Component Of Thick Filaments Research Articles

Visualization of cardiac thick filament dynamics in ex vivo heart preparations

RationaleCardiac muscle cells are terminally differentiated after birth and must beat continually throughout one's lifetime. This mechanical process is driven by the sliding of actin-based thin filaments along myosin-based thick filaments, organized within sarcomeres. Despite costly energetic demand, the half-life of the proteins that comprise the cardiac thick filaments is ∼10 days, with individual molecules being replaced stochastically, by unknown mechanisms. ObjectivesTo allow for the stochastic replacement of molecules, we hypothesized that the structure of thick filaments must be highly dynamic in vivo. Methods and resultsTo test this hypothesis in adult mouse hearts, we replaced a fraction of the endogenous myosin regulatory light chain (RLC), a component of thick filaments, with GFP-labeled RLC by adeno-associated viral (AAV) transduction. The RLC-GFP was properly localized to the heads of the myosin molecules within thick filaments in ex vivo heart preparations and had no effect on heart size or actin filament siding in vitro. However, the localization of the RLC-GFP molecules was highly mobile, changing its position within the sarcomere on the minute timescale, when quantified by fluorescence recovery after photobleaching (FRAP) using multiphoton microscopy. Interestingly, RLC-GFP mobility was restricted to within the boundaries of single sarcomeres. When cardiomyocytes were lysed, the RLC-GFP remained strongly bound to myosin heavy chain, and the intact myosin molecules adopted a folded, compact configuration, when disassociated from the filaments at physiological ionic conditions. ConclusionsThese data demonstrate that the structure of the thick filament is highly dynamic in the intact heart, with a rate of molecular exchange into and out of thick filaments that is ∼1500 times faster than that required for the replacement of molecules through protein synthesis or degradation.

Characterization of paramyosin protein structure and gene expression during myogenesis in Pacific oyster (Crassostrea gigas).

Paramyosin is a key component of thick filaments in invertebrate muscles. In this study, we isolated the full length cDNA of paramyosin from Pacific oyster (Crassostrea gigas), and determined its pattern of expression during myogenesis. The full length paramyosin (CgPM) cDNA contains an open reading frame (ORF) of 2586bp encoding a 861-amino acid protein. Sequence analysis revealed an assembly competence domain (ACD) and a heptad repeat (d-e-f-g-a-b-c) with 28-residue repeat zones in the CgPM primary structure, a characteristic of coiled-coil protein. Quantitative analysis of CgPM expression revealed a sharp increase in trochophore stage, and peaked at the D-shaped stage. Strong CgPM expression was found in smooth adductor muscle, followed by striated adductor muscle and mantle tissue. Whole-mount in situ hybridization (WISH) showed a restricted pattern of CgPM expression in adductor muscle, larval velum retractor and foot muscles at the umbo and eyed larval stages. These data indicate that CgPM is strongly expressed during larval myogenesis in C. gigas, which provides the basis for further functional studies of paramyosin in oyster to better understand the molecular and cellular mechanisms of muscle formation in mollusks.

High-resolution imaging of muscle attachment structures in Caenorhabditis elegans.

