Biomedical subjects

T Obinata

Publications and source records attributed to T Obinata.

At least 19 recordsLinked to original sources

PubMed2001

Induction of ascidian peripheral neuron by vegetal blastomeres.

Ascidian tadpole larvae have a similar dorsal tubular nervous system as vertebrates. The induction of brain formation from a4.2-derived (a-line) cells requires signals from the A4.1-derived (A-line) cells. However, little is known about the mechanism underlying the development of the larval peripheral nervous system due to the lack of a suitable molecular marker. Gelsolin, an actin-binding protein, is specifically expressed in epidermal sensory neurons (ESNs) that mainly constitute the entire peripheral nervous system of the ascidian young tadpoles. Here, we address the role of cell interactions in the specification of ESNs using immunostaining with an anti-gelsolin antibody. Animal half (a4.2- and b4.2-derived) embryos did not give rise to any gelsolin-positive neurons, indicating that differentiation of ESNs requires signals from vegetal cells. Cell isolation experiments showed that A4.1 blastomeres induce gelsolin-positive neurons from a-line cells but not from b4.2-derived (b-line) cells. On the other hand, B4.1 blastomeres induce gelsolin-positive neurons both from b-line cells and a-line cells. This is in sharp contrast to the specification of brain cells which is not affected by the ablation of B4.1-derived (B-line) cells. Furthermore, basic fibroblast growth factor (bFGF) induced ESNs from the a-line cells and b-line cells in the absence of vegetal cells. Their competence to form ESNs was lost between the 110-cell stage and the neurula stage. Our results suggested that the specification of the a-line cells and b-line cells into ESNs is controlled by distinct inducing signals from the anterior and posterior vegetal blastomeres. ESNs in the trunk appear to be derived from the a8.26 blastomeres aligning on the edge of presumptive neural region where ascidian homologue of Pax3 is expressed. These findings highlight the close similarity of ascidian ESNs development with that of vertebrate placode and neural crest.

Animals↗

PubMed2001

Dynamic reorientation of cultured cells and stress fibers under mechanical stress from periodic stretching.

Cell lines derived from rat aorta and frog kidney were cultured on elastic membrane, and mechanical stress was given to the cells by stretching the membrane periodically. Cell reorientation oblique to the direction of stretching occurred as a result of the rapid withdrawal of cell periphery located along the direction of stretching and gradual extension of the cell membrane toward the direction oblique to the direction of stretching. Dynamic reorganization of stress fibers in living cells was visualized by labeling stress fibers with TRITC(3)-actin or EGFP-tagged moesin fragments with actin-binding ability. Stress fibers aligned in the direction of stretching disappeared soon after the start of stretching and then obliquely reoriented stress fibers appeared. The stretch-induced reorientation of cultured cells was suppressed by an inhibitor of stretch-activated (SA) cation channels and by a Ca(2+) chelator. However, the rearrangement of stress fibers was not affected by these agents. From these results, we suggest that Ca(2+) influx via SA channels is involved in stretch-induced cell reorientation but stress fiber rearrangement is independent of SA channels. Therefore, cell reorientation does not simply depend on the arrangement of stress fibers but may be controlled by some additional mechanism(s) which is regulated by calcium signaling.

Actins↗

PubMed2001

Functional involvement of Xenopus LIM kinases in progression of oocyte maturation.

LIM kinases (LIMK), including LIMK1 and LIMK2, are unique LIM-family proteins containing a catalytic (kinase) domain. These kinases phosphorylate an actin-depolymerizing factor, cofilin, involved in the regulation of actin-filament dynamics. An unanswered question is the in vivo function of LIMK and how they contribute to development. When we cloned Xenopus homologues of mammalian LIMK, Xlimk1 and Xlimk2, we found that their mRNA and products were abundantly expressed in oocytes. In addition, we obtained evidence for the functional involvement of Xlimk1/2 during oocyte maturation. The microinjection of Xlimk1/2 mRNA into progesterone-treated oocytes significantly inhibited the appearance of a white maturation spot (WMS), an indicator of entry into meiosis. In oocytes lacking a WMS, the organization and/or migration of the microtubule-derived precursor of the meiotic spindle was predominantly affected. We also found that the ectopic expression of Xlimk1/2 clearly prevented dephosphorylation (activation) of Xenopus cofilin (XAC) during oocyte maturation. Furthermore, co-injection of Xlimk1/2 with the constitutively active type of XAC overcame the inhibitory effects by Xlimk1/2, suggesting that XLIMK-induced abnormality in oocyte maturation was mediated by XAC inactivation. Based on these findings, we propose that XLIMK is a putative regulator of cytoskeletal rearrangements during oocyte maturation, and the interaction between XLIMK activity and microtubule dynamics seems highly likely.

