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Abstract: The complete gene giant muscle protein obscurin, a modular protein composed largely of tandem Ig-domains, GDP/GTP exchange factor domains (GEF) for small G-proteins, and differentially spliced kinase domains, was analysed. The splice donor and acceptor sites of the 117 exons give important clues for potential splice pathways. The fusion of the conventional obscurin A, containing only the GEF domain, and obscurin B, fusing into the 3′ kinase exons, was experimentally confirmed and analysed. The linker between the two kinases contains multiple predicted phosphorylation sites, as well as a predicted NFX zinc finger domain. Both kinases show only weak homology to either myosin light chain kinases or other giant muscle protein kinases, suggesting that they are functionally distinct.

Mutations of the human telethonin gene have recently been shown to cause limb girdle muscular dystrophy type 2G in three Brazilian families. The mRNA has been shown to be dynamically regulated in animals, however, the fate of the protein in human muscle is unknown. In order to assess the expression of telethonin in more frequently encountered myopathological conditions we generated and characterized a rabbit antiserum raised against the C-terminal end of telethonin by immunoblotting and immunogold EM. Indirect immunofluorescence analysis of a wide variety of neuromuscular disorders including dystrophinopathies, metabolic myopathies, denervation disorders, congenital and inflammatory myopathies revealed that the characteristic Z-band staining of telethonin was preserved in all disease entities included in our study. However, a reduced telethonin immunoreactivity was observed in up to 10% of type II fibers in 10 cases of neurogenic atrophy. A decreased telethonin staining was more frequently observed in early stages of fiber atrophy than in type II fibers displaying normal or highly atrophic fiber diameters. Hence, not only the telethonin transcript is rapidly downregulated in denervated muscle but the protein itself undergoes dynamic changes while its known sarcomeric binding partner titin remains unaltered. Beyond its role as a static component of Z-bands, these findings indicate that telethonin protein levels seems to be at least in part regulated by neuronal activity and is thus linked to the dynamic control of myofibrillogenesis and muscle turnover in human skeletal muscle.

The giant molecule titin/connectin was demonstrated to connect the ends of thick filaments with the Z-disks and thus to provide an elastic connection that seems to be responsible for passive tension in striated muscle. To investigate the physiological limits of I-band titin extension in skeletal muscle, we have measured sarcomere lengths of a number of mouse postural and clonal muscles in situ under the constraints imposed by the skeletal, ligamentous and tendinous components of the motile apparatus. These values now give upper limits for the extension of the I-band and therefore for the maximal degree of titin extension under physiological constraints. We find that I-band extension in all muscles investigated does not exceed a factor of 2.5 in situ, which is well below values obtainable in isolated fibre preparations. Approach to the yield-point is therefore prevented by extramuscular mechanisms. Sarcomere lengths near the tendinous junction and within the muscle are virtually identical in extended muscle, suggesting that a major function of titin in intact muscle is to ensure uniform sarcomere lengths over the entire muscle length and thus to prevent localized myofibril overstretch during isometric contraction

The giant molecule titin (also called connectin) provides an elastic connection in the I-band between the Z-disk and A-band of striated muscle. This region is assembled in a tissue-specific way by extensive differential splicing events. We have raised monoclonal antibodies against the two N2-line isoforms of titin and demonstrate that both forms of cardiac I-band titin are constitutively co-expressed in atrial and ventricular muscle. In developing mouse embryos, the expression of the cardiac N2-B isoform remains strictly cardiac-specific and is linked to the expression of the ubiquitous N2-A isoform. The mechanical function of the cardiac N2-line region was investigated ultrastructurally. Immunoelectron microscopy reveals that the N2-B region separates two mechanically distinct sections of titin with a hyperextensible segment spanning the distance to the Z-disk. The formation of a plateau in the extension of cardiac titin rules out that Ig-domains can be unfolded as a mechanism of elasticity.

We have investigated the interaction of a 6-repeat recombinant human nebulin fragment (S6R2R7) with F-actin, with Mg2+-induced actin paracrystals, and G-actin, respectively. This fragment corresponds to super-repeat 6, repeat 2 to 7 of human nebulin, and is located in the N-terminal part of the super-repeat region of the nebulin molecule. The S6R2R7 fragment included an immuno-tag of three amino-acid residues (EEF) at one end which was detectable by a monoclonal anti-tubulin YL1/2. By a cosedimentation assay, interaction between F-actin and S6R2R7 was observed. Electron microscopy revealed the formation of large bundle-like aggregates containing highly parallelized actin filaments, apparently caused by actin bundling of the nebulin fragment. Compared with Mg2+-induced actin paracrystals where the helices of the actin filaments are arranged in register, the filaments in the actin–nebulin bundles seem to be packed in a different way and show no obvious periodicity. The bundles were also visible in the light microscope, and immunofluorescence microscopy revealed binding of the nebulin fragment S6R2R7 to both preformed Mg2+paracrystals and to F-actin. We also analyzed the effect of S6R2R7 on actin under non-polymerizing conditions by cosedimentation assays and pyrene actin fluorimetry, as well as fluorescence microscopy and electron microscopy. Nebulin-induced actin polymerization was observed with an enhancement of the nucleation step indicating a stabilization of actin nuclei by S6R2R7. Light and electron microscopy revealed bundle-like actin–nebulin aggregates similar to those formed by pre-assembled F-actin and S6R2R7. Thus, even in the absence of salt, S6R2R7 promotes actin polymerization and induces formation of tightly packed actin filament bundles. We assume that the actin filaments are crosslinked by the nebulin fragments, indicating a rather low cooperativity of binding to a single filament.