Replacement of troponin components in myofibrils.

The Ca(2+)-sensitive ATPase activity of rabbit skeletal myofibrils was desensitized by treatment with excess troponin T and was found to be activated irrespective of the Ca2+ concentrations. A SDS-gel electrophoretic study showed that both troponin C and troponin I were removed from the myofibrils on treatment with troponin T. The Ca(2+)- and Sr(2+)- sensitivities of the ATPase of troponin T-treated myofibrils reconstituted with troponin C. I were the same as in the intact myofibrils. The Ca(2+)-activated ATPase of rabbit skeletal myofibrils was also desensitized on treatment with chicken breast troponin T or its 26K fragment. The SDS-gel electrophoretic study revealed that troponin T, in addition to troponin C and troponin I, was also removed from the myofibrils and, instead, chicken breast troponin T or its 26K fragment was incorporated into the myofibrils. The Ca(2+)- sensitivity of myofibrils treated with chicken breast troponin T or its 26K fragment was then regained on reconstitution with troponin C.I. These findings indicate that the change in composition of myofibrils on treatment with troponin T or its 26K fragment is due to the selective replacement of the troponin C.I.T complex in the myofibrils as a whole with troponin T or its 26K fragment.     

Effect of substitution of troponin C in cardiac myofibrils with skeletal troponin C or calmodulin on the Ca2+- and Sr2+-sensitive ATPase activity

Troponin C was removed almost completely from the porcine cardiac myofibrils by the same extraction procedure using CDTA as that previously reported for the rabbit skeletal myofibrils (Morimoto, S. & Ohtsuki, I. (1987) J. Biochem. 101, 291-301), and the effects of substitution of troponin C in cardiac myofibrils with rabbit skeletal troponin C or bovine brain calmodulin were examined. While the ATPase activity of intact cardiac myofibrils or cardiac troponin C-reconstituted cardiac myofibrils was activated at only a little higher concentration of Sr2+ than Ca2+, the skeletal troponin C-substituted cardiac myofibrils, as well as intact rabbit skeletal myofibrils, required more than 10 times higher concentration of Sr2+ than Ca2+ for activation of the myofibrillar ATPase activity. However, the concentrations of Ca2+ and Sr2+ required for the activation of the ATPase activity of the skeletal troponin C-substituted cardiac myofibrils were both about 5 times higher than those of intact skeletal myofibrils. The skeletal troponin C-substituted cardiac myofibrils, as well as intact skeletal myofibrils, also showed higher cooperativity in the Ca2+-activation of the ATPase activity than intact or cardiac troponin C-reconstituted cardiac myofibrils. The ATPase activity of calmodulin-substituted cardiac myofibrils was activated at a several times lower concentration of Ca2+ or Sr2+ than that of calmodulin-substituted skeletal myofibrils, while the ratios of the concentration of Sr2+ to Ca2+ required for activation were almost the same in both cases.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Elevated levels of a calcium-activated muscle protease in rapidly atrophying muscles from vitamin E-deficient rabbits.

A Ca2+-activated proteolytic enzyme that partially degrades myofibrils was isolated from hind limb muscles of normal rabbits and rabbits undergoing rapid muscle atrophy as a result of vitamin E deficiency. Extractable Ca2+-activated protease activity was 3.6 times higher in muscle tissue from vitamin E-deficient rabbits than from muscle tissue of control rabbits. Ultrastructural studies of muscle from vitamin E-deficient rabbits showed that the Z disk was the first myofibrillar structure to show degradative changes in atrophying muscle. Myofibrils prepared from muscles from vitamin E-deficient rabbits showed partial or complete loss of Z-disk density. Sodium dodecyl sulfate polyacrylamide gel electrophoresis showed that the amount of troponin-T (37 000 daltons) and alpha-actinin (96 000 daltons) was reduced in myofibrils from atrophying muscle as compared to myofibrils prepared from control muscle. In vitro treatment of purified myofibrils with purified Ca2+-activated proteolytic enzyme produced alterations in myofibrillar ultrastructure that were identical to the initial alterations occurring in myofibrils from atrophying muscle (i.e. weakening and subsequent removal of Z disks). Additonally the electrophoretic banding pattern of Ca2+-activated proteolytic enzyme-treated myofibrils is very similar to that of myofibrils prepared from muscles atrophying as a result of nutritional vitamin E deficiency. The possible role of Ca2+-activated proteolytic enzyme in disassembly and degradation of the myofibril is discussed.     

