scholarly journals Nonmuscle Myosin IIA Regulates Platelet Contractile Forces Through Rho Kinase and Myosin Light-Chain Kinase

2016 ◽  
Vol 138 (10) ◽  
Author(s):  
Shirin Feghhi ◽  
Wes W. Tooley ◽  
Nathan J. Sniadecki
Keyword(s):  
Atpase Activity ◽  
Light Chain ◽  
Myosin Light Chain Kinase ◽  
Myosin Light Chain ◽  
Rho Kinase ◽  
Myosin Atpase ◽  
Nonmuscle Myosin ◽  
Myosin Phosphorylation ◽  
Myosin Atpase Activity ◽  
Contractile Forces

Platelet contractile forces play a major role in clot retraction and help to hold hemostatic clots against the vessel wall. Platelet forces are produced by its cytoskeleton, which is composed of actin and nonmuscle myosin filaments. In this work, we studied the role of Rho kinase, myosin light-chain kinase, and myosin in the generation of contractile forces by using pharmacological inhibitors and arrays of flexible microposts to measure platelet forces. When platelets were seeded onto microposts, they formed aggregates on the tips of the microposts. Forces produced by the platelets in the aggregates were measured by quantifying the deflection of the microposts, which bent in proportion to the force of the platelets. Platelets were treated with small molecule inhibitors of myosin activity: Y-27632 to inhibit the Rho kinase (ROCK), ML-7 to inhibit myosin light-chain kinase (MLCK), and blebbistatin to inhibit myosin ATPase activity. ROCK inhibition reduced platelet forces, demonstrating the importance of the assembly of actin and myosin phosphorylation in generating contractile forces. Similarly, MLCK inhibition caused weaker platelet forces, which verifies that myosin phosphorylation is needed for force generation in platelets. Platelets treated with blebbistatin also had weaker forces, which indicates that myosin's ATPase activity is necessary for platelet forces. Our studies demonstrate that myosin ATPase activity and the regulation of actin–myosin assembly by ROCK and MLCK are needed for the generation of platelet forces. Our findings illustrate and explain the importance of myosin for clot compaction in hemostasis and thrombosis.

The roles of myosin ATPase activity and myosin light chain relative content in the slowing of type IIB fibers with hindlimb unweighting in rats

2007 ◽  
Vol 293 (2) ◽  
pp. C723-C728 ◽  
Author(s):  
Sheng Zhong ◽  
LaDora V. Thompson
Keyword(s):  
Atpase Activity ◽  
Light Chain ◽  
Myosin Light Chain ◽  
Relative Content ◽  
Myosin Atpase ◽  
Shortening Velocity ◽  
Essential Light Chain ◽  
Myosin Atpase Activity ◽  
Semimembranosus Muscle ◽  
Relative Change

We tested the hypothesis that slowing of shortening velocity generated by type IIB fibers from hindlimb-unweighted (HU) rats resulted from a reduced ATPase activity and/or a reduction in the relative content of myosin light chain 3f isoform content (MLC3f). After 2, 3, and 4 wk of HU, maximal unloaded shortening velocity ( Vo) of single permeabilized semimembranosus muscle fibers was determined by the slack test. Subsequently, the myosin heavy chain and the relative content of MLC were determined by SDS-PAGE. The ratio of MLC3f to MLC2f was determined by densitometric analysis. In addition, myofibrils were prepared from permeabilized fibers (soleus and semimembranosus muscles) and assayed for resting myosin ATPase and Ca2+-activated myosin ATPase. After HU, Vo declined by 28–40% and the MLC3f/MLC2f ratio decreased by 32 to 48%. A significant correlation between the relative amount of MLC3f and Vo was found ( r = 0.48, P < 0.05). Resting myosin ATPase rates were not different between myofibrils prepared from corresponding muscles of control and HU rats ( P = 0.86). Ca2+-activated myosin ATPase activities also were not different between myofibrils prepared from corresponding muscles of control and HU rats ( P = 0.13). These data suggest that the slowing of maximal unloaded shortening velocity in type IIB fibers with HU is, at least in part, due to a relative change in the essential light chain composition, a decrease in the relative amount of MLC3f and most likely a concomitant increase in MLC1f. However, this reduction in Vo is independent of myosin ATPase activity.

Uncoupling of actin-activated myosin ATPase activity from actin binding by a monoclonal antibody directed against the N-terminus of myosin light chain 1

Biochemistry ◽  
1992 ◽  
Vol 31 (16) ◽  
pp. 4090-4095 ◽  
Author(s):  
Wylinn Boey ◽  
Alan W. Everett ◽  
John Sleep ◽  
John Kendrick-Jones ◽  
Cristobal G. Dos Remedios
Keyword(s):  
Monoclonal Antibody ◽  
Atpase Activity ◽  
Light Chain ◽  
Myosin Light Chain ◽  
Myosin Atpase ◽  
Actin Binding ◽  
Myosin Atpase Activity ◽  
N Terminus

Polylysine activates smooth muscle myosin ATPase activity via induction of a 10S to 6S transition

