RESUMEN
Non-muscle myosin II has diverse functions in cell contractility, cytokinesis and locomotion, but the specific contributions of its different isoforms have yet to be clarified. Here, we report that ablation of the myosin IIA isoform results in pronounced defects in cellular contractility, focal adhesions, actin stress fibre organization and tail retraction. Nevertheless, myosin IIA-deficient cells display substantially increased cell migration and exaggerated membrane ruffling, which was dependent on the small G-protein Rac1, its activator Tiam1 and the microtubule moter kinesin Eg5. Myosin IIA deficiency stabilized microtubules, shifting the balance between actomyosin and microtubules with increased microtubules in active membrane ruffles. When microtubule polymerization was suppressed, myosin IIB could partially compensate for the absence of the IIA isoform in cellular contractility, but not in cell migration. We conclude that myosin IIA negatively regulates cell migration and suggest that it maintains a balance between the actomyosin and microtubule systems by regulating microtubule dynamics.
Asunto(s)
Actomiosina/metabolismo , Movimiento Celular/fisiología , Microtúbulos/metabolismo , Miosina Tipo IIA no Muscular/fisiología , Aminoquinolinas/farmacología , Animales , Azepinas/farmacología , Células COS , Adhesión Celular/fisiología , Movimiento Celular/efectos de los fármacos , Chlorocebus aethiops , Células Madre Embrionarias/citología , Células Madre Embrionarias/efectos de los fármacos , Células Madre Embrionarias/metabolismo , Inhibidores Enzimáticos/farmacología , Fibroblastos/citología , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Factores de Intercambio de Guanina Nucleótido/genética , Factores de Intercambio de Guanina Nucleótido/metabolismo , Compuestos Heterocíclicos de 4 o más Anillos/farmacología , Humanos , Cinesinas/antagonistas & inhibidores , Cinesinas/genética , Cinesinas/metabolismo , Ratones , Microtúbulos/efectos de los fármacos , Naftalenos/farmacología , Nocodazol/farmacología , Miosina Tipo IIA no Muscular/antagonistas & inhibidores , Miosina Tipo IIA no Muscular/genética , Miosina Tipo IIB no Muscular/antagonistas & inhibidores , Miosina Tipo IIB no Muscular/genética , Miosina Tipo IIB no Muscular/fisiología , Pirimidinas/farmacología , ARN Interferente Pequeño/genética , Proteína 1 de Invasión e Inducción de Metástasis del Linfoma-T , Tionas/farmacología , Transfección , Vinblastina/farmacología , Proteína de Unión al GTP rac1/antagonistas & inhibidores , Proteína de Unión al GTP rac1/genética , Proteína de Unión al GTP rac1/metabolismoRESUMEN
The innate immune system responds to endogenous molecules released during cellular stress or those that have undergone modifications normally absent in healthy tissue. These structures are detected by pattern-recognition receptors, alerting the immune system to "danger." In this study, we looked for early signals that direct immune cells to cells undergoing stress before irreversible damage takes place. To avoid detecting signals emanating from apoptotic or necrotic cells we exposed fibroblasts to sublethal oxidative stress. Our results indicate that both nonenzymatic chemical reactions and aldehyde dehydrogenase-2-mediated enzymatic activity released signals from fibroblasts that selectively attracted CD14(+) monocytes but not T, NK, and NKT cells or granulocytes. Splenocytes from MyD88(-/-) mice did not migrate, and treatment with an inhibitory peptide that blocks MyD88 dimerization abrogated human monocyte migration. Monocyte migration was accompanied by downmodulation of CD14 expression and by the phosphorylation of IL-1R-associated kinase 1, a well-known MyD88-dependent signaling molecule. The scavenger receptor inhibitors, dextran sulfate and fucoidan, attenuated monocyte migration toward stressed cells and IL-1R-associated kinase 1 phosphorylation. Surprisingly, although monocyte migration was MyD88 dependent, it was not accompanied by inflammatory cytokine secretion. Taken together, these results establish a novel link between scavenger receptors and MyD88 that together function as sensors of oxidation-associated molecular patterns and induce monocyte motility. Furthermore, the data indicate that MyD88 independently regulates monocyte activation and motility.
