| Budget Amount *help |
¥3,500,000 (Direct Cost: ¥3,500,000)
Fiscal Year 2006: ¥1,400,000 (Direct Cost: ¥1,400,000)
Fiscal Year 2005: ¥2,100,000 (Direct Cost: ¥2,100,000)
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| Research Abstract |
An actin associate protein, fascin, bundles filamentous actin. The binding of fascin to f-actin is regulated by phosphorylation. Here, we examined the regulatory mechanism of binding of fascin to f-actin by using mutant fascins in which Ser-39 (site of phosphorylation) is replaced by Ala (Fascin Ala) or by Asp (fascin-Asp). Fascin-Ala and-Asp correspond to the state of dephosphorylation and of phosphorylation, respectively. We examined binding of wild type and of these mutant fascins to f-actin.
We expressed EGFP wild type-fascin, EGFP fascin-ala, and EGFP fascin-Asp in growth cones of NG108 cells. EGFP wild-fascin localized at cytosol and actin bundles. EGFP fascin-Ala localized at actin bundles while EGFP fascin-Asp localized at cytosol. Thus, we found localization of fascin is controlled by the state of phosphorylation in living growth cones. Moreover, we recorded fluorecence of single GFP molecule of wild type fascin, fascin-Ala, and fascin-Asp in living growth cones. Now, we are analyzing dynamics of fascin with single molecule level. Moreover, we visualized dynamics of almost all kinds of actin associate proteins of growth cones, which are determined by proteomics.
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