Amino Acid Sequence↗

PubMed2001

Changes in gelsolin expression during ascidian metamorphosis.

Gelsolin is an actin regulatory protein that is expressed in a wide variety of tissues and is especially abundant in muscle and blood cells. The role of gelsolin during structural reorganization of the body, such as during metamorphosis or regeneration, is poorly understood. We analyzed changes in gelsolin expression during ascidian embryogenesis and metamorphosis using nucleic acid probes and a monoclonal antibody (AS23) specific for ascidian gelsolin; our results indicated that gelsolin is maternally provided and that its de novo gene transcription is initiated during the neurula stage. In the larva, gelsolin was detectable in specific types of nerve cells, i.e. the adhesive papillae, motor neurons and epidermal sensory neurons. During metamorphosis, the expression of gelsolin changes markedly: the expression is suppressed in nerve tissues after tail resorption but is induced in mesodermal tissues. Gelsolin accumulated in mesenchyme cells until the onset of tail resorption, and following tail resorption, these cells migrated to the tunic and differentiated into tunic cells with many fine processes. Migration of the mesenchyme cells into the tunic was completely inhibited by treatment with cytochalasin B. Gelsolin was colocalized with actin in tunic cells, suggesting that it is involved in the rearrangement of actin filaments during cell locomotion or morphogenesis.

Animals↗

PubMed2001

Molecular cloning and characterization of neural activity-related RING finger protein (NARF): a new member of the RBCC family is a candidate for the partner of myosin V.

Activity-dependent synaptic plasticity has been thought to be a cellular basis of memory and learning. The late phase of long-term potentiation (L-LTP), distinct from the early phase, lasts for up to 6 h and requires de novo synthesis of mRNA and protein. Many LTP-related genes are enhanced in the hippocampus during pentyrenetetrazol (PTZ)- and kainate (KA)-mediated neural activation. In this study, mice were administered intraperitoneal injections of PTZ 10 times, once every 48 h, and showed an increase in seizure indexes. Genes related to plasticity were efficiently induced in the mouse hippocampus. We used a PCR-based cDNA subtraction method to isolate genes that are expressed in the hippocampus of repeatedly PTZ-treated mice. One of these genes, neural activity-related RING finger protein (NARF), encodes a new protein containing a RING finger, B-box zinc finger, coiled-coil (RBCC domain) and beta-propeller (NHL) domain, and is predominantly expressed in the brain, especially in the hippocampus. In addition, KA up-regulated the expression of NARF mRNA in the hippocampus. This increase correlated with the activity of the NMDA receptor. By analysis using GFP-fused NARF, the protein was found to localize in the cytoplasm. Enhanced green fluorescent protein-fused NARF was also localized in the neurites and growth cones in neuronal differentiated P19 cells. The C-terminal beta-propeller domain of NARF interacts with myosin V, which is one of the most abundant myosin isoforms in neurons. The NARF protein increases in hippocampal and cerebellar neurons after PTZ-induced seizure. These observations indicated that NARF expression is enhanced by seizure-related neural activities, and NARF may contribute to the alteration of neural cellular mechanisms along with myosin V.

Amino Acid Sequence↗

PubMed2001

Enhancement of branching efficiency by the actin filament-binding activity of N-WASP/WAVE2.

