PubMed Health⌕ Search

Biomedical subjects

Yoshimi Takai

Publications and source records attributed to Yoshimi Takai.

At least 37 records · Page 2Linked to original sources
PubMed2005

PKA-catalyzed phosphorylation of tomosyn and its implication in Ca2+-dependent exocytosis of neurotransmitter.

Neurotransmitter is released from nerve terminals by Ca2+-dependent exocytosis through many steps. SNARE proteins are key components at the priming and fusion steps, and the priming step is modulated by cAMP-dependent protein kinase (PKA), which causes synaptic plasticity. We show that the SNARE regulatory protein tomosyn is directly phosphorylated by PKA, which reduces its interaction with syntaxin-1 (a component of SNAREs) and enhances the formation of the SNARE complex. Electrophysiological studies using cultured superior cervical ganglion (SCG) neurons revealed that this enhanced formation of the SNARE complex by the PKA-catalyzed phosphorylation of tomosyn increased the fusion-competent readily releasable pool of synaptic vesicles and, thereby, enhanced neurotransmitter release. This mechanism was indeed involved in the facilitation of neurotransmitter release that was induced by a potent biological mediator, the pituitary adenylate cyclase-activating polypeptide, in SCG neurons. We describe the roles and modes of action of PKA and tomosyn in Ca2+-dependent neurotransmitter release.

Adenylyl Cyclases↗
PubMed2005

RA-RhoGAP, Rap-activated Rho GTPase-activating protein implicated in neurite outgrowth through Rho.

Rap1 and Rho small G proteins have been implicated in the neurite outgrowth, but the functional relationship between Rap1 and Rho in the neurite outgrowth remains to be established. Here we identified a potent Rho GTPase-activating protein (GAP), RA-RhoGAP, as a direct downstream target of Rap1 in the neurite outgrowth. RA-RhoGAP has the RA and GAP domains and showed GAP activity specific for Rho, which was enhanced by the binding of the GTP-bound active form of Rap1 to the RA domain. Overexpression of RA-RhoGAP induced inactivation of Rho for promoting the neurite outgrowth in a Rap1-dependent manner. Knockdown of RA-RhoGAP reduced the Rap1-induced neurite outgrowth. These results indicate that RA-RhoGAP transduces a signal from Rap1 to Rho and regulates the neurite outgrowth.

Cell Line↗
PubMed2005

Expression of nectin-2 in mouse granulosa cells.

OBJECTIVE: The development and maturation of the ovarian follicles are characterized by structural changes that require components involved in cell-cell adhesion systems. Recently a novel group of cell adhesion molecules named nectins has been identified. The present study examined expression and cell-specific localization of nectins during mouse follicular development. STUDY DESIGN: Expression of nectins in mouse ovary was investigated by immnuoblot analysis and immunohistochemistry. More precise localization was determined by electron microscopy. RESULTS: Immunoblot analysis revealed expression of nectin-2 and nectin-3 but not nectin-1 in ovarian granulosa cells. Immunohistochemistry demonstrated expression of nectin-2 at cell-cell adhesion sites of granulosa cell layer in the primary and preantral follicles. Especially, intense immunoreactivity of nectin-2 accumulated around the zona pellucida. In antral follicles, the intensity of nectin-2 expression on granulosa cells was decreased. By electron microscopy nectin-2 was detected not only on thin extensions of granulosa cells penetrating the zona pellucida, but also on the attachment sites between thin extensions of granulosa cells and oocyte surface. CONCLUSION: The restricted expression of nectin-2 in the granulosa cells of primary and preantral follicles might reflect some of the molecular changes in cell-cell adhesion during early follicular development.

Animals↗
PubMed2005

MAGI1 recruits Dll1 to cadherin-based adherens junctions and stabilizes it on the cell surface.

