Tropomyosin isoforms in developing chicken gizzard smooth muscle

In the embryonic smooth muscle of chicken gizzards we found 4 high-Mr-type and 5 low-Mr-type tropomyosin isoforms in addition to alpha- and beta-isoforms reported already. The criteria by which they were identified as tropomyosin isoforms were as follows: 1) anomalous reduction of electrophoretic mobility in the presence of urea, 2) cross reactivity with antisera against tropomyosins, 3) inclusion in a tropomyosin preparation obtained by the usual method for tropomyosin purification, and 4) binding ability to skeletal muscle actin. At the early stages of development, the amounts of these isoforms were larger than those of alpha- and beta-isoforms, but they gradually decreased at later stages and finally disappeared completely after hatching. Our previous study of gizzard smooth muscle showed that the amount ratio of accumulated tropomyosin to gamma-actin was reasonably constant in the development after hatching, while, at the earlier embryonic stages (7-14 d of incubation), it was lower than expected. The isoforms found in this study were present in amounts large enough to bring the ratio at the earlier stages up to the constant amount ratio observed after hatching. Therefore, the coordinate accumulation of actin and tropomyosin was suggested to occur even at the embryonic stages.     

Differences in cellular contractile protein contents among porcine smooth muscles: evidence for variation in the contractile system

Cellular myosin, actin, and tropomyosin contents and ratios were determined for arterial (carotid, aorta, and coronary), intestinal (circular and longitudinal), esophageal, uterine, and tracheal smooth muscles inthe pig. Tissue protein contents were estimated by densitometry of polyacrylamide gels after electrophoresis of sodium dodecyl sulfate-treated tissue homogenates. Cellular contractile protein contents were estimated by correction for extracellular spaces. Cellular myosin contents were similar in each tissue (average +/- 1 SEM = 19.6 +/- 0.8 mg/g cell wet wt). However, the cellular contents of the thin filament proteins, actin and tropomyosin, were significantly higher in the arteries than in the nonarterial tissues. The calculated weight ratios of actin: myosin averaged 2.6 +/- 0.2 in the three arterial tissues and 1.5 +/- 0.1 in the nonarterial tissues, which may be compared with 0.36 in vertebrate striated muscles. The actin:tropomyosin weight ratios for all tissues were 3.7 +/- 0.1, a value comparable to the skeletal muscle ratio. The physiological implications of variations in the cellular thin filament protein contents are unknown, but these variations probably contribute to the observed differences in contractile function among various smooth muscles.     

Contents of myofibrillar proteins in cardiac, skeletal, and smooth muscles

The in situ contents of myosin, actin, alpha-actinin, tropomyosin, troponin, desmin were estimated in dog cardiac, rabbit skeletal, and chicken smooth muscles. Whole muscle tissues were dissolved with 8 M guanidine hydrochloride and subjected to two-dimensional gel electrophoresis, which is a nonequilibrium pH gradient electrophoresis (Murakami, U. & Uchida, K. (1984) J. Biochem. 95, 1577-1584) with some modification. The amount of protein in a spot on a slab gel was determined by quantification of the extracted dye. Dye binding capacity of individual myofibrillar proteins was determined by using the purified protein. Myosin contents were 82 +/- 7 pmol/mg wet weight in cardiac muscle, 105 +/- 10 pmol/mg wet weight in skeletal muscle, and 45 +/- 4 pmol/mg wet weight in smooth muscle. Actin contents were 339 +/- 15 pmol/mg wet weight in cardiac muscle, 625 +/- 27 pmol/mg wet weight in skeletal muscle, and 742 +/- 13 pmol/mg wet weight in smooth muscle. The subunit stoichiometry of myosin in the three types of muscles was two heavy chains and four light chains, and there was one light chain 2 for every heavy chain. The molar ratio of actin to tropomyosin was 7/1 in the three types of muscles. Striking differences were seen in the molar ratio of myosin to actin: 1.0/4.1 in cardiac muscle, 1.0/6.0 in skeletal muscle, and 1.0/16.5 in smooth muscle.     

