ABSTRACT
Nebulette and nebulin are homologous proteins associated with the Z-lines of cardiac and skeletal muscle sarcomeres. Although these proteins share 70% sequence homology and an identical domain layout, nebulette is one-tenth the size of nebulin. To define structurally important features of these proteins in terms of the Z-line architecture, we have analyzed the primary structure of nebulette and nebulin from a variety of species and developmental stages. Alignment of the 35 residue nebulin-like modules from both proteins demonstrates that the individual modules share 30-90% homology across the six proteins analyzed. In addition, this analysis demonstrates a number of areas in which the identity across the six proteins is as high as 75%. These areas may be important signals for Z-line assembly and function in the striated muscles. Significantly, most of the areas of high identity also coincide with consensus phosphorylation sites. To evaluate if nebulette, like nebulin, exhibits tissue-specific and developmental specific polymorphism, a series of immunoblot assays were performed. These data demonstrate that nebulettes from different portions of the heart are the same size. Comparison of nebulette from embryonic and adult cardiac muscle also demonstrates that this protein does not appear to vary in size with developmental stage. Consistent with the large number of consensus phosphorylation sites identified in the nebulette primary structure, we find that nebulette is phosphorylated in the cardiac muscle at serine and threonine residues. These data and sequence analyses are discussed in terms of current models for Z-line architecture.
Subject(s)
Actin Cytoskeleton/genetics , Muscle Proteins/genetics , Sarcomeres/genetics , Amino Acid Sequence/genetics , Animals , Carrier Proteins , Chickens , Cytoskeletal Proteins , Humans , Isoelectric Point , LIM Domain Proteins , Mice , Molecular Sequence Data , Phosphorylation , Polymorphism, Genetic/genetics , Protein Isoforms/genetics , Rabbits , Sequence Alignment , Sequence Homology, Amino Acid , Species SpecificityABSTRACT
Nebulette, a 107 kDa protein associated with the I-Z-I complex of cardiac myofibrils, may play an important role in the assembly of the Z-line. Determination of the complete primary structure of 1011 residue human fetal nebulette reveals a four-domain layout similar to skeletal muscle nebulin: a short N-terminal domain, followed by 22 nebulin-like repeats that are linked to a C-terminal Src homology 3 (SH3) domain via a short linker domain. To elucidate the mechanisms of assembly for nebulette in the Z-line, the complete coding sequence or fusions of nebulette domains with green fluorescent protein (GFP) were expressed in cardiomyocytes and fibroblasts. The complete protein localized to Z-lines in cardiac cells and to dense bodies in nonmuscle cells. The GFP-repeat domain forms bundles that are associated with actin filaments in both cell types and disrupts the microfilament network. In contrast, the GFP-repeat plus linker shows limited interaction with dense bodies in nonmuscle cells and the Z-lines of cardiomyocytes. Interestingly, the tagged linker or SH3 is diffusely distributed in nonmuscle cells, but localizes to the Z-lines in cardiomyocytes. Supporting the cellular localization work, recombinant nebulette fragments bind to actin, tropomyosin, and alpha-actinin in in vitro binding assays. These results suggest the repeat domain contains actin binding functions and that the linker domain may target this interaction to Z-lines and dense bodies. Our data also indicate that the linker and SH3 domains can distinguish between dense bodies and Z-lines, suggesting that the ligands for their interactions are specific to these muscular substructures.
