At least six different actins are expressed in a higher mammal: An analysis based on the amino acid sequence of the amino-terminal tryptic peptide

The protein chemical characterization of the amino-terminal tryptic peptide of actin from different bovine tissues shows that at least six different actin structural genes are expressed in this mammal.Unique amirio acid sequences are found for actin from skeletal muscle, for actin from heart muscle, for two different actin species from smooth muscle, and for two different actin species typical of non-muscle tissues such as brain and thymus. The presence of more than one actin species in the same tissue (e.g. nonmuscle tissues and smooth muscles) is demonstrated by different amino-terminal peptides which, however, are closely related. The actins from the sarcomeric muscles (e.g. skeletal muscle and heart muscle) show unique but extremely similar amino-terminal peptides. A limited comparison of bovine and avian actins involving smooth and skeletal muscles emphasizes that among higher vertebrates actin divergence involves tissue rather than species specificity.For the lower eukaryotic organism Physarum polycephalum a single actin amino-terminal peptide is found, indicating that only one actin species is present during the plasmodial stage. The amino acid sequence of this peptide although unique reveals a high degree of homology with the corresponding mammalian cytoplasmic actin peptides.Different actin extraction and purification procedures have been compared by the relative yields of the different amino-terminal peptides. The results indicate that the various actin species obtained by the current purification procedures are a true reflection of the actual actins present in the tissue. In addition we compare the resolution provided by either isoelectric focusing analysis of different actins or by the protein chemical characterization of the amino-terminal peptides of different actins. We show that the latter procedure is more suitable for recording changes in actin expression during evolution and differentiation.     

Comparative studies on actins from various sources. Fragments of actins from Ascaris muscle cleaved at cysteinyl residues in comparison with those of other actins.

Pure actins were obtained from various animal muscles: Vertebrata (skeletal, smooth, and cardiac muscles), Prochordata (smooth muscle), Nematoda (obliquely striated muscle), and Mollusca (striated, smooth and obliquely striated muscles). These actins were all identical in apparent molecular weight on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. All actins treated with 2-nitro-5-thiocyanobenzoic acid yielded four major (about 33,000, 26,000, 24,000, and less than 10,000 daltons) and three minor (22,000, 17,000, and 10,000 daltons) bands in addition to intact actin on gel electrophoresis. The results suggest that all actins from various types of muscle have cysteinyl residues at similar positions on the primary structure.     

Insect muscle actins differ distinctly from invertebrate and vertebrate cytoplasmic actins

Summary Invertebrate actins resemble vertebrate cytoplasmic actins, and the distinction between muscle and cytoplasmic actins in invertebrates is not well established as for vertebrate actins. However, Bombyx and Drosophila have actin genes specifically expressed in muscles. To investigate if the distinction between muscle and cytoplasmic actins evidenced by gene expression analysis is related to the sequence of corresponding genes, we compare the sequences of actin genes of these two insect species and of other Metazoa. We find that insect muscle actins form a family of related proteins characterized by about 10 muscle-specific amino acids. Insect muscle actins have clearly diverged from cytoplasmic actins and form a monophyletic group emerging from a cluster of closely related proteins including insect and vertebrate cytoplasmic actins and actins of mollusc, cestode, and nematode. We propose that muscle-specific actin genes have appeared independently at least twice during the evolution of animals: insect muscle actin genes have emerged from an ancestral cytoplasmic actin gene within the arthropod phylum, whereas vertebrate muscle actin genes evolved within the chordate lineage as previously described.Offprint requests to.: N. Mounier     

Actin amino-acid sequences. Comparison of actins from calf thymus, bovine brain, and SV40-transformed mouse 3T3 cells with rabbit skeletal muscle actin.

