Visualization of the polarity of isolated titin molecules: a single globular head on a long thin rod as the M band anchoring domain?

TII, the extractable form of titin, was purified from myofibrils and separated by high resolution gel permeation chromatography into two fractions (TIIA and TIIB). Novel specimen orientation methods used before metal shadowing and EM result in striking pictures of the two forms. Molecules layered on mica become uniformly oriented when subjected to centrifugation. TIIB comprises a very homogeneous fraction. All molecules reveal a single globular head at one end on a long and very thin rod of uniform diameter. The lengths of the rods have a very narrow distribution (900 +/- 50 nm). TIIA molecules seem lateral oligomers of TIIB, attached to each other via the head regions. While dimers are the predominant species, trimers and some higher oligomers can also be discerned. Mild proteolysis destroys the heads and converts TIIA and TIIB into TIIB-like rods. Similar molecules also result from titin purified from myofibrils by certain established purification schemes. Headless titin molecules show in gel electrophoresis only the TII band, while head bearing molecules give rise to two additional polypeptides at 165 and 190 kD. Immunoelectron microscopy of myofibrils identifies both titin-associated proteins as M band constituents. We speculate that in the polar images of TII the globular head region corresponds to the M band end of the titin molecules. This hypothesis is supported by immunoelectron micrographs of TIIB molecules using titin antibodies of known epitope location in the half sarcomere. This proposal complements our previous immunoelectron microscopic data on myofibrils. They showed that epitopes present only on the nonextractable TI species locate to the Z line and its immediately adjacent region (Fürst, D. O., M. Osborn, R. Nave, and K. Weber. 1988. J. Cell Biol. 106:1563-1572). Thus, the two distinct ends of the titin molecule attach to Z and M band material respectively.     

Architecture of the sarcomere matrix of skeletal muscle: immunoelectron microscopic evidence that suggests a set of parallel inextensible nebulin filaments anchored at the Z line

Nebulin, a giant myofibrillar protein (600-800 kD) that is abundant (3%) in the sarcomere of a wide range of skeletal muscles, has been proposed as a component of a cytoskeletal matrix that coexists with actin and myosin filaments within the sarcomere. Immunoblot analysis indicates that although polypeptides of similar size are present in cardiac and smooth muscles at low abundance, those proteins show no immunological cross-reactivity with skeletal muscle nebulin. Gel analysis reveals that nebulins in various skeletal muscles of rabbit belong to at least two classes of size variants. A monospecific antibody has been used to localize nebulin by immunoelectron microscopy in a mechanically split rabbit psoas muscle fiber preparation. Labeled split fibers exhibit six pairs of stripes of antibody-imparted transverse densities spaced at 0.1-1.0 micron from the Z line within each sarcomere. These epitopes maintain a fixed distance to the Z line irrespective of sarcomere length and do not exhibit the characteristic elastic stretch-response of titin epitopes within the I band domain. It is proposed that nebulin constitutes a set of inextensible filaments attached at one end to the Z line and that nebulin filaments are in parallel, and not in series, with titin filaments. Thus the skeletal muscle sarcomere may have two sets of nonactomyosin filaments: a set of I segment-linked nebulin filaments and a set of A segment-linked titin filaments. This four-filament sarcomere model raises the possibility that nebulin and titin might act as organizing templates and length- determining factors for actin and myosin respectively.     