Subject(s)
Actins/metabolism , Muscle Proteins/genetics , Repetitive Sequences, Nucleic Acid , src Homology Domains , Amino Acid Sequence , Animals , COS Cells , Carrier Proteins , Cells, Cultured , Chick Embryo , Cytoskeletal Proteins , DNA, Complementary/chemistry , DNA, Complementary/genetics , Gene Expression , Gene Library , Green Fluorescent Proteins , Humans , LIM Domain Proteins , Luminescent Proteins/genetics , Microscopy, Fluorescence , Molecular Sequence Data , Muscle Proteins/physiology , Muscle, Skeletal/metabolism , Myocardium/cytology , Myocardium/metabolism , Myofibrils/metabolism , Protein Binding , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Sequence Alignment , Sequence Analysis, DNA , Sequence Homology, Amino AcidABSTRACT
To investigate the roles of thiol proteases such as cathepsins and calpains in muscle differentiation, we have treated primary cultures of pectoralis muscle with a variety of protease inhibitors and examined the effects these agents have on myoblast fusion and myofibrillogenesis. We have found that a membrane-permeable inhibitor, E64D, has dramatic effects on both events of muscle differentiation. Cells treated with this inhibitor display gross morphological changes, severe delays in myofibril assembly, and reduced ability to fuse in culture. These morphological changes are correlated with a build up of beta1-integrin throughout the cytoplasm. These effects could also be produced using NH4Cl, a lysosomotrophic agent. In addition, we show that two nonpermeable inhibitors (leupeptin and E64) slightly decrease myoblast fusion, but have no effects on the ability of the cells to form mature myofibrils. These results are discussed in terms of their relevance to the inheritable disease of muscular dystrophy and I-cell disease (mucolipodosis II).
Subject(s)
Cysteine Endopeptidases/physiology , Muscle, Skeletal/physiology , Myofibrils/physiology , Animals , Cell Differentiation/physiology , Cell Fusion/physiology , Cells, Cultured , Chickens , Cysteine Proteinase Inhibitors/pharmacology , Muscle, Skeletal/cytology , Muscle, Skeletal/enzymologyABSTRACT
Myosin couples ATP hydrolysis to the translocation of actin filaments to power many forms of cellular motility. A striking feature of the structure of the muscle myosin head domain is a 9-nm long "lever arm" that has been postulated to produce a 5-10-nm power stroke. This motion must be coupled to conformational changes around the actin and nucleotide binding sites. The linkage of these sites to the lever arm has been analyzed by site-directed mutagenesis of a conserved glycine residue (G699) found in a bend joining two helices containing the highly reactive and mobile cysteine residues, SH1 and SH2. Alanine mutagenesis of this glycine (G699A) dramatically alters the motor activity of skeletal muscle myosin, inhibiting the velocity of actin filament movement by > 100-fold. Analysis of the defect in the G699A mutant myosin is consistent with a marked slowing of the transition within the motor domain from a strong binding to a weak binding interaction with actin. This result is interpreted in terms of the role of this residue (G699) as a pivot point for motion of the lever arm. The recombinant myosin used in these experiments has been produced in a unique expression system. A shuttle vector containing a regulated muscle-specific promoter has been developed for the stable expression of recombinant myosin in C2C12 cells. The vector uses the promoter/enhancer region, the first two and the last five exons of an embryonic rat myosin gene, to regulate the expression of an embryonic chicken muscle myosin cDNA. Stable cell lines transfected with this vector express the unique genetically engineered myosin after differentiation into myotubes. The myosin assembles into myofibrils, copurifies with the endogenous myosin, and contains a complement of muscle-specific myosin light chains. The functional activity of the recombinant myosin is readily analyzed with an in vitro motility assay using a species-specific anti-S2 mAb to selectively assay the recombinant protein. This expression system has facilitated manipulation and analysis of the skeletal muscle myosin motor domain and is also amenable to a wide range of structure-function experiments addressing questions unique to the muscle-specific cytoarchitecture and myosin isoforms.
