Purification and characterization of an alpha-actinin-like protein from porcine kidney

An alpha-actinin-like protein was partially purified from the Triton-insoluble cytoskeleton of porcine kidney by 0.6 M MgCl2 treatment, ammonium sulfate fractionation, DEAE-cellulose chromatography and hydroxyapatite chromatography. Apparent purity of the kidney protein was approximately 90% by quantitative densitometry of Coomassie-stained polyacrylamide gels. The kidney alpha-actinin-like protein is very similar to muscle alpha-actinins by the following criteria: (1) both kidney protein and muscle alpha-actinins bind to F-actin at a similar ratio; (2) both proteins demonstrate no difference in the actomyosin turbidity assay and the ATPase assay for alpha-actinin activity; (3) both native proteins contain a large core of identical molecular weight resistant to trypsin; (4) on two-dimensional gels, both kidney protein and muscle alpha-actinins have similar isoelectric points of 5.9-6.1. However, kidney alpha-actinin-like protein is not identical in every respect with muscle alpha-actinins. Electrophoretic mobility of the kidney protein is slightly greater than that of chicken gizzard alpha-actinin and is identical to that of a component of chicken skeletal muscle alpha-actinin. One-dimensional peptide mappings of the kidney protein and muscle alpha-actinins were significantly different from each other. The interaction between kidney alpha-actinin-like protein and F-actin is sensitive to Ca2+. Similar Ca2+-sensitivity was observed with other non-muscle cell alpha-actinins.     

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).     

Molecular evolution and structure of alpha-actinin

The N-terminal actin-binding domain of alpha-actinin is connected to the C-terminal EF-hands by a rod domain. Because of its ability to form dimers, alpha-actinin can cross-link actin filaments in muscle cells as well as in nonmuscle cells. In the prototypic alpha-actinins, the rod domain contains four triple helical bundles, or so-called spectrin repeats. We have found some atypical alpha-actinins in early diverging organisms, such as protozoa and yeast, where the rod domain contains one and two spectrin repeats, respectively. This implies that the four repeats present in modern alpha-actinins arose after two consecutive intragenic duplications from an alpha-actinin with a single repeat. Further, the evolutionary gene tree of alpha-actinins shows that the appearance of four distinct alpha-actinin isoforms may have occurred after the vertebrate-invertebrate split. The topology of the tree lends support to the hypothesis that two rounds (2R) of genome duplication occurred early in the vertebrate radiation. The phylogeny also considers these atypical isoforms as the most basal to alpha-actinins of vertebrates and other eukaryotes. The analysis also positioned alpha-actinin of the fungi Encephalitozoo cuniculi close to the protozoa, supporting the suggestion that microsporidia are early eukaryotes. Because alpha-actinin is considered the basal member of the spectrin family, our studies will improve the understanding of the origin and evolution of this superfamily.     

Polymorphism of alpha-actinin. Electrophoretic and immunological studies of rabbit skeletal muscle alpha-actinins

Heterogeneity of alpha-actinins from rabbit skeletal muscles was studied. Polyacrylamide gel electrophoresis in the presence and absence of sodium dodecyl sulfate has made it possible to distinguish two closely related alpha-actinins from rabbit fast, white muscle. One isoprotein (designated type I alpha-actinin) appears to be preferentially located in the psoas muscle, while the other (designated type II alpha-actinin) appears to be preferentially located in the longissimus dorsi muscle. Electrophoretic analyses have further shown that the two isoproteins are present as mixtures in most rabbit white, fast-twitch muscles. A standard polyacrylamide gel--sodium dodecyl sulfate/polyacrylamide gel sequential electrophoretic procedure was developed to resolve the different alpha-actinin dimers and to determine their subunit compositions. By this technique, both type I and type II alpha-actinins appeared to be homodimers. No heterodimeric species of alpha-actinin were detected. alpha-Actinin of red, slow-twitch muscles was similar to type II alpha-actinin of fast, white muscle on one-dimensional and two dimensional gels. However, slow, red muscle alpha-actinin was significantly different from fast, white muscle alpha-actinins in terms of one-dimensional peptide mapping and immunological cross-reactivity.     

