RESUMEN
Non-muscle myosin II (NMII) is a conserved force-producing cytoskeletal enzyme with important but poorly understood roles in cell migration. To investigate myosin heavy chain (MHC) phosphorylation roles in 3D migration, we expressed GFP-tagged NMIIA wild-type or mutant constructs in cells depleted of endogenous NMIIA protein. We find that individual mutation or double mutation of Ser-1916 or Ser-1943 to alanine potently blocks recruitment of GFP-NM-IIA filaments to leading edge protrusions in 2D, and this in turn blocks maturation of anterior focal adhesions. When placed in 3D collagen gels, cells expressing wild-type GFP MHC-IIA behave like parental cells, displaying robust and active formation and retraction of protrusions. However, cells depleted of NMIIA or cells expressing the mutant GFP MHC-IIA display severe defects in invasion and in stabilizing protrusions in 3D. These studies reveal an NMIIA-specific role in 3D invasion that requires competence for NMIIA phosphorylation at Ser-1916 and Ser-1943. In sum, these results demonstrate a critical and previously unrecognized role for NMIIA phosphorylation in 3D invasion.
Asunto(s)
Adhesión Celular , Movimiento Celular , Cadenas Pesadas de Miosina/metabolismo , Miosina Tipo IIA no Muscular/metabolismo , Animales , Células COS , Línea Celular , Línea Celular Tumoral , Chlorocebus aethiops , Humanos , Ratones , Cadenas Pesadas de Miosina/análisis , Miosina Tipo IIA no Muscular/análisis , FosforilaciónRESUMEN
Pro-fibrotic mesenchymal cells are known to be the key effector cells of fibroproliferative disease, but the specific matrix signals and the induced cellular responses that drive the fibrogenic phenotype remain to be elucidated. The key mediators of the fibroblast fibrogenic phenotype were characterized using a novel assay system that measures fibroblast behavior in response to actual normal and fibrotic lung tissue. Using this system, we demonstrate that normal lung promotes fibroblast motility and polarization, while fibrotic lung immobilizes the fibroblast and promotes myofibroblast differentiation. These context-specific phenotypes are surprisingly both mediated by myosin II. The role of myosin II is supported by the observation of an increase in myosin phosphorylation and a change in intracellular distribution in fibroblasts on fibrotic lung, as compared with normal lung. Moreover, loss of myosin II activity has opposing effects on protrusive activity in fibroblasts on normal and fibrotic lung. Loss of myosin II also selectively inhibits myofibroblast differentiation in fibroblasts on fibrotic lung. Importantly, these findings are recapitulated by varying the matrix stiffness of polyacrylamide gels in the range of normal and fibrotic lung tissue. Comparison of the effects of myosin inhibition on lung tissue with that of polyacrylamide gels suggests that matrix fiber organization drives the fibroblast phenotype under conditions of normal/soft lung, while matrix stiffness drives the phenotype under conditions of fibrotic/stiff lung. This work defines novel roles for myosin II as a key regulatory effector molecule of the pro-fibrotic phenotype, in response to biophysical properties of the matrix.
Asunto(s)
Fibroblastos/fisiología , Miosina Tipo II/fisiología , Fibrosis Pulmonar/metabolismo , Animales , Diferenciación Celular , Línea Celular , Movimiento Celular , Polaridad Celular , Forma de la Célula , Matriz Extracelular/fisiología , Femenino , Humanos , Pulmón/metabolismo , Pulmón/patología , Ratones Endogámicos C57BL , Fenotipo , Fibrosis Pulmonar/patologíaRESUMEN
Our prior work identified the mammalian target of rapamycin complex 2 (mTORC2) as a key regulator of bladder cancer cell migration and invasion, although upstream growth factor mediators of this pathway in bladder cancer have not been well delineated. We tested whether transforming growth factor (TGF)-ß, which can function as a promotility factor in bladder cancer cells, could regulate mTORC2-dependent bladder cancer cell motility and invasion. In human bladder cancers, the highest levels of phosphorylated SMAD2, a TGF-ß signaling intermediate, were present in high-grade invasive bladder cancers and associated with more frequent recurrence and decreased disease-specific survival. Increased expression of TGF-ß isoforms, receptors, and signaling components was detected in invasive high-grade bladder cancer cells that expressed Vimentin and lacked E-cadherin. Application of TGF-ß induced phosphorylation of the Ser473 residue of AKT, a selective target of mTORC2, in a SMAD2- and SMAD4-independent manner and increased bladder cancer cell migration in a modified scratch wound assay and invasion through Matrigel. Inhibition of TGF-ß receptor I using SB431542 ablated TGF-ß-induced migration and invasion. A similar effect was seen when Rictor, a key mTORC2 component, was selectively silenced. Our results suggest that TGF-ß can induce bladder cancer cell invasion via mTORC2 signaling, which may be applicable in most bladder cancers.