1993 ◽  
Vol 265 (2) ◽  
pp. C379-C386 ◽  
Author(s):  
P. T. Szymanski ◽  
D. G. Ferguson ◽  
R. J. Paul
Keyword(s):  
Smooth Muscle ◽  
Atpase Activity ◽  
Light Chain ◽  
Myosin Light Chain ◽  
Myosin Atpase ◽  
Solution Viscosity ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Myosin Atpase Activity ◽  
Muscle Myosin

Polylysine (10-13 kDa) stimulates contraction in smooth muscle skinned fibers and activates actomyosin adenosinetriphosphatase (ATPase) activity in the absence of myosin light chain phosphorylation [P. T. Szymanski and R. J. Paul. Adv. Exp. Med. 304: 363-368, 1991; P. T. Szymanski, J. D. Strauss, G. Doerman, J. DiSalvo, and R. J. Paul. Am J. Physiol. 262 (Cell Physiol. 31): C1445-C1455, 1992]. To provide further information on the mechanism of polylysine action on contractility in smooth muscle, we investigated its effect on ATPase activity and conformation of purified gizzard myosin. We report here that polylysine directly stimulates myosin ATPase activity in a concentration-dependent manner. This stimulation could be completely abolished with the addition of heparin, a negatively charged heteropolysaccharide. Polylysine (10 microM) increases myosin ATPase activity to a level similar to that of myosin phosphorylation. Addition of 10 microM polylysine to phosphorylated myosin [with myosin light chain kinase and adenosine 5'-O-(3-thiotriphosphate) (ATP gamma S), to approximately 1.9 mol P/mol myosin], however, did not further stimulate ATPase activity. At 0.2 M KCl (the salt concentration at which myosin exists primary in the 10S form), the addition of polylysine increases myosin ATPase activity to a level comparable to that of untreated myosin in 0.3 M KCl. These changes parallel the increase in solution viscosity elicited by polylysine. These results suggest that polylysine induces a transition in myosin conformation from the 10S to the 6S form, and this was confirmed by electron microscopy.(ABSTRACT TRUNCATED AT 250 WORDS)

scholarly journals Thrombin-induced contraction in alveolar epithelial cells probed by traction microscopy

2006 ◽  
Vol 101 (2) ◽  
pp. 512-520 ◽  
Author(s):  
Núria Gavara ◽  
Raimon Sunyer ◽  
Pere Roca-Cusachs ◽  
Ramon Farré ◽  
Mar Rotger ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Actin Cytoskeleton ◽  
Light Chain ◽  
Myosin Light Chain Kinase ◽  
Myosin Light Chain ◽  
Rho Kinase ◽  
Alveolar Epithelial Cells ◽  
Alveolar Epithelial ◽  
Contractile Forces ◽  
Traction Microscopy

Contractile tension of alveolar epithelial cells plays a major role in the force balance that regulates the structural integrity of the alveolar barrier. The aim of this work was to study thrombin-induced contractile forces of alveolar epithelial cells. A549 alveolar epithelial cells were challenged with thrombin, and time course of contractile forces was measured by traction microscopy. The cells exhibited basal contraction with total force magnitude 55.0 ± 12.0 nN (mean ± SE, n = 12). Traction forces were exerted predominantly at the cell periphery and pointed to the cell center. Thrombin (1 U/ml) induced a fast and sustained 2.5-fold increase in traction forces, which maintained peripheral and centripetal distribution. Actin fluorescent staining revealed F-actin polymerization and enhancement of peripheral actin rim. Disruption of actin cytoskeleton with cytochalasin D (5 μM, 30 min) and inhibition of myosin light chain kinase with ML-7 (10 μM, 30 min) and Rho kinase with Y-27632 (10 μM, 30 min) markedly depressed basal contractile tone and abolished thrombin-induced cell contraction. Therefore, the contractile response of alveolar epithelial cells to the inflammatory agonist thrombin was mediated by actin cytoskeleton remodeling and actomyosin activation through myosin light chain kinase and Rho kinase signaling pathways. Thrombin-induced contractile tension might further impair alveolar epithelial barrier integrity in the injured lung.

scholarly journals MicroRNA Regulation of Nonmuscle Myosin Light Chain Kinase Expression in Human Lung Endothelium

2013 ◽  
Vol 49 (1) ◽  
pp. 58-66 ◽  
Author(s):  
Djanybek M. Adyshev ◽  
Nurgul Moldobaeva ◽  
Brandon Mapes ◽  
Venkate Elangovan ◽  
Joe G. N. Garcia
Keyword(s):  
Light Chain ◽  
Myosin Light Chain Kinase ◽  
Myosin Light Chain ◽  
Human Lung ◽  
Nonmuscle Myosin ◽  
Microrna Regulation ◽  
Lung Endothelium ◽  
Kinase Expression

scholarly journals Nonmuscle myosin light-chain kinase mediates neutrophil transmigration in sepsis-induced lung inflammation by activating β2 integrins

2008 ◽  
Vol 9 (8) ◽  
pp. 880-886 ◽  
Author(s):  
Jingsong Xu ◽  
Xiao-Pei Gao ◽  
Ramaswamy Ramchandran ◽  
You-Yang Zhao ◽  
Stephen M Vogel ◽  
...  
Keyword(s):  
Light Chain ◽  
Lung Inflammation ◽  
Myosin Light Chain Kinase ◽  
Myosin Light Chain ◽  
Nonmuscle Myosin ◽  
Β2 Integrins
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