Drosophila alpha-actinin in ovarian follicle cells is regulated by EGFR and Dpp signalling and required for cytoskeletal remodelling

alpha-Actinin is an evolutionarily conserved actin filament crosslinking protein with functions in both muscle and non-muscle cells. In non-muscle cells, interactions between alpha-actinin and its many binding partners regulate cell adhesion and motility. In Drosophila, one non-muscle and two muscle-specific alpha-actinin isoforms are produced by alternative splicing of a single gene. In wild-type ovaries, alpha-actinin is ubiquitously expressed. The non-muscle alpha-actinin mutant Actn(Delta233), which is viable and fertile, lacks alpha-actinin expression in ovarian germline cells, while somatic follicle cells express alpha-actinin at late oogenesis. Here we show that this latter population of alpha-actinin, termed FC-alpha-actinin, is absent from the dorsoanterior follicle cells, and we present evidence that this is the result of a negative regulation by combined Epidermal growth factor receptor (EGFR) and Decapentaplegic signalling. Furthermore, EGFR signalling increased the F-actin bundling activity of ectopically expressed muscle-specific alpha-actinin. We also describe a novel morphogenetic event in the follicle cells that occurs during egg elongation. This event involves a transient repolarisation of the basal actin fibres and the assembly of a posterior beta-integrin-dependent adhesion site accumulating alpha-actinin and Enabled. Clonal analysis using Actn null alleles demonstrated that although alpha-actinin was not necessary for actin fibre formation or maintenance, the cytoskeletal remodelling was perturbed, and Enabled did not localise in the posterior adhesion site. Nevertheless, epithelial morphogenesis proceeded normally. This work provides the first evidence that alpha-actinin is involved in the organisation of the cytoskeleton in a non-muscle tissue in Drosophila.     

Alpha-actinin expression during avian myogenesis in vivo. Evidence for the existence of an embryo-specific isoform of alpha-actinin

The isoforms of skeletal muscle alpha-actinin present during chick embryogenesis were analyzed by two-dimensional electrophoresis in combination with the immunoblot technique. Chicken embryonic muscles at 8-15 days contain an embryo-specific isoform of alpha-actinin. The embryonic alpha-actinin isoform has a molecular mass of 112 kDa and an isoelectric point of 5.8, whereas the values for the adult isoform of alpha-actinin were 100 kDa and 5.85, respectively. To characterize the two classes of alpha-actinin polypeptides we have compared the two proteins by 125I-labeled two-dimensional peptide mapping. The embryonic isoform is highly similar to, but exhibited extensive peptide differences to, the adult isoform of alpha-actinin. The developmental sequence of the expression of the alpha-actinins was also studied. In extracts of skeletal muscle from 8-10-day-old embryos, only the embryonic isoform was detected. In extracts from 15-day-old embryos, both the embryonic and the adult isoforms coexisted. However by 21 days, the embryonic isoform had disappeared and only the adult isoform was detected. These data suggested that the embryonic and the adult isoform of alpha-actinins are distinct proteins and that during skeletal myogenesis in ovo one class of alpha-actinin is replaced by a new class of alpha-actinin polypeptides, and that the latter is maintained into adulthood.     

The interaction of actin with dystrophin

Proton NMR spectroscopy of synthetic peptides corresponding to defined regions of human dystrophin has been employed to study the interaction with F-actin. No evidence of interaction with a C-terminal region corresponding to amino acid residues 3429-3440 was obtained. F-actin restricted the mobility of residues 19-27 in a synthetic peptide corresponding to residues 10-32. This suggests that this is a site of F-actin interaction in the intact dystrophin molecule. Identical sequences to that of residues 19-22 in dystrophin, namely Lys-Thr-Phe-Thr are also present in the N-terminal regions of the alpha-actinins implying this is also a site of F-actin interaction with alpha-actinin.     

