Tropomodulin assembles early in myofibrillogenesis in chick skeletal muscle: evidence that thin filaments rearrange to form striated myofibrils.
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Actin filament lengths in muscle and nonmuscle cells are believed to depend on the regulated activity of capping proteins at both the fast growing (barbed) and slow growing (pointed) filament ends. In striated muscle, the pointed end capping protein, tropomodulin, has been shown to maintain the lengths of thin filaments in mature myofibrils. To determine whether tropomodulin might also be involved in thin filament assembly, we investigated the assembly of tropomodulin into myofibrils during differentiation of primary cultures of chick skeletal muscle cells. Our results show that tropomodulin is expressed early in differentiation and is associated with the earliest premyofibrils which contain overlapping and misaligned actin filaments. In addition, tropomodulin can be found in actin filament bundles at the distal tips of growing myotubes, where sarcomeric alpha-actinin is not always detected, suggesting that tropomodulin caps actin filament pointed ends even before the filaments are cross-linked into Z bodies by alpha-actinin. Tropomodulin staining exhibits an irregular punctate pattern along the length of premyofibrils that demonstrate a smooth phalloidin staining pattern for F-actin. Strikingly, the tropomodulin dots often appear to be located between the closely spaced, dot-like Z bodies that are stained for (&agr;)-actinin. Thus, in the earliest premyofibrils, the pointed ends of the thin filaments are clustered and partially aligned with respect to the Z bodies (the location of the barbed filament ends). At later stages of differentiation, the tropomodulin dots become aligned into regular periodic striations concurrently with the appearance of striated phalloidin staining for F-actin and alignment of Z bodies into Z lines. Tropomodulin, together with the barbed end capping protein, CapZ, may function from the earliest stages of myofibrillogenesis to restrict the lengths of newly assembled thin filaments by capping their ends; thus, transitions from nonstriated to striated myofibrils in skeletal muscle are likely due principally to filament rearrangements rather than to filament polymerization or depolymerization. Rearrangements of actin filaments capped at their pointed and barbed ends may be a general mechanism by which cells restructure their actin cytoskeletal networks during cell growth and differentiation. (+info)
Tropomodulin isolated from rabbit skeletal muscle inhibits filament formation of actin in the presence of tropomyosin and troponin.
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Tropomodulin is a tropomyosin-binding protein, originally isolated from human erythrocytes. Tropomodulin is currently regarded as the sole actin pointed-end capping protein [Weber, A., Pennise, C.R., Babcock, G.G. & Fowler, V.M. (1994) J. Cell Biol. 127, 1627-1635]. This work first describes a procedure for the purification of tropomodulin from rabbit skeletal muscle. Tropomodulin almost completely inhibited filament formation of actin in the presence of tropomyosin and troponin. For the maximal inhibition of actin polymerization, approximately 0.10, 0.12 and 0.003 mol of tropomyosin, troponin and tropomodulin per mol of actin were required, respectively. Fluorescence-intensity measurements, electron-microscopy and sedimentation experiments revealed that only very short fragments and amorphous aggregates, but not filaments, were formed when actin was copolymerized with tropomyosin, troponin and tropomodulin by the addition of 50 mM KCl at pH 8.0. The effects of tropomyosin, troponin and tropomodulin were more remarkable on Ca-actin than on Mg-actin. It appears that tropomodulin caps both the pointed and barbed ends of tropomyosin- and troponin-bound actin filaments. (+info)
Identification of a novel tropomodulin isoform, skeletal tropomodulin, that caps actin filament pointed ends in fast skeletal muscle.
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Tropomodulin (E-Tmod) is an actin filament pointed end capping protein that maintains the length of the sarcomeric actin filaments in striated muscle. Here, we describe the identification and characterization of a novel tropomodulin isoform, skeletal tropomodulin (Sk-Tmod) from chickens. Sk-Tmod is 62% identical in amino acid sequence to the previously described chicken E-Tmod and is the product of a different gene. Sk-Tmod isoform sequences are highly conserved across vertebrates and constitute an independent group in the tropomodulin family. In vitro, chicken Sk-Tmod caps actin and tropomyosin-actin filament pointed ends to the same extent as does chicken E-Tmod. However, E- and Sk-Tmods differ in their tissue distribution; Sk-Tmod predominates in fast skeletal muscle fibers, lens, and erythrocytes, while E-Tmod is found in heart and slow skeletal muscle fibers. Additionally, their expression is developmentally regulated during chicken breast muscle differentiation with Sk-Tmod replacing E-Tmod after hatching. Finally, in skeletal muscle fibers that coexpress both Sk- and E-Tmod, they are recruited to different actin filament-containing cytoskeletal structures within the cell: myofibrils and costameres, respectively. All together, these observations support the hypothesis that vertebrates have acquired different tropomodulin isoforms that play distinct roles in vivo. (+info)
Tropomodulin increases the critical concentration of barbed end-capped actin filaments by converting ADP.P(i)-actin to ADP-actin at all pointed filament ends.
