RESUMO
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.
Assuntos
Actomiosina/metabolismo , Movimento Celular/fisiologia , Microtúbulos/metabolismo , Miosina não Muscular Tipo IIA/fisiologia , Aminoquinolinas/farmacologia , Animais , Azepinas/farmacologia , Células COS , Adesão Celular/fisiologia , Movimento Celular/efeitos dos fármacos , Chlorocebus aethiops , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/efeitos dos fármacos , Células-Tronco Embrionárias/metabolismo , Inibidores Enzimáticos/farmacologia , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Fatores de Troca do Nucleotídeo Guanina/genética , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Compostos Heterocíclicos de 4 ou mais Anéis/farmacologia , Humanos , Cinesinas/antagonistas & inibidores , Cinesinas/genética , Cinesinas/metabolismo , Camundongos , Microtúbulos/efeitos dos fármacos , Naftalenos/farmacologia , Nocodazol/farmacologia , Miosina não Muscular Tipo IIA/antagonistas & inibidores , Miosina não Muscular Tipo IIA/genética , Miosina não Muscular Tipo IIB/antagonistas & inibidores , Miosina não Muscular Tipo IIB/genética , Miosina não Muscular Tipo IIB/fisiologia , Pirimidinas/farmacologia , RNA Interferente Pequeno/genética , Proteína 1 Indutora de Invasão e Metástase de Linfoma de Células T , Tionas/farmacologia , Transfecção , Vimblastina/farmacologia , Proteínas rac1 de Ligação ao GTP/antagonistas & inibidores , Proteínas rac1 de Ligação ao GTP/genética , Proteínas rac1 de Ligação ao GTP/metabolismoRESUMO
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.
Assuntos
Movimento Celular , Fibroblastos/fisiologia , Monócitos/fisiologia , Fator 88 de Diferenciação Mieloide/fisiologia , Estresse Oxidativo/fisiologia , Receptores Depuradores/fisiologia , Aldeído Desidrogenase , Animais , Fibroblastos/metabolismo , Humanos , Receptores de Lipopolissacarídeos , Macrófagos , Camundongos , Fator 88 de Diferenciação Mieloide/metabolismo , Receptores Depuradores/metabolismo , Transdução de SinaisRESUMO
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.
Assuntos
Movimento Celular/fisiologia , Matriz Extracelular/fisiologia , Modelos Biológicos , Animais , Membrana Basal/metabolismo , Membrana Basal/fisiologia , Matriz Extracelular/metabolismo , Humanos , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Proteínas Monoméricas de Ligação ao GTP/fisiologia , Transdução de Sinais/fisiologiaRESUMO
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.
Assuntos
Arginase/fisiologia , Citocinas/antagonistas & inibidores , Células-Tronco Embrionárias/enzimologia , Células-Tronco Embrionárias/imunologia , Ativação Linfocitária/imunologia , Subpopulações de Linfócitos T/enzimologia , Subpopulações de Linfócitos T/imunologia , Adulto , Animais , Arginina/metabolismo , Complexo CD3/imunologia , Complexo CD3/metabolismo , Linhagem Celular , Movimento Celular/genética , Movimento Celular/imunologia , Células Cultivadas , Técnicas de Cocultura , Citocinas/metabolismo , Células-Tronco Embrionárias/transplante , Regulação Enzimológica da Expressão Gênica/imunologia , Humanos , Leucócitos Mononucleares/citologia , Leucócitos Mononucleares/enzimologia , Leucócitos Mononucleares/imunologia , Camundongos , Receptores de Antígenos de Linfócitos T/antagonistas & inibidores , Receptores de Antígenos de Linfócitos T/biossíntese , Subpopulações de Linfócitos T/citologia , Teratoma/enzimologia , Teratoma/imunologia , Teratoma/patologiaRESUMO
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.
Assuntos
Camadas Germinativas , Células-Tronco Pluripotentes , Animais , Humanos , Células-Tronco Pluripotentes/metabolismo , Embrião de Mamíferos , Linha Primitiva , Diferenciação Celular , Via de Sinalização Wnt , MamíferosRESUMO
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.
