Transmitter release at synapses between nerve cells is spatially restricted to active zones, where synaptic vesicle docking, priming, and Ca2+-dependent fusion take place in a temporally highly coordinated manner. Munc13s are essential for priming synaptic vesicles to a fusion competent state, and their specific active zone localization contributes to the active zone restriction of transmitter release and the speed of excitation-secretion coupling. However, the molecular mechanism of the active zone recruitment of Munc13s is not known. We show here that the active zone recruitment of Munc13 isoforms Munc13-1 and ubMunc13-2 is regulated by their binding to the Rab3A-interacting molecule RIM1alpha, a key determinant of long term potentiation of synaptic transmission at mossy fiber synapses in the hippocampus. We identify a single point mutation in Munc13-1 and ubMunc13-2 (I121N) that, depending on the type of assay used, strongly perturbs or abolishes RIM1alpha binding in vitro and in cultured fibroblasts, and we demonstrate that RIM1alpha binding-deficient ubMunc13-2(I121) is not efficiently recruited to synapses. Moreover, the levels of Munc13-1 and ubMunc13-2 levels are decreased in RIM1alpha-deficient brain, and Munc13-1 is not properly enriched at active zones of mossy fiber terminals of the mouse hippocampus if RIM1alpha is absent. We conclude that one function of the Munc13/RIM1alpha interaction is the active zone recruitment of Munc13-1 and ubMunc13-2.
This report is concerned with an inverse acoustic scattering problem for shape identification. The inverse problem under consideration is to determine the shape of an obstacle in the fluid from a knowledge of the time-harmonic incident wave and the far field pattern of the scattered wave. The integral equation which is based on the relation between the small modification of the boundary shape and sound scattering field is introduced. Using the integral equation and Schffer's theorem, a numerical method for inverse acoustic scattering problem is presented. Numerical examples are given showing the practicality of the method.
The chemical synapse transmits information from a neuron to another neuron in the neuronal network in the brain. The efficacy of synaptic transmission changes by modifying the number or size of synapses dynamically in cognitive functions. Thus, morphological analyses of synapses are of particular importance in neuroscience research. In the current study, we applied super-resolved three-dimensional stimulated emission depletion (3D-STED) microscopy for the morphological analyses of synapses. This approach allowed us to estimate the precise number of excitatory and inhibitory synapses in the mouse hippocampal tissue. Using this method, we discovered a region-specific increase in excitatory synapses in a model mouse of autism spectrum disorder, Neuroligin-3 KO. We detected an increase in excitatory synapses at the stratum oriens of hippocampal area CA1, although such an increase was not detected by conventional confocal microscopy. Our approach to estimating the synapse number will open a new field in developmental neuroscience.
Aim: The waist circumference (WC) cut-off values in the diagnostic criteria of metabolic syndrome (MetS) established in Japan (Japanese criteria) differ from those established by the International Diabetes Federation (IDF) for Asians (IDF criteria).Methods: To settle this contradiction, a cross-sectional study of Japanese aged 20-65 years was performed. After excluding subjects suffering from significant diseases other than those constituting MetS, excessive drinkers, and regular smokers, 835 males and 1,304 females were examined. WC was measured at the umbilical level (UWC) and midway between the ribs and iliac crest (MWC) according to both criteria in each subject.Results: Upper limits of reference intervals of MWC estimated in subjects free from MetS were 84.6 and 84.3 cm in older (40-65 years) and younger (20-39) males, and 78.4 and 70.5 cm in older and younger females, respectively; those of UWC were 86.4, 86.2, 87.9 and 78.9 cm, respectively. Receiver operating characteristic (ROC) curves for MWC to predict UWC reproduced the relationships of the two types of cut-off values in each population.Conclusion: WC cut-off values in the Japanese and IDF criteria have the potential to be valid as cut-off values of UWC and MWC in Japanese, respectively. Their difference can be explained by the variation in the WC definition, and they can stand together without inconsistency. Acceptance of the recently prevailing view that the WC standard in IDF for Asian males should be 85 cm and the introduction of new criteria for younger females in consideration of their generation differences in both criteria could facilitate their higher compatibility.
