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: (1) gizzard-type γ-actin begins to be synthesized late on the 7th day of embryogenesis and increases in amount until hatching, (2) nonmuscle-type γ-actin exists only at earlier stages (before 15 days of embryogenesis), and (3) the amount of β-actin increases in proportion to the decrease of nonmuscle type γ-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 β-actin and nonmuscle-type γ-actin. These observations indicate that developmental process of gizzard smooth muscle cells are classified as three stages: nonmuscle, intermediate and smooth muscle stages.     

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.     

Cytoskeletal remodeling in differentiated vascular smooth muscle is actin isoform dependent and stimulus dependent

Dynamic remodeling of the actin cytoskeleton plays an essential role in the migration and proliferation of vascular smooth muscle cells. It has been suggested that actin remodeling may also play an important functional role in nonmigrating, nonproliferating differentiated vascular smooth muscle (dVSM). In the present study, we show that contractile agonists increase the net polymerization of actin in dVSM, as measured by the differential ultracentrifugation of vascular smooth muscle tissue and the costaining of single freshly dissociated cells with fluorescent probes specific for globular and filamentous actin. Furthermore, induced alterations of the actin polymerization state, as well as actin decoy peptides, inhibit contractility in a stimulus-dependent manner. Latrunculin pretreatment or actin decoy peptides significantly inhibit contractility induced by a phorbol ester or an alpha-agonist, but these procedures have no effect on contractions induced by KCl. Aorta dVSM expresses alpha-smooth muscle actin, beta-actin, nonmuscle gamma-actin, and smooth muscle gamma-actin. The incorporation of isoform-specific cell-permeant synthetic actin decoy peptides, as well as isoform-specific probing of cell fractions and two-dimensional gels, demonstrates that actin remodeling during alpha-agonist contractions involves the remodeling of primarily gamma-actin and, to a lesser extent, beta-actin. Taken together, these results show that net isoform- and agonist-dependent increases in actin polymerization regulate vascular contractility.     

Use of actin isoform-specific antibodies to probe the domain structure in three smooth muscles

We investigated the location of actin isoforms in relation to each other and to filament attachment sites by studying the edge-to-edge distribution of both immunofluorescence and immunogold probes in smooth muscle cells from three sources. Antibodies to alpha- or alpha,gamma-actin labeled uniformly across smooth muscle cells from each source. Antibodies to beta-cytoplasmic actin were concentrated on and near dense bodies, especially in gizzard smooth muscle, but were also located throughout the filament compartment. Double immunofluorescent labeling with antibodies to alpha- or alpha/gamma- and to beta-actin shows overlap of label at dense bodies and attachment plaques. Double immunofluorescent labeling with antibodies to alpha-actinin and to beta-actin identified dense bodies and attachment plaques as sites of colocalization. Immunogold labeling with anti-desmin was most prominent near dense bodies in the gizzard and was widely dispersed in vas deferens and arterial smooth muscle cells. Our results indicate that there is extensive overlap between the locations of contractile and cytoskeletal elements and, thus, do not support the two-domain model of smooth muscle structure. Tissue-specific organizational motif differences were seen when gizzard, vas deferens, and artery were compared and suggest that one model may not apply to these three smooth muscles.     

Polarized distribution of actin isoforms in gastric parietal cells.