Asunto(s)
Movimiento Celular , Fibroblastos/fisiología , Monocitos/fisiología , Factor 88 de Diferenciación Mieloide/fisiología , Estrés Oxidativo/fisiología , Receptores Depuradores/fisiología , Aldehído Deshidrogenasa , Animales , Fibroblastos/metabolismo , Humanos , Receptores de Lipopolisacáridos , Macrófagos , Ratones , Factor 88 de Diferenciación Mieloide/metabolismo , Receptores Depuradores/metabolismo , Transducción de SeñalRESUMEN
The ability of cells to migrate within the extracellular matrix and to remodel it depends as much on the physical and biochemical characteristics of a particular matrix as on cellular properties. Analyzing the different modes of migration of cells in matrices, and how cells switch between these modes, is vital for understanding a variety of physiological and pathological processes. Recent work provides new insights, but also raises some debates about the mechanisms and regulation of cell migration in three-dimensional matrices.
Asunto(s)
Movimiento Celular/fisiología , Matriz Extracelular/fisiología , Modelos Biológicos , Animales , Membrana Basal/metabolismo , Membrana Basal/fisiología , Matriz Extracelular/metabolismo , Humanos , Proteínas de Unión al GTP Monoméricas/metabolismo , Proteínas de Unión al GTP Monoméricas/fisiología , Transducción de Señal/fisiologíaRESUMEN
Human embryonic stem cells (hESCs) can proliferate extensively in culture and give rise to progeny of the three germ layers. Several reports suggested that mouse and hESCs may attenuate immune responses. In this study, we focused on the mechanism by which hESCs inhibit T cell responses. Using coculture experiments, we demonstrate that hESCs inhibit cytokine secretion and T cell proliferation in response to potent T cell activators. Furthermore, we show that hESCs downmodulate the TCR-associated CD3-zeta chain. These effects are maintained when hESCs are replaced by their conditioned media and can be restored by the addition of L-arginine to hESC-conditioned media or by treatment of hESCs with a specific arginase inhibitor. Moreover, we show arginase-I expression and activity in hESCs. We further demonstrate that mouse ESCs (mESCs) similarly inhibit T cell activation via arginase I, suggesting an evolutionary conserved mechanism of T cell suppression by ESCs. In addition, we demonstrate that arginase I expression is not limited to ESCs in culture, but can also be detected in the inner cell mass and the trophectoderm of preimplantation mouse embryos and hESC-derived trophectoderm cells. Finally, T cells infiltrating ESC-derived teratomas have significantly lower levels of CD3-zeta chain. Collectively, the data indicate a role for ESC-arginase I activity in the attenuation of T cell activation.
Asunto(s)
Arginasa/fisiología , Citocinas/antagonistas & inhibidores , Células Madre Embrionarias/enzimología , Células Madre Embrionarias/inmunología , Activación de Linfocitos/inmunología , Subgrupos de Linfocitos T/enzimología , Subgrupos de Linfocitos T/inmunología , Adulto , Animales , Arginina/metabolismo , Complejo CD3/inmunología , Complejo CD3/metabolismo , Línea Celular , Movimiento Celular/genética , Movimiento Celular/inmunología , Células Cultivadas , Técnicas de Cocultivo , Citocinas/metabolismo , Células Madre Embrionarias/trasplante , Regulación Enzimológica de la Expresión Génica/inmunología , Humanos , Leucocitos Mononucleares/citología , Leucocitos Mononucleares/enzimología , Leucocitos Mononucleares/inmunología , Ratones , Receptores de Antígenos de Linfocitos T/antagonistas & inhibidores , Receptores de Antígenos de Linfocitos T/biosíntesis , Subgrupos de Linfocitos T/citología , Teratoma/enzimología , Teratoma/inmunología , Teratoma/patologíaRESUMEN
In the mammalian embryo, a formative pluripotent phase is proposed to exist at the early post-implantation period, during the transition from the pre-implantation naive-to the post-implantation primed-epiblast. By recapitulating a laminin component of the extracellular matrix niche during embryonic formative transition, and defined culture conditions, we generated cultures highly enriched for self-renewing human pluripotent stem cells (hPSCs), exhibiting properties of early post-implantation epiblast cells. These hPSCs display post-implantation-epiblast gene expression profiles. FGF and TGF-ß signaling maintain their self-renewal for multiple passages. They have inactive canonical Wnt signaling, do not express primitive streak markers, and are competent to initiate differentiation toward germline and somatic fates. hPSCs exhibiting early post-implantation epiblast properties may shed light on human embryonic PSCs development and may serve for initiating somatic and germ cell specification.