Assuntos
Movimento Celular/fisiologia , Proteínas rac de Ligação ao GTP/metabolismo , Técnicas de Cultura de Células , Extensões da Superfície Celular/metabolismo , Células Cultivadas , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Fibroblastos/citologia , Fibroblastos/metabolismo , Fibronectinas/metabolismo , Guanosina Trifosfato/metabolismo , Humanos , Integrinas/genética , Integrinas/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Distribuição Aleatória , Transdução de Sinais/fisiologia , Proteína cdc42 de Ligação ao GTP/genética , Proteína cdc42 de Ligação ao GTP/metabolismo , Proteínas rac de Ligação ao GTP/genética , Proteínas rho de Ligação ao GTP/genética , Proteínas rho de Ligação ao GTP/metabolismoRESUMO
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.
Assuntos
Fibrina/química , Géis , Modelos Biológicos , Humanos , Microscopia de Força AtômicaRESUMO
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.
Assuntos
Interferência de RNA/fisiologia , Proteínas rho de Ligação ao GTP/antagonistas & inibidores , Linhagem Celular , DNA Complementar/genética , Humanos , RNA Interferente Pequeno/fisiologia , Transfecção/métodos , Proteínas rho de Ligação ao GTP/genéticaAssuntos
Integrinas/metabolismo , Proteínas , Receptores de Fatores de Crescimento/metabolismo , Animais , Fibronectinas/metabolismo , Quinase 1 de Adesão Focal , Proteína-Tirosina Quinases de Adesão Focal , Humanos , Ligantes , Sistema de Sinalização das MAP Quinases , Modelos Biológicos , Fosfoproteínas/metabolismo , Fosforilação , Proteínas Tirosina Quinases/metabolismo , Proteína p130 Retinoblastoma-Like , Transdução de Sinais , Tirosina/metabolismoRESUMO
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.
Assuntos
Transformação Celular Neoplásica , Neoplasias/irrigação sanguínea , Neovascularização Patológica/fisiopatologia , Receptores de Trombina/fisiologia , Células 3T3 , Animais , Northern Blotting , Fatores de Crescimento Endotelial/genética , Fatores de Crescimento Endotelial/metabolismo , Expressão Gênica , Genes ras/genética , Genes src/genética , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/genética , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Linfocinas/genética , Linfocinas/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Transplante de Neoplasias , Neoplasias/genética , Neoplasias Experimentais/genética , Neoplasias Experimentais/patologia , Oncogenes/genética , Fosfatidilinositol 3-Quinases/metabolismo , Proteína Quinase C/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Receptor PAR-1 , Receptores de Trombina/genética , Transfecção , Transplante Heterólogo , Células Tumorais Cultivadas , Fator A de Crescimento do Endotélio Vascular , Fatores de Crescimento do Endotélio Vascular , Quinases da Família src/metabolismoRESUMO
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.
Assuntos
Células Epiteliais/metabolismo , Invasividade Neoplásica/fisiopatologia , Metástase Neoplásica/fisiopatologia , Receptor PAR-1/fisiologia , Animais , Mama/irrigação sanguínea , Mama/crescimento & desenvolvimento , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Divisão Celular , Citoesqueleto/ultraestrutura , Células Epiteliais/patologia , Feminino , Humanos , Integrinas/fisiologia , Camundongos , Camundongos Knockout , Morfogênese , Neovascularização Patológica/fisiopatologia , Oligonucleotídeos Antissenso/farmacologia , Placenta/irrigação sanguínea , Gravidez , Receptores de Vitronectina/fisiologia , Trombina/fisiologia , Fator A de Crescimento do Endotélio Vascular/biossíntese , Fator A de Crescimento do Endotélio Vascular/genéticaRESUMO
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.
Assuntos
Dano ao DNA/imunologia , Macrófagos/imunologia , Animais , Células Cultivadas , Quebras de DNA de Cadeia Dupla , Reparo do DNA por Junção de Extremidades/imunologia , Receptores ErbB/imunologia , Receptores ErbB/farmacologia , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/imunologia , Fator de Crescimento Semelhante a EGF de Ligação à Heparina/imunologia , Fator de Crescimento Semelhante a EGF de Ligação à Heparina/farmacologia , Humanos , Fígado/efeitos dos fármacos , Fígado/imunologia , Fígado/fisiologia , Macrófagos/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Monócitos/efeitos dos fármacos , Monócitos/imunologia , Oxirredução , Transdução de Sinais/imunologiaRESUMO
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.
