RESUMEN
Directional motility assays make use of Boyden chambers or Transwell culture inserts with porous membranes that separate cells seeded in the upper chamber from a chemoattractant supplied in a lower chamber. These assays are often time-consuming and are associated with several limitations due to manual counting and inconsistent results; low signal-to-noise ratio and fluorescence interference; and high cost and the need for specific equipment. Here, we describe a simple, direct, and easy ATP luminescence-based motility assay (ALMA), which can be used for 96-well plate quantification.
Asunto(s)
Adenosina Trifosfato/metabolismo , Técnicas de Cultivo de Célula/instrumentación , Técnicas de Cultivo de Célula/métodos , Ensayos de Migración Celular , Luminiscencia , Neoplasias de la Mama/patología , Línea Celular Tumoral , Medio de Cultivo Libre de Suero , Femenino , Humanos , Sensibilidad y EspecificidadRESUMEN
BACKGROUND INFORMATION: The appropriate regulation of cell-cell adhesion is an important event in the homoeostasis of different cell types. In epithelial cells, tight adhesion mediated by E-cadherin receptors is essential for the differentiation and functionality of epithelial sheets. Upon assembly of cadherin-mediated cell-cell contacts, it is well established that the small GTPases Rho and Rac are activated and are necessary for junction stability. However, the role of the small GTPase Cdc42 in cadherin adhesion is less clear. Cdc42 can be activated by E-cadherin in a breast tumour cell line, but the requirement for Cdc42 function for new junction assembly or maintenance has been contradictory. Cdc42 participation in cell-cell contacts has been inferred from the presence of filopodia, the typical F-actin structure induced by Cdc42 activation, as cells approach each other to establish cell-cell contacts. Yet, under these conditions, the contribution of migration to filopodia protrusion cannot be excluded and the results are difficult to interpret. RESULTS: In the present study, we set out to address (a) whether Cdc42 is activated by new E-cadherin cell-cell contacts when junction assembly occurs without prior migration and (b) whether Cdc42 function is necessary for cadherin stability. We found that junction formation in confluent keratinocytes or upon E-cadherin clustering decreased Cdc42-GTP levels. In the absence of serum- and migration-induced Cdc42 activation, we demonstrated that cell-cell contacts do not induce filopodia or require Cdc42 function to assemble. CONCLUSION: We conclude that Cdc42 does not participate in the early events that initiate stable cadherin adhesion in keratinocytes. Yet, it is feasible that Cdc42 may be activated at later time points or by other receptors. Cdc42 can then participate in additional functions during polarization, such as Golgi re-positioning or basolateral trafficking.
Asunto(s)
Cadherinas/metabolismo , Uniones Intercelulares/metabolismo , Queratinocitos/metabolismo , Seudópodos/metabolismo , Proteína de Unión al GTP cdc42/metabolismo , Animales , Adhesión Celular/fisiología , Línea Celular , Células Cultivadas , Técnica del Anticuerpo Fluorescente , Humanos , Ratones , Transducción de SeñalRESUMEN
The mechanisms underlying functional interactions between ERM (ezrin, radixin, moesin) proteins and Rho GTPases are not well understood. Here we characterized the interaction between ezrin and a novel Rho guanine nucleotide exchange factor, PLEKHG6. We show that ezrin recruits PLEKHG6 to the apical pole of epithelial cells where PLEKHG6 induces the formation of microvilli and membrane ruffles. These morphological changes are inhibited by dominant negative forms of RhoG. Indeed, we found that PLEKHG6 activates RhoG and to a much lesser extent Rac1. In addition we show that ezrin forms a complex with PLEKHG6 and RhoG. Furthermore, we detected a ternary complex between ezrin, PLEKHG6, and the RhoG effector ELMO. We demonstrate that PLEKHG6 and ezrin are both required in macropinocytosis. After down-regulation of either PLEKHG6 or ezrin expression, we observed an inhibition of dextran uptake in EGF-stimulated A431 cells. Altogether, our data indicate that ezrin allows the local activation of RhoG at the apical pole of epithelial cells by recruiting upstream and downstream regulators of RhoG and that both PLEKHG6 and ezrin are required for efficient macropinocytosis.