Asunto(s)
Complejos Multiproteicos/fisiología , Serina-Treonina Quinasas TOR/fisiología , Factor de Crecimiento Transformador beta/fisiología , Neoplasias de la Vejiga Urinaria/patología , Benzamidas/farmacología , Cadherinas/metabolismo , Movimiento Celular/fisiología , Dioxoles/farmacología , Humanos , Diana Mecanicista del Complejo 2 de la Rapamicina , Invasividad Neoplásica , Fosforilación/fisiología , Receptores de Factores de Crecimiento Transformadores beta/antagonistas & inhibidores , Transducción de Señal/fisiología , Proteína Smad2/metabolismo , Proteína Smad4/metabolismo , Células Tumorales Cultivadas , Regulación hacia Arriba/fisiología , Neoplasias de la Vejiga Urinaria/fisiopatología , Vimentina/metabolismoRESUMEN
In studies initially focused on roles of nonmuscle myosin IIA (NMIIA) in the developing mouse epidermis, we have discovered that a previously described cytokeratin 5 (K5)-Cre gene construct is expressed in early embryo development. Mice carrying floxed alleles of the nonmuscle myosin II heavy chain gene (NMHC IIA(flox/flox)) were crossed with the K5-Cre line. The progeny of newborn pups did not show a Mendelian genotype distribution, suggesting embryonic lethality. Analysis of post-implantation conceptuses from embryonic day (E)9.5 to E13.5 revealed poorly developed embryos and defective placentas, with significantly reduced labyrinth surface area and blood vessel vascularization. These results suggested the novel possibility that the bovine K5 promoter-driven Cre-recombinase was active early in trophoblast-lineage cells that give rise to the placenta. To test this possibility, K5-Cre transgenic mice were crossed with the mT/mG reporter mouse in which activation of GFP expression indicates Cre transgene expression. We observed activation of K5-Cre-driven GFP expression in the ectoplacental cone, in the extraembryonic ectoderm, and in trophoblast giant cells in the E6.5 embryo. In addition, we observed GFP expression at E11.5 to E13.5 in both the labyrinth of the placenta and the yolk sac. NMIIA expression was detected in these same cell types in normal embryos, as well as in E13.5 yolk sac and labyrinth. These findings taken together suggest that NMHC IIA may play critical roles in the early trophoblast-derived ectoplacental cone and extraembryonic ectoderm, as well as in the yolk sac and labyrinth tissues that form later. Our findings are consistent with phenotypes of constitutive NMIIA knockout mice made earlier, that displayed labyrinth and yolk sac-specific defects, but our findings extend those observations by suggesting possible NMIIA roles in trophoblast lineages as well. These results furthermore demonstrate that K5-Cre gene constructs, previously reported to be activated starting at approximately E12.5 in the forming epidermis, may be widely useful as drivers for activation of cre/lox based gene excision in early embryo extraembronic trophoblast tissues as well.