Myogenesis in the mouse embryo: differential onset of expression of myogenic proteins and the involvement of titin in myofibril assembly

Antibodies to muscle-specific proteins were used in immunofluorescence to monitor the development of skeletal muscle during mouse embryogenesis. At gestation day (g.d.) 9 a single layer of vimentin filament containing cells in the myotome domain of cervical somites begins to stain positively for myogenic proteins. The muscle-specific proteins are expressed in a specific order between g.d. 9 and 9.5. Desmin is detected first, then titin, then the muscle specific actin and myosin heavy chains, and finally nebulin. At g.d. 9.5 fibrous desmin structures are already present, while for the other myogenic proteins no structure can be detected. Some prefusion myoblasts display at g.d. 11 and 12 tiny and immature myofibrils. These reveal a periodic pattern of myosin, nebulin, and those titin epitopes known to occur at and close to the Z line. In contrast titin epitopes, which are present in mature myofibrils along the A band and at the A-I junction, are still randomly distributed. We propose, that the Z line connected structures and the A bands (myosin filaments) assemble independently, and that the known interaction of the I-Z-I brushes with the A bands occurs at a later developmental stage. After fusion of myoblasts to myotubes at g.d. 13 and 14 all titin epitopes show the myofibrillar banding pattern. The predominantly longitudinal orientation of desmin filaments seen in myoblasts and in early myotubes is transformed at g.d. 17 and 18 to distinct Z line connected striations. Vimentin, still present together with desmin in the myoblasts, is lost from the myotubes. Our results indicate that the putative elastic titin filaments act as integrators during skeletal muscle development. Some developmental aspects of eye and limb muscles are also described.     

The structure of the sarcomeric M band: localization of defined domains of myomesin, M-protein, and the 250-kD carboxy-terminal region of titin by immunoelectron microscopy

The M band of vertebrate cross-striated myofibrils has remained an enigmatic structure. In addition to myosin thick filaments, two major structural proteins, myomesin and M-protein, have been localized to the M band. Also, titin is expected to be anchored in this structure. To begin to understand the molecular layout of these three proteins, a panel of 16 polyclonal and monoclonal antibodies directed against unique epitopes of defined sequence was assembled, and immunoelectron microscopy was used to locate the position of the epitopes at the sarcomere level. The results allow the localization and orientation of defined domains of titin, myomesin, and M-protein at high resolution. The 250-kD carboxy-terminal region of titin clearly enters the M band with the kinase domain situated approximately 52 nm from the central M1- line. The positions of three additional epitopes are compatible with the view that the titin molecule reaches approximately 60 nm into the opposite sarcomere half. Myomesin also seems to bridge the central M1- line and is oriented parallel to the long axis of the myofibril. The neighboring molecules are oriented in an antiparallel and staggered fashion. The amino-terminal portion of the protein, known to contain a myosin binding site, seems to adopt a specific three-dimensional arrangement. While myomesin is present in both slow and fast fibers, M- protein is restricted to fast fibers. It appears to be organized in a fundamentally different manner: the central portion of the polypeptide is around the M1-line, while the terminal epitopes seem to be arranged along thick filaments. This orientation fits the conspicuously stronger M1-lines in fast twitch fibers. Obvious implications of this model are discussed.     

Monoclonal antibodies distinguish titins from heart and skeletal muscle

Murine monoclonal antibodies specific for titin have been elicited using a chicken heart muscle residue as antigen. The three antibodies T1, T3, and T4 recognize both bands of the titin doublet in immunoblot analysis on polypeptides from chicken breast muscle. In contrast, on chicken cardiac myofibrils two of the antibodies (T1, T4) react only with the upper band of the doublet indicating immunological differences between heart and skeletal muscle titin. This difference is even more pronounced for rat and mouse. Although all three antibodies react with skeletal muscle titin, T1 and T4 did not detect heart titin, whereas T3 reacts with this titin both in immunofluorescence microscopy and in immunoblots. Immunofluorescence microscopy of myofibrils and frozen tissues from a variety of vertebrates extends these results and shows that the three antibodies recognize different epitopes. All three titin antibodies decorate at the A-I junction of the myofibrils freshly prepared from chicken skeletal muscle and immunoelectron microscopy using native myosin filaments demonstrates that titin is present at the ends of the thick filaments. In chicken heart, however, antibodies T1 and T4 stain within the I-band rather than at the A-I junction. The three antibodies did not react with any of the nonmuscle tissues or permanent cell lines tested and do not decorate smooth muscle. In primary cultures of embryonic chicken skeletal muscle cells titin first appears as longitudinal striations in mononucleated myoblasts and later at the myofibrillar A-I junction of the myotubes.     