Subject(s)
Cell Movement/physiology , Glycine/physiology , Muscle, Skeletal/physiology , Myosins/physiology , Animals , Antibody Specificity , Base Sequence , Cell Line , Chick Embryo , Chickens , Cysteine , Genetic Vectors , Mice , Molecular Sequence Data , Muscle Fibers, Skeletal , Muscle, Skeletal/cytology , Mutagenesis, Site-Directed , Myosins/biosynthesis , Myosins/chemistry , Myosins/genetics , Myosins/isolation & purification , Point Mutation/physiology , Protein Structure, Secondary , Rats , Recombinant Fusion Proteins/biosynthesis , Recombinant Fusion Proteins/isolation & purificationABSTRACT
In developing muscle, relatively little is known about the synthesis and incorporation of the large actin binding protein, nebulin, into the sarcomere. To determine the temporal pattern of nebulin assembly into the myofibrils of differentiating skeletal muscle cells, myofibril assembly was examined by immunofluorescence microscopy. The distribution of nebulin was compared to other myofibrillar and cytoskeletal proteins (myosin, titin, actin, desmin, tubulin). At the onset of differentiation, we observed that nebulin is first seen in a diffuse distribution throughout the cytoplasm. At this time, muscle specific myosin and titin are also distributed in this manner. Myosin and titin become associated with the nascent myofibrils prior to the addition of nebulin. The mature striated pattern of myosin and titin also preceded the development of striations with nebulin. After nebulin becomes organized into a striated pattern, actin filaments separate across the A-band and form thin filaments of uniform length. These patterns of assembly suggest that nebulin is required for restricting the lengths of the thin filaments. We have employed the strategy of using ethyl methane sulfonate and taxol to perturb myofibril assembly to examine interactions critical for the addition of nebulin to the developing sarcomeres. The same temporal pattern of assembly seen in the normal cultures was observed in the ethyl methane sulfonate treated cultures, but at a much slower rate. In cultures treated with the microtubule stabilizing drug taxol, the amount of stress fibers and nascent I-bands was greatly diminished as previously reported by others; however, nebulin was found associated with myofibrils in a mature striated distribution. In addition, our results indicate that the taxol treated cultures contain remnants of the Z-line. These results suggest that nebulin assembly into the myofibril requires interactions or anchorage at the Z-line and within the A-band.
Subject(s)
Muscle Proteins/metabolism , Muscle, Skeletal/metabolism , Sarcomeres/metabolism , Animals , Chickens , Humans , Immunoblotting , Myofibrils/metabolismABSTRACT
A 107-kD protein has been identified in primary cultures of chicken embryonic cardiomyocytes by immunoprecipitations with certain anti-nebulin monoclonal antibodies (mAbs). These mAbs, prepared against a fragment of human skeletal muscle nebulin located near the carboxyl terminus, detect a 107-kD protein in extracts of adult chicken heart, adult mouse heart, and adult rabbit heart by immunoblot analysis. A partial cDNA corresponding to this protein has been isolated by immunological screening of a chicken heart cDNA expression vector library. The partial cDNA encodes a 380-amino acid open reading frame composed entirely of nebulin-like 35-residue modules marked by the highly conserved sequence motifs: SXXXYK and TPD. The open reading frame exhibits 60-85% homology with skeletal muscle nebulins from a variety of species. This cDNA recognizes an approximately 8-kb transcript in cardiac RNA and does not hybridize to skeletal muscle RNAs by northern analysis. Immunofluorescence localization of this nebulin-like protein in primary cultures of chicken cardiomyocytes and embryonic chicken cardiac myofibrils indicates that the protein is localized to the I-Z-I complex of the myofibrils, extending approximately 25% of the thin filament length. Comparisons of the distribution of this protein relative to actin, myosin, and titin in spreading cardiomyocytes suggest that the cardiac nebulin-like protein becomes aligned with the nascent myofibrils early during myofibrillogenesis. To distinguish this petite nebulin-like protein from the 600-900 kD skeletal muscle nebulin, we have named it nebulette.