Delta-Notch signaling plays an essential role in cell fate determination in many tissue types, including the central nervous system. Although the signaling mechanism of Notch has been extensively studied, the behaviors of its ligands are not well understood. In the present study, we found that, in the developing neural tube, Dll1(Delta-like 1) was mainly localized on the processes extending from nascent neurons toward both the pia and the ventricle and accumulated at apical termini, where adherens junctions (AJs) were formed. To understand the mechanism of Dll1 localization, we searched for binding proteins for Dll1 and identified a scaffolding molecule, MAGI1. In the developing spinal cord, MAGI1 mRNA was highly expressed in the ventricular zone, where Dll1 mRNA was expressed. MAGI1 protein accumulated at the AJs formed around the termini of apically extending processes and was partially colocalized with Dll1. MAGI1 bound not only to Dll1 but also to N-cadherin-beta-catenin complexes. In cultured AJ-forming fibroblasts, MAGI1 was localized at AJs, and Dll1 was recruited to these AJs through binding to MAGI1. In addition, Dll1 was stabilized on the cell surface by MAGI1. Taken together, these results suggest that Dll1 is presented on the surface of AJs formed at the apical termini of processes through interaction with MAGI1 to activate Notch on neighboring cells in the developing central nervous system.

Adaptor Proteins, Signal Transducing↗
PubMed2005

Regulation of E-cadherin endocytosis by nectin through afadin, Rap1, and p120ctn.

Adherens junctions (AJs) are a major cell-cell adhesion structure in epithelial cells that are formed by two major cell-cell adhesion molecules, E-cadherin and nectin. We have previously shown that nectin first forms cell-cell adhesion and then recruits non-trans-interacting E-cadherin to the nectin-based cell-cell adhesion sites, which gradually trans-interact there, eventually forming AJs. We have examined here the effect of trans-interacting nectin on non-trans-interacting E-cadherin endocytosis. Trans-interacting nectin capable of associating with afadin, but not trans-interacting nectin mutant incapable of associating with afadin, inhibited non-trans-interacting E-cadherin endocytosis in intact cells. Afadin is a nectin- and actin filament-binding protein that connects nectin to the actin cytoskeleton. Studies on the mode of action of the nectin-afadin system using cell-free assay revealed that afadin associated with nectin bound Rap1 activated by trans-interacting nectin, interacted with p120ctn, and strengthened the binding of p120ctn to E-cadherin, eventually reducing non-trans-interacting E-cadherin endocytosis. Afadin, which did not bind Rap1, was inactive in this capacity. These results indicate that trans-interacting nectin inhibits non-trans-interacting E-cadherin endocytosis through afadin, Rap1, and p120ctn and thereby further accumulates non-trans-interacting E-cadherin to the nectin-based cell-cell adhesion sites for the formation of AJs.

Cadherins↗
PubMed2005

Migrating postmitotic neural precursor cells in the ventricular zone extend apical processes and form adherens junctions near the ventricle in the developing spinal cord.

Postmitotic neural precursors are generated in the ventricular zone (VZ) of the developing neural tube and immediately migrate to the mantle layer (ML) where they differentiate into mature neurons. Although the regulation of neuronal differentiation and migration has extensively been studied, the behavior of the early postmitotic precursors migrating toward the ML is largely unknown. In this study, we have identified Neph3 as a specific marker for early postmitotic neural precursors in the VZ of the developing spinal cord. Analysis of Neph3 localization by immunofluorescence and immunoelectron microscopy revealed that early neural precursors in the VZ possessed not only pia-connected processes but also ones that reached the ventricle. This apical extension of processes was confirmed by analyzing another early postmitotic marker, Dll1 mRNA, which was actively transported toward the ventricle and accumulated at the termini of the processes. Furthermore, adherens junctions (AJs) were formed around the apical end of processes extending from Neph3- and Dll1 mRNA-positive postmitotic precursors. Taken together, these observations suggest that migrating early postmitotic neural precursors in the VZ of the developing spinal cord form a neuroepithelial cell-like bipolar morphology and communicate with their neighboring cells through AJs.

Adherens Junctions↗
PubMed2005

Molecular perspective on tight-junction assembly and epithelial polarity.

