be seen in non-muscle brushborder cells; one of the components of their cytoskeleton is a giant titin-like protein (Eilertsen and Keller III, 1992) which is constructed of a known pattern of repeating motifs characteristic for muscular titin and mini-titins (IgC2 and FnIII, Benian et al., 1989; Einheber and Fischman, 1990, 1991). Only the newly identified kettin (M = 500 kDa; Lakey et al., 1993)-though of an obviously modular character-seems to be an exception up to now. It is constructed exclusively of domains of the IgC2 type which are connected by short motifs of various sequences. As in mini-titins, however, the molecular weights of kettins differ among species. In addition, the cross-reacting epitopes for /I-connectin-antibodies in nematode dense bodies (Matsuno et al., 1989) indicate a kettin-related protein in these muscles too. This makes it reasonable that variations in the molecular weights of mini-titins might reflect an evolutionary adaptation to the variability of sarcomere lengths, to the lengths of A-I-Z-junctions and to the lengths of thick filaments in invertebrate muscles. The modular principle of mini-titin construction would then be valuable for varying the length of proteins according to different constructions of sarcomeres or variations in cellular shape. The construction of C-, X-, H-protein, myomesin, M-protein and skelemin, which are components of the vertebrate skeletal muscle (Benian et al., 1989; Einheber and Fischman, 1990; Epstein and Fischman, 1991; Price, 1987; Price et al., 1990; Price and Gomer, 1993; Nave and Weber, 1990; Nave et al., 1991; Lakey et al.,
1990, 1993; Ayme-Southgate et al., 1991; Fyrberg et al., 1992; Okagaki et al., 1993; Fiirst et al., 1992; Tan et al., 1993), might also follow this modular approach. Until recently no C-protein has been detected in invertebrate muscles, but even the molecular shapes of titin, C-protein, twitchin, mini-titins, projectin and connectin are nearly identical (compare Fiirst et al., 1988, 1989a, 1992; Hu et al., 1990; Nave et al., 1989, 1991; Ziegler et al., 1990; Vibert et al., 1993; Manabe et al., 1993). Considering that the ratio of sarcomere length/length of C-protein is about 40 in vertebrate muscles and that the ratio sarcomere length/length of mini-titins is about 40 in the very long sarcomeres of insect leg, and crayfish claw muscles too, it seems likely that the mini-titins play a role as “giant C-proteins”, located in the A-bands of these non-tight invertebrate muscles. Neither a single molecule of the minititins nor one of the unstretched invertebrate connectin molecule, however, would be able to span half the lengths of the sarcomeres of the respective muscles (Hu et al., 1990; Nave and Weber, 1990; Nave et al., 1991; Ziegler et al., 1990; Manabe et al., 1993). Thus, the co-operation of other proteins (like paramyosin) seems essential for the function of minititins in invertebrate muscles. Reasons for this proposal include: (1) Mini-titins are able to promote binding of paramyosin as well as of myosin in vitro. (2) The kinase domain of twitchin, which has been suggested as a regulatory factor in invertebrate muscle contraction, exhibits only autophosphorylation at this time. (3) Even the unstretched 3000 kDa connectin would not be able to reach from the Z-line to the M-line in the very long sarcomeres of crayfish claw muscles. (4) Mini-titins are apparently components of connecting filaments in insect flight muscles, but they do not reach to the M-line either. Even this latter Z-line-bound type of mini-titins, however, is a component of the synchronous non-fibrillar and not stretchactivated flight muscles of the locust (Locusta migratoria, Fig. la; Nave and Weber, 1990; Ziegler et al., 1990). The role of invertebrate mini-titins in stretch
Titin-related proteins in invertebrate muscles
activation, proposed since their discovery (Auber and Couteaux, 1962, 1963; Garamvtilgyi, 1965a,b), seems, therefore, to be only one aspect of their multiple functions.
References Ashton F. T., Beinbrech G. and Pepe F. A. (1987) Subfilament organization in myosin filaments of the fast abdominal muscles of the lobster Homarus americanus. Tiss. Cell 19, 51-63. Auber J. and Couteaux R. (1962) L’attache des myofilaments secondaires au niveau de la strie Z dans les muscles de dipteres. Compt. Rend. Acad. Sci. 254, 3425-3429. Auber J. and Couteaux R. (1963) Ultrastructure de la strie Z dans les muscles de dipteres. J. Microsc. (Paris) 2, 309-324. Ayme-Southgate A., Vigoreaux J., Benian G. and Pardue M. L. (1991) Drosophila has a twitchin/titin related gene that appears to encode projectin. Proc. natn. Acad. Sci. U.S.A. 88, 7973-7977. BBhler M., Eppenberger H. M. and Wallimann T. (1985a) Novel thick filament protein of chicken pectoralis muscle: the 86 kd protein I. Purification and characterization. J. Molec. Biol. 186,381-391. BHhler M., Eppenberger H. M. and Wallimann T. (1985b) Novel thick filament protein of chicken pectoralis muscle: the 86 kd protein II. Distribution and localization. J. Molec. Biol. 186, 393401. Becker K. D., O’Donnell P. T., Heitz J. M., Vito M. and Bernstein S. I. (1992) Analysis of Drosophila paramyosin: Identification of a novel isoform which is restricted to a subset of adult muscles. J. Cell Biol. 116, 669-681. Beinbrech G., Ashton F. T. and Pepe F. A. (1988) Invertebrate myosin filament: subfilament arrangement in the wall of tubular filaments of insect flight muscles. J. Molec. Biol. 201, 557-565. Beinbrech G., Ashton F. T. and Pepe F. A. (1992) The invertebrate myosin filament: subfilament arrangement of the solid filaments of insect flight muscles. Biophys. J. 61, 1495-1512. Benian G. M., Kiff J. E., Neckelmann N., Moermann D. G. and Waterston R. H. (1989) Sequence of an unusually large protein implicated in regulation of myosin activity in C. elegans. Nature 324, 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-l 104. Bernstein S. I., Mogami K., Donady J. J. and Emerson C. P. Jr (1983) Drosophila muscle myosin heavy chain is encoded by a single gene in a cluster of muscle mutations. Nature 302, 393-397. Bullard B., Hammond K. and Luke B. M. (1977) The site of an unidentified protein between myosin filaments and Z line. J. Molec. Biol. 115, 41740. Dlugosz A. A., Antin P. B., Nachmias V. T. and Holtzer H. (1984) The relationship between stress fiber-like structures and nascent myofibrils in
831
cultured cardiac myocytes. J. Cell Biol. 99, 2268-2278. Eilertsen K. J. and Keller III T. C. S. (1992) Identification and characterization of two huge protein components of the brush border cytoskeleton: evidence for a cellular isoform of titin. J. Cefl Biol. 119, 549-557. Einheber S. and Fischman D. A. (1990) Isolation and characterization of a cDNA clone encoding avian skeletal muscle C-protein: an intracellular member of the immunoglobulin superfamily. Proc. natn. Acad. Sci. U.S.A. 87, 2157-2161. Epstein H. F., Casey D. L. and Ortiz I. (1993) Myosin and paramyosin of Caenorhabditis elegans embryos assemble into nascent structures distinct from thick filaments and multi-filament assemblages. J. Cell Biol. 122, 845-858. Epstein H. F. and Fischman D. A. (1991) Molecular analysis of protein assembly in muscle development. Science 251, 1039-1044. Epstein H. F., Waterston R. H. and Brenner S. (1974) A mutant affecting the heavy chain of myosin in Caenorhabditis elegans. J. Molec. Biol. 90,291-300. Francis G. R. and Waterston R. H. (1985) Muscle organization in Cuenorhabditis eIegans: localization of proteins implicated thin filament attachment and I-band organization. J. Cell Biol. 101, 1532-1549. Francis R. and Waterston R. H. (1991) Muscle cell attachment in Caenorhabditis elegans. J. Cell Biol. 114, 465479. Fiirst D. O., Nave R., Osborn M. and Weber K. (1989a) Repetitive titin epitopes with a 42 nm spacing coincide in relative position with known A band striations also identified by major myosinassociated proteins. J. Cell Sci. 94, 119-125. Fiirst D. O., Osborn M., Nave R. and Weber K. (1988) The organization of titin filaments in the half sarcomere revealed by monoclonal antibodies in immunoelectron microscopy: a map of ten non-repetitive epitopes starting at the Z-line extends close to the M-line. J. Cell Biol. 106, 1563-1572. Fiirst D. O., Osborn M. and Weber K. (1989b) Myogenesis in the mouse embryo: differential onset of expression of myogenic proteins and the involvement of titin in myofibril assembly. J. Cell Biol. 109, 5 17-527. Fiirst D. O., Vinkemeier U. and Weber K. (1992) Mammalian striated muscle C-Protein: purification from bovine muscle, binding to titin and the characterization of a full length human c-DNA. J. Cell Sci. 102, 769-778. Fyrberg C. C., Labeit S., Bullard B., Leonard K. and Fyrberg E. (1992) Drosophila-projectin-relatedness to titin and twitchin and correlation with lethal (4) 102 CDs and bent-dominant mutants. Proc. R. Sot. London Ser. B 249, 33-40. Garamvijlgyi N. (1965a) The arrangement of the myofilaments in the insect flight muscle I. J. ultrastruct. Res. 13, 409424. Garamvijlgyi N. (1965b) The arrangement of the myofilaments in the insect flight muscle II. J. ultrastruct. Res. 13, 425434. Goldfine S. M., Einheber S. and Fischman D. A. (1991) Cell-free incorporation of newly synthesized
