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Yoshimi Takai

Publications and source records attributed to Yoshimi Takai.

At least 19 recordsLinked to original sources
PubMed2006

Defective chemokine-directed lymphocyte migration and development in the absence of Rho guanosine diphosphate-dissociation inhibitors alpha and beta.

Rho family small GTP-binding proteins, including Rho, Rac, and Cdc42, are key determinants of cell movement and actin-dependent cytoskeletal morphogenesis. Rho GDP-dissociation inhibitor (GDI) alpha and Rho GDIbeta (or D4/Ly-GDI), closely related regulators for Rho proteins, are both expressed in hemopoietic cell lineages. Nevertheless, the functional contributions of Rho GDIs remain poorly understood in vivo. In this study, we report that combined disruption of both the Rho GDIalpha and Rho GDIbeta genes in mice resulted in reduction of marginal zone B cells in the spleen, retention of mature T cells in the thymic medulla, and a marked increase in eosinophil numbers. Furthermore, these mice showed lower CD3 expression and impaired CD3-mediated proliferation of T cells. While B cells showed slightly enhanced chemotactic migration in response to CXCL12, peripheral T cells showed markedly reduced chemotactic migration in response to CCL21 and CCL19 associated with decreased receptor levels of CCR7. Overall, Rho protein levels were reduced in the bone marrow, spleen, and thymus but sustained activation of the residual part of RhoA, Rac1, and Cdc42 was detected mainly in the bone marrow and spleen. Rho GDIalpha and Rho GDIbeta thus play synergistic roles in lymphocyte migration and development by modulating activation cycle of the Rho proteins in a lymphoid organ-specific manner.

Animals↗
PubMed2006

Involvement of up-regulated Necl-5/Tage4/PVR/CD155 in the loss of contact inhibition in transformed NIH3T3 cells.

Normal cells show contact inhibition of cell movement and proliferation, but this is lost following transformation. We found that Necl-5, originally identified as a poliovirus receptor and up-regulated in many cancer cells, enhances growth factor-induced cell movement and proliferation. We showed that when cells contact other cells, Necl-5 interacts in trans with nectin-3 and is removed by endocytosis from the cell surface, resulting in a reduction of cell movement and proliferation. We show here that up-regulation of the gene encoding Necl-5 by the oncogene V12-Ki-Ras causes enhanced cell movement and proliferation. Upon cell-cell contact, de novo synthesis of Necl-5 exceeds the rate of Necl-5 endocytosis, eventually resulting in a net increase in the amount of Necl-5 at the cell surface. In addition, expression of the gene encoding nectin-3 is markedly reduced in transformed cells. Thus, up-regulation of Necl-5 following transformation contributes to the loss of contact inhibition in transformed cells.

Animals↗
PubMed2006

Interneurite affinity is regulated by heterophilic nectin interactions in concert with the cadherin machinery.

Neurites recognize their specific partners during the formation of interneuronal connections. In hippocampal pyramidal neurons, axons attach to dendrites for their synaptogenesis, but the dendrites do not form stable contacts with each other, suggesting the presence of a mechanism to allow their selective associations. Nectin-1 (N1), an immunoglobulin domain adhesive protein, is preferentially localized in axons, and its heterophilic partner, N3, is present in both axons and dendrites; we tested their potential roles in interneurite recognition. The overexpression of N1, causing its mislocalization to dendrites, induced atypical dendrodendritic as well as excessive axodendritic associations. On the contrary, the genetic deletion of N1 loosened the contacts between axons and dendritic spines. Those actions of nectins required cadherin-catenin activities, but the overexpression of cadherin itself could not accelerate neurite attachment. These results suggest that the axon-biased localization of N1 and its trans-interaction with N3 in cooperation with the cadherin machinery is critical for the ordered association of axons and dendrites.

Adherens Junctions↗
PubMed2006

Rab3 GTPase-activating protein regulates synaptic transmission and plasticity through the inactivation of Rab3.