Apical-basal polarity and a highly organized actin cytoskeleton are main characteristics of epithelial cells that support exchange of ions and nutrients from one body compartment to another. The junctional complexes, localized to the apical end of the basolateral domain of the plasma membrane, are not simply epithelial barriers in paracellular transport or fences preventing diffusion of integral proteins in the plasma membrane, but also contain proteins involved in the maintenance of the physiologic epithelial cell state and signal transduction. Claudin-based tight junctions and E-cadherin-based adherens junctions have been extensively studied. Nectins, along with a unique scaffolding protein, afadin, form homophilic and heterophilic trans-dimers and play a key role in identifying cell partners in the primordial cell-cell adhesion. Nectin-based cell-cell adhesion participates in the epithelial morphogenesis, both independently and cooperatively with claudin-based tight junctions and cadherin-based adherens junctions. This review discusses how these adhesion systems interact with each other to form apical junctional complexes, and how they reorganize the actin cytoskeleton in a multistage process of cell adhesion, migration, and polarization.

Animals↗
PubMed2005

Transcriptional activation of the mouse Necl-5/Tage4/PVR/CD155 gene by fibroblast growth factor or oncogenic Ras through the Raf-MEK-ERK-AP-1 pathway.

Necl-5/Tage4/poliovirus receptor/CD155 is the poliovirus receptor and upregulated in rodent and human carcinoma. We have recently shown that mouse Necl-5 is upregulated by an oncogenic Ki-Ras (V12Ki-Ras) in NIH3T3 cells and enhances cell movement induced by growth factors, including platelet-derived growth factor and fibroblast growth factor (FGF), in an integrin alpha(v)beta(3)-dependent manner in wild type and V12Ki-Ras-transformed NIH3T3 cells. In addition, it enhances the growth factor-induced cell proliferation. We examined here how mouse Necl-5 was upregulated by V12Ki-Ras in NIH3T3 cells. Expression of the luciferase reporter gene fused to the Necl-5 promoter was induced by V12Ki-Ras in NIH3T3 cells. This induction was mediated through the Raf-MEK-ERK pathway. The Necl-5 promoter has an AP-1-binding site and this site was required for the V12Ki-Ras-induced activation of the Necl-5 promoter. Expression of the luciferase reporter gene fused to the Necl-5 promoter was also induced by FGF through the Raf-MEK-ERK-AP-1 pathway in NIH3T3 cells. These results indicate that the expression of mouse Necl-5 is induced by FGF or V12Ki-Ras through the Raf-MEK-ERK-AP-1 pathway.

3T3 Cells↗
PubMed2005

Nectin-like molecule-1/TSLL1/SynCAM3: a neural tissue-specific immunoglobulin-like cell-cell adhesion molecule localizing at non-junctional contact sites of presynaptic nerve terminals, axons and glia cell processes.

Nectins are Ca2+-independent immunoglobulin-like cell-cell adhesion molecules and comprise a family of four members. At the mossy fiber terminals of hippocampus, nectin-1 and nectin-3 localize at the presynaptic and postsynaptic sides of synaptic junctions, respectively, and their trans-interactions play a role in formation of synapses in cooperation with N-cadherin. Nectins are associated with the actin cytoskeleton through afadin, a nectin- and actin-filament-binding protein. Five nectin-like molecules (Necls) which have domain structures similar to those of nectins have been identified and here we characterize Necl-1/TSLL1/SynCAM3, from now on referred to as Necl-1. Tissue distribution analysis showed that Necl-1 was specifically expressed in the neural tissue. Immunofluorescence and immunoelectron microscopy revealed that Necl-1 localized at the contact sites among axons, their terminals, and glia cell processes that cooperatively formed synapses, axon bundles and myelinated axons. Necl-1 showed Ca2+-independent homophilic cell-cell adhesion activity. It furthermore showed Ca2+-independent heterophilic cell-cell adhesion activity with Necl-2/IGSF4/RA175/SgIGSF/TSLC1/SynCAM1 from now on referred to as Necl-2, nectin-1 and nectin-3, but not with Necl-5 or nectin-2. The C-terminal cytoplasmic region of Necl-1 did not bind afadin but bound membrane-associated guanylate kinase subfamily members that contain the L27 domain, including Dlg3, Pals2 and CASK. These results indicate that Necl-1 is a neural-tissue-specific Ca2+-independent immunoglobulin-like cell-cell adhesion molecule which potentially has membrane-associated guanylate kinase subfamily member-binding activity and localizes at the non-junctional cell-cell contact sites.

