A Network of Transverse and Longitudinal Filaments Is Associated with Sarcomeres of Adult Vertebrate Skeletal Muscle Intermediate

An extensive network of transverse and longitudinal filamentous bridges was revealed when small myofibril bundles, prepared from Triton-EGTA-treated rabbit skeletal muscles, were extracted with KI to remove the majority of thin and thick filaments. Transmission and scanning electron microscopic studies of these salt-resistant cytoskeletal residues indicated: (a) small bundles of short transverse filaments connect adjacent myofibrils by forming Z to Z and M to M bridges; (b) parallel, continuous longitudinal filaments connect the peripheries of successive Z-disks and ensheath the sarcomere. These transverse and longitudinal filaments have the characteristic morphology of intermediate filaments; (c) two rings of tightly interwoven and tangled filaments, connected laterally by short filaments, encircle each Z disk. This doublering also encircles a weblike meshwork which penetrates the sarcomeric space. From the peripheries of these rings, transverse and longitudinal intermediate filaments emerge; and (d) a massive amount of, material translocated and accumulated near Z disks during KI extraction. The residues were fairly resistant to solubilization by urea and SDS, and complete dissolution was achieved only with guanidinium chloride. SDS PAGE indicated that the residues consisted mainly of titin, nebulin, and variable amounts of residual myosin and actin. Desmin represented only a few percent of total residual proteins; however, it may be a major component of the intermediate filament network. We suggest that the intermediate filament should be considered an integral sarcomeric component that may play important cytoskeletal roles in muscle structure and mechanics. The characteristic cross-striated appearance of skeletal and cardiac muscles arises as a result of the transverse alignment of sarcomeric striations of neighboring myofibrils. The maintenance of this alignment has been attributed to the existence of filamentous bridges between Z disks and between M-lines across the fiber axis (for reviews, see references 16, 17). Most studies have focused on the fdamentous bridges that connect neighboring myofibrils--i.e., the transverse, interfibrillar type. However, little attention has been paid to the possible existence of longitudinal, intrafibrillar bridges that may connect adjacent sarcomeric structures of the same myofibril. Residual longitudinal filaments spanning the gaps between IZI brushes or between Z structures ~ of the same myofibril have been l ln this paper the term "Z structure" is used to refer to the dense residual structure appearing after KI extraction at the position of "Z disk" of intact muscle. This distinction is considered important because repeatedly detected in myofibrils that were depleted of thin and thick filaments by selective salt extractions (e.g. 3, 9, 13, 22), but the identity of these residual filaments remains unresolved. During our investigations oft i t in and n e b u l i n a group of large, major myofibrillar proteins (32-35)--we became intrigued by these residual longitudinal filaments and considered it possible that they represent a new type of myofilament consisting of these proteins. We have, therefore, investigated the t'me structure and spatial organization of these fdaments in Kl-extracted rabbit skeletal myofibrils. We report that the longitudinal fdaments appear to be parallel, continuous, interKI extracts much of the density of Z disks and causes salt-resistant non-Z proteins to translocate and to accumulate near the remains of Z disks. Therefore, the Z structure may bear only partial resemblance in structure and in composition to the Z disk of intact muscle. (K. Wang, unpublished observations). THE JOURNAL OF CLLL BIOLOGY-VOLUME 96 FEBRUARY 1983 562-570 562 © The Rockefeller University Press . 0021-9525/83/02/0562/09 $1.00 on O cber 0, 2017 jcb.rress.org D ow nladed fom mediate filaments that form a sleeve surrounding each myofibril and connect the peripheries of successive Z disks. Preliminary reports of this work have appeared in several abstracts (24, 32, 33). MATERIALS AND METHODS Preparation of Rabbit Skeletal Myofibrils and Myofibril Bundles The following procedures were carried out at 4°C in a cold room. A New Zealand white rabbit (~6 mo old) was injected intravenously with a sublethal dose of nembutal (3 ml of 50 mg/ml) and then exsanguinated. Back muscle strips (1-3 mm thick) were tied to plastic rods either at resting length or after gentle stretching to 150% of resting length and then excised. They were soaked twice in a Triton X-t00-containing chemical skinning solution (1% (wt/ vol) Triton X-IOO, 0.1 M KCL 1 mM MgCI2, 6.67 mM potassium phosphate, 5 mM EGTA, 0.1 mM DTT, pH 7.0) for 60 min each time, then washed three times with the skinning buffer without Triton X-IOO for 30 min each time. All procedures were performed in an apparatus designed according to the principle of the Bio-Rad tube gel diffusion destainer (Bio-Rad Laboratories, Richmond, CA) which circulates