Irradiations of rabbit myofibrils with an ultraviolet microbeam. II. Phalloidin protects actin in solution but not in myofibrils from depolymerization by ultraviolet light

We tested whether phalloidin protects actin in myofibrils from depolymerization by ultraviolet light (UV). I bands in glycerinated rabbit psoas myofibrils were irradiated with a UV microbeam in the presence and absence of phalloidin. We used the retention of contractility of the irradiated I band as the assay for protection of actin by phalloidin, since previous experiments indicated that UV blocks contraction of an irradiated I band by depolymerizing the thin filaments. The I bands of myofibrils incubated in phalloidin were as sensitive to UV as control I bands, indicating that phalloidin did not protect the thin filaments. However, phalloidin did protect F-actin in solution from depolymerization by UV. This apparent contradiction between F-actin in myofibrils and F-actin in solution was resolved by observing unirradiated myofibrils that were stained with rhodamine-phalloidin. It was found that phalloidin does not bind uniformly to the thin filaments, though as the fluorescence image is observed over time the staining pattern changes until it does appear to bind uniformly. We conclude that phalloidin does not protect F-actin in myofibrils from depolymerization by UV because it does not bind uniformly to the filaments.     

Ca2+- and Sr2+-sensitivity of the ATPase activity of rabbit skeletal myofibrils: effect of the complete substitution of troponin C with cardiac troponin C, calmodulin, and parvalbumins

The Ca2+-sensitive ATPase activity of rabbit skeletal myofibrils disappeared completely after treatment with a solution containing CDTA, a strong divalent cation chelator, at a low ionic strength. A gel electrophoretic study revealed that all troponin C and about half of myosin light chain 2 were removed from the myofibrils by the CDTA treatment. The CDTA-treated myofibrils, when reconstituted with skeletal troponin C, showed almost exactly the same Ca2+- or Sr2+-sensitive ATPase activity as that of intact myofibrils. The CDTA-treated myofibrils reconstituted with porcine cardiac troponin C showed the same Ca2+- or Sr2+-sensitivity of the ATPase as that of porcine cardiac myofibrils; Sr2+-sensitivity relative to Ca2+-sensitivity was about ten times higher than, and the maximal slope of the activation curve was about half that of skeletal myofibrils. These findings indicate that these characteristic features of divalent cation regulation in the contraction of skeletal and cardiac muscles are determined solely by the species of troponin C. Bovine brain calmodulin hardly activated the ATPase activity of the CDTA-treated myofibrils even in the presence of Ca2+. Excess calmodulin, however, was found to give Ca2+- or Sr2+-sensitivity to the ATPase activity of the CDTA-treated myofibrils. Frog skeletal parvalbumins 1 and 2, even in excess, did not affect the ATPase activity of the CDTA-treated myofibrils.     

Induction of contractile activity by rigor complexes in myofibrils

The relation between ATPase rate and substrate concentration was investigated for myofibrils with varying amounts of added HMM. There was a biphasic, 3 to 5-fold increase in ATPase in the absence of Ca⁺⁺. In the absence of added HMM, the peak activity occurred at ≤ 0.1 mM MgATP. With increasing concentrations of HMM, the position and magnitude of the ATPase peak shifted to larger substrate concentrations and higher rates. The cofactor activity of regulated actin in myofibrils is activated to a similar degree by Ca⁺⁺ as by HMM (rigor links). SDS gel electrophoretic patterns of myofibrils mixed with HMM indicated the soluble HMM binds to myofibrils at 0.1 mM MgATP and is dissociated at higher MgATP concentrations. Thus, in well-regulated myofibrils in the absence of Ca⁺⁺ actin cofactor activity can be activated by rigor complexes.     

Post-translational incorporation of actin into myofibrils in vitro: evidence for isoform specificity.