Actin was purified from calf thymus, bovine brain and SV40-transformed mouse 3T3 cells grown in tissue culture. Isoelectric focusing analysis showed the presence of the two actin polypeptides beta and gamma typical for non-muscle actins in all three actins. Tryptic and thermolytic peptides accounting for the complete amino-acid sequence of the cytoplasmic actins were separated and isolated by preparative fingerprint techniques. All peptides were characterized by amino-acid analysis and compared with the corresponding peptides from rabbit skeletal muscle actin. Peptides which differed in amino-acid composition from the corresponding skeletal muscle actin peptides were subjected to sequence analysis in order to localize the amino-acid replacement. The results obtained show that all three mammalian cytoplasmic actins studied contain the same amino-acid exchanges indicating that mammalian cytoplasmic actins are very similar if not identical in amino-acid sequence. The presence of two different isoelectric species beta and gamma in cytoplasmic actins from higher vertebrates is acccounted for by the isolation of two very similar but not identical amino-terminal peptides in all three actin preparations. The nature of the amino-acid replacements in these two peptides not only accounts for the different isoelectric forms but also shows that beta and gamma cytoplasmic actins are the products of two different structural genes expressed in the same cell. The total number of amino-acid replacements so far detected in the comparison of these cytoplasmic actins and skeletal muscle actin is 25 for the beta chain and 24 for the gamma chain. With the exception of the amino-terminal three or four residues, which are responsible for the isoelectric differences, the replacements do not involve charged amino acids. The exchanges are not randomly distributed. No replacements were detected in regions 18--75 and 299--356 while the regions between residues 2--17 and 259--298 show a high number of replacements. In addition documentation for a few minor revisions of the amino acid sequence of rabbit skeletal muscle actin is provided.     

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.     

Antigenic reactivity of a monoclonal antibody against Amoeba proteus actin

The reactivity of a monoclonal antibody against actin of Amoeba proteus with actins from other sources was examined. The monoclonal antibody cross-reacted with actins from vertebrate muscles, human erythrocytes, and Acanthamoeba castellanii, but it did not react with Naegleria gruberi actin. The amoeba actin was resolved into 3 bands with isoelectric points of 5.96, 6.03 and 6.10 in electrofocusing gels and they corresponded to 3 peptide spots reacting with the antibody on 2-dimensional immunoblots.     

Actins from plant and animal sources tend not to form heteropolymers in vitro and function differently in plant cells

Summary. Pollen and skeletal muscle actins were purified and labeled with fluorescent dyes that have different emission wavelengths. Observation by electron microscopy shows that the fluorescent actins are capable to polymerize into filamentous actin in vitro, bind to myosin S-1 fragments, and have a critical concentration similar to unlabeled actin, indicating that they are functionally active. The globular actins from two sources were mixed and polymerized by the addition of ATP and salts. The copolymerization experiment shows that when excited by light of the appropriate wavelength, both red actin filaments (pollen actin) and green actin filaments (muscle actin) can be visualized under the microscope, but no filaments exhibiting both green and red colors are detected. Furthermore, coprecipitations of labeled pollen actin with unlabeled pollen and skeletal muscle actin were performed. Measurements of fluorescent intensity show that the amount of labeled pollen actin precipitating with pollen actin was much higher than that with skeletal muscle actin, indicating that pollen and muscle actin tend not to form heteropolymers. Injection of labeled pollen actin into living stamen hair cells results in the formation of normal actin filaments in transvacuolar strands and the cortical cytoplasm. In contrast, labeled skeletal muscle actin has detrimental effects on the cellular architecture. The results from coinjection of the actin-disrupting reagent cytochalasin D with pollen actin show that overexpression of pollen actin prolongs the displacement of the nucleus and facilitates the recovery of the nuclear position, actin filament architecture, and transvacuolar strands. However, muscle actin perturbs actin filaments when injected into stamen hair cells. Moreover, nuclear displacement occurs more rapidly when cytochalasin D and muscle actin are coinjected into the cell. It is concluded that actins from plant and animal sources behave differently in vitro and in vivo and that they are functionally not interchangeable.     

Sodium dodecyl sulfate gel electrophoresis studies of connectin-like high molecular weight proteins of various types of vertebrate and invertebrate muscles

Using an SDS gel electrophoresis method, connectin, very high molecular weight (approximately 10(6) dalton) protein, was detected in an SDS extract of whole tissues of various types of muscles of vertebrates and invertebrates. Connectin bands were clearly recognized in all the types of striated muscles (skeletal and cardiac) of the vertebrates examined: rabbit, chicken, turtle, snake, newt, frog, and fish. This was also the case with skeletal muscle of prochordate, Amphioxus. In invertebrates, the situation was much complicated. Connectin-like protein bands were detected in C. elegans (nematode), but not in earthworm (annelid). Smaller sizes of proteins (approximately 10(6)) were faintly found in molluscan adductor muscles. In arthropods, connectin-like proteins were clearly detected in some muscles (e.g., claw muscles of crab and crayfish; leg muscles of several insects) but not at all in other muscles (e.g., tail muscles of crayfish and shrimp; thoracic muscles of some insects). These peculiar observations might be related to the presence of such specific elastic proteins as projectin in honeybee flight muscle. The present study has revealed that connectin is an elastic protein of vertebrate striated muscle, skeletal and cardiac muscles.     

