Tropomodulin is associated with the free (pointed) ends of the thin filaments in rat skeletal muscle

The length and spatial organization of thin filaments in skeletal muscle sarcomeres are precisely maintained and are essential for efficient muscle contraction. While the major structural components of skeletal muscle sarcomeres have been well characterized, the mechanisms that regulate thin filament length and spatial organization are not well understood. Tropomodulin is a new, 40.6-kD tropomyosin-binding protein from the human erythrocyte membrane skeleton that binds to one end of erythrocyte tropomyosin and blocks head-to-tail association of tropomyosin molecules along actin filaments. Here we show that rat psoas skeletal muscle contains tropomodulin based on immunoreactivity, identical apparent mobility on SDS gels, and ability to bind muscle tropomyosin. Results from immunofluorescence labeling of isolated myofibrils at resting and stretched lengths using anti-erythrocyte tropomodulin antibodies indicate that tropomodulin is localized at or near the free (pointed) ends of the thin filaments; this localization is not dependent on the presence of myosin thick filaments. Immunoblotting of supernatants and pellets obtained after extraction of myosin from myofibrils also indicates that tropomodulin remains associated with the thin filaments. 1.2-1.6 copies of muscle tropomodulin are present per thin filament in myofibrils, supporting the possibility that one or two tropomodulin molecules may be associated with the two terminal tropomyosin molecules at the pointed end of each thin filament. Although a number of proteins are associated with the barbed ends of the thin filaments at the Z disc, tropomodulin is the first protein to be specifically located at or near the pointed ends of the thin filaments. We propose that tropomodulin may cap the tropomyosin polymers at the pointed end of the thin filament and play a role in regulating thin filament length.     

Transverse elasticity of myofibrils of rabbit skeletal muscle studied by atomic force microscopy

Surface structure of myofibrils of rabbit skeletal muscle and their transverse elasticity were studied by atomic force microscopy. Images of myofibrils had a periodic structure characteristic of sarcomeres of skeletal muscle fibers. The transverse elasticity distribution in the sarcomere was determined based on force-distance curves measured at various loci of single myofibrils. The Z-line in rigor myofibrils was the most rigid in all the loci of myofibrils studied under various physiological conditions. The overall transverse elasticity of myofibrils decreased in the order in rigor solution > +AMPPNP solution > relaxing solution. The "apparent" transverse Young's modulus of myofibrils estimated at the overlap region between thin and thick filaments was 84.0 +/- 18.1, 37.5 +/- 14.0, and 11.5 +/- 3.5 kPa in rigor, +AMPPNP, and relaxing solution respectively.     

Ultrastructurally different muscle cell types in Eisenia foetida (Annelida,Oligochaeta)

Muscles in the body wall, intestinal wall, and contractile hemolymphatic vessels (pseudohearts) of an oligochaete anelid (Eisenia foetida) were studied by electron microscopy. The muscle cells in all locations, except for the outer layer of the pseudohearts, are variants of obliquely striated muscle cells. Cells comprising the circular layer of the body wall possess single, peripherally located myofibrils that occupy most of the cytoplasm and surround other cytoplasmic organelles. The nuclei of the cells lie peripherally to the myofibrils. The sarcomeres consist of thin and thick myofilaments that are arranged in parallel arrays. In one plane of view, the filaments appear to be oriented obliquely to Z bands. Thin myofilaments measure 5–6 nm in diameter. Thick myofilaments are fusiform in shape and their width decreases from their centers (40–45 nm) to their tips (23–25 nm). The thin/thick filament ratio in the A bands is 10. The Z bands consist of Z bars alternating with tubules of the sarcoplasmic reticulum. Subsarcolemmal electron-dense plaques are found frequently. The cells forming the longitudinal layer of the body wall musculature are smaller than the cells in the circular layer and their thick filaments are smaller (31–33 nm centrally and 21–23 nm at the tips). Subsarcolemmal plaques are less numerous. The cells forming the heart wall inner layer, the large hemolymphatic vessels, and the intestinal wall are characterized by their large thick myofilaments (50–52 nm centrally and 27–28 nm at the tips) and abundance of mitochondria. The cells forming the outer muscular layer of the pseudohearts are smooth muscle cells. These cells are richer in thick filaments than vertebrate smooth muscle cells. They differ from obliquely striated muscle cells by possessing irregularly distributed electron-dense bodies for filament anchorage rather than sarcomeres and Z bands and by displaying tubules of smooth endoplasmic reticulum among the bundles of myofilaments. © 1995 Wiley-Liss, Inc.     

