Regulation of cellular contractile force in response to mechanical stretch by diphosphorylation of myosin regulatory light chain via RhoA signaling cascade.
Fibroblasts regulate their contractile force in response to external stretch; however, the detailed mechanism by which the force is regulated is unclear. Here, we show that diphosphorylation and dephosphorylation of myosin regulatory light chain (MRLC) are involved in the stretch-induced force response. Cellular stiffness, which reflects the cellular contractile force, under external stretch was measured by mechanical-scanning probe microscopy. Fibroblasts (NIH-3T3) expressing green fluorescent protein (GFP)-tagged mutant-type MRLC (MRLC(T18A)-GFP), which cannot be diphosphorylated, did not show any stretch-induced stiffness response, whereas the stiffness in cells expressing GFP-tagged wild-type MRLC (MRLC(WT)-GFP) increased immediately after the stretch and subsequently decreased after 1-2 h. Urea-PAGE western blot analysis showed that the proportion of diphosphorylated MRLC (PP-MRLC) transiently increased after the stretch and decreased after 1-2 h. Dominant-negative RhoA (RhoA(N19))-expressing cells did not show the stiffness response to the stretch, whereas wild-type RhoA-expressing cells did. It was concluded that the cellular force response originates in the stretch-induced diphosphorylation and dephosphorylation of MRLC and is regulated via the RhoA signaling cascade.
Mizutani
Takeomi
T
Division of Biological Sciences, Graduate School of Science, Hokkaido University, Kita-ku, Sapporo, Japan. mizutani@sci.hokudai.ac.jp
Kawabata
Kazushige
K
Koyama
Yoshikazu
Y
Takahashi
Masayuki
M
Haga
Hisashi
H
D000818:Animals; D002460:Cell Line; D005347:Fibroblasts / Q000166:cytology / Q000502:physiology; D051379:Mice; D009119:Muscle Contraction / Q000502:physiology*; D050505:Mutant Proteins / Q000378:metabolism*; D018994:Myosin Light Chains / Q000235:genetics / Q000378:metabolism*; D041681:NIH 3T3 Cells; D010766:Phosphorylation / Q000502:physiology; D015398:Signal Transduction / Q000502:physiology; D013314:Stress, Mechanical; D020741:rho GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D020742:rhoA GTP-Binding Protein
D050505:Mutant Proteins; D018994:Myosin Light Chains; C526380:RhoA protein, mouse; D020741:rho GTP-Binding Proteins; D020742:rhoA GTP-Binding Protein