scholarly journals Apoptotic Membrane Blebbing Is Regulated by Myosin Light Chain Phosphorylation

1998 ◽  
Vol 140 (3) ◽  
pp. 627-636 ◽  
Author(s):  
Jason C. Mills ◽  
Nicole L. Stone ◽  
Joseph Erhardt ◽  
Randall N. Pittman
Keyword(s):  
Light Chain ◽  
Myosin Light Chain ◽  
Kinase Inhibitors ◽  
Morphological Changes ◽  
Myosin Ii ◽  
Model System ◽  
Membrane Blebbing ◽  
Execution Phase ◽  
Fragmentation Mechanisms ◽  
Mlc Phosphorylation

The evolutionarily conserved execution phase of apoptosis is defined by characteristic changes occurring during the final stages of death; specifically cell shrinkage, dynamic membrane blebbing, condensation of chromatin, and DNA fragmentation. Mechanisms underlying these hallmark features of apoptosis have previously been elusive, largely because the execution phase is a rapid event whose onset is asynchronous across a population of cells. In the present study, a model system is described for using the caspase inhibitor, z-VAD-FMK, to block apoptosis and generate a synchronous population of cells actively extruding and retracting membrane blebs. This model system allowed us to determine signaling mechanisms underlying this characteristic feature of apoptosis. A screen of kinase inhibitors performed on synchronized blebbing cells indicated that only myosin light chain kinase (MLCK) inhibitors decreased blebbing. Immunoprecipitation of myosin II demonstrated that myosin regulatory light chain (MLC) phosphorylation was increased in blebbing cells and that MLC phosphorylation was prevented by inhibitors of MLCK. MLC phosphorylation is also mediated by the small G protein, Rho. C3 transferase inhibited apoptotic membrane blebbing, supporting a role for a Rho family member in this process. Finally, blebbing was also inhibited by disruption of the actin cytoskeleton. Based on these results, a working model is proposed for how actin/myosin II interactions cause cell contraction and membrane blebbing. Our results provide the first evidence that MLC phosphorylation is critical for apoptotic membrane blebbing and also implicate Rho signaling in these active morphological changes. The model system described here should facilitate future studies of MLCK, Rho, and other signal transduction pathways activated during the execution phase of apoptosis.

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 Distinct roles of MLCK and ROCK in the regulation of membrane protrusions and focal adhesion dynamics during cell migration of fibroblasts

2004 ◽  
Vol 164 (3) ◽  
pp. 427-439 ◽  
Author(s):  
Go Totsukawa ◽  
Yue Wu ◽  
Yasuharu Sasaki ◽  
David J. Hartshorne ◽  
Yoshihiko Yamakita ◽  
...  
Keyword(s):  
Cell Migration ◽  
Light Chain ◽  
Myosin Light Chain ◽  
Myosin Ii ◽  
Focal Adhesions ◽  
Cell Periphery ◽  
Membrane Ruffling ◽  
Regulatory Myosin Light Chain ◽  
Membrane Protrusions ◽  
Mlc Phosphorylation

We examined the role of regulatory myosin light chain (MLC) phosphorylation of myosin II in cell migration of fibroblasts. Myosin light chain kinase (MLCK) inhibition blocked MLC phosphorylation at the cell periphery, but not in the center. MLCK-inhibited cells did not assemble zyxin-containing adhesions at the periphery, but maintained focal adhesions in the center. They generated membrane protrusions all around the cell, turned more frequently, and migrated less effectively. In contrast, Rho-associated kinase (ROCK) inhibition blocked MLC phosphorylation in the center, but not at the periphery. ROCK-inhibited cells assembled zyxin-containing adhesions at the periphery, but not focal adhesions in the center. They moved faster and more straight. On the other hand, inhibition of myosin phosphatase increased MLC phosphorylation and blocked peripheral membrane ruffling, as well as turnover of focal adhesions and cell migration. Our results suggest that myosin II activated by MLCK at the cell periphery controls membrane ruffling, and that the spatial regulation of MLC phosphorylation plays critical roles in controlling cell migration of fibroblasts.

