Rac regulates phosphorylation of the myosin-II heavy chain, actinomyosin disassembly and cell spreading

Leeuwen, Frank N. van; Delft, Sanne van; Kain, Hendrie E.T.; Kammen, Rob A. van der; Collard, John G.

doi:10.1038/12068

Cited by 197 publications

(162 citation statements)

References 40 publications

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“…A single mechanism that might account for these paradoxical effects involves increased intracellular tension. Rho-mediated intracellular tension has been shown to decrease cell spreading (Jalink et al, 1994;Hirose et al, 1998;van Leeuwen et al, 1999), an effect that is supported by theoretical models (Palecek et al, 1999). At the same time, Rho-mediated intracellular tension has also been shown to increase stress fiber formation, integrin density, and FA formation in many studies Ballestrem et al, 2001).…”

Section: Kda (mentioning

confidence: 59%

“…To investigate whether signaling through ROCK was necessary for the VE-cadherin-mediated changes in cell spreading, we inhibited ROCK with Y-27632 in cells plated at different densities ( Figure 5B). Treatment with Y-27632 shifted the baseline to a higher degree of cell spreading even at low seeding densities, consistent with inactivation of signaling through RhoA (Jalink et al, 1994;van Leeuwen et al, 1999). Inhibiting ROCK activity also abrogated the ability of cell-cell contact to decrease cell spreading.…”

Section: Kda (mentioning

confidence: 75%

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Vascular Endothelial-Cadherin Regulates Cytoskeletal Tension, Cell Spreading, and Focal Adhesions by Stimulating RhoA

Nelson

Pirone

Tan

et al. 2004

MBoC

164

137

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Changes in vascular endothelial (VE)-cadherin-mediated cell-cell adhesion and integrin-mediated cell-matrix adhesion coordinate to affect the physical and mechanical rearrangements of the endothelium, although the mechanisms for such cross talk remain undefined. Herein, we describe the regulation of focal adhesion formation and cytoskeletal tension by intercellular VE-cadherin engagement, and the molecular mechanism by which this occurs. Increasing the density of endothelial cells to increase cell-cell contact decreased focal adhesions by decreasing cell spreading. This contact inhibition of cell spreading was blocked by disrupting VE-cadherin engagement with an adenovirus encoding dominant negative VE-cadherin. When changes in cell spreading were prevented by culturing cells on a micropatterned substrate, VE-cadherin-mediated cell-cell contact paradoxically increased focal adhesion formation. We show that VE-cadherin engagement mediates each of these effects by inducing both a transient and sustained activation of RhoA. Both the increase and decrease in cell-matrix adhesion were blocked by disrupting intracellular tension and signaling through the Rho-ROCK pathway. In all, these findings demonstrate that VE-cadherin signals through RhoA and the actin cytoskeleton to cross talk with cell-matrix adhesion and thereby define a novel pathway by which cell-cell contact alters the global mechanical and functional state of cells. INTRODUCTIONCoordinated changes between cell adhesion to the extracellular matrix (ECM) and to neighboring cells are crucial for the many physical transformations that cells must undergo during development, tissue homeostasis, and wound healing. In the endothelium, where a single layer of cells separates tissues from their blood supply, the concerted regulation of cell-cell and cell-ECM adhesion drives rapid changes in cell shape and vascular architecture essential to vascular remodeling in both normal and disease processes (Vestweber, 2000;Dudek and Garcia, 2001). Dynamic changes in cell-cell and cell-matrix adhesion and mechanical forces are responsible for regulating vascular permeability, and agents that alter permeability invariably affect both types of adhesion complexes (Dudek and Garcia, 2001). During blood vessel sprouting and migration in angiogenesis, endothelial cells must disassemble and reform their cell-cell and cell-ECM contacts in synchrony to generate appropriate structures. These two adhesion systems each regulate their functional effects through both biochemical and mechanical signals.Changes in integrin binding to ECM not only alter signals that regulate cell proliferation, migration, and survival (Assoian and Schwartz, 2001;Hood and Cheresh, 2002;Stupack and Cheresh, 2002) but also modulate cell mechanics by affecting focal adhesion (FA) maturation, adhesion strength, cytoskeletal contractility, and cell shape (Geiger and Bershadsky, 2002;Juliano, 2002). Likewise, changes in the engagement of vascular endothelial (VE)-cadherin, the principal junctional molecule in endothe...

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Section: Kda (mentioning

confidence: 59%

Section: Kda (mentioning

confidence: 75%

Vascular Endothelial-Cadherin Regulates Cytoskeletal Tension, Cell Spreading, and Focal Adhesions by Stimulating RhoA

Nelson

Pirone

Tan

et al. 2004

MBoC

164

137

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“…It is therefore likely that the phosphorylation of MLC 20 and MHC by PKC isoforms are differentially involved in the reorganization of actomyosin filaments during the cell motile events modulated based on the types of extracellular stimuli. Interestingly, it was reported that bradykinin and EGF-mediated MHC-IIA and -IIB phosphorylation is associated with a loss of cortical myosin II (van Leeuwen et al, 1999;Straussman et al, 2001). These previous works also suggest that MHC phosphorylation is involved in the regulation of microfilament disassembly in the cell cortex.…”