The incorporation of actin into myofibrils has been examined in a cell-free system [Bouché et al.: Journal of Cell Biology 107:587-596, 1988; Goldfine et al.: Cellular and Molecular Biology of Muscle Development, 1989]. Actin was translated in a reticulocyte lysate in the presence of 35S-methionine (35S-actin) or purified from muscle and labeled with fluorescein-5-isothiocyanate (FITC-actin). Myofibrils were incubated with either 35S-actin or FITC-actin and then analyzed by gel electrophoresis or fluorescence microscopy. When myofibrils were incubated with FITC-actin monomer in the reticulocyte lysate buffer, strong fluorescent labeling was observed in Z-band regions and less so in I-bands. No fluorescence was detected in non-overlap regions of A-bands. Confocal microscopic analysis of these myofibrils indicated that FITC-actin was distributed evenly across the diameter of the myofibrils. These observations suggest that actin incorporation in the reticulocyte lysate buffer occurred at sites in the sarcomere which contain actin. In contrast, FITC-actin showed a variety of non-physiological incorporation patterns when incubated with myofibrils in the presence of an isotonic buffer (I-buffer). However, when ATP was added to I-buffer, FITC-actin showed a pattern of incorporation into myofibrils similar to that seen in the reticulocyte lysate buffer. Immunoblots indicated that actin of native size was released from myofibrils during incubation in the reticulocyte lysate buffer. No actin release was detected when the myofibrils were incubated in I-buffer lacking ATP. We used this system to compare the incorporation of actin isoforms into myofibrils. Both alpha- and beta-actins exhibited incorporation into the myofibrils but there was a three-fold greater incorporation of the alpha isoform. We propose that the differential affinities of actin isoforms for myofibrils and other cytoskeletal structures could provide a mechanism for actin isoform targeting within the cytoplasm.     

FITC-labeled I-protein specifically binds to A-bands and/or Z-lines of glycerinated myofibrils of chicken breast muscle

Ion-exchange column-purified I-protein was labeled by fluorescein isothiocyanate (FITC) at an equimolar ratio. When FITC-labeled I-protein was reacted with glycerinated myofibrils of chicken breast muscle in a phosphate-buffered saline, fluorescence was observed at the A-band and/or the Z-line of the sarcomere. However, FITC-labeled I-protein did not stain freshly prepared myofibrils. When FITC-I-protein was reacted with a nitrocellulose paper sheet on which muscle proteins were blotted after SDS-polyacrylamide gel electrophoresis, some peptide bands, including connectin and nebulin, were fluorescent. These facts can explain why anti-I-protein antibodies stain the A-I junctional region of fresh myofibrils and A-bands and/or Z-lines of glycerinated myofibrils. It is very likely that I-protein is transferred from the A-I junctions of myofibrils and translocates to A-bands and Z-lines, where some components that can bind to I-protein are localized, as myofibrils are degraded during the glycerination.     

Biochemical and ultrastructural aspects of Mr 165,000 M-protein in cross-striated chicken muscle

To better understand the relationship between the Mr 165,000 M-line protein (M-protein) and H-zone structure in skeletal and in cardiac muscle, as well as the possible interaction of M-protein with another skeletal muscle M-line component, the homodimeric creatine kinase isoenzyme composed of two M subunits (MM-CK), we performed biochemical, immunological, and ultrastructural studies on myofibrils extracted by different procedures. In contrast to MM-CK, M-protein could not be completely removed from myofibrils by low ionic strength extraction. Fab-fragments of antibodies against M-protein could not release M- protein quantitatively from either breast or heart myofibrils but remained bound to the myofibrillar structure, whereas monovalent antibodies against MM-CK cause the specific release of MM-CK and the concomitant disappearance of the M-line from chicken skeletal muscle myofibrils. When MM-CK was removed from skeletal myofibrils by low ionic strength extraction or, more specifically, by incubation with anti-MM-CK Fab, M-protein was still not released quantitatively upon treatment with anti-M-protein Fab as judged from immunofluorescence data. In the ultrastructural investigation of low ionic strength extracted muscle fibers, M protein could be localized in two stripes on both sides of the former M-line, suggesting a reduced attachment to the residual H-zone structure, whereas the specific removal of MM-CK resulted in the same dense staining pattern for M-protein within the M- line as observed in untreated fibers. However, the binding of M-protein to the residual M-line structure seemed to be reduced, as a considerable amount of this protein could be identified in the supernate of sequentially incubated myofibrils. The results indicate a strong binding of M-protein within the H-zone structure of skeletal as well as heart myofibrils.     

