Nonmuscle Isoforms Research Articles

Caldesmon inhibits nonmuscle cell contractility and interferes with the formation of focal adhesions.

Caldesmon is known to inhibit the ATPase activity of actomyosin in a Ca(2+)-calmodulin-regulated manner. Although a nonmuscle isoform of caldesmon is widely expressed, its functional role has not yet been elucidated. We studied the effects of nonmuscle caldesmon on cellular contractility, actin cytoskeletal organization, and the formation of focal adhesions in fibroblasts. Transient transfection of nonmuscle caldesmon prevents myosin II-dependent cell contractility and induces a decrease in the number and size of tyrosine-phosphorylated focal adhesions. Expression of caldesmon interferes with Rho A-V14-mediated formation of focal adhesions and stress fibers as well as with formation of focal adhesions induced by microtubule disruption. This inhibitory effect depends on the actin- and myosin-binding regions of caldesmon, because a truncated variant lacking both of these regions is inactive. The effects of caldesmon are blocked by the ionophore A23187, thapsigargin, and membrane depolarization, presumably because of the ability of Ca(2+)-calmodulin or Ca(2+)-S100 proteins to antagonize the inhibitory function of caldesmon on actomyosin contraction. These results indicate a role for nonmuscle caldesmon in the physiological regulation of actomyosin contractility and adhesion-dependent signaling and further demonstrate the involvement of contractility in focal adhesion formation.

Mutations in the skeletal muscle alpha-actin gene in patients with actin myopathy and nemaline myopathy.

Muscle contraction results from the force generated between the thin filament protein actin and the thick filament protein myosin, which causes the thick and thin muscle filaments to slide past each other. There are skeletal muscle, cardiac muscle, smooth muscle and non-muscle isoforms of both actin and myosin. Inherited diseases in humans have been associated with defects in cardiac actin (dilated cardiomyopathy and hypertrophic cardiomyopathy), cardiac myosin (hypertrophic cardiomyopathy) and non-muscle myosin (deafness). Here we report that mutations in the human skeletal muscle alpha-actin gene (ACTA1) are associated with two different muscle diseases, 'congenital myopathy with excess of thin myofilaments' (actin myopathy) and nemaline myopathy. Both diseases are characterized by structural abnormalities of the muscle fibres and variable degrees of muscle weakness. We have identified 15 different missense mutations resulting in 14 different amino acid changes. The missense mutations in ACTA1 are distributed throughout all six coding exons, and some involve known functional domains of actin. Approximately half of the patients died within their first year, but two female patients have survived into their thirties and have children. We identified dominant mutations in all but 1 of 14 families, with the missense mutations being single and heterozygous. The only family showing dominant inheritance comprised a 33-year-old affected mother and her two affected and two unaffected children. In another family, the clinically unaffected father is a somatic mosaic for the mutation seen in both of his affected children. We identified recessive mutations in one family in which the two affected siblings had heterozygous mutations in two different exons, one paternally and the other maternally inherited. We also identified de novo mutations in seven sporadic probands for which it was possible to analyse parental DNA.

Two skeletal α-tropomyosin transcripts with distinct 3′UTR have different temporal and spatial patterns of expression in the striated muscle lineages of Xenopus laevis

The Xenopus laevis α-tropomyosin (TM) gene, like its vertebrates counterparts, encodes muscle and non-muscle isoforms through two promoters and alternatively spliced exons. In the present study we describe a cDNA clone (XTM α7) encoding a skeletal muscle isoform of the gene that differs from the previously described skeletal TM transcript (XTM α2) by its 3′UTR sequence. The two skeletal α-TM encoding mRNAs are generated through distinct 3′end processing using different polyA signals and distinct patterns of exon splicing. Using RNAse protection and RNA in situ hybridization, we have analysed the developmental and spatial expression of the two transcripts. Both are expressed in the embryo, but XTM α7 is by far the most prevalent of the two. In contrast, only XTM α2 is expressed in adult striated muscle tissues. In the embryo, the spatial expression of XTM α7 is restricted to the somites whereas XTM α2 is expressed in both somites and embryonic heart.