Section: Discussionmentioning

confidence: 99%

The Phosphorylation of Myosin II at the Ser1 and Ser2 Is Critical for Normal Platelet-derived Growth Factor–induced Reorganization of Myosin Filaments

Komatsu

Ikebe

2007

MBoC

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Phosphorylation of the regulatory light chain of myosin II (MLC 20 ) at the activation sites promotes both the motor activity and the filament formation of myosin II, thus playing an important role in various cell motile processes. In contrast, the physiological function of phosphorylation of MLC 20 at the inhibitory sites is unknown. Here we report for the first time the function of the inhibitory site phosphorylation in the cells. We successfully produced the antibodies specifically recognizing the phosphorylation sites of MLC 20 at Ser1, and the platelet-derived growth factor (PDGF)-induced change in the phosphorylation at the Ser1 was monitored. The phosphorylation of MLC 20 at the Ser1 significantly increased during the PDGF-induced actin cytoskeletal reorganization. PDGF disassembled the stress fibers, and this was attenuated with the expression of unphosphorylatable MLC 20 at the Ser1/Ser2 phosphorylation sites. The present results suggest that the down-regulation of myosin II activity achieved by the phosphorylation at the Ser1/Ser2 sites plays an important role in the normal reorganization of actomyosin filaments triggered by PDGF receptor stimulation. INTRODUCTIONCell migration plays a key role in both the physiological and the pathophysiological function of the cells including development, wound healing, immunity, and metastasis (Lauffenburger and Horwitz, 1996). Reorganization of actomyosin filaments is an essential process for these cell behaviors. It has been thought that myosin II plays a fundamental role in various types of cellular motility.In vitro biochemical studies have revealed that the function of smooth muscle and nonmuscle myosin II is regulated by the phosphorylation of MLC 20 (Sellers, 1991;Tan et al., 1992). A number of studies have shown that the phosphorylation of MLC 20 at Thr18 and Ser19 activates its motor activity and increases filament stability. On the other hand, it has been known that MLC 20 can be phosphorylated at other sites different from the activation sites. Originally, it was found that protein kinase C (PKC) phosphorylates Ser1/Ser2 and Thr9 of MLC 20 . This phosphorylation decreases the affinity of myosin II phosphorylated at the activation sites for actin and the affinity of MLC 20 for myosin light-chain kinase (MLCK; Nishikawa et al., 1984;Bengur et al., 1987;Ikebe et al., 1987;Ikebe and Reardon, 1990). In other words, the phosphorylation of MLC 20 at these sites inhibits rather than activates myosin II.It was reported that the phosphorylation of MLC 20 at the Thr9 inhibits myosin II motor activity and the phosphorylation of MLC 20 by MLCK (Nishikawa et al., 1984;Turbedsky et al., 1997); however, phosphorylation of the inhibitory sites in nonmuscle cells has only been observed on Ser1 and/or Ser2, but not on Thr9 in vivo (Kawamoto et al., 1989;Yamakita et al., 1994), and this is because the Ser1/Ser2 sites are resistant to dephosphorylation by myosin light chain phosphatases while phospho-Thr9 is readily dephosphorylated (Ikebe et al., 1999). Furthermore, it has b...

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“…The mechanisms regulating cellular function have thus been widely investigated in various disciplines. For example, it has previously been demonstrated that the Rho family of small GTPases including Rho, Rac, and Cdc42 is a key regulator of cytoskeletal dynamics, which are essential for achieving coordinated and directed cellular migration [1][2][3]. When specific receptors on the cellular surface bind to growth factors or to extracellular matrix (ECM), these small GTPases are activated by guanine nucleotide exchange factors (GEFs) [4].…”

Section: Introductionmentioning

confidence: 99%

Contribution of cellular contractility to spatial and temporal variations in cellular stiffness

Nagayama

Haga

Takahashi

et al. 2004

Experimental Cell Research

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Scanning probe microscopy and immunofluorescence observations indicated that cellular stiffness was attributed to a contractile network structure consisting of stress fibers. We measured temporal variations in cellular stiffness when cellular contractility was regulated by dosing with lysophosphatidic acid or Y-27632. This experiment reveals a clear relation between cellular stiffness and contractility: Increases in contractility cause cells to stiffen. On the other hand, decreases in contractility reduce cellular stiffness. In both cases, not only the stiffness of the stress fibers but also that of the whole of the cell varies. Immunofluorescence observations of myosin II and vinculin indicated that the stiffness variations induced by the regulation of cellular contractility were mainly due to rearrangements of the contractile actin network on the dorsal surface. Taken together, our findings provide evidence that the actin cytoskeletal network and its contractility features provide and modulate the mechanical stability of adherent cells.

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Rac regulates phosphorylation of the myosin-II heavy chain, actinomyosin disassembly and cell spreading

Cited by 197 publications

References 40 publications

Vascular Endothelial-Cadherin Regulates Cytoskeletal Tension, Cell Spreading, and Focal Adhesions by Stimulating RhoA

Vascular Endothelial-Cadherin Regulates Cytoskeletal Tension, Cell Spreading, and Focal Adhesions by Stimulating RhoA

The Phosphorylation of Myosin II at the Ser1 and Ser2 Is Critical for Normal Platelet-derived Growth Factor–induced Reorganization of Myosin Filaments

Contribution of cellular contractility to spatial and temporal variations in cellular stiffness

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