Isolation of cDNA clones for mouse cytoskeletal gamma-actin and differential expression of cytoskeletal actin mRNAs in mouse cells.

We described the structures of mouse cytoskeletal gamma-actin cDNA clones and showed that there is strong conservation of the untranslated regions with human gamma-actin cDNA. In addition, we found that the expression levels of beta- and gamma-actin mRNAs are differentially controlled in various mouse tissues and cell types but are coordinately increased in the cellular growing state. These results suggest that there are multiple regulatory mechanisms of cytoskeletal actin genes and are consistent with the argument that beta- and gamma-actins might have functional diversity in mammalian cells.     

Regulation of alpha-smooth muscle actin and other polypeptides in proliferating and density-arrested vascular smooth muscle cells

We have examined alpha-smooth muscle actin (alpha-SM actin) protein and mRNA levels in proliferating and density-arrested rabbit vascular smooth muscle cells (SMC) and also studied overall polypeptide synthesis in these cells by two-dimensional (2-D) gel electrophoresis. Of the approximately 1,000 cellular polypeptides resolved by 2-D gel analysis, we consistently detected increased expression of 12 polypeptides in growth-arrested SMC. These polypeptides, with apparent molecular weights of 24,000 to 55,000 exhibited relative increases of between fourfold to greater than tenfold. Three of these polypeptides were expressed at undetectable levels in proliferating SMC. We also detected 12 secreted polypeptides that were expressed at higher levels in growth-arrested SMC. More changes were associated with the secreted polypeptides, since they represented approximately 4% of the total resolved secreted polypeptides, while only 1% of the cellular polypeptides were increased in high-density growth-arrested cells. Under these conditions we observed no change in relative alpha-SM actin protein content as determined by 2-D gel analysis and Western blots. This was corroborated by high levels of alpha-SM actin mRNA levels in both proliferating and high-density growth-arrested SMC. These results indicate rabbit vascular SMC maintain a high level of expression of a smooth muscle differentiation marker (alpha-SM actin) in a proliferation- and density-independent manner. We also examined polypeptide synthesis in SMC isolated by enzymatic digestion of the aorta vs. cells isolated by the explant method. We found that although overall protein patterns were remarkably similar, several differences were observed. These differences were not due to increased contamination by fibroblasts, since both enzymatically- and explant-derived SMC contained high levels of alpha-SM actin as determined by immunofluorescence and by Northern analysis.     

X-ray diffraction studies on oriented gels of vertebrate smooth muscle thin filaments.