Expression of the 71 kDa dystrophin isoform (Dp71) evaluated by gene targeting

To investigate the function of the major non-muscle dystrophin isoform, Dp71, we substituted a β-galactosidase (βgal) reporter gene for Dp71 by homologous recombination in embryonic stem cells. Staining for βgal activity in chimeric mice revealed Dp71 promoter activity in glial cells in the CNS, in neurons of the inner nuclear and inner plexiform layers of the retina, and in the kidney tubules and collecting ducts. Our observations demonstrate that Dp71 is widely expressed in the adult CNS (retina, cerebellum, cerebral cortex, ependyma, and choroid) as well as the adult kidney epithelium and suggest a broad function for Dp71 in differentiated tissues.

A Single Human Myosin Light Chain Kinase Gene (MLCK; MYLK)Transcribes Multiple Nonmuscle Isoforms

The myosin light chain kinase (MLCK) gene, a muscle member of the immunoglobulin gene superfamily, yields both smooth muscle and nonmuscle cell isoforms. Both isoforms are known to regulate contractile activity via calcium/calmodulin-dependent myosin light chain phosphorylation. We previously cloned from a human endothelial cell (EC) cDNA library a high-molecular-weight nonmuscle MLCK isoform (EC MLCK (MLCK 1) with an open reading frame that encodes a protein of 1914 amino acids. We now describe four novel nonmuscle MLCK isoforms (MLCK 2, 3a, 3b, and 4) that are the alternatively spliced variants of an mRNA precursor that is transcribed from a single human MLCK gene. The primary structure of the cDNA encoding the nonmuscle MLCK isoform 2 is identical to the previously published human nonmuscle MLCK (MLCK 1) (J. G. N. Garciaet al.,1997,Am. J. Respir. Cell Mol. Biol.16, 489–494) except for a deletion of nucleotides 1428–1634 (D2). The full nucleotide sequence of MLCK isoforms 3a and 3b and partial sequence for MLCK isoform 4 revealed identity to MLCK 1 except for deletions at nucleotides 5081–5233 (MLCK 3a, D3), double deletions of nucleotides 1428–1634 and 5081–5233 (MLCK 3b), and nucleotide deletions 4534–4737 (MLCK 4, D4). Northern blot analysis demonstrated the extended expression pattern of the nonmuscle MLCK isoform(s) in both human adult and human fetal tissues. RT-PCR using primer pairs that were designed to detect specifically nonmuscle MLCK isoforms 2, 3, and 4 deletions (D2, D3, and D4) confirmed expression in both human adult and human fetal tissues (lung, liver, brain, and kidney) and in human endothelial cells (umbilical vein and dermal). Furthermore, relative quantitative expression studies demonstrated that the nonmuscle MLCK isoform 2 is the dominant splice variant expressed in human tissues and cells. Further analysis of the human MLCK gene revealed that the MLCK 2 isoform represents the deletion of an independent exon flanked by 5′ and 3′ neighboring introns of 0.6 and 7.0 kb, respectively. Together these studies demonstrate for the first time that the human MLCK gene yields multiple nonmuscle MLCK isoforms by alternative splicing of its transcribed mRNA precursor with differential distribution of these isoforms in various human tissues and cells.

Polypyrimidine tract binding protein functions as a repressor to regulate alternative splicing of alpha-actinin mutally exclusive exons.

The smooth muscle (SM) and nonmuscle (NM) isoforms of alpha-actinin are produced by mutually exclusive splicing of an upstream NM exon and a downstream SM-specific exon. A rat alpha-actinin genomic clone encompassing the mutually exclusive exons was isolated and sequenced. The SM exon was found to utilize two branch points located 382 and 386 nucleotides (nt) upstream of the 3' splice site, while the NM exon used a single branch point 191 nt upstream. Mutually exclusive splicing arises from the proximity of the SM branch points to the NM 5' splice site, and this steric repression could be relieved in part by the insertion of spacer elements. In addition, the SM exon is repressed in non-SM cells and extracts. In vitro splicing of spacer-containing transcripts could be activated by (i) truncation of the transcript between the SM polypyrimidine tract and exon, (ii) addition of competitor RNAs containing the 3' end of the actinin intron or regulatory sequences from alpha-tropomyosin (TM), and (iii) depletion of the splicing extract by using biotinylated alpha-TM RNAs. A number of lines of evidence point to polypyrimidine tract binding protein (PTB) as the trans-acting factor responsible for repression. PTB was the only nuclear protein observed to cross-link to the actinin RNA, and the ability of various competitor RNAs to activate splicing correlated with their ability to bind PTB. Furthermore, repression of alpha-actinin splicing in the nuclear extracts depleted of PTB by using biotinylated RNA could be specifically restored by the addition of recombinant PTB. Thus, alpha-actinin mutually exclusive splicing is enforced by the unusual location of the SM branch point, while constitutive repression of the SM exon is conferred by regulatory elements between the branch point and 3' splice site and by PTB.