Subcellular sorting of isoactins: selective association of gamma actin with skeletal muscle mitochondria

We describe two subpopulations of actin antibodies isolated by affinity chromatography from a polyclonal antibody to chicken gizzard actin. One subpopulation recognizes gamma actins from smooth muscle and nonmuscle cells, but does not recognize alpha actin from skeletal muscle. The other subpopulation recognizes determinants that are common to alpha actin from skeletal muscle and the two gamma actin isotypes. Neither antibody recognizes cytoplasmic beta actin. Both antibodies recognize only actins or molecules with determinants that are also present in actins. By immunofluorescence we found that the anti-gamma actin colocalizes with mitochondria in fibers of mouse diaphragm, and that it does not bind detectably to the 1 bands of sarcomeres. The antibody that recognizes both alpha and gamma actins stains 1 bands intensely, as expected. We interpret these observations as preliminary evidence for selective association of gamma actin with skeletal muscle mitochondria and, more broadly, as evidence for subcellular sorting of isoactins.     

Correlation between polymerizability and conformation in scallop beta-like actin and rabbit skeletal muscle alpha-actin.

In order to investigate the structural basis for functional differences among actin isoforms, we have compared the polymerization properties and conformations of scallop adductor muscle beta-like actin and rabbit skeletal muscle alpha-actin. Polymerization of scallop Ca(2+)-actin was slower than that of skeletal muscle Ca(2+)-actin. Cleavage of the actin polypeptide chain between Gly-42 and Val-43 with Escherichia coli protease ECP 32 impaired the polymerization of scallop Mg(2+)-actin to a greater extent than skeletal muscle Mg(2+)-actin. When monomeric scallop and skeletal muscle Ca(2+)-actins were subjected to limited proteolysis with trypsin, subtilisin, or ECP 32, no differences in the conformation of actin subdomain 2 were detected. At the same time, local differences in the conformations of scallop and skeletal muscle actin subdomains 1 were revealed as intrinsic fluorescence differences. Replacement of tightly bound Ca(2+) with Mg(2+) resulted in more extensive proteolysis of segment 61-69 of scallop actin than in the case of skeletal muscle actin. Furthermore, segment 61-69 was more accessible to proteolysis with subtilisin in polymerized scallop Ca(2+)-actin than in polymerized skeletal muscle Ca(2+)-actin, indicating that, in the polymeric form, the nucleotide-containing cleft is in a more open conformation in beta-like scallop actin than in skeletal muscle alpha-actin. We suggest that this difference between scallop and skeletal muscle actins is due to a less efficient shift of scallop actin subdomain 2 to the position it has in the polymer. The possible consequences of amino acid substitutions in actin subdomain 1 in the allosteric regulation of the actin cleft, and hence in the different stabilities of polymers formed by different actins, are discussed.     

Identical distribution of fluorescently labeled brain and muscle actins in living cardiac fibroblasts and myocytes

We have investigated whether living muscle and nonmuscle cells can discriminate between microinjected muscle and nonmuscle actins. Muscle actin purified from rabbit back and leg muscles and labeled with fluorescein isothiocyanate, and nonmuscle actin purified from lamb brain and labeled with lissamine rhodamine B sulfonyl chloride, were co-injected into chick embryonic cardiac myocytes and fibroblasts. When fluorescence images of the two actins were compared using filter sets selective for either fluorescein isothiocyanate or lissamine rhodamine B sulfonyl chloride, essentially identical patterns of distribution were detected in both muscle and nonmuscle cells. In particular, we found no structure that, at this level of resolution, shows preferential binding of muscle or nonmuscle actin. In fibroblasts, both actins are associated primarily with stress fibers and ruffles. In myocytes, both actins are localized in sarcomeres. In addition, the distribution of structures containing microinjected actins is similar to that of structure containing endogenous F-actin, as revealed by staining with fluorescent phalloidin or phallacidin. Our results suggest that, at least under these experimental conditions, actin-binding sites in muscle and nonmuscle cells do not discriminate among different forms of actins.     

