Molecular mechanism of localized activation of myosin II isoforms in cell
| Project/Area Number |
18570144
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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 |
Biophysics
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| Research Institution | Hokkaido University |
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Principal Investigator |
MASAYUKI Takahashi Hokkaido University, Faculty of Science, Hokkaido University, Associate Professor (50241295)
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| Co-Investigator(Kenkyū-buntansha) |
MICHIO Yazawa Hokkaido University, Faculty of Advanced Life Science, Professor (50101134)
AKIKO Nakatomi Hokkaido University, Faculty of Advanced Life Science, Assistant Professor (20360894)
AKIHIKO Yamagishi Ochanomizu University, Faculty of Science, Visiting Professor (70001865)
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| Project Period (FY) |
2006 – 2007
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| Project Status |
Completed (Fiscal Year 2007)
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| Budget Amount *help |
¥3,950,000 (Direct Cost: ¥3,500,000、Indirect Cost: ¥450,000)
Fiscal Year 2007: ¥1,950,000 (Direct Cost: ¥1,500,000、Indirect Cost: ¥450,000)
Fiscal Year 2006: ¥2,000,000 (Direct Cost: ¥2,000,000)
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| Keywords | myosin / filament / subcellular localization / phosphorylation / cell motility |
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| Research Abstract |
Nonmuscle myosin II plays a role in a number of cell motility processes. The activities of myosin II are regulated by phosphorylation of its regulatory light chain (RLC). To function in the cell, nonmuscle myosin II molecules assemble into filaments through their C-terminal tails. In mammalian cells, there are three isoforms of nonmuscle myosin II (IIA IIB and IIC). They most likely assemble in an isoform-specific mode to form homo-filaments in cells. In order to clarify the molecular mechanism of the localized activation of each myosin II isoform in the cell, we performed the following investigations in this project.
1. Identification of the essential regions for filament assembly:
The charge-reversal mutants of the previously proposed three charge clusters (Ni, E.1743-E1753; P1, K1842-K1852; P2, K1874-R1884) of EGFP-MHC-JIB were expressed in the MRC-5 SV1 TG1 cells. The P1 and P2 mutants were diffused in the cell, indicating their importance for the filament assembly.
2. Analyses of the filament mode of myosin II isoforms:
Using FCCS analysis, we found that addition of Mtsl (S100A4), specifically stripped IIA rod fragments away from the pre-formed hetero-assemblies, and consequently, homo-assemblies of IIB rod fragments were formed.
We demonstrated that the C-terminal 305-residue rod fragment of the myosin IIB heavy chain (BRF305) in SV1 cells induced unstable morphology like MHC-IIB^<-/-> fibroblasts. Furthermore, we proposed that the N-57 and C-63 of BRF305 are involved in self-recognition when myosin IIB molecules assemble into-homo-filamen
3. Mechanism of the localized diphosphorylation of RLC in cell spreading:
Immunofluorescence observation of the phosphorylated RLC and F-actin revealed that RLC of myosin IIA isoform was diphosphorylated at the transverse bundles in the lamella. Furthermore, it was indicated that Rho-kinase is involved in this diphosphorylation.
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Report
(3 results)
Research Products
(34 results)