832
C. Zie,gler
myosin subunits into thick myofilaments. J. Muscle Res. Cell Motil. 12, 161-170. Heumann H.-G. and Zebe E. (1967) Uber Feinbau und Funktionsweise der Fasern aus dem Hautmuskelschlauch des Regenwurms Lumbricus ter restris L. Z. Zellforsch. 78, 131-150. Hill C. S., Duran S., Lin Z., Weber K. and Holtzer H. (1986) Titin and myosin, but not desmin are linked during myofibrillogenesis in post mitotic mononucleated myoblasts. J. Cell Biol. 103, 2185-2196. Honda S. and Epstein H. F. (1990) Modulation of muscle gene expression in Cuenorhubditis elegans : differential levels of transcripts, mRNAs and polypeptides for thick filament proteins during nematode development. Proc. natn. Acad. Sci. U.S.A. 87, 876880. Hresko M. C., Williams B. D. and Waterston R. H. (1994) Assembly of body wall muscle and muscle cell attachment structures in Caenorhabditis elegans. J. Cell Biol. 124, 491-506. 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, 148551492. 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, 497-5 11. Kiff J. E., Moerman D. G., Schriefer L. A. and Waterston R. H. (1988) Transposon-induced deletions in tow-22 of C. eleguns associated with almost normal gene activity. Nature 331, 631-633. Kolsch B., Ziegler C. and Beinbrech G. (1992) Length determination of coaggregates of locust myosin and mollusc paramyosin by projectin. J. Muscle Res. Cell Motil. 13, 240. Kolsch B., Ziegler C. and Beinbrech G. Length determination of synthetic thick filaments by cooperation of paramyosin and projectin (mini-titin). (submitted). Labeit S., Lakey A. and Trinick J. (1992) Towards a molecular understanding of titin. EMBO J. 11, 1711-1716. 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. Lakey A., Labeit S., Gautel M., Ferguson C., Barlow D. P., Leonard K. and Bullard B. (1993) Kettin, a large modular protein in the Z-disc of insect muscles. EMBO J. 12, 2863-2871. Locker R. H. and Wild D. J. C. (1986) A comparative study of high molecular weight proteins in various types of muscle across the animal kingdom. J. Biochem. 99, 147331484. 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. Maroto M., Vines J., Marco R. and Cervera M. (1992) Autophosphorylating protein kinase
activity in titin-like arthropod projectin. J. Molec. Biol. 224, 287-291. Maruyama K., Cage P. E. and Bell J. L. (1977a) The role of connectin in elastic properties of insect flight muscle. Comp. Biochem. Physiol. 61A, 623-627. Maruyama K., Kimura S., Ohashi K. and Kuwano Y. (1981) Connectin, an elastic protein of muscle. Identification of “titin” with connectin. J. Biochem. (Tokyo) 89, 701-710. Maruyama K., Murakami F. and Ohashi K. (1977b) Connectin, an elastic protein of muscle. J. Biochem. (Tokyo) 82, 339-345. Maruyama K., Natori R. and Nonomura Y. (1976) A new elastic protein from muscle. Nature 262, 58-60. Matsuno A., Takano-Ohmuro H., Itoh Y., Matsuura T., Shibata M., Nakae H., Kaminuma T. and Maruyama K. (1989) Anti-connectin monoclonal antibodies that react with the unc-22 gene product bind dense bodies of Caenorhabditis (nematode) body wall muscles. Tiss. Cell 21, 495-505. Moerman D. G., Benian G. M., Barstead R. J., Schriefer L. A. and Waterston R. H. (1988) Identification and intracellular localization of the urn-22 gene product of Caenorhabditis elegans. Genes Dez;l. 2, 93-105. Moerman D. G., Benian G. M. and Waterston R. H. (1986) Molecular cloning of the muscle gene unc-22 in Caenorhabditis elegans by Tel transposon tagging. Proc. natn. Acad. Sci. U.S.A. 83, 2579-2583. Mori I., Benian G. M., Moerman D. G. and Waterston R. H. (1988) Transposable element Tel of Caenorhabditis elegans recognizes specific target sequences for integration. Proc. natn. Acad. Sci. 85, 861-864. Nave R., Fiirst D. O., Vinkemeier U. and Weber K. (1991) Purification and physical properties of nematode mini-titins and their relation to twitchin. J. Cell Sci. 98, 491496. Nave R., Fiirst D. 0. and Weber K. (1989) Visualization of the polarity of isolated titin molecules: a single globular head on a long thin rod as the M band anchoring domain? J. Cell Biol. 109, 2177-2187. 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. Offer G., Moos C. and Starr R. (1973) A new protein of the thick filaments of vertebrate skeletal myofibrils. J. Molec. Biol. 74, 653-676. Okagaki T., Weber F. E., Fischman D. A.. Vaughan K. T.. Mikawa T. and Reinach F. C. (1993) The major myosin-binding domain of skeletal muscle MyBP-C(C-protein) resides in the COOH-terminal immunoglobulin C2 motif. J. Cell Biol. 123, 619-626. Olson N. J., Pearson R. B., Needleman D. S., Hurwitz M. Y., Kemp B. E. and Means A. R. (1990) Regulatory and structural motifs of chicken gizzard myosin light chain kinase. Proc. natn. Acad. Sci. U.S.A. 87, 22842288. Pardo J. V., Sicilian0 J. d’A. and Craig S. W. (1983) A vinculin-containing cortical lattice in skeletal muscle: transverse lattice elements (“costameres”)
Titin-related proteins in invertebrate mm&s mark sites of attachment between myofibrils and sarcolemma. Proc. natn. Acad. Sci. U.S.A. 80, 1008-1012. Price M. G. (1987) Skelemins: cytoskeletal proteins located at the periphery of M-discs in striated muscle. J. Cell Biol. 104, 1325-I 336. Price M. G., Brooks C. A. and Gomer R. H. (1990) Skelemins are members of a family of myosinassociated proteins with immunoglobulins superfamily C2 and fibronectin type III domains. J. Cell Biol. 111, 170a. Price M. G. and Gomer R. H. (1993) Skelemin, cytoskeletal M-disc protein, a periphery contains motifs of adhesion/recognition and intermediate filament proteins. J. biol. Chem. 268, 21,800-21,810. Pringle J. W. S. (1977) The mechanical characteristics of insect fibrillar muscle. In Insect Flight Muscle (Edited by Tregear R.), pp. 9-14. North Holland ~blishing Company, Amsterdam. Rozek C. E. and Davidson N. (1983) Drosophila has one myosin heavy chain gene with three developmentally regulated transcripts. Cell 32, 23-34. Saide J. D. (1981) Identification of a connecting filament protein in insect fibrillar flight muscle. J. Molec. 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 of leg muscle of the honeybee. J. Muscle Res. Ceil 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. Celi Biol. 109, 2157-2167. Schultheiss T., Lin Z., Lu M.-H., Murray J., Fischman D. A., Weber K., Masaki M., Imamura M. and Holtzer H. (1990) Differential distribution of subsets of myofibrillar proteins in cardiac nonstriated and striated myofib~ls. J. Cell Biol. 110, 1159-l 172. Shoemaker M. O., Lau W., Shattuck R. L., Kwiatkowski A. P., Matrisian P. E., Guerra-Santos L., Wilson E., Lukas T. J., VanEldik L. J. and Watterson D. M. (1990) Use of DNA sequence and mutant analyses and antisense oligodeoxynucleotides to examine the molecular basis of nonmuscle myosin light chain kinase autoinhibition, calmodulin recognition, and activity. J. Cell Biol. 111, 1107-I 125. Somerville L. L. and Wang K. (1987) In aivo phosphorylation of titin and nebulin in frog-skeletal muscle. Biochem. biophys. Res. Commun. 147, 986-992.