The actin genes encode several structurally similar, but perhaps functionally different, protein isoforms that mediate contractile function in muscle cells and determine the morphology and motility in nonmuscle cells. To reveal the isoform profile in the gastric monomeric actin pool, we purified actin from the cytosol of gastric epithelial cells by DNase I affinity chromatography followed by two-dimensional gel electrophoresis. Actin isoforms were identified by Western blotting with a monoclonal antibody against all actin isoforms and two isoform-specific antibodies against cytoplasmic beta-actin and gamma-actin. Densitometry revealed a ratio for beta-actin/gamma-actin that equaled 0.73 +/- 0.09 in the cytosol. To assess the distribution of actin isoforms in gastric glandular cells in relation to ezrin, a putative membrane-cytoskeleton linker, we carried out double immunofluorescence using actin-isoform-specific antibodies and ezrin antibody. Immunostaining confirmed that ezrin resides mainly in canaliculi and apical plasma membrane of parietal cells. Staining for the beta-actin isoform was intense along the entire gland lumen and within the canaliculi of parietal cells, thus predominantly near the apical membrane of all gastric epithelial cells, although lower levels of beta-actin were also identified near the basolateral membrane. The gamma-actin isoform was distributed heavily near the basolateral membrane of parietal cells, with much less intense staining of parietal cell canaliculi and no staining of apical membranes. Within parietal cells, the cellular localization of beta-actin, but not gamma-actin, isoform superimposed onto that of ezrin. In a search for a possible selective interaction between actin isoforms and ezrin, we carried out immunoprecipitation experiments on gastric membrane extracts in which substantial amounts of actin were co-eluted with ezrin from an anti-ezrin affinity column. The ratio of beta-actin/gamma-actin in the immunoprecipitate (beta/gamma = 2.14 +/- 0.32) was significantly greater than that found in the cytosolic fraction. In summary, we have shown that beta- and gamma-actin isoforms are differentially distributed in gastric parietal cells. Furthermore, our data suggest a preferential, but not exclusive, interaction between beta-actin and ezrin in gastric parietal cells. Finally, our results suggest that the beta- and gamma-actin-based cytoskeleton networks might function separately in response to the stimulation of acid secretion.     

Transfection of nonmuscle beta- and gamma-actin genes into myoblasts elicits different feedback regulatory responses from endogenous actin genes

We have examined the role of feedback-regulation in the expression of the nonmuscle actin genes. C2 mouse myoblasts were transfected with the human beta- and gamma-actin genes. In gamma-actin transfectants we found that the total actin mRNA and protein pools remained unchanged. Increasing levels of human gamma-actin expression resulted in a progressive down-regulation of mouse beta- and gamma-actin mRNAs. Transfection of the beta-actin gene resulted in an increase in the total actin mRNA and protein pools and induced an increase in the levels of mouse beta-actin mRNA. In contrast, transfection of a beta-actin gene carrying a single-point mutation (beta sm) produced a feedback-regulatory response similar to that of the gamma-actin gene. Expression of a beta-actin gene encoding an unstable actin protein had no impact on the endogenous mouse actin genes. This suggests that the nature of the encoded actin protein determines the feedback-regulatory response of the mouse genes. The role of the actin cytoskeleton in mediating this feedback-regulation was evaluated by disruption of the actin network with Cytochalasin D. We found that treatment with Cytochalasin D abolished the down-regulation of mouse gamma-actin in both the gamma- and beta sm-actin transfectants. In contrast, a similar level of increase was observed for the mouse beta-actin mRNA in both control and transfected cells. These experiments suggest that the down-regulation of mouse gamma-actin mRNA is dependent on the organization of the actin cytoskeleton. In addition, the mechanism responsible for the down-regulation of beta-actin may be distinct from that governing gamma-actin. We conclude that actin feedback-regulation provides a biochemical assay for differences between the two nonmuscle actin genes.     

The Complete Amino Acid Sequence of Actins from Bovine Aorta, Bovine Heart, Bovine Fast Skeletal Muscle, and Rabbit, Slow Skeletal Muscle

