RESUMEN

Toll-like receptors are essential for animal development and survival, with conserved roles in innate immunity, tissue patterning, and cell behavior. The mechanisms by which Toll receptors signal to the nucleus are well characterized, but how Toll receptors generate rapid, localized signals at the cell membrane to produce acute changes in cell polarity and behavior is not known. We show that Drosophila Toll receptors direct epithelial convergent extension by inducing planar-polarized patterns of Src and PI3-kinase (PI3K) activity. Toll receptors target Src activity to specific sites at the membrane, and Src recruits PI3K to the Toll-2 complex through tyrosine phosphorylation of the Toll-2 cytoplasmic domain. Reducing Src or PI3K activity disrupts planar-polarized myosin assembly, cell intercalation, and convergent extension, whereas constitutive Src activity promotes ectopic PI3K and myosin cortical localization. These results demonstrate that Toll receptors direct cell polarity and behavior by locally mobilizing Src and PI3K activity.

Asunto(s)

Desarrollo Embrionario/genética , Fosfatidilinositol 3-Quinasas/genética , Receptores Toll-Like/genética , Familia-src Quinasas/genética , Actomiosina/metabolismo , Animales , Membrana Celular/genética , Polaridad Celular/genética , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Drosophila melanogaster/crecimiento & desarrollo , Morfogénesis/genética

RESUMEN

Cells display dynamic and diverse morphologies during development, but the strategies by which differentiated tissues achieve precise shapes and patterns are not well understood. Here we identify a developmental program that generates a highly ordered square cell grid in the Drosophila embryo through sequential and spatially regulated cell alignment, oriented cell division, and apicobasal cell elongation. The basic leucine zipper transcriptional regulator Cnc is necessary and sufficient to produce a square cell grid in the presence of a midline signal provided by the EGF receptor ligand Spitz. Spitz orients cell divisions through a Pins/LGN-dependent spindle-positioning mechanism and controls cell shape and alignment through a transcriptional pathway that requires the Pointed ETS domain protein. These results identify a strategy for producing ordered square cell packing configurations in epithelia and reveal a molecular mechanism by which organized tissue structure is generated through spatiotemporally regulated responses to EGF receptor activation.

Asunto(s)

Drosophila melanogaster/genética , Desarrollo Embrionario , Receptores ErbB/genética , Morfogénesis/genética , Animales , División Celular/genética , Polaridad Celular/genética , Forma de la Célula/genética , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/embriología , Factor de Crecimiento Epidérmico/genética , Factor de Crecimiento Epidérmico/metabolismo , Células Epiteliales/citología , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Transducción de Señal/genética

RESUMEN

The embryonic epidermis displays a remarkable ability to repair wounds rapidly. Embryonic wound repair is driven by the evolutionary conserved redistribution of cytoskeletal and junctional proteins around the wound. Drosophila has emerged as a model to screen for factors implicated in wound closure. However, genetic screens have been limited by the use of manual analysis methods. We introduce MEDUSA, a novel image-analysis tool for the automated quantification of multicellular and molecular dynamics from time-lapse confocal microscopy data. We validate MEDUSA by quantifying wound closure in Drosophila embryos, and we show that the results of our automated analysis are comparable to analysis by manual delineation and tracking of the wounds, while significantly reducing the processing time. We demonstrate that MEDUSA can also be applied to the investigation of cellular behaviors in three and four dimensions. Using MEDUSA, we find that the conserved nonreceptor tyrosine kinase Abelson (Abl) contributes to rapid embryonic wound closure. We demonstrate that Abl plays a role in the organization of filamentous actin and the redistribution of the junctional protein ß-catenin at the wound margin during embryonic wound repair. Finally, we discuss different models for the role of Abl in the regulation of actin architecture and adhesion dynamics at the wound margin.

Asunto(s)

Automatización , Drosophila melanogaster/embriología , Procesamiento de Imagen Asistido por Computador , Proteínas Proto-Oncogénicas c-abl/metabolismo , Cicatrización de Heridas , Actinas/metabolismo , Algoritmos , Animales , Rastreo Celular , Drosophila melanogaster/anatomía & histología , Drosophila melanogaster/enzimología , Embrión no Mamífero/citología , Embrión no Mamífero/enzimología , Microscopía Confocal , Modelos Biológicos , Seudópodos/metabolismo , Reproducibilidad de los Resultados , beta Catenina/metabolismo