Nebulin as a length regulator of thin filaments of vertebrate skeletal muscles: correlation of thin filament length, nebulin size, and epitope profile

Nebulin, a family of giant proteins with size-variants from 600 to 900 kD in various skeletal muscles, have been proposed to constitute a set of inextensible filaments anchored at the Z line (Wang, K., and J. Wright. 1988. J. Cell Biol. 107:2199-2212). This newly discovered filament of the skeletal muscle sarcomere is an attractive candidate for a length-regulating template of thin filaments. To evaluate this hypothesis, we address the question of coextensiveness of nebulin and the thin filament by searching for a correlation between the size of nebulin variants and the length distribution of the thin filaments in several skeletal muscles. A positive linear correlation indeed exists for a group of six skeletal muscles that display narrow thin filament length distributions. To examine the molecular and architectural differences of nebulin size-variants, we carried out immunoelectron microscopic studies to map out epitope profiles of nebulin variants in these muscles. For this purpose, a panel of mAbs to distinct nebulin epitopes was produced against rabbit nebulin purified by an improved protocol. Epitope profiles of nebulin variants in three skeletal muscles revealed that (a) nebulin is inextensible since nebulin epitopes maintain a fixed distance to the Z line irrespective of the degree of sarcomere stretch; (b) a single nebulin polypeptide spans a minimal distance of 0.9 microns from the Z line; (c) nebulin contains repeating epitopes that are spaced at 40 nm or its multiples; (d) nebulin repeats coincide with thin filament periodicity; (e) nebulin variants differ mainly at either or both ends; and (f) nebulin remains in the sarcomere in actin-free sarcomeres produced by gelsolin treatment. Together, these data suggest that nebulin is an inextensible full-length molecular filament that is coextensive with thin filaments in skeletal muscles. We propose that nebulin acts as a length-regulating template that determines thin filament length by matching its large number of 40-nm repeating domains with an equal number of helical repeats of the actin filaments.     

Organization of connectin/titin filaments in sarcomeres of differentiating chicken skeletal muscle cells

Very long, elastic connectin/titin molecules position the myosin filaments at the center of a sarcomere by linking them to the Z line. The behavior of the connectin filaments during sarcomere formation in differentiating chicken skeletal muscle cells was observed under a fluorescent microscope using the antibodies to the N terminal (located in the Z line), C terminal (M line), and C zone (myosin filament) regions of connectin and was compared to the incorporation of -actinin and myosin into forming sarcomeres. In early stages of differentiating muscle cells, the N terminal region of connectin was incorporated into a stress fiber-like structure (SFLS) together with -actinin to form dots, whereas the C terminal region was diffusely distributed in the cytoplasm. When both the C and N terminal regions formed striations in young myofibrils, the epitope to the C zone of A-band region, that is the center between the A-I junction and the M-line, initially was diffuse in appearance and later formed definite striations. It appears that it took some time for the N and C terminal regions of connectin to form a regular organization in a sarcomere. Thus the two ends of the connectin filaments were first fixed followed by the specific binding of the middle portion onto the myosin filament during sarcomere formation.     