Angiotensin II type 1 receptor antagonist downregulates nonmuscle myosin heavy chains in spontaneously hypertensive rat aorta.

The aim of this study was to clarify the differences between the angiotensin II type 1 (AT1) receptor antagonist and the angiotensin-converting enzyme (ACE) inhibitor on smooth muscle and nonmuscle myosin heavy chain isoforms in aortic smooth muscle cells of Wistar-Kyoto rats and spontaneously hypertensive rats. All 4 myosin heavy chain isoforms are heterogeneously expressed in the smooth muscle cells of the aortic tunica media in 20-week-old rats, and the contractile-type myosin heavy chains are highly expressed in smooth muscle cells of the aortic tunica media compared with the synthetic-type myosin heavy chains. Both the AT1 receptor antagonist and the ACE inhibitor had the same effects on hemodynamics, smooth muscle cell hypertrophy and proliferation, fibrosis, and vascular remodeling in spontaneously hypertensive rats. However, the AT1 receptor antagonist had a more potent effect on the downregulation of the synthetic-type myosin heavy chains than the ACE inhibitor in spontaneously hypertensive rat aortic tunica media. In contrast, these effects of the AT1 receptor antagonist and the ACE inhibitor on hemodynamics, morphology, fibrosis, and expression of myosin heavy chain isoforms in smooth muscle cells of the aortic tunica media were not observed in Wistar-Kyoto rats. Thus, within 6 weeks, the AT1 receptor antagonist might modulate the cellular composition of myosin heavy chain isoforms in smooth muscle cells more efficiently than the ACE inhibitor, without morphological changes in the spontaneously hypertensive rat aorta.

Specific Isoforms of Actin-binding Proteins on Distinct Populations of Golgi-derived Vesicles

Golgi membranes and Golgi-derived vesicles are associated with multiple cytoskeletal proteins and motors, the diversity and distribution of which have not yet been defined. Carrier vesicles were separated from Golgi membranes, using an in vitro budding assay, and different populations of vesicles were separated using sucrose density gradients. Three main populations of vesicles labeled with beta-COP, gamma-adaptin, or p200/myosin II were separated and analyzed for the presence of actin/actin-binding proteins. beta-Actin was bound to Golgi cisternae and to all populations of newly budded vesicles. Centractin was selectively associated with vesicles co-distributing with beta-COP-vesicles, while p200/myosin II (non-muscle myosin IIA) and non-muscle myosin IIB were found on different vesicle populations. Isoforms of the Tm5 tropomyosins were found on selected Golgi-derived vesicles, while other Tm isoforms did not colocalize with Tm5 indicating the association of specialized actin filaments with Golgi-derived vesicles. Golgi-derived vesicles were shown to bind to F-actin polymerized from cytosol with Jasplakinolide. Thus, newly budded, coated vesicles derived from Golgi membranes can bind to actin and are customized for differential interactions with microfilaments by the presence of selective arrays of actin-binding proteins.

Subcutaneous tissue fibroblasts transfected with muscle and nonmuscle actins: A good in vitro model to study fibroblastic cell plasticity.

Cultured fibroblasts develop several biochemical and morphological properties of smooth muscle cells, particularly the expression of alpha-smooth muscle actin, the actin isoform typical of vascular smooth muscle cells. They resemble modified fibroblasts or myofibroblasts observed in granulation tissue during wound repair and in fibrotic situations. We have analysed by immunolabeling the fate of exogenous epitope-tagged actin isoforms by transfection of the corresponding cDNAs into fibroblasts cultured from rat subcutaneous tissue. Tagged muscle actins were efficiently integrated into stress fibers and did not produce obvious changes in cell shape of transfected cells. Transfected nonmuscle actins in contrast changed the morphology and were not or poorly incorporated into stress fibers. These cultured subcutaneous fibroblasts behave similarly to smooth muscle cells when transfected with the same actin encoding cDNAs, indicating another common characteristic of these two cell types in sorting and targeting actin isoforms. Subcutaneous fibroblasts transfected with muscle and nonmuscle actin isoforms provide a good in vitro model to analyze the intracellular sorting of isoactins and to improve our knowledge of myofibroblast characterization and differentiation during tissue repair as well as to understand the relationships between modifications of actin cytoskeleton, adhesion and extracellular matrix proteins.