Actins↗
PubMed2005

Roles of cell-adhesion molecules nectin 1 and nectin 3 in ciliary body development.

Nectins are Ca2+-independent immunoglobulin-like cell-cell-adhesion molecules consisting of four members. Nectins homophilically and heterophilically trans-interact to form a variety of cell-cell junctions, including cadherin-based adherens junctions in epithelial cells and fibroblasts in culture, synaptic junctions in neurons, and Sertoli cell-spermatid junctions in the testis, in cooperation with, or independently of, cadherins. To further explore the function of nectins, we generated nectin 1-/- and nectin 3-/-)mice. Both nectin 1-/- and nectin 3-/- mice showed a virtually identical ocular phenotype, microphthalmia, accompanied by a separation of the apex-apex contact between the pigment and non-pigment cell layers of the ciliary epithelia. Immunofluorescence and immunoelectron microscopy revealed that nectin 1 and nectin 3, but not nectin 2, localized at the apex-apex junctions between the pigment and non-pigment cell layers of the ciliary epithelia. However, nectin 1-/- and nectin 3-/- mice showed no impairment of the apicolateral junctions between the pigment epithelia where nectin 1, nectin 2 and nectin 3 localized, or of the apicolateral junctions between the non-pigment epithelia where nectin 2 and nectin 3, but not nectin 1, localized. These results indicate that the heterophilic trans-interaction between nectin 1 and nectin 3 plays a sentinel role in establishing the apex-apex adhesion between the pigment and non-pigment cell layers of the ciliary epithelia that is essential for the morphogenesis of the ciliary body.

Animals↗
PubMed2005

Direct binding of Lgl2 to LGN during mitosis and its requirement for normal cell division.

The Drosophila tumor suppressor protein lethal (2) giant larvae (l(2)gl) is involved in asymmetric cell division during development and epithelial cell polarity through interaction with the aPKC.Par-6 complex. We showed here that Lgl2, a mammalian homolog of l(2)gl, directly bound to LGN, a mammalian homolog of Partner of inscuteable in HEK293 cells. The C-terminal tail of Lgl2 bound to LGN with a K(d) value of about 56 nm. Endogenous Lgl2 formed a complex with aPKC, Par-6, and LGN. This complex formation was enhanced in metaphase of the synchronized cells by treatment with thymidine and nocodazole. Immunofluorescence staining of the complex was the strongest at the cell periphery of the metaphase cells. Overexpression of the C-terminal tail of Lgl2 induced mis-localization of the nuclear mitotic apparatus protein NuMA and disorganization of the mitotic spindle during mitosis, eventually causing formation of multiple micronuclei. Knockdown of endogenous Lgl (Lgl1 and Lgl2) also induced disorganization of the mitotic spindle, thereby causing formation of multiple micronuclei. The binding between Lgl2 and LGN played a role in the mitotic spindle organization through regulating formation of the LGN.NuMA complex. These results indicate that Lgl2 forms a Lgl2.Par-6.aPKC.LGN complex, which responds to mitotic signaling to establish normal cell division.

Antigens, Nuclear↗
PubMed2005

Purification and properties of Rab3 GEP (DENN/MADD).