We used structured illumination microscopy (SIM) to obtain super-resolution images of muscle attachment structures in Caenorhabditis elegans striated muscle. SIM imaging of M-line components revealed two patterns: PAT-3 (β-integrin) and proteins that interact in a complex with the cytoplasmic tail of β-integrin and localize to the basal muscle cell membrane [UNC-112 (kindlin), PAT-4 (ILK), UNC-97 (PINCH), PAT-6 (α-parvin), and UNC-95], are found in discrete, angled segments with gaps. In contrast, proteins localized throughout the depth of the M-line (UNC-89 (obscurin) and UNC-98) are imaged as continuous lines. Systematic immunostaining of muscle cell boundaries revealed that dense body components close to the basal muscle cell membrane also localize at cell boundaries. SIM imaging of muscle cell boundaries reveal "zipper-like" structures. Electron micrographs reveal electron dense material similar in appearance to dense bodies located adjacent to the basolateral cell membranes of adjacent muscle cells separated by ECM. Moreover, by EM, there are a variety of features of the muscle cell boundaries that help explain the zipper-like pattern of muscle protein localization observed by SIM. Short dense bodies in atn-1 mutants that are null for α-actinin and lack the deeper extensions of dense bodies, showed "zipper-like" structures by SIM similar to cell boundary structures, further indicating that the surface-proximal components of dense bodies form the "zipper-like" structures at cell boundaries. Moreover, mutants in thin and thick filament components do not have "dot-like" dense bodies, suggesting that myofilament tension is required for assembly or maintenance of proper dense body shape.

Genetic mutations and mechanisms in dilated cardiomyopathy

Genetic mutations account for a significant percentage of cardiomyopathies, which are a leading cause of congestive heart failure. In hypertrophic cardiomyopathy (HCM), cardiac output is limited by the thickened myocardium through impaired filling and outflow. Mutations in the genes encoding the thick filament components myosin heavy chain and myosin binding protein C (MYH7 and MYBPC3) together explain 75% of inherited HCMs, leading to the observation that HCM is a disease of the sarcomere. Many mutations are "private" or rare variants, often unique to families. In contrast, dilated cardiomyopathy (DCM) is far more genetically heterogeneous, with mutations in genes encoding cytoskeletal, nucleoskeletal, mitochondrial, and calcium-handling proteins. DCM is characterized by enlarged ventricular dimensions and impaired systolic and diastolic function. Private mutations account for most DCMs, with few hotspots or recurring mutations. More than 50 single genes are linked to inherited DCM, including many genes that also link to HCM. Relatively few clinical clues guide the diagnosis of inherited DCM, but emerging evidence supports the use of genetic testing to identify those patients at risk for faster disease progression, congestive heart failure, and arrhythmia.

Lasp regulates Actin Filament Dynamics in Drosophila Myofibrils Assembly

The actin cytoskeleton plays a key role in a number of motile and morphogenetic processes. The coordinated assembly and disassembly of actin filaments promotes cell shape changes, mediates motility, contractility, and many other processes. In each case, actin dynamics is finely regulated by a large number of actin-binding proteins (ABPs) that control actin filament polymerization, nucleation and crosslinking.One group of ABPs is the nebulin family. To date, this family comprises 5 members, each containing from 2 to 185 actin-binding nebulin repeats. In our previous work, we showed that Lasp is the single member of this family in Drosophila. Lasp has an N-terminal LIM domain, two nebulin repeats, and a C-terminal SH3 domain. Lasp null mutants are homozygous viable, but male sterile. The stem cell niche is not properly anchor and actin cone migration is impaired, resulting in failure of spermatid individualization. Recently, we discovered that Lasp also functions in myofibril assembly. In Lasp mutants, sarcomere length is reduced and muscle contractility is weaker than in wild type flies. These results are consistent with a function of nebulin family proteins as scaffolding and actin filament organizing proteins.Using a double-tagged Lasp, I performed a pull down assay to identify interacting partners. As expected Lasp binds muscle-specific actin isoforms. More surprisingly, most of the isolated proteins are components of thick filaments, suggesting new functions for Lasp. We will report which domain of Lasp regulates actin dynamics, protein interactions and proper localization in sarcomere assembly. By deciphering Lasp function in both Z-disc and A-band, we will obtain further insights into the mechanism of myogenesis.