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The pointed end capping protein, tropomodulin, increases the critical concentration of barbed end capped actin, i.e. it lowers the apparent affinity of pointed ends for actin monomers. We show here that this is due to the conversion of pointed end ADP. P(i)-actin (low critical concentration) to ADP-actin (high critical concentration) when 70-98% of the ends are capped by tropomodulin. We propose that this is due to the low affinity of tropomodulin for pointed ends (K(d) approximately 0.3 microM), which allows tropomodulin to rapidly exchange binding sites and transiently block access of actin monomers to all pointed ends. This leaves time for ATP hydrolysis and phosphate release to go to completion between successive monomer additions to the pointed end. When the affinity of tropomodulin for pointed ends was increased about 1000-fold by the presence of tropomyosin (K(d) < 0.05 nM), capping of 95% of the ends by tropomodulin did not alter the critical concentration. However, the critical concentration did increase when the tropomodulin concentration was raised to the high values effective in the absence of tropomyosin. This may reflect transient tropomodulin binding to tropomyosin-free actin molecules at the pointed ends of the tropomyosin-actin filaments without a high affinity tropomodulin cap, i.e. the ends that determine the value of the actin critical concentration. (+info)
Pathogenesis of dilated cardiomyopathy: molecular, structural, and population analyses in tropomodulin-overexpressing transgenic mice.
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Dilated cardiomyopathy is characterized by decreased contractile function and loss of myofibril organization. Previously unexplored structural and molecular events that precede and initiate dilation can now be studied in tropomodulin-overexpressing transgenic (TOT) mice exhibiting progressive dilated cardiomyopathy. Onset of dilation did not correspond to a change in transgene expression levels, which were more than threefold above normal at birth and remained elevated throughout postnatal life. Similarly, mitogen-activated protein kinase activation (p38, ERK1/ERK2, JNK1/JNK2) was not associated with dilation. In contrast, calcineurin was activated before dilation, presumably due to doubling of intracellular diastolic calcium levels in TOT cardiomyocytes. Amplitude of systolic calcium transients was greatly increased as well, demonstrating the novel and unique calcium handling profile of TOT cardiomyocytes. Loss of myofibril organization was not apparent by confocal microscopy until over 1 week after birth, although neonatal sarcomeric abnormalities were revealed by ultrastructural analysis. Rapid postnatal increases in heart:body weight ratio at 1.5 weeks were followed by two waves of mortality between 2 and 3 weeks after birth coincident with maturational stress. Ultimately, TOT pathogenesis is a compensatory response to altered sarcomeric structure driven by calcineurin activation within days after birth, making TOTs an excellent paradigm for studying the role of calcium overload in dilated cardiomyopathy. (+info)
Tropomodulin and tropomyosin mediate lens cell actin cytoskeleton reorganization in vitro.