Unique expression patterns of myosin heavy chain genes in the ductus arteriosus and uterus of rabbits.

In smooth muscle tissue, two smooth muscle myosin heavy chain (MHC) isoforms (SM1, SM2) and two non-muscle MHC isoforms (NMA, NMB) have been identified. The purpose of our study was to clarify whether smooth muscle MHC mRNA expression reflects the physiological and functional state of the muscle. We studied the expression pattern of MHC mRNAs, using the S1-nuclease mapping procedure, in functionally and morphologically changeable organs; the ductus arteriosus (DA) during development (25 and 29 days of gestation, and from 3-day-old neonates) and uteri from virgin, day-10 pregnant (P10) and day-29 pregnant (P29) rabbits. The results demonstrated that SM2 expression was greater in the fetal DA than in the fetal aortic and pulmonary arteries, but that it decreased significantly following closure of DA. In the gravid uterus, SM1 expression was significantly (P<0.05) strong compared to other MHC mRNAs from virgin to P10 rabbits. During pregnancy, NMB expression showed a tendency to increase until P10, and after P10, SM2 expression increased dramatically and NMB expression decreased to give almost a mirror image of the SM2 expression. Smooth muscle type (SM1, SM2) was significantly (P<0.05) strong compared to non-muscle type expression (NMA, NMB) at P29. These data suggest that smooth muscle MHC mRNA, especially SM2 expression reflects the physiological and functional state of the smooth muscle.

Characterization of pI Cln binding proteins: identification of p17 and assessment of the role of acidic domains in mediating protein–protein interactions

pI Cln is a ubiquitous and abundant 27 kDa soluble protein that is localized primarily to the cytoplasm. The protein has been proposed to be a swelling-activated anion channel or a channel regulator. Recent studies, however, have cast significant doubt on these hypotheses, and the function of pI Cln therefore remains unknown. To further characterize the physiological role of pI Cln, we have begun to identify the proteins that bind to it and the amino acid domains that mediate pI Cln protein–protein interactions. Using affinity assays and immunoprecipitation we have identified three proteins in C6 glioma cells with molecular masses of 17 kDa, 29 kDa and 72 kDa that bind selectively to pI Cln. Microsequencing revealed that p17 is the non-muscle isoform of the alkali myosin light chain. pI Cln contains three acidic amino acid domains termed AD1, AD2 and AD3. Mutation of AD1 and/or AD2 had no effect on p17, p29 and p72 binding. However, binding of p72 was lost when four acidic amino acid residues were mutated in AD3, which is located at the carboxy terminus. A truncation peptide containing the last 29 amino acids of pI Cln was able to bind p72 normally. These results indicate that the carboxy terminus is necessary for p72–pI Cln interaction. Based on these and other findings, we propose that pI Cln is a protein responsible for regulating the structure and function of the cytoskeleton, and/or a protein involved in mediating interactions between components of intracellular signal transduction pathways.

Splicing of Two Internal and Four Carboxyl-terminal Alternative Exons in Nonmuscle Tropomyosin 5 Pre-mRNA Is Independently Regulated during Development

Four nonmuscle tropomyosin isoforms have been reported to be produced from the rat Tm5 gene by alternative splicing (Beisel, K. W., and Kennedy, J. E. (1994) Gene (Amst.) 145, 251-256). In order to detect additional isoforms that might be expressed from that gene, we used reverse transcriptase-polymerase chain reaction assays and evaluated the presence of all product combinations of two alternative internal exons (6a and 6b) and four carboxyl-terminal exons (9a, 9b, 9c, and 9d) in developing and adult rat brain. We identified five different combinations for exon 9 (9a + 9b, 9a + 9c, 9a + 9d, 9c, and 9d), and the exon combinations 9a + 9c and 9a + 9d were previously unreported. Each of these combinations existed with both exon 6a and exon 6b. Thus, the rat brain generates at least 10 different isoforms from the Tm5 gene. Northern blot hybridization with alternative exon-specific probes revealed that these isoforms were also expressed in a number of different adult rat tissues, although some exons are preferentially expressed in particular tissues. Studies of regulation of the 10 different Tm5 isoform mRNAs during rat brain development indicated that no two isoforms are coordinately accumulated. Furthermore, there is a developmental switch in the use of exon 6a to exon 6b from embryonic to adult isoforms. TM5 protein isoforms show a differential localization in the adult cerebellum.