scholarly journals Actin-myosin–based contraction is responsible for apoptotic nuclear disintegration

2005 ◽  
Vol 168 (2) ◽  
pp. 245-255 ◽  
Author(s):  
Daniel R. Croft ◽  
Mathew L. Coleman ◽  
Shuixing Li ◽  
David Robertson ◽  
Teresa Sullivan ◽  
...  
Keyword(s):  
Atpase Activity ◽  
Myosin Light Chain ◽  
Wild Type ◽  
Membrane Blebbing ◽  
Nuclear Disintegration ◽  
Execution Phase ◽  
Apoptosis Inhibition ◽  
Microtubular Cytoskeleton ◽  
Mlc Phosphorylation ◽  
Rock Activity

Membrane blebbing during the apoptotic execution phase results from caspase-mediated cleavage and activation of ROCK I. Here, we show that ROCK activity, myosin light chain (MLC) phosphorylation, MLC ATPase activity, and an intact actin cytoskeleton, but not microtubular cytoskeleton, are required for disruption of nuclear integrity during apoptosis. Inhibition of ROCK or MLC ATPase activity, which protect apoptotic nuclear integrity, does not affect caspase-mediated degradation of nuclear proteins such as lamins A, B1, or C. The conditional activation of ROCK I was sufficient to tear apart nuclei in lamin A/C null fibroblasts, but not in wild-type fibroblasts. Thus, apoptotic nuclear disintegration requires actin-myosin contractile force and lamin proteolysis, making apoptosis analogous to, but distinct from, mitosis where nuclear disintegration results from microtubule-based forces and from lamin phosphorylation and depolymerization.

scholarly journals FFAR1 activation attenuates histamine-induced myosin light chain phosphorylation and cortical tension development in human airway smooth muscle cells

2020 ◽  
Vol 21 (1) ◽  
Author(s):  
Shengjie Xu ◽  
Anthony Schwab ◽  
Nikhil Karmacharya ◽  
Gaoyuan Cao ◽  
Joanna Woo ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Airway Smooth Muscle ◽  
Light Chain ◽  
Myosin Light Chain ◽  
Airway Hyperreactivity ◽  
Human Airway Smooth Muscle ◽  
Tension Development ◽  
Cortical Tension ◽  
Human Airway ◽  
Mlc Phosphorylation

Abstract Background Activation of free fatty acid receptors (FFAR1 and FFAR4) which are G protein-coupled receptors (GPCRs) with established (patho)physiological roles in a variety of obesity-related disorders, induce human airway smooth muscle (HASM) cell proliferation and shortening. We reported amplified agonist-induced cell shortening in HASM cells obtained from obese lung donors. We hypothesized that FFAR1 modulate excitation–contraction (EC) coupling in HASM cells and play a role in obesity-associated airway hyperresponsiveness. Methods In HASM cells pre-treated (30 min) with FFAR1 agonists TAK875 and GW9508, we measured histamine-induced Ca2+ mobilization, myosin light chain (MLC) phosphorylation, and cortical tension development with magnetic twisting cytometry (MTC). Phosphorylation of MLC phosphatase and Akt also were determined in the presence of the FFAR1 agonists or vehicle. In addition, the effects of TAK875 on MLC phosphorylation were measured in HASM cells desensitized to β2AR agonists by overnight salmeterol treatment. The inhibitory effect of TAK875 on MLC phosphorylation was compared between HASM cells from age and sex-matched non-obese and obese human lung donors. The mean measurements were compared using One-Way ANOVA with Dunnett’s test for multiple group comparisons or Student’s t-test two-group comparison. For cortical tension measurements by magnetic twisted cytometry, mixed effect model using SAS V.9.2 was applied. Means were considered significant when p ≤ 0.05. Results Unexpectedly, we found that TAK875, a synthetic FFAR1 agonist, attenuated histamine-induced MLC phosphorylation and cortical tension development in HASM cells. These physiological outcomes were unassociated with changes in histamine-evoked Ca2+ flux, protein kinase B (AKT) activation, or MLC phosphatase inhibition. Of note, TAK875-mediated inhibition of MLC phosphorylation was maintained in β2AR-desensitized HASM cells and across obese and non-obese donor-derived HASM cells. Conclusions Taken together, our findings identified the FFAR1 agonist TAK875 as a novel bronchoprotective agent that warrants further investigation to treat difficult-to-control asthma and/or airway hyperreactivity in obesity.

