|Budget Amount *help
¥4,020,000 (Direct Cost: ¥3,600,000、Indirect Cost: ¥420,000)
Fiscal Year 2007: ¥1,820,000 (Direct Cost: ¥1,400,000、Indirect Cost: ¥420,000)
Fiscal Year 2006: ¥2,200,000 (Direct Cost: ¥2,200,000)
To establish the MDCK cells which constitutively express FRET probes, the plasmids containing retroviral components are generated. In a previous version, the FRET probes, which consisted of YFP-small GTPase-the effector-CFP, are driven by actin promotor. Since the YFP and CFP are the fluorescent proteins similarly derived from GFP (isolated from jelly fish Aequarea victoria) and their amino acid sequences are similar, it was difficult to obtain the cells both expressing YFP and CFP probably due to homologous recombination between YFP and CFP when the DNA integrates to the genome. In stead of CFP, I utilized another fluorescent protein, mAG (Azami-Green from the stony coral, Galaxeidae) to replace CFP in the FRET probes. Although the MDCK cells expressing the FRET probes were established, the fluorescence intensity was not enough to be observed with our microscopy system. Next, recently-isolated TFP (teal fluorescent protein) from coral Clavularia sp. replaced CFP in the RhoA, Rac1, and Cdc42probes. A series of experiments indicated that replacement of CFP by TFP did not perturb the conformation of the RhoA, Rac1, and Cdc42 probes in migrating cells. MDCK cells expressing those probes have been established. Culturing them in Matrigel induced the cyst formation, and the confocal microscopy observation showed that the activation of Cdc42, but not Rac and RhoA, was higher in apical membrane than baso-lateral one. The FRET probes to detect the distribution of phosphatidylinositides and its derivative diacylglycerol, have been also established and named Pippi (Phosphatidylinositol phosphate indicator), to found that PIP3, PI(4,5) P2, PI(3,4) P2, but not PI(4) P, are increased in the front of the migrating MDCK cells. Now the Pippi-probes using TFP have been established to examine the polarity formation in the 3-dimensional culture system.