The α-Actinin Gene Family: A Revised Classification

The sequencing of a genome is the first stage of its complete characterization. Subsequent work seeks to utilize available sequence data to gain a better understanding of the genes which are found within a genome. Gene families comprise large portions of the genomes of higher vertebrates, and the available genomic data allow for a reappraisal of gene family evolution. This reappraisal will clarify relatedness within and between gene families. One such family, the alpha-actinin gene family, is part of the spectrin superfamily. There are four known loci, which encode alpha-actinins 1, 2, 3, and 4. Of the eight domains in alpha-actinin, the actin-binding domain is the most highly conserved. Here we present evidence gained through phylogenetic analyses of the highly conserved actin-binding domain that alpha-actinin 2 was the first of the four alpha-actinins to arise by gene duplication, followed by the divergence of alpha-actinin 3 and then alpha-actinins 1 and 4. Resolution of the gene tree for this gene family has allowed us to reclassify several alpha-actinins which were previously given names inconsistent with the most widely accepted nomenclature for this gene family. This reclassification clarifies previous discrepancies in the public databases as well as in the literature, thus eliminating confusion caused by continued misclassification of members of the alpha-actinin gene family. In addition, the topology found for this gene family undermines the 2R hypothesis theory of two rounds of genome duplication early in vertebrate evolution.     

Drosophila non-muscle alpha-actinin is localized in nurse cell actin bundles and ring canals, but is not required for fertility

The single copy Drosophila alpha-actinin gene is alternatively spliced to generate three different isoforms that are expressed in larval muscle, adult muscle and non-muscle cells, respectively. We have generated novel alpha-actinin alleles, which specifically remove the non-muscle isoform. Homozygous mutant flies are viable and fertile with no obvious defects. Using a monoclonal antibody that recognizes all three splice variants, we compared alpha-actinin distribution in wild type and mutant embryos and ovaries. We found that non-muscle alpha-actinin was present in young embryos and in the embryonic central nervous system. In ovaries, non-muscle alpha-actinin was localized in the nurse cell subcortical cytoskeleton, cytoplasmic actin cables and ring canals. In the mutant, alpha-actinin expression remained in muscle tissues, but also in a subpopulation of epithelial cells in both embryos and ovaries. This suggests that various populations of non-muscle cells regulate alpha-actinin expression in different ways. We also show that ectopically expressed adult muscle-specific alpha-actinin localizes to all F-actin containing structures in the nurse cells in the absence of endogenous non-muscle alpha-actinin.     

Differential expression and distribution of chicken skeletal- and smooth-muscle-type alpha-actinins during myogenesis in culture

Antibodies to chicken fast skeletal muscle (pectoralis) alpha-actinin and to smooth muscle (gizzard) alpha-actinin were absorbed with opposite antigens by affinity chromatography, and four antibody fractions were thus obtained: common antibodies reactive with both pectoralis and gizzard alpha-actinins ([C]anti-P alpha-An and [C]anti-G alpha-An), antibody specifically reactive with pectoralis alpha-actinin ([S]anti-P alpha-An), and antibody specifically reactive with gizzard alpha-actinin ([S]anti-G alpha-An). In indirect immunofluorescence microscopy, (C)anti-P alpha-An, (S)anti-P alpha-An, and (C)anti-G alpha- An stained Z bands of skeletal muscle myofibrils, whereas (S)anti-G alpha-An did not. Although (S)anti-G alpha-An and two common antibodies stained smooth muscle cells, (S)anti-P alpha-An did not. We used (S)anti-P alpha-An and (S)anti-G alpha-An for immunofluorescence microscopy to investigate the expression and distribution of skeletal- and smooth-muscle-type alpha-actinins during myogenesis of cultured skeletal muscle cells. Skeletal-muscle-type alpha-actinin was found to be absent from myogenic cells before fusion but present in them after fusion, restricted to Z bodies or Z bands. Smooth-muscle-type alpha- actinin was present diffusely in the cytoplasm and on membrane- associated structures of mononucleated and fused myoblasts, and then confined to membrane-associated structures of myotubes. Immunoblotting and peptide mapping by limited proteolysis support the above results that skeletal-muscle-type alpha-actinin appears at the onset of fusion and that smooth-muscle-type alpha-actinin persists throughout the myogenesis. These results indicate (a) that the timing of expression of skeletal-muscle-type alpha-actinin is under regulation coordination with other major skeletal muscle proteins; (b) that, with respect to expression and distribution, skeletal-muscle-type alpha-actinin is closely related to alpha-actin, whereas smooth-muscle-type alpha- actinin is to gamma- and beta-actins; and (c) that skeletal- and smooth- muscle-type alpha-actinins have complementary distribution and do not co-exist in situ.     

