| Project/Area Number |
14580637
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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 |
Functional biochemistry
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| Research Institution | HOKKAIDO UNIVERSITY |
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Principal Investigator |
TAKAHASHI Masayuki Hokkaido Univ., Grad. School of Sci., Asso. Prof., 大学院・理学研究科, 助教授 (50241295)
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| Co-Investigator(Kenkyū-buntansha) |
YAZAWA Michiko Hokkaido Univ., Grad. School of Sci., Prof., 大学院・理学研究科, 教授 (50101134)
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| Project Period (FY) |
2002 – 2003
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| Project Status |
Completed (Fiscal Year 2003)
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| Budget Amount *help |
¥2,900,000 (Direct Cost: ¥2,900,000)
Fiscal Year 2003: ¥1,300,000 (Direct Cost: ¥1,300,000)
Fiscal Year 2002: ¥1,600,000 (Direct Cost: ¥1,600,000)
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| Keywords | nonmuscle myosin II / myosin heavy chain / regulatory light chain / myosin II filament / stress fiber / focal adhesion / 非筋細胞 / ミオシンII / アクチン / 細胞骨格 / 細胞運動 / フィラメント |
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| Research Abstract |
1. Critical regions for filament formation of vertebrate nonmuscle myosin II (termed N1, P1, P2) were identified in the α-helical coiled-coil rod portion of myosin IIB heavy chain by biochemical studies and a molecular modeling. We proposed that the antiparallel interaction between N1 and P2 located within these regions is essential for the nucleation step of nonmuscle myosin II assembly and the parallel interaction between N1 and P1 is important for the elongation step. The composition of filament formed in the mixture of two myosin II isoforms, HA and IIB, was analyzed by FCS or FCCS methods using each rod fragments. The results indicated that heterofilaments were formed in vitro and the exchange of monomer fragments occurred among the formed filaments. 2. The subcellular localization of two kinds of regulatory light chain was examined by the fluorescence observation of nmRLC-GFP or smRLC-GFP expressed in MRC-5 SV1. Both isoforms were localized to stress fibers and no difference of localization was observed between two isoforms. 3. Blot overlay was performed to identify proteins interacting with myosin IIB rod from rat brain extracts. 125 kDa protein was found as a candidate. 4. MRC-5 cells were cultured on the patterned silicon surface, and observed cell morphology and cytoskeletal structure in the cell. In the cell striding over several pits, focal adhesions were formed at the edge of partition between two pits, and stress fibers stretched side by side from those points to the focal adhesions at the edge of the other side. Diphosphorylated RLCs were particularly localized at the peripheral stress fibers close to the partitions, indicating myosin II close to focal adhesions on the partitions were highly activated and produced tension in the cell on the micropit arrays.
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