Biomedical subjects

T Shirao

Publications and source records attributed to T Shirao.

At least 37 records · Page 2Linked to original sources

PubMed1996

Modulatory role of drebrin on the cytoskeleton within dendritic spines in the rat cerebral cortex.

Morphological changes in the dendritic spines have been postulated to participate in the expression of synaptic plasticity. The cytoskeleton is likely to play a key role in regulating spine structure. Here we examine the molecular mechanisms responsible for the changes in spine morphology, focusing on drebrin, an actin-binding protein that is known to change the properties of actin filaments. We found that adult-type drebrin is localized in the dendritic spines of rat forebrain neurons, where it binds to the cytoskeleton. To identify the cytoskeletal proteins that associated with drebrin, we isolated drebrin-containing cytoskeletons using immunoprecipitation with a drebrin antibody. Drebrin, actin, myosin, and gelsolin were co-precipitated. We next examined the effect of drebrin on actomyosin interaction. In vitro, drebrin reduced the sliding velocity of actin filaments on immobilized myosin and inhibited the actin-activated ATPase activity of myosin. These results suggest that drebrin may modulate the actomyosin interaction within spines and may play a role in the structure-based plasticity of synapses.

Actins↗

PubMed1996

Stabilization of adhesion plaques by the expression of drebrin A in fibroblasts.

The expression of drebrin A was induced in mouse fibroblasts (L cells) after transformation of cells with a vector that carried cDNA for rat drebrin A (developmentally regulated brain protein A) under the control of the promoter of the gene for metallothionein-I. When drebrin was expressed in the transformed cells (MTI-5 cells), the organization of actin filaments changed such that stress fibers were converted to a mesh-like structure. After subsequent treatment with 5 micrograms/ml cytochalasin D (a reagent that depolymerizes actin filaments), MTI-5 cells maintained their shape, while cells of a drebrin-negative cell line, MTI-11, formed retraction processes. Simultaneously, actin filaments changed into patchy dot-like aggregates in the cytoplasm of both MTI-5 and MTI-11 cells. These aggregates are known as cytoplasmic pools. In MTI-5 cells, adhesion plaques that were resistant to treatment with cytochalasin D appeared upon expression of drebrin. These adhesion plaques were immunostained with vinculin-specific antibodies, while those in MTI-11 cells were hardly immunostained. The amount of vinculin in MTI-5 cells increased in parallel with increase in the level of drebrin. These results suggest that expression of drebrin A induces changes in the assembly of actin filaments and adhesion plaques, with resultant modulation of cellular adhesion to the substratum.

Blotting, Western↗

PubMed1996

Disappearance of actin-binding protein, drebrin, from hippocampal synapses in Alzheimer's disease.

The actin-binding protein drebrin is localized in postsynaptic terminals in adult brain and is considered to be related to synaptic plasticity. Immunocytochemical study demonstrated that widespread drebrin immunoreactivity was observed in hippocampal formations of control human brains, while Alzheimer's disease (AD) brains showed remarkable reductions in this immunoreactivity. Western blot analysis demonstrated that drebrin E (116kD) as well as drebrin A (125 kD) presented in adult human brains, and that these isoforms were decreased in parallel in AD brains. On the other hand, synaptic vesicle-specific 38-kD protein (SVP-38), a presynaptic marker was not so changed in AD brains in comparison with control brains by both techniques. These findings suggest that drebrin E and A in the adult human brain may be co-localized in postsynaptic terminals, and that drebrin may be more sensitive as a marker of synaptic damage than SVP-38, and that the disappearance of drebrin may contribute to the pathogenesis of memory disturbance in AD.

Aged↗

PubMed1996

Inhibition by drebrin of the actin-bundling activity of brain fascin, a protein localized in filopodia of growth cones.

The purification of drebrin, an actin-binding protein that is specifically expressed in embryonic rat brain, was described previously. During the purification of drebrin, we found that an actin-binding protein of 54 kDa was also expressed at high levels in embryonic brain, and this protein was identified by immunoblotting as fascin. To explore the roles of fascin in brain development, we purified fascin from brains of infant rats and characterized it. We found that the actin-binding activity of fascin was strongly inhibited by drebrin. Fascin caused formation of actin bundles, a process that was inhibited in the presence of drebrin, as confirmed by electron microscopy and a low-speed centrifugation assay. In PC12 cells, fascin was localized in the filopodia of growth cones, whereas drebrin was localized in the basal region of growth cones. Our results suggest that fascin might play an important role in the organization of actin in filopodia and that this organization might be regulated by drebrin.

