Phosphorylation of myosin II regulatory light chain is necessary for migration of HeLa cells but not for localization of myosin II at the leading edge
Phosphorylation of myosin II regulatory light chain is necessary for migration of HeLa cells but not for localization of myosin II at the leading edge
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DOI：
10.1042/bj20021559
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发表时间：
2003-03-01
影响因子：
4.1
通讯作者：
Hosoya, H
中科院分区：
文献类型：
--
作者：
Fumoto, K;Uchimura, T;Hosoya, H
为了研究磷酸化肌球蛋白 11 调节轻链 (MRLC) 在活细胞迁移中的作用，我们对这些突变型 MRLC 进行了改造并将其引入 HeLa 细胞中。突变型MRLC包括不可磷酸化形式，其中Thr-18和Ser-19均被Ala取代（AA-MRLC），以及假磷酸化形式，其中Thr-18和Ser-19分别被Ala和Asp取代（AD-MRLC），Thr-18和Ser-19均被Asp取代（DD-MRLC）。通过刮擦对表达突变 MRLC 的单层细胞进行机械刺激，迫使细胞向给定方向迁移。在这种伤口愈合测定中，表达 AA-MRLC 的细胞比表达野生型 MRLC 的细胞迁移得慢得多。在DD-MRLC和AD-MRLC表达细胞的情况下，与野生型MRLC表达细胞相比，在其迁移速度方面没有观察到显着差异。间接免疫荧光染色显示，尽管 AA-MRLC 已掺入肌球蛋白重链并位于前缘，但在表达 AA-MRLC 的细胞中未观察到内源二磷酸化 MRLC 在前缘的积累。总之，我们提出 MRLC 的磷酸化对于产生细胞迁移的驱动力是必需的，但对于肌球蛋白 II 在前缘的定位不是必需的。
To investigate the role of phosphorylated myosin 11 regulatory light chain (MRLC) in living cell migration, these mutant MRLCs were engineered and introduced into HeLa cells. The mutant MRLCs include an unphosphorylatable form, in which both Thr-18 and Ser-19 were substituted with Ala (AA-MRLC), and pseudophosphorylarted forms, in which Thr-18 and Ser-19 were replaced with Ala and Asp, respectively (AD-MRLC), and both Thr-18 and Ser-19 were replaced with Asp (DD-MRLC). Mutant MRLC-expressing cell monolayers were mechanically stimulated by scratching, and the cells were forced to migrate in a given direction. In this wound-healing assay, the AA-MRLC-expressing cells migrated much more slowly than the wildtype MRLC-expressing cells. In the case of DD-MRLC- and AD-MRLC-expressing cells, no significant differences compared with wild-type MRLC-expressing cells were observed in their migration speed. Indirect immunofluorescence staining showed that the accumulation of endogenous diphosphorylated MRLC at the leading edge was not observed in AA-MRLC-expressing cells, although AA-MRLC was incorporated into myosin heavy chain and localized at the leading edge. In conclusion, we propose that the phosphorylation of MRLC is required to generate the driving force in the migration of the cells but not necessary for localization of myosin II at the leading edge.