Ultrastructure of Developing Flight Muscle in Drosophila. I. Assembly of Myofibrils

In order to evaluate the effects of specific mutations on sarcomere assembly and function in vivo, we describe the course of normal development of Drosophila indirect flight muscle (IFM) in staged pupae using electron microscopy. We find that no contractile assemblies remain in larval muscle remnants invaded by imaginal myoblasts, establishing that myofibrils in IFM assemble de novo. Stress-fiber-like structures or other template structures are not prominent before or during sarcomere assembly. By 42 hr pupation (eclosion 112 hr), thick and thin filaments have appeared simultaneously in slender, interdigitated arrays between regularly spaced Z-bodies. Each tiny, uniformly striated myofibril forms within a "sleeve" of microtubules, and both microtubules and myofibrils are attached to the cell membrane at each end of the fiber from the initial stages of assembly. Later in pupation, the microtubule "sleeves" disassemble. Sarcomere number appears to remain constant. We saw no evidence that terminal sarcomeres are sites for addition of new sarcomeres or that Z-lines split transversely, producing new, very short sarcomeres. Rather, initial thick and thin filaments and sarcomeres are much shorter than adult length. Sarcomere length increases smoothly and coordinately from 1.7 to 3.2 μm, reflecting increase in filament lengths and indicating that myosin and actin molecules must be incorporated into filaments after sarcomere formation. Myofilaments are not seen scattered in the cytoplasm at any time, nor do we detect filaments that could be in the process of being "trolleyed" along myofibrils into positions of lateral register. Myofibril diameter increases uniformly from 4-thick filaments to 36-thick filaments across, by peripheral addition of myofilaments. At each successive stage, all sarcomeres in a fiber attained similar length and diameter. Initial thick filaments are solid but within several hours these and all subsequently assembled thick filaments appear hollow. Initial Z-bodies do not show any internal lattice and are more irregularly shaped than adult Z-discs.     

Immunohistochemistry of chaetognath body wall muscles

Abstract. A light and electron immunohistochemical study was carried out on the body wall muscles of the chaetognath Sagitta friderici for the presence of a variety of contractile proteins (myosin, paramyosin, actin), regulatory proteins (tropomyosin, troponin), and structural proteins (α‐actinin, desmin, vimentin). The primary muscle (～80% of body wall volume) showed the characteristic structure of transversely striated muscles, and was comparable to that of insect asynchronous flight muscles. In addition, the body wall had a secondary muscle with a peculiar structure, displaying two sarcomere types (S1 and S2), which alternated along the myofibrils. S1 sarcomeres were similar to those in the slow striated fibers of many invertebrates. In contrast, S2 sarcomeres did not show a regular sarcomeric pattern, but instead exhibited parallel arrays of 2 filament types. The thickest filaments (～10–15 nm) were arranged to form lamellar structures, surrounded by the thinnest filaments (～6 nm). Immunoreactions to desmin and vimentin were negative in both muscle types. The primary muscle exhibited the classical distribution of muscle proteins: actin, tropomyosin, and troponin were detected along the thin filaments, whereas myosin and paramyosin were localized along the thick filaments; immunolabeling of α‐actinin was found at Z‐bands. Immunoreactions in the S1 sarcomeres of the secondary muscle were very similar to those found in the primary muscle. Interestingly, the S2 sarcomeres of this muscle were labeled with actin and tropomyosin antibodies, and presented no immunore‐actions to both myosin and paramyosin. α‐Actinin in the secondary muscle was only detected at the Z‐lines that separate S1 from S2. These findings suggest that S2 are not true sarcomeres. Although they contain actin and tropomyosin in their thinnest filaments, their thickest filaments do not show myosin or paramyosin, as the striated muscle thick myofilaments do. These peculiar S2 thick filaments might be an uncommon type of intermediate filament, which were labeled neither with desmin or vimentin antibodies.     