Actins↗

PubMed1995

K252a, a potent inhibitor of protein kinases, inhibits the migration of cerebellar granule cells in vitro.

In order to elucidate the cellular mechanisms of migrating neurons, we developed an assay system in vitro, using an aggregation culture of developing granule cells from the rat cerebellum. This assay system allowed us to eliminate the effects of various factors other than neurons and to examine the direct effects of individual molecules on neuronal migration. In this assay system, we examined the effects of several protein kinase inhibitors on cerebellar granule cell migration, and revealed that K252a, an inhibitor of protein kinases and of the actions of neurotrophins, inhibited the migration. Within 5 min after the addition of K252a to the culture medium, most of the migrating spindle-shaped cells changed into non-migrating large and polygonal cells, which had many microspikes. Staining with rhodamine-phalloidin revealed the appearance of actin bundles that resembled stress fibers within these large cells. On the other hand, extension of neurites was not severely inhibited by the addition of K252a. These results suggest that the migration is regulated by a different mechanism from that of neurite growth.

Animals↗

PubMed1995

Effect of a neuron-specific actin-binding protein, drebrin A, on cell-substratum adhesion.

Drebrin A expression was induced in non-neuronal L cells via transfection with a vector containing the cDNA of rat drebrin A. Following treatment with colcemid (5 micrograms/ml) and cytochalasin D (0.5 micrograms/ml), most L cells collapsed into round cells, while drebrin expressing cells were resistant to the treatment, keeping their cell shapes. Simultaneously, actin filaments and microtubules were disrupted in both cell lines. By quantitative analysis, in the presence of cytochalasin D, the extent of cell spreading and cell attachment in drebrin expressing cells was significantly higher than that in control cells. These results suggest that drebrin A modulates cell-substratum adhesion.

Actins↗

PubMed1995

The roles of microfilament-associated proteins, drebrins, in brain morphogenesis: a review.

The cytoskeleton has been suggested to be one of the important endogenous factors that control neuronal morphogenesis. Analysis of the developmental changes in the protein composition of the brain led to the discovery of novel developmentally regulated actin-binding proteins, drebrins. Drebrins exhibit a number of characteristics that one might expect for an intracellular regulator of neuronal morphogenesis. Drebrin has three isoforms and the mRNA of each isoform is transcribed from a single gene through alternative RNA splicing mechanisms. The expression pattern of each isoform is regulated spatially and temporally in the developing brain. Drebrin and tropomyosin competitively bind to actin filaments, and the exclusion of tropomyosin from actin filaments by overexpression of drebrin in fibroblasts results in the appearance of thick, curving bundles of actin filaments, and the formation of cell processes. Taken together, these data indicate that drebrin is one of the intracellular regulators of the neuronal morphogenesis.

Actins↗

PubMed1994

Drebrin, a development-associated brain protein from rat embryo, causes the dissociation of tropomyosin from actin filaments.

Drebrin is a development-associated neuroprotein whose cDNA into fibroblasts causes the formation of dendrite-like structures (Shirao, T., Kojima, N., and Obata, K. (1992) Neuroreport 3, 109-112). To explore molecular functions of drebrin during brain development, we purified drebrin from brains of rat embryos. Drebrin bound to actin filaments at a stoichiometry of 1:5 with a dissociation constant (Kd) of 1.2 x 10(-7) M. It strongly inhibited the actin binding activity of tropomyosin. Excess amounts of tropomyosin also inhibited the drebrin binding to actin filaments, suggesting that drebrin and tropomyosin competitively bind to actin filaments. Further, drebrin inhibited not only the actin binding activity of alpha-actinin but also the actin cross-linking activity of alpha-actinin. Gene transfection experiments revealed that tropomyosin was dissociated from actin filaments in drebrin-overexpressing fibroblasts. Thus we hypothesize that drebrin may destabilize actin filaments by dissociating tropomyosin and alpha-actinin from actin filaments, resulting in the formation of axon and dendrites during neuronal development.

Actin Cytoskeleton↗

PubMed1994

Actin-binding protein, drebrin, accumulates in submembranous regions in parallel with neuronal differentiation.