Asunto(s)
Proteínas del Citoesqueleto/metabolismo , Citoesqueleto/metabolismo , Células Epiteliales/metabolismo , Factores de Intercambio de Guanina Nucleótido/metabolismo , Proteínas de Unión al GTP rho/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Secuencia de Aminoácidos , Animales , Línea Celular , Secuencia Conservada , Proteínas del Citoesqueleto/genética , Factores de Intercambio de Guanina Nucleótido/química , Factores de Intercambio de Guanina Nucleótido/genética , Humanos , Ratones , Datos de Secuencia Molecular , Unión Proteica , Alineación de Secuencia , Proteínas de Unión al GTP rho/genéticaRESUMEN
Gem is a protein of the Ras superfamily that plays a role in regulating voltage-gated Ca2+ channels and cytoskeletal reorganization. We now report that GTP-bound Gem interacts with the membrane-cytoskeleton linker protein Ezrin in its active state, and that Gem binds to active Ezrin in cells. The coexpression of Gem and Ezrin induces cell elongation accompanied by the disappearance of actin stress fibers and collapse of most focal adhesions. The same morphological effect is elicited when cells expressing Gem alone are stimulated with serum and requires the expression of ERM proteins. We show that endogenous Gem down-regulates the level of active RhoA and actin stress fibers. The effects of Gem downstream of Rho, i.e., ERM phosphorylation as well as disappearance of actin stress fibers and most focal adhesions, require the Rho-GAP partner of Gem, Gmip, a protein that is enriched in membranes under conditions in which Gem induced cell elongation. Our results suggest that Gem binds active Ezrin at the plasma membrane-cytoskeleton interface and acts via the Rho-GAP protein Gmip to down-regulate the processes dependent on the Rho pathway.
Asunto(s)
Proteínas del Citoesqueleto/metabolismo , Proteínas Activadoras de GTPasa/metabolismo , Proteínas de Unión al GTP Monoméricas/metabolismo , Proteínas de Unión al GTP rho/metabolismo , Células 3T3 , Actinas/metabolismo , Actinas/ultraestructura , Animales , Adhesión Celular , Membrana Celular/metabolismo , Tamaño de la Célula , Proteínas del Citoesqueleto/genética , Citoesqueleto/metabolismo , Regulación hacia Abajo , Proteínas Activadoras de GTPasa/genética , Células HeLa , Humanos , Proteínas Inmediatas-Precoces/genética , Proteínas Inmediatas-Precoces/metabolismo , Ratones , Proteínas de Unión al GTP Monoméricas/genética , Transducción de Señal , Técnicas del Sistema de Dos Híbridos , Levaduras/genéticaRESUMEN
Gene silencing by RNA interference (RNAi) has proven to be a powerful tool for investigating gene function in mammalian cells. Combination of several short interfering RNA (siRNA) targeting the same gene is commonly used to improve RNA interference. However, in contrary to the well-described mechanism of RNAi, efficiency of single siRNA compared to pool remains poorly documented. We addressed this issue using several active and inactive siRNA targeting Eg5, a kinesin-related motor involved in mitotic spindle assembly. These siRNA, used alone or in combination, were tested for their silencing efficiency in several cancer cell lines. Here we show that presence of inactive Eg5 siRNA in a pool dramatically decreases knockdown efficacy in a cell line- and dose-dependent manner. Lack of inhibition by unrelated siRNA suggests that a competition may occur during siRNA incorporation into RNA-induced silencing complexes (RISCs) along with the target mRNA. Altogether, our results, which need to be confirmed with additional inactive siRNA, indicate that combination of siRNA may not increase but instead decrease silencing efficiency.