833
Starr R. and Offer G. (1983) H-protein and X-protein. Two new components of the thick filaments of vertebrate skeletal muscle. J. Nolee. Biol. 170, 675698. Takano-Ohmuro H., Nakauchi Y., Kimura S. and Maruyama K. (1992) Autophosphoryiation of /Iconnectin (titin 2) in vitro. Biochem. biophys. Res. Commun. 183, 31-35. Tan K. O., Sater G. R., Myers A. M., Robson R. M. and Huiatt T. W. (1993) Molecular characterization of avian muscle titin. J. biol. Chem. 268, 22,90&22,907. Vibert P., Edelstein S. M., Castellani L. and Elliott B. W. Jr (1993) Mini-titins in striated and smooth molluscan muscles: structure, location and immunological cross-reactivity. J. Muscle Res. Cell Motif. 14, 598-607. Vigoreaux J. O., Saide J. D. and Pardue M. L. (1991) Structurally different ~rosophiZa striated muscles utilize distinct variants of Z-band-associated proteins. J. Muscle Res. Cell Motil. 12, 34&354. Vigoreaux J. O., Saide J. D., Valgeirsdottir K. and Pardue M. L. (1993) Flightin, a novel myofibrillar protein of Drosophila stretch-activated muscles. J. Cell Biol. 121, 587-598. Vinkemeier U., Obe~ann W., Weber K. and Fiirst D. 0. (1993) The globular head domain of titin extends into the center of the sarcomeric M-band-cDNA cloning, epitope mapping and immunoelectron microscopy of two titin-associated proteins. J. Ceil Sci. 106, 319-330. Wang K., McClure J. and Tu A. (1979) Titin: major myofibriilar components of striated muscle. Proc. natn. Acad. Sci. U.S.A. 76, 3698-3702. Williams B. D. and Waterston R. H. (1994) Genes critical for muscle development and function in Caenorhabditis elegans identified through lethal mutations. J. Cell Biol. 124, 475-490. Ziegler C., Ader G. and Beinbrech G. (1994) Insect paramyosin and projectin: simple and rapid purification and their binding to myosin. J. Muscle Res. Cell Motil. 15, 200-201. Ziegler C., Hinterding U. and Beinbrech G. (1992) In vitro stability and protein composition of thick filaments from insect flight muscles. ~“~perientiu48, 6062. Ziegler C., Meyer H. E. and Beinbrech G. (1990) Identification and immunological localization of a high molecular weight protein in arthropod muscles. “Muscle and Motility” (Edited by Mar&ha1 G. and Carraro U.), pp. 9-14. Intercept, Andover, Hampshire.
REVIEW Titin-related
proteins in invertebrate
muscles
Clemens Ziegler Institute of Animal Physiology, Hindenburgplatz
55, D-48143 Miinster, F.R.G.
The localization of filaments connecting the Z-line and the A-band in insect flight muscles and the identification of very large proteins as their components is reviewed. The characterization of twitchin in the obliquely striated muscles of Cuenorhabditis ekgans is reported and the deductions made from its amino acid sequence are considered. The characterization of mini-titins in obliquely striated molhrscan muscles is compared, The identification of projectin in the muscles of Drosophila mefunogaster by anti-twit&in-antibodies, its sequence analysis and the characterization of mini-titins in arthropod and mollusc fast-striated muscles are summarized. The possible biological functions of the different proteins in various invertebrate muscles are discussed. Key words: Twitchin;
Titin; Mini-titin; Projectin; Connectin; Connecting filaments; Insect flight muscle; Obliquely striated muscle; Drosophila melanogaster; Caenorhabditis elegans. Comp. Biochem. Physiol. 109A, 823-833,
1994.
Introduction into a group of direct and indirect flight muscles. The indirect flight muscles may then be further subdivided into a group of non-fibrillar synchronous and a group of fibrillar, stretch-activated asynchronous flight muscles. The direct flight muscles are not considered in this review. The second main group comprises the obliquely striated muscles of worms and molluscs which-in the case of the wormscan be subdivided into the vulval, anal, body wall and the pharynx muscles, and, in the case of the molluscs, into the motor muscles (for instance byssus retractor) and the very specialized “catch” muscle which is able to develop strong tension with a minimum of ATPase activity. It was among the fibrillar, asynchronous Correspondence to: C. Ziegler, Institute of Animal flight muscles of insects where a third Physiology, Hindenburgplatz 55, D-48143 Miinfilament system was detected that was ster, F.R.G. Tel. 251833878; Fax 251838371. to constitute the morphological Received 22 January 1994; revised 4 July 1994; thought accepted 18 July 1994. basis of stretch activation (Auber and
A survey of the invertebrate titin-relatives first necessitates a general review of the different muscle constructions in the invertebrates. In this review, most of the known invertebrate muscles are therefore assigned to various groups. The first main group is that of striated muscles which may be subdivided into a group of the flight and a group of the non-flight muscles. To the latter, the claw, leg and the other striated muscles of arthropods, the supercontractile gut muscles and the fast striated muscles of molluscs will be added. The group of insect flight muscles may be further subdivided
823
824
C. Ziegler
Couteaux, 1962,1963). The filaments of this system could be seen as elastic structures of variable length, connecting the ends of the thick filaments and the Z-line (Garamviilgyi, 1965a,b). This very instructive concept led to the discovery of titin in vertebrate muscles (Wang et al., 1979, “Connectin”, Maruyama et al., 1976,1977a,b, 1981). This giant protein connects the A-band and the Z-line and reaches up to the M-line of vertebrate skeletal muscles. In these muscles, the accessory C-, X- and H-proteins and an 86 kDa protein were shown to share epitopes with titin when probed with the respective monospecific or monoclonal antibodies (Offer et al., 1973; Starr and Offer, 1983; BBhler et al., 1985a,b; Fiirst et al., 1988, 1989a; Nave et al., 1989; Vinkemeier et al., 1993). Meanwhile, attempts to characterize the proteins from invertebrate muscles that are able to maintain myofibrillar elasticity and muscle cell structure have led to different conclusions depending on the type of muscle which was studied. Genomic analysis, immunohistochemistry as well as characterization of purified proteins, led to an enhanced but often contradictory understanding of the possible functions of the previously described connecting proteins and their close relatives from other invertebrate muscles. As a result of these different approaches and the multiplicity of more or less specialized invertebrate muscles, the denomination of proteins which are thought to play a role in the formation of connecting filaments and related structures is manifold. “Projectin” and “mini-titin” (Saide, 1981; Nave and Weber, 1990; Ziegler et al., 1990) have been identified in fibrillar and non-fibrillar insect flight muscles (classification according to Pringle, 1977). The very closely related proteins from the invertebrate non-fibrillar, nonflight muscles which rarely exhibit connecting filaments, and from the obliquely straited muscles, which, until recently, were shown to lack connecting filaments, are also called “projectin” and “mini-titin” as well as “connectin” or “twitchin” (Benian et al., 1989; Hu et al., 1990; Saide et al., 1989, 1990; Vigoreaux et al., 1991; Nave et al., 1991; Manabe et al., 1993; Vibert et al., 1993). When sequences of these proteins, as well as those from vertebrate M-protein,
skelemin, myosin light chain kinase, myomesin and nematode twitchin, are compared, it is possible to assign all of them to a large group of very similar proteins which are apparently responsible for the development of cellular shape and are related to cell adhesion and recognition proteins. the proteins of this Characteristically, group are characterized by a pattern of two repeating motifs (Benian et al., 1989; Einheber and Fischman, 1990, 1991; Okagaki et al., 1993; Tan et al., 1993; Price, 1987; Price et al., 1990; Price and Gomer, 1993; Olson et al., 1990; Shoemaker et al., 1990; Lakey et al., 1993; Hill et al., 1986; Fi_irst et al., 1989b; Schultheiss et al., 1990). Past studies on the identification and characterization of various invertebrate proteins from this group are reviewed, and their significance in the functioning of invertebrate muscles is considered.
