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Biomedical subjects

K Sobue

Publications and source records attributed to K Sobue.

At least 91 records · Page 5Linked to original sources
PubMed1989

Alpha-actinins, calspectin (brain spectrin or fodrin), and actin participate in adhesion and movement of growth cones.

We have used biochemical and immunocytochemical techniques to investigate the possible involvement of membrane cytoskeletal elements such as alpha-actinin, calspectin (brain spectrin or fodrin), and actin in growth cone activities. During NGF-induced differentiation of PC12 cells, alpha-actinin increased in association with neurite outgrowth and was predominantly distributed throughout the entire growth cone and the distal portion of neurites. Filopodial movements were sensitive to Ca2+ flux. Two types of alpha-actinin, with Ca2(+)-sensitive and -insensitive actin binding abilities, were identified in the differentiated cells. Ca2(+)-sensitive alpha-actinin and actin filaments were concentrated in filopodia. The Ca2(+)-insensitive protein was distributed from the body of the growth cone to the distal portion of neurites, corresponding to the substratum-adhesive sites. The location of calspectin in growth cones was similar to that of the Ca2(+)-insensitive alpha-actinin. These results are consistent with the hypothesis that Ca2(+)-sensitive alpha-actinin and actin filaments are involved in Ca2(+)-dependent filopodial movement and Ca2(+)-insensitive alpha-actinin and calspectin are associated with adhesion of growth cones.

Actinin↗
PubMed1989

The cytoskeletal architecture of the presynaptic terminal and molecular structure of synapsin 1.

We have examined the cytoskeletal architecture and its relationship with synaptic vesicles in synapses by quick-freeze deep-etch electron microscopy (QF.DE). The main cytoskeletal elements in the presynaptic terminals (neuromuscular junction, electric organ, and cerebellar cortex) were actin filaments and microtubules. The actin filaments formed a network and frequently were associated closely with the presynaptic plasma membranes and active zones. Short, linking strands approximately 30 nm long were found between actin and synaptic vesicles, between microtubules and synaptic vesicles. Fine strands (30-60 nm) were also found between synaptic vesicles. Frequently spherical structures existed in the middle of the strands between synaptic vesicles. Another kind of strand (approximately 100 nm long, thinner than the actin filaments) between synaptic vesicles and plasma membranes was also observed. We have examined the molecular structure of synapsin 1 and its relationship with actin filaments, microtubules, and synaptic vesicles in vitro using the low angle rotary shadowing technique and QF.DE. The synapsin 1, approximately 47 nm long, was composed of a head (approximately 14 nm diam) and a tail (approximately 33 nm long), having a tadpole-like appearance. The high resolution provided by QF.DE revealed that a single synapsin 1 cross-linked actin filaments and linked actin filaments with synaptic vesicles, forming approximately 30-nm short strands. The head was on the actin and the tail was attached to the synaptic vesicle or actin filament. Microtubules were also cross-linked by a single synapsin 1, which also connected a microtubule to synaptic vesicles, forming approximately 30 nm strands. The spherical head was on the microtubules and the tail was attached to the synaptic vesicles or to microtubules. Synaptic vesicles incubated with synapsin 1 were linked with each other via fine short fibrils and frequently we identified spherical structures from which two or three fibril radiated and cross-linked synaptic vesicles. We have examined the localization of synapsin 1 using ultracryomicrotomy and colloidal gold-immunocytochemistry of anti-synapsin 1 IgG. Synapsin 1 was exclusively localized in the regions occupied by synaptic vesicles. Statistical analyses indicated that synapsin 1 is located mostly at least approximately 30 nm away from the presynaptic membrane. These data derived via three different approaches suggest that synapsin 1 could be a main element of short linkages between actin filaments and synaptic vesicles, and between microtubules and synaptic vesicles, and between synaptic vesicles in the nerve terminals.(ABSTRACT TRUNCATED AT 400 WORDS)

Actins↗
PubMed1989

Immunochemical evidence that myosin I heavy chain-like protein is identical to the 110-kilodalton brush-border protein.