Asunto(s)
Ectodermo/embriología , Pérdida del Embrión/patología , Integrasas/metabolismo , Queratina-5/metabolismo , Miosina Tipo IIA no Muscular/metabolismo , Trofoblastos/metabolismo , Trofoblastos/patología , Alelos , Animales , Animales Recién Nacidos , Vasos Sanguíneos/embriología , Vasos Sanguíneos/metabolismo , Bovinos , Linaje de la Célula , Proliferación Celular , Cruzamientos Genéticos , Ectodermo/metabolismo , Ectodermo/patología , Pérdida del Embrión/metabolismo , Embrión de Mamíferos/metabolismo , Embrión de Mamíferos/patología , Desarrollo Embrionario , Epidermis/embriología , Epidermis/metabolismo , Epidermis/patología , Femenino , Eliminación de Gen , Genotipo , Antígeno Ki-67/metabolismo , Masculino , Ratones , Ratones Noqueados , Embarazo , Saco Vitelino/metabolismoRESUMEN
Behavioral analyses of the deletion mutants of the four known myosin II heavy chain (Mhc) kinases of Dictyostelium discoideum revealed that all play a minor role in the efficiency of basic cell motility, but none play a role in chemotaxis in a spatial gradient of cAMP generated in vitro. However, the two kinases MhckA and MhckC were essential for chemotaxis in a spatial gradient of Ca(2+), shear-induced directed movement, and reorientation in the front of waves of cAMP during natural aggregation. The phenotypes of the mutants mhckA(-) and mhckC(-) were highly similar to that of the Ca(2+) channel/receptor mutant iplA(-) and the myosin II phosphorylation mutant 3XALA, which produces constitutively unphosphorylated myosin II. These results demonstrate that IplA, MhckA and MhckC play a selective role in chemotaxis in a spatial gradient of Ca(2+), but not cAMP, and suggest that Ca(2+) chemotaxis plays a role in the orientation of cells in the front of cAMP waves during natural aggregation.
Asunto(s)
Proteínas Quinasas Dependientes de Calcio-Calmodulina , Calcio , Movimiento Celular , Dictyostelium , Proteínas Protozoarias , Calcio/metabolismo , Proteínas Quinasas Dependientes de Calcio-Calmodulina/genética , Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Agregación Celular/genética , Movimiento Celular/genética , Movimiento Celular/fisiología , Quimiotaxis/genética , Quimiotaxis/fisiología , AMP Cíclico/metabolismo , Dictyostelium/genética , Dictyostelium/metabolismo , Dictyostelium/fisiología , Humanos , Cadenas Pesadas de Miosina/genética , Cadenas Pesadas de Miosina/metabolismo , Fosforilación , Proteínas Protozoarias/genética , Proteínas Protozoarias/metabolismo , Seudópodos/metabolismo , Eliminación de SecuenciaRESUMEN
Despite functional significance of nonmuscle myosin II in cell migration and invasion, its role in epithelial-mesenchymal transition (EMT) or TGF-ß 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-ß 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-ß 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.
Asunto(s)
Transición Epitelial-Mesenquimal , Miosina Tipo II/metabolismo , Isoformas de Proteínas/metabolismo , Factor de Crecimiento Transformador beta/fisiología , Animales , Línea Celular , Ratones , FosforilaciónRESUMEN
In Dictyostelium discoideum, myosin II resides predominantly in a soluble pool as the result of phosphorylation of the myosin heavy chain (MHC), and dephosphorylation of the MHC is required for myosin II filament assembly, recruitment to the cytoskeleton, and force production. Protein phosphatase type 2A (PP2A) was identified in earlier studies in Dictyostelium as a key biochemical activity that can drive MHC dephosphorylation. We report here gene targeting and cell biological studies addressing the roles of candidate PP2A B regulatory subunits (phr2aBα and phr2aBß) in myosin II assembly control in vivo. Dictyostelium phr2aBα- and phr2aBß-null cells show delayed development, reduction in the assembly of myosin II in cytoskeletal ghost assays, and defects in cytokinesis when grown in suspension compared to parental cell lines. These results demonstrate that the PP2A B subunits phr2aBα and phr2aBß contribute to myosin II assembly control in vivo, with phr2aBα having the predominant role facilitating MHC dephosphorylation to facilitate filament assembly.