Rab3A, a member of the Rab3 small GTP-binding protein (G protein) family, regulates Ca(2+)-dependent exocytosis of neurotransmitter. Rab3A cycles between the GDP-bound inactive and GTP-bound active forms, and the former is converted to the latter by the action of a GDP/GTP exchange protein (GEP). We have previously purified a GEP from rat brain with lipid-modified Rab3A as a substrate. Purified Rab3 GEP is active on all the Rab3 subfamily members including Rab3A, -3B, -3C, and -3D. Purified Rab3 GEP is active on the lipid-modified form, but not on the lipid-unmodified form. Purified Rab3 GEP is inactive on Rab3A complexed with Rab GDI. The recombinant protein is prepared from the Rab3 GEP-expressed Spodoptera frugiperda cells (Sf9 cells). The properties of recombinant Rab3 GEP, including the requirement for lipid modifications of Rab3A, the substrate specificity, and the sensitivity to Rab GDI, are similar to those of purified Rab3 GEP. Overexpression of Rab3 GEP inhibits Ca(2+)-dependent exocytosis from PC12 cells. On the other hand, Rab3 GEP is identical to a protein named DENN/MADD: differentially expressed in normal versus neoplastic (DENN)/mitogen-activated protein kinase-activating death domain (MADD). Here, we describe the purification method for recombinant Rab3 GEP from Sf9 cells and the functional properties of Rab3 GEP in Ca(2+)-dependent exocytosis by use of the human growth hormone coexpression assay system of PC12 cells.

Animals↗
PubMed2005

Purification and properties of rabconnectin-3.

Rab3A, a member of the Rab3 small GTP-binding protein (G protein) family, regulates Ca(2+)-dependent exocytosis of neurotransmitter. The cyclical activation and inactivation of Rab3A are essential for the Rab3A action in exocytosis. GDP-Rab3A is activated to GTP-Rab3A by Rab3 GDP/GTP exchange protein (Rab3 GEP), and GTP-Rab3A is inactivated to GDP-Rab3A by Rab3 GTPase-activating protein (Rab3 GAP). We) have found a novel protein, named rabconnectin-3, that is coimmunoprecipitated with Rab3 GEP or GAP from the crude synaptic vesicle fraction of rat brain. Rabconnectin-3 constitutes a subunit structure consisting of alpha and beta subunits and localizes at synaptic vesicles. Overexpression of the C-terminal fragment of rabconnectin-3alpha inhibits Ca(2+)-dependent exocytosis from PC12 cells. We describe the purification method for native rabconnectin-3alpha and -3beta from rat brain and the functional properties of rabconnectin-3alpha in Ca(2+)-dependent exocytosis by use of human growth hormone coexpression assay system of PC12 cells.

Adaptor Proteins, Signal Transducing↗
PubMed2005

Mutations of the catalytic subunit of RAB3GAP cause Warburg Micro syndrome.

Warburg Micro syndrome (WARBM1) is a severe autosomal recessive disorder characterized by developmental abnormalities of the eye and central nervous system and by microgenitalia. We identified homozygous inactivating mutations in RAB3GAP, encoding RAB3 GTPase activating protein, a key regulator of the Rab3 pathway implicated in exocytic release of neurotransmitters and hormones, in 12 families with Micro syndrome. We hypothesize that the underlying pathogenesis of Micro syndrome is a failure of exocytic release of ocular and neurodevelopmental trophic factors.

Catalytic Domain↗
PubMed2005

Common signaling pathway is used by the trans-interaction of Necl-5/Tage4/PVR/CD155 and nectin, and of nectin and nectin during the formation of cell-cell adhesion.

Nectin is a Ca2+-independent Ig-like cell-cell adhesion molecule that forms homo- and hetero-trans-dimers (trans-interaction). Nectin first forms cell-cell adhesions and then recruits cadherin to the nectin-based cell-cell adhesion sites to form AJ cooperatively with cadherin. In addition, the trans-interaction of nectin and nectin induces the activation of Cdc42 and Rac small G proteins, which enhances the formation of AJ. The activation of Cdc42 and Rac by the trans-interaction of nectin and nectin is mediated by c-Src, another small G protein, Rap1, a Cdc42-GEF, FRG, and a Rac-GEF, Vav2. Necl-5/Tage4/PVR/CD155 is another Ca2+-independent Ig-like molecule, which does not homophilically trans-interact, but heterophilically trans-interacts with nectin-3, one member of the nectin family. We show here that the trans-interaction of Necl-5 and nectin-3 bidirectionally induces the activation of Cdc42 and Rac. Similarly to the activation of Cdc42 and Rac by the trans-interaction of nectin and nectin, the trans-interaction of Necl-5 and nectin-3 first recruits and activates c-Src at the Necl-5/nectin-3-based cell-cell contact sites. c-Src then phosphorylates FRG and Vav2, and the tyrosine-phosphorylated FRG and Vav2 are recruited to the Necl-5/nectin-3-based cell-cell contact sites. The trans-interaction of Necl-5 and nectin-3 also activates Rap1 through C3G, a Rap-GEF, and this activation of Rap1 is required for the activation of Cdc42 and Rac. These results indicate that the trans-interactions of Necl-5 and nectin-3 and of nectin and nectin induce the activation of Cdc42 and Rac through the common signaling molecules c-Src, Rap1, FRG, and Vav2.

