Differential localization of myosin and myosin phosphatase subunits in smooth muscle cells and migrating fibroblasts
Differential localization of myosin and myosin phosphatase subunits in smooth muscle cells and migrating fibroblasts
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DOI：
10.1091/mbc.8.4.663
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发表时间：
1997-04-01
影响因子：
3.3
通讯作者：
Brautigan, DL
中科院分区：
文献类型：
--
作者：
Murata, K;Hirano, K;Brautigan, DL
肌球蛋白II轻链（MLC20）由一种Ca2 +/钙调蛋白激活的激酶磷酸化，并由一种磷酸酶去磷酸化，该磷酸酶已被纯化为一种三聚体，包含1型催化亚基的δ异构体（PP1Cδ）、一个与肌球蛋白结合的130 kDa亚基（M130）和一个20 kDa亚基。通过免疫荧光显微镜和差异提取后的免疫印迹，在平滑肌细胞和成纤维细胞中检测了M130和PP1C以及肌球蛋白II的分布。在通透化的细胞中，肌球蛋白和M130与肌动蛋白应力纤维共定位。然而，在未通透化的细胞中，肌球蛋白和M130的染色不同，肌球蛋白主要位于细胞周边，而M130则弥漫分布于整个细胞质。因此，在细胞通透化过程中，大部分M130在可溶性组分中被回收，但所使用的条件影响了M130和肌球蛋白两者的溶解性。PP1Cα异构体与M130共定位，并且也在细胞核中，而PP1Cδ异构体显著定位于细胞核和粘着斑。在迁移细胞中，M130集中在尾缘，并从细胞的前半部分耗尽，双重染色显示肌球蛋白II存在于此。因为已知迁移细胞的尾缘含有磷酸化的肌球蛋白，所以细胞迁移可能需要抑制肌球蛋白LC20磷酸酶，可能是通过M130亚基的磷酸化来实现。
Myosin II light chains (MLC20) are phosphorylated by a Ca2+/calmodulin-activated kinase and dephosphorylated by a phosphatase that has been purified as a trimer containing the delta isoform of type 1 catalytic subunit (PP1C delta), a myosin-binding 130-kDa subunit (M130) and a 20-kDa subunit. The distribution of M130 and PP1C as well as myosin II was examined in smooth muscle cells and fibroblasts by immunofluorescence microscopy and immunoblotting after differential extraction. Myosin and M130 colocalized with actin stress fibers in permeabilized cells. However, in nonpermeabilized cells the staining for myosin and M130 was different, with myosin mostly at the periphery of the cell and the M130 appearing diffusely throughout the cytoplasm. Accordingly, most M130 was recovered in a soluble fraction during permeabilization of cells, but the conditions used affected the solubility of both M130 and myosin. The PP1C alpha isoform colocalized with M130 and also was in the nucleus, whereas the PP1C delta isoform was localized prominently in the nucleus and in focal adhesions. In migrating cells, M130 concentrated in the tailing edge and was depleted from the leading half of the cell, where double staining showed myosin II was present. Because the tailing edge of migrating cells is known to contain phosphorylated myosin, inhibition of myosin LC20 phosphatase, probably by phosphorylation of the M130 subunit, may be required for cell migration.