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T Obinata

Publications and source records attributed to T Obinata.

At least 55 records · Page 3Linked to original sources
PubMed1995

Effects of cofilin on actin filamentous structures in cultured muscle cells. Intracellular regulation of cofilin action.

The previous investigation (Abe et al. (1989) J. Biochem. 106, 696-702) suggested that cofilin is deeply involved in the regulation of actin assembly in developing skeletal muscle. In this study, to examine further the function of cofilin in living myogenic cells in culture, recombinant cofilin having extra Cys residues at the N terminus was produced in Escherichia coli and was labeled with tetramethylrhodamine-iodoacetamide (IATMR). When the cofilin labeled with IATMR (IATMR-cofilin) was introduced into myogenic cells, actin filaments in the cytoplasm or nascent myofibrils were promptly disrupted, and many cytoplasmic rods which contained both IATMR-cofilin and actin were generated. Sarcomeric myofibrillar structures were not disrupted but tropomyosin was dissociated from the structures by the exogenous cofilin, and the IATMR-cofilin became localized in I-band regions. 24 hours after the injection, however, the actin-cofilin rods disappeared completely and the IATMR-cofilin became diffused in the cytoplasm as endogenous cofilin. Concomitantly, actin filaments were recovered and tropomyosin was re-associated with sarcomeric I-bands. At this point, the IATMR-cofilin in the cells still retained the functional activity to form intranuclear actin-cofilin rods in response to stimulation by DMSO just as endogenous cofilin. FITC-labeled actin introduced into myogenic cells at first failed to assemble into filamentous structures in the presence of the exogenous cofilin, but was gradually incorporated into myofibrils with time. The drastic effects of the exogenous cofilin on actin assembly were suppressed by phosphatidylinositol 4,5-bisphosphate (PIP2). These results indicate that the exogenous cofilin is active and alters actin dynamics remarkably in muscle cells, but its activity in the cytoplasm gradually becomes regulated by the action of some factors including PIP2-binding.

Actin Depolymerizing Factors↗
PubMed1995

Assembly of cardiac C-protein during myofibrillogenesis in myogenic cells in culture.

To examine the function of C-protein, a thick filament-associated protein of vertebrate striated muscles, during myofibrillogenesis, the cDNA encoding chicken cardiac C-protein and the truncated cDNA were subcloned into a expression vector and introduced into mouse C2 myogenic cells. The expression and assembly of the C-protein was investigated by immunofluorescence methods. When the cDNA containing the entire open reading frame was introduced, in C2 myoblasts, the transiently expressed exogenous cardiac C-protein existed only diffusely in the cytoplasm, but it became localized in striated structures together with sarcomeric myosin heavy chains (MHC) in myotubes. To clarify the functional domains of C-protein, the cDNA constructs that lack the regions encoding the C-terminal immunoglobulin (Ig) C2 motif or the N-terminal Ig C2 motif were introduced into C2 cells to produce mutant proteins. The truncated chicken cardiac C-protein, which lacked the C-terminal Ig C2 motif, apparently lost the ability to bind to myosin filaments; the protein was not assembled into myofibrils but diffused in the cytoplasm even in the myotubes. The protein without N-terminal Ig C2 motif, however, was assembled into sarcomeric structures just as complete protein molecules. From these results, we conclude that 1) the assembly of sarcomeric MHC into myofibrils in myotubes is accompanied with that of cardiac C-protein, and 2) the C-terminal Ig C2 motif is necessary for assembly of cardiac C-protein in sarcomeric structures in the cytoplasm.

Amino Acid Sequence↗
PubMed1994

Characterization of a novel cofilin isoform that is predominantly expressed in mammalian skeletal muscle.

Cofilin is an actin-modulating protein of 20 kDa, which is widely distributed throughout muscle and non-muscle cells. By means of immunoblotting combined with two-dimensional gel electrophoresis, we found that two cofilin variants, muscle type (M-type) and non-muscle type (NM-type), exist in mammals, while a single isoform exists in chickens. During in vitro myogenesis of mouse C2 cells, expression of the M-type cofilin was upregulated. To better understand the nature of the M-type cofilin, we cloned cDNAs encoding M-type cofilin from the cDNA library of C2 myotubes and determined the entire sequence. The deduced peptide sequence contained a nuclear localization signal and a putative actin-binding sequence as reported in NM-type cofilin. The sequence showed 81% identity in the amino acid residues with the mouse NM-type cofilin sequence and, interestingly, higher homology (96% identity) with that of chicken cofilin. The mRNA encoding M-type cofilin, though it contains two variants that differ in the size of their 3'-non-coding sequences, was detected predominantly in heart, skeletal muscle, C2 myotubes, and testis by Northern blotting, while the mRNA for NM-type cofilin was seen in a variety of non-muscle tissues. The presence of the muscle type isoform of cofilin strongly suggests that cofilin is deeply involved in the regulation of actin function not only in non-muscle cells but also in muscle cells.