Animals↗
PubMed2005

Recruitment of E-cadherin associated with alpha- and beta-catenins and p120ctn to the nectin-based cell-cell adhesion sites by the action of 12-O-tetradecanoylphorbol-13-acetate in MDCK cells.

The formation of tight junctions (TJs) is dependent on the formation of adherens junctions (AJs) in MDCK cells. E-Cadherin and nectin are major cell-cell adhesion molecules (CAMs) at AJs, whereas claudin, occludin and junctional adhesion molecule (JAM) are major CAMs at TJs. When MDCK cells precultured at 2 microm Ca(2+) are cultured at 2 mm Ca(2+), nectin first forms cell-cell adhesion and recruits E-cadherin to the nectin-based cell-cell adhesion sites to form AJs. Thereafter, nectin recruits first JAM-A and then claudin-1 and occludin to the apical side of AJs to form TJs. In contrast, when MDCK cells precultured at 2 microm Ca(2+) are cultured at 2 microm Ca(2+) in the presence of a phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA), a TJ-like structure is formed without the formation of the E-cadherin-based AJs. We showed here that GFP-E-cadherin, which did not trans-interact due to 2 microm Ca(2+) but associated with alpha- and beta-catenins and p120(ctn), was recruited to the nectin-based cell-cell adhesion sites by the action of TPA. The nectin inhibitors, which inhibited the trans-interaction of nectin, inhibited the recruitment of GFP-E-cadherin and their associating catenins by the action of TPA. Microbeads coated with the extracellular fragment of nectin recruited not only cellular nectin but also GFP-E-cadherin and their associating catenins by the action of TPA. These results indicate that when the TJ-like structure is formed by the action of TPA, non-trans-interacting E-cadherin and its associating catenins are recruited to the nectin-based cell-cell adhesion sites and that the trans-interaction of E-cadherin is not essential for the formation of TJs.

Animals↗
PubMed2004

An activating mutant of Cdc42 that fails to interact with Rho GDP-dissociation inhibitor localizes to the plasma membrane and mediates actin reorganization.

Cdc42 is a member of the Rho family of GTPases and plays an important role in the regulation of actin cytoskeletal organization. Activation of Cdc42 and associated signal transduction cascades are dependent upon proper localization of this GTPase. The studies described herein address the hypothesis that Rho GDP-dissociation inhibitor, RhoGDI, plays an essential role in the translocation of Cdc42 to signaling complexes at the plasma membrane and is essential for Cdc42-mediated actin cytoskeletal rearrangements. An activating mutant of Cdc42 that is RhoGDI-binding defective (Cdc42(G12V/R66E)) is characterized and used as a tool to study the functional importance of the Cdc42-RhoGDI interaction. Overexpression of mycCdc42(G12V/R66E) in COS-7 cells results in actin cytoskeletal rearrangements that are indistinguishable from those stimulated by overexpression of mycCdc42(G12V). In addition, the G12V activating mutant of Cdc42 was overexpressed in mesangial cells that are null for RhoGDI expression. MycCdc42(G12V) stimulation of filopodia formation in these cells was indistinguishable from that observed in wild-type mesangial cells. Taken together, the results presented herein indicate that although RhoGDI is a critical regulator of guanine nucleotide binding, cycling of Cdc42 between membranes and the cytosol and cellular transformation, it is not essential for Cdc42-mediated organization of the actin cytoskeleton.

Actins↗