Function of Nonmuscle Myosin II for Formation of Contractile Apparatus in Cell

非肌肉肌球蛋白 II 在细胞收缩装置形成中的功能

• 批准号: 14580637
• 负责人: TAKAHASHI Masayuki
• 金额: $ 1.86万
• 依托单位: HOKKAIDO UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2002
• 资助国家: 日本
• 起止时间: 2002 至 2003
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-14580637/
• 关键词: 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

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.

1.通过生物化学研究和分子模拟，在肌球蛋白IIB重链的α-螺旋卷曲螺旋杆部分确定了脊椎动物非肌肉肌球蛋白II（称为N1，P1，P2）细丝形成的关键区域。我们提出位于这些区域内的N1和P2之间的反平行相互作用对于非肌肉肌球蛋白II组装的成核步骤是必不可少的，N1和P1之间的平行相互作用对于延伸步骤是重要的。用FCS或FCCS方法分析了两种肌球蛋白II亚型HA和IIB混合物中形成的肌丝的组成。结果表明，在离体培养条件下形成了异丝，异丝之间发生了单体片段的交换。2.通过对MRC-5 SV 1中表达的nmRLC-GFP或smRLC-GFP的荧光观察，检测两种调节轻链的亚细胞定位。两种亚型均定位于应力纤维，两种亚型之间的定位无差异。3.进行印迹覆盖以鉴定来自大鼠脑提取物的与肌球蛋白IIB杆相互作用的蛋白质。发现125 kDa蛋白作为候选蛋白。4. MRC-5细胞在图案化硅表面上培养，观察细胞形态和细胞骨架结构。在跨越多个凹陷的细胞中，在两个凹陷之间的间隔边缘形成局灶性粘连，并且应力纤维从这些点并排延伸到另一侧边缘的局灶性粘连。二磷酸化的RLC特别定位于靠近分区的外周应力纤维处，表明靠近分区上的粘着斑的肌球蛋白II被高度激活，并在微坑阵列上的细胞中产生张力。

项目成果

Tojima, T. et al.: "Dual regulation of LIM kinase 1 expression by cyclic AMP and calcium determines cofilin phosphorylation states during neuritogenesis in NG108-15 cells."Brain Research. 985. 43-55 (2003)

Tojima, T. 等人：“环 AMP 和钙对 LIM 激酶 1 表达的双重调节决定了 NG108-15 细胞神经突发生过程中丝切蛋白的磷酸化状态。”大脑研究。

Hibi, M. et al.: "Dictyostelium discoideum talin A is crucial for myosin II-independent and adhesion-dependent cytokinesis."Journal of Muscle Research and Cell Motility. (in press). (2004)

Hibi, M. 等人：“盘基网柄菌 talin A 对于不依赖于肌球蛋白 II 和粘附依赖性的胞质分裂至关重要。”《肌肉研究与细胞运动杂志》。

Tojima, T.et al.: "Dual regulation of LIM kinase 1 expression by cyclic AMP and calcium determines cofilin phosphorylation states during neuritogenesis in NG108-15 cells"Brain Research. 985・1. 43-55 (2004)

Tojima, T. 等人：“环 AMP 和钙对 LIM 激酶 1 表达的双重调节决定了 NG108-15 细胞神经突发生过程中的丝切蛋白磷酸化状态”Brain Research 985·1 (2004)。

Tojima T. et al.: "Expression by cyclic AMP and calcium determines coffilin phosphorylation states during neuritogenesis in NG108-15 cells"Brain Research. 985-1. 43-55 (2003)

Tojima T. 等人：“环 AMP 和钙的表达决定了 NG108-15 细胞神经突发生过程中的 coffilin 磷酸化状态”Brain Research。