The ATPase activity of acto-myosin subfragment 1 (S1) at low ratios of S1 to actin in the presence of tropomyosin is dependent on the tropomyosin source and ionic conditions. Whereas skeletal muscle tropomyosin causes a 60% inhibitory effect at all ionic strengths, the effect of smooth muscle tropomyosin was found to be dependent on the ionic strength. At low ionic strength (20 mM) smooth muscle tropomyosin inhibits the ATPase activity by 60%, while at high ionic strength (120 mM) it potentiates the ATPase activity three- to five-fold. Therefore, the difference in the effect of smooth muscle and skeletal muscle tropomyosin on the acto-S1 ATPase activity was due to a greater fraction of the tropomyosin-actin complex being turned on in the absence of S1 with smooth muscle tropomyosin than with skeletal muscle tropomyosin. Using well-oriented gels of actin and of reconstituted specimens from vertebrate smooth muscle thin filament proteins suitable for X-ray diffraction, we localized the position of tropomyosin on actin under different levels of acto-S1 ATPase activity. By analysing the equatorial X-ray pattern of the oriented specimens in combination with solution scattering experiments, we conclude that tropomyosin is located at a binding radius of about 3.5 nm on the f-actin helix under all conditions studied. Furthermore, we find no evidence that the azimuthal position of tropomyosin is different for smooth muscle tropomyosin at various ionic strengths, or vertebrate tropomyosin, since the second actin layer-line intensity (at 17.9 nm axial and 4.3 nm radial spacing), which was shown in skeletal muscle to be a sensitive measure of this parameter, remains strong and unchanged. Differences in the ATPase activity are not necessarily correlated with different positions of tropomyosin on f-actin. The same conclusion is drawn from our observations that, although the regulatory protein caldesmon inhibits the ATPase activity in native and reconstituted vertebrate smooth muscle thin filaments at a molar ratio of actin/tropomyosin/caldesmon of 28:7:1, the second actin layer-line remains strong. Only adding caldesmon in excess reduces the intensity of the second actin layer-line, from which the binding radius of caldesmon can be estimated to be about 4 nm. The lack of predominant meridional reflections in oriented specimens, with caldesmon present, suggests that caldesmon does not project away from the thin filament as troponin molecules in vertebrate striated muscle in agreement with electron micrographs of smooth muscle thin filaments. In freshly prepared native smooth muscle thin filaments we observed a Ca(2+)-sensitive reversible bundling effect.(ABSTRACT TRUNCATED AT 400 WORDS)     

Structure, chromosome location, and expression of the human gamma-actin gene: differential evolution, location, and expression of the cytoskeletal beta- and gamma-actin genes.

The accumulation of the cytoskeletal beta- and gamma-actin mRNAs was determined in a variety of mouse tissues and organs. The beta-isoform is always expressed in excess of the gamma-isoform. However, the molar ratio of beta- to gamma-actin mRNA varies from 1.7 in kidney and testis to 12 in sarcomeric muscle to 114 in liver. We conclude that, whereas the cytoskeletal beta- and gamma-actins are truly coexpressed, their mRNA levels are subject to differential regulation between different cell types. The human gamma-actin gene has been cloned and sequenced, and its chromosome location has been determined. The gene is located on human chromosome 17, unlike beta-actin which is on chromosome 7. Thus, if these genes are also unlinked in the mouse, the coexpression of the beta- and gamma-actin genes in rodent tissues cannot be determined by gene linkage. Comparison of the human beta- and gamma-actin genes reveals that noncoding sequences in the 5'-flanking region and in intron III have been conserved since the duplication that gave rise to these two genes. In contrast, there are sequences in intron III and the 3'-untranslated region which are not present in the beta-actin gene but are conserved between the human gamma-actin and the Xenopus borealis type 1 actin genes. Such conserved noncoding sequences may contribute to the coexpression of beta- and gamma-actin or to the unique regulation and function of the gamma-actin gene. Finally, we demonstrate that the human gamma-actin gene is expressed after introduction into mouse L cells and C2 myoblasts and that, upon fusion of C2 cells to form myotubes, the human gamma-actin gene is appropriately regulated.     

Differential control of tropomyosin mRNA levels during myogenesis suggests the existence of an isoform competition-autoregulatory compensation control mechanism.

We have isolated tropomyosin cDNAs from human skeletal muscle and nonmuscle cDNA libraries and constructed gene-specific DNA probes for each of the four functional tropomyosin genes. These DNA probes were used to define the regulation of the corresponding mRNAs during the process of myogenesis. Tropomyosin regulation was compared with that of beta- and gamma-actin. No two striated muscle-specific tropomyosin mRNAs are coordinately accumulated during myogenesis nor in adult striated muscles. Similarly, no two nonmuscle tropomyosins are coordinately repressed during myogenesis. However, mRNAs encoding the 248 amino acid nonmuscle tropomyosins and beta- and gamma-actin are more persistent in adult skeletal muscle than those encoding the 284 amino acid nonmuscle tropomyosins. In particular, the nonmuscle tropomyosin Tm4 is expressed at similar levels in adult rat nonmuscle and striated muscle tissues. We conclude that each tropomyosin mRNA has its own unique determinants of accumulation and that the 248 amino acid nonmuscle tropomyosins may have a role in the architecture of the adult myofiber. The variable regulation of nonmuscle isoforms during myogenesis suggests that the different isoforms compete for inclusion into cellular structures and that compensating autoregulation of mRNA levels bring gene expression into alignment with the competitiveness of each individual gene product. Such an isoform competition-autoregulatory compensation mechanism would readily explain the unique regulation of each gene.     