Differential distribution of subsets of myofibrillar proteins in cardiac nonstriated and striated myofibrils

Cultured cardiac myocytes were stained with antibodies to sarcomeric alpha-actinin, troponin-I, alpha-actin, myosin heavy chain (MHC), titin, myomesin, C-protein, and vinculin. Attention was focused on the distribution of these proteins with respect to nonstriated myofibrils (NSMFs) and striated myofibrils (SMFs). In NSMFs, alpha-actinin is found as longitudinally aligned, irregular approximately 0.3-microns aggregates. Such aggregates are associated with alpha-actin, troponin-I, and titin. These I-Z-I-like complexes are also found as ectopic patches outside the domain of myofibrils in close apposition to the ventral surface of the cell. MHC is found outside of SMFs in the form of discrete fibrils. The temporal-spatial distribution and accumulation of the MHC-fibrils with respect to the I-Z-I-like complexes varies greatly along the length of the NSMFs. There are numerous instances of I-Z-I-like complexes without associated MHC-fibrils, and also cases of MHC-fibrils located many microns from I-Z-I-like complexes. The transition between the terminal approximately 1.7-microns sarcomere of any given SMF and its distal NSMF-tip is abrupt and is marked by a characteristic narrow alpha-actinin Z-band and vinculin positive adhesion plaque. A titin antibody T20, which localizes to an epitope at the Z-band in SMFs, precisely costains the 0.3-microns alpha-actinin aggregates in ectopic patches and NSMFs. Another titin antibody T1, which in SMFs localizes to an epitope at the A-I junction, typically does not stain ectopic patches and NSMFs. Where detectable, the T1-positive material is adjacent to rather than part of the 0.3-microns alpha-actinin aggregates. Myomesin and C-protein are found only in their characteristic sarcomeric locations (even in just perceptible SMFs). These A-band-associated proteins appear to be absent in ectopic patches and NSMFs.     

M line-deficient titin causes cardiac lethality through impaired maturation of the sarcomere

Titin, the largest protein known to date, has been linked to sarcomere assembly and function through its elastic adaptor and signaling domains. Titin's M-line region contains a unique kinase domain that has been proposed to regulate sarcomere assembly via its substrate titin cap (T-cap). In this study, we use a titin M line-deficient mouse to show that the initial assembly of the sarcomere does not depend on titin's M-line region or the phosphorylation of T-cap by the titin kinase. Rather, titin's M-line region is required to form a continuous titin filament and to provide mechanical stability of the embryonic sarcomere. Even without titin integrating into the M band, sarcomeres show proper spacing and alignment of Z discs and M bands but fail to grow laterally and ultimately disassemble. The comparison of disassembly in the developing and mature knockout sarcomere suggests diverse functions for titin's M line in embryonic development and the adult heart that not only involve the differential expression of titin isoforms but also of titin-binding proteins.     

Elastic filaments in situ in cardiac muscle: deep-etch replica analysis in combination with selective removal of actin and myosin filaments

To clarify the full picture of the connectin (titin) filament network in situ, we selectively removed actin and myosin filaments from cardiac muscle fibers by gelsolin and potassium acetate treatment, respectively, and observed the residual elastic filament network by deep-etch replica electron microscopy. In the A bands, elastic filaments of uniform diameter (6-7 nm) projecting from the M line ran parallel, and extended into the I bands. At the junction line in the I bands, which may correspond to the N2 line in skeletal muscle, individual elastic filaments branched into two or more thinner strands, which repeatedly joined and branched to reach the Z line. Considering that cardiac muscle lacks nebulin, it is very likely that these elastic filaments were composed predominantly of connectin molecules; indeed, anti-connectin monoclonal antibody specifically stained these elastic filaments. Further, striations of approximately 4 nm, characteristic of isolated connectin molecules, were also observed in the elastic filaments. Taking recent analyses of the structure of isolated connectin molecules into consideration, we concluded that individual connectin molecules stretched between the M and Z lines and that each elastic filament consisted of laterally-associated connectin molecules. Close comparison of these images with the replica images of intact and S1-decorated sarcomeres led us to conclude that, in intact sarcomeres, the elastic filaments were laterally associated with myosin and actin filaments in the A and I bands, respectively. Interestingly, it was shown that the elastic property of connectin filaments was not restricted by their lateral association with actin filaments in intact sarcomeres. Finally, we have proposed a new structural model of the cardiac muscle sarcomere that includes connectin filaments.     

