Hideo Nishioka

Tomosyn: a syntaxin-1-binding protein that forms a novel complex in the neurotransmitter release process.

Syntaxin-1 is a component of the synaptic vesicle docking and/or membrane fusion soluble N-ethylmaleimide-sensitive factor attachment receptor (SNARE) complex (7S and 20S complexes) in nerve terminals. Syntaxin-1 also forms a heterodimer with Munc18/n-Sec1/rbSec1 in a complex that is distinct from the 7S and 20S complexes. In this report, we identify a novel syntaxin-1-binding protein, tomosyn, that is capable of dissociating Munc18 from syntaxin-1 and forming a novel 10S complex with syntaxin-1, soluble N-etyhlmaleimide-sensitive factor attachment (SNAP) 25, and synaptotagmin. The 130 kDa isoform of tomosyn is specifically expressed in brain, where its distribution partly overlaps with that of syntaxin-1 in nerve terminals. High level expression of either syntaxin-1 or tomosyn results in a specific reduction in Ca2+-dependent exocytosis from PC12 cells. These results suggest that tomosyn is an important component in the neurotransmitter release process where it may stimulate SNARE complex formation.

Afadin: A key molecule essential for structural organization of cell-cell junctions of polarized epithelia during embryogenesis.

Afadin is an actin filament–binding protein that binds to nectin, an immunoglobulin-like cell adhesion molecule, and is colocalized with nectin at cadherin-based cell–cell adherens junctions (AJs). To explore the function of afadin in cell–cell adhesion during embryogenesis, we generated afadin−/− mice and embryonic stem cells. In wild-type mice at embryonic days 6.5–8.5, afadin was highly expressed in the embryonic ectoderm and the mesoderm, but hardly detected in the extraembryonic regions such as the visceral endoderm. Afadin−/− mice showed developmental defects at stages during and after gastrulation, including disorganization of the ectoderm, impaired migration of the mesoderm, and loss of somites and other structures derived from both the ectoderm and the mesoderm. Cystic embryoid bodies derived from afadin−/− embryonic stem cells showed normal organization of the endoderm but disorganization of the ectoderm. Cell–cell AJs and tight junctions were improperly organized in the ectoderm of afadin−/− mice and embryoid bodies. These results indicate that afadin is highly expressed in the ectoderm- derived cells during embryogenesis and plays a key role in proper organization of AJs and tight junctions of the highly expressing cells, which is essential for proper tissue morphogenesis.

Progressive impairment of kidneys and reproductive organs in mice lacking Rho GDIα

The Rho small G protein family members regulate various actin cytoskeleton-dependent cell functions. The Rho GDI (GDP dissociation inhibitor) family, consisting of Rho GDIα, -β, and -γ, is a regulator that keeps the Rho family members in the cytosol as the GDP-bound inactive form and translocates the GDP-bound form from the membranes to the cytosol after the GTP-bound form accomplishes their functions. Rho GDIα is ubiquitously expressed in mouse tissues and shows GDI activity on all the Rho family members in vitro. We have generated mice lacking Rho GDIα by homologous recombination to clarify its in vivo function. Rho GDIα −/− mice showed several abnormal phenotypes. Firstly, Rho GDIα −/− mice were initially viable but developed massive proteinuria mimicking nephrotic syndrome, leading to death due to renal failure within a year. Histologically, degeneration of tubular epithelial cells and dilatation of distal and collecting tubules were readily detected in the kidneys. Secondly, Rho GDIα −/− male mice were infertile and showed impaired spermatogenesis with vacuolar degeneration of seminiferous tubules in their testes. Thirdly, Rho GDIα −/− embryos derived from Rho GDIα −/− female mice were defective in the postimplantation development. In addition, these morphological and functional abnormalities showed age-dependent progression. These results suggest that the signaling pathways of the Rho family members regulated by Rho GDIα play important roles in maintaining the structure and physiological function of at least kidneys and reproductive systems in adult mice.

Localization of membrane-associated guanylate kinase (MAGI)-1/BAI-associated protein (BAP) 1 at tight junctions of epithelial cells.

