RESUMEN
The Abl-interactor (Abi) family of adaptor proteins has been linked to signaling pathways involving the Abl tyrosine kinases and the Rac GTPase. Abi proteins localize to sites of actin polymerization in protrusive membrane structures and regulate actin dynamics in vitro. Here we demonstrate that Abi2 modulates cell morphogenesis and migration in vivo. Homozygous deletion of murine abi2 produced abnormal phenotypes in the eye and brain, the tissues with the highest Abi2 expression. In the absence of Abi2, secondary lens fiber orientation and migration were defective in the eye, without detectable defects in proliferation, differentiation, or apoptosis. These phenotypes were consistent with the localization of Abi2 at adherens junctions in the developing lens and at nascent epithelial cell adherens junctions in vitro. Downregulation of Abi expression by RNA interference impaired adherens junction formation and correlated with downregulation of the Wave actin-nucleation promoting factor. Loss of Abi2 also resulted in cell migration defects in the neocortex and hippocampus, abnormal dendritic spine morphology and density, and severe deficits in short- and long-term memory. These findings support a role for Abi2 in the regulation of cytoskeletal dynamics at adherens junctions and dendritic spines, which is critical for intercellular connectivity, cell morphogenesis, and cognitive functions.
Asunto(s)
Movimiento Celular/genética , Espinas Dendríticas/genética , Proteínas de Homeodominio/genética , Aprendizaje , Memoria , Morfogénesis/genética , Proteínas Adaptadoras Transductoras de Señales , Uniones Adherentes/metabolismo , Animales , Línea Celular , Dendritas/genética , Perros , Embrión de Mamíferos/citología , Fibroblastos/metabolismo , Eliminación de Gen , Células HeLa , Hipocampo/citología , Proteínas de Homeodominio/metabolismo , Homocigoto , Humanos , Cristalino/embriología , Cristalino/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Neocórtex/citología , Interferencia de ARN , ARN Interferente Pequeño/metabolismoRESUMEN
The recent development of real-time PCR allows for the rapid and accurate quantitation of gene expression in cells and tissues. Real-time PCR instrumentation is designed for the simultaneous quantitation of gene expression from a few samples up to 384 samples. The normal tissue expression profile of a gene can provide useful insights into its potential role in normal physiological processes. When combined with the tissue expression profile of the gene in diseased tissues, information concerning the potential role in pathological processes can be determined. This unit describes a protocol to determine the relative abundance of mRNA in a panel of human tissues using real-time PCR.