Rab3A small G protein is a member of the Rab family and is most abundant in the brain, where it is localized on synaptic vesicles. Evidence is accumulating that Rab3A plays a key role in neurotransmitter release and synaptic plasticity. Rab3A cycles between the GDP-bound inactive and GTP-bound active forms, and this change in activity is associated with the trafficking cycle of synaptic vesicles at nerve terminals. Rab3 GTPase-activating protein (GAP) stimulates the GTPase activity of Rab3A and is expected to determine the timing of the dissociation of Rab3A from synaptic vesicles, which may be coupled with synaptic vesicle exocytosis. Rab3 GAP consists of two subunits: the catalytic subunit p130 and the noncatalytic subunit p150. Recently, mutations in p130 were found to cause Warburg Micro syndrome with severe mental retardation. Here, we generated p130-deficient mice and found that the GTP-bound form of Rab3A accumulated in the brain. Loss of p130 in mice resulted in inhibition of Ca(2+)-dependent glutamate release from cerebrocortical synaptosomes and altered short-term plasticity in the hippocampal CA1 region. Thus, Rab3 GAP regulates synaptic transmission and plasticity by limiting the amount of the GTP-bound form of Rab3A.

Animals↗
PubMed2006

Beta8 integrin binds Rho GDP dissociation inhibitor-1 and activates Rac1 to inhibit mesangial cell myofibroblast differentiation.

Alpha(v)beta8 integrin expression is restricted primarily to kidney, brain, and placenta. Targeted alpha(v) or beta8 deletion is embryonic lethal due to defective placenta and brain angiogenesis, precluding investigation of kidney alpha(v)beta8 function. We find that kidney beta8 is localized to glomerular mesangial cells, and expression is decreased in mouse models of glomerulosclerosis, suggesting that beta8 regulates normal mesangial cell differentiation. To interrogate beta8 signaling pathways, yeast two-hybrid and co-precipitation studies demonstrated beta8 interaction with Rho guanine nucleotide dissociation inhibitor-1 (GDI). Selective beta8 stimulation enhanced beta8-GDI interaction as well as Rac1 (but not RhoA) activation and lamellipodia formation. Mesangial cells from itgb8-/- mice backcrossed to a genetic background that permitted survival, or gdi-/- mice, which develop glomerulosclerosis, demonstrated RhoA (but not Rac1) activity and alpha-smooth muscle actin assembly, which characterizes mesangial cell myofibroblast transformation in renal disease. To determine whether Rac1 directly modulates RhoA-associated myofibroblast differentiation, mesangial cells were transduced with inhibitory Rac peptide fused to human immunodeficiency virus-Tat, resulting in enhanced alpha-smooth muscle actin organization. We conclude that the beta8 cytosolic tail in mesangial cells organizes a signaling complex that culminates in Rac1 activation to mediate wild-type differentiation, whereas decreased beta8 activation shifts mesangial cells toward a RhoA-dependent myofibroblast phenotype.

Animals↗
PubMed2006

Interaction of integrin alpha(v)beta3 with nectin. Implication in cross-talk between cell-matrix and cell-cell junctions.

Cell-matrix and cell-cell junctions cross-talk together, and these two junctions cooperatively regulate cell movement, proliferation, adhesion, and polarization. However, the mechanism of this cross-talk remains unknown. An immunoglobulin-like cell-cell adhesion molecule nectin first trans-interacts with each other to form cell-cell adhesion and induces activation of Rap1, Cdc42, and Rac small G proteins through c-Src. Trans-interacting nectin then recruits another cell-cell adhesion molecule cadherin to the nectin-based cell-cell adhesion sites and forms adherens junctions (AJs). Here, we show that integrin alpha(v)beta3 functionally and physically associates with nectin. Integrin alpha(v)beta3 colocalized with nectin at the nectin-based cell-cell adhesion sites. The association of integrin alpha(v)beta3 with nectin was direct and was mediated through their extracellular regions. This interaction was necessary for the nectin-induced signaling. Focal adhesion kinase, which relays the integrin-initiated outside-in signals to the intracellular signaling molecules, was also involved in the nectin-induced signaling. During the formation of AJs, the high affinity form of integrin alpha(v)beta3 co-localized with nectin at the primordial cell-cell contact sites, and then after the establishment of AJs, this high affinity form of integrin alpha(v)beta3 was converted to the low affinity form, which continued to co-localize with nectin. Thus, integrin alpha(v)beta3 and nectin play pivotal roles in the cross-talk between cell-matrix and cell-cell junctions and the formation of cadherin-based AJs.