Membrane-associated guanylate kinase (MAGI)-1/BAI-associated protein (BAP) 1 and Synapse-associated protein (SAP) 97/human Discs-large tumor suppressor gene (hDLG) are ubiquitous isoforms of synaptic scaffolding molecule (S-SCAM) and Postsynaptic density (PSD)-95/SAP90, both of which are implicated in the structures of synapses, respectively. SAP97/hDLG is localized at epithelial junctions and may function as a scaffolding protein, but the subcellular localization or the function of MAGI-1/BAP1 has not been clarified. In intestinal epithelial cells, MAGI-1/BAP1 was localized at tight junctions, whereas SAP97/hDLG was localized diffusely at cell – cell junctions. In Madine Darby canine kidney (MDCK) cells, MAGI-1/BAP1 was colocalized with ZO-1, whereas SAP97/hDLG was colocalized with E-cadherin. In MDCK cells, dominant active and negative mutants of Rac1 small G protein changed the amounts of SAP97/hDLG at cell – cell junctions, but not that of MAGI-1/BAP1. When MDCK cells were switched to a low Ca2+ medium, E-cadherin disappeared from the plasma membrane, and cells were dissociated. The phorbol 12-myristate 13-acetate-treatment after the low Ca2+ switch induced a tight junction-like structure. MAGI-1/BAP1 was recruited with ZO-1 to this structure, but SAP97/hDLG or E-cadherin was not. These findings suggest that MAGI-1/BAP1 is a component of tight junctions of epithelial cells, and that its role is different from that of SAP97/hDLG.

PHYSICAL AND FUNCTIONAL INTERACTION OF RABPHILIN-3A WITH ALPHA -ACTININ

Rabphilin-3A is a downstream target molecule of Rab3A small GTP-binding protein and implicated in Ca2+-dependent neurotransmitter release. Here we have isolated a rabphilin-3A-interacting molecule from a human brain cDNA library by the yeast two-hybrid method and identified it to be α-actinin, known to cross-link actin filaments into a bundle. α-Actinin interacts with the N-terminal region of rabphilin-3A, with which GTP-Rab3A interacts, and this interaction stimulates the activity of α-actinin to cross-link actin filaments into a bundle. The interaction of rabphilin-3A with α-actinin is inhibited by guanosine 5′-(3-O-thio)triphosphate-Rab3A. These results suggest that the Rab3A-rabphilin-3A system regulates the α-actinin-regulated reorganization of actin filaments. It has been shown that reorganization of actin filaments is also involved in Ca2+-dependent exocytosis. Therefore, rabphilin-3A may serve as a linker for Rab3A and cytoskeleton.

Involvement of Cdc42 small G protein in cell-cell adhesion, migration and morphology of MDCK cells

The Rho small G protein family consists of the Rho, Rac, and Cdc42 subfamilies and regulates various cell functions through reorganization of the actin cytoskeleton. We previously showed that the Rho subfamily regulates the formation of stress fibers and focal adhesions whereas the Rac subfamily regulates the E-cadherin-based cell-cell adhesion in MDCK cells. We studied here the function of the Cdc42 subfamily, consisting of two members, Cdc42Hs and G25k, in cell adhesion, migration, and morphology of MDCK cells. For this purpose, we made and used MDCK cell lines stably expressing each of dominant active mutants of Cdc42Hs (sMDCK-Cdc42HsDA) and G25K (sMDCK-G25KDA). Actin filaments at the cell-cell adhesion sites increased in both sMDCK-Cdc42HsDA and -G25KDA cells. Both E-cadherin and β-catenin, adherens junctional proteins, at the cell-cell adhesion sites also increased in both sMDCK-Cdc42HsDA and -G25KDA cells. Electron microscopic analysis revealed that sMDCK-Cdc42HsDA cells tightly contacted with each other throughout the lateral membranes. Moreover, both the HGF- and TPA-induced disruption of the cadherin-based cell-cell adhesion and the subsequent cell migration were inhibited in both sMDCK-Cdc42HsDA and -G25KDA cells. Co-expression of the dominant negative mutant of Rac1, a member of the Rac subfamily, with the dominant active mutant of Cdc42Hs did not inhibit the increased accumulation of actin filaments at the cell-cell adhesion sites. These results suggest that the Cdc42 subfamily is involved in the cadherin-based cell-cell adhesion in a manner independent of the Rac subfamily. Furthermore, the cells were frequently enveloped by the large multinuclear cells in both sMDCK-Cdc42HsDA and -G25KDA cells. Video microscopic analysis revealed that the cells were engulfed by the large cells during cytokinesis.