Asunto(s)
Cinesinas/antagonistas & inhibidores , Cinesinas/genética , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/farmacología , Secuencia de Bases , Línea Celular Tumoral , ADN Complementario/genética , Humanos , Mitosis/efectos de los fármacos , Mitosis/genética , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , TransfecciónRESUMEN
Ral GTPase activity is a crucial cell-autonomous factor supporting tumor initiation and progression. To decipher pathways impacted by Ral, we have generated null and hypomorph alleles of the Drosophila melanogaster Ral gene. Ral null animals were not viable. Reduced Ral expression in cells of the sensory organ lineage had no effect on cell division but led to postmitotic cell-specific apoptosis. Genetic epistasis and immunofluorescence in differentiating sensory organs suggested that Ral activity suppresses c-Jun N-terminal kinase (JNK) activation and induces p38 mitogen-activated protein (MAP) kinase activation. HPK1/GCK-like kinase (HGK), a MAP kinase kinase kinase kinase that can drive JNK activation, was found as an exocyst-associated protein in vivo. The exocyst is a Ral effector, and the epistasis between mutants of Ral and of msn, the fly ortholog of HGK, suggest the functional relevance of an exocyst/HGK interaction. Genetic analysis also showed that the exocyst is required for the execution of Ral function in apoptosis. We conclude that in Drosophila Ral counters apoptotic programs to support cell fate determination by acting as a negative regulator of JNK activity and a positive activator of p38 MAP kinase. We propose that the exocyst complex is Ral executioner in the JNK pathway and that a cascade from Ral to the exocyst to HGK would be a molecular basis of Ral action on JNK.
Asunto(s)
Apoptosis , Drosophila melanogaster/citología , Drosophila melanogaster/embriología , Proteínas de Unión al GTP/metabolismo , Proteína Quinasa 9 Activada por Mitógenos/metabolismo , Alelos , Animales , Diferenciación Celular , Linaje de la Célula , Drosophila melanogaster/enzimología , Drosophila melanogaster/genética , Embrión no Mamífero , Activación Enzimática , Epistasis Genética , Proteínas de Unión al GTP/genética , Eliminación de Gen , Regulación del Desarrollo de la Expresión Génica , Genes Esenciales , Genes de Insecto , Inmunohistoquímica , MAP Quinasa Quinasa 4/metabolismo , Microscopía por Video , Proteínas Serina-Treonina Quinasas/metabolismo , Órganos de los Sentidos/embriología , Órganos de los Sentidos/crecimiento & desarrollo , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
The E-Cadherin-catenin complex plays a critical role in epithelial cell-cell adhesion, polarization, and morphogenesis. Here, we have analyzed the mechanism of Drosophila E-Cadherin (DE-Cad) localization. Loss of function of the Drosophila exocyst components sec5, sec6, and sec15 in epithelial cells results in DE-Cad accumulation in an enlarged Rab11 recycling endosomal compartment and inhibits DE-Cad delivery to the membrane. Furthermore, Rab11 and Armadillo interact with the exocyst components Sec15 and Sec10, respectively. Our results support a model whereby the exocyst regulates DE-Cadherin trafficking, from recycling endosomes to sites on the epithelial cell membrane where Armadillo is located.
Asunto(s)
Cadherinas/metabolismo , Membrana Celular/metabolismo , Proteínas de Drosophila/metabolismo , Endosomas/metabolismo , Proteínas de la Membrana/metabolismo , Oocitos/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Animales , Proteínas del Dominio Armadillo , Proteínas Portadoras/metabolismo , Drosophila/metabolismo , Drosophila/ultraestructura , Proteínas de Drosophila/genética , Células Epiteliales/metabolismo , Exocitosis/fisiología , Proteínas de la Membrana/genética , Proteínas de Transporte de Membrana , Modelos Biológicos , Oocitos/ultraestructura , Transporte de Proteínas/fisiología , Transactivadores/metabolismo , Factores de Transcripción , Proteínas de Transporte Vesicular/genéticaRESUMEN
The Drosophila (fruit fly) model system has been instrumental in our current understanding of human biology, development, and diseases. Here, we used a high-throughput yeast two-hybrid (Y2H)-based technology to screen 102 bait proteins from Drosophila melanogaster, most of them orthologous to human cancer-related and/or signaling proteins, against high-complexity fly cDNA libraries. More than 2300 protein-protein interactions (PPI) were identified, of which 710 are of high confidence. The computation of a reliability score for each protein-protein interaction and the systematic identification of the interacting domain combined with a prediction of structural/functional motifs allow the elaboration of known complexes and the identification of new ones. The full data set can be visualized using a graphical Web interface, the PIMRider (http://pim.hybrigenics.com), and is also accessible in the PSI standard Molecular Interaction data format. Our fly Protein Interaction Map (PIM) is surprisingly different from the one recently proposed by Giot et al. with little overlap between the two data sets. Analysis of the differences in data sets and methods suggests alternative strategies to enhance the accuracy and comprehensiveness of the post-genomic generation of broad-scale protein interaction maps.