Tight junctions (TJs) serve as a barrier that prevents solutes and water from passing through the paracellular pathway, and as a fence between the apical and basolateral plasma membranes in epithelial cells. TJs consist of transmembrane proteins (claudin, occludin, and JAM) and many peripheral membrane proteins, including actin filament (F-actin)-binding scaffold proteins (ZO-1, -2, and -3), non-F-actin-binding scaffold proteins (MAGI-1), and cell polarity molecules (ASIP/PAR-3 and PAR-6). We identified here a novel peripheral membrane protein at TJs from a human cDNA library and named it Pilt (for protein incorporatedlater into TJs), because it was incorporated into TJs later after the claudin-based junctional strands were formed. Pilt consists of 547 amino acids with a calculatedM r of 60,704. Pilt has a proline-rich domain. In cadherin-deficient L cells stably expressing claudin or JAM, Pilt was not recruited to claudin-based or JAM-based cell-cell contact sites, suggesting that Pilt does not directly interact with claudin or JAM. The present results indicate that Pilt is a novel component of TJs. Tight junctions (TJs) serve as a barrier that prevents solutes and water from passing through the paracellular pathway, and as a fence between the apical and basolateral plasma membranes in epithelial cells. TJs consist of transmembrane proteins (claudin, occludin, and JAM) and many peripheral membrane proteins, including actin filament (F-actin)-binding scaffold proteins (ZO-1, -2, and -3), non-F-actin-binding scaffold proteins (MAGI-1), and cell polarity molecules (ASIP/PAR-3 and PAR-6). We identified here a novel peripheral membrane protein at TJs from a human cDNA library and named it Pilt (for protein incorporatedlater into TJs), because it was incorporated into TJs later after the claudin-based junctional strands were formed. Pilt consists of 547 amino acids with a calculatedM r of 60,704. Pilt has a proline-rich domain. In cadherin-deficient L cells stably expressing claudin or JAM, Pilt was not recruited to claudin-based or JAM-based cell-cell contact sites, suggesting that Pilt does not directly interact with claudin or JAM. The present results indicate that Pilt is a novel component of TJs. tight junction adherens junction actin filament Src homology 3 guanylate kinase enhanced green fluorescent protein amino acid(s) glutathione S-transferase membrane-associated guanylate kinase homologues bovine serum albumin antibody Cell-cell junctions play crucial roles in various cell functions, including cell adhesion, growth, and polarization (for reviews, see Refs. 1Takeichi M. Science. 1991; 251: 1451-1455Crossref PubMed Scopus (2978) Google Scholar and 2Gumbiner B.M. Cell. 1996; 84: 345-357Abstract Full Text Full Text PDF PubMed Scopus (2917) Google Scholar). In polarized epithelial cells, cell-cell junctions form a specialized membrane structure, comprising TJs,1 AJs, and desmosomes, which is known as the junctional complex. These three junctional structures are aligned from the apical side to the basal side of the lateral membrane, although desmosomes are independently distributed in other areas. TJs function as a barrier preventing solutes and water from passing freely through the paracellular pathway (for reviews, see Refs. 3Tsukita S. Furuse M. Itoh M. Curr. Opin. Cell Biol. 1999; 11: 628-633Crossref PubMed Scopus (265) Google Scholar and4Tsukita S. Furuse M. Itoh M. Nat. Rev. Mol. Cell. Biol. 2001; 2: 285-293Crossref PubMed Scopus (2004) Google Scholar). TJs also serve as a fence between the apical and basolateral plasma membranes to form and maintain cell polarity (3Tsukita S. Furuse M. Itoh M. Curr. Opin. Cell Biol. 1999; 11: 628-633Crossref PubMed Scopus (265) Google Scholar, 4Tsukita S. Furuse M. Itoh M. Nat. Rev. Mol. Cell. Biol. 2001; 2: 285-293Crossref PubMed Scopus (2004) Google Scholar). Recent studies have revealed the molecular architecture of TJs (3Tsukita S. Furuse M. Itoh M. Curr. Opin. Cell Biol. 1999; 11: 628-633Crossref PubMed Scopus (265) Google Scholar, 4Tsukita S. Furuse M. Itoh M. Nat. Rev. Mol. Cell. Biol. 2001; 2: 285-293Crossref PubMed Scopus (2004) Google Scholar). TJs consist of transmembrane proteins and many peripheral membrane proteins. The peripheral membrane proteins include F-actin, F-actin-binding scaffold proteins, non-F-actin-binding scaffold proteins, and cell polarity and signaling molecules. As a major transmembrane protein, claudin forms TJ strands and plays a crucial role in the formation and maintenance of TJs (3Tsukita S. Furuse M. Itoh M. Curr. Opin. Cell Biol. 1999; 11: 628-633Crossref PubMed Scopus (265) Google Scholar, 4Tsukita S. Furuse M. Itoh M. Nat. Rev. Mol. Cell. Biol. 2001; 2: 285-293Crossref PubMed Scopus (2004) Google Scholar). Occludin also forms TJ strands, but the physiological function remains to be clarified (3Tsukita S. Furuse M. Itoh M. Curr. Opin. Cell Biol. 1999; 11: 628-633Crossref PubMed Scopus (265) Google Scholar, 4Tsukita S. Furuse M. Itoh M. Nat. Rev. Mol. Cell. Biol. 2001; 2: 285-293Crossref PubMed Scopus (2004) Google Scholar). At the cytoplasmic face, claudin and occludin interact with F-actin-binding scaffold molecules, ZO-1, -2, and -3 (5Stevenson B.R. Siliciano J.D. Mooseker M.S. Goodenough D.A. J. Cell Biol. 1986; 103: 755-766Crossref PubMed Scopus (1276) Google Scholar, 6Gumbiner B. Lowenkopf T. Apatira D. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 3460-3464Crossref PubMed Scopus (428) Google Scholar, 7Itoh M. Nagafuchi A. Yonemura S. Kitani-Yasuda T. Tsukita S. Tsukita S. J. Cell Biol. 1993; 121: 491-502Crossref PubMed Scopus (496) Google Scholar, 8Furuse M. Itoh M. Hirase T. Nagafuchi A. Yonemura S. Tsukita S. Tsukita S. J. Cell Biol. 1994; 127: 1617-1626Crossref PubMed Scopus (804) Google Scholar, 9Jesaitis L.A. Goodenough D.A. J. Cell Biol. 1994; 124: 949-961Crossref PubMed Scopus (386) Google Scholar, 10Itoh M. Nagafuchi A. Moroi S. Tsukita S. J. Cell Biol. 1997; 138: 181-192Crossref PubMed Scopus (567) Google Scholar, 11Fanning A.S. Jameson B.J. Jesaitis L.A. Anderson J.M. J. Biol. 