scholarly journals Blockade of ROCK inhibits migration of human primary keratinocytes and malignant epithelial skin cells by regulating actomyosin contractility

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Srisathya Srinivasan ◽  
Sreya Das ◽  
Vishakha Surve ◽  
Ankita Srivastava ◽  
Sushant Kumar ◽  
...  
Keyword(s):  
Light Chain ◽  
Myosin Light Chain ◽  
Focal Adhesions ◽  
Primary Keratinocytes ◽  
Skin Cells ◽  
Physiological Processes ◽  
Actomyosin Contractility ◽  
Malignant Skin ◽  
And Migration ◽  
Mlc Phosphorylation

AbstractActomyosin contractility, crucial for several physiological processes including migration, is controlled by the phosphorylation of myosin light chain (MLC). Rho-associated protein kinase (ROCK) and Myosin light chain kinase (MLCK) are predominant kinases that phosphorylate MLC. However, the distinct roles of these kinases in regulating actomyosin contractility and their subsequent impact on the migration of healthy and malignant skin cells is poorly understood. We observed that blockade of ROCK in healthy primary keratinocytes (HPKs) and epidermal carcinoma cell line (A-431 cells) resulted in loss of migration, contractility, focal adhesions, stress fibres, and changes in morphology due to reduction in phosphorylated MLC levels. In contrast, blockade of MLCK reduced migration, contractile dynamics, focal adhesions and phosphorylated MLC levels of HPKs alone and had no effect on A-431 cells due to the negligible MLCK expression. Using genetically modified A-431 cells expressing phosphomimetic mutant of p-MLC, we show that ROCK dependent phosphorylated MLC controls the migration, focal adhesion, stress fibre organization and the morphology of the cells. In conclusion, our data indicate that ROCK is the major kinase of MLC phosphorylation in both HPKs and A-431 cells, and regulates the contractility and migration of healthy as well as malignant skin epithelial cells.

scholarly journals A Novel Regulatory Mechanism of Myosin Light Chain Phosphorylation via Binding of 14-3-3 to Myosin Phosphatase

2008 ◽  
Vol 19 (3) ◽  
pp. 1062-1071 ◽  
Author(s):  
Yasuhiko Koga ◽  
Mitsuo Ikebe
Keyword(s):  
Light Chain ◽  
Myosin Light Chain ◽  
Regulatory Mechanism ◽  
Kinase Inhibitor ◽  
Myosin Ii ◽  
Critical Role ◽  
Rho Kinase ◽  
Myosin Phosphatase ◽  
Dependent Cell ◽  
Direct Binding

Myosin II phosphorylation–dependent cell motile events are regulated by myosin light-chain (MLC) kinase and MLC phosphatase (MLCP). Recent studies have revealed myosin phosphatase targeting subunit (MYPT1), a myosin-binding subunit of MLCP, plays a critical role in MLCP regulation. Here we report the new regulatory mechanism of MLCP via the interaction between 14-3-3 and MYPT1. The binding of 14-3-3β to MYPT1 diminished the direct binding between MYPT1 and myosin II, and 14-3-3β overexpression abolished MYPT1 localization at stress fiber. Furthermore, 14-3-3β inhibited MLCP holoenzyme activity via the interaction with MYPT1. Consistently, 14-3-3β overexpression increased myosin II phosphorylation in cells. We found that MYPT1 phosphorylation at Ser472 was critical for the binding to 14-3-3. Epidermal growth factor (EGF) stimulation increased both Ser472 phosphorylation and the binding of MYPT1-14-3-3. Rho-kinase inhibitor inhibited the EGF-induced Ser472 phosphorylation and the binding of MYPT1-14-3-3. Rho-kinase specific siRNA also decreased EGF-induced Ser472 phosphorylation correlated with the decrease in MLC phosphorylation. The present study revealed a new RhoA/Rho-kinase–dependent regulatory mechanism of myosin II phosphorylation by 14-3-3 that dissociates MLCP from myosin II and attenuates MLCP activity.