Animals↗
PubMed2006

SAD: a presynaptic kinase associated with synaptic vesicles and the active zone cytomatrix that regulates neurotransmitter release.

A serine/threonine kinase SAD-1 in C. elegans regulates synapse development. We report here the isolation and characterization of mammalian orthologs of SAD-1, named SAD-A and SAD-B, which are specifically expressed in the brain. SAD-B is associated with synaptic vesicles and, like the active zone proteins CAST and Bassoon, is tightly associated with the presynaptic cytomatrix in nerve terminals. A short conserved region (SCR) in the COOH-terminus is required for the synaptic localization of SAD-B. Overexpression of SAD-B in cultured rat hippocampal neurons significantly increases the frequency of miniature excitatory postsynaptic current but not its amplitude. Introduction of SCR into presynaptic superior cervical ganglion neurons in culture significantly inhibits evoked synaptic transmission. Moreover, SCR decreases the size of the readily releasable pool measured by applying hypertonic sucrose. Furthermore, SAD-B phosphorylates the active zone protein RIM1 but not Munc13-1. These results suggest that mammalian SAD kinase presynaptically regulates neurotransmitter release.

Animals↗
PubMed2006

Active zone protein CAST is a component of conventional and ribbon synapses in mouse retina.

CAST is a novel cytomatrix at the active zone (CAZ)-associated protein. In conventional brain synapses, CAST forms a large molecular complex with other CAZ proteins, including RIM, Munc13-1, Bassoon, and Piccolo. Here we investigated the distribution of CAST and its structurally related protein, ELKS, in mouse retina. Immunofluorescence analyses revealed that CAST and ELKS showed punctate signals in the outer and inner plexiform layers of the retina that were well-colocalized with those of Bassoon and RIM. Both proteins were found presynaptically at glutamatergic ribbon synapses, and at conventional GABAergic and glycinergic synapses. Moreover, immunoelectron microscopy revealed that CAST, like Bassoon and RIM, localized at the base of synaptic ribbons, whereas ELKS localized around the ribbons. Both proteins also localized in the vicinity of the presynaptic plasma membrane of conventional synapses in the retina. These results indicated that CAST and ELKS were novel components of the presynaptic apparatus of mouse retina.

ATP-Binding Cassette Transporters↗
PubMed2006

Involvement of the Ras-Ras-activated Rab5 guanine nucleotide exchange factor RIN2-Rab5 pathway in the hepatocyte growth factor-induced endocytosis of E-cadherin.

E-cadherin is a key cell-cell adhesion molecule at adherens junctions (AJs) and undergoes endocytosis when AJs are disrupted by the action of an extracellular signal, such as hepatocyte growth factor (HGF)/scatter factor. Rab5 small G protein has been implicated in the HGF-induced endocytosis of E-cadherin, but the molecular mechanism for the regulation of Rab5 activity remains unknown. We first studied this mechanism by using the cell-free assay system for the endocytosis of E-cadherin of the AJ-enriched fraction from rat livers. HGF induced activation of Ras small G protein, which then bound to RIN2, a Rab5 GDP/GTP exchange factor with the Vps9p-like guanine nucleotide exchange factor and Ras association domains, and activated it. Activated RIN2 then activated Rab5, eventually inducing the endocytosis of E-cadherin. We then studied whether RIN2 was involved in the HGF-induced endocytosis of E-cadherin in intact Madin-Darby canine kidney cells. RIN2 localized at the cell-cell adhesion sites, and its guanine nucleotide exchange factor activity was required for the HGF-induced endocytosis of E-cadherin in Madin-Darby canine kidney cells. These results indicate that RIN2 connects Ras to Rab5 in the HGF-induced endocytosis of E-cadherin.

Animals↗
PubMed2006

Activation of Rap1, Cdc42, and rac by nectin adhesion system.

