2004 Fiscal Year Final Research Report Summary
Visualization of phosphorylated myosin II in living mammalian cultured cells
| Project/Area Number |
15570161
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Cell biology
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| Research Institution | HIROSHIMA UNIVERSITY |
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Principal Investigator |
KOSAKA Toshikazu Hiroshima University, Graduate School of Science, Associate Professor, 大学院・理学研究科, 助教授 (10033903)
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| Co-Investigator(Kenkyū-buntansha) |
HOSOYA Hiroshi Hiroshima University, Graduate School of Science, Professor, 大学院・理学研究科, 教授 (90183102)
SAKO Yasushi Osaka University, Graduate School of Life Science, Associate Professor, 大学院生命科学研究科, 助教授 (20215700)
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| Project Period (FY) |
2003 – 2004
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| Keywords | myosin II / migration / HeLa cell / phosphorylation / wound healing / leading edge / 細胞移動 |
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| Research Abstract |
To investigate the role of phosphorylated myosin II regulatory light chain (MRLC) in living cell migration, mutant MRLCs were engineered and introduced into HeLa cells. The mutant MRLCs include an unphosphorylatable for, m, in which both Thr-18 and Ser-19 were substituted with Ala (AA-MRLC), and pseudophosphorylated forms, in which Thr-18 and Ser-19 were replaced with Ala and Asp, respectively (AD-MRLC), and both Thr-18 and Ser-19 were replaced with Asp (DD-MRLC). Mutant MRLC-expressing cell monolayers were mechanically stimulated by scratching, and the cells were forced to migrate in a given direction. In this wound-healing assay, the AA-MRLC-expressing cells migrated much more slowly than the wild-type MRLC-expressing cells. In the case of DD-MRLC- and AD-MRLC-expressing cell, no significant differences compared with wild-type MRLC-expressing cells were observed in their migration speed. Indirect immunofluorescence staining showed that the accumulation of endogenous diphosphorylated MRLC at the leading edge was not observed in AA-MRLC-expressing cells, although AA-MRLC was incorporated into myosin heavy chain and localized at the leading edge. In conclusion, we propose that the phosphorylation of MRLC is required to generate the driving source in the migration of the cells but not necessary for localization of myosin II at the leading edge.
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