Asunto(s)
Estratos Germinativos , Células Madre Pluripotentes , Animales , Humanos , Células Madre Pluripotentes/metabolismo , Embrión de Mamíferos , Línea Primitiva , Diferenciación Celular , Vía de Señalización Wnt , MamíferosRESUMEN
Directional migration moves cells rapidly between points, whereas random migration allows cells to explore their local environments. We describe a Rac1 mechanism for determining whether cell patterns of migration are intrinsically random or directionally persistent. Rac activity promoted the formation of peripheral lamellae that mediated random migration. Decreasing Rac activity suppressed peripheral lamellae and switched the cell migration patterns of fibroblasts and epithelial cells from random to directionally persistent. In three-dimensional rather than traditional two-dimensional cell culture, cells had a lower level of Rac activity that was associated with rapid, directional migration. In contrast to the directed migration of chemotaxis, this intrinsic directional persistence of migration was not mediated by phosphatidylinositol 3'-kinase lipid signaling. Total Rac1 activity can therefore provide a regulatory switch between patterns of cell migration by a mechanism distinct from chemotaxis.
Asunto(s)
Movimiento Celular/fisiología , Proteínas de Unión al GTP rac/metabolismo , Técnicas de Cultivo de Célula , Extensiones de la Superficie Celular/metabolismo , Células Cultivadas , Células Epiteliales/citología , Células Epiteliales/metabolismo , Fibroblastos/citología , Fibroblastos/metabolismo , Fibronectinas/metabolismo , Guanosina Trifosfato/metabolismo , Humanos , Integrinas/genética , Integrinas/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Inhibidores de las Quinasa Fosfoinosítidos-3 , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Distribución Aleatoria , Transducción de Señal/fisiología , Proteína de Unión al GTP cdc42/genética , Proteína de Unión al GTP cdc42/metabolismo , Proteínas de Unión al GTP rac/genética , Proteínas de Unión al GTP rho/genética , Proteínas de Unión al GTP rho/metabolismoRESUMEN
Multiple cell types have an inherent ability to contract the extracellular matrix to which they are attached and grow on. Cells exert contractile forces on a compliant substrate, increase the tension, and deform it. Numerous intracellular as well as environmental factors are involved in determination of cellular contractility, which can be precisely measured by atomic force microscopy, laser tweezers, or other complex apparatus. These, however, are far from being standard equipment in most cell biology labs. Fibrin gels provide a simple and affordable alternative for evaluation of changes in cell contractility by either quantitation of end-point gel contraction or in a dynamic mode by time-lapse imaging. They also provide a flexible system in which the physical properties, such as density and compliance, as well as their biochemical composition can easily be altered to suit the special requirements of various cell types and experimental models.
Asunto(s)
Fibrina/química , Geles , Modelos Biológicos , Humanos , Microscopía de Fuerza AtómicaRESUMEN
Selective down-modulation or silencing of individual members of the Rho-GTPase family is now practical using RNA interference. Transfection of mammalian cells with an individual siRNA duplex or siRNA pools can suppress expression of a specific isoform to understand its function. By adjusting the dose of siRNA, intermediate levels of suppression can be attained to test the biological role of different levels of a GTPase such as Rac. Nevertheless, there are significant potential pitfalls, including "off-target" effects of the siRNA on other genes. Besides demonstrating successful, noncytotoxic suppression of protein and activity levels of a specific GTPase, controls are essential to establish specificity. In this chapter, we provide methods for selective knockdown of expression by siRNA and confirmation of the effectiveness of Rho GTPase silencing, as well as descriptions and some examples of controls for specificity that include evaluations of dose-response, negative and positive controls, GTPase specificity, confirmation by using more than one siRNA for the same gene, rescue by a mutated siRNA-resistant cDNA encoding the target gene, and complementary supporting evidence. Selective silencing of specific Rho family GTPases should provide increasing insight into the regulatory and functional roles of each isoform in a wide variety of biological processes.
