RESUMEN
The AF6/afadin protein is a component of cell membranes at specialized sites of cell-cell contact. Two main splice variants exist, known as l- and s-afadin, respectively. L-afadin is widely expressed in cells of epithelial origin, whilst s-afadin expression is restricted to the brain. Here we demonstrate that the short form of AF6/s-afadin is a dual residency protein able to localize to the plasma membrane or nucleus whilst the long form of AF6, l-afadin is unable to localize to the nucleus. AF6/s-afadin clusters in a distinctive speckled pattern in the nucleus, but is unable to do so when cell cycle progression is inhibited at the G(1)/S or G(2)/M checkpoints. The formation of AF6/s-afadin nuclear bodies is also sensitive to the transcriptional activity of the cell with inhibition of RNA polymerase activity abolishing AF6/s-afadin nuclear clustering. AF6/s-afadin nuclear bodies localize to a novel subnuclear compartment, failing to colocalize with other known nuclear bodies. Formation of the AF6/s-afadin nuclear foci can be regulated by specific growth factor receptor mediated signaling events and by cytoplasmic tyrosine kinases, but does not correlate with tyrosine phosphorylation of AF6/s-afadin. AF6/s-afadin is a candidate for mediating control of cellular growth processes by regulated translocation to the nucleus.
Asunto(s)
Membrana Celular/metabolismo , Núcleo Celular/metabolismo , Cinesinas/metabolismo , Proteínas de Microfilamentos/metabolismo , Miosinas/metabolismo , Empalme Alternativo , Animales , Ciclo Celular/efectos de los fármacos , Línea Celular , Estructuras del Núcleo Celular/metabolismo , ARN Polimerasas Dirigidas por ADN/antagonistas & inhibidores , Perros , Proteínas Fluorescentes Verdes/genética , Humanos , Cinesinas/genética , Proteínas con Dominio LIM , Proteínas de Microfilamentos/genética , Moduladores de la Mitosis/farmacología , Miosinas/genética , Inhibidores de la Síntesis del Ácido Nucleico/farmacología , Oligopéptidos , Péptidos/genética , Fosforilación , Transporte de Proteínas/efectos de los fármacos , Proteínas Tirosina Quinasas/genética , Proteínas Tirosina Quinasas/metabolismo , Ratas , Proteínas Recombinantes/metabolismo , Transducción de Señal , Factores de Tiempo , Transcripción Genética/efectos de los fármacos , TransfecciónRESUMEN
Paired helical filaments (PHF) are the principal pathologic components of neurofibrillary tangles in Alzheimer's disease (AD). To reproduce the formation of PHF in tissue culture, we stably expressed human tau with and without pathogenic mutations in human SH-SY5Y cells and exposed them for 5 days to aggregated synthetic beta-amyloid peptide (A beta 42). This caused a decreased solubility of tau along with the generation of PHF-like tau-containing filaments. These were 20 nm wide and had periodicities of 130-140 nm in the presence of P301L mutant tau or 150-160 nm in the presence of wild-type tau. Mutagenesis of the phosphoepitope serine 422 of tau prevented both the A beta 42-mediated decrease in solubility and the generation of PHF-like filaments, suggesting a role of serine 422 or its phosphorylation in tau filament formation. Together, our data underscore a role of A beta 42 in the formation of PHF-like filaments. Our culture system will be useful to map phosphoepitopes of tau involved in PHF formation and to identify and characterize modifiers of the tau pathology. Further adaptation of the system may allow the screening and validation of compounds designed to prevent PHF formation.