Drebrins are developmentally regulated actin-binding proteins. In this study, we analyzed subcellular distribution of drebrin E in neuroblastoma cells (SH-SY5Y) in culture, especially in terms of its relationship to actin filaments. In undifferentiated cells, drebrin E was scattered as flocculus small dots along the stress fibers and also accumulated at adhesion plaques. In parallel with the neuronal differentiation following retinoic acid treatment, drebrin E was accumulated, accompanying filamentous (F) actin, in the submembranous cortical cytoplasm. Similar submembranous localization of drebrins was observed in primary cultured neurons. In the presence of drebrin E F-actin was more stable against cytochalasin D than F-actin lacking drebrin E. These results suggest that drebrin E plays a role in neuronal morphological differentiation by changing its subcellular localization with stabilized F-actin.

Actins↗

PubMed1994

Formation of thick, curving bundles of actin by drebrin A expressed in fibroblasts.

Drebrin A is a neuron-specific protein, the expression of which is regulated during development. Upon transfection of fibroblasts with drebrin A cDNA, the protein is expressed at high levels in fibroblasts and the outgrowth of highly branched, neurite-like cell processes is induced. In this report, we describe a biochemical examination of the binding of drebrin A to actin filaments. We also demonstrate by an immunocytochemical method that, when drebrin A is expressed in transfected cells, it binds to actin filaments and is concentrated in cell processes. Furthermore, we provide evidence that thick, curving bundles of actin together with drebrin are formed in some of the transfected cells. Our results suggest that the actin filaments that bind drebrin might be a novel class of actin filaments and might play a role in neuronal morphogenesis.

Actins↗

PubMed1993

Molecular cloning of cDNA encoding human drebrin E and chromosomal mapping of its gene.

Drebrins are novel actin-binding proteins in the brain which are developmentally regulated. Three isoforms: two embryonic types (E1 and E2) and an adult type (A) are generated by alternative RNA splicing from a single debrin gene in the chicken brain. A full length cDNA clone of human drebrin E has been isolated from a cDNA library of human fetus brain. The clone is 2596 base pairs in length and contains an open reading frame of 1947 nucleotides encoding a protein of 649 amino acids. The deduced amino acid sequence, except for the internal 138-nucleotide sequence (ins2), exhibits 88% homology with rat drebrin A. Spot blot hybridization using flow-sorted human chromosomes provides evidence that the gene encoding human drebrin protein locates on human chromosome 5.

Amino Acid Sequence↗

PubMed1993

Molecular cloning of a developmentally regulated brain protein, chicken drebrin A and its expression by alternative splicing of the drebrin gene.

Drebrins are developmentally regulated proteins found in the chicken brain and are classified into three forms, E1, E2 and A. Previously we isolated two cDNAs corresponding to the embryonic drebrin mRNAs from a chick embryo cDNA library. They differed in that an internal 129-nucleotide sequence, designated ins1, was inserted in the cDNA encoding drebrin E2 and was deleted in the other cDNA encoding drebrin E1. To search for the cDNA clone encoding drebrin A, a cDNA library of 1-day-old chick brains was screened using embryonic drebrin cDNA fragments as probes. Consequently, a novel cDNA was isolated, the sequence of which was entirely identical with that of drebrin E2 except for the insertion of a 138-nucleotide sequence, designated ins2, in the 5' direction immediately upstream from ins1. Since the translation product of the entire coding region was similar to that of drebrin A, this cDNA should correspond to the mRNA for drebrin A. Sequencing analysis of three drebrin cDNAs clearly indicated that the heterogeneity of chicken drebrins was caused by the insertion or deletion of the two sequences, ins1 and ins2. The amino-terminal half region including ins2 and two short sequences in the carboxyl-terminal region of the predicted drebrin A were highly evolutionarily conserved. Cloning and sequencing of the drebrin gene revealed that ins1 and ins2 were independently encoded by separate exons and three drebrin isoforms were thought to arise by alternative splicing from a single drebrin gene. The difference in the time course of expression and tissue distribution of each drebrin suggests that the machinery of alternative splicing site selection of the drebrin gene is regulated in a developmental stage-dependent and tissue-specific manner.

Alternative Splicing↗

PubMed1992

Lesions of nigrostriatal pathway reduce expression of tyrosine hydroxylase gene in residual dopaminergic neurons of substantia nigra.

The effects of unilateral mechanical transection of the nigrostriatal bundle of rat brain on the level of tyrosine hydroxylase (TH) mRNA and on the activity of TH enzyme in the substantia nigra (SN) were examined. Lesions resulted, by 14 days, in reductions of TH mRNA level to 10% of control and of TH enzyme activity to 39% of control in the ipsilateral SN. The percentage of TH mRNA is lower than either the percentage of surviving dopaminergic neurons or the remaining TH enzyme activity. In situ hybridization analyses also demonstrated the reduction of TH mRNA concentration in surviving dopaminergic neurons in the ipsilateral SN.