Localization of paratropomyosin in skeletal muscle myofibrils and its translocation during postmortem storage of muscles

Using polyclonal antibodies against paratropomyosin, which is believed to modify the actin-myosin interaction in postrigor skeletal muscles, we studied the localization of paratropomyosin in chicken breast muscle myofibrils. Intact myofibrils stained with fluorescent antibodies showed that paratropomyosin was exclusively located at the A-I junction region of sarcomeres. In stretched myofibrils (3.7 micron in sarcomere length), the approximate width of the fluorescent stripes and their relation to the A band remained constant. Removal of the A band from myofibrils led to loss of stainability. During postmortem storage of muscles, on the other hand, paratropomyosin was translocated from its original position at the A-I junction region onto thin filaments. The translocation of paratropomyosin was successfully induced with a calcium ion concentration of 10(-4) M in the presence of protease inhibitors. We therefore conclude that in postrigor muscles, paratropomyosin is released from the A-I junction region following the increase in the sarcoplasmic calcium ion concentration to 10(-4) M, and then binds to thin filaments, which results in weakening of rigor linkages formed between actin and myosin.     

Structural evidence for a possible role of reversible disulphide bridge formation in the elasticity of the muscle protein titin

BACKGROUND: The giant muscle protein titin contributes to the filament system in skeletal and cardiac muscle cells by connecting the Z disk and the central M line of the sarcomere. One of the physiological functions of titin is to act as a passive spring in the sarcomere, which is achieved by the elastic properties of its central I band region. Titin contains about 300 domains of which more than half are folded as immunoglobulin-like (Ig) domains. Ig domain segments of the I band of titin have been extensively used as templates to investigate the molecular basis of protein elasticity. RESULTS: The structure of the Ig domain I1 from the I band of titin has been determined to 2.1 A resolution. It reveals a novel, reversible disulphide bridge, which is neither required for correct folding nor changes the chemical stability of I1, but it is predicted to contribute mechanically to the elastic properties of titin in active sarcomeres. From the 92 Ig domains in the longest isoform of titin, at least 40 domains have a potential for disulphide bridge formation. CONCLUSIONS: We propose a model where the formation of disulphide bridges under oxidative stress conditions could regulate the elasticity of the I band in titin by increasing sarcomeric resistance. In this model, the formation of the disulphide bridge could refrain a possible directed motion of the two beta sheets or other mechanically stable entities of the I1 Ig domain with respect to each other when exposed to mechanical forces.     

Nebulin and titin expression in Duchenne muscular dystrophy appears normal

Monoclonal antibodies which recognize different epitopes on either titin or nebulin show normal staining patterns on frozen sections of three muscle biopsies of Duchenne muscular dystrophy (DMD). Gel electrophoresis and immunoblotting performed on two of these muscle biopsies show the normal pattern of titin and nebulin polypeptides. Since the donor of one of these biopsies has a large deletion of the 5'-region of the DMD gene, our results argue against the recent proposal that nebulin is the gene mutated in DMD.     

Solution structure of ZASP PDZ domain; implications for sarcomere ultrastructure and enigma family redundancy

Z band alternately spliced PDZ-containing protein (ZASP) is a sarcomere Z disk protein expressed in human cardiac and skeletal muscle that is thought to be involved in a dominant familial dilated cardiomyopathy. The N-terminal PDZ domain of ZASP interacts with the C terminus of alpha-actinin-2, the major component of the Z disk, probably by forming a ternary complex with titin Z repeats. We have determined the structure of ZASP PDZ by NMR and showed that it is a classical class 1 PDZ domain that recognizes the carboxy-terminal sequence of an alpha-actinin-2 calmodulin-like domain with micromolar affinity. We also characterized the role of each component in the ternary complex ZASP/alpha-actinin-2/titin, showing that the alpha-actinin-2/ZASP PDZ interaction involves a binding surface distinct from that recognized by the titin Z repeats. ZASP PDZ structure was used to model other members of the enigma family by homology and to predict their abilities to bind alpha-actinin-2.     