Nectin is an immunoglobulin-like cell-cell adhesion molecule and forms adherens junctions cooperatively with cadherin. Trans-interaction of nectin induces activation of Rap1, Cdc42, and Rac small G proteins. The activity of these small G proteins can be analyzed by the pull-down assay using GST-Ral-GDS fusion protein for Rap1 and GST-PAK-CRIB for Cdc42 and Rac. The fluorescent resonance energy transfer (FRET) system is also available to spatially and temporally detect the activity of these small G proteins in the living cells. In addition to these assays, the activity of Cdc42 and Rac is indirectly, but easily, evaluated by the cell-spreading assay to examine formation of filopodia and lamellipodia, respectively. To clearly explore the effect of trans-interacting nectin on the small G proteins, we use L fibroblasts stably expressing nectin-1 (nectin-1-L cells) and the extracellular domain of nectin-3 fused to human IgG Fc (Nef-3). Treatment of nectin-1-L cells with Nef-3 remarkably increases both the amount of the GTP-bound form and the FRET efficiency of all Rap1, Cdc42, and Rac small G proteins. In the cell-spreading assay, Cdc42 and Rac activated in this way promote the formation of filopodia and lamellipodia, respectively. Here, we focus on how the activity of Cdc42 and Rac induced by trans-interacting nectin is examined by use of the pull-down and the cell-spreading assays.

Animals↗
PubMed2006

ELKS, a protein structurally related to the active zone protein CAST, is involved in Ca2+-dependent exocytosis from PC12 cells.

The active zone protein CAST binds directly to the other active zone proteins RIM, Bassoon and Piccolo, and it has been suggested that these protein-protein interactions play an important role in neurotransmitter release. To further elucidate the molecular mechanism, we attempted to examine the function of CAST using PC12 cells as a model system. Although PC12 cells do not express CAST, they do express ELKS, a protein structurally related to CAST. Endogenous and exogenously expressed ELKS, RIM2 and Bassoon were colocalized in punctate signals in PC12 cells. Over-expression of full-length ELKS resulted in a significant increase in stimulated exocytosis of human growth hormone (hGH) from PC12 cells, similar to the effect of full-length RIM2. This increase was not observed following over-expression of deletion constructs of ELKS that lacked either the last three amino acids (IWA) required for binding to RIM2 or a central region necessary for binding to Bassoon. Moreover, over-expression of the NH(2)-terminal RIM2-binding domain of Munc13-1, which is known to inhibit the binding between RIM and Munc13-1, inhibited the stimulated increase in hGH secretion by full-length RIM2. Furthermore, this construct also inhibited the stimulated increase in hGH secretion induced by full-length ELKS. These results suggest that ELKS is involved in Ca(2+)-dependent exocytosis from PC12 cells at least partly via the RIM2-Munc13-1 pathway.

Adaptor Proteins, Signal Transducing↗
PubMed2006

Role of cell adhesion molecule nectin-3 in spermatid development.

Seminiferous epithelia of the testes contain two types of intercellular junctions: Sertoli-Sertoli junctions and Sertoli-spermatid junctions. The former junctions are equipped with tight and adherens junctions while the latter junctions are not. Ca2+ -independent immunoglobulin-like cell-cell adhesion molecules, nectin-2 and nectin-3, asymmetrically localize at the Sertoli cell side and at the spermatid side of Sertoli-spermatid junctions, respectively. They heterophilically trans-interact to make contact between the two cells. Nectin-2(-/-) mice have shown male-specific infertility, disrupted Sertoli-spermatid junctions and morphologically impaired spermatid development. Here we report testicular phenotypes of nectin-3(-/-) mice exhibiting male-specific infertility. Nectin-3(-/-) mice had defects in the later steps of sperm morphogenesis including distorted nuclei and abnormal distribution of mitochondria, as well as in localization of nectin-2 at the Sertoli-spermatid junctions. Transplantation of wild-type spermatogenic stem cells into the nectin-3(-/-) testes partially rescued these defects in sperm morphogenesis. These results indicate that the heterophilic trans-interaction between nectin-2 and nectin-3 is essential for the formation and maintenance of Sertoli-spermatid junctions that plays a critical role in spermatid development.

Animals↗
PubMed2006

Tomosyn is expressed in beta-cells and negatively regulates insulin exocytosis.