buffer (400 ml for 12 strips) efficiently around the firmly attached muscle strips. The skinned muscle strips were frequently used immediately for myofibril preparation or after storage at 2 0 ° C in 500 (vol/vol) glycerol in Triton X-loo-free skinning buffer for no more than 2 wk. The muscle strips were chopped into 2-ram pieces with a scalpel, suspended in 10-20 ml of the 50% (vol/vol) glycerol solution, and then blended in a VirTis Model 23 blade homogenizer (VirTis Co., Inc., Gardiner, NY) at 30-s bursts for a total of 2-10 min at a medium speed setting. Fragmentation was monitored with phase contrast microscopy. For the purpose of detecting interfibrillar connections, the blending was stopped when the majority of the myofibrils were stilt linked together as bundles consisting of two to five myofibrils. The preparation was washed one time with five volumes of Triton X-loo-free skinning solution by centrifuging at 5,000 rpm (3,000 gin,,) for 5 min in a Sorvall (DuPont Instruments-Sorvall Biomedical Div., Newtown, CT) SS-34 rotor and resuspended to 2-6 mg/mI. Preparation and Structural Studies of PotassiumIodide-extracted Myofibrils LIGHT MICROSCOPY; A drop ofmyofibri l suspension was placed between a slide and a coverslip for a few minutes and a drop of Triton X-t00-free skinning solution was added to one side of the cover slip while the buffer was withdrawn with a piece of filter paper on the other side to wash away nonadherent myofibrlls. Extraction with potassium iodide solution (0.6 M KI, 0.1 M Tris-Cl, 3 mM EGTA, 3 mM MgCI,2, 3 mM Na4P207, 5 mM Na2S203, 0.l mM DTT, pH 7.5) was performed immediately at room temperature by the same irrigation procedure. Individual myofibrils varied in the lag period before the commencement of extraction. Once started, however, the extraction process was rapid and was completed within 5-30 s. It was essential to perform the extraction within 10 min from the onset of the attachment of myofibrils to cover slips, because myofibrils became increasingly resistant to such extraction after prolonged attachment to the glass surface (>30 rain), despite the fact that nonadherent myofibrils in solution respond normally to such a treatment. Photomicrographs were taken with a Zeiss Universal microscope and a x 100 phase-contrast lens on Kodak Pan-X film and were developed with Microdol-X. ELECTRON MICROSCOPY: A drop of myofibril suspension o n a piece of parafilm was mixed gently with an equal volume of a 2x concentrated KI solution for 2 min at room temperature. A loo-mesh grid, coated with a glowdischarged carbon film deposited on Formvar, was then inverted on top o f the droplet. The grid was removed after 2 min, rinsed gently with a few drops of Ix KI solution, and fixed in 2% glutaraldehyde in 25 mM cacodylate, pH 7.2, for 4 min. The fixed sample was rinsed twice with 0.l M KCI, 0.01 M Tris-maleate, 2 mM MgCI.,, 2 mM EGTA, 0.1 mM DTT, pH 6.8, stained with a drop of 0.5% uranyl acetate for 5 s, and air-dried before observing it with a JEOL looCX transmission electron microscope operated at 80 kV. For scanning electron microscopy: small pieces of freshly cleaved mica were placed on top of the KIextracted myofibril suspension, rinsed, and fixed as described above. The fixed sample was rinsed thoroughly with water, dehydrated in ethanol, transferred through an amyl acetate in ethanol series, then critical-point dried. The samples were coated with 20 ~, of Au /Pd and examined with a JEOL 100CX equipped with a high resolution scanning attachment. The microscope was operated at 40 kV and calibrated for magnification with a Fullam calibration grating (463 nm/ line). SDS GEL ELECTROPHORESIS: Samples for SDS gel electrophoresis were prepared by dissolving myofibrils or Kl-residue (extracted in suspension for 14 min at room temperature and pelleted by centrifuging at 15,000 rpm for 10 min in a Sorvall SS-34 rotor) in a guanidinium chloride buffer (6 M guanidinium chloride, 0.05 M Tris.Cl, 5 mM EDTA, 10 mM DTT, pH 8.0) for 30 min at room temperature. The solubilized samples were dialyzed against 7 M urea buffer (7 M urea, 0.05 M Tris.Cl, 5 mM EDTA, 0.l M DTT, pH 8.0) to remove guanidinium chloride and then prepared for SDS gel electrophoresis by the addition of an appropriate volume of 3x SDS sample buffer (0.03 M Tris. el , 3 mM EDTA, 0.12 M DTT, 3% [wt/vol] SDS, 300 [vot/vol] glycerol, 30 #g/ml Pyronin Y., pH 8.0), followed by incubation at 50 °C for 15 min. Guanidinium chloride was necessary for consistent gel patterns representative of the entire sample. The KI-residue frequently aggregated, especially after vigorous stirring or centrifugation, into a gellike material that could be solubilized only partially in SDS or urea. Both titin and nebulin resisted solubilization. The KI-residue, however, was completely dissolved in guanidinium chloride and remained soluble after being dialyzed into urea solution. SDS samples were electrophoresed on gradient polyacrylamide gels (2-12% linear gradient) and were stained with Coomassie Blue as described (27, cf. reference 5). DESMIN PREPARATION: Chicken gizzard desmin was purified according to