The actin-related protein (Arp) 2/3 complex is an essential regulator of de novo actin filament formation. Arp2/3 nucleates the polymerization of actin and creates branched actin filaments when activated by Arp2/3-complex activating domain (VCA) of Wiskott-Aldrich syndrome proteins (WASP family proteins). We found that the branching of actin filaments on pre-existing ADP filaments mediated by the Arp2/3 complex is twice as efficient when Arp2/3 was activated by wild-type neural WASP (N-WASP) or WASP-family verprolin-homologous protein (WAVE) 2 than when activated by the VCA domain alone. By contrast, there was no difference between wild-type N-WASP or WAVE2 and VCA in the branching efficiency on de novo filaments, which are thought to consist mainly of ADP-phosphate filaments. This increased branching efficiency on ADP filaments is due to the basic region located in the center of N-WASP and WAVE2, which was found to associate with ADP actin filaments. Actin filaments and phosphatidylinositol bisphosphate (PIP2) associate with N-WASP at different sites. This association of N-WASP and WAVE2 with actin filaments enhanced recruitment of Arp2/3 to the pre-existing filaments, presumably leading to efficient nucleation and branch formation on pre-existing filaments. These data together suggest that the actin filament binding activity of N-WASP and WAVE2 in the basic region increases the number of barbed ends created on pre-existing filaments. Efficient branching on ADP filaments may be important for initiation of actin-based motility.

Actin Cytoskeleton↗

PubMed2000

Expression of cofilin isoforms during development of mouse striated muscles.

Cofilin (CF) is an actin regulatory protein that plays a critical role in actin filament dynamics in a variety of cells. Two cofilin isoforms. muscle-type (M-CF) and nonmuscle-type (NM-CF) encoded by different genes, exist in mammals; in the adult, the former is predominantly expressed in muscle tissues, while the latter is distributed in various non-muscle tissues (Ono et al., 1994). In this study, we examined cofilin isoform expression during skeletal and cardiac muscle development in mice using cDNA probes and antibodies which distinguish the isoforms. We found that the expression of M-CF was initiated in terminally differentiated myogenic cells in both the myotome and limb buds. In myogenic cell cultures, its expression occurred coupled with myotube formation. NM-CF was expressed in developing skeletal and cardiac muscles but disappeared from skeletal muscle during postnatal development, while its expression persisted in the heart, even in adult mice. A similar situation was observed in the heart of other mammals. Thus, it is likely that the both cofilin isoforms are involved in the regulation of actin assembly during myofibrillogenesis. Only M-CF could be involved in actin dynamics in mature skeletal muscle, while both isoforms could be in the mature heart.

Actin Depolymerizing Factors↗

PubMed2000

Developmental relationship of myosin binding proteins (myomesin, connectin and C-protein) to myosin in chicken somites as studied by immunofluorescence microscopy.

The developmental relationship of myosin binding proteins (myomesin, connectin and C-protein) to myosin was studied in chicken cervical somites by immunofluorescence microscopy. Muscle and non-muscle myosins initially appeared as slender rods at the same sites, and then, fused to form non-striated fibrils. As muscle myosin formed striated structures (A bands), non-muscle myosin disappeared from this structure. Myomesin (reactive with monoclonal antibodies MyB4 and MyBB78) and connectin (carboxy terminal region, reactive with monoclonal antibody T51) were seen as dots in the center of these myosin rods. These proteins then formed characteristic mature striations on non-striated fibrils of myosin. Earlier alignment of these myosin binding proteins rather than myosin indicates that the correct assembly of these proteins seems to be related to the formation of initial myosin rods as well as subsequent linear and periodic alignment of myosin molecules to form early A bands. Connectin spots reactive with 9D10 were scattered around myosin rods/myomesin dots/connectin T51 dots. These spots may represent radiating connectin filaments from these rods/dots to link myosin rods to the I-Z-I structures of myofibrils to be incorporated. Since the slow isoform of C-protein formed its characteristic bands ("doublets") prior to H zone formation within A bands by myosin, this isoform may help to precisely align myosin filaments within the A band region. The presence of the slow, then the slow and the cardiac, and finally the co-existence of the slow and the fast isoforms of C-protein may interfere with the incorporation and co-polymerization of non-adult isoforms into myofibrils.

Animals↗

PubMed1999

Signaling from Rho to the actin cytoskeleton through protein kinases ROCK and LIM-kinase.

