Roles of cofilin in regulation of actin cytoskeleton dynamics
| Project/Area Number |
17570152
|
|---|
| Research Category |
Grant-in-Aid for Scientific Research (C)
|
| Allocation Type | Single-year Grants |
|---|
| Section | 一般 |
|---|
| Research Field |
Cell biology
|
|---|
| Research Institution | Tohoku University |
|---|
Principal Investigator |
OHASHI Kazumasa Tohoku University, Graduate School of Life Sciences, Associate Professor, 大学院生命科学研究科, 助教授 (10312539)
|
|---|
| Project Period (FY) |
2005 – 2006
|
| Project Status |
Completed (Fiscal Year 2006)
|
| Budget Amount *help |
¥3,500,000 (Direct Cost: ¥3,500,000)
Fiscal Year 2006: ¥1,500,000 (Direct Cost: ¥1,500,000)
Fiscal Year 2005: ¥2,000,000 (Direct Cost: ¥2,000,000)
|
|---|
| Keywords | Actin cytoskeleton / cofilin / LIM-kinase / Slingshot / GFP / Phosphorylation / Inhibitor / イメージング / ダイナミクス |
|---|
| Research Abstract |
Actin cytoskeletal dynamics and reorganization play essential roles in various intracellular events, including cell migration, cytokinesis, endocytosis, and polarity formation. Actin dynamics and reorganization are spatiotemporally regulated by a large number of actin-binding proteins and upstream signaling molecules. Cofilin, a key regulator of actin filament dynamics, stimulates depolymerization and severing of actin filaments. Cofilin is inactivated by phosphorylation of Ser-3 by LIM-kinase (LIMK). The Ser-3-phosphorylated cofilin (P-cofilin) is dephosphorylated and reactivated by the Slingshot phosphatase. The purposes of this project were to reveal the function of cofilin in actin cytoskeleton reorganization. In the present study, we have developed a new probe that detects the actin-binding activity of cofilin using a bimolecular fluorescence complementation (BiFC) method. The probe was improved higher intensity and sensitivity in this study. We expressed and purified these probe proteins in insect cells using a baculovirus expressing system, and developed methods to measure the actin-binding activity of cofilin, the kinase activity of LIMK, and the phosphatase activity of Slingshot with the fluorescence of complemented-GFP. We screened the inhibitors of cofilin, LIMK and Slingshot with over five hundred chemical compounds. As a consequence, LIMK was inhibited two compounds which was known as Src tyrosine kinase inhibitor. The compounds inhibited the hyper-polymerization of actin in LIMK overexpressing cells. It was reported that one of the compounds inhibited cell migration and neurite extension at the concentration of LIMK inhibition. We are tring to analyze the role of cofilin phosphorylation in the actin cytoskeleton reorganization using the compounds in living cells. We are going to screen more specific inhibitors for LIMK in derivative forms of the compounds. These inhibitors will be seeds of the drugs for inhibition of metastasis and inflammation.
|
Report
(3 results)
Research Products
(23 results)