3T3 Cells↗
PubMed1994

Effect of thyroid hormone on developmental transition of myosin light chains during flounder metamorphosis.

Thyroidal control of the transition of myosin isoforms during flounder metamorphosis was examined by the administration of either only thiourea (TU), a potent inhibitor of thyroid hormone synthesis, or thyroxine (T4) together with TU into premetamorphic larvae. Immersion of premetamorphic larvae in 400 microM of TU inhibited the appearance of the adult-type DTNB (5,5'-dithio-bis-nitrobenzoic acid) light chain, LC2, whereas administration of T4 with TU induced a precocious appearance of LC2 and decreased the relative amount of the larval-type DTNB light chain, LC2*. TU was administered into juveniles just after completion of metamorphosis. The treatment did not affect the composition of the myosin light chains. These results suggest that thyroid hormone irreversibly turns on the switch for transition of the DTNB light chains from larval to adult type during metamorphosis of the flounder.

Animals↗
PubMed1993

Ascidian entactin/nidogen. Implication of evolution by shuffling two kinds of cysteine-rich motifs.

Entactin/nidogen, a major component of the basement membrane, has a domain structure comprising three globular domains, and thread-like and rod-like domains connecting them. It contains six epidermal-growth-factor-(EGF)-like motifs and one thyroglobulin-like motif. In the present study, ascidian entactin/nidogen has been identified by a monoclonal antibody technique. We prepared anti-(ascidian entactin/nidogen)IgG, named anti-AsEnt1, then cloned the cDNA of ascidian entactin/nidogen using anti-AsEnt1 as a probe, and determined its entire sequence. Mainly because the deduced amino acid sequence exhibited high similarity to mouse entactin and human nidogen, and because the antigen localized in basement membrane of ascidian body-wall muscle, we have concluded that the antigen anti-AsEnt1 corresponds to the ascidian entactin/nidogen homologue. The deduced amino acid sequence of ascidian entactin/nidogen clearly showed that the ascidian homologue also has a domain structure. However, the ascidian homologue lacked the thread-like domain, and the rod-like domain differed from that of mouse entactin in composition, consisting of two kinds of cysteine-rich motifs, that is, the EGF-like motif and the thyroglobulin-like motif. These results suggest that entactin/nidogen have evolved by modifying the domains, especially by shuffling the two kinds of cysteine-rich motifs.

Amino Acid Sequence↗
PubMed1993

Cytoplasmic localization and nuclear transport of cofilin in cultured myotubes.

We used immunofluorescence methods to examine the cellular distribution of cofilin in chicken myotubes in primary culture. Cofilin showed mainly diffuse distribution in the cytoplasm except for rather strong staining around the nuclei and faint striated patterns along myofibrils, but did not stain inside the nuclei. Neither stress fiber-like structures nor myofibrils were clearly stained. In the presence of 10% dimethyl sulfoxide (DMSO), intranuclear actin-cofilin rods, which were composed of alpha-actin isoform and cofilin, were formed in all the nuclei of individual myotubes. In the cofilin sequence, a putative nuclear localization signal (NLS) was observed. We examined the NLS activity of this portion by using a synthetic peptide corresponding to the putative NLS. When the NLS peptide conjugated with bovine serum albumin was microinjected into the cytoplasm of myotubes, it was rapidly accumulated into the nuclei. The same result was obtained with in vitro a nuclear protein import assay system with digitonin-permeabilized myotubes. Therefore, we suggest that this portion is responsible for the nuclear transport of cofilin. In myotubes, the majority of cofilin was present in an unphosphorylated form and this form remained unchanged after the DMSO treatment. Thus, we suggest that the phosphorylation of cofilin itself is not directly involved in its nuclear transport at least in myotubes.

Actin Depolymerizing Factors↗
PubMed1993

Colocalization of ADF and cofilin in intranuclear actin rods of cultured muscle cells.

