scholarly journals Spatial Localization of Mono-and Diphosphorylated Myosin II Regulatory Light Chain at the Leading Edge of Motile HeLa Cells

2002 ◽  
Vol 27 (6) ◽  
pp. 479-486 ◽  
Author(s):  
Takashi Uchimura ◽  
Katsumi Fumoto ◽  
Yoshihiro Yamamoto ◽  
Kozue Ueda ◽  
Hiroshi Hosoya
Keyword(s):  
Light Chain ◽  
Hela Cells ◽  
Myosin Ii ◽  
Leading Edge ◽  
Spatial Localization ◽  
Regulatory Light Chain

scholarly journals 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

2003 ◽  
Vol 370 (2) ◽  
pp. 551-556 ◽  
Author(s):  
Katsumi FUMOTO ◽  
Takashi UCHIMURA ◽  
Takahiro IWASAKI ◽  
Kozue UEDA ◽  
Hiroshi HOSOYA
Keyword(s):  
Light Chain ◽  
Hela Cells ◽  
Myosin Ii ◽  
Leading Edge ◽  
Regulatory Light Chain ◽  
Wild Type ◽  
Wound Healing Assay ◽  
Cell Monolayers ◽  
Indirect Immunofluorescence Staining

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.

Characterization of myosin II regulatory light chain isoforms in HeLa cells

Cytoskeleton ◽  
2015 ◽  
Vol 72 (12) ◽  
pp. 609-620 ◽  
Author(s):  
Tomo Kondo ◽  
Morihiro Okada ◽  
Kayo Kunihiro ◽  
Masayuki Takahashi ◽  
Yoshio Yaoita ◽  
...  
Keyword(s):  
Light Chain ◽  
Hela Cells ◽  
Myosin Ii ◽  
Regulatory Light Chain

scholarly journals A fluorescent protein biosensor of myosin II regulatory light chain phosphorylation reports a gradient of phosphorylated myosin II in migrating cells.

1995 ◽  
Vol 6 (12) ◽  
pp. 1755-1768 ◽  
Author(s):  
P L Post ◽  
R L DeBiasio ◽  
D L Taylor
Keyword(s):  
Energy Transfer ◽  
Light Chain ◽  
Fluorescent Protein ◽  
Myosin Ii ◽  
Leading Edge ◽  
Living Cells ◽  
Regulatory Light Chain ◽  
Actin Binding ◽  
Transfer Ratio ◽  
Regulatory Light Chain Phosphorylation

Phosphorylation of the regulatory light chain by myosin light chain kinase (MLCK) regulates the motor activity of smooth muscle and nonmuscle myosin II. We have designed reagents to detect this phosphorylation event in living cells. A new fluorescent protein biosensor of myosin II regulatory light chain phosphorylation (FRLC-Rmyosin II) is described here. The biosensor depends upon energy transfer from fluorescein-labeled regulatory light chains to rhodamine-labeled essential and/or heavy chains. The energy transfer ratio increases by up to 26% when the regulatory light chain is phosphorylated by MLCK. The majority of the change in energy transfer is from regulatory light chain phosphorylation by MLCK (versus phosphorylation by protein kinase C). Folding/unfolding, filament assembly, and actin binding do not have a large effect on the energy transfer ratio. FRLC-Rmyosin II has been microinjected into living cells, where it incorporates into stress fibers and transverse fibers. Treatment of fibroblasts containing FRLC-Rmyosin II with the kinase inhibitor staurosporine produced a lower ratio of rhodamine/fluorescein emission, which corresponds to a lower level of myosin II regulatory light chain phosphorylation. Locomoting fibroblasts containing FRLC-Rmyosin II showed a gradient of myosin II phosphorylation that was lowest near the leading edge and highest in the tail region of these cells, which correlates with previously observed gradients of free calcium and calmodulin activation. Maximal myosin II motor force in the tail may contribute to help cells maintain their polarized shape, retract the tail as the cell moves forward, and deliver disassembled subunits to the leading edge for incorporation into new fibers.

scholarly journals CONCENTRATION OF SINGLY PHOSPHORYLATED MYOSIN II REGULATORY LIGHT CHAIN ALONG THE CLEAVAGE FURROW OF DIVIDING HeLa CELLS

1998 ◽  
Vol 19 (2) ◽  
pp. 111-115 ◽  
Author(s):  
MAKI MURATA-HORI ◽  
NORIO MURAI ◽  
SATOSHI KOMATSU ◽  
YOSHIE UJI ◽  
HIROSHI HOSOYA
Keyword(s):  
Light Chain ◽  
Hela Cells ◽  
Myosin Ii ◽  
Regulatory Light Chain ◽  
Cleavage Furrow

Adenosine induces dephosphorylation of myosin II regulatory light chain in cultured bovine corneal endothelial cells

2004 ◽  
Vol 79 (4) ◽  
pp. 543-551 ◽  
Author(s):  
S.P. Srinivas ◽  
M. Satpathy ◽  
P. Gallagher ◽  
E. Larivière ◽  
W. Van Driessche
Keyword(s):  
Endothelial Cells ◽  
Light Chain ◽  
Myosin Ii ◽  
Regulatory Light Chain ◽  
Corneal Endothelial Cells

Telokin/KRP differentially modulates myosin II filament assembly and regulatory light chain phosphorylation in fibroblasts

BIOPHYSICS ◽  
2006 ◽  
Vol 51 (5) ◽  
pp. 764-770
Author(s):  
D. V. Serebryanaya ◽  
O. V. Shcherbakova ◽  
T. V. Dudnakova ◽  
V. P. Shirinsky ◽  
A. V. Vorotnikov
Keyword(s):  
Light Chain ◽  
Myosin Ii ◽  
Regulatory Light Chain ◽  
Filament Assembly ◽  
Regulatory Light Chain Phosphorylation
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