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Lee J. Gu Q. Lasky L.A. J. Biol. Chem. 2000; 275: 21477-21485Abstract Full Text Full Text PDF PubMed Scopus (231) Google Scholar) and a GDP/GTP exchange protein for Rap small G protein (23Ohtsuka T. Hata Y. Ide N. Yasuda T. Inoue E. Inoue T. Mizoguchi A. Takai Y. Biochem. Biophys. Res. Commun. 1999; 265: 38-44Crossref PubMed Scopus (92) Google Scholar). As cell polarity molecules, ASIP/PAR-3 and PAR-6 are concentrated at TJs (24Joberty G. Petersen C. Gao L. Macara I.G. Nat. Cell Biol. 2000; 2: 531-539Crossref PubMed Scopus (757) Google Scholar, 25Lin D. Edwards A.S. Fawcett J.P. Mbamalu G. Scott J.D. Pawson T. Nat. Cell Biol. 2000; 2: 540-547Crossref PubMed Scopus (47) Google Scholar). Recently, ASIP/PAR-3 has been shown to interact with JAM (26Ebnet K. Suzuki A. Horikoshi Y. Hirose T. Meyer Zu Brickwedde M.K. Ohno S. Vestweber D. EMBO J. 2001; 20: 3738-3748Crossref PubMed Scopus (325) Google Scholar, 27Itoh M. Sasaki H. Furuse M. Ozaki H. Kita T. Tsukita S. J. Cell Biol. 2001; 154: 491-497Crossref PubMed Scopus (319) Google Scholar). Furthermore, several peripheral membrane proteins, cingulin, 7H6 antigen, and symplekin, have been shown to localize at TJs (28Citi S. Sabanay H. Jakes R. Geiger B. Kendrick-Jones J. Nature. 1988; 333: 272-276Crossref PubMed Scopus (402) Google Scholar, 29Zhong Y. Saitoh T. Minase T. Sawada N. Enomoto K. Mori M. J. Cell Biol. 1993; 120: 477-483Crossref PubMed Scopus (244) Google Scholar, 30Keon B.H. Schafer S.S. Kuhn C. Grund C. Franke W.W. J. Cell Biol. 1996; 134: 1003-1018Crossref PubMed Scopus (267) Google Scholar). Recently, cingulin has been shown to interact with ZO-1, -2, and -3 (31Cordenonsi M. D'Atri F. Hammar E. Parry D.A. Kendrick-Jones J. Shore D. Citi S. J. Cell Biol. 1999; 147: 1569-1582Crossref PubMed Scopus (240) Google Scholar). Several molecules involved in intracellular vesicle trafficking, such as Rab3B small G protein and mammalian homologues of yeast SEC6 and -8 gene products, are also concentrated at TJs (32Weber E. Berta G. Tousson A. St. John P. Green M.W. Gopalokrishnan U. Jilling T. Sorscher E.J. Elton T.S. Abrahamson D.R. Kirk K.L. J. Cell Biol. 1994; 125: 583-594Crossref PubMed Scopus (168) Google Scholar, 33Grindstaff K.K. Yeaman C. Anandasabapathy N. Hsu S.C. Rodriguez-Boulan E. Scheller R.H. Nelson W.J. Cell. 1998; 93: 731-740Abstract Full Text Full Text PDF PubMed Scopus (446) Google Scholar, 34Hsu S.C. Hazuka C.D. Foletti D.L. Scheller R.H. Trends Cell Biol. 1999; 9: 150-153Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar). AJs consist of cell adhesion molecules and many peripheral membrane proteins including F-actin, F-actin-binding proteins, and non-F-actin-binding scaffold proteins. As a major cell adhesion molecule, cadherin plays a crucial role in the formation and maintenance of AJs (1Takeichi M. Science. 1991; 251: 1451-1455Crossref PubMed Scopus (2978) Google Scholar, 2Gumbiner B.M. Cell. 1996; 84: 345-357Abstract Full Text Full Text PDF PubMed Scopus (2917) Google Scholar). Another cell adhesion molecule, nectin, localizes at AJs and regulates the formation of AJs in cooperation with cadherin (35Takahashi K. Nakanishi H. Miyahara M. Mandai M. Satoh K. Satoh A. Nishioka H. Aoki J. Nomoto A. Mizoguchi A. Takai Y. J. Cell Biol. 1999; 145: 539-549Crossref PubMed Scopus (441) Google Scholar, 36Tachibana K. Nakanishi H. Mandai K. Ozaki K. Ikeda W. Yamamoto Y. Nagafuchi A. Tsukita S. Takai Y. J. Cell Biol. 2000; 150: 1161-1171Crossref PubMed Scopus (225) Google Scholar). At the cytoplasmic face, cadherin and nectin interact with F-actin-binding proteins, α-catenin and afadin, respectively (37Rimm D.L. Koslov E.R. Kebriaei P. Cianci C.D. Morrow J.S. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 8813-8817Crossref PubMed Scopus (631) Google Scholar, 38Mandai K. Nakanishi H. Satoh A. Obaishi H. Wada M. Nishioka H. Itoh M. Mizoguchi A. Aoki T. Fujimoto T. Matsuda Y. Tsukita S. Takai Y. J. Cell Biol. 1997; 139: 517-528Crossref PubMed Scopus (393) Google Scholar). α-Catenin furthermore interacts with other F-actin-binding proteins, such as vinculin and α-actinin (1Takeichi M. Science. 1991; 251: 1451-1455Crossref PubMed Scopus (2978) Google Scholar, 2Gumbiner B.M. Cell. 1996; 84: 345-357Abstract Full Text Full Text PDF PubMed Scopus (2917) Google Scholar). A vinculin- and afadin-binding protein, ponsin, also localizes at AJs (39Mandai K. Nakanishi H. Satoh A. Takahashi T. Satoh K. Nishioka H. Mizoguchi A. Takai Y. J. Cell Biol. 1999; 144: 1001-1017Crossref PubMed Scopus (213) Google Scholar). As a non-F-actin-binding scaffold protein, hDlg/SAP97 localizes at AJs (40Muller B.M. Kistner U. Veh R.W. Cases-Langhoff C. Becker B. Gundelfinger E.D. Garner C.C. J. Neurosci. 