Isolation and characterization of a novel actin filament-binding protein from Saccharomyces cerevisiae

We purified a novel actin filament (F-actin)-binding protein from the soluble fraction of Saccharomyces cerevisiae by successive column chromatographies by use of the 125I-labeled F-actin blot overlay method. The purified protein showed a minimum Mr of about 140 kDa on SDS-polyacrylamide gel electrophoresis and we named it ABP140. A search with the partial amino acid sequences of ABP140 against the Saccharomyces Genome Database revealed that the open reading frame of the ABP140 gene (ABP140) corresponded to YOR239W fused with YOR240W by the +1 translational frame shift. The encoded protein consisted of 628 amino acids with a calculated Mr of 71,484. The recombinant protein interacted with F-actin and showed the activity to crosslink F-actin into a bundle. Indirect immunofluorescence study demonstrated that ABP140 was colocalized with both cortical actin patches and cytoplasmic actin cables in intact cells. However, elimination of ABP140 by gene disruption did not show a deleterious effect on cell growth or affect the organization of F-actin. These results indicate that ABP140 is not required for cell growth but may be involved in the reorganization of F-actin in the budding yeast.

Frabin, a Novel FGD1-related Actin Filament-binding Protein Capable of Changing Cell Shape and Activating c-Jun N-terminal Kinase

We purified from rat brain a novel F-actin-binding protein with a M r of about 105,000 (p105), which was estimated by SDS-polyacrylamide gel electrophoresis. We cloned its cDNA from a rat brain cDNA library and characterized it. p105 was a protein of 766 amino acids and showed a calculated M r of 86,449. p105 consisted of one F-actin-binding domain at the N-terminal region, one Dbl homology domain and one pleckstrin homology domain at the middle region, and one cysteine-rich domain at the C-terminal region. This domain organization of p105 was similar to that of FGD1, which has been determined to be the genetic locus responsible for faciogenital dysplasia or Aarskog-Scott syndrome. We therefore named p105 frabin (FGD1-related F-actin-binding protein). Frabin bound along the sides of F-actin and showed F-actin-cross-linking activity. Overexpression of frabin in Swiss 3T3 cells and COS7 cells induced cell shape change and c-Jun N-terminal kinase activation, respectively, as described for FGD1. Because FGD1 has been shown to serve as a GDP/GTP exchange protein for Cdc42 small G protein, it is likely that frabin is a direct linker between Cdc42 and the actin cytoskeleton.

MAGUIN, a novel neuronal membrane-associated guanylate kinase-interacting protein.

Postsynaptic density (PSD)-95/Synapse-associated protein (SAP) 90 and synaptic scaffolding molecule (S-SCAM) are neuronal membrane-associated guanylate kinases. Because PSD-95/SAP90 and S-SCAM function as synaptic scaffolding proteins, identification of ligands for these proteins is important to elucidate the structure of synaptic junctions. Here, we report a novel protein interacting with the PDZ domains of PSD-95/SAP90 and S-SCAM and named it MAGUIN-1 (membrane-associated guanylate kinase-interacting protein-1). MAGUIN-1 has one sterile α motif, one PDZ, and one plekstrin homology domain. MAGUIN-1 is localized at the plasma membranevia the plekstrin homology domain and the C-terminal region and interacts with PSD-95/SAP90 and S-SCAM via a C-terminal PDZ domain-binding motif. MAGUIN-1 has a short isoform, MAGUIN-2, which lacks a PDZ domain-binding motif. MAGUINs are expressed in neurons and localized in the cell body and neurites and are coimmunoprecipitated with PSD-95/SAP90 and S-SCAM from rat crude synaptosome. MAGUIN-1 may play an important role with PSD-95/SAP90 and S-SCAM to assemble the components of synaptic junctions.

Isolation and characterization of mammalian homologues of Caenorhabditis elegans lin-7: localization at cell-cell junctions.

In Caenorhabditis elegans, the vulval induction is mediated by the let-23 receptor tyrosine kinase (RTK)/Ras signaling pathway. The precise localization of the let-23 RTK at the epithelial junctions is essential for the vulval induction, and requires three genes including lin-2, -7, and -10. The mammalian homologue of lin-2 has been identified as a protein interacting with a neuronal adhesion molecule, neurexin, and named CASK. CASK has recently been reported to interact with syndecans and an actin-binding protein, band 4.1, at epithelial and synaptic junctions, and to play central roles in the formation of cell-cell junctions. The product of C. elegans lin-7 directly interacts with let-23 RTK and localize it at epithelial junctions. Here, we report three rat homologues of lin-7 ubiquitously expressed in various tissues. These homologues are accumulated at the junctional complex region in cultured Madin-Darby canine kidney cells, and are also localized at the synaptic junctions in neurons. The mammalian homologues of lin-7 may be implicated in the formation of cell-cell junctions.