Mitosis and intermediate-sized filaments in developing skeletal muscle

A new class of filaments intermediate in diameter between actin and myosin filaments has been demonstrated in skeletal muscle cells cultured from chick embryos. These filaments, which account for the majority of free filaments, average 100 A in diameter. They may run for more than 2 µ in a single section and can be distinguished in size and appearance from the thick and thin filaments assembled into myofibrils. The 100-A filaments are seen scattered throughout the sarcoplasm at all stages of development and show no obvious association with the myofibrils. The 100-A filaments are particularly conspicuous in myotubes fragmented by the mitotic inhibitors, colchicine and Colcemid. In addition, filaments similar in size and appearance to those found in myotubes are present in fibroblasts, chondrocytes, and proliferating mononucleated myoblasts. The 100-A filaments are present in cells arrested in metaphase by mitotic inhibitors. Definitive thick (about 150 A) or thin (about 60 A) myofilaments are not found in skeletal myogenic cells arrested in metaphase. Myogenic cells arrested in metaphase do not bind fluorescein-labeled antibody directed against myosin or actin. For these reasons, it is concluded that not all "thin" filaments in myogenic cells are uniquely associated with myogenesis.     

Fine Structure of Cardiac Sarcomeres in the Black Widow Spider Latrodectus mactans

Fine structural characteristics of the cardiac muscle and its sarcomere organization in the black widow spider, Latrodectus mactans were examined using transmission electron microscopy. The arrangement of cardiac muscle fibers was quite similar to that of skeletal muscle fibers, but they branched off at the ends and formed multiple connections with adjacent cells. Each cell contained multiple myofibrils and an extensive dyadic sarcotubular system consisting of sarcoplasmic reticulum and T‐tubules. Thin and thick myofilaments were highly organized in regular repetitive arrays and formed contractile sarcomeres. Each repeating band unit of the sarcomere had three apparent striations, but the H‐zone and M‐lines were not prominent. Myofilaments were arranged into distinct sarcomeres defined by adjacent Z‐lines with relatively short lengths of 2.0 μm to 3.3 μm. Cross sections of the A‐band showed hexagon‐like arrangement of thick filaments, but the orbit of thin filaments around each thick filament was different from that seen in other vertebrates. Although each thick filament was surrounded by 12 thin filaments, the filament ratio of thin and thick myofilaments varied from 3:1 to 5:1 because thin filaments were shared by adjacent thick filaments.     

Scaffolds and chaperones in myofibril assembly: putting the striations in striated muscle

Sarcomere assembly in striated muscles has long been described as a series of steps leading to assembly of individual proteins into thick filaments, thin filaments and Z-lines. Decades of previous work focused on the order in which various structural proteins adopted the striated organization typical of mature myofibrils. These studies led to the view that actin and α-actinin assemble into premyofibril structures separately from myosin filaments, and that these structures are then assembled into myofibrils with centered myosin filaments and actin filaments anchored at the Z-lines. More recent studies have shown that particular scaffolding proteins and chaperone proteins are required for individual steps in assembly. Here, we review the evidence that N-RAP, a LIM domain and nebulin repeat protein, scaffolds assembly of actin and α-actinin into I-Z-I structures in the first steps of assembly; that the heat shock chaperone proteins Hsp90 & Hsc70 cooperate with UNC-45 to direct the folding of muscle myosin and its assembly into thick filaments; and that the kelch repeat protein Krp1 promotes lateral fusion of premyofibril structures to form mature striated myofibrils. The evidence shows that myofibril assembly is a complex process that requires the action of particular catalysts and scaffolds at individual steps. The scaffolds and chaperones required for assembly are potential regulators of myofibrillogenesis, and abnormal function of these proteins caused by mutation or pathological processes could in principle contribute to diseases of cardiac and skeletal muscles.     