Fibrillar Insect Flight Muscles as Objects of Identification and Localization of Connecting Filament Proteins Since their detection in the asynchronous flight muscles of the fly Calliphora erythrocephala (Auber and Couteaux, 1962, 1963), biochemical studies on the proteins from invertebrate muscles have been typically aggravated by limited amounts of muscle tissue. As a result, the molecular basis of connecting filaments in such muscles was primarily studied in the water-bug (Lethocerus maximus), the dung beetle (Heliocopris japetus) and the honey-bee (Apis mellijica). These species are particularly useful because of their size and the predominance of the flight muscles which permit isolation of reasonable amounts of homogeneous myofibers. Dung beetle fibrillar flight muscles, which were used for paramyosin purification and then stained with anti-paramyosin antibodies, exhibited epitopes at the M-line and at the ends of the thick filaments. Only staining in the centre of the sarcomeres was thought to show epitopes for paramyosin. This led to the conclusion that paramyosin, which constitutes a considerable proportion of the filament mass, should be localized within the core of the thick filaments. The staining at the ends was thought to be
Titin-related proteins in invertebrate muscles
caused by an additional protein that was identified as an impurity of the paramyosin preparation (Bullard et al., 1977). Parallel with the characterization of purified proteins from various different muscles, another line of investigation used purified Z-discs from the asynchronous fibrillar flight muscles of the honey-bee. These structures proved to be a valuable tool for making antibodies. As a result, a protein that projects from the periphery of the Z-lines into the (short) I-band was identified. This so-called “projectin” is arranged in the region which was earlier thought to be the binding site for connecting filaments in the flight muscles of the fly and for the protein associated with the paramyosin from the flight muscles of the dung beetle (Auber and Coutaux, 1962, 1963; Bullard et al., 1977; Saide, 1981). This protein is larger than myosin and is related to a set of very large proteins which are components of almost all invertebrate muscles. In SDS-PAGE it could be seen that the mobilities of these types of proteins vary signi~cantly among a wide variety of invertebrate muscles (Locker and Wild, 1986; Hu et al., 1986). Thus, the study of relationship between connecting the filament proteins, the huge proteins from other invertebrate muscles and vertebrate titin was a distinct goal of research in this area. Since then, many proteins have been identified in various invertebrate muscles that exhibit a very close relationship to projectin and to the vertebrate titin.
825
tangular direction (see Heumann and Zebe, 1967; Francis and Waterston, 1985). The quickly developing nematode Caenorhabditis elegans provides a powerful tool for using a genetic approach to investigate these muscles (Epstein et al., 1974). Therefore, this species is frequently used for the molecular characterization of proteins when the development of contractile structures and of muscle attachment is studied (Moerman et al., 1986, 1988; Mori et af., 1988; Francis and Waterston, 1985, 1991; Epstein et al., 1993; Williams and Waterston, 1994; Hresko ef al., 1994). A useful tool involves the disruption of normal larval development of the nematode by application of mutagenic agents. Thus, the genes which are responsible for biosynthesis of contractile proteins and for muscle development and assembly are well known (see Hresko et al., 1994; Williams and Waterston, 1994). One of the mutations, unc-22, has been shown to be responsible for a constant twitching of the body wall muscles in C. elegans (Moerman et al., 1988) and thus the product of this gene-a very large protein-was called “twitchin”. The myofibrillar lattice is disrupted in such zdnc-22-muscles, but the numbers of thick filaments are nearly normal. Provided that certain mutations within the myosin head are present, even the assembly of these filaments into the sarcomere is almost the same as in the unaffected muscle. The mutant phenotype is relatively normal, probably because of the redundancy of motifs within the gene which might be able to mask the absence of several units. As a result, this gene (more than 20 kb) is able to Characterization of Various Titin- tolerate mutations. Furthermore, a 4.3 kb Related Proteins as Components of open reading frame exhibits several sites for transposon insertions which may result in a Numerous Invertebrate Muscles distinct plasticity of the gene (Kiff et al., Titin -like proteins in obliquely striated 1988; Mori et al., 1988). Consequently, muscles however, the molecular weight of the (a) Twitchin in the muscles of nematodes. product is not always well defined: two The obliquely striated muscles of worms possible starting sites for its m-RNA have been identified, and a molecular weight of and molluscs are characteristically different from striated muscles. The thin filaments about 668 or 753 kDa may be calculated. are attached to conical dense bodies which Twitchin was the first example of an intraare arranged in oblique strings within the cellular protein whose complete sequence muscle cell and are attached to the cuticle. was demonstrated to be composed of a regular repeating pattern of two motifs. Therefore, the muscle appears to be smooth when viewed from one direction but Each motif has a length of about 100 amino obliquely striated when viewed from a rec- acids, and the sequences reveal similarity
826
C. Ziegler
not only to the C2-subset of immunoglobulins (the class of IgC2 domains), but also to the type III-repeats of fibronectin (the class of Fn III-domains). A single kinase domain which is encoded within its sequence may act as a novel regulating factor in muscle contraction of the nematode (Benian et al., 1989, 1993). Monospecific antibodies against an in vitro-expressed peptide that represented part of its sequence, identified the product of the genes as a component of the A-band. The antibodies stained the body wall, anal and vulva1 muscles and the pharynx of wild-type animals but only the pharynx in unc-22-mutants (Moerman et al., 1988). Therefore, the epitopes in the pharynx are thought to be the result of a similar protein from a different gene. Staining with monoclonal antibodies against p-connectin, a proteolytic product of a-connectin (Maruyama et al., 1981) with a sequence very similar to that of twitchin, labelled not only the A-band of the nematode muscles but the dense bodies as well (Matsuno et al., 1989). Therefore, a very closely related modular protein is most probably present in these structures. Probing the epitopes in the body wall muscles of C. elegans with antibodies against a mini-titin from insect striated muscles showed a pattern of cross-reactions quite similar to the anti-twitchin-antibodies (Nave et al., 1991). Research with antibodies against this mini-titin had demonstrated earlier the presence of closely related, if not identical, proteins in a wide variety of different invertebrate muscles (Nave and Weber, 1990; compare Locker and Wild, 1986; Hu et al., 1986). Mini-titins from nematode and molluscan muscles, as well as mini-titins and projectins from crayfish and insect muscles, are flexible, rod-like molecules, about 260 nm long and about 5 nm wide. Characteristically, the proteins from all these muscles are composed of a large number of P-sheets which makes them closely related to vertebrate C-, X- and H-protein and to titin (Starr and Offer, 1983; Hu et al., 1990; Nave and Weber, 1990; Ziegler et al., 1990; Nave et al., 1991; Vibert et al., 1993; Fiirst et al., 1992). (b) Mini-titins in mollusc obliquely striated muscles. Molluscs make use of striated
as well as obliquely striated muscles, and in
both types of muscles, epitopes for insect mini-titins have been found exclusively in the A-band and most prominently in the A-I-junction. Because the specialization of their obliquely striated muscles is significantly different from the muscles of worms, the relationship between the molluscan mini-titins and twitchin was carefully studied. Comparing reactions of the antibodies against mollusc-mini-titin in the muscles of scallop and mussel with their cross-reaction in the myofibrils of crayfish, Limulus and the giant water-bug, demonstrated that the analogous proteins of arthropods seem to be slightly larger than the molluscan minititins. In addition, the molecular weights of the mini-titins from molluscs differed slightly from those of nematode muscles. Antibodies against twitchin’s repetitive epitopes stain molluscan mini-titins in a similar pattern as was found with twitchin in nematode muscles. Also, use of monoclonal antibodies localized the kinase domain of twitchin near the M-line of the striated muscles of the muscular system of molluscs (Vibert et al., 1993). Additionally, the molecular shape of molluscan mini-titins proved to be identical to that from nematode or arthropod muscles and evidence for an ability to bind to the myosin rods has been found (Vibert et al., 1993; Nave et al., 1991). This behaviour is reminiscent of projectin in arthropod non-flight muscles where even the whole A-band may be stained after suitable preparation of the myofibrils (Saide et al., 1990; Vigoreaux et al., 1991; Fig. lb, b’). The slight differences among twitchins, projectins and relatives in many invertebrate muscles, however, seem to indicate a distinct functional diversity (Table 1). Titin-like proteins muscles
in arthropod striated
For several of its contractile proteins, melunoguster makes use of single copy genes (Bernstein et al., 1983; Rozek and Davidson, 1983; Becker et al., 1992); one of these genes (sited 102 C/D) encodes a protein which has been detected by probes for the twitchin gene (Moerman et al., 1988; Benian et al., 1989; AymeSouthgate et al., 1991). This protein is very similar and closely related to twitchin and to vertebrate titin. It is expressed in every Drosophila
1200
900
360 600
800
Projectin
Locust-HMWP
Projectin
P800
500 700
668 753
600800
> 500/700
600-900
Kettin
Twitchin
Mini-titin
Twitchin/ mini-titin
Projectin
nd.