Complete amino acid sequences for four mammalian muscle actins are reported: bovine skeletal muscle actin, bovine cardiac actin, the major component of bovine aorta actin, and rabbit slow skeletal muscle actin. The number of different actins in a higher mammal for which full amino acid sequences are now available is therefore increased from two to five. Screening of different smooth muscle tissues revealed in addition to the aorta type actin a second smooth muscle actin, which appears very similar if not identical to chicken gizzard actin. Since the sequence of chicken gizzard actin is known, six different actins are presently characterized in a higher mammal.
The two smooth muscle actins—bovine aorta actin and chicken gizzard actin—differ by only three amino acid substitutions, all located in the amino-terminal end. In the rest of their sequences both smooth muscle actins share the same four amino acid substitutions, which distinguish them from skeletal muscle actin. Cardiac muscle actin differs from skeletal muscle actin by only four amino acid exchanges. No amino acid substitutions were found when actins from rabbit fast and slow skeletal muscle were compared.
In addition we summarize the amino acid substitution patterns of the six different mammalian actins and discuss their tissue specificity. The results show a very close relationship between the four muscle actins in comparison to the nonmuscle actins. The amino substitution patterns indicate that skeletal muscle actin is the highest differentiated actin form, whereas smooth muscle actins show a noticeably closer relation to nonmuscle actins. By these criteria cardiac muscle actin lies between skeletal muscle actin and smooth muscle actins.     

The complete amino acid sequence of actins from bovine aorta, bovine heart, bovine fast skeletal muscle, and rabbit slow skeletal muscle. A protein-chemical analysis of muscle actin differentiation.

Complete amino acid sequences for four mammalian muscle actins are reported: bovine skeletal muscle actin, bovine cardiac actin, the major component of bovine aorta actin, and rabbit slow skeletal muscle actin. The number of different actins in a higher mammal for which full amino acid sequences are now available is therefore increased from two to five. Screening of different smooth muscle tissues revealed in addition to the aorta type actin a second smooth muscle actin, which appears very similar if not identical to chicken gizzard actin. Since the sequence of chicken gizzard actin is known, six different actins are presently characterized in a higher mammal. The two smooth muscle actins--bovine aorta actin and chicken gizzard actin--differ by only three amino acid substitutions, all located in the amino-terminal end. In the rest of their sequences both smooth muscle actins share the same four amino acid substitutions, which distinguish them from skeletal muscle actin. Cardiac muscle actin differs from skeletal muscle actin by only four amino acid exchanges. No amino acid substitutions were found when actins from rabbit fast and slow skeletal muscle were compared. In addition we summarize the amino acid substitution patterns of the six different mammalian actins and discuss their tissue specificity. The results show a very close relationship between the four muscle actins in comparison to the nonmuscle actins. The amino substitution patterns indicate that skeletal muscle actin is the highest differentiated actin form, whereas smooth muscle actins show a noticeably cloer relation to nonmuscle actins. By these criteria cardiac muscle actin lies between skeletal muscle actin and smooth muscle actins.     

A smooth muscle-specific monoclonal antibody recognizes smooth muscle actin isozymes

Injection of chicken gizzard actin into BALB/c mice resulted in the isolation of a smooth muscle-specific monoclonal antibody designated CGA7. When assayed on methanol-Carnoy's fixed, paraffin-embedded tissue, it bound to smooth muscle cells and myoepithelial cells, but failed to decorate striated muscle, endothelium, connective tissue, epithelium, or nerve. CGA7 recognized microfilament bundles in early passage cultures of rat aortic smooth muscle cells and human leiomyosarcoma cells but did not react with human fibroblasts. In Western blot experiments, CGA7 detected actin from chicken gizzard and monkey ileum, but not skeletal muscle or fibroblast actin. Immunoblots performed on two-dimensional gels demonstrated that CGA7 recognizes gamma-actin from chicken gizzard and alpha- and gamma-actin from rat colon muscularis. This antibody was an excellent tissue-specific smooth muscle marker.     

Molecular properties and functions in vitro of chicken smooth-muscle alpha-actinin in comparison with those of striated-muscle alpha-actinins