In situ reconstitution of myosin filaments within the myosin-extracted myofibril in cultured skeletal muscle cells

We studied the in situ reconstitution of myosin filaments within the myosin-extracted myofibrils in cultured chick embryo skeletal muscle cells using the electron microscope and polarization microscope. Myosin was first extracted from the myofibrils in glycerinated muscle cells with a high-salt solution containing 0.6 M KCl. When rabbit skeletal muscle myosin was added to the myosin-extracted cells in the high-salt solution, thin filaments in the ghost myofibrils were bound with myosin to form arrowhead complexes. Subsequent dilution of KCl in the myosin solution to 0.1 M resulted in the formation of thick myosin filaments within the myofibrils, increasing the birefringence of the myofibrils. When Mg-ATP was added such myosin-reassembled myofibrils were induced either to form supercontraction bands or to restore the sarcomeric arrangement of thick and thin filaments. Under the polarization microscope, vibrational movement of the myofibrils was seen transiently upon addition of Mg-ATP, often resulting in a regular arrangement of myofibrils in register. These myofibrils, with reconstituted myosin filaments, structurally and functionally resembled the native myofibrils. The findings are discussed with special reference to the myofibril formation in developing muscle cells.     

Measurement of thin filament lengths by distributed deconvolution analysis of fluorescence images

The lengths of the actin (thin) filaments in sarcomeres directly influence the physiological properties of striated muscle. Although electron microscopy techniques provide the highest precision and accuracy for measuring thin filament lengths, significant obstacles limit their widespread use. Here, we describe distributed deconvolution, a fluorescence-based method that determines the location of specific thin filament components such as tropomodulin (Tmod) or probes such as phallacidin (a phalloidin derivative). Using Tmod and phallacidin fluorescence, we were able to determine the thin filament lengths of isolated chicken pectoralis major myofibrils with an accuracy and precision comparable to electron microscopy. Additionally, phallacidin fluorescence intensity at the Z line provided information about the width of Z lines. Furthermore, we detected significant variations in thin filaments lengths among individual myofibrils from chicken posterior latissimus dorsai and embryonic chick cardiac myocytes, suggesting that a ruler molecule (e.g., nebulin) does not strictly determine thin filament lengths in these muscles. This versatile method is applicable to myofibrils in living cells that exhibit significant variation in sarcomere lengths, and only requires a fluorescence microscope and a CCD camera.     

Ultrastructure of the aortic diverticula of the adult dragonfly Sympetrum danae (Odonata: anisoptera)

Summary The aorta of Sympetrum danae possesses two dorsal diverticula: one in the mesothorax and one in the metathorax. They are very similar in form and position. Each diverticulum has a dorsal valve through which blood is pumped from the wings down into the aorta. The wall of the aortic diverticula consists of two simple cell layers: an outer epidermis-like layer and an inner muscle layer. The nuclei of the muscle cells are situated close to the lumen of the diverticula. The mitochondria are evenly dispersed between the myofibrils and are often paired up on either side of the Z-band. The Z-bands are thick and fragmented. The length of the sarcomeres varies from 3.3 to 6.1 . The A-band length is about 3 . The myofibrils consist of thick (250 Å) and thin (85 Å) filaments. Each thick filament is surrounded by 9–12 thin filaments. The sarcoplasmic reticulum is well developed and separates the myofibrils with one or two layers. The T-tubules are flattened and branch irregularly like a two-dimensional tree between the lamellar myofibrils. Intercalated discs are observed.The peculiarities of the muscle of aortic diverticula in S. danae are discussed in relation to various muscles of other insects and arthropods.     

Sarcomere formation occurs by the assembly of multiple latent protein complexes

The stereotyped striation of myofibrils is a conserved feature of muscle organization that is critical to its function. Although most components that constitute the basic myofibrils are well-characterized biochemically and are conserved across the animal kingdom, the mechanisms leading to the precise assembly of sarcomeres, the basic units of myofibrils, are poorly understood. To gain insights into this process, we investigated the functional relationships of sarcomeric protein complexes. Specifically, we systematically analyzed, using either RNAi in primary muscle cells or available genetic mutations, the organization of myofibrils in Drosophila muscles that lack one or more sarcomeric proteins. Our study reveals that the thin and thick filaments are mutually dependent on each other for striation. Further, the tension sensor complex comprised of zipper/Zasp/α-actinin is involved in stabilizing the sarcomere but not in its initial formation. Finally, integrins appear essential for the interdigitation of thin and thick filaments that occurs prior to striation. Thus, sarcomere formation occurs by the coordinated assembly of multiple latent protein complexes, as opposed to sequential assembly.     

Relation of nebulin and connectin (titin) to dynamics of actin in nascent myofibrils of cultured skeletal muscle cells.