A sea urchin gene encoding dystrophin-related proteins.

The gene which is defective in Duchenne muscular dystrophy (DMD) is the largest known gene. The product of the gene in muscle, dystrophin, is a 427 kDa protein. The same gene encodes at least six additional products: two non-muscle dystrophin isoforms transcribed from promoters located in the 5'-end region of the gene and four smaller proteins transcribed from internal promoters located further downstream. Several other genes, encoding evolutionarily related proteins, have been identified. These include a structurally very similar gene in vertebrates encoding utrophin (DRP1), which is closely related to dystrophin, and a number of small and simple genes in vertebrates or invertebrates encoding proteins similar to some of the small products of the DMD gene. We have isolated a sea urchin gene showing very strong sequence and structural homology with the DMD and utrophin genes. Sequence and intron/exon structure similarities suggest that this gene is related to a precursor of both the DMD gene and the gene encoding utrophin. The sea urchin gene has the unique complex structure of the DMD gene. There is at least one, and possibly more, product(s) transcribed from internal promoters, as well as a large product of >300 kDa containing at least three of the four major domains of dystrophin. The small product seems to be evolutionarily related to Dp116, one of the small products of the human DMD gene. Partial characterization of this gene helped us to construct an evolutionary tree connecting the vertebrate dystrophin gene family with related genes in invertebrates. The constructed evolutionary tree also implies that the vertebrate small and simple structured gene encoding a Dp71-like protein, called DRP2 , evolved from the dystrophin/utrophin ancestral large and complex gene by a duplication of only a small part of the gene.

Exchangeability of actin in cardiac myocytes and fibroblasts as determined by fluorescence photobleaching recovery

Rhodamine (Rho)-labeled muscle and non-muscle actins were microinjected into cultured embryonic chicken cardiac myocytes and fibroblasts. After incorporation of the fluorescent actin analog into cellular structures, small areas of labeled structures were photobleached with a laser pulse, and fluorescence recovery (FR) was measured to determine the exchangeability of isoactins in these structures. With both Rho-muscle and Rho-non-muscle actins, the FR rate in any part of stress fibers was consistently faster than that observed in any part of myofibrils. Thus, although non-striated (proximal and terminal) portions of nascent myofibrils are similar in appearance and composition to stress fibers, our data clearly revealed differences in actin stability between these two structures. Further, although cardiomyocytes were incapable of discriminating between the incorporation of muscle and non-muscle actin isoforms into myofibrils, FR after photobleaching of Rho-muscle actin was faster than that of Rho-non-muscle actin in immature non-striated portions. This indicates that actin molecules in cardiac myofibrils cannot be readily exchanged by heterotypic non-muscle actin. Fluorescently labeled actin incorporated into non-striated (proximal and terminal) portions of myofibrils and terminal portions of stress fibers was found to be more stable than α-actinin. The relative stability of actin could facilitate the formation of nascent Z-bands of myofibrils and the reorganization of stress fibers at these portions.

MyoD Is Indispensable for Muscle-specific Alternative Splicing in Mouse Mitochondrial ATP Synthase γ-Subunit Pre-mRNA

Muscle-specific alternative RNA splicing is an essential step during myogenesis. In this paper, we report that a muscle-specific transcription factor, MyoD, plays a central role in the induction of muscle-specific alternative splicing during myogenesis. Recently, we reported that muscle and nonmuscle isoforms of the mitochondrial ATP synthase gamma-subunit (F1gamma) were generated by alternative splicing and that acidic stimulation promoted this muscle-specific alternative splicing (Endo, H., Matsuda, C., and Kagawa, Y. (1994) J. Biol. Chem. 269, 12488-12493). In this report, mouse myoblasts are shown to express the muscle-specific isoform of F1gamma after induction with low-serum medium (differentiation medium) or acidic medium, although myotube formation was not detected after acidic induction. RNA blot analysis revealed that the expression levels of both MEF2 and myogenin were increased by low-serum induction, but not by acidic induction. High expression of MyoD mRNA was observed after both types of induction. Overexpression of exogenous MyoD in fibroblasts showed that MyoD was necessary for muscle-specific alternative splicing in both types of induction. Exogenous Id, a negative regulator of MyoD, blocked muscle-specific alternative splicing of F1gamma pre-mRNA by both types of induction. In addition, MyoD induced several muscle-specific alternative splicings, including structural protein pre-mRNAs such as beta-tropomyosin and neural-cell adhesion molecule and transcriptional protein pre-mRNAs such as MEF2A and MEF2D. Our analysis of the two induction systems shows a common MyoD-dependent mechanism of muscle-specific alternative splicing in several genes, independent of MEF2 and myogenin.