Role of stress fiber-like structures in assembling nascent myofibrils in myosheets recovering from exposure to ethyl methanesulfonate

When day 1 cultures of chick myogenic cells were exposed to the mutagenic alkylating agent ethyl methanesulfonate (EMS) for 3 d, 80% of the replicating cells were killed, but postmitotic myoblasts survived. The myoblasts fused to form unusual multinucleated "myosheets": extraordinarily wide, flattened structures that were devoid of myofibrils but displayed extensive, submembranous stress fiber-like structures (SFLS). Immunoblots of the myosheets indicated that the carcinogen blocked the synthesis and accumulation of the myofibrillar myosin isoforms but not that of the cytoplasmic myosin isoform. When removed from EMS, widely spaced nascent myofibrils gradually emerged in the myosheets after 3 d. Striking co-localization of fluorescent reagents that stained SFLS and those that specifically stained myofibrils was observed for the next 2 d. By both immunofluorescence and electron microscopy, individual nascent myofibrils appeared to be part of, or juxtaposed to, preexisting individual SFLS. By day 6, all SFLS had disappeared, and the definitive myofibrils were displaced from their submembranous site into the interior of the myosheet. Immunoblots from recovering myosheets demonstrated a temporal correlation between the appearance of the myofibrillar myosin isoforms and the assembly of thick filaments. The assembly of definitive myofibrils did not appear to involve desmin intermediate filaments, but a striking aggregation of sarcoplasmic reticulum elements was seen at the level of each I-Z-band. Our findings suggest that SFLS in the EMS myosheets function as early, transitory assembly sites for nascent myofibrils.     

Elevated levels of a calcium-activated muscle protease in rapidly atrophying muscles from vitamin E-deficient rabbits

A Ca²⁺-activated proteolytic enzyme ¹ that partially degrades myofibrials was isolated from hind limb muscles of normal rabbits and rabbits undergoing rapid muscle atrophy as a result of vitamin E deficiency. Extractable Ca²⁺-activated protease activity was 3.6 times higher in muscle tissue from vitamin E-deficient rabbits than from muscle tissue of control rabbits. Ultrastructural studies of muscle from vitamin E-deficient rabbits showed that the Z disk was the first myofibrillar structure to show degradative changes in atrophying muscle. Myofibris prepared from muscles vitamin E-deficient rabbits showed partial or complete loss of Z-disk density. Sodium dodecyl sulfate polyacrylamide gel electrophoresis showed that the amount of troponin-T (37 000 daltons) and α-actinin (96 000 daltons) was reduced in myofibrils from atrophying muscle as compared to myofibrils prepared from control muscle. In vitro treatment of purified myofibrils with purified Ca²⁺-activated proteolytic enzyme produced alterations in myofibrillar ultrastructure that were identical to the initial alterations occuring in myofibrils from atrophying muscle (i.e. weakening and subsequent removal of Z disks). Additionally the electrophoretic banding pattern of Ca²⁺-activated proteolytic enzyme-treated myofibrils is very similar to that of myofibrils prepared from muscles atrophying as a result of nutritional vitamin E deficiency. The possible role of Ca²⁺-activated proteolytic enzyme in disassembly and degradation of the myofibril is discussed.     

Phosphorylated cardiac myofibrils and their effect on ATPase activity.

Control guinea pig cardiac myofibrils were isolated in the presence of Triton X-100. Experimental myofibrils, prepared in the presence of Triton X-100, NaF, cyclic AMP and ATP, possessed a reduced myofibrillar ATPase activity. When myofibrils isolated under control conditions were incubated for two hours at 25°C with NaF, ATP and cyclic AMP, the ATPase activity was also decreased; however, the ATPase activity was not reduced as much as that of myofibrils isolated under experimental conditions. Incubation of myofibrils with $\text{E. coli}$ aklaline phosphatase and guinea pig heart phosphoprotein phosphatase resulted in an increase in ATPase activity and a decrease in phosphoprotein phosphate. Thus there appeared to be an inverse relationship between myofibrillar ATPase activity and phosphoprotein phosphate content. The results indicated that a protein kinase is associated with the Triton X-100 purified myofibrils and supports the notion that intact myofibrils can exist in at least two catalytic forms.     