Asunto(s)
Dictyostelium/metabolismo , Miosina Tipo II/metabolismo , Proteína Fosfatasa 2/metabolismo , Subunidades de Proteína/metabolismo , Proteínas Protozoarias/metabolismo , Animales , Movimiento Celular/fisiología , Citocinesis/fisiología , Dictyostelium/citología , Dictyostelium/genética , Marcación de Gen , Miosina Tipo II/genética , Fosforilación , Proteína Fosfatasa 2/genética , Subunidades de Proteína/genética , Proteínas Protozoarias/genéticaRESUMEN
The contractile vacuole (CV) is a dynamic organelle that enables Dictyostelium amoeba and other protist to maintain osmotic homeostasis by expelling excess water. In the present study, we have uncovered a mechanism that coordinates the mechanics of the CV with myosin II, regulated by VwkA, an unconventional protein kinase that is conserved in an array of protozoa. Green fluorescent protein (GFP)-VwkA fusion proteins localize persistently to the CV during both filling and expulsion phases of water. In vwkA null cells, the established CV marker dajumin still localizes to the CV, but these structures are large, spherical and severely impaired for discharge. Furthermore, myosin II cortical localization and assembly are abnormal in vwkA null cells. Parallel analysis of wild-type cells treated with myosin II inhibitors or of myosin II null cells also results in enlarged CVs with impaired dynamics. We suggest that the myosin II cortical cytoskeleton, regulated by VwkA, serves a critical conserved role in the periodic contractions of the CV, as part of the osmotic protective mechanism of protozoa.
Asunto(s)
Amoeba/metabolismo , Miosina Tipo II/metabolismo , Presión Osmótica , Proteínas Quinasas/metabolismo , Amoeba/enzimología , Animales , Proteínas Fluorescentes Verdes/genética , Miosina Tipo II/genética , Proteínas Quinasas/genética , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismoRESUMEN
BACKGROUND: Phosphorylation of non-muscle myosin II regulatory light chain (RLC) at Thr18/Ser19 is well established as a key regulatory event that controls myosin II assembly and activation, both in vitro and in living cells. RLC can also be phosphorylated at Ser1/Ser2/Thr9 by protein kinase C (PKC). Biophysical studies show that phosphorylation at these sites leads to an increase in the Km of myosin light chain kinase (MLCK) for RLC, thereby indirectly inhibiting myosin II activity. Despite unequivocal evidence that PKC phosphorylation at Ser1/Ser2/Thr9 can regulate myosin II function in vitro, there is little evidence that this mechanism regulates myosin II function in live cells. RESULTS: The purpose of these studies was to investigate the role of Ser1/Ser2/Thr9 phosphorylation in live cells. To do this we utilized phospho-specific antibodies and created GFP-tagged RLC reporters with phosphomimetic aspartic acid substitutions or unphosphorylatable alanine substitutions at the putative inhibitory sites or the previously characterized activation sites. Cell lines stably expressing the RLC-GFP constructs were assayed for myosin recruitment during cell division, the ability to complete cell division, and myosin assembly levels under resting or spreading conditions. Our data shows that manipulation of the activation sites (Thr18/Ser19) significantly alters myosin II function in a number of these assays while manipulation of the putative inhibitory sites (Ser1/Ser2/Thr9) does not. CONCLUSIONS: These studies suggest that inhibitory phosphorylation of RLC is not a substantial regulatory mechanism, although we cannot rule out its role in other cellular processes or perhaps other types of cells or tissues in vivo.