Alpha-tropomyosin gene expression in Xenopus laevis: differential promoter usage during development and controlled expression by myogenic factors.

Tropomyosins (TMs) constitute a group of contractile proteins encoded by a multigene family showing distinct cell-type-specific and developmental expression patterns. In mammals and birds, the alpha-TM gene is the most complex and can produce several muscle and non-muscle isoforms. We report here the characterization of the 5' region of the Xenopus laevis alpha-TM gene and its developmental expression. The 5' region of the gene is structurally related to the avian and mammalian cognates and presents two promoters flanking a pair of alternatively spliced exons, 2a/2b, where exon 2a is a smooth-muscle-specific exon. The internal promoter is used to generate a non-muscle low molecular weight TM whilst muscle TM isoforms originate from the distal promoter. RNase protection analysis shows that the two promoters have distinct temporal programs of activation. The internal promoter is activated early in oogenesis and non-muscle transcripts are found throughout oogenesis, embryogenesis and in adult tissues. Only low molecular weight non-muscle TM-encoding mRNAs are expressed in oogenesis. The distal promoter is silent during oogenesis, and the skeletal muscle alpha-TM transcripts accumulate from stage 15 in the embryo and are expressed in adult striated muscle tissues. In situ hybridization indicates that these transcripts are expressed in both the somites and heart of the embryo. Ectopic expression of myogenic factors, but not the MEF2 myocyte-specific enhancer factor 2 factors SL1 and SL2, can induce the expression of the alpha-TM gene suggesting that the gene is a direct target for myogenic but not for MEF2 factors. The amphibian alpha-TM gene constitutes a gene marker for studying the developmental control expression of muscle genes in the different myogenic lineages.

Functional nonequivalence of Drosophila actin isoforms.

We show that different Drosophila actin isoforms are not interchangeable. We sequenced the six genes that encode conventional Drosophila actins and found that they specify amino acid replacements in 27 of 376 positions. To test the significance of these changes we used directed mutagenesis to introduce 10 such conversions, independently, into the Act88F flight muscle-specific actin gene. We challenged these variant actins to replace the native protein by transforming germline chromosomes of a Drosophila strain lacking flight muscle actin. Only one of the 10 reproducibly perturbed myofibrillar function, demonstrating that most isoform-specific amino acid replacements are of minor significance. In order to establish the consequences of multiple amino acid replacements, we substituted portions of the Drosophila Act88F actin gene with corresponding regions of genes encoding other isoforms. Only one of five constructs tested engendered normally functioning flight muscles, and the severity of myofibrillar defects correlated with the number of replacements within the chimeric genes. Finally, we completely converted the flight muscle actin-encoding gene to one specifying a nonmuscle isoform, a change entailing a total of 18 amino acid replacements. Transformation of flies with this construct resulted in disruption of flight muscle structure and function. We conclude that actin isoform sequences are not equivalent and that effects of the amino acid replacements, while minor individually, collectively confer unique properties.

Deuterostomic actin genes and the definition of the chordates: cDNA cloning and gene organization for cephalochordates and hemichordates.

The evolutionary relationship of muscle and nonmuscle actin isoforms in deuterostomia was studied by the isolation and characterization of two actin genes from the cephalochordate Branchiostoma lanceolatum and two from the hemichordate Saccoglossus kowalevskii The Branchiostoma genes specify a muscle and a nonmuscle actin type, respectively. Together with earlier results on muscle actins from vertebrates and urochordates, a N-terminal sequence signature is defined for chordate muscle actins. These diagnostic amino acid residues separate the chordates from the echinoderms and other metazoa. Although the two Saccoglossus actins characterized so far lack the diagnostic residues, in line with the presumptive phylogenetic position of hemichordates outside the chordates, a definitive conclusion can only be expected once the full complement of actin genes of Saccoglossus is established. Comparison of the intron patterns of the various deuterostomic actin genes shows that intron 330-3, which is present in all vertebrate genes, is conspicuously absent from nonvertebrate genes. The possible origin of this intron is discussed.

