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.

Lanthanum chloride causes blood–brain barrier disruption through intracellular calcium-mediated RhoA/Rho kinase signaling and myosin light chain kinase

Metallomics ◽  
2020 ◽  
Author(s):  
Jie Wu ◽  
Jinghua Yang ◽  
Miao Yu ◽  
Wenchang Sun ◽  
Yarao Han ◽  
...  
Keyword(s):  
Actin Cytoskeleton ◽  
Intracellular Calcium ◽  
Light Chain ◽  
Myosin Light Chain Kinase ◽  
Myosin Light Chain ◽  
Rho Kinase ◽  
Endothelial Barrier ◽  
Blood Brain Barrier Disruption ◽  
Intracellular Ca ◽  
Brain Barrier Disruption

Lanthanum caused endothelial barrier hyperpermeability, loss of VE-cadherin and rearrangement of the actin cytoskeleton, though intracellular Ca2+-mediated RhoA/ROCK and MLCK pathways.

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.

Physiological regulation of epithelial tight junctions is associated with myosin light-chain phosphorylation

1997 ◽  
Vol 273 (4) ◽  
pp. C1378-C1385 ◽  
Author(s):  
Jerrold R. Turner ◽  
Brian K. Rill ◽  
Susan L. Carlson ◽  
Denise Carnes ◽  
Rachel Kerner ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Tight Junction ◽  
Tight Junctions ◽  
Light Chain ◽  
Myosin Light Chain Kinase ◽  
Myosin Light Chain ◽  
Kinase Inhibitors ◽  
Tight Junction Regulation ◽  
Tight Junction Permeability ◽  
Mlc Phosphorylation

Tight junctions serve as the rate-limiting barrier to passive movement of hydrophilic solutes across intestinal epithelia. After activation of Na+-glucose cotransport, the permeability of intestinal tight junctions is increased. Because previous analyses of this physiological tight junction regulation have been restricted to intact mucosae, dissection of the mechanisms underlying this process has been limited. To characterize this process, we have developed a reductionist model consisting of Caco-2 intestinal epithelial cells transfected with the intestinal Na+-glucose cotransporter, SGLT1. Monolayers of SGLT1 transfectants demonstrate physiological Na+-glucose cotransport. Activation of SGLT1 results in a 22 ± 5% fall in transepithelial resistance (TER) ( P< 0.001). Similarly, inactivation of SGLT1 by addition of phloridzin increases TER by 24 ± 2% ( P < 0.001). The increased tight junction permeability is size selective, with increased flux of small nutrient-sized molecules, e.g., mannitol, but not of larger molecules, e.g., inulin. SGLT1-dependent increases in tight junction permeability are inhibited by myosin light-chain kinase inhibitors (20 μM ML-7 or 40 μM ML-9), suggesting that myosin regulatory light-chain (MLC) phosphorylation is involved in tight junction regulation. Analysis of MLC phosphorylation showed a 2.08-fold increase after activation of SGLT1 ( P< 0.01), which was inhibited by ML-9 ( P < 0.01). Thus monolayers incubated with glucose and myosin light-chain kinase inhibitors are comparable to monolayers incubated with phloridzin. ML-9 also inhibits SGLT1-mediated tight junction regulation in small intestinal mucosa ( P < 0.01). These data demonstrate that epithelial cells are the mediators of physiological tight junction regulation subsequent to SGLT1 activation. The intimate relationship between tight junction regulation and MLC phosphorylation suggests that a critical step in regulation of epithelial tight junction permeability may be myosin ATPase-mediated contraction of the perijunctional actomyosin ring and subsequent physical tension on the tight junction.

scholarly journals Myosin light chain kinase regulates cell polarization independently of membrane tension or Rho kinase

2015 ◽  
Vol 209 (2) ◽  
pp. 275-288 ◽  
Author(s):  
Sunny S. Lou ◽  
Alba Diz-Muñoz ◽  
Orion D. Weiner ◽  
Daniel A. Fletcher ◽  
Julie A. Theriot
Keyword(s):  
Cell Polarity ◽  
Light Chain ◽  
Myosin Light Chain Kinase ◽  
Myosin Light Chain ◽  
Rho Kinase ◽  
Model System ◽  
Cell Polarization ◽  
Membrane Tension ◽  
Specific Mechanism