Ubiquitylation by Trim32 causes coupled loss of desmin, Z-bands, and thin filaments in muscle atrophy

During muscle atrophy, myofibrillar proteins are degraded in an ordered process in which MuRF1 catalyzes ubiquitylation of thick filament components (Cohen et al. 2009. J. Cell Biol. http://dx.doi.org/10.1083/jcb.200901052). Here, we show that another ubiquitin ligase, Trim32, ubiquitylates thin filament (actin, tropomyosin, troponins) and Z-band (α-actinin) components and promotes their degradation. Down-regulation of Trim32 during fasting reduced fiber atrophy and the rapid loss of thin filaments. Desmin filaments were proposed to maintain the integrity of thin filaments. Accordingly, we find that the rapid destruction of thin filament proteins upon fasting was accompanied by increased phosphorylation of desmin filaments, which promoted desmin ubiquitylation by Trim32 and degradation. Reducing Trim32 levels prevented the loss of both desmin and thin filament proteins. Furthermore, overexpression of an inhibitor of desmin polymerization induced disassembly of desmin filaments and destruction of thin filament components. Thus, during fasting, desmin phosphorylation increases and enhances Trim32-mediated degradation of the desmin cytoskeleton, which appears to facilitate the breakdown of Z-bands and thin filaments.

Phosphorylation motifs in the nonhelical domains of myosin heavy chain and paramyosin may negatively regulate assembly in Caenorhabditis elegans striated muscle

We are interested in mechanisms that establish and maintain the highly ordered contractile apparatus of striated muscle. The homologous proteins myosin and paramyosin are the major structural components of thick filaments in invertebrate animals. In Caenorhabditis elegans, both proteins contain a homologous, small nonhelical domain that is known to be phosphorylated in paramyosin. In this report, we show that a proposed phosphorylation motif (S_S_A), which is present in several copies in the nonhelical regions of both myosin and paramyosin, is highly conserved among nematodes. We used in vivo assays to examine the assembly properties of proteins in which one or more motifs were targeted by point mutagenesis or deletion. In all cases, expression of mutant proteins improved the phenotype of the corresponding null mutant animals, but produced variable structural defects, including birefringent aggregates in adults and abnormal localization in embryos. Point mutation, but not deletion, of the myosin A nonhelical tailpiece produced ectopic structures that appeared as masses of jumbled filaments by TEM. Antibody labeling showed that aggregates of either mutant protein did not recruit the endogenous version of the other. Analysis of mutant embryos lacking either paramyosin or myosin A (the essential isoform at the thick filament center) indicated that both wild-type proteins can independently localize and initiate assembly, although the structures produced are abnormal. Our results suggest that muscle cells actively restrict myosin and paramyosin assembly through phosphorylation of the S_S_A motifs and that each protein is regulated independently.

Determination of AMP-activated protein kinase phosphorylation sites in recombinant protein expressed using the pET28a vector: A cautionary tale

AMP-activated protein kinase (AMPK) is responsible for sensing of the cell’s energetic status and it phosphorylates numerous substrates involved in anabolic and catabolic processes as well as interacting with signaling cascades. Mutations in the gene encoding the γ2 regulatory subunit have been shown to cause hypertrophic cardiomyopathy (HCM) with conduction abnormalities. As part of a study to examine the role of AMPK in the heart, we tested whether specific domains of the thick filament component cardiac myosin binding protein-C (cMyBP-C) were good in vitro AMPK substrates. The commercially available pET28a expression vector was used to generate a recombinant form of the cMyBP-C C8 domain as a fusion protein with a hexahistidine tag. In vitro phosphorylation with activated kinase showed that the purified fusion protein was a good AMPK substrate, phosphorylated at a similar rate to the control SAMS peptide and with phosphate incorporation specifically in serine residues. However, subsequent analysis of alanine replacement mutants and thrombin digestion revealed that the strong AMPK phosphorylation site was contained within the thrombin cleavage sequence encoded by the vector. As this sequence is common to many commercial pET vectors, caution is advised in the mapping of AMPK phosphorylation sites when this sequence is present.