alpha-Actinin purified from chicken gizzard smooth muscle was characterized in comparison with alpha-actinins from chicken striated muscles, or fast-skeletal muscle, slow-skeletal muscle, and cardiac muscle. The gizzard alpha-actinin molecule consisted of two apparently identical subunits with a molecular weight of 100,000 on SDS-polyacrylamide gel electrophoresis, as do striated-muscle alpha-actinins. Its isoelectric points in the presence of urea were similar to the striated-muscle counterparts. Despite these similarities, distinctive amino acid sequences between smooth-muscle alpha-actinin and striated-muscle alpha-actinins were revealed by peptide mapping using limited proteolysis in SDS. Gizzard alpha-actinin was immunologically distinguished from striated-muscle alpha-actinins. Gizzard alpha-actinin formed bundles of gizzard F-actin as well as of skeletal-muscle F-actin, but could not form any cross-bridges between adjacent actin filaments under conditions where skeletal-muscle alpha-actinin could. Temperature-dependent competition between gizzard alpha-actinin and tropomyosin on binding to gizzard thin filaments was demonstrated by electron microscopic observations. Gizzard alpha-actinin promoted Mg2+-ATPase activity of reconstituted skeletal actomyosin, gizzard acto-skeletal myosin, and gizzard actomyosin. This promoting effect was depressed by the addition of gizzard tropomyosin. These findings imply that, despite structural differences between gizzard and striated-muscle alpha-actinin molecules, they function similarly in vitro, and that gizzard alpha-actinin can interact not only with smooth-muscle actin (gamma- and beta-actin) but also with skeletal-muscle actin (alpha-actin).     

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.     

Vascular smooth muscle caldesmon

Caldesmon, a major actin- and calmodulin-binding protein, has been identified in diverse bovine tissues, including smooth and striated muscles and various nonmuscle tissues, by denaturing polyacrylamide gel electrophoresis of tissue homogenates and immunoblotting using rabbit anti-chicken gizzard caldesmon. Caldesmon was purified from vascular smooth muscle (bovine aorta) by heat treatment of a tissue homogenate, ion-exchange chromatography, and affinity chromatography on a column of immobilized calmodulin. The isolated protein shared many properties in common with chicken gizzard caldesmon: immunological cross-reactivity, Ca2+-dependent interaction with calmodulin, Ca2+-independent interaction with F-actin, competition between actin and calmodulin for caldesmon binding only in the presence of Ca2+, and inhibition of the actin-activated Mg2+-ATPase activity of smooth muscle myosin without affecting the phosphorylation state of myosin. Maximal binding of aorta caldesmon to actin occurred at 1 mol of caldesmon: 9-10 mol of actin, and binding was unaffected by tropomyosin. Half-maximal inhibition of the actin-activated myosin Mg2+-ATPase occurred at approximately 1 mol of caldesmon: 12 mol of actin. This inhibition was also unaffected by tropomyosin. Caldesmon had no effect on the Mg2+-ATPase activity of smooth muscle myosin in the absence of actin. Bovine aorta and chicken gizzard caldesmons differed in several respects: Mr (149,000 for bovine aorta caldesmon and 141,000 for chicken gizzard caldesmon), extinction coefficient (E1%280nm = 19.5 and 5.0 for bovine aorta and chicken gizzard caldesmon, respectively), amino acid composition, and one-dimensional peptide maps obtained by limited chymotryptic and Staphylococcus aureus V8 protease digestion. In a competitive enzyme-linked immunosorbent assay, using anti-chicken gizzard caldesmon, a 174-fold molar excess of bovine aorta caldesmon relative to chicken gizzard caldesmon was required for half-maximal inhibition. These studies establish the widespread tissue and species distribution of caldesmon and indicate that vascular smooth muscle caldesmon exhibits physicochemical differences yet structural and functional similarities to caldesmon isolated from chicken gizzard.     

Accumulation of tropomyosin in developing chicken gizzard smooth muscle

Smooth muscle of chicken embryonic gizzards has been shown to contain 9 tropomyosin isoforms (E1, E2, E3, E4, E5, E6, E7, E8, and E9) in addition to alpha and beta isoforms (Hosoya et al. (1989) J. Biochem. 105, 712-717). At the early stages of development, the amount of these isoforms was larger than those of alpha and beta isoforms. However, they gradually decreased at later stages and finally disappeared completely after hatching. By using two-dimensional gel electrophoresis and an image analyzing system, we examined the process of tropomyosin accumulation in gizzard smooth muscle development. The accumulation patterns of tropomyosin isoforms and their relative molar ratios to actin in embryonic development were different from those in the stages after hatching. The relative molar ratio of tropomyosin to actin in the thin filament preparation of embryonic gizzards was lower than that of adult, and it gradually increased in the course of embryonic development.     