Immunofluorescence microscopy revealed that two actin-binding proteins of low molecular weight with different functional activity, ADF and cofilin, are transported into nuclei of cultured myogenic cells to form rod structures there together with actin, when the cells were incubated in medium containing dimethylsulfoxide. In most cases, ADF and cofilin colocalized in the same nuclear actin rods, but ADF appeared to predominate in mononucleated cells, while cofilin was present in multinucleated myotubes. In some mononucleated cells, the nuclear actin rods were composed of ADF and actin but devoid of cofilin. An ADF homologue in mammals, destrin, was also translocated into nuclear actin rods under similar conditions. As a nuclear transport signal sequence exists in cofilin and ADF but not in actin, ADF and/or cofilin may be responsible for the nuclear import of actin in myogenic cells under certain conditions.

Actin Depolymerizing Factors↗
PubMed1993

Increased expression of cofilin in dystrophic chicken and mouse skeletal muscles.

A monoclonal antibody (McAb) specific for actin depolymerizing factor (ADF) was prepared. With this and previously prepared anti-cofilin McAb (MAB-22) and other antibodies, the expression of cofilin and ADF in the muscles of dystrophic (NH-413) chicken and dystrophic (C57BL/6J dy/dy) mice was compared with that in normal control animals by immunoblotting and immunocytochemical methods. Since cofilin expression is down-regulated during normal postnatal development of skeletal muscles [Abe et al. (1989) J. Biochem. 106, 696-702], cofilin was detected in the breast (pectoralis) muscle of normal adult chicken and the leg (femoris and tibialis anterior) muscles of normal mice only at a low level. ADF was not detectable in adult skeletal muscles. However, a significant increase of cofilin amount, but not of ADF amount, was observed in these muscles of the dystrophic animals, when the symptom of muscular dystrophy became evident. In order to localize cofilin in individual muscle fibers, serial cryosections of the dystrophic chicken muscle were examined with anti-cofilin antibody (MAB-22). The antibody stained cells of different size in the dystrophic muscle, indicating that cofilin expression was induced in the regenerating muscle cells as well as in the pre-existing myofibers. We suggest that cofilin is involved in disassembly or reorganization of actin in the dystrophic muscle.

Actin Depolymerizing Factors↗
PubMed1991

Transfection of chicken skeletal muscle alpha-actinin cDNA into nonmuscle and myogenic cells: dimerization is not essential for alpha-actinin to bind to microfilaments.

alpha-Actinins from striated muscle, smooth muscle, and nonmuscle cells are distinctive in their primary structure and Ca2+ sensitivity for the binding to F-actin. We isolated alpha-actinin cDNA clones from a cDNA library constructed from poly(A)+ RNA of embryonic chicken skeletal muscle. The amino acid sequence deduced from the nucleotide sequence of these cDNAs was identical to that of adult chicken skeletal muscle alpha-actinin. To examine whether the differences in the structure and Ca2+ sensitivity of alpha-actinin molecules from various tissues are responsible for their tissue-specific localization, the cDNA cloned into a mammarian expression vector was transfected into cell lines of mouse fibroblasts and skeletal muscle myoblasts. Immunofluorescence microscopy located the exogenous alpha-actinin by use of an antibody specific for skeletal muscle alpha-actinin. When the protein was expressed at moderate levels, it coexisted with endogenous alpha-actinin in microfilament bundles in the fibroblasts or myoblasts and in Z-bands of sarcomeres in the myotubes. These results indicate that Ca2+ sensitivity or insensitivity of the molecules does not determine the tissue-specific localization. In the cells expressing high levels of the exogenous protein, however, the protein was diffusely present and few microfilament bundles were found. Transfection with cDNAs deleted in their 3' portions showed that the expressed truncated proteins, which contained the actin-binding domain but lacked the domain responsible for dimerization, were able to localize, though less efficiently in microfilament bundles. Thus, dimer formation is not essential for alpha-actinin molecules to bind to microfilaments.

Actin Cytoskeleton↗
PubMed1991

Thyroid hormone regulates developmental changes in muscle during flounder metamorphosis.