Subject(s)
Muscle Proteins/analysis , Muscle Proteins/genetics , Myocardium/chemistry , Animals , Antibodies, Monoclonal , Antibody Specificity , Base Sequence , Carrier Proteins , Cells, Cultured/physiology , Chick Embryo , Cross Reactions , Cytoskeletal Proteins , DNA, Complementary/genetics , Gene Library , Genetic Testing , Humans , Immunoblotting , LIM Domain Proteins , Mice , Mice, Inbred BALB C , Molecular Sequence Data , Molecular Weight , Muscle Proteins/immunology , Muscle, Skeletal/chemistry , Myocardium/cytology , Myofibrils/chemistry , Rabbits , Sarcomeres/chemistry , Sequence Analysis, DNA , Sequence Homology, Amino AcidABSTRACT
Skeletal muscle myosin cDNAs were expressed in a simian kidney cell line (COS) and a mouse myogenic cell line to investigate the mechanisms controlling early stages of myosin filament assembly. An embryonic chicken muscle myosin heavy chain (MHC) cDNA was linked to constitutive promoters from adenovirus or SV40 and transiently expressed in COS cells. These cells accumulate hybrid myosin molecules composed of muscle MHCs and endogenous, nonmuscle, myosin light chains. The muscle myosin is found associated with a Triton insoluble fraction from extracts of the COS cells by immunoprecipitation and is detected in 2.4 +/- 0.8-micron-long filamentous structures distributed throughout the cytoplasm by immunofluorescence microscopy. These structures are shown by immunoelectron microscopy to correspond to loosely organized bundles of 12-16-nm-diameter myosin filaments. The muscle and nonmuscle MHCs are segregated in the transfected cells; the endogenous nonmuscle myosin displays a normal distribution pattern along stress fibers and does not colocalize with the muscle myosin filament bundles. A similar assembly pattern and distribution are observed for expression of the muscle MHC in a myogenic cell line. The myosin assembles into filament bundles, 1.5 +/- 0.6 micron in length, that are distributed throughout the cytoplasm of the undifferentiated myoblasts and segregated from the endogenous nonmuscle myosin. In both cell lines, formation of the myosin filament bundles is dependent on the accumulation of the protein. In contrast to these results, the expression of a truncated MHC that lacks much of the rod domain produces an assembly deficient molecule. The truncated MHC is diffusely distributed throughout the cytoplasm and not associated with cellular stress fibers. These results establish that the information necessary for the segregation of myosin isotypes into distinct cellular structures is contained within the primary structure of the MHC and that other factors are not required to establish this distribution.
Subject(s)
Myosins/biosynthesis , Actin Cytoskeleton/physiology , Actin Cytoskeleton/ultrastructure , Animals , Cell Line , Chick Embryo , Cloning, Molecular , Cytarabine/pharmacology , DNA Replication/drug effects , DNA, Complementary , Gene Expression , Haplorhini , Kidney , Mice , Muscles/cytology , Muscles/metabolism , Myosins/analysis , Myosins/genetics , Myosins/metabolism , Nucleotide Mapping , Radioimmunoprecipitation Assay , TransfectionABSTRACT
The interaction of two monoclonal antibodies (mAbs) with actin has been characterized to map the epitopes defined by these mAbs and to determine the accessibility of these sites in the actin filament (F-actin). Both mAbs react specifically with actin in radioimmunoassays and Western blot assays, and by immunoprecipitation. The location of the epitopes within the primary structure of actin has been determined using limited proteolysis of actin and Western blots, or using immunoprecipitation of truncated actin fragments synthesized in a cell free translation assay. Both mAbs bind to the C-terminal fragment of actin (residues 68-375) produced by chymotrypsin cleavage. One epitope is further localized to a 9.9 kD peptide corresponding to residues 5-93. Therefore, the epitope defined by this mAb (2G11.4) lies between residues Lys68 and Glu93 of actin. The location of the other epitope was determined by immunoprecipitation of actin fragments synthesized in vitro. Removal of residues 356-365 from the C-terminus of actin completely abolished the binding of mAb 4E3.adl. Therefore, this mAb defines an epitope that involves residues between Trp356 and Ala365. The accessibility of these epitopes in native F-actin was determined with solution binding assays and characterized by immunoelectron microscopy. Monoclonal antibody 4E3.adl binds strongly to filaments, resulting in bundling or decoration of F-actin depending on the valency of the mAb, and indicating that the epitope is readily accessible in F-actin. In contrast, mAb 2G11.4 disrupts F-actin structure, resulting in the formation of an amorphous immunoprecipitate. These results place constraints on models of actin filament structure.