700
Mini-titin
Twitchin/projection
3000
(E)
Connectin
Term
Mollusc Annelid
Mollusc (Scallop, mussell) (Nematode (C. elegans)
Nematode (C. elegans)
Arthropod (Water-bug) Insect (D. ~e~ano~~ter) Arthropod (Water-bug, Fruitfly)
Arthropod Annelid Nematode Arthropod (Crayfish, Honey-bee) Arthropod (Locust, Crayfish, FleshBy) Arthropod (Honey-bee, Fruitfly)
Crayfish claw
Animals
Immunostaining Molecular Shape Immunostaining/ Immunostaining Molecular shape Immunostaining
Sequence Immunostaining
Partial Sequence in situ hybridization Sequence
Immunostaining
Immunostaining Molecular shape Immunostaining Physical data Molecular shape Immunostaining Molecular shape Immunostaining Molecular shape Immunostaining
Relation to titin as revealed by
+
4 +
non-flight
non-flight
+
non-flight
non-flight
+
+ + flight non-flight flight non-flight flight non-flight
+ + Z-d. n.d.
Hu et al. (1990)
+ + +
flight non-flight tlight non-thght flight non-flight
Matsuno et al. (1989) Nave et al. (1991) Hu et al. (1990)
d.b. -
-
Moerman et al. (1988) Benian et al. (1989) Benian et al. (1993) Vibert et al. (1993)
-
Ayme-Southgate et al. (1991) Lakey et al. (1993)
Ziegler et al. (1990) Kolsch et al. (subm.) Saide (1981) Saide et al. (1989) Saide et a/. (1990) Lakey et al. (1990)
Nave and Weber (1990) Nave et al. (1991)
flight non-flight
-
References Manabe et al. (1993)
-
Localization A-b. A-I-Z +
non-flight
Muscle
Table 1. Titin-like proteins as identified in various invertebrate muscles. The designations are those, used by the different authors for the identified proteins. nd. = not determined, MW = molecular weight. d.b. = dense bodies, A-b. = A-band, Z-d. = Z-disc
828
C. Ziegler
stage of development, except those stages and tissues lacking muscles, yet it is considerably similar to projectin from the muscles of the honey-bee (Saide et al., 1990; Ayme-Southgate et al., 1991; Fryberg et al., 1992). Its biological activity seems to stabilize muscle function: a mutant phenotype in the larval stage results in animals that suffer from a characteristic weakness of the body muscles (Fryberg et al., 1992). The fibrillar flight muscles of Drosophila exhibit epitopes for the monospecific antibodies against projectin and for the antibodies against twitchin’s peptides. These epitopes represent connections between the A-band and the Z-line at the same position as in the previously mentioned flight muscles of the honey-bee and the waterbug. The non-fibrillar muscles of these insects, the striated muscles of other arthropods and the fast striated muscles of molluscs, however, display the targetprotein predominantly in the A-band without any visible connection to the Z-line (Hu et al., 1990; Lakey et al., 1990; Saide et al., 1989, 1990; Vigoreaux et al., 1991; Vibert et al., 1993). The only exception up to now is the flight muscle of locusts, Though these muscles are non-fibrillar, their mini-titins bind to the Z-lines; in isolated thick filaments, monospecific antibodies against this mini-titin stained the protein in that region where the additional protein in dung beetle flight muscles had been previously localized (Bullard et al., 1977; Ziegler et al., 1990; Nave and Weber, 1990; Fig. la). In some other non-fibrillar muscles, a connection to the Z-line is apparently accomplished via a much larger protein, called “connectin”, which has most recently been identified in the giant sarcomeres of crayfish claw muscles. Projectin, on the other hand, is bound within the A-band predominantly in the A-I-junction in these muscles (Manabe et al., 1993; Hu et al., 1990; Ziegler et al., 1990; Table 1). The functional role of projectin in the non-flight muscles of the arthropods, as well as in the flight muscles of insects, may be outlined as follows: in the asynchronous fibrillar as well as the synchronous nonfibrillar flight muscles, projectin is part of the structures connecting the A-band and the Z-line. No regulatory activities have been described for these structures. In the
striated non-flight muscles, projectin is thought to act as a regulatory component of contraction quite similar to twitchin in the obliquely striated muscles (Saide et al., 1989, 1990; Vigoreaux et al., 1991; Benian et al., 1989, 1993). As a hint of function, Drosophila homogenates projectin in becomes labelled when incubated in viz,0 with [“PI. Also, projectin from the myofibers of crayfish muscles performs autophosphorylation which is thought to be catalysed by a kinase domain within the This activity is Mg’+same sequence. and/or Mn2+ -dependent and phosphorylates serine and threonine residues (Maroto et al., 1992). Crayfish muscles are exclusively non-fibrillar, and the kinase activity confirms the functional predictions. The small size of the fruitflies, however, makes it usually very difficult to separate the fibrillar flight muscles from the non-fibrillar non-flight muscles. Therefore, discrimination between possible fibrillar and nonfibrillar projectin (iso)forms cannot yet be made definitively. Although the target of these kinase activities is not yet established, a solely autophosphorylating activity, as also observed for titin, seems unlikely (Takano-Ohmuro et al., 1992; Somerville and Wang, 1987; Benian et al., 1989). Therefore, twitchin, projectin and related mini-titins may have many diverse roles, e.g. in anchoring thick filaments to the Z-discs, in filament formation and in regulation of contraction (Fig. 1).
