| Budget Amount *help |
¥14,200,000 (Direct Cost: ¥14,200,000)
Fiscal Year 2004: ¥5,900,000 (Direct Cost: ¥5,900,000)
Fiscal Year 2003: ¥8,300,000 (Direct Cost: ¥8,300,000)
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| Research Abstract |
1.Improvement of the fluorescence localization-based retrovirus-mediated expression cloning for identification of novel proteins localized at epithelial cell structures.
We constructed cDNA libraries of cultured epithelial cell lines MDCK and CSG using a mouse retrovirus expression vector. As host epithelial cells for retrovirus infection, MDCK cells overexpressing the retrovirus receptor were. In small-scale screenings, we confirmed that claudins, occludin, JAM, etc. can be cloned in this system. During several trials, we improved the protocol for effective screening.
2.Identification of novel proteins concentrated in epithelial cell-cell junctions.
In the middle-scale screening using the CSG cells-derived cDNA library, homer2, abLIM3, and a novel coiled-coil protein have been identified as novel proteins localizing at epithelial cell-cell junctions. We produced antibodies against these proteins and analyzed their tissue expression pattern. We also determined the domains required for junction allocalization for each protein. Among them, alLIM3 is a novel subtype of abLIM, which is known to function in neuron orretin a by interacting with actin filaments. We found that abLIM3 is localized at cell-cell junctions in endothelial cells in lung and muscle tissues, and interact with actin filaments in vitro. Tissue expression pattern of abLIM3 suggest some roles of this protein in cell-cell junctions in endothelial cells that receive extension and shrinkage.
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