Tetrahymena actin. Cloning and sequencing of the Tetrahymena actin gene and identification of its gene product

Actin is ubiquitous in eukaryotes, nevertheless its existence has not yet been clearly proven in Tetrahymena. Here we report the cloning and sequencing of an actin gene from the genomic library of Tetrahymena pyriformis using a Dictyostelium actin gene as a probe. The Tetrahymena actin gene has no intron. The predicted actin is composed of 375 amino acids like other actins and its molecular weight is estimated as 41,906. Both T. pyriformis and T. thermophila possess a single species of actin genes which differ in their restriction patterns. Northern hybridization analysis revealed that the actin gene was actively transcribed in vivo. To detect the gene product, we synthesized an N-terminal peptide of the deduced sequence and prepared its antibody. Using an immunoblotting technique, we identified Tetrahymena actin on a two-dimensional gel electrophoretic plate. The actin spot migrated near an added spot of rabbit skeletal muscle actin, but clearly differed from the latter in its isoelectric point and apparent molecular weight. The primary structure of Tetrahymena actin shares about 75% homology equally with those of other representative actins. This value is extremely low as a homology rate between known actins. Tetrahymena actin diverges not only in relatively variable regions of other actins, but also in relatively constant regions. The hydrophilicity levels of two regions (residues 190 to 200 and residues 225 to 235) are also quite different between the Tetrahymena actin and skeletal muscle actin. Thus, we conclude that actin is present in Tetrahymena, but it is one of the most unique actins among the actins known hereto.     

Brain Actin Synthesized In Vitro Undergoes Two Different and Sequential Posttranslational Modifications

Abstract: We have studied the posttranslational processing of actin molecules synthesized in a cell-free system. The results of these experiments indicate that during the in vivo synthesis of the actins from rat brain the primary translational products undergo two different and sequential posttranslational modifications. These modifications are accompanied by slight changes in the isoelectric points of the proteins and can be detected by isoelectric focusing analysis. The same posttranslational modifications can be detected during the in vitro synthesis of chick embryo skeletal muscle actin. The evidence presented suggest that the first posttranslational modification may correspond to the methylation of a histidine residue, and the second modification most likely corresponds to the acetylation of the NH₂-terminal amino acid residues of actin molecules.     

Switching of actin isoforms in skeletal muscle differentiation using mouse ES cells

Among six actin isoforms, α-skeletal and α-cardiac actins have similar amino acid components and are highly conserved. Although skeletal muscles essentially express α-skeletal actins in the adult tissue, α-cardiac isoform actin is prominent in the embryonic muscle tissue. Switching of actin isoforms from α-cardiac to α-skeletal actin occurs during skeletal muscle differentiation. The cardiac type α-actin is expressed in the regeneration and patho-physiological states of the skeletal muscles as well. In the present study, we demonstrate the morphological switching of α-type actin isoforms from α-cardiac to α-skeletal actin in vitro using mouse ES cells for the first time. Immunofluorescent double staining with two specific antibodies revealed that α-cardiac actin appeared first in myoblasts. After cell fusion to form myotubes, the cardiac type actin decreased and α-skeletal actin conversely increased. Finally, the α-skeletal isoform remained as a main actin component in the fully mature skeletal muscle fibers. The exchange of isoforms is not directly linked to the sarcomere formation. As a result, ES cells provide a useful in vitro system for exploring skeletal muscle differentiation.     