RESUMEN

Interactions between epithelial cells are mediated by adherens junctions that are dynamically regulated during development. Here we show that the turnover of ß-catenin is increased at cell interfaces that are targeted for disassembly during Drosophila axis elongation. The Abl tyrosine kinase is concentrated at specific planar junctions and is necessary for polarized ß-catenin localization and dynamics. abl mutant embryos have decreased ß-catenin turnover at shrinking edges, and these defects are accompanied by a reduction in multicellular rosette formation and axis elongation. Abl promotes ß-catenin phosphorylation on the conserved tyrosine 667 and expression of an unphosphorylatable ß-catenin mutant recapitulates the defects of abl mutants. Notably, a phosphomimetic ß-catenin(Y667E) mutation is sufficient to increase ß-catenin turnover and rescue axis elongation in abl deficient embryos. These results demonstrate that the asymmetrically localized Abl tyrosine kinase directs planar polarized junctional remodeling during Drosophila axis elongation through the tyrosine phosphorylation of ß-catenin.

Asunto(s)

Proteínas de Drosophila/fisiología , Drosophila melanogaster/genética , Embrión no Mamífero/patología , Proteínas Tirosina Quinasas/fisiología , Tirosina/metabolismo , beta Catenina/metabolismo , Actomiosina/metabolismo , Uniones Adherentes/metabolismo , Animales , Animales Modificados Genéticamente , Western Blotting , Polaridad Celular , Drosophila melanogaster/embriología , Drosophila melanogaster/metabolismo , Embrión no Mamífero/metabolismo , Femenino , Técnicas para Inmunoenzimas , Inmunoprecipitación , Masculino , Morfogénesis , Contracción Muscular , Mutación/genética , Fosforilación , Unión Proteica , ARN Interferente Pequeño/genética , beta Catenina/antagonistas & inhibidores , beta Catenina/genética

RESUMEN

Cell rearrangements shape the Drosophila embryo via spatially regulated changes in cell shape and adhesion. We show that Bazooka/Par-3 (Baz) is required for the planar polarized distribution of myosin II and adherens junction proteins and polarized intercalary behavior is disrupted in baz mutants. The myosin II activator Rho-kinase is asymmetrically enriched at the anterior and posterior borders of intercalating cells in a pattern complementary to Baz. Loss of Rho-kinase results in expansion of the Baz domain, and activated Rho-kinase is sufficient to exclude Baz from the cortex. The planar polarized distribution of Baz requires its C-terminal domain. Rho-kinase can phosphorylate this domain and inhibit its interaction with phosphoinositide membrane lipids, suggesting a mechanism by which Rho-kinase could regulate Baz association with the cell cortex. These results demonstrate that Rho-kinase plays an instructive role in planar polarity by targeting Baz/Par-3 and myosin II to complementary cortical domains.

Asunto(s)

Polaridad Celular , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/embriología , Embrión no Mamífero/fisiología , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Quinasas Asociadas a rho/fisiología , Animales , Animales Modificados Genéticamente , Western Blotting , Tipificación del Cuerpo , Núcleo Celular/genética , Núcleo Celular/metabolismo , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Femenino , Regulación del Desarrollo de la Expresión Génica , Técnicas para Inmunoenzimas , Péptidos y Proteínas de Señalización Intracelular/genética , Masculino , Miosina Tipo II/genética , Miosina Tipo II/metabolismo , Fosforilación , Proteína Quinasa C/genética , Proteína Quinasa C/metabolismo , ARN Mensajero/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transgenes/fisiología

RESUMEN

Epithelial cell-cell interactions require localized adhesive interactions between E-cadherin on opposing membranes and the activation of downstream signaling pathways that affect membrane and actin dynamics. However, it is not known whether E-cadherin engagement and activation of these signaling pathways are locally coordinated or whether signaling is sustained or locally down-regulated like other receptor-mediated pathways. To obtain high spatiotemporal resolution of immediate-early signaling events upon E-cadherin engagement, we used laser tweezers to place beads coated with functional E-cadherin extracellular domain on cells. We show that cellular E-cadherin accumulated rapidly around beads, reaching a sustained plateau level in 1-3 min. Phosphoinositides and Rac1 co-accumulated with E-cadherin, reached peak levels with E-cadherin, but then rapidly dispersed. Both E-cadherin and Rac1 accumulated independently of Rac1 GTP binding/hydrolysis, but these activities were required for Rac1 dispersal. E-cadherin accumulation was dependent on membrane dynamics and actin polymerization, but actin did not stably co-accumulate with E-cadherin; mathematical modeling showed that diffusion-mediated trapping could account for the initial E-cadherin accumulation. We propose that initial E-cadherin accumulation requires active membrane dynamics and involves diffusion-mediated trapping at contact sites; to propagate further contacts, phosphatidylinositol 3-kinase and Rac1 are transiently activated by E-cadherin engagement and initiate a new round of membrane dynamics, but they are subsequently suppressed at that site to allow maintenance of weak E-cadherin mediated adhesion.

