| Budget Amount *help |
¥16,490,000 (Direct Cost: ¥14,600,000、Indirect Cost: ¥1,890,000)
Fiscal Year 2007: ¥8,190,000 (Direct Cost: ¥6,300,000、Indirect Cost: ¥1,890,000)
Fiscal Year 2006: ¥8,300,000 (Direct Cost: ¥8,300,000)
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| Research Abstract |
To clarify the molecular mechanism behind the regulation of epithelial cell-cell adhesion, we looked for novel proteins that are localized at tight and adherens junctions by a novel expression cloning method based on subcellular localization, namely the fluorescence localization-based retrovirus-mediated expression cloning (FL-REX). Initially, we confirmed that cDNAs for various known components associated with tight and adherens junctions were indeed identified in this screening by using cDNA libraries generated from various epithelial cell lines. Then, we found that ARHGAP12 and SPAL3, potential GTPase activating proteins for small G proteins identified in this method, are concentrated into epithelial adherens junctions by immunoflureoscence staining. When SPAL3, which has a Rap-GAP domain, was overexpressed in cultured fibroblasts, the cells remarkably elongated. Furthermore, cultured epithelial cells overexpressing SPAL3 exhibited retardation in cell-cell junction formation, suggesting that SPAL3 is involved in the regulation of epithelial cell-cell adhesion via actin filaments. In addition, by using the FL-REX method, we have succeeded in identification of a novel membrane protein TRCX, which is localized at tricellular junctions and is involved in the barrier function of the epithelial cellular sheet. This result is expected to contribute to the future analyses of the molecular mechanism behind the formation and physiological functions of tricellular junctions, which have not been well analyzed yet.
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