Asunto(s)
Cadenas Ligeras de Miosina/metabolismo , Miosina Tipo II/metabolismo , Serina/metabolismo , Treonina/metabolismo , Dominio Catalítico , División Celular , Células Cultivadas , Células HeLa , Humanos , Cadenas Ligeras de Miosina/química , Miosina Tipo II/química , Fosforilación , Serina/química , Treonina/químicaRESUMEN
During cell division, the mechanisms by which myosin II is recruited to the contractile ring are not fully understood. Much recent work has focused on a model in which spatially restricted de novo filament assembly occurs at the cell equator via localized myosin II regulatory light chain (RLC) phosphorylation, stimulated by the RhoA-activating centralspindlin complex. Here, we show that a recombinant myosin IIA protein that assembles constitutively and is incapable of binding RLC still displays strong localization to the furrow in mammalian cells. Furthermore, this RLC-deficient myosin II efficiently drives cytokinesis, demonstrating that centralspindlin-based RLC phosphorylation is not necessary for myosin II localization during furrowing. Myosin II truncation analysis further reveals two distinct myosin II tail properties that contribute to furrow localization: a central tail domain mediating cortical furrow binding to heterologous binding partners and a carboxyl-terminal region mediating co-assembly with existing furrow myosin IIA or IIB filaments.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Citocinesis , Proteínas Asociadas a Microtúbulos/metabolismo , Miosina Tipo II/metabolismo , Fosfoproteínas/metabolismo , Animales , Células COS , Chlorocebus aethiops , Eliminación de Gen , Antígenos HLA-D/genética , Antígenos HLA-D/metabolismo , Células HeLa , Humanos , Cadenas Ligeras de Miosina/deficiencia , Cadenas Ligeras de Miosina/genética , Cadenas Ligeras de Miosina/metabolismo , Miosina Tipo II/genética , Fosforilación , Transporte de Proteínas , Proteínas Recombinantes de Fusión/metabolismoRESUMEN
To study the roles of nonmuscle myosin II (NM-II) during invasive cell migration, microfluidic migration chambers have been designed and fabricated using photo- and soft-lithography microfabrication techniques. The chamber consists of two channels separated by a vertical barrier with multiple bays of pores with widths varying from 6 microm to 16 microm, and lengths varying from 25 microm to 50 microm. The cells are plated in the channel on one side of the barrier while a chemoattractant is flowed through the channel on the other side of the barrier. In these chambers, cells can be observed with transmitted light or fluorescence optics while they chemotax through various sized pores that impose differential mechanical resistance to transmigration. As an initial test of this device, we compared breast-cancer cell chemotactic transmigration through different pore sizes with and without inhibition of NM-II. Two distinct rates were observed as cells attempted to pull their nucleus through the smaller pores, and the faster nuclear transit mode was critically dependent on NM-II motor activity. The ability to monitor cells as they chemotax through pores of different dimensions within a single experimental system provides novel information on how pore size affects cell morphology and migration rate, providing a dramatic improvement of imaging potential relative to other in vitro transmigration systems such as Boyden chambers.
Asunto(s)
Neoplasias de la Mama/patología , Neoplasias de la Mama/fisiopatología , Quimiotaxis , Citometría de Flujo/instrumentación , Técnicas Analíticas Microfluídicas/instrumentación , Microscopía/instrumentación , Miosina Tipo II/metabolismo , Técnicas de Cultivo de Célula/instrumentación , Línea Celular Tumoral , Separación Celular/instrumentación , Diseño de Equipo , Análisis de Falla de Equipo , Análisis de Inyección de Flujo/instrumentación , HumanosRESUMEN
We have identified a new protein kinase in Dictyostelium discoideum that carries the same conserved class of "alpha-kinase" catalytic domain as reported previously in myosin heavy chain kinases (MHCKs) in this amoeba but that has a completely novel domain organization. The protein contains an N-terminal von Willebrand factor A (vWFA)-like motif and is therefore named VwkA. Manipulation of VwkA expression level via high copy number plasmids (VwkA++ cells) or gene disruption (vwkA null cells) results in an array of cellular defects, including impaired growth and multinucleation in suspension culture, impaired development, and alterations in myosin II abundance and assembly. Despite sequence similarity to MHCKs, the purified protein failed to phosphorylate myosin II in vitro. Autophosphorylation activity, however, was enhanced by calcium/calmodulin, and the enzyme can be precipitated from cellular lysates with calmodulin-agarose, suggesting that VwkA may directly bind calmodulin. VwkA is cytosolic in distribution but enriched on the membranes of the contractile vacuole and Golgi-like structures in the cell. We propose that VwkA likely acts indirectly to influence myosin II abundance and assembly behavior and possibly has broader roles than previously characterized alpha kinases in this organism, which all seem to be MHCKs.
