|Budget Amount *help
¥3,700,000 (Direct Cost : ¥3,700,000)
Fiscal Year 2005 : ¥1,100,000 (Direct Cost : ¥1,100,000)
Fiscal Year 2004 : ¥1,100,000 (Direct Cost : ¥1,100,000)
Fiscal Year 2003 : ¥1,500,000 (Direct Cost : ¥1,500,000)
Iba1 is a macrophage/microglia-specific calcium binding protein, which is concerned with monomeric G protein Rac to regulate actin reorganization in motility and phagocytosis of activated microglia. To find out Iba1-binding protein, we performed a yeast two-hybrid screening, and then we got some clones encoding an actin bundling protein fimbrin. Fimbrin biochemically and directly bound to Iba1. In immunocytochemical studies of a microglia cell line MG5, Iba1 and fimbrin were shown to colocalized in membrane ruffles induced by M-CSF stimulation and in phagocytic cups formed during zymosan ingestion. Further, Iba1 was shown to enhance actin bundling activity of fimbrin. These facts suggest that binding of Iba1 and fimbrin is important for regulation of the actin cytoskeleton in motility and phagocytosis of activated microglia.
To visualize microglia in living animals and cells, we generated Iba1 promoter-driving-EGFP transgenic mice. To determine Iba1 promoter region, we isolated an Iba1 genomic clone, and found that the Iba1 gene spans within about 5.5kbp region, and that the promoter activity is possesses in a 1kbp fragment. Then, we constructed an Iba1-promoter-EGFP construct, and microinjected the construct into mouse one cell embryos. After that, we established seven lines of the transgenic mice, and among them, one line was found to express microglia-specific fluorescence signals. The mice will be very useful for microglia researches. We are now trying to generate other Iba1 promoter trangenic mice that express enzymes other than EGFP.