Spontaneous oscillation of tension and sarcomere length in skeletal myofibrils. Microscopic measurement and analysis.

We have devised a simple method for measuring tension development of single myofibrils by micromanipulation with a pair of glass micro-needles. The tension was estimated from the deflection of a flexible needle under an inverted phase-contrast microscope equipped with an image processor, so that the tension development is always accompanied by the shortening of the myofibril (auxotonic condition) in the present setup. The advantage of this method is that the measurement of tension (1/30 s for time resolution and about 0.05 micrograms for accuracy of tension measurement; 0.05 microns as a spatial resolution for displacement of the micro-needle) and the observation of sarcomere structure are possible at the same time, and the technique to hold myofibrils, even single myofibrils, is very simple. This method has been applied to study the tension development of glycerinated skeletal myofibrils under the condition where spontaneous oscillation of sarcomeres is induced, i.e., the coexistence of MgATP, MgADP and inorganic phosphate without free Ca2+. Under this condition, we found that the tension of myofibrils spontaneously oscillates accompanied by the oscillation of sarcomere length with a main period of a few seconds; the period was lengthened and shortened with stretch and release of myofibrils. A possible mechanism of the oscillation is discussed.     

Ca2+ binding to skeletal muscle troponin C in skeletal and cardiac myofibrils

Ca2+ binding to skeletal muscle troponin C in skeletal or cardiac myofibrils was measured by the centrifugation method using 45Ca. The specific Ca2+ binding to troponin C was obtained by subtracting the amount of Ca2+ bound to the CDTA-treated myofibrils (troponin C-depleted myofibrils) from that to the myofibrils reconstituted with troponin C. Results of Ca2+ binding measurement at various Ca2+ concentrations showed that skeletal troponin C had two classes of binding sites with different affinity for Ca2+. The Ca2+ binding of low-affinity sites in cardiac myofibrils was about eight times lower than that in skeletal myofibrils, while the high-affinity sites of troponin C in skeletal or cardiac myofibrils showed almost the same affinity for Ca2+. The Ca2+ sensitivity of the ATPase activity of skeletal troponin C-reconstituted cardiac myofibrils was also about eight times lower than that of skeletal myofibrils reconstituted with troponin C. These findings indicated that the difference in the sensitivity to Ca2+ of the ATPase activity between skeletal and cardiac CDTA-treated myofibrils reconstituted with skeletal troponin C was mostly due to the change in the affinity for Ca2+ of the low-affinity sites on the troponin C molecule.     

Differential distribution of myosin isoforms among the myofibrils of individual developing muscle fibers

Myosin was localized in situ in the posthatch chicken pectoralis using isoform-specific mAbs. The distribution among myofibrils was demonstrated by immunofluorescence and by immunogold EM. Fluorescein- or rhodamine-labeled antibody (12C5) specific for the head region (S1) of myosin was used as a marker to identify "embryonic" myosin. In longitudinal semithin frozen sections, a minority population of myofibrils stained intensely with 12C5. All other myofibrils in the same cell stained only weakly. Similarly, in Lowicryl-embedded ultrathin sections prepared for EM, a minority population reacted preferentially with gold-labeled 12C5. An antibody (5B4) specific for the rod portion of "neonatal" myosin reacted strongly with nearly all myofibrils, and this was evident by light and electron microscopy. A few of the fibrils that reacted strongly with 12C5 reacted weakly with 5B4. These observations demonstrate that an epitope reacting with 12C5 is more abundant in some myofibrils than in others within the same cell. Three categories of myofibrils can be identified by their relative proportions of embryonic and neonatal forms of myosin: in nearly all fibrils, a neonatal isoform predominates; in a minority population, embryonic and neonatal isoforms are both abundant; and in a few fibrils, an embryonic isoform predominates. It is concluded that there are distinct populations of myofibrils in which specific isoforms are segregated within an individual cell.     