Structural role of tropomyosin in muscle regulation: analysis of the x-ray diffraction patterns from relaxed and contracting muscles

Previous work has shown that there are significant differences in the X-ray diffraction patterns obtained from relaxed and contracting muscles. We show that some of these changes can be explained in terms of a small movement (~ 5 to 15 Å) of the tropomyosin molecules in the groove of the actin helix. The position of the tropomyosin in relaxed skeletal muscle is such that it might physically block or at least structurally alter the cross-bridge attachment site on actin, whereas in contracting skeletal muscle the tropomyosin moves to a position well clear of the attachment site. The movement of the tropomyosin molecules is apparently smaller in molluscan muscles during tonic contraction than in vertebrate skeletal muscle. We suggest a possible relationship between the smaller movement of the tropomyosin and the “catch” response of molluscan muscles.We also show that any increase of intensity on the 59 Å and 51 Å layer-lines is most likely to be associated with some extra mass (HMM S-1) attaching to the actin molecules. Such a change cannot be explained in terms of a change in tropomyosin structure or in the order within the thin filaments. Since changes on these two layer-lines have been observed during contraction, this provides good evidence for cross-bridge attachment to actin in contracting muscles.     

Developmental change of protein constituents in chicken gizzards

Developmental change of protein constituents of chick gizzard smooth muscle was described by the fluorescent antibody technique and two-dimensional polyacrylamide gel electrophoresis. Myosin heavy chain, tropomyosin, and desmin were immunohistologically detected in 5-day-old gizzard primordia, but myoglobin was detected after 19 days of incubation. Two-dimensional polyacrylamide gel electrophoresis revealed that most structural proteins including beta- and gamma-actin are synthesized almost simultaneously in the primordium, and accumulate in three patterns by which the proteins examined are classified: (1) gradually increasing protein (gamma-actin, tropomyosin, desmin), (2) abruptly increasing protein at a certain stage (myosin, myoglobin), (3) decreasing or constantly kept protein (tubulin, beta-actin). Based on the quantitative analysis of protein constituents, the nature of regulatory system of protein synthesis in smooth muscle and the possible functional difference between beta- and gamma-actin are discussed.     

Change of actin isomers during differentiation of smooth muscle

Changes of actin isomers during development and differentiation of chicken gizzard were investigated by polyacrylamide gel electrophoresis. The two-dimensional gel electrophoresis with SDS-polyacrylamide gels in the presence of urea as the second dimension clearly separated three actin isomers which appear during development of the smooth muscle. The three actin isomers change the relative concentrations during development as follows: gizzard-type gamma-actin begins to be synthesized late on the 7th day of embryogenesis and increases in amount until hatching, nonmuscle-type gamma-actin exists only at earlier stages (before 15 days of embryogenesis), and the amount of beta-actin increases in proportion to the decrease of nonmuscle type gamma-actin, the amount of nonmuscle actin in gizzards then becoming constant. Actin composition of gizzard before 7 days of embryonic age was nonmuscle type and consisted of beta-actin and nonmuscle-type gamma-actin. These observations indicate that developmental process of gizzard smooth muscle cells are classified as three stages: nonmuscle, intermediate and smooth muscle stages.     

Structure, chromosome location, and expression of the human smooth muscle (enteric type) gamma-actin gene: evolution of six human actin genes.