Morphological and biochemical changes in the muscular tissue of metamorphosing flounder were studied in relation to the regulatory role of thyroid hormone. Premetamorphic larvae were reared in seawater alone or seawater containing either thyroxine (T4) or an antithyroid drug (thiourea, TU). Histological changes in the muscle were examined and biochemical changes in the muscle proteins were evaluated by SDS-PAGE and immunoblotting for troponin T (TNT). The muscle tissue of premetamorphic larvae was characterized by abundant vacuoles and basophilic sarcoplasm. In control fish, the larval muscle transformed into the adult type during metamorphic climax; the fibers were filled with abundant myofibrils and the vacuoles disappeared. Analysis by SDS-PAGE showed that the bands at 41.5, 35.5, 34.0, 33.5, 25.5, 23.0, 20.0, and 19.0 kDa clearly increased in density from the climax stage. Premetamorphic larvae possessed two immunoreactive TNT isoforms of 41.5 and 34.0 kDa, the former being predominant. At the climax stage an additional isoform appeared at 33.5 kDa, and the 34.0- and 33.5-kDa TNT became predominant. The administration of T4 precociously induced these histological and biochemical changes in the muscle tissue of flounder larvae. In contrast, TU treatment inhibited these developmental changes in the larval muscle. Our results suggest that the developmental changes in the muscular tissue of metamorphosing flounder are regulated by thyroid hormone.

Animals↗
PubMed1991

Radixin, a barbed end-capping actin-modulating protein, is concentrated at the cleavage furrow during cytokinesis.

Radixin is a barbed end-capping actin-modulating protein which was first identified in isolated cell-to-cell adherens junctions from rat liver (Tsukita, Sa., Y. Hieda, and Sh. Tsukita, 1989. J. Cell Biol. 108:2369-2382). In the present study, we have analyzed the distribution of radixin in dividing cells. For this purpose, an mAb specific for radixin was obtained using chicken gizzard radixin as an antigen. By immunofluorescence microscopy with this mAb and a polyclonal antibody obtained previously, it was clearly shown in rat fibroblastic cells (3Y1 cells) that radixin was highly concentrated at the cleavage furrow during cytokinesis. Radixin appeared to accumulate rapidly at the cleavage furrow at the onset of furrowing, continued to be concentrated at the furrow during anaphase and telophase, and was finally enriched at the midbody. This concentration of radixin at the cleavage furrow was detected in all other cultured cells we examined: bovine epithelial cells (MDBK cells), mouse myeloma cells (P3 cells), rat kangaroo Ptk2 cells, mouse teratocarcinoma cells, and chicken fibroblasts. Furthermore, it became clear that the epitope for the mAb was immunofluorescently masked in the cell-to-cell adherens junctions. Together, these results lead us to conclude that radixin is present in the undercoat of the cell-to-cell adherens junctions and that of the cleavage furrow, although their respective molecular architectures are distinct. The possible roles of radixin at the cleavage furrow are discussed with special reference to the molecular mechanism of the actin filament-plasma membrane interaction at the furrow.

Animals↗
PubMed1990

Myogenin contains two domains conserved among myogenic factors.

Previously, three structurally related proteins, MyoD, myogenin, and Myf5, have been identified, and each of them was found to convert C3H10T1/2 fibroblasts to myoblasts when their respective cDNAs were expressed under the control of a viral promoter. Here, we describe the cloning and DNA sequencing of myogenin cDNAs from chicken and mouse. They encode polypeptides highly homologous to each other, but the polypeptide sequences we have obtained are not homologous in the carboxyl-terminal 70 amino acids with those previously reported for mouse, rat, and human because of a single base deletion in the previously reported cDNAs. Determination of genomic sequence coding for myogenin revealed that the mouse myogenin cDNA presented here corresponds to a correct transcript of the gene, and the nucleotide sequence of the previously reported cDNA is incorrect. A comparison of chicken and mouse myogenins with other myogenic regulatory factors, MyoD and Myf-5, identified a domain with an interesting feature located in the carboxyl terminus of these proteins in addition to the myc homology domain previously reported.

Amino Acid Sequence↗
PubMed1990

Sequence of cDNAs encoding actin depolymerizing factor and cofilin of embryonic chicken skeletal muscle: two functionally distinct actin-regulatory proteins exhibit high structural homology.

Two actin-regulatory proteins of 19 and 20 kDa are involved in the regulation of actin assembly in developing chicken skeletal muscle. They are homologous with actin depolymerizing factor (ADF) and cofilin, a pH-dependent actin-modulating protein, which were originally discovered in chicken and mammalian brain, respectively. In this study, full-length cDNA clones were isolated by screening a lambda gt11 cDNA library constructed from poly(A+) RNA of embryonic chicken skeletal muscle with the antibodies specific for each protein, and their complete sequences were determined. The chicken cofilin cDNA encoded a protein of 166 amino acids, the sequence of which had over 80% identity with that of porcine brain cofilin. The amino acid sequence of the ADF was 165 amino acids and showed about 70% identity with either chicken or mammalian cofilin, in spite of the fact that ADF and cofilin are functionally distinct. Like chicken and mammalian cofilin, ADF contained a sequence similar to the nuclear transport signal sequence of SV40 large T antigen. ADF and cofilin shared a hexapeptide identical with the amino-terminal sequence of tropomyosin as well as the regions homologous to other actin-regulatory proteins, including depactin, gelsolin, and profilin. The overall nucleotide sequences and Southern blot analysis of genomic DNA, however, indicated that the two proteins were derived from different genes.