The actin cytoskeleton undergoes extensive remodeling during cell morphogenesis and motility. The small guanosine triphosphatase Rho regulates such remodeling, but the underlying mechanisms of this regulation remain unclear. Cofilin exhibits actin-depolymerizing activity that is inhibited as a result of its phosphorylation by LIM-kinase. Cofilin was phosphorylated in N1E-115 neuroblastoma cells during lysophosphatidic acid-induced, Rho-mediated neurite retraction. This phosphorylation was sensitive to Y-27632, a specific inhibitor of the Rho-associated kinase ROCK. ROCK, which is a downstream effector of Rho, did not phosphorylate cofilin directly but phosphorylated LIM-kinase, which in turn was activated to phosphorylate cofilin. Overexpression of LIM-kinase in HeLa cells induced the formation of actin stress fibers in a Y-27632-sensitive manner. These results indicate that phosphorylation of LIM-kinase by ROCK and consequently increased phosphorylation of cofilin by LIM-kinase contribute to Rho-induced reorganization of the actin cytoskeleton.

Actin Cytoskeleton↗

PubMed1999

Genomic organization, expression, and analysis of the troponin C gene pat-10 of Caenorhabditis elegans.

We have cloned and characterized the troponin C gene, pat-10 of the nematode Caenorhabditis elegans. At the amino acid level nematode troponin C is most similar to troponin C of Drosophila (45% identity) and cardiac troponin C of vertebrates. Expression studies demonstrate that this troponin is expressed in body wall muscle throughout the life of the animal. Later, vulval muscles and anal muscles also express this troponin C isoform. The structural gene for this troponin is pat-10 and mutations in this gene lead to animals that arrest as twofold paralyzed embryos late in development. We have sequenced two of the mutations in pat-10 and both had identical two mutations in the gene; one changes D64 to N and the other changes W153 to a termination site. The missense alteration affects a calcium-binding site and eliminates calcium binding, whereas the second mutation eliminates binding to troponin I. These combined biochemical and in vivo studies of mutant animals demonstrate that this troponin is essential for proper muscle function during development.

Amino Acid Sequence↗

PubMed1999

Differential expression of C-protein isoforms in developing and degenerating mouse striated muscles.

With the aim of clarifying the roles of C-protein isoforms in developing mammalian skeletal muscle, we cloned the complementary DNA (cDNAs) encoding mouse fast (F) and slow (S) skeletal muscle C-proteins and determined their entire sequences. Northern blotting with these cDNAs together with mouse cardiac (C) C-protein cDNA was performed. It revealed that in adult mice, C, F, and S isoforms are expressed in a tissue-specific fashion, although the messages for both F and S isoforms are transcribed in extensor digitorum longus muscle, which has been categorized as a fast muscle. In addition, although C isoform is expressed first and transiently during development of chicken skeletal muscles, C isoform is not expressed in mouse skeletal muscles at all through the developmental stages; S isoform is first expressed, followed by the appearance of F isoform. Finally, in dystrophic mouse skeletal muscles, the expression of S isoform is increased as it is in dystrophic chicken muscle. These observations suggest that mutations in C isoform (MyBP-C) do not lead to any disturbance in skeletal muscle, although they may lead to familial hypertrophic cardiomyopathy. We also suggest that the expression of S isoform may be stimulated in degenerating human dystrophic muscles.

Amino Acid Sequence↗

PubMed1999

BDM (2,3-butanedione monoxime), an inhibitor of myosin-actin interaction, suppresses myofibrillogenesis in skeletal muscle cells in culture.

During the initial phase of myofibrillogenesis in developing muscle cells, the majority of thin filaments lie parallel to, and exhibit correct polarity and spatial position with thick filaments, as in mature myofibrils. Since myosin is known to function as an accelerator of actin polymerization in vitro, it has been postulated that myosin-actin interaction is important in the initial phase of myofibrillogenesis. To clarify further the role of actin-myosin interaction in myofibril formation during development, BDM (2, 3-butanedione 2-monoxime), an inhibitor of myosin ATPase, was applied to primary cultures of skeletal muscle to inhibit myosin activity during myofibrillogenesis, and myofibril formation was examined. When 10 mM BDM was added to the myotubes just after fusion and the cultures were maintained for a further 4 days, cross-striated myofibrils were scarcely observed by fluorescence microscopy when examined by staining with antibodies to actin, myosin, troponin and alpha-actinin, whereas in the control myotubes not exposed to BDM, typical sarcomeric structures were detected. Electron microscopy revealed a disorganized arrangement of myofilaments and incomplete sarcomeric structures in the BDM-treated myotubes. Thus, formation of cross-striated myofibrils was remarkably suppressed in the BDM-treated myotubes. When the myotubes cultured in BDM-containing media were transferred to control media, sarcomeric structures were formed in 2-3 days, suggesting that the inhibitory effect of BDM on myotubes is reversible. These results suggest that actin-myosin interaction plays a critical role in the early process of myofibrillogenesis.