Epitopes on the outer surface protein A of Borrelia burgdorferi recognized by antibodies and T cells of patients with Lyme disease.

We have characterized immunogenic epitopes of the 31-kDa outer surface protein A (OspA) protein of Borrelia burgdorferi, which is a major surface Ag of the spirochete causing Lyme disease. Full length and truncated forms of rOspA proteins were expressed in Escherichia coli, and their reactivities with antibodies and human T cell clones isolated from patients with Lyme disease were determined. The epitopes recognized by three of four OspA-reactive T cell clones are contained within the 60 COOH-terminal amino acids. Each of the four OspA-reactive T cell clones has a different HLA class II molecule involved in Ag recognition and recognizes a distinct epitope. One T cell clone promiscuously recognized an epitope in the context of different HLA-DQ molecules. In addition, the binding of a murine monoclonal anti-OspA antibody, as well as antibodies in sera of three of five patients with Lyme disease, was dependent upon the amino acids in the carboxy-terminal protion of this protein. Taken together, our results indicate that the 60 COOH-terminal amino acids of OspA contain epitopes recognized by human antibodies and T cells.     

Sarcomere matrix of striated muscle: in vivo phosphorylation of titin and nebulin in mouse diaphragm muscle

Titin and nebulin are two major protein components of a cytoskeletal matrix that coexists with thick and thin filaments within the sarcomere of a wide range of striated muscles. Purified titin and nebulin from mouse diaphragm muscle are similar in size, in relative abundance, and in amino acid composition to analogous proteins from other mammals or avians. Phosphate analysis of these nucleic-acid-free proteins indicated that both proteins contain substantial amounts of protein-bound phosphate: about 12 mol of phosphate per mole of titin subunit and 11 mol of phosphate per mole of nebulin subunit. Incubation of intact, excised mouse diaphragm with radioactive inorganic phosphate resulted in significant incorporation of radiophosphate into titin and nebulin. The identification of titin and nebulin phosphorylation was facilitated by a simple salt fractionation and nuclease digestion procedure that effectively separated titin and nebulin from radiolabeled nucleic acids. Such in vivo phosphorylation studies indicated that approximately 2 mol of phosphate per titin subunit and 5 to 7 mol of phosphate per nebulin subunit were incorporated within 5 h of incubation. The incorporation nearly doubled when the beta-adrenergic agonist, isoproterenol, or a phosphodiesterase inhibitor, theophylline, was present in the medium. For both proteins, phosphorylation occurred mainly on serine residues. Nebulin also appears to possess a smaller number of threonine sites. Taken together, our data indicate that a small proportion (20 to 40%) of the steady-state titin phosphates are rapidly turning over. In contrast, most of the nebulin phosphates (50 to 100%) are readily exchanged. The modulation of turnover by external stimuli that increase cytosolic cAMP raises the possibility that at least a portion of the multiple phosphorylation sites of titin and nebulin may be involved in the functional regulation of the sarcomere matrix.     

Obscurin, a giant sarcomeric Rho guanine nucleotide exchange factor protein involved in sarcomere assembly.

Vertebrate-striated muscle is assumed to owe its remarkable order to the molecular ruler functions of the giant modular signaling proteins, titin and nebulin. It was believed that these two proteins represented unique results of protein evolution in vertebrate muscle. In this paper we report the identification of a third giant protein from vertebrate muscle, obscurin, encoded on chromosome 1q42. Obscurin is approximately 800 kD and is expressed specifically in skeletal and cardiac muscle. The complete cDNA sequence of obscurin reveals a modular architecture, consisting of >67 intracellular immunoglobulin (Ig)- or fibronectin-3-like domains with multiple splice variants. A large region of obscurin shows a modular architecture of tandem Ig domains reminiscent of the elastic region of titin. The COOH-terminal region of obscurin interacts via two specific Ig-like domains with the NH(2)-terminal Z-disk region of titin. Both proteins coassemble during myofibrillogenesis. During the progression of myofibrillogenesis, all obscurin epitopes become detectable at the M band. The presence of a calmodulin-binding IQ motif, and a Rho guanine nucleotide exchange factor domain in the COOH-terminal region suggest that obscurin is involved in Ca(2+)/calmodulin, as well as G protein-coupled signal transduction in the sarcomere.     