Asunto(s)
Interferencia de ARN/fisiología , Proteínas de Unión al GTP rho/antagonistas & inhibidores , Línea Celular , ADN Complementario/genética , Humanos , ARN Interferente Pequeño/fisiología , Transfección/métodos , Proteínas de Unión al GTP rho/genéticaAsunto(s)
Integrinas/metabolismo , Proteínas , Receptores de Factores de Crecimiento/metabolismo , Animales , Fibronectinas/metabolismo , Quinasa 1 de Adhesión Focal , Proteína-Tirosina Quinasas de Adhesión Focal , Humanos , Ligandos , Sistema de Señalización de MAP Quinasas , Modelos Biológicos , Fosfoproteínas/metabolismo , Fosforilación , Proteínas Tirosina Quinasas/metabolismo , Proteína p130 Similar a la del Retinoblastoma , Transducción de Señal , Tirosina/metabolismoRESUMEN
The formation of new blood vessels is a critical determinant of tumor progression. We find that Par1 gene expression plays a central role in blood vessel recruitment in animal models. By in vivo injection of either Matrigel plugs containing Par1-expressing cells or of rat prostatic carcinoma cells transfected with tetracycline-inducible Par1 expression vectors, we show that Par1 significantly enhances both angiogenesis and tumor growth. Several vascular endothelial growth factor (VEGF) splice forms are induced in cells expressing Par1. Activation of PAR1 markedly augments the expression of VEGF mRNAs and of functional VEGFs as determined by in vitro assays for endothelial tube alignment and bovine aortic endothelial cell proliferation. Because neutralizing anti-VEGF antibodies potently inhibited Par1-induced endothelial cell proliferation, we conclude that Par1-induced angiogenesis requires VEGF. Specific inhibitors of protein kinase C (PKC), Src, and phosphatidylinositol 3-kinase (PI3K) inhibit Par1-induced VEGF expression, suggesting the participation of these kinases in the process. We also show that oncogenic transformation by genes known to be part of PAR1 signaling machinery is sufficient to increase VEGF expression in NIH 3T3 cells. These data support the novel notion that initiation of cell signaling either by activating PAR1 or by the activated forms of oncogenes is sufficient to induce VEGF and hence angiogenesis.
Asunto(s)
Transformación Celular Neoplásica , Neoplasias/irrigación sanguínea , Neovascularización Patológica/fisiopatología , Receptores de Trombina/fisiología , Células 3T3 , Animales , Northern Blotting , Factores de Crecimiento Endotelial/genética , Factores de Crecimiento Endotelial/metabolismo , Expresión Génica , Genes ras/genética , Genes src/genética , Humanos , Péptidos y Proteínas de Señalización Intercelular/genética , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Linfocinas/genética , Linfocinas/metabolismo , Masculino , Ratones , Ratones Endogámicos BALB C , Trasplante de Neoplasias , Neoplasias/genética , Neoplasias Experimentales/genética , Neoplasias Experimentales/patología , Oncogenes/genética , Fosfatidilinositol 3-Quinasas/metabolismo , Proteína Quinasa C/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ratas , Receptor PAR-1 , Receptores de Trombina/genética , Transfección , Trasplante Heterólogo , Células Tumorales Cultivadas , Factor A de Crecimiento Endotelial Vascular , Factores de Crecimiento Endotelial Vascular , Familia-src Quinasas/metabolismoRESUMEN
While protease-activated receptors (PARs) play a traditional role in vascular biology, they emerge with surprisingly new assignments in tumor biology. PAR1 expression correlates with the invasion properties of breast carcinoma, whereas human PAR1 antisense reduces their ability to migrate through Matrigel. Part of the molecular mechanism of PAR1 invasion involves the formation of focal contact complexes on PAR1 activation. PAR1 induces angiogenesis in animal models in vivo and exhibits an oncogenic phenotype of enhanced ductal complexity when overexpressed in mouse mammary glands.