Recombinant phages that carry the human smooth muscle (enteric type) gamma-actin gene were isolated from human genomic DNA libraries. The amino acid sequence deduced from the nucleotide sequence matches those of cDNAs but differs from the protein sequence previously reported at one amino acid position, codon 359. The gene containing one 5' untranslated exon and eight coding exons extends for 27 kb on human chromosome 2. The intron between codons 84 and 85 (site 3) is unique to the two smooth muscle actin genes. In the 5' flanking region, there are several CArG boxes and E boxes, which are regulatory elements in some muscle-specific genes. Hybridization with the 3' untranslated region, which is specific for the human smooth muscle gamma-actin gene, suggests the single gene in the human genome and specific expressions in enteric and aortic tissues. From characterized molecular structures of the six human actin isoform genes, we propose a hypothesis of evolutionary pathway of the actin gene family. A presumed ancestral actin gene had introns at least sites 1, 2, and 4 through 8. Cytoplasmic actin genes may have directly evolved from it through loss of introns at sites 5 and 6. However, through duplication of the ancestral actin gene with substitutions of many amino acids, a prototype of muscle actin genes had been created. Subsequently, striated muscle actin and smooth muscle actin genes may have evolved from this prototype by loss of an intron at site 4 and acquisition of a new intron at site 3, respectively.     

Effect of muscle and non-muscle tropomyosins in reconstituted skeletal muscle actomyosin

Smooth and non-muscle tropomyosins were found to produce a 2-3-fold Ca-insensitive stimulation of the ATPase activity of reconstituted skeletal muscles actomyosin at normal MgATP concentrations and physiological ratios of myosin to actin. Under the same conditions skeletal muscles tropomyosin had no effect. Similar effects of these three tropomyosins were observed for the low myosin/F-actin ratios necessary for kinetic measurements. Since it could be established that this actomyosin system, with or without tropomyosin, obeyed Michaelian kinetics, the tropomyosin effects could be interpreted in terms of their influence on maximal turnover (V) or on the affinity of myosin for actin (Kapp). Accordingly, gizzard tropomyosin had practically no effect on the affinity and reduced only slightly the value of V, compared to pure actin. In contrast to gizzard tropomyosin, brain tropomyosin produced an approximately twofold increase in both Kapp and V; i.e. it increased the turnover rate but decreased the affinity. It is apparent from the data that brain tropomyosin acts as an uncompetitive activator with respect to pure actin, while having the same V as the actin plus gizzard tropomyosin complex. Further studies on these tropomyosins show that only skeletal and smooth muscle tropomyosin have similar functional properties with respect to troponin inhibition and the activation of the ATPase at low ATP concentrations. It is suggested that the noted increases in V by tropomyosin are caused by the acceleration of the dissociation of the myosin head from actin at the end point of the cross bridge movement.     

Role of tropomyosin in smooth muscle contraction: effect of tropomyosin binding to actin on actin activation of myosin ATPase

The binding of gizzard tropomyosin to gizzard F-actin is highly dependent on free Mg2+ concentration. At 2 mM free Mg2+, a concentration at which actin-activated ATPase activity was shown to be Ca2+ sensitive, a molar ratio of 1:3 (tropomyosin:actin monomer) is required to saturate the F-actin with tropomyosin to the stoichiometric ratio of 1 mol of tropomyosin to 7 mol of actin monomer. Increasing the Mg2+ could decrease the amount of tropomyosin required for saturating the F-actin filament to the stoichiometric level. Analysis of the binding of smooth muscle tropomyosin to smooth muscle actin by the use of Scatchard plots indicates that the binding exhibits strong positive cooperativity at all Mg2+ concentrations. Calcium has no effect on the binding of tropomyosin to actin, irrespective of the free Mg2+ concentration. However, maximal activation of the smooth muscle actomyosin ATPase in low free Mg2+ requires the presence of Ca2+ and stoichiometric binding of tropomyosin to actin. The lack of effect of Ca2+ on the binding of tropomyosin to actin shows that the activation of actomyosin ATPase by Ca2+ in the presence of tropomyosin is not due to a calcium-mediated binding of tropomyosin to actin.     