Maximal stimulation-induced in situ myosin light chain kinase activity is upregulated in fetal compared with adult ovine carotid arteries

Postnatal decreases in vascular reactivity involve decreases in the thick filament component of myofilament calcium sensitivity, which is measured as the relationship between cytosolic calcium concentration and myosin light chain (MLC20) phosphorylation. The present study tests the hypothesis that downregulation of thick filament reactivity is due to downregulation of myosin light chain kinase (MLCK) activity in adult compared with fetal arteries. Total MLCK activity, calculated as %MLC20 phosphorylated per second in intact arteries during optimal inhibition of myosin light chain phosphatase activity, was significantly less in adult (6.56+/-0.29%) than in fetal preparations (7.39+/-0.53%). In situ MLC20 concentrations (microM) in adult (198+/-28) and fetal arteries (236+/-44) did not differ significantly. In situ MLCK concentrations (microM), however, were significantly greater in adult (8.21+/-0.59) than in fetal arteries (1.83+/-0.13). In situ MLCK activities (ng MLC20 phosphorylated.s(-1).ng MLCK(-1)) were significantly less in adult (0.26+/-0.01) than in fetal arteries (1.52+/-0.11). In contrast, MLCK activities in adult (15.8+/-1.5) and fetal artery homogenates (17.3+/-1.3) were not significantly different. When in situ fractional activation was calculated, adult values (1.72+/-0.17%) were significantly less than fetal values (9.08+/-0.83%). Together, these results indicate that decreased thick filament reactivity in adult compared with fetal ovine carotid arteries is due at least in part to greater MLCK activity in fetal arteries, which in turn cannot be explained by differences in MLCK, MLC20, or calmodulin concentrations. Instead, this difference appears to involve age-related differences in fractional activation of the MLCK enzyme.

CF2 activity and enhancer integration are required for proper muscle gene expression in Drosophila

The creation of the contractile apparatus in muscle involves the co-activation of a group of genes encoding muscle-specific proteins and the production of high levels of protein in a short period of time. We have studied the transcriptional control of six Drosophila muscle genes that have similar expression profiles and we have compared these mechanisms with those employed to control the distinct expression profiles of other Drosophila genes. The regulatory elements controlling the transcription of co-expressed muscle genes share an Upstream Regulatory Element and an Intronic Regulatory Element. Moreover, similar clusters of MEF2 and CF2 binding sites are present in these elements. Here, we demonstrate that CF2 depletion alters the relative expression of thin and thick filament components. We propose that the appropriate rapid gene expression responses during muscle formation and the maintenance of each muscle type is guaranteed in Drosophila by equivalent duplicate enhancer-like elements. This mechanism may be exceptional and restricted to muscle genes, reflecting the specific requirement to mediate rapid muscle responses. However, it may also be a more general mechanism to control the correct levels of gene expression during development in each cell type.

Upregulation of In Situ Myosin Light Chain Kinase Activity In Fetal Compared to Adult Ovine Carotid Arteries

Vascular reactivity changes dramatically during postnatal maturation due in large part to developmetal changes in myofilament calcium sensitivity. In turn, our recent findings suggest that reactivity of the thick filament component of calcium sensitivity, which is measured as the relation between cytosolic calcium concentration and the extent of myosin light chain (MLC) phosphorylation, is upregulated in fetal compared to adult arteries. The present study tests the hypothesis that upregulation of fetal thick filament reactivity is due to upregulation of myosin light chain kinase (MLCK) activity. Western blot analysis revealed that MLCK abundance was 6.5±0.4 fold greater in adult than in fetal arteries. Total MLCK activity, estimated as the rate of phosphorylation of the substrate myosin light chain in intact arteries, was greater in fetal (7.9 ± 0.3 %/sec) than in adult (6.6 ± 0.3 %/sec) preparations. When total MLCK activity was normalized relative to MLCK abundance to estimate the specific activity of MLCK (%phosphorylation/sec/μg MLCK), these estimates were dramatically greater in fetal (45.5 ± 2.3) than in adult (5.8 ± 0.5) arteries. Together, these results indicate that upregulation of MLCK specific activity contributes significantly to upregulation of thick‐filament reactivity in fetal compared to adult ovine carotid arteries. Supported by NIH HD31226, HL54120, and HL64867.

