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1.
Dev Biol ; 373(2): 267-80, 2013 Jan 15.
Artículo en Inglés | MEDLINE | ID: mdl-23195221

RESUMEN

During lung development, proper epithelial cell arrangements are critical for the formation of an arborized network of tubes. Each tube requires a lumen, the diameter of which must be tightly regulated to enable optimal lung function. Lung branching and lumen morphogenesis require close epithelial cell-cell contacts that are maintained as a result of adherens junctions, tight junctions and by intact apical-basal (A/B) polarity. However, the molecular mechanisms that maintain epithelial cohesion and lumen diameter in the mammalian lung are unknown. Here we show that Scribble, a protein implicated in planar cell polarity (PCP) signalling, is necessary for normal lung morphogenesis. Lungs of the Scrib mouse mutant Circletail (Crc) are abnormally shaped with fewer airways, and these airways often lack a visible, 'open' lumen. Mechanistically we show that Scrib genetically interacts with the core PCP gene Vangl2 in the developing lung and that the distribution of PCP pathway proteins and Rho mediated cytoskeletal modification is perturbed in Scrib(Crc/Crc) lungs. However A/B polarity, which is disrupted in Drosophila Scrib mutants, is largely unaffected. Notably, we find that Scrib mediates functions not attributed to other PCP proteins in the lung. Specifically, Scrib localises to both adherens and tight junctions of lung epithelia and knockdown of Scrib in lung explants and organotypic cultures leads to reduced cohesion of lung epithelial cells. Live imaging of Scrib knockdown lungs shows that Scrib does not affect bud bifurcation, as previously shown for the PCP protein Celsr1, but is required to maintain epithelial cohesion. To understand the mechanism leading to reduced cell-cell association, we show that Scrib associates with ß-catenin in embryonic lung and the sub-cellular distribution of adherens and tight junction proteins is perturbed in mutant lung epithelia. Our data reveal that Scrib is required for normal lung epithelial organisation and lumen morphogenesis by maintaining cell-cell contacts. Thus we reveal novel and important roles for Scrib in lung development operating via the PCP pathway, and in regulating junctional complexes and cell cohesion.


Asunto(s)
Comunicación Celular , Células Epiteliales/citología , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Pulmón/citología , Pulmón/embriología , Mamíferos/embriología , Morfogénesis , Uniones Adherentes/efectos de los fármacos , Uniones Adherentes/metabolismo , Animales , Comunicación Celular/efectos de los fármacos , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Polaridad Celular/efectos de los fármacos , Citoesqueleto/efectos de los fármacos , Citoesqueleto/metabolismo , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Epitelio/efectos de los fármacos , Epitelio/embriología , Epitelio/metabolismo , Técnicas de Silenciamiento del Gen , Imagenología Tridimensional , Pulmón/efectos de los fármacos , Pulmón/metabolismo , Ratones , Modelos Biológicos , Morfogénesis/efectos de los fármacos , Morfolinos/farmacología , Proteínas del Tejido Nervioso/metabolismo , Unión Proteica/efectos de los fármacos , Transporte de Proteínas/efectos de los fármacos , Receptores Acoplados a Proteínas G/metabolismo , Uniones Estrechas/efectos de los fármacos , Uniones Estrechas/metabolismo , Proteína de la Zonula Occludens-2/metabolismo , beta Catenina/metabolismo , Proteína de Unión al GTP rhoA/metabolismo
2.
Dev Dyn ; 242(6): 622-37, 2013 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-23483685

RESUMEN

BACKGROUND: Lung-branching morphogenesis is a fundamental developmental process, yet the cellular dynamics that occur during lung development and the molecular mechanisms underlying recent postulated branching modes are poorly understood. RESULTS: Here, we implemented a time-lapse video microscopy method to study the cellular behavior and molecular mechanisms of planar bifurcation and domain branching in lung explant- and organotypic cultures. Our analysis revealed morphologically distinct stages that are shaped at least in part by a combination of localized and orientated cell divisions and by local mechanical forces. We also identified myosin light-chain kinase as an important regulator of bud bifurcation, but not domain branching in lung explants. CONCLUSIONS: This live imaging approach provides a method to study cellular behavior during lung-branching morphogenesis and suggests the importance of a mechanism primarily based on oriented cell proliferation and mechanical forces in forming and shaping the developing lung airways.