1995; 15: 2354-2366Crossref PubMed Google Scholar). In addition, growth factor receptors, such as the hepatocyte growth factor and epidermal growth factor receptors, are concentrated at AJs (41Maratos-Flier E. Kao C.Y. Verdin E.M. King G.L. J. Cell Biol. 1987; 105: 1595-1601Crossref PubMed Scopus (95) Google Scholar, 42Crepaldi T. Pollack A.L. Prat M. Zborek A. Mostov K. Comoglio P.M. J. Cell Biol. 1994; 125: 313-320Crossref PubMed Scopus (109) Google Scholar). hDlg has three isoforms, PSD-95/SAP90, PSD-93/chapsyn, and SAP102 (for reviews, see Refs. 43Craven S.E. Bredt D.S. Cell. 1998; 93: 495-498Abstract Full Text Full Text PDF PubMed Scopus (426) Google Scholar and 44Fanning A.S. Anderson J.M. Curr. Opin. Cell Biol. 1999; 11: 432-439Crossref PubMed Scopus (273) Google Scholar). Like ZO-1, -2, and -3, these isoforms belong to the MAGUKs (43Craven S.E. Bredt D.S. Cell. 1998; 93: 495-498Abstract Full Text Full Text PDF PubMed Scopus (426) Google Scholar, 44Fanning A.S. Anderson J.M. Curr. Opin. Cell Biol. 1999; 11: 432-439Crossref PubMed Scopus (273) Google Scholar). The MAGUKs contain several PDZ domains, one SH3 domain, and one GK domain. Of these hDlg isoforms, PSD-95/SAP90, a neuron-specific isoform, has most extensively been characterized, and its many binding molecules have been identified. The PDZ domains of PSD-95/SAP90 interact with theN-methyl-d-aspartate receptor, K+channels, neuroligins, synGAP, Citron, MAGUIN, APC, and CRIPT (43Craven S.E. Bredt D.S. Cell. 1998; 93: 495-498Abstract Full Text Full Text PDF PubMed Scopus (426) Google Scholar, 44Fanning A.S. Anderson J.M. Curr. Opin. Cell Biol. 1999; 11: 432-439Crossref PubMed Scopus (273) Google Scholar, 45Matsumine A. Ogai A. Senda T. Okumura N. Satoh K. Baeg G.H. Kawahara T. Kobayashi S. Okada M. Toyoshima K. Akiyama T. Science. 1996; 272: 1020-1023Crossref PubMed Scopus (407) Google Scholar, 46Yao I. Hata Y. Ide N. Hirao K. Deguchi M. Nishioka H. Mizoguchi A. Takai Y. J. Biol. Chem. 1999; 274: 11889-11896Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). The GK domain interacts with SAPAP/GKAP/DAP and BEGAIN (44Fanning A.S. Anderson J.M. Curr. Opin. Cell Biol. 1999; 11: 432-439Crossref PubMed Scopus (273) Google Scholar, 47Takeuchi M. Hata Y. Hirao K. Toyoda A. Irie M. Takai Y. J. Biol. Chem. 1997; 272: 11943-11951Abstract Full Text Full Text PDF PubMed Scopus (306) Google Scholar, 48Deguchi M. Hata Y. Takeuchi M. Ide N. Hirao K. Yao I. Irie M. Toyoda A. Takai Y. J. Biol. Chem. 1998; 273: 26269-26272Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar). hDlg is ubiquitously expressed including epithelial cells. Dlg1, aDrosophila counterpart of hDlg, plays a critical role in the formation of cell-cell junctions of epithelial cells and the polarity of neuroepithelial cells in the embryo ofDrosophila (49Bilder D. Li M. Perrimon N. Science. 2000; 289: 113-116Crossref PubMed Scopus (723) Google Scholar, 50Ohshiro T. Yagami T. Zhang C. Matsuzaki F. Nature. 2000; 408: 593-596Crossref PubMed Scopus (268) Google Scholar, 51Peng C.Y. Manning L. Albertson R. Doe C.Q. Nature. 2000; 408: 596-600Crossref PubMed Scopus (287) Google Scholar). Although the function of hDlg in mammalian epithelial cells is not clear, it could be a core protein in the formation of cell-cell junctions. However, little is known about hDlg-binding molecules or the mechanism of the localization of this molecule at AJs. In this study, we attempted to identify an hDlg-binding protein using the yeast two-hybrid screening and identified a novel protein from a human cDNA library. However, this protein was a component of TJs rather than AJs. We also found that the protein was incorporated into TJs after TJ strands were formed, and therefore named it Pilt (protein incorporated later intoTJs). We describe here the identification and characterization of Pilt. A bait vector, pBTM116HA hDlg-2–2, was constructed by subcloning the insert encoding aa 581–926 of hDlg-2 into pBTM116HA (52Fujiwara T. Tanaka K. Mino A. Kikyo M. Takahashi K. Shimizu K. Takai Y. Mol. Biol. Cell. 1998; 9: 1221-1233Crossref PubMed Scopus (143) Google Scholar). A mouse 11-day embryo yeast two-hybrid library was purchased from CLONTECH and screened as described previously (47Takeuchi M. Hata Y. Hirao K. Toyoda A. Irie M. Takai Y. J. Biol. Chem. 1997; 272: 11943-11951Abstract Full Text Full Text PDF PubMed Scopus (306) Google Scholar). A cDNA of Pilt (NT2RP3003185; GenBank™/EMBL/DDBL accession no. AK024269) was kindly supplied from Dr. T. Isogai (Helix Research Institute Inc., Chiba, Japan). A cDNA of hDlg-2 was kindly supplied from Dr. T. Akiyama (Tokyo University, Tokyo, Japan). Various expression vectors were constructed in pClneo Myc (53Hirao K. Hata Y. Ide N. Takeuchi M. Irie M. Yao I. Deguchi M. Toyoda A. Südhof T.C. Takai Y. J. Biol. Chem. 1998; 273: 21105-21110Abstract Full Text Full Text PDF PubMed Scopus (253) Google Scholar), pMXII-EGFPC, and pGex4T-1 (Amersham Biosciences, Inc.). pClneo Myc was designed to express an N-terminal Myc-tagged protein. pMXII-EGFPC was constructed by inserting a cDNA fragment encoding EGFP into pMXII (54Ono Y. Nakanishi H. Nishimura M. Kakizaki M. Takahashi K. Miyahara M. Satoh-Horikawa K. Mandai K. Takai Y. Oncogene. 2000; 19: 3050-3058Crossref PubMed Scopus (55) Google Scholar) to express an N-terminal EGFP-tagged protein. Various constructs of Pilt and hDlg-2 contained the following aa: pClneo Myc Pilt, aa 1–547 (full-length); pMXII-EGFPC Pilt, aa 1–547 (full-length); pGex4T-1 Pilt-2, aa 1–260; and pGex4T-1 hDlg-2–4, aa 560–926 (SH3 and GK domains). A rabbit polyclonal Ab was raised against GST-Pilt-2. A mouse monoclonal anti-human JAM Ab (55Ozaki H. Ishii K. Horiuchi H. Arai H. Kawamoto T. Okawa K. Iwamatsu A. Kita T. J. Immunol. 