Primary structure of chicken skeletal muscle and fibroblast alpha-actinins deduced from cDNA sequences

The complete 897-amino-acid sequence of chicken skeletal muscle alpha-actinin and the 856-amino-acid sequence (97% of the entire sequence) of chicken fibroblast alpha-actinin have been determined by cloning and sequencing the cDNAs. Genomic Southern analysis with the cDNA sequences shows that skeletal and fibroblast alpha-actinins are encoded by separate single-copy genes. RNA blot analyzes show that the skeletal alpha-actinin gene is expressed in the pectoralis muscle and that the fibroblast gene is expressed in the gizzard smooth muscle as well as in the fibroblast. The deduced skeletal alpha-actinin molecule has a calculated Mr of 104 x 10(3), and each alpha-actinin can be divided into three domains: (1) the NH2-terminal highly conserved actin-binding domain, which shows similarity to the product of the Duchenne's muscular dystrophy locus; (2) the middle rod-shaped dimer-forming domain, which contains the spectrin-type repeat units; and (3) the COOH-terminal two EF-hand consensus regions. Comparison of the skeletal alpha-actinin sequence with the fibroblast and smooth muscle alpha-actinin sequences demonstrated that the EF-hand structure was conserved in all of these alpha-actinin sequences, despite the reported variability of the Ca2+ sensitivities of the actin-gelation by various alpha-actinin isoforms.     

Analysis of the actin-binding domain of alpha-actinin by mutagenesis and demonstration that dystrophin contains a functionally homologous domain

To define the actin-binding site within the NH2-terminal domain (residues 1-245) of chick smooth muscle alpha-actinin, we expressed a series of alpha-actinin deletion mutants in monkey Cos cells. Mutant alpha-actinins in which residues 2-19, 217-242, and 196-242 were deleted still retained the ability to target to actin filaments and filament ends, suggesting that the actin-binding site is located within residues 20-195. When a truncated alpha-actinin (residues 1-290) was expressed in Cos cells, the protein localized exclusively to filament ends. This activity was retained by a deletion mutant lacking residues 196-242, confirming that these are not essential for actin binding. The actin-binding site in alpha-actinin was further defined by expressing both wild-type and mutant actin-binding domains as fusion proteins in E. coli. Analysis of the ability of such proteins to bind to F-actin in vitro showed that the binding site was located between residues 108 and 189. Using both in vivo and in vitro assays, we have also shown that the sequence KTFT, which is conserved in several members of the alpha-actinin family of actin-binding proteins (residues 36-39 in the chick smooth muscle protein) is not essential for actin binding. Finally, we have established that the NH2-terminal domain of dystrophin is functionally as well as structurally homologous to that in alpha-actinin. Thus, a chimeric protein containing the NH2-terminal region of dystrophin (residues 1-233) fused to alpha-actinin residues 244-888 localized to actin-containing structures when expressed in Cos cells. Furthermore, an E. coli-expressed fusion protein containing dystrophin residues 1-233 was able to bind to F-actin in vitro.     

A novel nonmuscle alpha-actinin. Purification and characterization of chicken lung alpha-actinin.

Two distinct alpha-actinin-like proteins were detected in chicken lung extract by immunoblot analysis with monoclonal antibodies against alpha-actinin. The mobilities of these proteins on SDS-polyacrylamide gel electrophoresis are very close (approximately 100 kDa). On SDS-polyacrylamide gel electrophoresis in the presence of 6 M urea, however, one of the proteins migrates at 115 kDa and is clearly separated from the other protein (105 kDa). The 115-kDa protein was purified and shown to have at least three unique amino acid sequences which were not detected in other kinds of alpha-actinins: one locates at the extreme NH2-terminal region, and the others locate at the COOH-terminal half region. Immunoblot and proteolytic cleavage analyses revealed that the 115-kDa protein has structural divergence at the COOH-terminal region that includes Ca(2+)-binding EF-hand motifs. Falling-ball viscometric studies showed that although the 115-kDa protein-induced gelation of F-actin is sensitive to Ca2+, the gelation activity of the 115-kDa protein is much higher than that of Ca(2+)-insensitive gizzard alpha-actinin regardless of Ca2+. This indicates that the 115-kDa protein is distinct from other nonmuscle alpha-actinins by its Ca2+ sensitivity.     