Actins↗

PubMed1999

XAIP1: a Xenopus homologue of yeast actin interacting protein 1 (AIP1), which induces disassembly of actin filaments cooperatively with ADF/cofilin family proteins.

We carried out affinity column chromatography using Xenopus ADF/cofilin (XAC), identified several polypeptides in oocytes specifically bound to this column with actin, and isolated a full-length cDNA clone for a 65 kDa protein in this fraction. The predicted amino acid sequence revealed that the 65 kDa protein has seven obvious WD repeats and exhibits striking homology with yeast actin interacting protein 1 (AIP1). Thus, we designated this protein Xenopus AIP1 (XAIP1). We purified XAIP1 from Xenopus oocytes, and its interaction with actin was characterized by a pelleting assay, photometrical analysis and electron microscopy. Although XAIP1 itself cosedimented with F-actin and increased unsedimented actin to some extent, it induced a rapid, drastic disassembly of actin filaments associated with XAC. Electron microscopic observation revealed that XAIP1 severs actin filaments in the presence of XAC. To elucidate the in vivo effects of XAIP1, the purified protein was injected into blastomeres at the two-cell stage. Although the localization of XAIP1 was similar to that of XAC, at the cortical cytoskeleton and diffusely in the cytoplasm, injection of a large amount of XAIP1 arrested development and abolished the strong cortical staining of both actin and XAC. From these results, we concluded that XAIP1 regulates the dynamics of the cortical actin cytoskeleton cooperatively with XAC in eggs.

Actin Depolymerizing Factors↗

PubMed1999

HP33: hepatocellular carcinoma-enriched 33-kDa protein with similarity to mitochondrial N-acyltransferase but localized in a microtubule-dependent manner at the centrosome.

Using a new subtraction method and chemically induced rat hepatocellular carcinomas, we identified a hepatocellular carcinogenesis and hepatocyte proliferation-related gene designated hp33 that encoded a 33-kDa protein. The predicted protein was similar to the bovine aralkyl N-acyltransferase and arylacetyl N-acyltransferase. HP33 was restrictively expressed in the liver and kidney, and its gene expression was stimulated in the regenerating liver as well as in hepatocellular carcinoma. Interestingly, it was demonstrated in various hepatic cells that HP33 was localized in regions surrounding the centrosome, where mitochondria were not concentrated. Moreover, its centrosomal localization was evident in the interphase but not in the mitotic phase of the cell cycle. The centrosomal localization of HP33 was dependent on microtubules, and ectopically expressed HP33 was seen at centrosomes even in fibroblasts, which do not exhibit a typical staining pattern of HP33. The centrosomal localization of HP33 became invisible by nocodazole treatment, whereas the mitochondrial staining pattern was not affected by it. In vitro cosedimentation experiments using purified microtubules indicated that HP33 bound to MTs directly and that its MT-binding ability was dependent on the C-terminal basic domain of the protein. These results suggest that, different from early predictions based on its primary structure, HP33 has a growth- and carcinogenesis-related function that may be independent of mitochondrial function.

Acyltransferases↗

PubMed1999

Organization of connectin/titin filaments in sarcomeres of differentiating chicken skeletal muscle cells.

Very long, elastic connectin/titin molecules position the myosin filaments at the center of a sarcomere by linking them to the Z line. The behavior of the connectin filaments during sarcomere formation in differentiating chicken skeletal muscle cells was observed under a fluorescent microscope using the antibodies to the N terminal (located in the Z line), C terminal (M line), and C zone (myosin filament) regions ofconnectin and was compared to the incorporation of alpha-actinin and myosin into forming sarcomeres. In early stages of differentiating muscle cells, the N terminal region of connectin was incorporated into a stress fiber-like structure (SFLS) together with alpha-actinin to form dots, whereas the C terminal region was diffusely distributed in the cytoplasm. When both the C and N terminal regions formed striations in young myofibrils, the epitope to the C zone of A-band region, that is the center between the A-I junction and the M-line, initially was diffuse in appearance and later formed definite striations. It appears that it took some time for the N and C terminal regions of connectin to form a regular organization in a sarcomere. Thus the two ends of the connectin filaments were first fixed followed by the specific binding of the middle portion onto the myosin filament during sarcomere formation.