Purification and characterization of an alpha 1 beta 2 isoform of CapZ from human erythrocytes: cytosolic location and inability to bind to Mg2+ ghosts suggest that erythrocyte actin filaments are capped by adducin.

CapZ ("capping protein") is a heterodimeric actin capping protein that blocks actin filament assembly and disassembly at the fast growing (barbed) filament ends and is proposed to function in regulating actin filament dynamics as well as in stabilizing actin filament lengths in muscle and nonmuscle cells. We show here that erythrocytes contain a nonmuscle isoform of capZ (EcapZ) that is present exclusively in the cytosol and is not associated with the short actin filaments in the erythrocyte membrane skeleton. This is unlike other cell types where capZ is associated with cytoskeletal actin filaments and suggests that cytosolic EcapZ may be inactive, or alternatively, that the barbed ends are capped by adducin, a membrane skeleton protein that was shown recently to cap actin filament barbed ends in vitro [Kuhlman, P. A., Hughes, C. A., Bennett, V., & Fowler, V. M. (1996) J. Biol. Chem. 271, 7986]. To distinguish between these possibilities, we purified EcapZ from erythrocyte cytosol and characterized its biochemical and functional properties. Two-dimensional gel electrophoresis and western blotting reveals the EcapZ subunit composition to be alpha1beta2, as described for capZ from many other nonmuscle cells, with no evidence for posttranslational modifications. Purified EcapZ is fully functional in blocking actin elongation from barbed filament ends (Kcap approximately 1-5 nM) as well as in nucleating actin polymerization. Furthermore, cytosolic EcapZ binds to actin filament barbed ends, indicating that sequestering of EcapZ by a cytosolic inhibitory factor or insufficient amounts of EcapZ in cytosol also cannot account for its absence from the membrane skeleton. To test directly whether the barbed ends of the erythrocyte actin filaments were already capped, we measured binding of purified EcapZ to isolated membranes. Purified EcapZ does not cosediment with membranes prepared by hypotonic lysis in the presence of magnesium, suggesting that the barbed ends of the erythrocyte actin filaments are capped under these conditions but not by EcapZ. In contrast, purified EcapZ stoichiometrically reassociates with all the actin filament barbed ends in membranes prepared by hypotonic lysis in 5 mM sodium phosphate, pH 8.0 (5P8), conditions in which the barbed filament ends were previously reported to be uncapped. Comparison of the amounts of adducin associated with membranes prepared in the presence and absence of magnesium reveals that 60-80% of the adducin dissociates from the membrane during hemolysis and washing in 5P8 buffer, suggesting that the barbed ends become artifactually uncapped due to loss of adducin. The erythrocyte actin filaments may thus represent a specialized class of membrane-associated actin filaments that are capped by adducin instead of capZ.

Isolation of the Embryonic Form of Smooth Muscle Myosin Heavy Chain (SMemb/NMHC-B) Gene and Characterization of Its 5′-Flanking Region

To examine the molecular mechanisms that regulate the expression of the SMemb/NMHC-B gene, a nonmuscle myosin heavy chain isoform predominantly expressed in fetal aorta, we have isolated and characterized the 5′-flanking region of the rabbit SMemb/NMHC-B gene. Transient transfection experiments demonstrated that 105 base pairs of 5′-flanking sequence was necessary to direct high level transcription in C2/2 cells, vascular smooth muscle cells derived from rabbit aorta. An essential cis-regulatory element was localized between −100 and −91 base pairs from the transcription start site based on the results that replacement mutagenesis within this region significantly reduced promoter activity. Sequence of this region is completely conserved between mouse and rabbit and fits no known DNA binding consensus. Gel mobility shift assays revealed that a specific DNA-protein complex was formed at this site with nuclear extracts from C2/2 cells, which can be competed by H-2KbCCAAT box but not by Hsp70 CCAAT box or other CCAAT-containing sequences. We conclude that expression of the SMemb/NMHC-B gene is regulated through an interaction between a sequence element located at −100 and a distinct member of CCAAT-binding proteins.