Alpha smooth muscle actin (α-SM actin) in normal human ovaries,in ovarian stromal hyperplasia and in ovarian neoplasms

An immunohistochemical investigation of alpha-smooth muscle actin (alpha-SM actin) using the monoclonal anti-alpha-SM-1 antibody was carried out in 15 normal ovaries, in three ovaries with stromal hyperplasia and in 27 neoplastic ovaries. In selected cases the pattern of actin isoforms was examined by means of 2 D-gel electrophoresis. In addition, the tissues were stained for vimentin and desmin. In normal ovaries alpha-SM actin was found in the inner cortex and in the theca externa. In ovarian stromal hyperplasia expression of alpha-SM actin was minimal or absent. In primary and metastatic epithelial tumors there was positive stromal staining for alpha-SM actin, especially in the vicinity of epithelial elements. This tended to be more widespread in malignant neoplasms. Thecomas did not express alpha-SM-actin and could thus be differentiated from leiomyomas which stained intensely for alpha-SM actin. Only focal stromal staining of alpha-SM actin was observed in granulosa and germ cell tumors. In all the tissues studied blood vessels were strongly positive for alpha-SM actin. Desmin, although present in the stroma of most of the specimens, was less abundant than alpha-SM actin. We concluded that alpha-SM actin is a component of the normal human ovary where it may contribute to the contractility of its stroma. Its absence in the normal outer cortex and theca interna, and in stromal hyperplasia and thecoma implies that sex hormones do not constitute a stimulus for alpha-SM actin production in the ovary. Among neoplasms it is most widely represented in the stroma of epithelial tumors in which it may reflect stromal stimulation mediated by neoplastic epithelium.     

Three-dimensional reconstruction of caldesmon-containing smooth muscle thin filaments

Caldesmon is known to inhibit actomyosin ATPase and filament sliding in vitro, and may play a role in modulating smooth muscle contraction as well as in diverse cellular processes including cytokinesis and exocytosis. However, the structural basis of caldesmon action has not previously been apparent. We have recorded electron microscope images of negatively stained thin filaments containing caldesmon and tropomyosin which were isolated from chicken gizzard smooth muscle in EGTA. Three-dimensional helical reconstructions of these filaments show actin monomers whose bilobed shape and connectivity are very similar to those previously seen in reconstructions of frozen-hydrated skeletal muscle thin filaments. In addition, a continuous thin strand of density follows the long-pitch actin helices, in contact with the inner domain of each actin monomer. Gizzard thin filaments treated with Ca2+/calmodulin, which dissociated caldesmon but not tropomyosin, have also been reconstructed. Under these conditions, reconstructions also reveal a bilobed actin monomer, as well as a continuous surface strand that appears to have moved to a position closer to the outer domain of actin. The strands seen in both EGTA- and Ca2+/calmodulin-treated filaments thus presumably represent tropomyosin. It appears that caldesmon can fix tropomyosin in a particular position on actin in the absence of calcium. An influence of caldesmon on tropomyosin position might, in principle, account for caldesmon's ability to modulate actomyosin interaction in both smooth muscles and non-muscle cells.     

Tropomyosin-caldesmon/actomyosin systems in platelets and arterial smooth muscle: results from exchange experiments