Asunto(s)
Dictyostelium/enzimología , Proteínas Quinasas/química , Proteínas Quinasas/metabolismo , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Calmodulina/metabolismo , Clonación Molecular , ADN Complementario/genética , ADN Protozoario/genética , Dictyostelium/genética , Dictyostelium/crecimiento & desarrollo , Expresión Génica , Genes Protozoarios , Aparato de Golgi/enzimología , Datos de Secuencia Molecular , Miosina Tipo II/metabolismo , Proteínas Quinasas/genética , Proteínas Protozoarias/química , Proteínas Protozoarias/genética , Proteínas Protozoarias/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/aislamiento & purificación , Proteínas Recombinantes de Fusión/metabolismo , Homología de Secuencia de Aminoácido , Vacuolas/enzimología , Factor de von Willebrand/química , Factor de von Willebrand/genéticaRESUMEN
Myosin II filament assembly in Dictyostelium discoideum is regulated via phosphorylation of residues located in the carboxyl-terminal portion of the myosin II heavy chain (MHC) tail. A series of novel protein kinases in this system are capable of phosphorylating these residues in vitro, driving filament disassembly. Previous studies have demonstrated that at least three of these kinases (MHCK A, MHCK B, and MHCK C) display differential localization patterns in living cells. We have created a collection of single, double, and triple gene knockout cell lines for this family of kinases. Analysis of these lines reveals that three MHC kinases appear to represent the majority of cellular activity capable of driving myosin II filament disassembly, and reveals that cytokinesis defects increase with the number of kinases disrupted. Using biochemical fractionation of cytoskeletons and in vivo measurements via fluorescence recovery after photobleaching (FRAP), we find that myosin II overassembly increases incrementally in the mutants, with the MHCK A(-)/B(-)/C(-) triple mutant showing severe myosin II overassembly. These studies suggest that the full complement of MHC kinases that significantly contribute to growth phase and cytokinesis myosin II disassembly in this organism has now been identified.
Asunto(s)
Proteínas Quinasas Dependientes de Calcio-Calmodulina/fisiología , Citocinesis/fisiología , Citoesqueleto/enzimología , Dictyostelium/enzimología , Miosina Tipo II/fisiología , Animales , Proteínas Quinasas Dependientes de Calcio-Calmodulina/genética , Línea Celular , Dictyostelium/crecimiento & desarrollo , Genes Reporteros , Cadenas Pesadas de Miosina/metabolismo , Miosina Tipo II/genética , Miosina Tipo II/metabolismo , Fosforilación , Procesamiento Proteico-Postraduccional , Proteínas Protozoarias , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismoRESUMEN
Initial stages of tumor cell metastasis involve an epithelial-mesenchyme transition that involves activation of amoeboid migration and loss of cell-cell adhesion. The actomyosin cytoskeleton has fundamental but poorly understood roles in these events. Myosin II, an abundant force-producing protein, has roles in cell body translocation and retraction of the posterior of the cell during migration. Recent studies have suggested that this protein may also have roles in leading edge protrusive events. The metastasis-promoting protein metastasin-1, a regulator of myosin II assembly, colocalizes with myosin IIA at the leading edge of cancer cells, suggesting direct roles for myosin II in metastatic behavior. We have assessed the roles of specific myosin II isoforms during lamellar spreading of MDA-MB-231 breast cancer cells on extracellular matrix. We find that the two major myosin II isoforms IIA and IIB are both expressed in these cells, and both are recruited dramatically to the lamellar margin during active spreading on fibronectin. There is also a transient increase in regulatory light chain phosphorylation that correlates the recruitment of myosin IIA and myosin IIB into this spreading margin. Pharmacologic inhibition of myosin II or myosin light chain kinase dramatically reduced spreading. Depletion of myosin IIA via small interfering RNA impaired migration but enhanced lamellar spreading, whereas depletion of myosin IIB impaired not only migration but also impaired initial rates of lamellar spreading. These results indicate that both isoforms are critical for the mechanics of cell migration, with myosin IIB seeming to have a preferential role in the mechanics of lamellar protrusion.