In a previous study, we identified a new mammalian myosin heavy chain, termed myosin I heavy chain-like protein (MIHC), by molecular cloning of a bovine intestinal cDNA clone. In this investigation, we examined the relationship between MIHC and the 110-kDa intestinal brush-border protein, which possesses a myosin-like ATPase activity. We raised antibodies against a chemically synthesized oligopeptide representing a part of the MIHC sequence. These antibodies reacted specifically in immunoblots with the 110-kDa protein in both purified 110-kDa protein-calmodulin complex and crude microvillar protein extracts. Staining of tissue sections with these antibodies was specifically localized to the brush-border microvilli of small intestines, indicating an identical cellular localization for both MIHC and the 110-kDa protein. Furthermore, analysis of the MIHC sequence revealed two putative calmodulin-binding sites, which is consistent with the fact that the 110-kDa protein forms a complex with calmodulin. These results strongly support the conclusion that MIHC is identical to the 110-kDa protein and suggest that not only the conventional myosin system but also the MIHC (110-kDa protein)-calmodulin complex may play an important role in ATP-dependent and Ca2+-induced brush-border contraction.

Acanthamoeba↗
PubMed1989

Localization of 4.1 related proteins in cerebellar neurons.

Localization of 4.1 related proteins in neurons was studied with immunofluorescence microscopy and with immunoelectron microscopy on ultrathin cryosections. In rat cerebellum, 4.1 immunoreactive proteins were demonstrated in Purkinje cell bodies, dendrites and other neurons in the cerebellar cortex. Some glial cells showed staining, but no labeling was found in myelinated axons of the white matter and of the glomeruli in the granule cell layer. At the ultrastructural level, the 4.1 related proteins were localized mainly in the cytoplasmic matrix, while some labeling was found underneath the plasma membrane. To determine whether 4.1 related proteins in neuronal cytoplasm exist as part of the cytoskeleton or not, PC12 cells cultured in the presence of nerve growth factor were stained with the anti-4.1 antibody. Since cytoplasmic staining was retained after detergent treatment, the 4.1 related proteins seem to exist as a component of the neural cell cytoskeleton. Localization of 4.1 related proteins during the postnatal development of the cerebellum was also studied. In Purkinje cells, localization of 4.1 related proteins changed according to the stages of the postnatal development. The present data suggest that 4.1 related proteins in neurons localized mainly in the cytoplasm and may play some role in organizing cytoskeletal networks in the cytomatrix. Their distribution is developmentally regulated in some neurons, possibly in relationship to their maturation in the cytoskeleton.

Animals↗
PubMed1988

Localization of pp60c-src in growth cone of PC12 cell.

By immunocytochemical and biochemical techniques, we observed the localization and expression of pp60c-src in nerve growth factor (NGF)-treated PC12 cells. Immunostaining of pp60c-src is detected in the neuronal soma and the tips of neurites (growth cones). Immunofluorescence in the neurites is less significant. High-resolution microscopy reveals that the location of pp60c-src in growth cone is in good agreement with the adhesive site of growth cone to the substratum. The pp60c-src kinase activity and the pp60c-src protein level increase 3.1- to 3.5-fold and 2.0-fold during differentiation of PC12 cells, respectively. The pp60c-src levels in the neurite fraction are also higher than those in the neuronal soma fraction. These results support the immunocytochemical finding that pp60c-src is localized in growth cones of differentiated PC12 cells. Furthermore, we discuss the possible role of pp60c-src in growth cone.

Adrenal Gland Neoplasms↗
PubMed1988

Immunohistochemical localization of a membrane-associated, 4.1-like protein in the rat visual cortex during postnatal development.