Subject(s)
Actins/ultrastructure , Antibodies, Monoclonal , Actins/chemistry , Actins/immunology , Amino Acid Sequence , Animals , Clone Cells , Cross Reactions , Epitopes/analysis , Mice , Models, Molecular , Molecular Sequence Data , Molecular Weight , Peptide Fragments/analysis , Sequence Homology, Amino AcidABSTRACT
Argininosuccinate synthetase and argininosuccinate lyase catalyze the synthesis of arginine from citrulline in kidney and also serve as components of the urea cycle in liver of ureotelic animals. Dietary and hormonal regulation of mRNAs encoding these enzymes have been well studied in liver but not in kidney. Messenger RNAs for these enzymes are localized within the renal cortex. Starvation and extreme variations in dietary protein content (0% vs 60% casein) produced 2.6- to 3.5-fold increases in mRNA abundance for these two enzymes in rat kidney. Argininosuccinate lyase mRNA was not induced by dibutyryl cAMP, dexamethasone, or a combination of the two agents. In contrast, argininosuccinate synthetase mRNA was induced 2-fold by dibutyryl cAMP but was unresponsive to dexamethasone. Thus, diet and hormones regulate levels of these mRNAs in rat kidney, but the responses are both qualitatively and quantitatively distinct from the responses previously reported for rat liver.
Subject(s)
Argininosuccinate Lyase/genetics , Argininosuccinate Synthase/genetics , Kidney/enzymology , Ligases/genetics , Lyases/genetics , RNA, Messenger/analysis , Animals , Argininosuccinate Lyase/metabolism , Argininosuccinate Synthase/metabolism , Blotting, Northern , Blotting, Southern , Bucladesine/pharmacology , Chickens , Cloning, Molecular , DNA Probes , Dexamethasone/pharmacology , Dietary Proteins/administration & dosage , Enzyme Induction , Food Deprivation , Kidney/drug effects , Kidney Cortex/enzymology , Liver/enzymology , Rats , Rats, Inbred Strains , Restriction MappingABSTRACT
Chromosomal deletions at and around the albino locus on chromosome 7 of the mouse affect the enzyme activities and steady-state levels of mRNAs for five urea-cycle enzymes in liver. In newborn c3H homozygotes, activities of these enzymes were 43-62% of normal, while corresponding mRNA levels were 14-29% of normal. c14CoS deletion homozygotes expressed mRNA levels for these enzymes which were 32-48% of normal. However, transcription rates of these genes in hepatic nuclei of c3H/c3H mice were reduced only to 57-84% of normal. Since effects of the deletions had previously been noted in the kidney, mRNA levels for three enzymes expressed also in the kidney were examined. Mice homozygous for the c3H deletion, shown previously to have drastically reduced mRNA levels for phosphoenolpyruvate carboxykinase in the liver, expressed the same deficiency in the kidney, while mRNA levels for argininosuccinate synthetase and argininosuccinate lyase were reduced in the liver but remained unaffected in the kidney. However, mRNA levels for phosphoenolpyruvate carboxykinase, carbamyl phosphate synthetase I, and ornithine transcarbamylase were unaffected in the intestine of c3H homozygotes. The results suggest that a regulatory factor(s) encoded in the DNA encompassed by the deletion is involved in the normal developmental maturation of hepatocytes and certain cells in the kidney.