1999; 15: 553-557Google Scholar) was kindly supplied from Drs. T. Kita and H. Ozaki (Kyoto University, Kyoto, Japan). A mouse monoclonal anti-ZO-1 Ab (56Itoh M. Yonemura S. Nagafuchi A. Tsukita S. Tsukita S. J. Cell Biol. 1991; 115: 1449-1462Crossref PubMed Scopus (204) Google Scholar) was kindly supplied from Drs. S. Tsukita and M. Itoh (Kyoto University, Kyoto, Japan). A rat monoclonal anti-nectin-2 Ab was prepared as described (35Takahashi K. Nakanishi H. Miyahara M. Mandai M. Satoh K. Satoh A. Nishioka H. Aoki J. Nomoto A. Mizoguchi A. Takai Y. J. Cell Biol. 1999; 145: 539-549Crossref PubMed Scopus (441) Google Scholar). Rabbit polyclonal anti-claudin-1, mouse monoclonal anti-Myc, and mouse monoclonal anti-PSD-95 family Abs were obtained from Zymed Laboratories Inc., American Type Culture Collection, and Upstate Biotechnology, respectively. COS7, MTD-1A, and L cells were cultured in Dulbecco's modified Eagle's medium with 10% fetal calf serum. COS7 cells were transfected with the DEAE dextran method (57Hata Y. Südhof T.C. J. Biol. Chem. 1995; 270: 13022-13028Abstract Full Text Full Text PDF PubMed Scopus (167) Google Scholar). MTD-1A cells stably expressing EGFP-Pilt were prepared using retrovirus-mediated gene transfection (54Ono Y. Nakanishi H. Nishimura M. Kakizaki M. Takahashi K. Miyahara M. Satoh-Horikawa K. Mandai K. Takai Y. Oncogene. 2000; 19: 3050-3058Crossref PubMed Scopus (55) Google Scholar, 58Asakura T. Nakanishi H. Sakisaka T. Takahashi K. Mandai K. Nishimura M. Sasaki T. Takai Y. Genes Cells. 1999; 4: 573-581Crossref PubMed Scopus (81) Google Scholar). Claudin-L and JAM-L cells were kindly supplied by Drs. S. Tsukita, M. Furuse, and M. Itoh (Kyoto University, Kyoto, Japan). COS7 cells on two 10-cm dishes were transfected with pClneo Myc Pilt. The cells were then sonicated in 0.2 ml of Buffer A (20 mm Tris/Cl at pH 7.5, 150 mmNaCl, 1 mm EDTA, 1 mm dithiothreitol, 10 μm α-amidinophenylmethanesulfonyl fluoride hydrochloride, 10 μg/ml aprotinin, and 10 μg/ml leupeptin) containing 1% (w/v) Triton X-100, followed by ultracentrifugation at 100,000 × g for 15 min. The supernatant was incubated with GST or GST-hDlg-2–4 (2 nmol each) immobilized on 50 μl (wet volume) of glutathione-Sepharose beads (Amersham Biosciences, Inc.). After the beads were extensively washed with Buffer A containing 0.3% (w/v) Triton X-100. the bound proteins were eluted by boiling the beads in an SDS sample buffer (60 mm Tris/Cl at pH 6.7, 3% SDS, 2% (v/v) 2-mercaptoethanol, and 5% glycerol). The sample was then subjected to SDS-PAGE (10% polyacrylamide gel), followed by Western blotting with the anti-Myc Ab or protein staining with Coomassie Brilliant Blue. Wound healing assay was performed as described (58Asakura T. Nakanishi H. Sakisaka T. Takahashi K. Mandai K. Nishimura M. Sasaki T. Takai Y. Genes Cells. 1999; 4: 573-581Crossref PubMed Scopus (81) Google Scholar, 59Yonemura S. Itoh M. Nagafuchi A. Tsukita S. J. Cell Sci. 1995; 108: 127-142Crossref PubMed Google Scholar, 60Ando-Akatsuka Y. Yonemura S. Itoh M. Furuse M. Tsukita S. J. Cell. Physiol. 1999; 179: 115-125Crossref PubMed Scopus (143) Google Scholar). Briefly, about 60% confluent MTD1-A cells stably expressing EGFP-Pilt on 35-mm dishes were detached with 0.25% trypsin and 1 mm EDTA, and 5 × 105cells were seeded on 35-mm grid tissue culture dishes. After the cells were incubated for about 48 h in Dulbecco's modified Eagle's medium containing 10% fetal calf serum to be confluent, the cells were scratched manually with the needle of a 10-μl syringe (Hamilton Co.) and further cultured for 3, 6, or 8 h. Other procedures, including subcellular fractionation of rat liver (61Tsukita S. Tsukita S. J. Cell Biol. 1989; 108: 31-41Crossref PubMed Scopus (93) Google Scholar), immunofluorescence microscopy of cultured cells and frozen sections (35Takahashi K. Nakanishi H. Miyahara M. Mandai M. Satoh K. Satoh A. Nishioka H. Aoki J. Nomoto A. Mizoguchi A. Takai Y. J. Cell Biol. 1999; 145: 539-549Crossref PubMed Scopus (441) Google Scholar, 38Mandai K. Nakanishi H. Satoh A. Obaishi H. Wada M. Nishioka H. Itoh M. Mizoguchi A. Aoki T. Fujimoto T. Matsuda Y. Tsukita S. Takai Y. J. Cell Biol. 1997; 139: 517-528Crossref PubMed Scopus (393) Google Scholar, 39Mandai K. Nakanishi H. Satoh A. Takahashi T. Satoh K. Nishioka H. Mizoguchi A. Takai Y. J. Cell Biol. 1999; 144: 1001-1017Crossref PubMed Scopus (213) Google Scholar), and immunoelectron microscopy (35Takahashi K. Nakanishi H. Miyahara M. Mandai M. Satoh K. Satoh A. Nishioka H. Aoki J. Nomoto A. Mizoguchi A. Takai Y. J. Cell Biol. 1999; 145: 539-549Crossref PubMed Scopus (441) Google Scholar, 38Mandai K. Nakanishi H. Satoh A. Obaishi H. Wada M. Nishioka H. Itoh M. Mizoguchi A. Aoki T. Fujimoto T. Matsuda Y. Tsukita S. Takai Y. J. Cell Biol. 1997; 139: 517-528Crossref PubMed Scopus (393) Google Scholar, 39Mandai K. Nakanishi H. Satoh A. Takahashi T. Satoh K. Nishioka H. Mizoguchi A. Takai Y. J. Cell Biol. 1999; 144: 1001-1017Crossref PubMed Scopus (213) Google Scholar), were performed as described. Protein concentrations were determined with BSA as a reference protein (62Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (214351) Google Scholar). SDS-PAGE was done as described (63Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (206602) Google Scholar). Prestained markers used in Western blotting were β-galactosidase (123 kDa), phosphorylase B (106 kDa), and BSA (77 kDa). Standard