Expression and localization of a membrane-associated protein, an analog of erythrocyte protein 4.1, in the visual cortex were immunohistochemically studied in the rat, ranging in age from newborn to adult. In the adult, dendrites and somas of layer V pyramidal cells were stained by the antiprotein 4.1 antibody. In most of these immunoreactive neurons, the plasma membrane seemed to be preferentially stained. Neurons located in layers II and III of the cortex were only faintly stained, and those in layers IV and VI were not stained. At birth, the immunoreactivity was already present in pyramidal cells located in the upper part of the cortical subplate. Immature neurons located in the cortical plate were not stained by the antibody, suggesting that the 4.1-like protein is expressed only in the neurons that have differentiated or are differentiating. At postnatal days 2-8, immunoreactive neurons were dramatically increased in layers V and VI and intense labeling was seen at the apical dendrites of layer V pyramidal cells. Most of the stained processes of these and other neurons showed a sign of rapid dendritic growth, i.e., growth cones and filopidia. At days 10-17, the basal dendrites of pyramidal cells in layers II and III became detectable, although still slender. At days 20-37, these dendrites in layers II, III, and V became intensely immunoreactive, and dendritic spines were visualized by the antibody. Throughout all the ages, axons of neurons and neuroglia were not stained by the antibody. Also, most of the neurons in layer IV of the cortex were not immunoreactive. These results suggest that the 4.1-like protein is abundantly expressed in growing parts of the dendrites and spines. A hypothesis that this protein may play a role in synaptic plasticity in the developing visual cortex is discussed.

Aging↗
PubMed1988

Caldesmon: a common actin-linked regulatory protein in the smooth muscle and nonmuscle contractile system.

Caldesmon was originally purified from gizzard smooth muscle as a major calmodulin-binding protein which also interacts with actin filaments. It has an alternative binding ability to either calmodulin or actin filaments depending upon the concentration of Ca2+ ("flip-flop binding"). Two forms of caldesmon (Mr's in the range of 120-150 kDa and 70-80 kDa) have been demonstrated in a wide variety of smooth muscles and nonmuscle cells. Immunohistochemical studies suggest that caldesmon is colocalized with actin filaments in vivo. Considering its abundance, the Ca2+-dependent flip-flop binding ability to either calmodulin or actin filaments, and its intracellular localization, caldesmon is expected to be involved in contractile events. Recent results from our laboratory have led to the conclusion that caldesmon regulates the smooth muscle and nonmuscle actin-myosin interaction and the smooth muscle actin-high Mr actin-binding protein (ABP or filamin) interactin in a flip-flop manner. It might function in cell motility by regulating the contractile system.

Animals↗
PubMed1988

Tumor promoter induces reorganization of actin filaments and calspectin (fodrin or nonerythroid spectrin) in 3T3 cells.

We have used immunofluorescence, differential-interference-contrast, and interference-reflection microscopy to examine the translocation of actin filaments and calspectin (fodrin or nonerythroid spectrin) in 3T3 cells induced by phorbol 12-myristate 13-acetate (PMA). The two cytoskeletal proteins were observed to localize in dot structures that corresponded to the cell-substratum contact sites (focal contact) of the cytoplasmic surface of the plasma membrane. The induction of these cytoskeletal changes was specific for tumor promoters. High-resolution microscopy revealed that calspectin was intensely concentrated in ring-like structures surrounding actin dots. It was also located within the areas of actin dots, but to a lesser extent. Trifluoperazine and other phenothiazine derivatives inhibited the formation of those dot structures that appeared after the addition of PMA. Some serine protease inhibitors were also demonstrated to influence cytoskeletal changes by PMA. Our results provide evidence that calspectin is closely associated with actin filaments in dot structures induced by PMA. Possible mechanisms for these cytoskeletal changes produced by PMA are discussed.

Actins↗
PubMed1988

Calmodulin fragments can not activate target enzymes.