Actin Depolymerizing Factors↗
PubMed1990

Detection of a fast isoform of C-protein with an antiserum directed against the N-terminal portion of dystrophin.

An antiserum raised against the N-terminal actin-binding portion of dystrophin cross-reacted with a 130-kDa protein in fast skeletal muscle. The results of purification and two-dimensional gel electrophoresis and its immunological properties demonstrated that this protein is identical to a 130-kDa basic isoform of fast-C-protein. These results suggest that the actin-binding domain of dystrophin shares one or more antigenic determinants with those of C-protein.

Antibodies↗
PubMed1990

Myogenesis and histogenesis of skeletal muscle on flexible membranes in vitro.

Primary muscle cell cultures consisting of single myocytes and fibroblasts are grown on flexible, optically clear biomembranes. Muscle cell growth, fusion and terminal differentiation are normal. A most effective membrane for these cultures is commercially available Saran Wrap. Muscle cultures on Saran will, once differentiated, contract vigorously and will deform the Saran which is pinned to a Sylgard base. At first, the muscle forms a two-dimensional network which ultimately detaches from the Saran membrane allowing an undergrowth of fibroblasts so that these connective tissue cells completely surround groups of muscle fibers. A three-dimensional network is thus formed, held in place through durable adhesions to stainless steel pins. This three-dimensional, highly contractile network is seen to consist of all three connective tissue compartments seen in vivo, the endomysium, perimysium and epimysium. Finally, this muscle shows advanced levels of maturation in that neonatal and adult isoforms of myosin heavy chain are detected together with high levels of myosin fast light chain 3.

Animals↗
PubMed1990

Characterization of C-protein isoforms expressed in developing, denervated, and dystrophic chicken skeletal muscles by two-dimensional gel electrophoresis.

C-Proteins in developing, denervated, and dystrophic chicken skeletal muscles were examined by means of two-dimensional (2D) gel electrophoresis in combination with immunoblotting. In this analysis, the electrophoresis system which was devised by Hirabayashi (Anal. Biochem. 117, 443-451, 1981) provided excellent resolution; three C-protein variants, one fast-type (Cf) and two slow-types (CS3 and CS4) with different Mrs and pIs, were distinguished on a 2D gel. In the neonatal breast muscle, both Cf and CS3 were detected, but during postnatal development, CS3 disappeared from this muscle and Cf became only the C-protein isoform in the adult muscle. In posterior latissimus dorsi (PLD) muscle, both Cf and CS3 were similarly detected at the neonatal stage, but CS3 was replaced by CS4 as this muscle developed. When the breast and PLD muscles were denervated or suffered from muscular dystrophy, both CS3 and CS4 were co-expressed in these muscles in addition to Cf. These results definitely show that the C-protein isoform pattern varies during development and degeneration of chicken skeletal muscles, and in addition the dystrophic or denervated muscle differs from the neonatal muscle with regard to C-protein isoform expression. We suggest that chicken skeletal muscle degenerating due to denervation or muscular dystrophy does not simply recapture the nature of the neonatal muscle, but shifts in a somewhat different direction.

Aging↗
PubMed1989

Cross-reactivity of an antineurofilament antibody with a troponin-T isoform.

Histochemical examination has revealed that a monoclonal antibody raised against the 200-kilodalton (kd) subunit of neurofilament reacts not only with neural tissue but with type 1 and type 2C fibers of skeletal muscle. To identify the cross-reactive substance(s), rat soleus muscle, which includes type 1 and type 2C fibers, was separated into a soluble fraction and an insoluble myofibrillar fraction. Western blot analysis demonstrated that the antibody reacted with 30-kd and 36-kd polypeptides in the soluble fraction and with a 38-kd polypeptide in the insoluble fraction. We isolated a fraction that contained the 38-kd polypeptide from the insoluble fraction. The material in this fraction bound to F-actin. Two-dimensional electrophoresis of the fraction showed that the 38-kd polypeptide was part of the group of isoforms of troponin-T, which was identified by a monoclonal antibody developed against troponin-T. Thus, we conclude that this antineurofilament antibody recognizes one of the isoforms of troponin-T in type 1 and type 2C fibers.

Animals↗