markers used in protein staining were phosphorylase B (97 kDa), BSA (66 kDa), ovalbumin (45 kDa), and carbonic anhydrase (31 kDa). To identify an hDlg-binding protein, we performed the yeast two-hybrid screening using the region of hDlg-2 containing the SH3 and GK domains (aa 581–926) as a bait. We obtained 31 positive clones from 1 × 106 clones of a mouse 11-day embryo yeast two-hybrid library. Twenty-three clones were overlapped and encoded an identical protein. These mouse cDNA clones had homology to the C-terminal portion of a human cDNA (NT2RP3003185; GenBank™/EMBL/DDBL accession no. AK024269) (74% identity of aa sequence). The full-length clone of this human cDNA encoded a protein composed of 547 aa and a calculated M r of 60,704 (Fig.1 A). We named this protein Pilt (protein incorporated later into TJs), because it was incorporated into TJs later after the claudin-based junctional strands were formed as described below. The aa sequences of the N- and C-terminal regions of Pilt were 43 and 51% identical to those of BEGAIN (48Deguchi M. Hata Y. Takeuchi M. Ide N. Hirao K. Yao I. Irie M. Toyoda A. Takai Y. J. Biol. Chem. 1998; 273: 26269-26272Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar), respectively, but other regions showed no homology to BEGAIN (Fig. 1 B). BEGAIN has been identified as a protein interacting with the GK domain of PSD-95/SAP90 (48Deguchi M. Hata Y. Takeuchi M. Ide N. Hirao K. Yao I. Irie M. Toyoda A. Takai Y. J. Biol. Chem. 1998; 273: 26269-26272Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar). Like BEGAIN, the software COILS version 2 predicted a coiled-coil structure in the N-terminal region of Pilt. Pilt has a proline-rich domain, whereas BEGAIN does not. BEGAIN has one nuclear localization signal, whereas Pilt does not. Pilt has no transmembrane segment. To confirm whether the isolated cDNA encodes the full-length of Pilt, COS7 cells were transfected with pClneo Myc Pilt. The extract was subjected to SDS-PAGE, followed by Western blotting with the anti-Pilt Ab. A protein with a molecular mass of ∼85 kDa was detected (Fig.1 C). When the extract of MTD-1A cells was subjected to SDS-PAGE, followed by Western blotting with the anti-Pilt Ab, a protein with almost the same molecular mass as that of Myc-tagged Pilt was detected. Therefore, we concluded that the isolated cDNA encodes the full length of Pilt. To confirm the interaction of Pilt with hDlg, the extract of COS7 cells expressing Myc-tagged Pilt was incubated with a GST fusion protein of hDlg (SH3 and GK domains) immobilized on glutathione-Sepharose beads. After washing the beads, the bound proteins were eluted and the half of the eluate was subjected to SDS-PAGE, followed by Western blotting with the anti-Myc Ab. The other half was subjected to SDS-PAGE, followed by protein staining with Coomassie Brilliant Blue. Myc-tagged Pilt indeed bound hDlg (Fig.2, A and B). Northern blot analysis using the full-length cDNA of Pilt as a probe detected ∼3.0-kb mRNA in all the human tissues examined, including heart, brain, placenta, lung, liver, skeletal muscle, kidney, pancreas, spleen, thymus, prostate, testis, ovary, small intestine, colon, and leukocytes (Fig. 3 A). Subcellular fractionation analysis of Pilt in rat liver indicated that it was enriched in the fraction rich in AJs and TJs, where hDlg was also enriched (Fig. 3 B). Because hDlg has been shown to localize at AJs (40Muller B.M. Kistner U. Veh R.W. Cases-Langhoff C. Becker B. Gundelfinger E.D. Garner C.C. J. Neurosci. 1995; 15: 2354-2366Crossref PubMed Google Scholar), we examined by immunofluorescence microscopy whether Pilt colocalized with hDlg at AJs in MTD-1A cells. MTD-1A cells are mouse mammary tumor cells (64Takeuchi K. Sato N. Kasahara H. Funayama N. Nagafuchi A. Yonemura S. Tsukita S. Tsukita S. J. Cell Biol. 1994; 125: 1371-1384Crossref PubMed Scopus (317) Google Scholar). Pilt and hDlg localized at cell-cell junctions, but the distribution pattern of Pilt was slightly different from that of hDlg (Fig.4 A). We therefore next examined whether Pilt colocalized with ZO-1 at TJs in MTD-1A cells. The two proteins colocalized at the cell-cell junctions (Fig.4 B). To further confirm the colocalization of Pilt and ZO-1 in MTD-1A cells, EGFP-tagged full-length Pilt was stably expressed in MTD-1A cells and the localization of the expressed protein was compared with that of endogenous ZO-1. Exogenously expressed Pilt colocalized with ZO-1 (Fig. 4 C). Because ZO-1 localizes at TJs in epithelial cells (5Stevenson B.R. Siliciano J.D. Mooseker M.S. Goodenough D.A. J. Cell Biol. 1986; 103: 755-766Crossref PubMed Scopus (1276) Google Scholar, 7Itoh M. Nagafuchi A. Yonemura S. Kitani-Yasuda T. Tsukita S. Tsukita S. J. Cell Biol. 1993; 121: 491-502Crossref PubMed Scopus (496) Google Scholar), these results suggest that Pilt localizes at TJs. It may be noted that Pilt was also stained at the perinuclear regions, most presumably the Golgi complex as estimated by the co-staining with Golgi 58-kDa protein (p58), a marker for the Golgi complex (65Bloom G.S. Brashear T.A. J. Biol. Chem. 1989; 264: 16083-16092Abstract Full Text PDF PubMed Google Scholar) (Fig. 4 D). To obtain the definitive evidence for the localization of Pilt at TJs, its localization was analyzed in small intestine absorptive epithelial cells, because TJs and AJs are well separated in