Under conditions where nM level of calmodulin was able to show full activation of myosin light chain kinase and cyclic-nucleotide phosphodiesterase, the fragments of calmodulin at concentrations as high as 20 microM failed to activate these enzymes in the presence of Ca2+. The fragments tested were Ala1-Lys75 (F12), Ala1-Arg74 (F12'), Lys75-Lys148 (F34'), Met76-Lys148 (F34'), Asp78-Lys148 (F34), Ala1-Arg106 (F123), and His107-Lys148 (F4). Purification of the proteolytic fragments through HPLC was necessary to remove contaminant calmodulin. Among the fragments, that corresponding to the C-terminal half domain inhibited myosin light chain kinase activity with the inhibition constant of 13 microM. The integrated structure of calmodulin consisting of N-terminal half domain, C-terminal half domain, and the linker peptide was indispensable for the enzyme activation. We discuss the functions of the two structural domains (N-domain and C-domain) in the activation of various enzymes.

2',3'-Cyclic-Nucleotide Phosphodiesterases↗
PubMed1987

Comparison of the microtubule formation induced by cytosynalin and MAPs.

A comparison was made of cytosynalin- and MAPs-induced microtubule formation. Cytosynalin (a 35 kDa cytoskeleton-interacting and calmodulin-binding protein) facilitated more rapid and extensive microtubule formation than MAPs. Electron microscopic examination revealed that the cytosynalin-induced microtubules were 24 nm tubules surrounded by a periodic nap structure along their length. Consequently, the apparent diameter of microtubules was seen as 34 nm. Cytosynalin maximally bound to tubulin dimer at a molar ratio of 1:1. The effect of cytosynalin on the microtubule formation was found to be more potent than that of MAP2 or tau factor as determined by electron microscopy and cosedimentation assay.

Animals↗
PubMed1987

Caldesmon regulates the three-dimensional contraction (myosin-dependent contraction of the actin binding protein-induced actin gel).

We managed to develop a three-dimensional contractile model system using gizzard smooth muscle contractile elements. Phosphorylation of myosin was prerequisite for contraction. A high Mr actin-binding protein (ABP, or filamin), which cross-links actin filaments into a three-dimensional meshwork, was an essential factor for the three-dimensional contraction. Caldesmon suppressed contraction through the inhibition of the actin-ABP and actin-myosin interactions. Further, it was found that calmodulin could overcome the inhibitory effects of caldesmon on the above interactions, resulting in contraction. The possibility of this contractile model system being applied to nonmuscle contractile event is also discussed.

Actins↗
PubMed1987

The role of tropomyosin in the interactions of F-actin with caldesmon and actin-binding protein (or filamin).

The interactions of actin filaments with actin-binding protein (filamin) and caldesmon under the influence of tropomyosin were studied in detail using falling-ball viscometry, binding assay and electron microscopy. Caldesmon decreased the binding constant of filamin with F-actin. In contrast, the maximum binding ability of filamin to F-actin was decreased by tropomyosin. The filamin-induced gelation of actin filaments was inhibited by caldesmon. Tropomyosin also inhibited this gelation. The effect of caldesmon became stronger under the influence of tropomyosin. Furthermore, both caldesmon and tropomyosin additionally decreased the filamin binding to F-actin. From these results, caldesmon and tropomyosin appeared to influence filamin binding to F-actin with different modes of actin. In addition, there was no sign of direct interactions between filamin, caldesmon and tropomyosin as judged from gel filtration. Under the influence of caldesmon and tropomyosin, calmodulin conferred Ca2+ sensitivity on the filamin-induced gelation of actin filaments.

Actins↗
PubMed1987

Identification of a new 84/82 kDa calmodulin-binding protein, which also interacts with actin filaments, tubulin and spectrin, as synapsin I.

A new 84/82 kDa calmodulin-binding protein, which also interacts with actin filaments, tubulin and spectrin, was purified from the bovine synaptosomal membrane. The binding of calmodulin to this protein was Ca2+-dependent, and was inhibited by trifluoperazine, the association constant being calculated to be 2.2 X 10(6) M-1. Maximally, 1 mol of calmodulin bound to 1 mol of the purified protein. This protein was phosphorylated by both kinase II (Ca2+- and calmodulin-dependent kinase) and cyclic AMP-dependent kinase. In addition, antibody against this protein was demonstrated to have an immunological crossreactivity with synapsin I in the synaptosomal membrane.

