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

1.通过生化研究和分子模拟，在脊椎动物非肌肉肌球蛋白II重链的α螺旋卷曲杆段确定了肌球蛋白II微丝形成的关键区域(N1、P1、P2)。我们认为，位于这些区域的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，而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的混合物中形成的细丝的组成。结果表明，在体外形成了异质纤维，并在形成的纤维之间发生了单体片段的交换。2.通过对mRC-5 SV1中表达的nmRLC-GFP或smRLC-GFP的荧光观察，检测两种调控轻链的亚细胞定位。两种异构体均定位于应力纤维，两种异构体的定位没有差异。3.用印迹重叠技术从大鼠脑提取液中鉴定与肌球蛋白IIB棒相互作用的蛋白质。筛选出125 kDa的候选蛋白。4.在图案化硅表面培养MRC-5细胞，观察细胞形态和细胞骨架结构。在跨越多个凹坑的细胞中，在两个凹坑之间的隔板边缘形成灶性粘连，应力纤维从这些点并排伸展到另一侧边缘的灶性粘连。二磷酸化的RLC尤其定位于靠近分隔区的外周应力纤维，这表明靠近分隔区焦点粘连的肌球蛋白II被高度激活，并在微坑阵列上的细胞中产生张力。

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.