Animals↗

PubMed1992

Changes of drebrin expression in the visual cortex of the cat during development.

The expression of and developmental changes in drebrin were studied in cat visual cortex using immunohistochemistry and immunoblot analysis. Drebrin is a developmentally regulated brain protein which in the chicken has characteristic changes in expression related to developmental stage. A monoclonal antibody (MAb M2F6) raised against drebrin, was found to label the neuropil of the kitten visual cortex in the early postnatal period. At 1-3 weeks of age, the staining was prominent in layer IV of the visual cortex. The immunoreactivity, however, was found to be dramatically decreased around the end of the sensitive period for ocular dominance plasticity (approximately 3 months of age). In the adult visual cortex, almost no immunostaining was observed. These developmental changes revealed by an immunohistochemical method were confirmed using immunoblot analysis. Upon immunoblot analysis after SDS-PAGE of protein from the kitten visual cortex, MAb M2F6 was found to recognize two protein bands with molecular weights of 130 kDa (drebrin E) and 140 kDa (drebrin A). The developmental profile of the intensity of the two bands of the drebin closely parallels in time the postnatal changes in cortical susceptibility to visual deprivation. These results indicate that the expression of drebrin in kitten visual cortex is restricted to the early postnatal period and suggest that it may play an important role in the experience-dependent modification of cortical circuitry during the sensitive period.

Aging↗

PubMed1992

Cloning of drebrin A and induction of neurite-like processes in drebrin-transfected cells.

The developmentally-regulated neuron-specific protein, drebrin A, is expressed first at the time of outgrowth and maturation of dendrites, and is localized within dendrites of the adult brain. A cDNA clone of adult rat drebrin A was isolated and sequenced. There is no overall homology with other reported protein sequences except chicken drebrins. We constructed the expression vector MIW-DA containing the drebrin A cDNA. Transfection of nonneuronal cells with MIW-DA induced the formation of highly branched neurite-like cell processes. In these process-bearing transfectants, expressed debrin A is concentrated in submembraneous regions of the cell. Furthermore, actin concentration is higher in these cells than other fibroblasts. These results suggest a possible role of drebrin A in neurite outgrowth.

Amino Acid Sequence↗

PubMed1992

[The role of neuronal cytoskeleton associated proteins in neuronal network formation].

There have been many studies to determine extrinsic factors that may regulate the neuronal migration and growth of axons and dendrites. However, the intracellular mechanism, especially the regulation of cytoskeleton, has not been clarified. It has been reported that actin filament crosslinking protein, MAR-CKS, play roles in cell motility through cytoskeletal rearrangement accompanied by rapid, PKC-dependent phosphorylation. Recently, we have demonstrated that neuron-specific actin binding protein, drebrin, changed the stability and distribution of microfilaments within the fibroblast and formed highly-branched dendrite-like cell processes from their cell perimeters. It has also been reported that overexpression of microtubule associated protein, tau, in a fibroblast induced long axon-like cellular processes. This review will focus on dynamic regulations of the microfilament by drebrin and those of the microtubules by MAP2 and tau. Since all kinds of cytoskeletons are related to each other, the binding ability of neurofilament H to microtubules and that of MAP2 to neurofilaments were also discussed.

Actin Cytoskeleton↗

PubMed1990

Expression of three drebrin isoforms in the developing nervous system.

Drebrins are developmentally regulated brain proteins which were first isolated from brains of 10-day chick embryos. They are classified into three forms, drebrins E1, E2 and A. Cloning of drebrin cDNAs revealed that each drebrin isoform is encoded in an independent mRNA. Genomic Southern blot analysis and cloning of a drebrin gene revealed that the mRNAs of three drebrin isoforms are generated by alternative RNA splicing from a single gene. Immunohistochemistry and in situ hybridization analysis of the embryonic cerebellum indicated that drebrin mRNA is first transcribed in postmitotic neurons and that there seem relations between cell migration and expression of drebrin E1.

Animals↗

PubMed1988

Molecular cloning of a cDNA for the developmentally regulated brain protein, drebrin.

A lambda gt11 cDNA library from 10-day-old chicken embryo was screened immunologically using an antiserum against drebrins E1, E2 and A, proteins previously designated S5, S6 and S54, respectively. A cDNA clone for a common domain of drebrin was isolated. Northern blot analysis of chicken brain indicated that drebrin mRNAs are about 2.7 kilobases in molecular size and that expression of these proteins is developmentally regulated.

Animals↗