this cell type (7Itoh M. Nagafuchi A. Yonemura S. Kitani-Yasuda T. Tsukita S. Tsukita S. J. Cell Biol. 1993; 121: 491-502Crossref PubMed Scopus (496) Google Scholar). Immunofluorescence microscopy showed that Pilt colocalized with ZO-1 at TJs in small intestine absorptive epithelial cells (Fig.5 A). It was also stained at the perinuclear regions, presumably the Golgi complex. Immunoelectron microscopy revealed that Pilt exclusively localized at TJs and were absent from AJs and desmosomes (Fig. 5 B). We examined whether Pilt directly interacts with claudin or JAM. For this purpose, we took advantage of cadherin-deficient L cells stably expressing claudin-1 or JAM (claudin-L and JAM-L cells, respectively) (27Itoh M. Sasaki H. Furuse M. Ozaki H. Kita T. Tsukita S. J. Cell Biol. 2001; 154: 491-497Crossref PubMed Scopus (319) Google Scholar, 66Furuse M Sasaki H Fujimoto K Tsukita S. J. Cell Biol. 1998; 143: 391-401Crossref PubMed Scopus (784) Google Scholar). In claudin-L cells, claudin-1 and ZO-1 were concentrated at cell-cell contact sites, but Pilt was not concentrated there (Fig.6 A). Pilt was stained at the perinuclear regions. In JAM-L cells, JAM was concentrated at cell-cell contact sites, but Pilt was not concentrated there (Fig.6 B). Pilt was again stained at the perinuclear regions. It has been shown that ZO-1 colocalizes with JAM (27Itoh M. Sasaki H. Furuse M. Ozaki H. Kita T. Tsukita S. J. Cell Biol. 2001; 154: 491-497Crossref PubMed Scopus (319) Google Scholar). Although we have not examined the in vitro binding of Pilt with claudin, JAM, or ZO-1, the results suggest that Pilt does not directly interact with these proteins. When confluent cultures of MTD-1A cells are scratched with a needle, very thin cellular protrusions begin to emerge from the front edge of the wound at the initial stage of wound healing process. At the next stage, small cell-cell junctions are formed at the tips of these cellular protrusions, which are regarded as spot-like primordial junctions as reported previously (59Yonemura S. Itoh M. Nagafuchi A. Tsukita S. J. Cell Sci. 1995; 108: 127-142Crossref PubMed Google Scholar, 60Ando-Akatsuka Y. Yonemura S. Itoh M. Furuse M. Tsukita S. J. Cell. Physiol. 1999; 179: 115-125Crossref PubMed Scopus (143) Google Scholar). Nectin-2 is concentrated at these small contact sites (58Asakura T. Nakanishi H. Sakisaka T. Takahashi K. Mandai K. Nishimura M. Sasaki T. Takai Y. Genes Cells. 1999; 4: 573-581Crossref PubMed Scopus (81) Google Scholar). We confirmed this earlier observation (Fig.7 A). Pilt was not accumulated at the spot-like junctions, although it was stained at cell-cell junctions at the non-wounding regions. At the next stage of this wound healing process, the spot-like junctions begin to be fused to form short line-like junctions (58Asakura T. Nakanishi H. Sakisaka T. Takahashi K. Mandai K. Nishimura M. Sasaki T. Takai Y. Genes Cells. 1999; 4: 573-581Crossref PubMed Scopus (81) Google Scholar, 59Yonemura S. Itoh M. Nagafuchi A. Tsukita S. J. Cell Sci. 1995; 108: 127-142Crossref PubMed Google Scholar, 60Ando-Akatsuka Y. Yonemura S. Itoh M. Furuse M. Tsukita S. J. Cell. Physiol. 1999; 179: 115-125Crossref PubMed Scopus (143) Google Scholar). Claudin was accumulated at this type of junction (Fig. 7 B). Pilt was not concentrated at the line-like junctions. At the later stage of this process, the line-like junctions grow up to complete cell-cell junctions. Pilt was finally stained at these junctions (Fig. 7 C). These results indicate that Pilt is incorporated into TJs at the very late stage. We have isolated here a novel protein, named Pilt, as an hDlg-binding protein. However, the immunofluorescence and immunoelectron microscopic analyses indicate that Pilt localizes at TJs but not at AJs in epithelial cells including cultured MTD-1A cells and small intestine absorptive epithelial cells, whereas hDlg localizes at AJs. Furthermore, Pilt is incorporated into TJs at the very late stage of wound healing process, whereas hDlg is incorporated into spot-like junctions at the initial stage. 2A. Fukuhara, K. Irie, and Y. Takai, unpublished observation. These results suggest that Pilt is not a physiological binding partner of hDlg in epithelial cells, although we do not exclude the possibility that Pilt directly interacts with hDlg in cells lacking TJs. The Northern blot analysis indicates that Pilt is highly expressed in tissues, such as skeletal muscle and spleen, which are not rich in TJs. Conversely, the expression of Pilt is relatively low in tissues that are rich in TJs. It remains to be clarified why Pilt shows such tissue distribution patterns. It remains unknown, as well, whether Pilt colocalizes with hDlg in cells lacking TJs. Further studies are necessary to conclude whether Pilt is the physiological binding partner of hDlg. Because Pilt has no transmembrane segment and localizes at TJs, it could bind to the peripheral membrane proteins at TJs. Pilt is not recruited to claudin-based or JAM-based contact sites in L cells, suggesting that Pilt does not directly interact with claudin, JAM, or ZO-1. The exact reason for the failure of Pilt to be recruited to these cell-cell contact sites is not known, but may be simply because many other components of TJs found in epithelial cells, such as ZO-2 (9Jesaitis L.A. Goodenough D.A. J. Cell Biol. 1994; 124: 949-961Crossref PubMed Scopus (386) Google Scholar), ZO-3 (12Haskins J. Gu L. Wittchen E.S. Hibabrd J. Stevenson B.R. J. Cell Biol. 1998; 141: 199-208Crossref PubMed Scopus (489) Google Scholar), MAGI-1/2/3 (20Ide N. Hata Y. Nishioka H. Hirao K. Yao I. Deguchi M. Mizoguchi A. Nishimori H. Tokino T. Nakamura Y. Takai Y. Oncogene. 