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.

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

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

Three-dimensional in vivo analysis of Dictyostelium mounds reveals directional sorting of prestalk cells and defines a role for the myosin II regulatory light chain in prestalk cell sorting and tip protrusion

Development ◽  
2000 ◽  
Vol 127 (12) ◽  
pp. 2715-2728 ◽  
Author(s):  
P.A. Clow ◽  
T. Chen ◽  
R.L. Chisholm ◽  
J.G. McNally
Keyword(s):  
Long Range ◽  
Light Chain ◽  
Cell Sorting ◽  
Myosin Ii ◽  
Regulatory Light Chain ◽  
Upward Movement ◽  
Wild Type ◽  
Vertical Column ◽  
Initial Cluster

During cell sorting in Dictyostelium, we observed that GFP-tagged prestalk cells (ecmAO-expressing cells) moved independently and directionally to form a cluster. This is consistent with a chemotaxis model for cell sorting (and not differential adhesion) in which a long-range signal attracts many of the prestalk cells to the site of cluster formation. Surprisingly, the ecmAO prestalk cluster that we observed was initially found at a random location within the mound of this Ax3 strain, defining an intermediate sorting stage not widely reported in Dictyostelium. The cluster then moved en masse to the top of the mound to produce the classic, apical pattern of ecmAO prestalk cells. Migration of the cluster was also directional, suggesting the presence of another long-range guidance cue. Once at the mound apex, the cluster continued moving upward leading to protrusion of the mound's tip. To investigate the role of the cluster in tip protrusion, we examined ecmAO prestalk-cell sorting in a myosin II regulatory light chain (RLC) null in which tips fail to form. In RLC-null mounds, ecmAO prestalk cells formed an initial cluster that began to move to the mound apex, but then arrested as a vertical column that extended from the mound's apex to its base. Mixing experiments with wild-type cells demonstrated that the RLC-null ecmAO prestalk-cell defect is cell autonomous. These observations define a specific mechanism for myosin's function in tip formation, namely a mechanical role in the upward movement of the ecmAO prestalk cluster. The wild-type data demonstrate that cell sorting can occur in two steps, suggesting that, in this Ax3 strain, spatially and temporally distinct cues may guide prestalk cells first to an initial cluster and then later to the tip.

scholarly journals On the Role of Myosin-II in Cytokinesis: Division ofDictyostelium Cells under Adhesive and Nonadhesive Conditions

1997 ◽  
Vol 8 (12) ◽  
pp. 2617-2629 ◽  
Author(s):  
Ji-Hong Zang ◽  
Guy Cavet ◽  
James H. Sabry ◽  
Peter Wagner ◽  
Sheri L. Moores ◽  
...  
Keyword(s):  
Cell Division ◽  
Light Chain ◽  
Binding Sites ◽  
Myosin Ii ◽  
Hydrophobic Surface ◽  
Wild Type ◽  
Daughter Cells ◽  
Adhesive Surface ◽  
Spatial And Temporal Changes

We have investigated the role of myosin in cytokinesis inDictyostelium cells by examining cells under both adhesive and nonadhesive conditions. On an adhesive surface, both wild-type and myosin-null cells undergo the normal processes of mitotic rounding, cell elongation, polar ruffling, furrow ingression, and separation of daughter cells. When cells are denied adhesion through culturing in suspension or on a hydrophobic surface, wild-type cells undergo these same processes. However, cells lacking myosin round up and polar ruffle, but fail to elongate, furrow, or divide. These differences show that cell division can be driven by two mechanisms that we term Cytokinesis A, which requires myosin, and Cytokinesis B, which is cell adhesion dependent. We have used these approaches to examine cells expressing a myosin whose two light chain-binding sites were deleted (ΔBLCBS-myosin). Although this myosin is a slower motor than wild-type myosin and has constitutively high activity due to the abolition of regulation by light-chain phosphorylation, cells expressing ΔBLCBS-myosin were previously shown to divide in suspension ( Uyeda et al., 1996 ). However, we suspected their behavior during cytokinesis to be different from wild-type cells given the large alteration in their myosin. Surprisingly, ΔBLCBS-myosin undergoes relatively normal spatial and temporal changes in localization during mitosis. Furthermore, the rate of furrow progression in cells expressing a ΔBLCBS-myosin is similar to that in wild-type cells.

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

scholarly journals Akt isoforms differentially regulate neutrophil functions

Blood ◽  
2010 ◽  
Vol 115 (21) ◽  
pp. 4237-4246 ◽  
Author(s):  
Jia Chen ◽  
Haiyang Tang ◽  
Nissim Hay ◽  
Jingsong Xu ◽  
Richard D. Ye
Keyword(s):  
Kinase Inhibitor ◽  
Nicotinamide Adenine Dinucleotide Phosphate ◽  
Leading Edge ◽  
Neutrophil Activation ◽  
Enzyme Release ◽  
Wild Type ◽  
Akt Isoforms ◽  
Phosphoinositide 3 Kinase ◽  
O2 Production

In neutrophils, the phosphoinositide 3-kinase/Akt signaling cascade is involved in migration, degranulation, and O2− production. However, it is unclear whether the Akt kinase isoforms have distinct functions in neutrophil activation. Here we report functional differences between the 2 major Akt isoforms in neutrophil activation on the basis of studies in which we used individual Akt1 and Akt2 knockout mice. Akt2−/− neutrophils exhibited decreased cell migration, granule enzyme release, and O2− production compared with wild-type and Akt1−/− neutrophils. Surprisingly, Akt2 deficiency and pharmacologic inhibition of Akt also abrogated phorbol ester-induced O2− production, which was unaffected by treatment with the phosphoinositide 3-kinase inhibitor LY294002. The decreased O2− production in Akt2−/− neutrophils was accompanied by reduced p47phox phosphorylation and its membrane translocation, suggesting that Akt2 is important for the assembly of phagocyte nicotinamide adenine dinucleotide phosphate oxidase. In wild-type neutrophils, Akt2 but not Akt1 translocated to plasma membrane upon chemoattractant stimulation and to the leading edge in polarized neutrophils. In the absence of Akt2, chemoattractant-induced Akt protein phosphorylation was significantly reduced. These results demonstrate a predominant role of Akt2 in regulating neutrophil functions and provide evidence for differential activation of the 2 Akt isoforms in neutrophils.

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