Influence of lipid environment on insulin binding in cultured hepatoma cells

Three mouse monoclonal antibodies (Mabs) to human apo A-I were produced using apolipoprotein A-I or HDL3 as immunogens. These monoclonal antibodies, 2G11, 4A12 and 4B11, were characterized for their reactivity with isolated apolipoprotein A-I and HDL in solution. The immunoblotting patterns of the HDL3 two-dimensional electrophoresis show that these three monoclonal antibodies reacted with all the polymorphic forms of apolipoprotein A-I. Cotitration experiments indicated that they correspond to three distinct epitopes. In order to locate these three antigenic determinants on the isolated apolipoprotein A-I, the reactivity of the three monoclonal antibodies has been studied on CNBr-cleaved apolipoprotein A-I. The monoclonal antibodies 2G11 and 4A12 addressed to the amino (CNBr 1) and carboxy (CNBr 4) terminal segments, respectively. In comparison with the monoclonal antibodies characterized by Weech et al. ((1985) Biochim. Biophys. Acta 835, 390-401), monoclonal antibody 4A12 is the only one described in the literature which is specific of the carboxy terminal segment of apolipoprotein A-I. Monoclonal antibody 4B11 does not react with any CNBr fragment, its binding is temperature dependent, it could be directed to a conformational epitope. Relative differences were demonstrated in the expression of the three epitopes in HDL subfractions isolated by density gradient ultracentrifugation. According to Curtiss and Edgington ((1985) J. Biol. Chem. 260, 2982-2993) our results indicate the existence of an immunochemical heterogeneity in the organization of apolipoprotein A-I at the surface of HDL particles as well as in the soluble form of apolipoprotein A-I.     

Titin, a huge, elastic sarcomeric protein with a probable role in morphogenesis.

Titin, the largest protein identified to date (over 1 micron long, almost 3 million daltons in mass) is the third most abundant component of the sarcomere. In the mature myofibril, titin molecules span from M line to Z line, forming a third filament system which provides sarcomeric alignment and elastic recoil. In the developing sarcomere, accumulating evidence from studies both in vivo and in vitro implicates titin as part of a morphogenetic scaffolding, upon which critical events in myofibrillogenesis are coordinated in a time- and space-dependent manner.     

Interaction of nebulin SH3 domain with titin PEVK and myopalladin: implications for the signaling and assembly role of titin and nebulin

Skeletal muscle nebulin is thought to determine thin filament length and regulate actomyosin interaction in a calcium/calmodulin or S100 sensitive manner. We have investigated the binding of nebulin SH3 with proline-rich peptides derived from the 28-mer PEVK modules of titin and the Z-line protein myopalladin, using fluorescence, circular dichroism and nuclear magnetic resonance techniques. Of the six peptides studied, PR2 of titin (VPEKKAPVAPPK) and myopalladin MyoP2 (646VKEPPPVLAKPK657) bind to nebulin SH3 with micromolar affinity (approximately 31 and 3.4 microM, respectively), whereas the other four peptides bind weakly (>100 microM). Sequence analysis of titins reveals numerous SH3 binding motifs that are highly enriched in the PEVK segments of titin isoforms. Our findings suggest that titin PEVK and myopalladin may play signaling roles in targeting and orientating nebulin to the Z-line during sarcomere assembly.