scholarly journals Increased Phosphorylation of Myosin Light Chain Prevents in Vitro Decidualization

Endocrinology ◽  
2007 ◽  
Vol 148 (7) ◽  
pp. 3176-3184 ◽  
Author(s):  
Ivanna Ihnatovych ◽  
WenYang Hu ◽  
Jody L. Martin ◽  
Asgerally T. Fazleabas ◽  
Primal de Lanerolle ◽  
...  
Keyword(s):  
Light Chain ◽  
Myosin Light Chain ◽  
Smooth Muscle Actin ◽  
Myosin Ii ◽  
Active Form ◽  
Adenoviral Infection ◽  
Decidual Cells ◽  
Cytoskeletal Dynamics ◽  
Mlc20 Phosphorylation

Differentiation of stromal cells into decidual cells, which is critical to successful pregnancy, represents a complex transformation requiring changes in cytoskeletal architecture. We demonstrate that in vitro differentiation of human uterine fibroblasts into decidual cells includes down-regulation of α-smooth muscle actin and β-tubulin, phosphorylation of focal adhesion kinase, and redistribution of vinculin. This is accompanied by varied adhesion to fibronectin and a modified ability to migrate. Cytoskeletal organization is determined primarily by actin-myosin II interactions governed by the phosphorylation of myosin light chain (MLC20). Decidualization induced by cAMP [with estradiol-17β (E) and medroxyprogesterone acetate (P)] results in a 40% decrease in MLC20 phosphorylation and a 55% decline in the long (214 kDa) form of myosin light-chain kinase (MLCK). Destabilization of the cytoskeleton by inhibitors of MLCK (ML-7) or myosin II ATPase (blebbistatin) accelerates decidualization induced by cAMP (with E and P) but inhibits decidualization induced by IL-1β (with E and P). Adenoviral infection of human uterine fibroblast cells with a constitutively active form of MLCK followed by decidualization stimuli leads to a 30% increase in MLC20 phosphorylation and prevents decidualization. These data provide evidence that the regulation of cytoskeletal dynamics by MLC20 phosphorylation is critical for decidualization.

scholarly journals Rho kinase inhibitors stimulate the migration of human cultured osteoblastic cells by regulating actomyosin activity

2011 ◽  
Vol 16 (2) ◽  
Author(s):  
Xuejiao Zhang ◽  
Cheng Li ◽  
Huiling Gao ◽  
Hiroaki Nabeka ◽  
Tetsuya Shimokawa ◽  
...  
Keyword(s):  
Light Chain ◽  
Myosin Light Chain ◽  
Kinase Inhibitors ◽  
Rho Kinase ◽  
Cell Types ◽  
Osteosarcoma Cells ◽  
Human Osteoblasts ◽  
Protein Levels ◽  
Human Osteosarcoma Cells ◽  
Joint Prostheses

AbstractWe investigated the effects of Rho-associated kinase (ROCK) on migration and cytoskeletal organization in primary human osteoblasts and Saos-2 human osteosarcoma cells. Both cell types were exposed to two different ROCK inhibitors, Y-27632 and HA-1077. In the improved motility assay used in the present study, Y-27632 and HA-1077 significantly increased the migration of both osteoblasts and osteosarcoma cells on plastic in a dose-dependent and reversible manner. Fluorescent images showed that cells of both types cultured with Y-27632 or HA-1077 exhibited a stellate appearance, with poor assembly of stress fibers and focal contacts. Western blotting showed that ROCK inhibitors reduced myosin light chain (MLC) phosphorylation within 5 min without affecting overall myosin light-chain protein levels. Inhibition of ROCK activity is thought to enhance the migration of human osteoblasts through reorganization of the actin cytoskeleton and regulation of myosin activity. ROCK inhibitors may be potentially useful as anabolic agents to enhance the biocompatibility of bone and joint prostheses.