1999; 18: 7810-7815Crossref PubMed Scopus (106) Google Scholar), cingulin (28Citi S. Sabanay H. Jakes R. Geiger B. Kendrick-Jones J. Nature. 1988; 333: 272-276Crossref PubMed Scopus (402) Google Scholar), 7H6 antigen (29Zhong Y. Saitoh T. Minase T. Sawada N. Enomoto K. Mori M. J. Cell Biol. 1993; 120: 477-483Crossref PubMed Scopus (244) Google Scholar), and symplekin (30Keon B.H. Schafer S.S. Kuhn C. Grund C. Franke W.W. J. Cell Biol. 1996; 134: 1003-1018Crossref PubMed Scopus (267) Google Scholar), may be absent in non-epithelial cells, such as L cells. Although we have not examined here a possible interaction of Pilt with all the known peripheral membrane proteins at TJs, immunoprecipitation analysis with the anti-FLAG Ab from the soluble fraction of MDCK cells stably expressing FLAG-tagged Pilt has not yet revealed any Pilt-binding protein (data not shown). Identification of Pilt-binding proteins is necessary for our understanding of the physiological function of Pilt and of the mechanism of the localization of Pilt at TJs. During the formation of epithelial cell-cell junctions, primordial spot-like junctions are first formed at the tips of the cellular protrusions radiating from adjacent cells (58Asakura T. Nakanishi H. Sakisaka T. Takahashi K. Mandai K. Nishimura M. Sasaki T. Takai Y. Genes Cells. 1999; 4: 573-581Crossref PubMed Scopus (81) Google Scholar, 59Yonemura S. Itoh M. Nagafuchi A. Tsukita S. J. Cell Sci. 1995; 108: 127-142Crossref PubMed Google Scholar, 60Ando-Akatsuka Y. Yonemura S. Itoh M. Furuse M. Tsukita S. J. Cell. Physiol. 1999; 179: 115-125Crossref PubMed Scopus (143) Google Scholar). Cadherin, nectin, JAM, afadin, and ZO-1 colocalize at the spot-like junctions where claudin and occludin are not concentrated (58Asakura T. Nakanishi H. Sakisaka T. Takahashi K. Mandai K. Nishimura M. Sasaki T. Takai Y. Genes Cells. 1999; 4: 573-581Crossref PubMed Scopus (81) Google Scholar,60Ando-Akatsuka Y. Yonemura S. Itoh M. Furuse M. Tsukita S. J. Cell. Physiol. 1999; 179: 115-125Crossref PubMed Scopus (143) Google Scholar). 3A. Fukuhara, K. Irie, A. Yamada, T. Katata, T. Honda, K. Shimizu, H. Nakanishi, and Y. Takai, manuscript in preparation. As cellular polarization proceeds, claudin and occludin gradually accumulate at the spot-like junctions to form TJs, and cadherin, nectin, and afadin are sorted out from claudin, occludin, JAM, and ZO-1 to form AJs. We have shown here that Pilt is incorporated into TJs at the very late stage. It remains to be clarified how Pilt is recruited to TJs at the very late stage, but this recruitment may be closely correlated with the function of Pilt. Several molecules involved in vesicle trafficking, such as Rab3B and mammalian homologues of yeast SEC6 and -8 gene products, are concentrated at the cytoplasmic face of TJs (32Weber E. Berta G. Tousson A. St. John P. Green M.W. Gopalokrishnan U. Jilling T. Sorscher E.J. Elton T.S. Abrahamson D.R. Kirk K.L. J. Cell Biol. 1994; 125: 583-594Crossref PubMed Scopus (168) Google Scholar, 33Grindstaff K.K. Yeaman C. Anandasabapathy N. Hsu S.C. Rodriguez-Boulan E. Scheller R.H. Nelson W.J. Cell. 1998; 93: 731-740Abstract Full Text Full Text PDF PubMed Scopus (446) Google Scholar, 34Hsu S.C. Hazuka C.D. Foletti D.L. Scheller R.H. Trends Cell Biol. 1999; 9: 150-153Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar). As SEC6 and -8 products are involved in vesicular targeting required for polarized budding in yeast (67Terbush D.R. Maurice T. Roth D. Novick P. EMBO J. 1996; 15: 6483-6494Crossref PubMed Scopus (676) Google Scholar), it is proposed that TJs function as a site for vesicular targeting and fusion to establish and/or maintain epithelial cell polarity (34Hsu S.C. Hazuka C.D. Foletti D.L. Scheller R.H. Trends Cell Biol. 1999; 9: 150-153Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar). We have shown here that Pilt localizes at the Golgi complex as well as at TJs. Therefore, Pilt may be involved in vesicle trafficking between the Golgi complex and TJs to establish and maintain cell polarity. We thank Dr. T. Isogai (Helix Research Institute Inc., Chiba, Japan) for providing us with the cDNA of Pilt. We also thank Dr. T. Akiyama (Tokyo University, Tokyo, Japan) for the cDNA of hDlg-2; Drs. S. Tsukita, M. Furuse, and M. Itoh (Kyoto University, Kyoto, Japan) for the anti-ZO-1 Ab and claudin-L and JAM-L cells; and Drs. T. Kita and H. Ozaki (Kyoto University, Kyoto, Japan) for the anti-JAM Ab.
Side by Side arrangement is considered for the LNG-FPSO offloading operations. In that case, two-body coupled effects are important for LNG-FPSO and LNGC motion and sloshing analysis. The present study is concerned with a ship motion and sloshing analysis considering two-body motion and sloshing-motion coupled effects. The methodology is based on three-dimensional potential theory on a coupled model of LNG-FPSO and LNGC in the frequency domain. To calculation sloshing impact pressures, the violent liquid motion inside tank is treated with three-dimensional numerical model adopting SOLA-VOF scheme.