Cultured embryonic chicken skeletal muscle cells microinjected with rhodamine (rh)-labeled actin were stained with antibodies against nebulin and connectin (titin). In premyofibril areas, nebulin was observed as dotted structures, many of which were arranged in a linear fashion. These structures were associated with injected rh-actin. Among these linearly arranged dots of nebulin and rh-actin, numerous small nebulin dots without rh-actin incorporation were scattered. It is probable that the dots of nebulin and/or its associated protein(s) represent a preformed scaffold upon which actin monomers accumulate; exogenously introduced actin associates initially with small nebulin dots, which in turn coalesce to form rh-actin dots and are arranged linearly. In developing myofibrils, two patterns of nebulin distribution were found: "singlets" and "doublets." Recovery of rh-actin's fluorescence after photobleaching was slowest in the nonstriated dotted portions, followed by the striated myofibrillar portions with nebulin singlets and those with doublets, in that order. Thus, the distribution patterns of nebulin seem to be related to the accessibility/exchangeability of actin into nascent myofibrils. It is possible that early nebulin filaments exhibiting singlets are not tightly associated with actin filaments and that this loose association allows myofibrils to exchange nonadult isoforms of actin and other proteins into adult types. Connectin formed a striated pattern before the formation of rh-actin/nebulin striations. It appears that connectin does not have any significant role in the accessibility of actin into nascent myofibrils.     

STRUCTURE OF LIMULUS STRIATED MUSCLE : The Contractile Apparatus at Various Sarcomere Lengths

The musculature of the telson of Limulus polyphemus L. consists of three dorsal muscles: the medial and lateral telson levators and the telson abductor, and one large ventral muscle; the telson depressor, which has three major divisions: the dorsal, medioventral, and lateroventral heads. The telson muscles are composed of one type of striated muscle fiber, which has irregularly shaped myofibrils. The sarcomeres are long, with discrete A and I and discontinuous Z bands. M lines are not present. H zones can be identified easily, only in thick (1.0 µm) longitudinal sections or thin cross sections. In lengthened fibers, the Z bands are irregular and the A bands appear very long due to misalignment of constituent thick filaments. As the sarcomeres shorten, the Z lines straighten somewhat and the thick filaments become more aligned within the A band, leading to apparent decrease in A band length. Further A band shortening, seen at sarcomere lengths below 7.4 µm may be a function of conformational changes of the thick filaments, possibly brought about by alterations in the ordering of their paramyosin cores.     

Extraction of Thick Filaments in Individual Sarcomeres Affects Force Production by Single Myofibrils

It has been accepted that the force produced by a skeletal muscle myofibril depends on its cross-sectional area but not on the number of active sarcomeres because they are arranged in series. However, a previous study performed by our group showed that blocking actomyosin interactions within an activated myofibril and depleting the thick filaments in one sarcomere unexpectedly reduced force production. In this study, we examined in detail how consecutive depletion of thick filaments in individual sarcomeres within a myofibril affects force production. Myofibrils isolated from rabbit psoas were activated and relaxed using a perfusion system. An extra microperfusion needle filled with a high-ionic strength solution was used to erase thick filaments in individual sarcomeres in real time before myofibril activation. The isometric forces were measured upon activation. The force produced by myofibrils with intact sarcomeres was significantly higher than the force produced by myofibrils with one or more sarcomeres lacking thick filaments (p < 0.0001) irrespective of the number of contractions imposed on the myofibrils and their initial sarcomere length. Our results suggest that the myofibril force is affected by intersarcomere dynamics and the number of active sarcomeres in series.     

Immunofluorescence staining of thin-filament sections not participating in actomyosin crossbridges: Studies by use of a monoclonal antibody specific to actin

Summary Monoclonal antibodies (mcab) were produced in vitro by fusing mouse X63-Ag8.653 plasmacytoma cells with spleen cells from a Balb/c mouse immunized with primary cultures of chick skeletal muscle (pmcc). After cloning on agar, stable clones were obtained, the antibodies of which stain specifically the I-band of myofibrils in the immunofluorescence (IF) procedure. For further characterization of these mcab their affinities to muscle proteins were tested by immunoblotting and by enzyme-linked immunosorbent assay (ELISA). Mcab specific for actin were revealed by these criteria. One of the anti-actin antibodies, mcab 647, reveals a variety of IF-staining patterns on myofibrils. On rest-length myofibrils the I-band is labeled only. However, at sarcomere lengths below 2 m, where the thin filaments meet in the middle of the A-band and form a region of double overlap, an additional fluorescent band appears in this position. The fluorescence intensity of this band is increased significantly in shorter sarcomeres. Finally, when the I-band has disappeared at a sarcomere length of 1.5 m, fluorescence is located exclusively in the middle of the A-band. These IF-staining patterns suggest that only those sections of the thin filament are stained that do not participate in actomyosin crossbridges.     