Cells polarize to a single front and rear to achieve rapid actin-based motility, but the mechanisms preventing the formation of multiple fronts are unclear. We developed embryonic zebrafish keratocytes as a model system for investigating establishment of a single axis. We observed that, although keratocytes from 2 d postfertilization (dpf) embryos resembled canonical fan-shaped keratocytes, keratocytes from 4 dpf embryos often formed multiple protrusions despite unchanged membrane tension. Using genomic, genetic, and pharmacological approaches, we determined that the multiple-protrusion phenotype was primarily due to increased myosin light chain kinase (MLCK) expression. MLCK activity influences cell polarity by increasing myosin accumulation in lamellipodia, which locally decreases protrusion lifetime, limiting lamellipodial size and allowing for multiple protrusions to coexist within the context of membrane tension limiting protrusion globally. In contrast, Rho kinase (ROCK) regulates myosin accumulation at the cell rear and does not determine protrusion size. These results suggest a novel MLCK-specific mechanism for controlling cell polarity via regulation of myosin activity in protrusions.

Rat pulmonary arterial smooth muscle myosin light chain kinase and phosphatase activities decrease with age

2006 ◽  
Vol 290 (3) ◽  
pp. L509-L516 ◽  
Author(s):  
J. Belik ◽  
Ewa Kerc ◽  
Mary D. Pato
Keyword(s):  
Smooth Muscle ◽  
Light Chain ◽  
Myosin Light Chain Kinase ◽  
Myosin Light Chain ◽  
Rho Kinase ◽  
Adult Tissue ◽  
Adult Rat ◽  
Pulmonary Arterial ◽  
Pulmonary Arterial Smooth Muscle ◽  
Specific Activities

We and others have shown that the fetal pulmonary arterial smooth muscle potential for contraction and relaxation is significantly reduced compared with the adult. Whether these developmental changes relate to age differences in the expression and/or activity of key enzymes regulating the smooth muscle mechanical properties has not been previously evaluated. Therefore, we studied the catalytic activities and expression of myosin light chain kinase (MLCK) and myosin light chain phosphatase (MLCP) catalytic (PP1cδ) and regulatory (MYPT) subunits in late fetal, early newborn, and adult rat intrapulmonary arterial tissues. In keeping with the greater force development and relaxation of adult pulmonary artery, Western blot analysis showed that the MLCK, MYPT, and PP1cδ contents increased significantly with age and were highest in the adult rat. In contrast, their specific activities (activity/enzyme content) were significantly higher in the fetal compared with the adult tissue. The fetal and newborn pulmonary arterial muscle relaxant response to the Rho-kinase inhibitor Y-27632 was greater than the adult tissue. In addition to the 130-kDa isoform of MLCK, we documented the presence of minor higher-molecular-weight embryonic isoforms in the fetus and newborn. During fetal life, the lung pulmonary arterial MLCK- and MLCP-specific activities are highest and appear to be related to Rho-kinase activation during lung morphogenesis.

373 Role of myosin light chain kinase and Rho kinase in carbachol-induced contractions of human detrusor smooth muscle

2012 ◽  
Vol 11 (1) ◽  
pp. e373
Author(s):  
C. Protzel ◽  
T. Kirschstein ◽  
K. Porath ◽  
T. Sellmann ◽  
R. Koehling ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Light Chain ◽  
Myosin Light Chain Kinase ◽  
Myosin Light Chain ◽  
Rho Kinase ◽  
Detrusor Smooth Muscle ◽  
Human Detrusor

Phosphorylation regulates interaction of 210-kDa myosin light chain kinase N-terminal domain with actin cytoskeleton

2015 ◽  
Vol 80 (10) ◽  
pp. 1288-1297 ◽  
Author(s):  
E. L. Vilitkevich ◽  
A. Y. Khapchaev ◽  
D. S. Kudryashov ◽  
A. V. Nikashin ◽  
J. P. Schavocky ◽  
...  
Keyword(s):  
Actin Cytoskeleton ◽  
Light Chain ◽  
Myosin Light Chain Kinase ◽  
Myosin Light Chain ◽  
Terminal Domain
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