Tissue-specific forms of actin in the developing chick

Actin has been identified in nonmuscle and muscle tissues as a highly conserved homogeneous protein. We have identified and characterized actin from embryonic and adult chick brain and muscle, and have compared these actins by SDS and urea/SDS gradient polyacrylamide gel electrophoresis. In the presence of SDS alone, embryonic or adult brain and muscle actin co-migrate as homogeneous polypeptides. Electrophoresis of both actins in the presence of urea and SDS, however, reveals that brain and muscle actins migrate with distinctly different mobilities. Actin from embryonic thigh muscle at different stages of development migrates as two separate components. In early muscle development, only the “brain” type actin is present. As muscle development progresses the “muscle” type actin becomes relatively more abundant, so that by day 20 of embryonic development, “muscle” actin becomes predominant. These results may be interpreted as due to differences in the primary structure of actin.     

Developmental changes in isoactin expression in rat aortic smooth muscle cells in vivo. Relationship between growth and cytodifferentiation

There is an inverse relationship between cellular proliferation and smooth muscle alpha-isoactin expression in cultured vascular smooth muscle cells (SMCs) (Owens, G.K., Loeb, A., Gordon, D., and Thompson, M.M. (1986) J. Cell Biol. 102, 343-352). In the present studies, changes in isoactin expression were studied during developmental growth of rat aortic SMCs (ages 1-180 days) to better understand interrelationships between growth and cytodifferentiation in these cells in vivo. Actin expression (i.e. content and synthesis) was evaluated by one- and two-dimensional gel electrophoresis and using isoactin-specific antibodies. The major actin present in cells from newborn rats was nonmuscle beta-actin (56% of total actin), whereas cells from adult animals contained principally smooth muscle alpha-actin (Sm-alpha-actin) (76% of total actin). Increases in Sm-alpha-actin content with increasing age were due, in part, to an increase in Sm-alpha-actin synthesis. However, in SMCs from 90- and 180-day-old rats, the fractional content of Sm-alpha-actin exceeded its fractional synthesis at a time when total Sm-alpha-actin content was increasing. This suggests that Sm-alpha-actin turns over more slowly in mature animals. Decreases in the frequency of SMCs undergoing DNA synthesis with age could not account for increases in Sm-alpha-actin expression with age. However, combined immunocytological and [3H]thymidine autoradiographic studies demonstrated that nearly 50% of the medial derived cells from newborn rat aortas did not show detectable staining with a monoclonal antibody to smooth muscle-specific isoactins, and the replicative frequency was much higher in these cells than in cells that contained Sm-alpha-isoactins. Taken together, the results of the present studies and previous studies in cultured SMCs support the hypothesis that cessation of proliferation during development is associated with the induction of Sm-alpha-actin expression, but that factors other than cellular growth state play an important role in determining the level of Sm-alpha-actin expression in fully differentiated SMCs.     

Absence of gamma-actin expression in the mouse fibroblast cell line, L

Cytoplasmic isoactins of mouse fibroblast L-cells were examined by two-dimensional gel electrophoresis. In contrast to other cultured cell lines, which contain both beta- and gamma-actin, L-cells contained only beta-actin species. This unique phenomenon was due neither to the transformed status of this cell line nor to the characteristic nature of adipose tissue fibroblasts of C3H mice. When RNA of L-cells was translated in a nuclease-treated reticulocyte lysate system, actin synthesized in vitro also appeared as only one species, beta-actin. Therefore, the genetic information for gamma-actin is absent at the level of translatable mRNA in L-cells.     