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PURPOSE: To determine the role of the actin cytoskeleton regulatory proteins tropomyosin and tropomodulin (Tmod) in the reorganization of the actin cytoskeleton during lens epithelial cell differentiation. METHODS: Primary cultures of chick lens epithelial cells were allowed to differentiate in vitro to form lentoid bodies. Localization of F-actin, Tmod, and tropomyosin were determined by immunofluorescent staining followed by confocal microscopy. Tropomyosin and Tmod isoform expression was determined by immunoprecipitation and western blot analysis. RESULTS: In undifferentiated epithelial cells F-actin was organized in polygonal arrays of stress fibers and was also associated with the adherens belt. In contrast, F-actin in differentiated cells was predominantly associated with membranes in a reticular or fibrillar pattern and was organized in curvilinear fibrils in the cytoplasm. Tmod was not detected in the undifferentiated epithelial cells but was expressed upon cell differentiation and assembled into F-actin and non-F-actin structures. Tmod isoforms expressed in the lens cell cultures were identical with those expressed in the embryonic chick lens fiber cells. Tropomyosin was associated with the polygonal arrays of stress fibers in the undifferentiated epithelial cells and was recruited to cortical F-actin at the cell periphery during differentiation. This occurred coincident with a shift in tropomyosin isoform expression. CONCLUSIONS: Expression and sequential assembly of low-molecular-weight tropomyosin and Tmod into the cortical actin cytoskeleton of differentiated lens cells may help to reorganize the actin cytoskeleton during morphogenetic differentiation. Moreover, lens epithelial cell differentiation may include the generation of novel Tmod-containing, non-F-actin cytoskeletal structures. (+info)
Tropomyosin isoform 5b is expressed in human erythrocytes: implications of tropomodulin-TM5 or tropomodulin-TM5b complexes in the protofilament and hexagonal organization of membrane skeletons.
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The human erythrocyte membrane skeleton consists of hexagonal lattices with junctional complexes containing F-actin protofilaments of approximately 33-37 nm in length. We hypothesize that complexes formed by tropomodulin, a globular capping protein at the pointed end of actin filaments, and tropomyosin (TM), a rod-like molecule of approximately 33-35 nm, may contribute to the formation of protofilaments. We have previously cloned the human tropomodulin complementary DNA and identified human TM isoform 5 (hTM5), a product of the gamma-TM gene, as one of the major TM isoforms in erythrocytes. We now identify TM5b, a product of the alpha-TM gene, to be the second major TM isoform. TM5a, the alternatively spliced isoform of the alpha-TM gene, which differs by 1 exon and has a weaker actin-binding affinity, however, is not present. TM4, encoded by the delta-TM gene, is not present either. In sodium dodecyl sulfate-polyacrylamide gel electrophoresis, hTM5 comigrated with the slower TM major species in erythrocyte membranes, and hTM5b comigrated with the faster TM major species. TM5b, like TM5, binds strongly to tropomodulin, more so than other TM isoforms. The 2 major TM isoforms, therefore, share several common features: They have 248 residues, are approximately 33-35 nm long, and have high affinities toward F-actin and tropomodulin. These common features may be the key to the mechanism by which protofilaments are formed. Tropomodulin-TM5 or tropomodulin-TM5b complexes may stabilize F-actin in segments of approximately 33-37 nm during erythroid terminal differentiation and may, therefore, function as a molecular ruler. TM5 and TM5b further define the hexagonal geometry of the skeletal network and allow actin-regulatory functions of TMs to be modulated by tropomodulin. (Blood. 2000;95:1473-1480) (+info)
Hypertrophic defect unmasked by calcineurin expression in asymptomatic tropomodulin overexpressing transgenic mice.
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OBJECTIVE: Dilation and hypertrophy often occur concurrently in cardiomyopathy, yet the interaction between these two functionally distinct conditions remains unknown. METHODS: Combinatorial effects of hypertrophy and dilation were investigated by cross-breeding of two cardiomyopathic transgenic mouse lines which develop either hypertrophy (calcineurin-mediated) or dilation (tropomodulin-mediated). RESULTS: Altering the intensity of signals driving hypertrophy and dilation in cross-bred litters resulted in novel disease phenotypes different from either parental line. Augmenting the calcineurin-dependent hypertrophic stimulus in tropomodulin overexpressing transgenics elevated heart:body weight ratios, increased ventricular wall thickness, and significantly accelerated mortality. These effects were evident in calcineurin cross-breeding to tropomodulin backgrounds of transgene homozygosity (severe dilation) or heterozygosity (mild dilation to asymptomatic). Molecular analyses indicated that tropomodulin and calcineurin signaling events in the first week after birth were critical for determination of disease outcome, substantiated by demonstration that temporary neonatal inhibition of tropomodulin expression prevents dilation. CONCLUSIONS: This study shows that postnatal timing of altered signaling in cardiomyopathic transgenic mouse models is a pivotal part of determining outcome. In addition, intensifying hypertrophic stimulation exacerbates dilated cardiomyopathy, supporting the concept of shared molecular signaling between hypertrophy and dilation. (+info)