A Proteomic Study of Myosin II Motor Proteins during Tumor Cell Migration

Betapudi, Venkaiah; Gokulrangan, Giridharan; Chance, Mark R.; Egelhoff, Thomas

doi:10.1016/j.jmb.2011.02.010

Cited by 35 publications

(28 citation statements)

References 30 publications

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“…Integrin engagement to fibronectin or collagen extracellular matrices (ECMs) induces Rac1 activation [11, 12], while fibronectin engagement promotes phosphorylation of MIIA HC on serine 1916 (pS1916 MIIA HC) [32]. We performed western blot analysis with pS1916 MIIA HC phospho-specific antibodies of lysates from cells with Rac1 activity manipulated that were plated on either fibronectin or collagen.…”

Section: Resultsmentioning

confidence: 99%

Rac1-Dependent Phosphorylation and Focal Adhesion Recruitment of Myosin IIA Regulates Migration and Mechanosensing

et al. 2015

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Summary Background Cell migration requires coordinated formation of focal adhesions (FAs) and assembly and contraction of the actin cytoskeleton. Non-muscle myosin II (MII) is a critical mediator of contractility and FA dynamics in cell migration. Signaling downstream of the small GTPase Rac1 also regulates FA and actin dynamics, but its role in regulation of MII during migration is less clear. Results We found that Rac1 promotes association of MIIA with FA. Live-cell imaging showed that while most MIIA at the leading edge assembled into dorsal contractile arcs, a substantial subset assembled in or was captured within maturing FA, and this behavior was promoted by active Rac1. Protein kinase C (PKC) activation was necessary and sufficient for integrin- and Rac1-dependent phosphorylation of MIIA heavy chain (HC) on serine1916 (S1916) and recruitment to FA. S1916 phosphorylation of MIIA HC and localization in FA was enhanced during cell spreading and ECM stiffness mechanosensing, suggesting up-regulation of this pathway during physiological Rac1 activation. Phospho-mimic and non-phosphorylatable MIIA HC mutants demonstrated that S1916 phosphorylation was necessary and sufficient for the capture and assembly of MIIA mini-filaments in FA. S1916 phosphorylation was also sufficient to promote the rapid assembly of FAs to enhance cell migration and for the modulation of traction force, spreading, and migration by ECM stiffness. Conclusions Our study reveals for the first time that Rac1 and integrin activation regulates MIIA HC phosphorylation through a PKC -dependent mechanism which promotes MIIA association with FAs, and acts as a critical modulator of cell migration and mechanosensing.

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Section: Resultsmentioning

confidence: 99%

Rac1-Dependent Phosphorylation and Focal Adhesion Recruitment of Myosin IIA Regulates Migration and Mechanosensing

et al. 2015

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“…2D). Because myosin II is a known effector of cell mechanics, both in Dictyostelium as well as in other organisms (8,(10)(11)(12)(13)(14), we next queried whether the increase in cortical localization would have an impact on the mechanical properties of the cell. Using micropipette aspiration (MPA) assays, we determined that acute treatment with 700 pM carbamate-7 led to a 1.4-fold increase in the cell's cortical tension ( Fig.…”

Section: Resultsmentioning

confidence: 99%

Pharmacological activation of myosin II paralogs to correct cell mechanics defects