Conclusions In invertebrate muscles, proteins with a similar size as titin have only been detected in several cases until recently (“Connectin”, about 3000 kDa, Manabe et al., 1993). Thus, the different invertebrate mini-titins may reflect the diversity of molecular and functional variations in invertebrate muscles. As a result, the nomenclature also varies considerably, and the wide range of molecular weights from 500-l 200 kDa possibly reflects the variability of invertebrate sarcomeric muscles (Table 1). This might be a result of distinct genetic differences, a variable splicing pattern or possibly a result of post-translational modifications, as presumed earlier for some other insect proteins (Honda and Epstein,
Titin-related
proteins
1990; “Flightin”, Vigoreaux et al., 1993). The latter modifications might lead to differential binding of the titin-related invertebrate proteins to other muscular structures. On the other hand, it has also been speculated that this behaviour might be the result of a kinase domain alternately encoded within the sequences of projectin from fibrillar and non-fibrillar muscles (Vigoreaux et al., 1991). The in vitro binding of mini-titins from molluscan muscles to myosin rods reveals
a
a’
b
b'
C
C' Fig. 1. Myofibrils from various muscles of the locust labelled with anti-mini-titin-antibodies. a, b, c: phase contrast and a’, b’, c’: immunofluorescent micrographs of myofibrils from the respective muscle. a and a’: stretched myofibrils from locust flight muscles. b, b’: myofibrils from locust leg muscles presoaked (24 hr) in a buffer of low NaCl-concentration (50 mM) in an ice bath. c, c’: myofibrils from locust leg muscles immediately stained after preparation. Staining was done according to established procedures. The arrows indicate the Z-lines; bar = 10pm.
in invertebrate
muscles
829
similarity to bacterially expressed motifs of vertebrate titin (Labeit et al., 1992; Vibert et al., 1993). This might indicate a distinct interaction between these proteins and other contractile proteins. Accordingly, purified mini-titins from insect flight muscles seem to co-operate with paramyosin in determining the lengths of synthetic thick filaments (Kolsch et al., 1992, submitted). In contrast, the role of twitchin in the assembly of invertebrate thick filaments is assumed to be negligible. This would be quite similar to the effect of vertebrate C-protein on the in vitro-formation of thick filaments from vertebrate myosin (Benian et al., 1989; Goldfine et al., 1991). Thus, in invertebrate muscles, the co-operation is most likely more complex. Paramyosin is a protein characteristically found in invertebrate muscles and is localized within the core of invertebrate thick filaments (Bullard et al., 1977). It might therefore be an essential part in their construction which is probably supported by mini-titins. A brief review of the literature on the inner structure of the thick filaments from the flight muscles of various insects is therefore of interest. Superimposed image analysis of crosssections from insect flight muscles suggests that myosin molecules are arranged at the surface of the thick filaments (Ashton et al., 1987; Beinbrech et al., 1988). The core, however, is very different (Beinbrech et al., 1992), and mini-titins have not yet been detected here, possibly because they bind exclusively to the ends of thick filaments in insect flight muscles (Bullard et al., 1977; Ziegler et al., 1990). This behaviour might be restricted to this type of muscle where no regulatory functions of mini-titins are presumed (Vigoreaux et al., 1991). The binding of the mini-titins, however, is not only a function of their primary structure or the experimental conditions used during investigations. It must be remembered that contractile and cytoskeletal proteins vary their binding according to even slight differences in dielectric properties of the surrounding solvent (Ziegler et al., 1990; Ziegler, unpublished). Generally, however, both paramyosin and insect mini-titin are capable of promoting the binding of myosin to a protein matrix in vitro (Ziegler et al., 1994). It is therefore
830
C. Zie:gler
reasonable that both proteins are essential in the construction of a framework of invertebrate muscle cells. This stroma might constitute a nucleation center for the formation of thick filaments, in a similar manner as the cytoskeleton attachment structures, which connect the plasma membrane with the cytoskeleton. These latter structures are directly involved in myofibrillar assembly (Pardo et al., 1983; Dlugosz et al., 1984; Francis and Waterston, 1991; Epstein et al., 1993; Hresko et al., 1994) and provide a basis for anchoring the contractile and regulatory proteins and for filament nucleation (Epstein and Fischman, 1991). This relation of titin to the cytoskeleton can most easily be seen in non-muscle brushborder cells; one of the components of their cytoskeleton is a giant titin-like protein (Eilertsen and Keller III, 1992) which is constructed of a known pattern of repeating motifs characteristic for muscular titin and mini-titins (IgC2 and FnIII, Benian et al., 1989; Einheber and Fischman, 1990, 1991). Only the newly identified kettin (M = 500 kDa; Lakey et al., 1993)-though of an obviously modular character-seems to be an exception up to now. It is constructed exclusively of domains of the IgC2 type which are connected by short motifs of various sequences. As in mini-titins, however, the molecular weights of kettins differ among species. In addition, the cross-reacting epitopes for /I-connectin-antibodies in nematode dense bodies (Matsuno et al., 1989) indicate a kettin-related protein in these muscles too. This makes it reasonable that variations in the molecular weights of mini-titins might reflect an evolutionary adaptation to the variability of sarcomere lengths, to the lengths of A-I-Z-junctions and to the lengths of thick filaments in invertebrate muscles. The modular principle of mini-titin construction would then be valuable for varying the length of proteins according to different constructions of sarcomeres or variations in cellular shape. The construction of C-, X-, H-protein, myomesin, M-protein and skelemin, which are components of the vertebrate skeletal muscle (Benian et al., 1989; Einheber and Fischman, 1990; Epstein and Fischman, 1991; Price, 1987; Price et al., 1990; Price and Gomer, 1993; Nave and Weber, 1990; Nave et al., 1991; Lakey et al.,
1990, 1993; Ayme-Southgate et al., 1991; Fyrberg et al., 1992; Okagaki et al., 1993; Fiirst et al., 1992; Tan et al., 1993), might also follow this modular approach. Until recently no C-protein has been detected in invertebrate muscles, but even the molecular shapes of titin, C-protein, twitchin, mini-titins, projectin and connectin are nearly identical (compare Fiirst et al., 1988, 1989a, 1992; Hu et al., 1990; Nave et al., 1989, 1991; Ziegler et al., 1990; Vibert et al., 1993; Manabe et al., 1993). Considering that the ratio of sarcomere length/length of C-protein is about 40 in vertebrate muscles and that the ratio sarcomere length/length of mini-titins is about 40 in the very long sarcomeres of insect leg, and crayfish claw muscles too, it seems likely that the mini-titins play a role as “giant C-proteins”, located in the A-bands of these non-tight invertebrate muscles. Neither a single molecule of the minititins nor one of the unstretched invertebrate connectin molecule, however, would be able to span half the lengths of the sarcomeres of the respective muscles (Hu et al., 1990; Nave and Weber, 1990; Nave et al., 1991; Ziegler et al., 1990; Manabe et al., 1993). Thus, the co-operation of other proteins (like paramyosin) seems essential for the function of minititins in invertebrate muscles. Reasons for this proposal include: (1) Mini-titins are able to promote binding of paramyosin as well as of myosin in vitro. (2) The kinase domain of twitchin, which has been suggested as a regulatory factor in invertebrate muscle contraction, exhibits only autophosphorylation at this time. (3) Even the unstretched 3000 kDa connectin would not be able to reach from the Z-line to the M-line in the very long sarcomeres of crayfish claw muscles. (4) Mini-titins are apparently components of connecting filaments in insect flight muscles, but they do not reach to the M-line either. Even this latter Z-line-bound type of mini-titins, however, is a component of the synchronous non-fibrillar and not stretchactivated flight muscles of the locust (Locusta migratoria, Fig. la; Nave and Weber, 1990; Ziegler et al., 1990). The role of invertebrate mini-titins in stretch
Titin-related proteins in invertebrate muscles
activation, proposed since their discovery (Auber and Couteaux, 1962, 1963; Garamvtilgyi, 1965a,b), seems, therefore, to be only one aspect of their multiple functions.