FK506 BINDING PROTEIN 8 (FKBP8) INTERACTIONS WITH MYOSIN LIGHT CHAIN KINASE 1 ARE REQUIRED FOR TNF-INDUCED TIGHT JUNCTION BARRIER LOSS

2021 ◽  
Vol 27 (Supplement_1) ◽  
pp. S25-S25
Author(s):  
Li Zuo ◽  
Feng Cao ◽  
Wei-Ting Kuo ◽  
Jerrold Turner
Keyword(s):  
Tight Junction ◽  
Light Chain ◽  
Myosin Light Chain Kinase ◽  
Myosin Light Chain ◽  
Intestinal Barrier ◽  
Intestinal Epithelial ◽  
Binding Interaction ◽  
Chaperone Protein ◽  
Tight Junction Permeability ◽  
Mlc Phosphorylation

Abstract Background Tumor necrosis factor (TNF) regulates intestinal epithelial tight junction permeability by activating myosin light chain kinase 1 (MLCK1) expression and enzymatic activity. MLCK1 recruitment to the apical perijunctional actomyosin ring (PAMR) is, however, required for barrier regulation; Divertin, a small molecule drug that blocks this recruitment, prevents barrier loss and attenuates both acute and chronic experimental diarrheal disease. We therefore hypothesized that MLCK1 recruitment to the PAMR requires interactions with as yet unidentified chaperone protein(s). Objective To identify binding partners and define the mechanisms by which they activate MLCK1 recruitment to the PAMR. Results We performed a yeast-2-hybrid (Y2H) screen using the MLCK1 domains required for PAMR recruitment as bait. FKBP8, which encodes a peptidyl-prolyl cis-trans isomerase (PPI), was recovered, and direct binding to the MLCK1 domains (Kd=~5mM) was confirmed using microscale thermophoresis (MST). This binding interaction required the FK506-binding PPI domain and was specifically inhibited by FK506 (tacrolimus). Immunofluorescent microscopy demonstrated partial colocalization of MLCK1 and FKBP8 within intestinal epithelial monolayers; TNF caused both to concentrate around the PAMR. To further characterize this interaction, we performed proximity ligation assays (PLA) and found that TNF increased interaction between MLCK1 and FKBP8 &gt; 2-fold. FK506 prevented TNF-induced increases in PLA signal. FK506 was also able to reverse TNF-induced increases in myosin light chain (MLC) phosphorylation and tight junction permeability. In Caco-2 monolayers, FKBP8 knockout blocked TNF-induced MLCK1 recruitment, MLC phosphorylation, and tight junction barrier loss; all of which were restored by FKBP8 re-expression. In mice, MLC phosphorylation and intestinal barrier loss triggered by acute, anti-CD3-induced, T cell activation were blocked by luminal FK506. Importantly, this local FK506 treatment did not prevent anti-CD3-induced increases in mucosal TNF, IL-1b, and IFNg. Immunostains of biopsies from IBD patients documented increased PAMR MLC phosphorylation, MLCK1 recruitment, FKBP8 recruitment, and MLCK1-FKBP8 PLA signal relative to control subjects. Conclusions FKBP8 is a chaperone protein required for TNF-induced MLCK1 recruitment and barrier loss. This requires direct interaction between MLCK1 and the PPI domain of FKBP8. FK506 binding to the PPI domain displaces MLCK1 thereby preventing recruitment to the PAMR and barrier loss. These activities are separate from the immunosuppressive effects of FK506. We speculate that molecular blockade of the FKBP8-MLCK1 interaction may be a novel approach to barrier restoration and therapy of diseases associated with intestinal barrier dysfunction. Support NIH (DK068271, DK061931) and the NNSF of China (81800464, 82070548).

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