Surcel¹,

Ng²,

West‐Foyle³

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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Current approaches to cancer treatment focus on targeting signal transduction pathways. Here, we develop an alternative system for targeting cell mechanics for the discovery of novel therapeutics. We designed a live-cell, high-throughput chemical screen to identify mechanical modulators. We characterized 4-hydroxyacetophenone (4-HAP), which enhances the cortical localization of the mechanoenzyme myosin II, independent of myosin heavy-chain phosphorylation, thus increasing cellular cortical tension. To shift cell mechanics, 4-HAP requires myosin II, including its full power stroke, specifically activating human myosin IIB (MYH10) and human myosin IIC (MYH14), but not human myosin IIA (MYH9). We further demonstrated that invasive pancreatic cancer cells are more deformable than normal pancreatic ductal epithelial cells, a mechanical profile that was partially corrected with 4-HAP, which also decreased the invasion and migration of these cancer cells. Overall, 4-HAP modifies nonmuscle myosin II-based cell mechanics across phylogeny and disease states and provides proof of concept that cell mechanics offer a rich drug target space, allowing for possible corrective modulation of tumor cell behavior. mechanical modulator | 3,4-dichloroaniline | 4-hydroxyacetophenone | myosin II | pancreatic cancer C ell shape change processes include cell growth, division, motility, and the formation of complex structures like tissues and organs, all of which are governed by the intersection of biochemistry, genetics, and mechanics. These three modules are integral not just for normal function in healthy cells but also in disease states. Pharmacological manipulation of some of these modules has already led to treatment strategies for inflammation and cancer (e.g., paclitaxel) (1, 2). However, many presently available therapies, which address only one aspect of cell shape change, typically either fail to abolish the disease completely or lead to compensatory regulatory changes, and therefore to drug resistance. Targeting cell mechanics remains an underused approach for drug development. In cancer, altered cell mechanics are a hallmark of metastatic efficiency: cell stiffness decreases up to 70% in many metastatic cancer cells (3-5). It is rational then that one therapeutic approach is to increase cellular elasticity, which would, in turn, reduce metastatic potential and act downstream of cancer-inducing genetic alterations.Known mechanical modulators (e.g., latrunculin, blebbistatin) are often lethal, have numerous off-site targets, and act to generate a softer and metastatic-like mechanical phenotype (6, 7). However, the field's ability to increase cellular elasticity on acute time scales is highly restricted. In an effort to close this gap and find modulators that stiffen cells, we leveraged our molecular and analytical understanding of cytokinesis, an evolutionarily conserved and highly mechanical cell shape change event, to establish an in vivo, large-scale, high-throughput chemical screen for small-molecule modulators of cell shap...

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“…To determine whether MHC IIA phosphorylation is differentially regulated in luminal vs. basal cell lines, we took advantage of our recently developed phospho-specific antibodies directed against two phosphorylated residues in the MHC IIA tail region, S1916 and S1943 (23). Western blot analysis revealed strongly elevated MHC phosphorylation at both target sites in all three basal-like cell lines, relative to the luminal lines (Fig.…”

Section: Myosin Heavy Chain Isoforms Are Differentially Expressed In mentioning

confidence: 99%

Myosin II isoform switching mediates invasiveness after TGF-β–induced epithelial–mesenchymal transition

Beach

Hussey

Miller

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

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103

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Despite functional significance of nonmuscle myosin II in cell migration and invasion, its role in epithelial-mesenchymal transition (EMT) or TGF-β signaling is unknown. Analysis of normal mammary gland expression revealed that myosin IIC is expressed in luminal cells, whereas myosin IIB expression is up-regulated in myoepithelial cells that have more mesenchymal characteristics. Furthermore, TGF-β induction of EMT in nontransformed murine mammary gland epithelial cells results in an isoform switch from myosin IIC to myosin IIB and increased phosphorylation of myosin heavy chain (MHC) IIA on target sites known to regulate filament dynamics (S1916, S1943). These expression and phosphorylation changes are downstream of heterogeneous nuclear ribonucleoprotein-E1 (E1), an effector of TGF-β signaling. E1 knockdown drives cells into a migratory, invasive mesenchymal state and concomitantly up-regulates MHC IIB expression and MHC IIA phosphorylation. Abrogation of myosin IIB expression in the E1 knockdown cells has no effect on 2D migration but significantly reduced transmigration and macrophage-stimulated collagen invasion. These studies indicate that transition between myosin IIC/myosin IIB expression is a critical feature of EMT that contributes to increases in invasive behavior.

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A Proteomic Study of Myosin II Motor Proteins during Tumor Cell Migration

Cited by 35 publications

References 30 publications

Rac1-Dependent Phosphorylation and Focal Adhesion Recruitment of Myosin IIA Regulates Migration and Mechanosensing

Rac1-Dependent Phosphorylation and Focal Adhesion Recruitment of Myosin IIA Regulates Migration and Mechanosensing

Pharmacological activation of myosin II paralogs to correct cell mechanics defects

Myosin II isoform switching mediates invasiveness after TGF-β–induced epithelial–mesenchymal transition

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