Thin filament length regulation in striated muscle sarcomeres: pointed-end dynamics go beyond a nebulin ruler

The actin (thin) filaments in striated muscle are highly regulated and precisely specified in length to optimally overlap with the myosin (thick) filaments for efficient myofibril contraction. Here, we review and critically discuss recent evidence for how thin filament lengths are controlled in vertebrate skeletal, vertebrate cardiac, and invertebrate (arthropod) sarcomeres. Regulation of actin polymerization dynamics at the slow-growing (pointed) ends by the capping protein tropomodulin provides a unified explanation for how thin filament lengths are physiologically optimized in all three muscle types. Nebulin, a large protein thought to specify thin filament lengths in vertebrate skeletal muscle through a ruler mechanism, may not control pointed-end actin dynamics directly, but instead may stabilize a large core region of the thin filament. We suggest that this stabilizing function for nebulin modifies the lengths primarily specified by pointed-end actin dynamics to generate uniform filament lengths in vertebrate skeletal muscle. We suggest that nebulette, a small homolog of nebulin, may stabilize a correspondingly shorter core region and allow individual thin filament lengths to vary according to working sarcomere lengths in vertebrate cardiac muscle. We present a unified model for thin filament length regulation where these two mechanisms cooperate to tailor thin filament lengths for specific contractile environments in diverse muscles.     

Extensibility of the myofilaments in vertebrate skeletal muscle as revealed by stretching rigor muscle fibers

The extensibility of the myofilaments in vertebrate skeletal muscle was studied by stretching glycerinated rabbit psoas muscle fibers in rigor state and examining the resulting extension of sarcomere structures under an electron microscope. Although stretches applied to rigor fibers produced a successive yielding of the weakest sarcomeres, the length of the remaining intact sarcomeres in many myofibrils was fairly uniform, being definitely longer than the sarcomeres in the control, nonstretched part of rigor fibers. The stretch-induced increase in sarcomere length was found to be taken up by the extension of the H zone and the I band, whereas the amount of overlap between the thick and thin filaments did not change appreciably with stretches of 10-20%. The thick filament extension in the H zone was localized in the bare regions, whereas the thin filament extension in the I band appeared to take place uniformly along the filament length. No marked increase in the Z-line width was observed even with stretches of 20-30%. These results clearly demonstrate the extensibility of the thick and thin filaments. The possible contribution of the myofilament compliance to the series elastic component (SEC) in vertebrate skeletal muscle fibers is discussed on the basis of the electron microscopic data and the force-extension curve of the SEC in rigor fibers.     

Fine structure of muscles in the tickHyalomma (Hyalomma) dromedarii (Ixodoidea: Ixodidae)

Skeletal and visceral muscles are distinguished in the unfed nymphHyalomma (Hyalomma) dromedarii according to position, structure and function. The skeletal muscles include the capitulum, dorsoventral and leg oblique muscles. Their muscle fibres have the striated pattern of successive sarcomeres whose thick myosin filaments are surrounded by orbitals of up to 12 thin actin filaments. The cell membrane invaginates into tubular system (T) extending deeply into the sarcoplasm and closely associated to cisternae of sarcoplasmic reticulum (SR). The T and SR forming two-membered dyads are considered to be the main route of calcium ions whose movements are synchronized with the motor impulse to control contraction and relaxation in most muscles. Two types of skeletal muscle fibres are recognized, and are suggested as representing different physiological phases.In the visceral-muscle fibres investing tick internal organs, the actin and myosin filaments are slightly interrupted, and the T and SR are well demonstrated. Both skeletal and visceral muscles are invaginated by tracheoles and innervated by nerve-axons containing synaptic vesicles.     

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.