Asunto(s)

Cadherinas/metabolismo , Membrana Celular/metabolismo , Células Epiteliales/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Transducción de Señal/fisiología , Proteína de Unión al GTP rac1/metabolismo , Actinas/metabolismo , Animales , Adhesión Celular/fisiología , Línea Celular , Perros , Células Epiteliales/citología , Microesferas , Pinzas Ópticas , Fosfatidilinositoles/metabolismo , Transporte de Proteínas/fisiología

RESUMEN

A multitude of guanine nucleotide exchange factors (GEFs) regulate Rap1 small GTPases, however, their individual functions remain obscure. Here, we investigate the in vivo function of the Rap1 GEF RA-GEF-1. The expression of RA-GEF-1 in wild-type mice starts at embryonic day (E) 8.5, and continues thereafter. RA-GEF-1(-/-) mice appear normal until E7.5, but become grossly abnormal and dead by E9.5. This mid-gestation death appears to be closely associated with severe defects in yolk sac blood vessel formation. RA-GEF-1(-/-) yolk sacs form apparently normal blood islands by E8.5, but the blood islands fail to coalesce into a primary vascular plexus, indicating that vasculogenesis is impaired. Furthermore, RA-GEF-1(-/-) embryos proper show severe defects in the formation of major blood vessels. These results suggest that deficient Rap1 signaling may lead to defective vascular morphogenesis in the yolk sac and embryos proper.

Asunto(s)

Vasos Sanguíneos/anomalías , Vasos Sanguíneos/metabolismo , Pérdida del Embrión/metabolismo , Factores de Intercambio de Guanina Nucleótido/metabolismo , Morfogénesis , Neovascularización Patológica/embriología , Neovascularización Patológica/metabolismo , Animales , Femenino , Silenciador del Gen , Edad Gestacional , Masculino , Ratones , Ratones Endogámicos C57BL

RESUMEN

Actomyosin contraction powers the sealing of epithelial sheets during embryogenesis and wound closure; however, the mechanisms are poorly understood. After laser ablation wounding of Madin-Darby canine kidney cell monolayers, we observed distinct steps in wound closure from time-lapse images of myosin distribution during resealing. Immediately upon wounding, actin and myosin II regulatory light chain accumulated at two locations: (1) in a ring adjacent to the tight junction that circumscribed the wound and (2) in fibers at the base of the cell in membranes extending over the wound site. Rho-kinase activity was required for assembly of the myosin ring, and myosin II activity was required for contraction but not for basal membrane extension. As it contracted, the myosin ring moved toward the basal membrane with ZO-1 and Rho-kinase. Thus, we suggest that tight junctions serve as attachment points for the actomyosin ring during wound closure and that Rho-kinase is required for localization and activation of the contractile ring.

Asunto(s)

Células Epiteliales/citología , Cadenas Ligeras de Miosina/química , Cadenas Ligeras de Miosina/metabolismo , Cicatrización de Heridas/fisiología , Actomiosina/metabolismo , Animales , Adhesión Celular , Polaridad Celular , Forma de la Célula , Pollos , Perros , Células Epiteliales/enzimología , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Estructura Cuaternaria de Proteína , Uniones Estrechas/metabolismo , Quinasas Asociadas a rho

RESUMEN

How physical force is sensed by cells and transduced into cellular signaling pathways is poorly understood. Previously, we showed that tyrosine phosphorylation of p130Cas (Cas) in a cytoskeletal complex is involved in force-dependent activation of the small GTPase Rap1. Here, we mechanically extended bacterially expressed Cas substrate domain protein (CasSD) in vitro and found a remarkable enhancement of phosphorylation by Src family kinases with no apparent change in kinase activity. Using an antibody that recognized extended CasSD in vitro, we observed Cas extension in intact cells in the peripheral regions of spreading cells, where higher traction forces are expected and where phosphorylated Cas was detected, suggesting that the in vitro extension and phosphorylation of CasSD are relevant to physiological force transduction. Thus, we propose that Cas acts as a primary force sensor, transducing force into mechanical extension and thereby priming phosphorylation and activation of downstream signaling.