Tropomyosin and caldesomon reciprocally control the actomyosin system in smooth muscle and some non-muscle cells. To compare this mechanism between arterial smooth muscle and platelets, we carried out extensive exchange experiments. Actin, myosin, tropomyosin from arterial smooth muscle cells and platelets were recombined and the effects of two species of caldesmon ('caldesmon77' and 'caldesmon140') on the ATPase activities of both systems were examined and analyzed by the method of analysis of variance. (a) The actomyosin system itself is different between artery and platelets, the difference being determined by myosin (P less than 0.05) and not by actin. (b) Platelet tropomyosin differentiates platelet actin from arterial actin (P less than 0.01), while arterial tropomyosin does not. Neither does tropomyosin differentiate myosin. (c) The effect of caldesmon77 differentiates the origins of myosin (P less than 0.01), actin (P less than 0.05) and tropomyosin (P less than 0.05). The effect of caldesmon140 differentiates the origin of myosin (P less than 0.05) and the actin-myosin 'interaction' (combination) (P less than 0.01), but not the origin of tropomyosin (P greater than 0.1). (1) It is concluded that actomyosin/tropomyosin-caldesmon system is distinguishable between platelets and artery. (2) It is suggested that caldesmon is an actomyosin inhibitor which may interact with myosin, in addition to actin and tropomyosin.     

Alpha-smooth muscle actin is expressed in a subpopulation of cultured and cloned fibroblasts and is modulated by gamma-interferon.

Clinical and experimental investigations have shown that, during wound healing and fibrocontractive diseases, fibroblasts acquire, more or less permanently according to the situation, morphological and biochemical features of smooth muscle (SM) cells including the expression of alpha-SM actin. Primary and passaged cultures of rat and human fibroblasts contain a subpopulation of cells expressing alpha-SM actin. These cells could derive from SM cells and/or pericytes present in the tissue from which cultures have been produced or represent bona fide fibroblasts. We have investigated the presence of alpha-SM actin in fibroblast cultures, clones, and subclones. In all cases the fibroblastic populations studied showed a proportion of alpha-SM actin expressing cells. Even after cloning, we never obtained populations negative for alpha-SM actin. We conclude that alpha-SM actin expression in fibroblastic cultures is not due to contaminant cells but is a feature of fibroblasts themselves. Our results support the view that fibroblastic cells are a heterogeneous population. It has been previously shown that gamma-interferon (gamma-IFN) decreases alpha-SM actin expression in SM cells. In rat and human fibroblasts, gamma-IFN decreases alpha-SM actin protein and mRNA expression as well as proliferation. The properties of this cytokine make it a good candidate for exerting an anti-fibrotic activity in vivo.     

Tropomyosin and actin isoforms modulate the localization of tropomyosin strands on actin filaments

Tropomyosin is present in virtually all eucaryotic cells, where it functions to modulate actin-myosin interaction and to stabilize actin filament structure. In striated muscle, tropomyosin regulates contractility by sterically blocking myosin-binding sites on actin in the relaxed state. On activation, tropomyosin moves away from these sites in two steps, one induced by Ca(2+) binding to troponin and a second by the binding of myosin to actin. In smooth muscle and non-muscle cells, where troponin is absent, the precise role and structural dynamics of tropomyosin on actin are poorly understood. Here, the location of tropomyosin on F-actin filaments free of troponin and other actin-binding proteins was determined to better understand the structural basis of its functioning in muscle and non-muscle cells. Using electron microscopy and three-dimensional image reconstruction, the association of a diverse set of wild-type and mutant actin and tropomyosin isoforms, from both muscle and non-muscle sources, was investigated. Tropomyosin position on actin appeared to be defined by two sets of binding interactions and tropomyosin localized on either the inner or the outer domain of actin, depending on the specific actin or tropomyosin isoform examined. Since these equilibrium positions depended on minor amino acid sequence differences among isoforms, we conclude that the energy barrier between thin filament states is small. Our results imply that, in striated muscles, troponin and myosin serve to stabilize tropomyosin in inhibitory and activating states, respectively. In addition, they are consistent with tropomyosin-dependent cooperative switching on and off of actomyosin-based motility. Finally, the locations of tropomyosin that we have determined suggest the possibility of significant competition between tropomyosin and other cellular actin-binding proteins. Based on these results, we present a general framework for tropomyosin modulation of motility and cytoskeletal modelling.