Asunto(s)
Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Animales , Secuencia de Bases , ADN Complementario/genética , Proteínas de Drosophila/química , Biblioteca de Genes , Genes de Insecto , Genes ras , Humanos , Unión Proteica , Estructura Terciaria de Proteína , Especificidad de la Especie , Técnicas del Sistema de Dos HíbridosRESUMEN
Gem is a Ras-related protein whose expression is induced in several cell types upon activation by extracellular stimuli. With the aim of isolating the cellular partners of Gem that mediate its biological activity we performed a yeast two-hybrid screen and identified a novel protein of 970 amino acids, Gmip, that interacts with Gem through its N-terminal half, and presents a cysteine-rich domain followed by a Rho GTPase-activating protein (RhoGAP) domain in its C-terminal half. The RhoGAP domain of Gmip stimulates in vitro the GTPase activity of RhoA, but is inactive towards other Rho family proteins such as Rac1 and Cdc42; it is also specific for RhoA in vivo. The same is true for the full-length protein, which is furthermore able to down-regulate RhoA-dependent stress fibres in Ref-52 rat fibroblasts. These findings suggest that the signalling pathways controlled by two proteins of the Ras superfamily, RhoA and Gem, are linked via the action of the RhoGAP protein Gmip (Gem-interacting protein).
Asunto(s)
Proteínas Activadoras de GTPasa/metabolismo , Proteínas Activadoras de GTPasa/fisiología , Proteínas Inmediatas-Precoces/química , Proteínas Inmediatas-Precoces/metabolismo , Proteínas Inmediatas-Precoces/fisiología , Proteínas de Unión al GTP Monoméricas/química , Proteínas de Unión al GTP Monoméricas/metabolismo , Proteínas de Unión al GTP Monoméricas/fisiología , Proteínas de Unión al GTP rho/química , Actinas/metabolismo , Secuencia de Aminoácidos , Animales , Northern Blotting , Línea Celular , Clonación Molecular , Citoesqueleto/metabolismo , ADN Complementario/metabolismo , Fibroblastos/metabolismo , Biblioteca de Genes , Guanosina Trifosfato/metabolismo , Humanos , Microscopía Fluorescente , Datos de Secuencia Molecular , Pruebas de Precipitina , Unión Proteica , Proteínas Serina-Treonina Quinasas/metabolismo , Estructura Terciaria de Proteína , Proteínas Proto-Oncogénicas c-akt , Ratas , Proteínas Recombinantes/química , Homología de Secuencia de Aminoácido , Factores de Tiempo , Distribución Tisular , Técnicas del Sistema de Dos Híbridos , Proteína de Unión al GTP cdc42/metabolismo , Proteínas de Unión al GTP rho/metabolismo , Proteína de Unión al GTP rhoA/química , Proteína de Unión al GTP rhoA/metabolismoRESUMEN
TAL-1 is a basic helix-loop-helix oncoprotein that is expressed in up to 30% of T-cell acute lymphoblastic leukemias but not in the T lineage. We have cloned a complementary DNA, called Human Immune Associated Nucleotide 1 (hIAN1), whose messenger RNA (mRNA) level expression is inversely correlated to the TAL-1 mRNA level in human leukemic T-cell lines. The hIAN1 encodes a 38-kd protein that belongs to a novel family of proteins conserved from plants to humans and characterized by motifs related to, but highly divergent from, the consensus motifs found in guanosine triphosphate (GTP)-binding proteins. Despite these divergent amino acids at positions involved in GTP/guanosine diphosphate (GDP) binding and guanosine triphosphatase (GTPase) activities, we found that hIAN1 specifically binds GDP (K(d) = 0.47 microM) and GTP (K(d) = 6 microM) and exhibits intrinsic GTPase activity. Among mature hematopoietic cells, hIAN1 is specifically expressed in resting T and B lymphocytes, and its expression level tremendously decreased at the protein but not the mRNA level during B- or T-lymphocyte activation, suggesting a specific role for this new type of GTPase during the immune response.