Expression of a muscle-type alpha-actinin cDNA clone in non-muscle cells

We have previously isolated a chick smooth muscle-type alpha-actinin cDNA clone (C17) from a chick embryo fibroblast cDNA library. As part of an investigation into a possible role for a muscle isoform of alpha-actinin in non-muscle cells, we have cloned C17 into a eucaryotic expression vector, pKCR3, and examined the distribution of the expressed protein in non-muscle, monkey COS cells. We report here that the muscle isoform of chick alpha-actinin encoded by C17, was found in focal contacts and periodically distributed along actin filaments.     

Brush-border alpha-actinin? Comparison of two proteins of the microvillus core with alpha-actinin by two-dimensional peptide mapping

The bundle of filaments within the intestinal microvillus contains four major polypeptides in addition to actin calmodulin, a 70-kdalton subunit and two polypeptides with molecular masses similar to that of the Z-line component alpha-actinin (95 and 105 kdaltons). Two- dimensional mapping of tryptic peptides indicates that (a) alpha- actinins from chicken skeletal, cardiac, and smooth muscle are similar but not identical proteins and that skeletal alpha-actinin in more similar to the cardiac subunit than to the alpha-actinin from gizzard; (b) the brush-border 95- and 105-kdalton subunits are closely related to each other, but the smaller subunit is not a proteolytic fragment of the 105-kdalton subunit; and (c) although there is considerable peptide overlap between the brush-border subunits and the three alpha-actinins, the peptide maps of the 95- and 105-kdalton proteins are substantially distinct from the various alpha-actinin maps, suggesting that neither brush-border subunit is a bona fide alpha-actinin. Nevertheless, on the basis of peptide mapping criteria alone, one cannot exclude the possibility that the brush-border subunits are "alpha-actinin-like." However, there is no immunological cross-reactivity between the brush- border subunits and alpha-actinins, using antibodies prepared against gizzard alpha actinin.     

A gene for speed? The evolution and function of α‐actinin‐3

The alpha-actinins are an ancient family of actin-binding proteins that play structural and regulatory roles in cytoskeletal organisation and muscle contraction. alpha-actinin-3 is the most-highly specialised of the four mammalian alpha-actinins, with its expression restricted largely to fast glycolytic fibres in skeletal muscle. Intriguingly, a significant proportion ( approximately 18%) of the human population is totally deficient in alpha-actinin-3 due to homozygosity for a premature stop codon polymorphism (R577X) in the ACTN3 gene. Recent work in our laboratory has revealed a strong association between R577X genotype and performance in a variety of athletic endeavours. We are currently exploring the function and evolutionary history of the ACTN3 gene and other alpha-actinin family members. The alpha-actinin family provides a fascinating case study in molecular evolution, illustrating phenomena such as functional redundancy in duplicate genes, the evolution of protein function, and the action of natural selection during recent human evolution.     

Cloning, sequencing, and mapping of an alpha-actinin gene from the nematode Caenorhabditis elegans.

The dense-bodies in the body wall muscle of the nematode Caenorhabditis elegans function to anchor the actin thin filaments to the adjacent sarcolemma. One of the major components of the dense-bodies is the actin-binding protein alpha-actinin. To facilitate a genetic analysis of alpha-actinin, we have cloned a cDNA encoding the nematode protein, identified its position on the nematode physical map, and developed a unique PCR based approach to test the position of the cloned gene relative to known genetic deletions. The peptide sequence deduced from the cDNA shows that, apart from a few exceptional regions, the nematode protein shows strong similarity to other known alpha-actinins. Its position on the genetic map shows that none of the known muscle affecting mutations identified in C. elegans are in this alpha-actinin gene. This gene has been given the name atn-1 (alpha-actinin-1).