Phosphorylation of myorod (catchin) by kinases tightly associated to molluscan and vertebrate smooth muscle myosins

Myorod, also known as catchin, a newly discovered component of molluscan smooth muscle thick filaments, is an alternative product of the myosin heavy chain gene. It contains a C-terminal rod part that is identical to that part of myosin and a unique N-terminal domain that is very small relative to the myosin head domain. The role of myorod in contraction or relaxation of this muscle type is unknown. In the present study we demonstrated that myorod was phosphorylated not only by a kinase endogenous to molluscan myosin and twitchin but also to vertebrate smooth muscle myosin light chain kinase (MLCK). The rates and maximal levels of phosphorylation were up to threefold higher than those observed by protein kinase A with clear optima at the physiological salt concentrations. Using a mild digestion with chymotrypsin we isolated an 11 kDa phosphopeptide and showed that the phosphorylation site was located at the N-terminal domain of myorod at Thr 141 position. The sequence around this site exhibited a high degree of similarity to that expected for the substrate recognition site of MLCK. The phosphorylation rates strongly depended on the ionic conditions indicating that this site could be readily sterically blocked during myorod polymerization. Another component of the thick filaments involved in regulation of the catch state, twitchin, was phosphorylated by MLCK and exhibited endogenous myorod kinase and MLCK activities. A possible role of these phosphorylation reactions in the regulation of molluscan smooth muscles is discussed.

Caenorhabditis elegansUNC-96 Is a New Component of M-Lines That Interacts with UNC-98 and Paramyosin and Is Required in Adult Muscle for Assembly and/or Maintenance of Thick Filaments

To gain further insight into the molecular architecture, assembly, and maintenance of the sarcomere, we have carried out a molecular analysis of the UNC-96 protein in the muscle of Caenorhabditis elegans. By polarized light microscopy of body wall muscle, unc-96 mutants display reduced myofibrillar organization and characteristic birefringent "needles." By immunofluorescent staining of known myofibril components, unc-96 mutants show major defects in the organization of M-lines and in the localization of a major thick filament component, paramyosin. In unc-96 mutants, the birefringent needles, which contain both UNC-98 and paramyosin, can be suppressed by starvation or by exposure to reduced temperature. UNC-96 is a novel approximately 47-kDa polypeptide that has no recognizable domains. Antibodies generated to UNC-96 localize the protein to the M-line, a region of the sarcomere in which thick filaments are cross-linked. By genetic and biochemical criteria, UNC-96 interacts with UNC-98, a previously described component of M-lines, and paramyosin. Additionally, UNC-96 copurifies with native thick filaments. A model is presented in which UNC-96 is required in adult muscle to promote thick filament assembly and/or maintenance.

Aberrant myofibril assembly in tropomodulin1 null mice leads to aborted heart development and embryonic lethality.

Tropomodulin1 (Tmod1) caps thin filament pointed ends in striated muscle, where it controls filament lengths by regulating actin dynamics. Here, we investigated myofibril assembly and heart development in a Tmod1 knockout mouse. In the absence of Tmod1, embryonic development appeared normal up to embryonic day (E) 8.5. By E9.5, heart defects were evident, including aborted development of the myocardium and inability to pump, leading to embryonic lethality by E10.5. Confocal microscopy of hearts of E8–8.5 Tmod1 null embryos revealed structures resembling nascent myofibrils with continuous F-actin staining and periodic dots of α-actinin, indicating that I-Z-I complexes assembled in the absence of Tmod1. Myomesin, a thick filament component, was also assembled normally along these structures, indicating that thick filament assembly is independent of Tmod1. However, myofibrils did not become striated, and gaps in F-actin staining (H zones) were never observed. We conclude that Tmod1 is required for regulation of actin filament lengths and myofibril maturation; this is critical for heart morphogenesis during embryonic development.