Evolution of the chordate muscle actin gene

Summary The ascidians Styela plicata, S. clava, and Mogula citrina are urochordates. The larvae of urochordates are considered to morphologically resemble the ancestral vertebrate. We asked whether larval and adult ascidian muscle actin sequences are nonmusclelike as in lower invertebrates, musclelike as in vertebrates, or possess characteristics of both. Nonmuscle and muscle actin cDNA clones from S. plicata were sequenced. Based on 27 diagnostic amino acids, which distinguish vertebrate muscle actin from other actins, we found that the deduced protein sequences of ascidian muscle actins exhibit similarities to both invertebrate and vertebrate muscle actins. A comparison to muscle actins from different vertebrate and invertebrate phylogenetic groups suggested that the urochordate muscle actins represent a transition from a nonmusclelike sequence to a vertebrate musclelike sequence. The ascidian adult muscle actin is more similar to skeletal actin and the larval muscle actin is more similar to cardiac actin, which indicates that the divergence of the skeletal and cardiac isoforms occurred before the emergence of urochordates. The muscle actin gene may be a powerful probe for investigating the chordate lineage. Offprint requests to: C.R. Tomlinson     

The isolation and characterization of actin from porcine brain

Highly purified porcine brain actin has been prepared by a procedure involving anion-exchange chromatography, polymerization-depolymerization, and gel filtration. Electrophoresis of purified brain actin on polyacrylamide gels in the presence of sodium dodecyl sulfate shows a single protein band corresponding to more than 95% of the applied protein and migrating with the relative mobility of skeletal muscle actin. The amino acid composition of brain actin is similar but not identical to that of rabbit skeletal muscle actin. Brain actin activates the low ionic strength Mg²⁺-ATPase of skeletal muscle myosin to the same extent that skeletal muscle actin potentiates the muscle ATPase. Although similar to its skeletal muscle counterpart, brain actin is distinctly different. Isoelectric focusing experiments indicate that brain actin consists of at least two species, each of which is more basic than the α-species of skeletal muscle actin. The polymerization of brain actin was followed by viscometry and sedimentation techniques as a function of protein concentration, temperature, and ionic conditions. The critical actin concentrations of both brain and skeletal muscle actins polymerized at low ionic strength in the presence of 2.0 mm MgCl₂ are similar and show little dependence upon temperature. When polymerized in the presence of 0. 1 m KCl, brain actin has a critical actin concentration that is higher and more dependent upon temperature than the corresponding critical concentration of skeletal muscle actin.     

Localization of the charge differences in the actins of rabbit skeletal muscle and chicken gizzard by two-dimensional gel electrophoretic analysis of tryptic fragments.

Partial tryptic cleavage products of pure actin from rabbit skeletal muscle and chicken gizzard are compared by two-dimensional electrophoresis in polyacrylamide gels with respect to isoelectric point and molecular weight. While the intact polypeptides (Mr 42,000) have different isoelectric points, two large cleavage products (Mr 35,000) generated from both both actin species have identical isoelectric points and identical molecular weights. These relatively trypsin-resistant cleavage products are presumably identical to the known "core actin" fragments which lack the aminoterminal region of the polypeptide chain. Therefore the differences that are responsible for the different isoelectric points of rabbit skeletal muscle actin and chicken gizzard actin seem to be restricted to the aminoterminal part of the actin polypeptide chains as was proposed on the basis of partial amino acid sequence data.     

Eyestalk ablation has little effect on actin and myosin heavy chain gene expression in adult lobster skeletal muscles

The organization of skeletal muscles in decapod crustaceans is significantly altered during molting and development. Prior to molting, the claw muscles atrophy dramatically, facilitating their removal from the base of the claw. During development, lobster claw muscles exhibit fiber switching over several molt cycles. Such processes may be influenced by the secretion of steroid molting hormones, known collectively as ecdysteroids. To assay the effects of these hormones, we used eyestalk ablation to trigger an elevation of circulating ecdysteroids and then quantified myofibrillar mRNA levels with real-time PCR and myofibrillar protein levels by SDS-PAGE. Levels of myosin heavy chain (MHC) and actin proteins and the mRNA encoding them were largely unaffected by eyestalk ablation, but in muscles from intact animals, myofibrillar gene expression was modestly elevated in premolt and postmolt animals. In contrast, polyubiquitin mRNA was significantly elevated (about 2-fold) in claw muscles from eyestalk-ablated animals with elevated circulating ecdysteroids. Moreover, patterns of MHC and actin gene expression are significantly different among slow and fast claw muscles. Consistent with these patterns, the three muscle types differed in the relative amounts of myosin heavy chain and actin proteins. All three muscles also co-expressed fast and slow myosin isoforms, even in fibers that are generally regarded as exclusively fast or slow. These results are consistent with other recent data demonstrating co-expression of myosin isoforms in lobster muscles.