Studies on the role of microtubules in myofibrillogenesis

Co-localization of microtubule (MT) and muscle myosin (MHC) myofibril immunofluoresoonoe in developing myotubes of chicken skeletal muscle cultures was observed by using double staining of tubulin and MHC indirect immunofluorescence.120-tetradecanoyl-phorbol-12-acetate (TPA) selectively and reversibly blocks myofibrillogenesis and alters the morphology of myotubes in to myosacs where MTs are present in radiating pattern.When the arrested myogenic cells recover and start myofibrillogenesis after released from TPA,prior to the emergence of myofibrils,the pre-ecisting MTs become bipolarly aligned coincidently with the tubular restoration of cell shape.Single nascent myofibrils overlapping with MTs extend into the base of growth tips where MTs go farther to the end of the tips.That MT might act as scaffold in guiding the bipolar elongation of the growing myofibrils was suggested.Taxol and colcemid disturbed MT polymerization and disposition,and interfered with the normal spatial assembly of myofibrils in developing myotubes.     

The relationship between stress fiber-like structures and nascent myofibrils in cultured cardiac myocytes

The topographical relationship between stress fiber-like structures (SFLS) and nascent myofibrils was examined in cultured chick cardiac myocytes by immunofluorescence microscopy. Antibodies against muscle-specific light meromyosin (anti-LMM) and desmin were used to distinguish cardiac myocytes from fibroblastic cells. By various combinations of staining with rhodamine-labeled phalloidin, anti-LMM, and antibodies against chick brain myosin and smooth muscle alpha-actinin, we observed the following relationships between transitory SFLS and nascent and mature myofibrils: (a) more SFLS were present in immature than mature myocytes; (b) in immature myocytes a single fluorescent fiber would stain as a SFLS distally and as a striated myofibril proximally, towards the center of the cell; (c) in regions of a myocyte not yet penetrated by the elongating myofibrils, SFLS were abundant; and (d) in regions of a myocyte with numerous mature myofibrils, SFLS had totally disappeared. Spontaneously contracting striated myofibrils with definitive Z-band regions were present long before anti-desmin localized in the I-Z-band region and long before morphologically recognizable structures periodically link Z-bands to the sarcolemma. These results suggest a transient one-on-one relationship between individual SFLS and newly emerging individual nascent myofibrils. Based on these and other relevant data, a complex, multistage molecular model is presented for myofibrillar assembly and maturation. Lastly, it is of considerable theoretical interest to note that mature cardiac myocytes, like mature skeletal myotubes, lack readily detectable stress fibers.     

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.     

Ectopic expression and dynamics of TPM1alpha and TPM1kappa in myofibrils of avian myotubes

From the four known vertebrate tropomyosin genes (designated TPM1, TPM2, TPM3, and TPM4) over 20 isoforms can be generated. The predominant TPM1 isoform, TPM1alpha, is specifically expressed in both skeletal and cardiac muscles. A newly discovered alternatively spliced isoform, TPM1kappa, containing exon 2a instead of exon 2b contained in TPM1alpha, was found to be cardiac specific and developmentally regulated. In this work, we transfected quail skeletal muscle cells with green fluorescent proteins (GFP) coupled to chicken TPM1alpha and chicken TPM1kappa and compared their localizations in premyofibrils and mature myofibrils. We used the technique of fluorescence recovery after photobleaching (FRAP) to compare the dynamics of TPM1alpha and TPM1kappa in myotubes. TPM1alpha and TPM1kappa incorporated into premyofibrils, nascent myofibrils, and mature myofibrils of quail myotubes in identical patterns. The two tropomyosin isoforms have a higher exchange rate in premyofibrils than in mature myofibrils. F-actin and muscle tropomyosin are present in the same fibers at all three stages of myofibrillogenesis (premyofibrils, nascent myofibrils, mature myofibrils). In contrast, the tropomyosin-binding molecule nebulin is not present in the initial premyofibrils. Nebulin is gradually added during myofibrillogenesis, becoming fully localized in striated patterns by the mature myofibril stage. A model of thin filament formation is proposed to explain the increased stability of tropomyosin in mature myofibrils. These experiments are supportive of a maturing thin filament and stepwise model of myofibrillogenesis (premyofibrils to nascent myofibrils to mature myofibrils), and are inconsistent with models that postulate the immediate appearance of fully formed thin filaments or myofibrils.