References Ashton F. T., Beinbrech G. and Pepe F. A. (1987) Subfilament organization in myosin filaments of the fast abdominal muscles of the lobster Homarus americanus. Tiss. Cell 19, 51-63. Auber J. and Couteaux R. (1962) L’attache des myofilaments secondaires au niveau de la strie Z dans les muscles de dipteres. Compt. Rend. Acad. Sci. 254, 3425-3429. Auber J. and Couteaux R. (1963) Ultrastructure de la strie Z dans les muscles de dipteres. J. Microsc. (Paris) 2, 309-324. Ayme-Southgate A., Vigoreaux J., Benian G. and Pardue M. L. (1991) Drosophila has a twitchin/titin related gene that appears to encode projectin. Proc. natn. Acad. Sci. U.S.A. 88, 7973-7977. BBhler M., Eppenberger H. M. and Wallimann T. (1985a) Novel thick filament protein of chicken pectoralis muscle: the 86 kd protein I. Purification and characterization. J. Molec. Biol. 186,381-391. BHhler M., Eppenberger H. M. and Wallimann T. (1985b) Novel thick filament protein of chicken pectoralis muscle: the 86 kd protein II. Distribution and localization. J. Molec. Biol. 186, 393401. Becker K. D., O’Donnell P. T., Heitz J. M., Vito M. and Bernstein S. I. (1992) Analysis of Drosophila paramyosin: Identification of a novel isoform which is restricted to a subset of adult muscles. J. Cell Biol. 116, 669-681. Beinbrech G., Ashton F. T. and Pepe F. A. (1988) Invertebrate myosin filament: subfilament arrangement in the wall of tubular filaments of insect flight muscles. J. Molec. Biol. 201, 557-565. Beinbrech G., Ashton F. T. and Pepe F. A. (1992) The invertebrate myosin filament: subfilament arrangement of the solid filaments of insect flight muscles. Biophys. J. 61, 1495-1512. Benian G. M., Kiff J. E., Neckelmann N., Moermann D. G. and Waterston R. H. (1989) Sequence of an unusually large protein implicated in regulation of myosin activity in C. elegans. Nature 324, 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-l 104. Bernstein S. I., Mogami K., Donady J. J. and Emerson C. P. Jr (1983) Drosophila muscle myosin heavy chain is encoded by a single gene in a cluster of muscle mutations. Nature 302, 393-397. Bullard B., Hammond K. and Luke B. M. (1977) The site of an unidentified protein between myosin filaments and Z line. J. Molec. Biol. 115, 41740. Dlugosz A. A., Antin P. B., Nachmias V. T. and Holtzer H. (1984) The relationship between stress fiber-like structures and nascent myofibrils in
831
cultured cardiac myocytes. J. Cell Biol. 99, 2268-2278. Eilertsen K. J. and Keller III T. C. S. (1992) Identification and characterization of two huge protein components of the brush border cytoskeleton: evidence for a cellular isoform of titin. J. Cefl Biol. 119, 549-557. Einheber S. and Fischman D. A. (1990) Isolation and characterization of a cDNA clone encoding avian skeletal muscle C-protein: an intracellular member of the immunoglobulin superfamily. Proc. natn. Acad. Sci. U.S.A. 87, 2157-2161. Epstein H. F., Casey D. L. and Ortiz I. (1993) Myosin and paramyosin of Caenorhabditis elegans embryos assemble into nascent structures distinct from thick filaments and multi-filament assemblages. J. Cell Biol. 122, 845-858. Epstein H. F. and Fischman D. A. (1991) Molecular analysis of protein assembly in muscle development. Science 251, 1039-1044. Epstein H. F., Waterston R. H. and Brenner S. (1974) A mutant affecting the heavy chain of myosin in Caenorhabditis elegans. J. Molec. Biol. 90,291-300. Francis G. R. and Waterston R. H. (1985) Muscle organization in Cuenorhabditis eIegans: localization of proteins implicated thin filament attachment and I-band organization. J. Cell Biol. 101, 1532-1549. Francis R. and Waterston R. H. (1991) Muscle cell attachment in Caenorhabditis elegans. J. Cell Biol. 114, 465479. Fiirst D. O., Nave R., Osborn M. and Weber K. (1989a) Repetitive titin epitopes with a 42 nm spacing coincide in relative position with known A band striations also identified by major myosinassociated proteins. J. Cell Sci. 94, 119-125. Fiirst D. O., Osborn M., Nave R. and Weber K. (1988) The organization of titin filaments in the half sarcomere revealed by monoclonal antibodies in immunoelectron microscopy: a map of ten non-repetitive epitopes starting at the Z-line extends close to the M-line. J. Cell Biol. 106, 1563-1572. Fiirst D. O., Osborn M. and Weber K. (1989b) Myogenesis in the mouse embryo: differential onset of expression of myogenic proteins and the involvement of titin in myofibril assembly. J. Cell Biol. 109, 5 17-527. Fiirst D. O., Vinkemeier U. and Weber K. (1992) Mammalian striated muscle C-Protein: purification from bovine muscle, binding to titin and the characterization of a full length human c-DNA. J. Cell Sci. 102, 769-778. Fyrberg C. C., Labeit S., Bullard B., Leonard K. and Fyrberg E. (1992) Drosophila-projectin-relatedness to titin and twitchin and correlation with lethal (4) 102 CDs and bent-dominant mutants. Proc. R. Sot. London Ser. B 249, 33-40. Garamvijlgyi N. (1965a) The arrangement of the myofilaments in the insect flight muscle I. J. ultrastruct. Res. 13, 409424. Garamvijlgyi N. (1965b) The arrangement of the myofilaments in the insect flight muscle II. J. ultrastruct. Res. 13, 425434. Goldfine S. M., Einheber S. and Fischman D. A. (1991) Cell-free incorporation of newly synthesized
832
C. Zie,gler
myosin subunits into thick myofilaments. J. Muscle Res. Cell Motil. 12, 161-170. Heumann H.-G. and Zebe E. (1967) Uber Feinbau und Funktionsweise der Fasern aus dem Hautmuskelschlauch des Regenwurms Lumbricus ter restris L. Z. Zellforsch. 78, 131-150. Hill C. S., Duran S., Lin Z., Weber K. and Holtzer H. (1986) Titin and myosin, but not desmin are linked during myofibrillogenesis in post mitotic mononucleated myoblasts. J. Cell Biol. 103, 2185-2196. Honda S. and Epstein H. F. (1990) Modulation of muscle gene expression in Cuenorhubditis elegans : differential levels of transcripts, mRNAs and polypeptides for thick filament proteins during nematode development. Proc. natn. Acad. Sci. U.S.A. 87, 876880. Hresko M. C., Williams B. D. and Waterston R. H. (1994) Assembly of body wall muscle and muscle cell attachment structures in Caenorhabditis elegans. J. Cell Biol. 124, 491-506. 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, 148551492. 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, 497-5 11. Kiff J. E., Moerman D. G., Schriefer L. A. and Waterston R. H. (1988) Transposon-induced deletions in tow-22 of C. eleguns associated with almost normal gene activity. Nature 331, 631-633. Kolsch B., Ziegler C. and Beinbrech G. (1992) Length determination of coaggregates of locust myosin and mollusc paramyosin by projectin. J. Muscle Res. Cell Motil. 13, 240. Kolsch B., Ziegler C. and Beinbrech G. Length determination of synthetic thick filaments by cooperation of paramyosin and projectin (mini-titin). (submitted). Labeit S., Lakey A. and Trinick J. (1992) Towards a molecular understanding of titin. EMBO J. 11, 1711-1716. 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. Lakey A., Labeit S., Gautel M., Ferguson C., Barlow D. P., Leonard K. and Bullard B. (1993) Kettin, a large modular protein in the Z-disc of insect muscles. EMBO J. 12, 2863-2871. Locker R. H. and Wild D. J. C. (1986) A comparative study of high molecular weight proteins in various types of muscle across the animal kingdom. J. Biochem. 99, 147331484. 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. Maroto M., Vines J., Marco R. and Cervera M. (1992) Autophosphorylating protein kinase