Asunto(s)
Regulación del Desarrollo de la Expresión Génica , Pulmón/embriología , Microscopía por Video , Morfogénesis , Animales , División Celular , Proliferación Celular , Epitelio/embriología , Proteínas Fluorescentes Verdes/metabolismo , Procesamiento de Imagen Asistido por Computador , Ratones , Ratones Transgénicos , Microscopía Fluorescente , Quinasa de Cadena Ligera de Miosina/metabolismo , Regiones Promotoras Genéticas , Estrés Mecánico , Factores de Tiempo
3.
Hum Mol Genet ; 19(11): 2251-67, 2010 Jun 01.
Artículo en Inglés | MEDLINE | ID: mdl-20223754

RESUMEN

The lungs are generated by branching morphogenesis as a result of reciprocal signalling interactions between the epithelium and mesenchyme during development. Mutations that disrupt formation of either the correct number or shape of epithelial branches affect lung function. This, in turn, can lead to congenital abnormalities such as cystadenomatoid malformations, pulmonary hypertension or lung hypoplasia. Defects in lung architecture are also associated with adult lung disease, particularly in cases of idiopathic lung fibrosis. Identifying the signalling pathways which drive epithelial tube formation will likely shed light on both congenital and adult lung disease. Here we show that mutations in the planar cell polarity (PCP) genes Celsr1 and Vangl2 lead to disrupted lung development and defects in lung architecture. Lungs from Celsr1(Crsh) and Vangl2(Lp) mouse mutants are small and misshapen with fewer branches, and by late gestation exhibit thickened interstitial mesenchyme and defective saccular formation. We observe a recapitulation of these branching defects following inhibition of Rho kinase, an important downstream effector of the PCP signalling pathway. Moreover, epithelial integrity is disrupted, cytoskeletal remodelling perturbed and mutant endoderm does not branch normally in response to the chemoattractant FGF10. We further show that Celsr1 and Vangl2 proteins are present in restricted spatial domains within lung epithelium. Our data show that the PCP genes Celsr1 and Vangl2 are required for foetal lung development thereby revealing a novel signalling pathway critical for this process that will enhance our understanding of congenital and adult lung diseases and may in future lead to novel therapeutic strategies.


Asunto(s)
Pulmón/embriología , Morfogénesis/genética , Morfogénesis/fisiología , Proteínas del Tejido Nervioso/genética , Receptores Acoplados a Proteínas G/genética , Mucosa Respiratoria/metabolismo , Transducción de Señal/genética , Animales , Polaridad Celular/genética , Polaridad Celular/fisiología , Immunoblotting , Inmunohistoquímica , Ratones , Modelos Biológicos , Mutación/genética , Proteínas del Tejido Nervioso/metabolismo , Proteínas del Tejido Nervioso/fisiología , Oligonucleótidos/genética , Receptores Acoplados a Proteínas G/metabolismo , Receptores Acoplados a Proteínas G/fisiología , Mucosa Respiratoria/embriología
4.
PLoS Biol ; 2(9): E261, 2004 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-15328530

RESUMEN

The small GTPase Rab5 is a key regulator of clathrin-mediated endocytosis. On early endosomes, within a spatially restricted domain enriched in phosphatydilinositol-3-phosphate [PI(3)P], Rab5 coordinates a complex network of effectors that functionally cooperate in membrane tethering, fusion, and organelle motility. Here we discovered a novel PI(3)P-binding Rab5 effector, Rabankyrin-5, which localises to early endosomes and stimulates their fusion activity. In addition to early endosomes, however, Rabankyrin-5 localises to large vacuolar structures that correspond to macropinosomes in epithelial cells and fibroblasts. Overexpression of Rabankyrin-5 increases the number of macropinosomes and stimulates fluid-phase uptake, whereas its downregulation inhibits these processes. In polarised epithelial cells, this function is primarily restricted to the apical membrane. Rabankyrin-5 localises to large pinocytic structures underneath the apical surface of kidney proximal tubule cells, and its overexpression in polarised Madin-Darby canine kidney cells stimulates apical but not basolateral, non-clathrin-mediated pinocytosis. In demonstrating a regulatory role in endosome fusion and (macro)pinocytosis, our studies suggest that Rab5 regulates and coordinates different endocytic mechanisms through its effector Rabankyrin-5. Furthermore, its active role in apical pinocytosis in epithelial cells suggests an important function of Rabankyrin-5 in the physiology of polarised cells.