Asunto(s)
Células Epiteliales/metabolismo , Invasividad Neoplásica/fisiopatología , Metástasis de la Neoplasia/fisiopatología , Receptor PAR-1/fisiología , Animales , Mama/irrigación sanguínea , Mama/crecimiento & desarrollo , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , División Celular , Citoesqueleto/ultraestructura , Células Epiteliales/patología , Femenino , Humanos , Integrinas/fisiología , Ratones , Ratones Noqueados , Morfogénesis , Neovascularización Patológica/fisiopatología , Oligonucleótidos Antisentido/farmacología , Placenta/irrigación sanguínea , Embarazo , Receptores de Vitronectina/fisiología , Trombina/fisiología , Factor A de Crecimiento Endotelial Vascular/biosíntesis , Factor A de Crecimiento Endotelial Vascular/genéticaRESUMEN
The DNA damage response (DDR) is a comprehensive and complex network of phosphorylation-mediated signaling pathways that originates endogenously from the DNA lesion and activates intrinsic DNA repair mechanisms. Here we describe a macrophage-dependent mechanism that regulates the response to DNA damage. We demonstrate that human monocytes, by releasing macrophage-derived HB-EGF, enhance DDR in neighboring cells suffering from DNA damage. Consequently, HB-EGF-treated cells exhibit higher double-strand break (DSB) rejoining and display lower levels of residual DSBs. Diethylnitrosamine (DEN) injection induce DSBs along with elevation in the number of macrophages and HB-EGF expression. Significantly, macrophage depletion or blocking HB-EGF activity results in higher levels of nonrepairable DSBs, suggesting that macrophages play a role in the resolution of DNA damage via HB-EGF. This study establishes that macrophages, acting through the activation of the EGFR cascade, constitute an important cell nonautonomous physiologic component of the DDR and points to a unique role played by immune cells in maintaining genome integrity.
Asunto(s)
Daño del ADN/inmunología , Macrófagos/inmunología , Animales , Células Cultivadas , Roturas del ADN de Doble Cadena , Reparación del ADN por Unión de Extremidades/inmunología , Receptores ErbB/inmunología , Receptores ErbB/farmacología , Fibroblastos/citología , Fibroblastos/efectos de los fármacos , Fibroblastos/inmunología , Factor de Crecimiento Similar a EGF de Unión a Heparina/inmunología , Factor de Crecimiento Similar a EGF de Unión a Heparina/farmacología , Humanos , Hígado/efectos de los fármacos , Hígado/inmunología , Hígado/fisiología , Macrófagos/efectos de los fármacos , Masculino , Ratones , Ratones Endogámicos C57BL , Monocitos/efectos de los fármacos , Monocitos/inmunología , Oxidación-Reducción , Transducción de Señal/inmunologíaRESUMEN
A splicing mutation in the IKBKAP gene causes Familial Dysautonomia (FD), affecting the IKAP protein expression levels and proper development and function of the peripheral nervous system (PNS). Here we found new molecular insights for the IKAP role and the impact of the FD mutation in the human PNS lineage by using a novel and unique human embryonic stem cell (hESC) line homozygous to the FD mutation originated by pre implantation genetic diagnosis (PGD) analysis. We found that IKBKAP downregulation during PNS differentiation affects normal migration in FD-hESC derived neural crest cells (NCC) while at later stages the PNS neurons show reduced intracellular colocalization between vesicular proteins and IKAP. Comparative wide transcriptome analysis of FD and WT hESC-derived neurons together with the analysis of human brains from FD and WT 12 weeks old embryos and experimental validation of the results confirmed that synaptic vesicular and neuronal transport genes are directly or indirectly affected by IKBKAP downregulation in FD neurons. Moreover we show that kinetin (a drug that corrects IKBKAP alternative splicing) promotes the recovery of IKAP expression and these IKAP functional associated genes identified in the study. Altogether, these results support the view that IKAP might be a vesicular like protein that might be involved in neuronal transport in hESC derived PNS neurons. This function seems to be mostly affected in FD-hESC derived PNS neurons probably reflecting some PNS neuronal dysfunction observed in FD.