Asunto(s)

Proteína Sustrato Asociada a CrK/metabolismo , Mecanotransducción Celular , Familia-src Quinasas/metabolismo , Anticuerpos/inmunología , Fenómenos Biomecánicos , Biotinilación , Proteína Sustrato Asociada a CrK/química , Citoesqueleto/metabolismo , Humanos , Modelos Biológicos , Fosforilación , Fosfotirosina/metabolismo , Polietilenglicoles/metabolismo , Estructura Terciaria de Proteína , Proteínas Recombinantes/metabolismo , Proteínas de Unión al GTP rap1/metabolismo

Asunto(s)

Integrinas/fisiología , Transducción de Señal/genética , Familia-src Quinasas/fisiología , Animales , Anoicis/genética , Adhesión Celular/genética , Matriz Extracelular/metabolismo , Humanos , Fosfotransferasas (Aceptor de Grupo Alcohol)/fisiología , Unión Proteica , Transducción de Señal/fisiología

RESUMEN

Cells sense and respond to mechanical force. However, the mechanisms of transduction of extracellular matrix (ECM) forces to biochemical signals are not known. After removing the cell membrane and soluble proteins by Triton X-100 extraction, we found that the remaining complex (Triton cytoskeletons) activated Rap1 upon stretch. Rap1 guanine nucleotide exchange factor, C3G, was required for this activation; C3G as well as the adaptor protein, CrkII, in cell extract bound to Triton cytoskeletons in a stretch-dependent manner. CrkII binding, which was Cas dependent, correlated with stretch-dependent tyrosine phosphorylation of proteins in Triton cytoskeletons including Cas at the contacts with ECM. These in vitro findings were compatible with in vivo observations of stretch-enhanced phosphotyrosine signals, accumulation of CrkII at cell-ECM contacts, and CrkII-Cas colocalization. We suggest that mechanical force on Triton cytoskeletons activates local tyrosine phosphorylation, which provides docking sites for cytosolic proteins, and initiates signaling to activate Rap1.

Asunto(s)

Proteínas del Citoesqueleto/metabolismo , Citoesqueleto/metabolismo , Transducción de Señal , Actinas/metabolismo , Animales , Línea Celular , Colágeno Tipo I/metabolismo , Proteínas del Citoesqueleto/fisiología , Citoesqueleto/efectos de los fármacos , Citoesqueleto/fisiología , Detergentes/farmacología , Técnica del Anticuerpo Fluorescente , Colorantes Fluorescentes , Factor 2 Liberador de Guanina Nucleótido/metabolismo , Células HeLa , Humanos , Hidrazinas , Células L , Membranas Artificiales , Ratones , Microscopía Confocal , Modelos Biológicos , Octoxinol/farmacología , Faloidina , Fosforilación , Conformación Proteica/efectos de los fármacos , Desnaturalización Proteica/efectos de los fármacos , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-crk , Silicio/química , Tirosina/metabolismo

RESUMEN

The suprachiasmatic nucleus (SCN) is the neuroanatomical locus of the mammalian circadian pacemaker. Here we demonstrate that an abrupt shift in the light/dark (LD) cycle disrupts the synchronous oscillation of circadian components in the rat SCN. The phases of the RNA cycles of the period genes Per1 and Per2 and the cryptochrome gene Cry1 shifted rapidly in the ventrolateral, photoreceptive region of the SCN, but were relatively slow to shift in the dorsomedial region. During the period of desynchrony, the animals displayed increased nighttime rest, the timing of which was inversely correlated with the expression of Per1 mRNA in the dorsomedial SCN. Molecular resynchrony required approximately 6 d after a 10 hr delay and 9 approximately 13 d after a 6 hr advance of the LD cycle and was accompanied by the reemergence of normal rest-activity patterns. This dissociation and slow resynchronization of endogenous oscillators within the SCN after an LD cycle shift suggests a mechanism for the physiological symptoms that constitute jet lag.

Asunto(s)

Trastornos Cronobiológicos/etiología , Ritmo Circadiano , Proteínas de Drosophila , Proteínas del Ojo , Fotoperiodo , Células Fotorreceptoras de Invertebrados , Animales , Conducta Animal/fisiología , Relojes Biológicos , Proteínas de Ciclo Celular , Ritmo Circadiano/fisiología , Criptocromos , Flavoproteínas/genética , Flavoproteínas/metabolismo , Masculino , Actividad Motora/fisiología , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteínas Circadianas Period , Estimulación Luminosa/métodos , Proteínas Proto-Oncogénicas c-fos/genética , ARN Mensajero/metabolismo , Ratas , Ratas Wistar , Receptores Acoplados a Proteínas G , Núcleo Supraquiasmático/metabolismo , Núcleo Supraquiasmático/fisiología , Factores de Tiempo , Factores de Transcripción