Asunto(s)
Endocitosis/fisiología , Regulación de la Expresión Génica , Péptidos y Proteínas de Señalización Intracelular/fisiología , Proteínas de Unión al GTP rab5/fisiología , Adenoviridae/genética , Androstadienos/farmacología , Animales , Línea Celular , Línea Celular Tumoral , Membrana Celular/metabolismo , Clonación Molecular , Perros , Regulación hacia Abajo , Endosomas/metabolismo , Inhibidores Enzimáticos/farmacología , Células Epiteliales/citología , Fibroblastos/metabolismo , GTP Fosfohidrolasas/química , Humanos , Fosfatos de Inositol/química , Péptidos y Proteínas de Señalización Intracelular/genética , Liposomas/metabolismo , Ratones , Microscopía Confocal , Microscopía por Video , Movimiento , Células 3T3 NIH , Pinocitosis , Plásmidos/metabolismo , Unión Proteica , Estructura Terciaria de Proteína , Interferencia de ARN , Proteínas Recombinantes/química , Factores de Tiempo , Wortmanina
5.
ACS Biomater Sci Eng ; 3(11): 2934-2942, 2017.
Artículo en Inglés | MEDLINE | ID: mdl-29202009

RESUMEN

The contraction and spreading of nonmuscle cells are important phenomena in a number of cellular processes such as differentiation, morphogenesis, and tissue growth. Recent experimental work has shown that the topology and the mechanical properties of the underlying substrate play a significant role in directing the cell's response. In this work, we introduce a multiscale model to understand the sensing, activation, and contraction of the actin cytoskeleton of nonmuscle cells based on the idea that acto-myosin cross-bridges display a catch-bond response. After investigating the respective roles of bond catchiness and acto-myosin assembly on the mechano-sensitivity of stress fibers, we present full simulations of cells laying on arrays of micropillars. Model predictions show good qualitative agreements with experimental observation, suggesting that acto-myosin catch bonds are a major mechano-sensing element in nonmuscle cells.

6.
PLoS One ; 7(9): e44871, 2012.
Artículo en Inglés | MEDLINE | ID: mdl-23028652

RESUMEN

The lung is a complex system in biology and medicine alike. Whereas there is a good understanding of the anatomy and histology of the embryonic and adult lung, less is known about the molecular details and the cellular pathways that ultimately orchestrate lung formation and affect its health. From a forward genetic approach to identify novel genes involved in lung formation, we identified a mutated Nubp1 gene, which leads to syndactyly, eye cataract and lung hypoplasia. In the lung, Nubp1 is expressed in progenitor cells of the distal epithelium. Nubp1(m1Nisw) mutants show increased apoptosis accompanied by a loss of the distal progenitor markers Sftpc, Sox9 and Foxp2. In addition, Nubp1 mutation disrupts localization of the polarity protein Par3 and the mitosis relevant protein Numb. Using knock-down studies in lung epithelial cells, we also demonstrate a function of Nubp1 in regulating centrosome dynamics and microtubule organization. Together, Nubp1 represents an essential protein for lung progenitor survival by coordinating vital cellular processes including cell polarity and centrosomal dynamics.


Asunto(s)
Proteínas de Unión al GTP/metabolismo , Pulmón/crecimiento & desarrollo , Pulmón/metabolismo , Morfogénesis , Células Madre/citología , Células Madre/metabolismo , Proteínas Adaptadoras Transductoras de Señales , Secuencia de Aminoácidos , Animales , Apoptosis , Secuencia de Bases , Catarata/genética , Moléculas de Adhesión Celular/metabolismo , Proteínas de Ciclo Celular , Proliferación Celular , Supervivencia Celular , Centrosoma/metabolismo , Cromosomas de los Mamíferos/genética , Epitelio/metabolismo , Femenino , Proteínas de Unión al GTP/química , Proteínas de Unión al GTP/genética , Regulación del Desarrollo de la Expresión Génica , Humanos , Péptidos y Proteínas de Señalización Intracelular , Pulmón/citología , Enfermedades Pulmonares/genética , Ratones , Datos de Secuencia Molecular , Mutagénesis , Mutación Puntual , Sindactilia/genética
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