Subject(s)
Chromosome Deletion , Chromosome Mapping , Genes , Intestines/enzymology , Kidney/enzymology , Liver/enzymology , Phosphoenolpyruvate Carboxykinase (GTP)/genetics , Urea/metabolism , Animals , Crosses, Genetic , Female , Homozygote , Male , Mice , Mice, Inbred StrainsABSTRACT
Adaptive changes in levels of urea cycle enzymes are largely coordinate in both direction and magnitude. In order to determine the extent to which these adaptive responses reflect coordinate regulatory events at the pretranslational level, measurements of hybridizable mRNA levels for all five urea cycle enzymes were carried out for rats subjected to various dietary regimens and hormone treatments. Changes in relative abundance of the mRNAs in rats with varying dietary protein intakes are comparable to reported changes in enzyme activities, indicating that the major response to diet occurs at the pretranslational level for all five enzymes and that this response is largely coordinate. In contrast to the dietary changes, variable responses of mRNA levels were observed following intraperitoneal injections of dibutyryl cAMP and dexamethasone. mRNAs for only three urea cycle enzymes increased in response to dexamethasone. Levels of all five mRNAs increased severalfold in response to dibutyryl cAMP at both 1 and 5 h after injection, except for ornithine transcarbamylase mRNA which showed a response at 1 h but no response at 5 h. Combined effects of dexamethasone and dibutyryl cAMP were additive for only two urea cycle enzyme mRNAs, suggesting independent regulatory pathways for these two hormones. Transcription run-on assays revealed that transcription of at least two of the urea cycle enzyme genes--carbamylphosphate synthetase I and argininosuccinate synthetase--is stimulated approximately four- to fivefold by dibutyryl cAMP within 30 min. The varied hormonal responses indicate that regulatory mechanisms for modulating enzyme concentration are not identical for each of the enzymes in the pathway.
Subject(s)
Cyclic AMP/physiology , Diet , Gene Expression Regulation , Glucocorticoids/physiology , Liver/enzymology , RNA, Messenger/metabolism , Urea/metabolism , Animals , Bucladesine/pharmacology , Dexamethasone/pharmacology , Gene Expression Regulation/drug effects , Rats , Rats, Inbred StrainsABSTRACT
The gene coding for the common alpha subunit of the bovine pituitary glycoprotein hormones was isolated from a bovine genomic library. The gene spans roughly 16.5 kbp, contains three intervening sequences, and codes for a message of approximately 730 nucleotides. The complete coding region of the gene was sequenced as well as 315 nucleotides of 5' flanking sequence and the entire intron C. Only a single base difference was found when the sequence of the gene was compared with that of the cDNA. Genomic blotting experiments suggest the presence of a single alpha subunit gene. Comparison of the bovine and human alpha subunit genes indicated that the high level of homology observed in the coding regions has been maintained throughout the 5' and 3' untranslated regions, and at least 90 nucleotides of the 5'flanking regions. Additionally, there is an 18 base pair sequence present in both the 5' flanking and 5' untranslated regions of the gene that is homologous to a region of the chick ovalbumin gene. This ovalbumin sequence has been suggested as a binding site for the progesterone receptor-complex.
Subject(s)
Genes , Glycoproteins/genetics , Pituitary Hormones, Anterior/genetics , Amino Acid Sequence , Animals , Base Composition , Base Sequence , Cattle , Cloning, Molecular , Codon , DNA/metabolism , DNA Restriction Enzymes , Macromolecular Substances , Oligodeoxyribonucleotides/chemical synthesisABSTRACT
A cDNA clone for the pre-alpha subunit of the pituitary glycoprotein hormones has been isolated from a bovine pituitary cDNA library through the use of a pool of synthetic oligodeoxynucleotide probes. This clone, designated pB alpha, contains a 564-base pair insert which includes a portion of the signal sequence, the entire coding sequence of the mature protein, and 224 base pairs of the 3'-untranslated sequence. As expected, the nucleotide and amino acid sequence of the mature bovine alpha subunit was homologous to the sequences reported for humans and rodents, with the most extensive homology occurring between bovine and rodents (85-90%). However, a comparison of the 3'-untranslated regions of pre-alpha subunit mRNA from three different mammalian species indicated that in bovine and rat, or in human and rat, these sequences have rapidly diverged, yielding respective homologies of 21 and 36%. In contrast, the sequence homology observed between the 3'-untranslated regions of bovine and human was 79%, which approaches the level of homology shared by their coding sequences. Thus, the conservation of the 3'-untranslated sequence in bovine and human pre-alpha subunit mRNA may be an indication that this region is functionally significant in these two species.