DIM-1, a novel immunoglobulin superfamily protein in Caenorhabditis elegans, is necessary for maintaining bodywall muscle integrity.

The UNC-112 protein is required during initial muscle assembly in C. elegans to form dense bodies and M-lines. Loss of this protein results in arrest at the twofold stage of embryogenesis. In contrast, a missense mutation in unc-112 results in viable animals that have disorganized bodywall muscle and are paralyzed as adults. Loss or reduction of dim-1 gene function can suppress the severe muscle disruption and paralysis exhibited by these mutant hermaphrodites. The overall muscle structure in hermaphrodites lacking a functional dim-1 gene is slightly disorganized, and the myofilament lattice is not as strongly anchored to the muscle cell membrane as it is in wild-type muscle. The dim-1 gene encodes two polypeptides that contain three Ig-like repeats. The short DIM-1 protein isoform consists entirely of three Ig repeats and is sufficient for wild-type bodywall muscle structure and stability. DIM-1(S) localizes to the region of the muscle cell membrane around and between the dense bodies, which are the structures that anchor the actin filaments and may play a role in stabilizing the thin rather than the thick filament components of the sarcomere.

Muscle-specific RING finger-1 interacts with titin to regulate sarcomeric M-line and thick filament structure and may have nuclear functions via its interaction with glucocorticoid modulatory element binding protein-1.

The COOH-terminal A168–170 region of the giant sarcomeric protein titin interacts with muscle-specific RING finger-1 (MURF-1). To investigate the functional significance of this interaction, we expressed green fluorescent protein fusion constructs encoding defined fragments of titin's M-line region and MURF-1 in cardiac myocytes. Upon expression of MURF-1 or its central region (containing its titin-binding site), the integrity of titin's M-line region was dramatically disrupted. Disruption of titin's M-line region also resulted in a perturbation of thick filament components, but, surprisingly, not of the NH2-terminal or I-band regions of titin, the Z-lines, or the thin filaments. This specific phenotype also was caused by the expression of titin A168–170. These data suggest that the interaction of titin with MURF-1 is important for the stability of the sarcomeric M-line region.MURF-1 also binds to ubiquitin-conjugating enzyme-9 and isopeptidase T-3, enzymes involved in small ubiquitin-related modifier–mediated nuclear import, and with glucocorticoid modulatory element binding protein-1 (GMEB-1), a transcriptional regulator. Consistent with our in vitro binding data implicating MURF-1 with nuclear functions, endogenous MURF-1 also was detected in the nuclei of some myocytes. The dual interactions of MURF-1 with titin and GMEB-1 may link myofibril signaling pathways (perhaps including titin's kinase domain) with muscle gene expression.

Purification of Native Myosin Filaments from Muscle

Analysis of the structure and function of native thick (myosin-containing) filaments of muscle has been hampered in the past by the difficulty of obtaining a pure preparation. We have developed a simple method for purifying native myosin filaments from muscle filament suspensions. The method involves severing thin (actin-containing) filaments into short segments using a Ca 2+-insensitive fragment of gelsolin, followed by differential centrifugation to purify the thick filaments. By gel electrophoresis, the purified thick filaments show myosin heavy and light chains together with nonmyosin thick filament components. Contamination with actin is below 3.5%. Electron microscopy demonstrates intact thick filaments, with helical cross-bridge order preserved, and essentially complete removal of thin filaments. The method has been developed for striated muscles but can also be used in a modified form to remove contaminating thin filaments from native smooth muscle myofibrils. Such preparations should be useful for thick filament structural and biochemical studies.