Quantitative changes in cytoskeletal beta- and gamma-actin mRNAs and apparent absence of sarcomeric actin gene transcripts in early mouse embryos

Actin is known to be synthesized both during oogenesis and in cleavage-stage embryos in mice. Cytoskeletal beta-actin appears to be the major component, followed by gamma-actin, but the synthesis of alpha-actin has also been inferred from protein electrophoretic patterns. We have studied the expression of cytoskeletal (beta- and gamma-) and sarcomeric (alpha-cardiac and alpha-skeletal) actin genes at the level of the individual mRNAs in blot hybridization experiments using isoform-specific RNA probes. The results show that there are about 2 x 10(4) beta-actin mRNA molecules in the fully grown oocyte; this number drops to about one-half in the egg and less than one-tenth in the late two-cell embryo but increases rapidly during cleavage to about 3 x 10(5) molecules in the late blastocyst. The amount of gamma-actin mRNA is similar to that of beta-actin in oocytes and eggs but only about 40% as much in late blastocysts, indicating a differential accumulation of these mRNAs during cleavage. The developmental pattern of beta- and gamma-actin mRNA provides a striking example of the transition from maternal to embryonic control that occurs at the two-cell stage and involves the elimination of most or all of the maternal actin mRNA. There was no detectable alpha-cardiac or alpha-skeletal mRNA (i.e., less than 1,000 molecules per embryo) at any stage from oocyte to late blastocyst, suggesting that the sarcomeric actin genes are silent during preimplantation development.     

Phenotype-dependent expression of alpha-smooth muscle actin in visceral smooth muscle cells

Alpha-Smooth muscle actin is one of the molecular markers for a phenotype of vascular smooth muscle cells, because the actin is a major isoform expressed in vascular smooth muscle cells and its expression is upregulated during differentiation. Here, we first demonstrate that the phenotype-dependent expression of this actin in visceral smooth muscles is quite opposite to that in vascular smooth muscles. This actin isoform is not expressed in adult chicken visceral smooth muscles including gizzard, trachea, and intestine except for the inner layer of intestinal muscle layers, whereas its expression is clearly detected in these visceral smooth muscles at early stages of the embryo (10-day-old embryo) and is developmentally downregulated. In cultured gizzard smooth muscle cells maintaining a differentiated phenotype, alpha-smooth muscle actin is not detected while its expression dramatically increases during serum-induced dedifferentiation. Promoter analysis reveals that a sequence (-238 to -219) in the promoter region of this actin gene acts as a novel negative cis-element. In conclusion, the phenotype-dependent expression of alpha-smooth muscle actin would be regulated by the sum of the cooperative contributions of the negative element and well-characterized positive elements, purine-rich motif, and CArG boxes and their respective transacting factors.     

Actin and tropomyosin variants in smooth muscles. Dependence on tissue type

Actin was found to be the major source of myofibrillar protein heterogeneity in smooth muscles. Three isoelectric variants, alpha-smooth muscle (alpha-SM), beta-non-muscle (beta-NM), and gamma-actins (gamma-SM and gamma-NM) were measured in 15 different smooth muscles, alpha-SM and gamma-actin contents displayed an inverse relationship in a given smooth muscle, some of which contained primarily alpha-SM actin while gamma-actins dominated in others. alpha-SM actin and gamma-actin distributions were tissue-specific, independent of species. A greater proportion of alpha-SM actin appears to be associated with tissues having a high degree of tonic activity. beta-Nonmuscle actin was a significant, and relatively constant, component of all smooth muscle tissues. The high NM-actin content of these tissues may reflect the importance of proliferative, synthetic, or secretory activities in smooth muscle, because the alpha-SM actin disappeared in tissue culture with a time course paralleling the modulation of phenotype from a contractile to a proliferative cell. Two tropomyosin subunits were present in approximately equal amounts in all smooth muscle tissues studied. One tropomyosin subunit exhibited identical mobility on two-dimensional gel electrophoresis, while the other was characterized by some species-specific variation which was unrelated to actin variant distribution. No variants of the 20,000-dalton regulatory light chain of myosin were observed. These results suggest that SM-specific actin variants are associated with functional diversity among smooth muscles.