Asunto(s)
Neoplasias de la Mama/patología , Movimiento Celular/fisiología , Miosina Tipo IIA no Muscular/fisiología , Miosina Tipo IIB no Muscular/fisiología , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Adhesión Celular/fisiología , Línea Celular Tumoral , Fibronectinas , Humanos , Péptidos y Proteínas de Señalización Intracelular , Quinasa de Cadena Ligera de Miosina/antagonistas & inhibidores , Quinasa de Cadena Ligera de Miosina/metabolismo , Miosina Tipo IIA no Muscular/antagonistas & inhibidores , Miosina Tipo IIA no Muscular/genética , Miosina Tipo IIA no Muscular/metabolismo , Miosina Tipo IIB no Muscular/antagonistas & inhibidores , Miosina Tipo IIB no Muscular/genética , Miosina Tipo IIB no Muscular/metabolismo , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Serina-Treonina Quinasas/metabolismo , ARN Interferente Pequeño/genética , Transfección , Quinasas Asociadas a rhoRESUMEN
Human mesenchymal stem cell (hMSC)-based chondrogenesis is a key process used to develop tissue engineered cartilage constructs from stem cells, but the resulting constructs have inferior biochemical and biomechanical properties compared to native articular cartilage. Transforming growth factor ß containing medium is commonly applied to cell layers of hMSCs, which aggregate upon centrifugation to form 3-D constructs. The aggregation process leads to a high cell density condition, which can cause nutrient limitations during long-term culture and, subsequently, inferior quality of tissue engineered constructs. Our objective is to modulate the aggregation process by targeting RhoA/ROCK signaling pathway, the chief modulator of actomyosin contractility, to enhance the end quality of the engineered constructs. Through ROCK inhibition, repression of cytoskeletal tension in chondrogenic hMSCs was achieved along with less dense aggregates with enhanced transport properties. ROCK inhibition also led to significantly increased cartilaginous extracellular matrix accumulation. These findings can be used to create an improved microenvironment for hMSC-derived tissue engineered cartilage culture. We expect that these findings will ultimately lead to improved cartilaginous tissue development from hMSCs.
Asunto(s)
Cartílago/enzimología , Condrogénesis , Matriz Extracelular/metabolismo , Células Madre Mesenquimatosas/enzimología , Transducción de Señal , Quinasas Asociadas a rho/antagonistas & inhibidores , Transporte Biológico Activo , Cartílago/citología , Células Cultivadas , Citoesqueleto/metabolismo , Humanos , Células Madre Mesenquimatosas/citología , Ingeniería de Tejidos , Quinasas Asociadas a rho/metabolismoRESUMEN
BACKGROUND: Cell migration requires coordinated formation of focal adhesions (FAs) and assembly and contraction of the actin cytoskeleton. Nonmuscle 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, whereas 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 upregulation of this pathway during physiological Rac1 activation. Phosphomimic and nonphosphorylatable MIIA HC mutants demonstrated that S1916 phosphorylation was necessary and sufficient for the capture and assembly of MIIA minifilaments 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 that promotes MIIA association with FAs and acts as a critical modulator of cell migration and mechanosensing.
Asunto(s)
Movimiento Celular , Adhesiones Focales/metabolismo , Proteínas Motoras Moleculares/genética , Cadenas Pesadas de Miosina/genética , Transducción de Señal , Proteína de Unión al GTP rac1/genética , Línea Celular , Humanos , Mecanotransducción Celular/fisiología , Proteínas Motoras Moleculares/metabolismo , Cadenas Pesadas de Miosina/metabolismo , Fosforilación , Proteína de Unión al GTP rac1/metabolismoRESUMEN
Non-muscle myosin II (NMII) is reported to play multiple roles during cell migration and invasion. However, the exact biophysical roles of different NMII isoforms during these processes remain poorly understood. We analyzed the contributions of NMIIA and NMIIB in three-dimensional (3D) migration and in generating the forces required for efficient invasion by mammary gland carcinoma cells. Using traction force microscopy and microfluidic invasion devices, we demonstrated that NMIIA is critical for generating force during active protrusion, and NMIIB plays a major role in applying force on the nucleus to facilitate nuclear translocation through tight spaces. We further demonstrate that the nuclear membrane protein nesprin-2 is a possible linker coupling NMIIB-based force generation to nuclear translocation. Together, these data reveal a central biophysical role for NMIIB in nuclear translocation during 3D invasive migration, a result with relevance not only to cancer metastasis but for 3D migration in other settings such as embryonic cell migration and wound healing.