Assuntos
Proteínas de Transporte/metabolismo , Regulação para Baixo , Disautonomia Familiar/genética , Células-Tronco Embrionárias Humanas/patologia , Neurônios/metabolismo , Sistema Nervoso Periférico/patologia , Vesículas Sinápticas/metabolismo , Transporte Biológico/efeitos dos fármacos , Transporte Biológico/genética , Proteínas de Transporte/genética , Diferenciação Celular/efeitos dos fármacos , Regulação para Baixo/efeitos dos fármacos , Disautonomia Familiar/metabolismo , Disautonomia Familiar/patologia , Feto , Células-Tronco Embrionárias Humanas/efeitos dos fármacos , Humanos , Cinetina/farmacologia , Masculino , Mutação , Crista Neural/efeitos dos fármacos , Crista Neural/patologia , Neurônios/efeitos dos fármacos , Sistema Nervoso Periférico/efeitos dos fármacos , Fenótipo , Vesículas Sinápticas/efeitos dos fármacos , Fatores de Elongação da TranscriçãoRESUMO
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.
Assuntos
Transformação Celular Neoplásica/genética , Células-Tronco Embrionárias , Células-Tronco Pluripotentes , RNA Longo não Codificante/genética , Animais , Western Blotting , Linhagem Celular Tumoral , Técnicas de Silenciamento de Genes , Xenoenxertos , Humanos , Imuno-Histoquímica , Camundongos , Camundongos SCID , Microscopia de Fluorescência , Reação em Cadeia da Polimerase , RNA Interferente Pequeno , TransfecçãoRESUMO
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.
Assuntos
Técnicas de Cultura de Células/métodos , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/fisiologia , Engenharia Tecidual/métodos , Diferenciação Celular , Proliferação de Células , Células Cultivadas , HumanosRESUMO
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.
Assuntos
Células-Tronco Embrionárias/citologia , Células Epiteliais/citologia , Epitélio Pigmentado da Retina/citologia , Receptores de Ativinas Tipo I/farmacologia , Receptores de Activinas Tipo II/farmacologia , Ativinas/farmacologia , Animais , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Transplante de Células , Células-Tronco Embrionárias/efeitos dos fármacos , Células-Tronco Embrionárias/ultraestrutura , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/ultraestrutura , Fator 2 de Crescimento de Fibroblastos/farmacologia , Citometria de Fluxo , Humanos , Imunofenotipagem , Microscopia Eletrônica de Transmissão , Microscopia de Contraste de Fase , Reação em Cadeia da Polimerase , Ratos , Fator de Crescimento Transformador beta/farmacologiaRESUMO
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.
Assuntos
Movimento Celular , Doenças Genéticas Inatas/metabolismo , Proteínas Motores Moleculares/metabolismo , Cadeias Pesadas de Miosina/metabolismo , Miosina não Muscular Tipo IIA/metabolismo , Animais , Adesão Celular/genética , Perda do Embrião/genética , Perda do Embrião/metabolismo , Doenças Genéticas Inatas/genética , Humanos , Isoenzimas/genética , Isoenzimas/metabolismo , Camundongos , Proteínas Motores Moleculares/genética , Cadeias Pesadas de Miosina/genética , Miosina não Muscular Tipo IIA/genéticaRESUMO
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.
Assuntos
Diferenciação Celular/fisiologia , Matriz Extracelular , Células-Tronco Mesenquimais/metabolismo , Técnicas de Cultura de Células , Linhagem da Célula , Elasticidade , Matriz Extracelular/química , Matriz Extracelular/metabolismo , HumanosRESUMO
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.
Assuntos
Núcleo Celular/metabolismo , Proteínas do Citoesqueleto/metabolismo , Glicoproteínas/metabolismo , Timosina/metabolismo , Transporte Ativo do Núcleo Celular , Linhagem Celular Tumoral , Proteínas do Citoesqueleto/análise , Glicoproteínas/análise , Humanos , Peptídeos , Transporte Proteico , Timosina/genética , ZixinaRESUMO
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.