Tomosyn, a syntaxin-binding protein, is capable of dissociating mammalian homolog of the Caenorhabditis elegans unc-18 gene from syntaxin and is involved in the regulation of exocytosis. We have investigated the expression, cellular localization, and functional role of tomosyn in pancreatic beta-cells. Western blotting revealed a 130-kDa protein corresponding to tomosyn in insulin-secreting beta-cell lines. RT-PCR amplification showed that b-, m-, and s-tomosyn isoform mRNAs are expressed in beta-cell lines and rat pancreatic islets. Immunohistochemistry revealed punctate tomosyn immunoreactivity in the cytoplasm of insulin-, glucagon-, pancreatic polypeptide-, and somatostatin-containing islet cells. Syntaxin 1 coimmunoprecipitated with tomosyn in extracts of insulin-secreting cells. Overexpression of m-tomosyn in mouse beta-cells significantly decreased exocytosis, whereas inhibition of tomosyn expression by small interfering RNA increased exocytosis. Hence, in the pancreatic beta-cell, tomosyn negatively regulates insulin exocytosis.

Animals↗
PubMed2005

Regulation of the assembly and adhesion activity of E-cadherin by nectin and afadin for the formation of adherens junctions in Madin-Darby canine kidney cells.

The Ca2+-independent immunoglobulin-like molecule nectin first forms cell-cell adhesion and then assembles cadherin at nectin-based cell-cell adhesion sites, resulting in the formation of adherens junctions (AJs). Afadin is a nectin- and actin filament-binding protein that connects nectin to the actin cytoskeleton. Here, we studied the roles and modes of action of nectin and afadin in the formation of AJs in cultured MDCK cells. The trans-interaction of nectin assembled E-cadherin, which associated with p120(ctn), beta-catenin, and alpha-catenin, at the nectin-based cell-cell adhesion sites in an afadin-independent manner. However, the assembled E-cadherin showed weak cell-cell adhesion activity and might be the non-trans-interacting form. This assembly was mediated by the IQGAP1-dependent actin cytoskeleton, which was organized by Cdc42 and Rac small G proteins that were activated by the action of trans-interacting nectin through c-Src and Rap1 small G protein in an afadin-independent manner. However, Rap1 bound to afadin, and this Rap1-afadin complex then interacted with p120(ctn) associated with non-trans-interacting E-cadherin, thereby causing the trans-interaction of E-cadherin. Thus, nectin regulates the assembly and cell-cell adhesion activity of E-cadherin through afadin, nectin signaling, and p120(ctn) for the formation of AJs in Madin-Darby canine kidney cells.

Actins↗
PubMed2005

Involvement of nectins in the formation of puncta adherentia junctions and the mossy fiber trajectory in the mouse hippocampus.

Synapses are specialized intercellular junctions whose specificity and plasticity are mediated by synaptic cell adhesion molecules. In hippocampus, the mossy fibers form synapses on the apical dendrites of the CA3 pyramidal cells where synaptic and puncta adherentia junctions (PAJs) are highly developed. Synaptic junctions are the sites of neurotransmission, while PAJs are regarded as mechanical adhesion sites. Cell-cell adhesion molecules nectin-1 and nectin-3 asymmetrically localize at the pre- and post-synaptic sides of PAJs, respectively. To reveal the definitive role of nectins, we analyzed nectin-1-/- and nectin-3(-/-) mice. In both the mutant mice, the number of PAJs at the synapses between the mossy fiber terminals and the dendrites of the CA3 pyramidal cells was reduced. In addition, the abnormal mossy fiber trajectory was observed. These results indicate that nectins are involved in the formation of PAJs, which maintain the proper mossy fiber trajectory.

Animals↗
PubMed2005

Inhibition of cell movement and proliferation by cell-cell contact-induced interaction of Necl-5 with nectin-3.

Immunoglobulin-like Necl-5/Tage4/poliovirus receptor (PVR)/CD155, originally identified as the PVR, has been shown to be up-regulated in cancer cells and to enhance growth factor-induced cell movement and proliferation. In addition, Necl-5 heterophilically trans-interacts with nectin-3, a cell-cell adhesion molecule known to form adherens junctions in cooperation with cadherin. We show here that Necl-5 was down-regulated from cell surface upon cell-cell contacts in NIH3T3 cells. This down-regulation of Necl-5 was initiated by its interaction with nectin-3 and was mainly mediated by clathrin-dependent endocytosis. Then, the down-regulation of Necl-5 induced in this way reduced movement and proliferation of NIH3T3 cells. These results indicate that the down-regulation of Necl-5 induced by its interaction with nectin-3 upon cell-cell contacts may be at least one mechanism underlying contact inhibition of cell movement and proliferation.

Animals↗