| Budget Amount *help |
¥3,600,000 (Direct Cost: ¥3,600,000)
Fiscal Year 2001: ¥1,100,000 (Direct Cost: ¥1,100,000)
Fiscal Year 2000: ¥1,200,000 (Direct Cost: ¥1,200,000)
Fiscal Year 1999: ¥1,300,000 (Direct Cost: ¥1,300,000)
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| Research Abstract |
We have identified a novel calcium-binding protein, Iba1, which is specifically expressed in macrophages/microglia. We further demonstrated that Iba1 is involved in a signaling pathway of Rac, a member of the Rho family of small GTPases, and that Iba1 regulates migration and phagocytosis of activated microglia by modulating reorganization of the actin cytoskeleton. In a microglia cell line, MG5, Iba1 was shown to translocate together with Rac and F-actin into membrane ruffles induced by M-CSF, and into phagocytic cups formed during zymosan phagocytosis. We also demonstrated that Rac was simultaneously activated during membrane ruffling and phagocytosis. Expression of suppressive Iba1 mutants in MG5 resulted in inhibition of membrane ruffling and phagocytosis, further, membrane ruffling induced by dominant active RacV12 was also suppressed by the Iba1 mutants. These observations indicate that Iba1 functions in a Rac signaling pathway.
We have furthermore demonstrated actin-binding capacity of Iba1.In the presence of F-actin, Iba1 was co-precipitated with F-actin by high speed centrifugation, indicating F-actin binding of Iba1. In the presence ofIba1, F-action was precipitated by low speed centrifugation, suggesting F-actin-crosslinking activity of Iba1. The F-actin crosslinking activity was further confirmed by electromicroscopic observations. The suppressive Iba1 mutant was also examined for the actin co-precipitation assays, in which the mutant was shown to possess the F-actin-binding capacity, but to lose the F- actin-crosslinking activity, suggesting that this properties of the mutant disrupt the coordinated actin architecture. These findings indicated that Iba1 is a key molecule for reorganization of the actin cytoskeleton induced by the signaling of Rac.
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