Actins↗
PubMed1987

Immunocytochemical localization of calspectin (a non-erythroid spectrin-like protein) in thyroid glands of normal and TSH-treated rats.

The localization of calspectin (fodrin, a non-erythroid spectrin-like protein), which is known to bind calmodulin and F-actin, was detected in the thyroid gland of normal and TSH-treated rats by means of light-microscopic immunocytochemistry. Calspectin was demonstrated in the cytoplasm of the follicle epithelial cells especially along the baso-lateral plasma membrane in normal rats. In TSH-treated animals, in addition to the baso-lateral plasma membrane region, the apical plasma membrane region of the follicle epithelial cells also showed positive reaction to the immunostaining. These results suggest that calspectin, in conjugation with calmodulin and actin, play a role in the secretory activities including reabsorption activity of colloid of the follicle epithelial cell.

Animals↗
PubMed1987

Postnatal development of immunohistochemically localized spectrin-like protein (calspectin or fodrin) in the rat visual cortex: its excessive expression in developing cortical neurons.

Postnatal development of the expression and localization of a membrane-associated cytoskeletal protein, calspectin (fodrin or brain spectrin), in the visual cortex, was immunohistochemically studied in newborn to adult rats, by using an anti-calspectin antibody. At birth, calspectin-immunoreactivity was already present at the plasma membrane and in the cytoplasm of neurons which were mostly pyramidal cells located in the upper part of the cortical subplate. Immature neurons located in the cortical plate were not stained by the antibody, suggesting that calspectin is expressed only in neurons which have differentiated or are differentiating. At postnatal days 2 to 7, immunoreactive neurons were dramatically increased in layers V and VI and very intense labelling was seen in the apical dendrites of layer V pyramidal cells. Most of the stained processes of these and other neurons showed signs of rapid dendritic growth, i.e. non-terminal as well as terminal growth cones and filopodia. At days 10 to 17, dendrites of pyramidal cells in layers II and III became clearly detectable, although still slender. At days 24 to 34, the basal dendrites of pyramidal cells in layers II, III and V became intensely immunoreactive and dendritic spines were visualized by the antibody. In the adult, however, the calspectin immunoreactivity became very weak and spines were not recognizable. At all the ages, axons and neuroglia were unstained. Also, most of the neurons in layer IV of the cortex were not immunoreactive. These results suggest that calspectin is most abundantly expressed in growing parts of the dendrites and spines. A hypothesis that calspectin may play a role in synaptic plasticity in the developing visual cortex is discussed.

Aging↗
PubMed1987

Expression of high and low molecular weight caldesmons during phenotypic modulation of smooth muscle cells.

We investigated the expression of two molecular weight forms of caldesmon in a wide range of tissues and cells. The distribution of high molecular weight caldesmon (h-caldesmon, Mr 120,000-150,000) was restricted to smooth muscles where it was found in large quantity. The low molecular weight protein (l-caldesmon, Mr 70,000-80,000) was widely distributed in nonmuscle tissues and cells. Therefore, the expression of h-caldesmon might be much more specific to smooth muscles. We then examined the expressional changes of two caldesmons during phenotypic modulation of smooth muscle cells (SMCs). In developing gizzards, the expression of caldesmons switched from the l- to the h-form. Contrarily, the expression turned from h- to l-caldesmon in association with dedifferentiation of aortic SMCs in primary culture. In agreement with these observations, the levels of those mRNAs that direct the synthesis of both caldesmons were apparently in proportion to the quantities of protein, as determined by use of an in vitro translation system. In addition, h-caldesmon in smooth muscle-like BC3H1 cells increased in its amount with a concomitant reduction of l-caldesmon following serum-depleted and contact-inhibited cytodifferentiation. These results suggest that the expressional changes of two caldesmons are closely correlated with the phenotypic modulation of SMCs.

Animals↗