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 II 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 pseudophosphorylated 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 wild-type 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.
Fumoto
Katsumi
K
Department of Biological Science, Graduate School of Science, Hiroshima University, Higashi-Hiroshima 739-8526, Japan.
Uchimura
Takashi
T
Iwasaki
Takahiro
T
Ueda
Kozue
K
Hosoya
Hiroshi
H
D002465:Cell Movement / Q000502:physiology*; D049452:Green Fluorescent Proteins; D006367:HeLa Cells / Q000502:physiology*; D006801:Humans; D008164:Luminescent Proteins / Q000378:metabolism; D009154:Mutation; D018994:Myosin Light Chains / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D013194:Staining and Labeling
D008164:Luminescent Proteins; C466107:MRLC2 protein, human; D018994:Myosin Light Chains; D049452:Green Fluorescent Proteins