Asunto(s)
Movimiento Celular , Núcleo Celular/fisiología , Miosina Tipo IIB no Muscular/fisiología , Transporte Activo de Núcleo Celular , Animales , Fenómenos Biomecánicos , Línea Celular Tumoral , Ratones , Proteínas del Tejido Nervioso/metabolismo , Proteínas Nucleares/metabolismoRESUMEN
Myosin heavy chain kinase A (MHCK A) modulates myosin II filament assembly in the amoeba Dictyostelium discoideum. MHCK A localization in vivo is dynamically regulated during chemotaxis, phagocytosis, and other polarized cell motility events, with preferential recruitment into anterior filamentous actin (F-actin)-rich structures. The current work reveals that an amino-terminal segment of MHCK A, previously identified as forming a coiled-coil, mediates anterior localization. MHCK A co-sediments with F-actin, and deletion of the amino-terminal domain eliminated actin binding. These results indicate that the anterior localization of MHCK A is mediated via direct binding to F-actin, and reveal the presence of an actin-binding function not previously detected by primary sequence evaluation of the coiled-coil domain.
Asunto(s)
Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Dictyostelium/enzimología , Actinas/metabolismo , Animales , Sitios de Unión , Proteínas Quinasas Dependientes de Calcio-Calmodulina/química , Polaridad Celular , Quimiotaxis , Dictyostelium/citología , Dictyostelium/genética , Dictyostelium/fisiología , Técnicas In Vitro , Miosina Tipo II/metabolismo , Estructura Terciaria de Proteína , Proteínas Protozoarias , Seudópodos/enzimología , Seudópodos/fisiología , Conejos , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismoRESUMEN
Gene targeting is a powerful molecular genetic technique that has been widely used to understand specific gene function in vivo. This technique allows the ablation of an endogenous gene by recombination between an introduced DNA fragment and the homologous target gene. However, when multiple gene disruptions are needed, the availability of only a limited number of marker genes becomes a complication. Here we describe a new approach to perform double gene disruptions in Dictyostelium discoideum by simultaneous transfection of two gene targeting cassettes followed by performing clonal selection against only one marker gene. The subsequent PCR-based screens of blasticidin-resistant clones revealed the integration of both the selected and the nonselected targeting cassettes at their original respective loci creating complete gene disruptions. For the genes we have tested in these studies (myosin heavy chain kinases B and C), the efficiency of the double gene targeting event is found in the range of 2%-5% of all blasticidin-resistant colonies following the transfection step. This approach for the simultaneous disruptions of multiple genes should prove to be a valuable tool for other laboratories interested in creating multiple gene disruptants in Dictyostelium or other organisms where a limited number of selectable markers are available.
Asunto(s)
Dictyostelium/genética , Marcación de Gen/métodos , Ingeniería Genética/métodos , Nucleósidos/genética , Animales , Antibacterianos/inmunología , Línea Celular , Dictyostelium/inmunología , Silenciador del Gen , Marcadores Genéticos/genética , Marcadores Genéticos/inmunología , Nucleósidos/inmunología , Transfección/métodosRESUMEN
Bladder cancer is the fourth most common cause of cancer in males in the United States. Invasive behavior is a major determinant of prognosis. In this study, we identified mammalian target of rapamycin complex 2 (mTORC2) as a central regulator of bladder cancer cell migration and invasion. mTORC2 activity was assessed by the extent of phosphorylation of Ser473 in AKT and determined to be approximately 5-fold higher in specimens of invasive human bladder cancer as opposed to non-invasive human bladder cancer. The immortalized malignant bladder cell lines, UMUC-3, J82 and T24 demonstrated higher baseline mTORC2 activity relative to the benign bladder papilloma-derived cell line RT4 and the normal urothelial cell line HU1. The malignant bladder cancer cells also demonstrated increased migration in transwell and denudation assays, increased invasion of matrigel, and increased capacity to invade human bladder specimens. Gene silencing of rictor, a critical component of mTORC2, substantially inhibited bladder cancer cell migration and invasion. This was accompanied by a significant decrease in Rac1 activation and paxillin phosphorylation. These studies identify mTORC2 as a major target for neutralizing bladder cancer invasion.