activity in titin-like arthropod projectin. J. Molec. Biol. 224, 287-291. Maruyama K., Cage P. E. and Bell J. L. (1977a) The role of connectin in elastic properties of insect flight muscle. Comp. Biochem. Physiol. 61A, 623-627. Maruyama K., Kimura S., Ohashi K. and Kuwano Y. (1981) Connectin, an elastic protein of muscle. Identification of “titin” with connectin. J. Biochem. (Tokyo) 89, 701-710. Maruyama K., Murakami F. and Ohashi K. (1977b) Connectin, an elastic protein of muscle. J. Biochem. (Tokyo) 82, 339-345. Maruyama K., Natori R. and Nonomura Y. (1976) A new elastic protein from muscle. Nature 262, 58-60. Matsuno A., Takano-Ohmuro H., Itoh Y., Matsuura T., Shibata M., Nakae H., Kaminuma T. and Maruyama K. (1989) Anti-connectin monoclonal antibodies that react with the unc-22 gene product bind dense bodies of Caenorhabditis (nematode) body wall muscles. Tiss. Cell 21, 495-505. Moerman D. G., Benian G. M., Barstead R. J., Schriefer L. A. and Waterston R. H. (1988) Identification and intracellular localization of the urn-22 gene product of Caenorhabditis elegans. Genes Dez;l. 2, 93-105. Moerman D. G., Benian G. M. and Waterston R. H. (1986) Molecular cloning of the muscle gene unc-22 in Caenorhabditis elegans by Tel transposon tagging. Proc. natn. Acad. Sci. U.S.A. 83, 2579-2583. Mori I., Benian G. M., Moerman D. G. and Waterston R. H. (1988) Transposable element Tel of Caenorhabditis elegans recognizes specific target sequences for integration. Proc. natn. Acad. Sci. 85, 861-864. Nave R., Fiirst D. O., Vinkemeier U. and Weber K. (1991) Purification and physical properties of nematode mini-titins and their relation to twitchin. J. Cell Sci. 98, 491496. Nave R., Fiirst D. 0. and Weber K. (1989) Visualization of the polarity of isolated titin molecules: a single globular head on a long thin rod as the M band anchoring domain? J. Cell Biol. 109, 2177-2187. 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. Offer G., Moos C. and Starr R. (1973) A new protein of the thick filaments of vertebrate skeletal myofibrils. J. Molec. Biol. 74, 653-676. Okagaki T., Weber F. E., Fischman D. A.. Vaughan K. T.. Mikawa T. and Reinach F. C. (1993) The major myosin-binding domain of skeletal muscle MyBP-C(C-protein) resides in the COOH-terminal immunoglobulin C2 motif. J. Cell Biol. 123, 619-626. Olson N. J., Pearson R. B., Needleman D. S., Hurwitz M. Y., Kemp B. E. and Means A. R. (1990) Regulatory and structural motifs of chicken gizzard myosin light chain kinase. Proc. natn. Acad. Sci. U.S.A. 87, 22842288. Pardo J. V., Sicilian0 J. d’A. and Craig S. W. (1983) A vinculin-containing cortical lattice in skeletal muscle: transverse lattice elements (“costameres”)
Titin-related proteins in invertebrate mm&s mark sites of attachment between myofibrils and sarcolemma. Proc. natn. Acad. Sci. U.S.A. 80, 1008-1012. Price M. G. (1987) Skelemins: cytoskeletal proteins located at the periphery of M-discs in striated muscle. J. Cell Biol. 104, 1325-I 336. Price M. G., Brooks C. A. and Gomer R. H. (1990) Skelemins are members of a family of myosinassociated proteins with immunoglobulins superfamily C2 and fibronectin type III domains. J. Cell Biol. 111, 170a. Price M. G. and Gomer R. H. (1993) Skelemin, cytoskeletal M-disc protein, a periphery contains motifs of adhesion/recognition and intermediate filament proteins. J. biol. Chem. 268, 21,800-21,810. Pringle J. W. S. (1977) The mechanical characteristics of insect fibrillar muscle. In Insect Flight Muscle (Edited by Tregear R.), pp. 9-14. North Holland ~blishing Company, Amsterdam. Rozek C. E. and Davidson N. (1983) Drosophila has one myosin heavy chain gene with three developmentally regulated transcripts. Cell 32, 23-34. Saide J. D. (1981) Identification of a connecting filament protein in insect fibrillar flight muscle. J. Molec. 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 of leg muscle of the honeybee. J. Muscle Res. Ceil 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. Celi Biol. 109, 2157-2167. Schultheiss T., Lin Z., Lu M.-H., Murray J., Fischman D. A., Weber K., Masaki M., Imamura M. and Holtzer H. (1990) Differential distribution of subsets of myofibrillar proteins in cardiac nonstriated and striated myofib~ls. J. Cell Biol. 110, 1159-l 172. Shoemaker M. O., Lau W., Shattuck R. L., Kwiatkowski A. P., Matrisian P. E., Guerra-Santos L., Wilson E., Lukas T. J., VanEldik L. J. and Watterson D. M. (1990) Use of DNA sequence and mutant analyses and antisense oligodeoxynucleotides to examine the molecular basis of nonmuscle myosin light chain kinase autoinhibition, calmodulin recognition, and activity. J. Cell Biol. 111, 1107-I 125. Somerville L. L. and Wang K. (1987) In aivo phosphorylation of titin and nebulin in frog-skeletal muscle. Biochem. biophys. Res. Commun. 147, 986-992.
833
Starr R. and Offer G. (1983) H-protein and X-protein. Two new components of the thick filaments of vertebrate skeletal muscle. J. Nolee. Biol. 170, 675698. Takano-Ohmuro H., Nakauchi Y., Kimura S. and Maruyama K. (1992) Autophosphoryiation of /Iconnectin (titin 2) in vitro. Biochem. biophys. Res. Commun. 183, 31-35. Tan K. O., Sater G. R., Myers A. M., Robson R. M. and Huiatt T. W. (1993) Molecular characterization of avian muscle titin. J. biol. Chem. 268, 22,90&22,907. Vibert P., Edelstein S. M., Castellani L. and Elliott B. W. Jr (1993) Mini-titins in striated and smooth molluscan muscles: structure, location and immunological cross-reactivity. J. Muscle Res. Cell Motif. 14, 598-607. Vigoreaux J. O., Saide J. D. and Pardue M. L. (1991) Structurally different ~rosophiZa striated muscles utilize distinct variants of Z-band-associated proteins. J. Muscle Res. Cell Motil. 12, 34&354. Vigoreaux J. O., Saide J. D., Valgeirsdottir K. and Pardue M. L. (1993) Flightin, a novel myofibrillar protein of Drosophila stretch-activated muscles. J. Cell Biol. 121, 587-598. Vinkemeier U., Obe~ann W., Weber K. and Fiirst D. 0. (1993) The globular head domain of titin extends into the center of the sarcomeric M-band-cDNA cloning, epitope mapping and immunoelectron microscopy of two titin-associated proteins. J. Ceil Sci. 106, 319-330. Wang K., McClure J. and Tu A. (1979) Titin: major myofibriilar components of striated muscle. Proc. natn. Acad. Sci. U.S.A. 76, 3698-3702. Williams B. D. and Waterston R. H. (1994) Genes critical for muscle development and function in Caenorhabditis elegans identified through lethal mutations. J. Cell Biol. 124, 475-490. Ziegler C., Ader G. and Beinbrech G. (1994) Insect paramyosin and projectin: simple and rapid purification and their binding to myosin. J. Muscle Res. Cell Motil. 15, 200-201. Ziegler C., Hinterding U. and Beinbrech G. (1992) In vitro stability and protein composition of thick filaments from insect flight muscles. ~“~perientiu48, 6062. Ziegler C., Meyer H. E. and Beinbrech G. (1990) Identification and immunological localization of a high molecular weight protein in arthropod muscles. “Muscle and Motility” (Edited by Mar&ha1 G. and Carraro U.), pp. 9-14. Intercept, Andover, Hampshire.