Animals↗

PubMed1999

Detection of a sequence involved in actin-binding and phosphoinositide-binding in the N-terminal side of cofilin.

Cofilin is an actin-binding protein of low molecular weight which is widely distributed in eukaryotes and is deeply involved in the dynamics of actin assembly in the cytoplasm. The actin-binding ability of cofilin is inhibited by inositol phosphates (PIP2), and the PIP2- and actin-binding site(s) has been localized in residues W104-M115 of the cofilin primary sequence (Yonezawa et al. 1991 ). In the present study, in order to further clarify the functional domains in cofilin molecule, we constructed expression vectors containing cDNAs of different size with deletion at the 3'-region of the open reading frame. The truncated cofilin molecules produced in E. coli were purified and examined for their actin-binding and PIP2-binding ability. We found that the truncated cofilin molecule without C-terminal residues #100-#166 including the previously-described actin-binding site could be cross-linked with actin by EDC, a zero-length cross-linker. In addition, these truncated peptides as well as synthetic peptides corresponding to the N-terminal sequence of cofilin suppressed the inhibitory action of PIP2 on actin-cofilin interaction. These results strongly suggest that additional actin- and PIP2-binding sites exist in the N-terminal region of cofilin.

Actin Depolymerizing Factors↗

PubMed1998

Functional characteristics and the complete primary structure of ascidian gelsolin.

The body wall of the ascidian is composed of unusual multi-nucleated smooth muscle cells enriched with thin actin filaments containing troponin-tropomyosin which run along the longitudinal cell axis without being organized into striated structures. We purified an actin-binding protein of 80 kDa, tentatively termed 80K protein, from the body wall muscle of ascidian, Halocynthia roretzi, and characterized the functional properties and molecular structures. In the presence of Ca2+, the 80K protein accelerated the initial phase of actin polymerization, namely the nucleation process, decreased the level of polymerization at the steady state, caused marked reduction in viscosity of an F-actin solution, and fragmented F-actin filaments, while in the absence of Ca2+, it remained associated with F-actin without severing the filaments. The interaction of the 80K protein with actin was inhibited by phosphatidylinositol 4,5-bisphosphate (PIP2). When actin was polymerized in the presence of acrosome actin bundles from horseshoe crab sperm, the 80K protein inhibited the growth of actin filaments at the barbed end but not at the pointed end, indicating that the 80K protein functions as a barbed-end capping protein. In order to characterize the molecular structure of the 80K protein, cDNAs encoding this protein were isolated from the lambda gt11 cDNA library of the ascidian muscle by using a monoclonal antibody (AS23) specific for this protein and the entire sequence was determined. The deduced peptide sequence showed about 44% homology in amino acid residues with the human gelsolin sequence, and in addition, 6 repeating segments were observed in the sequence of the 80K protein as has been described in the gelsolin sequence. These results indicate strongly that the 80K protein belongs to the gelsolin family.

Actins↗

PubMed1998

Differentiation of muscle-specific proteins in chicken somites as studied by immunofluorescence microscopy.

Fluorescence microscopy of chicken cervical somites revealed that muscle-specific proteins began to appear at stage 11 (Hamburger and Hamilton numbering), and the onset of the expression of all the proteins examined in the present study had occurred by stage 17. Muscle proteins were classified into six groups according to the stage of their appearance. Since all these proteins were expressed before emergence of nerve fibers in myotomes, switching-on of their synthesis does not seem to require neuronal influence. However, since isoproteins other than adult muscle types disappeared and diversification of muscle fiber types occurred coordinately with the clustering of acetylcholine receptors in cervical muscles, switching-off of the synthesis of the nonadult isoforms might have been accelerated by the formation of functional neuromuscular junctions. The absence of nebulin and C-protein in early stages seems to indicate that these proteins are not required for the initial assembly of myofilaments and/or myofibrils. Further, this absence might be considered to facilitate exchangeabilities of proteins in nascent myofibrils, thereby changing the isoforms to adult types.

Animals↗