Asunto(s)
Proteínas Portadoras/metabolismo , Regulación hacia Abajo , Disautonomía Familiar/genética , Células Madre Embrionarias Humanas/patología , Neuronas/metabolismo , Sistema Nervioso Periférico/patología , Vesículas Sinápticas/metabolismo , Transporte Biológico/efectos de los fármacos , Transporte Biológico/genética , Proteínas Portadoras/genética , Diferenciación Celular/efectos de los fármacos , Regulación hacia Abajo/efectos de los fármacos , Disautonomía Familiar/metabolismo , Disautonomía Familiar/patología , Feto , Células Madre Embrionarias Humanas/efectos de los fármacos , Humanos , Cinetina/farmacología , Masculino , Mutación , Cresta Neural/efectos de los fármacos , Cresta Neural/patología , Neuronas/efectos de los fármacos , Sistema Nervioso Periférico/efectos de los fármacos , Fenotipo , Vesículas Sinápticas/efectos de los fármacos , Factores de Elongación TranscripcionalRESUMEN
The function of imprinted H19 long non-coding RNA is still controversial. It is highly expressed in early embryogenesis and decreases after birth and re-expressed in cancer. To study the role of H19 in oncogenesis and pluripotency, we down-regulated H19 expression in vitro and in vivo in pluripotent human embryonic carcinoma (hEC) and embryonic stem (hES) cells. H19 knockdown resulted in a decrease in the expression of the pluripotency markers Oct4, Nanog, TRA-1-60 and TRA-1-81, and in the up-regulation of SSEA1; it further attenuated cell proliferation, decreased cell-matrix attachment, and up-regulated E-Cadherin expression. SCID-Beige mice transplanted with H19 down-regulated hEC cells exhibited slower kinetics of tumor formation, resulting in an increased animal survival. Tumors derived from H19 down-regulated cells showed a decrease in the expression of pluripotency markers and up-regulation of SSEA-1 and E-cadherin. Our results suggest that H19 oncogenicity in hEC cells is mediated through the regulation of the pluripotency state.
Asunto(s)
Transformación Celular Neoplásica/genética , Células Madre Embrionarias , Células Madre Pluripotentes , ARN Largo no Codificante/genética , Animales , Western Blotting , Línea Celular Tumoral , Técnicas de Silenciamiento del Gen , Xenoinjertos , Humanos , Inmunohistoquímica , Ratones , Ratones SCID , Microscopía Fluorescente , Reacción en Cadena de la Polimerasa , ARN Interferente Pequeño , TransfecciónRESUMEN
Undifferentiated human embryonic stem cells (hESCs) are currently propagated on a relatively small scale as monolayer colonies. Culture of hESCs as floating aggregates is widely used for induction of differentiation into embryoid bodies. Here we show that hESC lines can be derived from floating inner cell masses in suspension culture conditions that do not involve feeder cells or microcarriers. This culture system supports prolonged propagation of the pluripotent stem cells as floating clusters without their differentiation into embryoid bodies. HESCs cultivated as aggregates in suspension maintain the expression of pluripotency markers and can differentiate into progeny of the three germ layers both in vitro and in vivo. We further show the controlled differentiation of hESC clusters in suspension into neural spheres. These results pave the way for large-scale expansion and controlled differentiation of hESCs in suspension, which would be valuable in basic and applied research.
Asunto(s)
Técnicas de Cultivo de Célula/métodos , Células Madre Embrionarias/citología , Células Madre Embrionarias/fisiología , Ingeniería de Tejidos/métodos , Diferenciación Celular , Proliferación Celular , Células Cultivadas , HumanosRESUMEN
Dysfunction and loss of retinal pigment epithelium (RPE) leads to degeneration of photoreceptors in age-related macular degeneration and subtypes of retinitis pigmentosa. Human embryonic stem cells (hESCs) may serve as an unlimited source of RPE cells for transplantation in these blinding conditions. Here we show the directed differentiation of hESCs toward an RPE fate under defined culture conditions. We demonstrate that nicotinamide promotes the differentiation of hESCs to neural and subsequently to RPE fate. In the presence of nicotinamide, factors from the TGF-beta superfamily, which presumably pattern RPE development during embryogenesis, further direct RPE differentiation. The hESC-derived pigmented cells exhibit the morphology, marker expression, and function of authentic RPE and rescue retinal structure and function after transplantation to an animal model of retinal degeneration caused by RPE dysfunction. These results are an important step toward the future use of hESCs to replenish RPE in blinding diseases.