Myosin rod protein: a novel thick filament component of Drosophila muscle

Myosin rod protein (MRP), a 155 kDa protein encoded by a gene internal to the Drosophila muscle myosin heavy chain (Mhc) gene, contains the MHC rod domain, but has 77 unique N-terminal residues that exactly replace the MHC motor and light chain binding domains. Originally described as an abundant testis protein, we now demonstrate the MRP also is a major component of myofilaments in Drosophila. Specifically, the Mrp promoter directs the expression of a LacZ reporter transgene in somatic, cardiac and visceral muscles. MRP-specific antibodies detect the protein in detergent-insoluble fractions of muscle extracts and co-localize the protein with MHC to the sarcomeric A-band in immunostained muscles. Immunoblot analysis shows that in a set of adult direct flight muscles (DFM), the ratio of MRP to MHC is 1:3. Chemical cross-link and co-immunoprecipitation experiments using 0·5 M KCl-extracted thick filament proteins indicate that native MRP is a homodimer. Electron microscopy of DFM49, which has a high MRP content, shows in cross section, disordered myofilament packing and a variable thin to thick filament ratio and, in longitudinal section, severely bent thin filaments that are not well associated with thick filaments. In rigor, thick filaments from DFM49 consist of segments with cross bridges that are interspersed with smooth domains lacking cross bridges. These data indicate that MRP is a novel contractile protein that co-integrates with myosin into the thick filament, thereby changing structure and function of the sarcomere.

Assembly-dependent phosphorylation of myosin and paramyosin of native thick filaments in Caenorhabdiris elegans

Phosphorylation of the thick filament proteins myosin and paramyosin was studied in Caenorhabditis elegans. We have incubated partially purified, native thick filaments with [gamma 32P] ATP in the presence of 50-750 mM NaCl, pH 6.5-8.0. Myosin heavy chain and paramyosin were phosphorylatable only upon solubilization at 450 mM and higher NaCl concentrations. Under conditions preserving native structures, no phosphorylation of these proteins occurred. The phosphorylation required Mg2+ but was unaffected by cAMP, cGMP or Ca2+. The specific inhibitor of cAMP and cGMP kinase catalytic subunits, H8, inhibits the activity. Sedimentation experiments show that the kinase may associate with but is not an intrinsic component of thick filaments. In C. elegans, phosphorylation by the thick filament associated activity of myosin and paramyosin is dependent upon the state of their assembly.

Identification and characterization of Drosophila melanogaster paramyosin

Paramyosin, a major structural component of thick filaments in invertebrates has been isolated, purified and characterized from whole adult Drosophila melanogaster extracts and a specific polyclonal antibody against it has been prepared. Paramyosin has been identified on the basis of several criteria, including molecular weight, α-helicity, species distribution, capability of fiber formation in vitro and sequence. We have used the immunopurified polyclonal antibody to isolate eight clones from a λgtl1 expression library of Drosophila 1 to 22 h embryo cDNA. The largest clone (pJV9) has been sequenced and encodes the coiled-coil region of D. melanogaster paramyosin that is 47% identical to Caenorhabditis elegans paramyosin. Indirect immunofluorescence in semi-thin sections of adult flies show fluorescence mainly in tubular muscle. Freshly prepared tubular myofibrils decorated with the immunoabsorbed antibody show the A region in the sarcomere as the specific localization of paramyosin. The amount of paramyosin in tubular synchronous muscles of insects appears to be five times higher than in fibrillar insect muscles. There are at least three paramyosin isoforms as shown by isoelectrofocusing separation. The more acidic and less abundant form is phosphorylated as shown by 32P in vivo labeling experiments in adult flies. The developmental pattern of expression of Drosophila paramyosin is presented. This mesoderm-specific protein, immunologically undetectable during gastrulation and early phases of germ band formation, progressively increases during organogenesis to the adult stage. Interestingly, it is also expressed as a major maternal product in the insoluble cytoskeletal fraction of the mature oocyte.