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

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.