Asunto(s)
Células Madre Embrionarias/citología , Células Epiteliales/citología , Epitelio Pigmentado de la Retina/citología , Receptores de Activinas Tipo I/farmacología , Receptores de Activinas Tipo II/farmacología , Activinas/farmacología , Animales , Diferenciación Celular/efectos de los fármacos , Línea Celular , Trasplante de Células , Células Madre Embrionarias/efectos de los fármacos , Células Madre Embrionarias/ultraestructura , Células Epiteliales/efectos de los fármacos , Células Epiteliales/ultraestructura , Factor 2 de Crecimiento de Fibroblastos/farmacología , Citometría de Flujo , Humanos , Inmunofenotipificación , Microscopía Electrónica de Transmisión , Microscopía de Contraste de Fase , Reacción en Cadena de la Polimerasa , Ratas , Factor de Crecimiento Transformador beta/farmacologíaRESUMEN
Non-muscle myosin II has diverse functions in cell contractility, morphology, cytokinesis and migration. Mammalian cells have three isoforms of non-muscle myosin II, termed IIA, IIB and IIC, encoded by three different genes. These isoforms share considerable homology and some overlapping functions, yet they exhibit differences in enzymatic properties, subcellular localization, molecular interaction and tissue distribution.(1-6) Our studies have focused on the IIA isoform, and they reveal unique regulatory roles in cell-cell adhesion and cell migration that are associated with cross-talk of the actomyosin system with microtubules. In humans, various mutations in the MYH9 gene that encodes the myosin IIA heavy chain cause autosomal dominant disease, whereas in mice, the complete deficiency is embryonic lethal but heterozygous mice are nearly normal. We discuss here the differences between mouse and human phenotypes and how the wealth of mechanistic knowledge about myosin II based on in vitro studies and mouse models can help us understand the molecular and cellular pathophysiology of myosin IIA deficiency in humans.
Asunto(s)
Movimiento Celular , Enfermedades Genéticas Congénitas/metabolismo , Proteínas Motoras Moleculares/metabolismo , Cadenas Pesadas de Miosina/metabolismo , Miosina Tipo IIA no Muscular/metabolismo , Animales , Adhesión Celular/genética , Pérdida del Embrión/genética , Pérdida del Embrión/metabolismo , Enfermedades Genéticas Congénitas/genética , Humanos , Isoenzimas/genética , Isoenzimas/metabolismo , Ratones , Proteínas Motoras Moleculares/genética , Cadenas Pesadas de Miosina/genética , Miosina Tipo IIA no Muscular/genéticaRESUMEN
A key challenge in stem cell research is to learn how to direct the differentiation of stem cells toward specific fates. In this issue of Cell, Engler et al. (2006) identify a new factor regulating stem cell fate: the elasticity of the matrix microenvironment. By changing the stiffness of the substrate, human mesenchymal stem cells could be directed along neuronal, muscle, or bone lineages.
Asunto(s)
Diferenciación Celular/fisiología , Matriz Extracelular , Células Madre Mesenquimatosas/metabolismo , Técnicas de Cultivo de Célula , Linaje de la Célula , Elasticidad , Matriz Extracelular/química , Matriz Extracelular/metabolismo , HumanosRESUMEN
Thymosin beta4 is a 43-amino acid actin-binding protein that promotes cell migration and is important in angiogenesis, wound healing, and tumor metastasis. We searched for genes upregulated by thymosin beta4 and identified zyxin as increased in SiHa cells in the presence of exogenously added thymosin beta4 and when thymosin beta4 is overexpressed using adenoviral vectors. Both zyxin and thymosin beta4 show increased localization in the nucleus. We conclude that thymosin beta4 may exert some of its migration promoting activity via increased zyxin expression.
Asunto(s)
Núcleo Celular/metabolismo , Proteínas del Citoesqueleto/metabolismo , Glicoproteínas/metabolismo , Timosina/metabolismo , Transporte Activo de Núcleo Celular , Línea Celular Tumoral , Proteínas del Citoesqueleto/análisis , Glicoproteínas/análisis , Humanos , Péptidos , Transporte de Proteínas , Timosina/genética , ZixinaRESUMEN
Previous work has shown that ablation or mutation of nonmuscle myosin heavy chain II-B (NMHC II-B) in mice results in defects in the heart and brain with death occurring between embryonic day 14.5 (E14.5) and birth (Tullio, A. N., Accili, D., Ferrans, V. J., Yu, Z. X., Takeda, K., Grinberg, A., Westphal, H., Preston, Y. A., and Adelstein, R. S. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 12407-12412). Here we show that mice ablated for NMHC II-A fail to develop a normal patterned embryo with a polarized visceral endoderm by E6.5 and die by E7.5. Moreover, A(-)/A(-) embryoid bodies grown in suspension culture constantly shed cells. These defects in cell adhesion and tissue organization are explained by loss of E-cadherin and beta-catenin localization to cell adhesion sites in both cell culture and in the intact embryos. The defects can be reproduced by introducing siRNA directed against NMHC II-A into wild-type embryonic stem cells. Our results suggest an essential role for a single, specific nonmuscle myosin isoform in maintaining cell-cell adhesions in the early mammalian embryo.