RESUMEN
We report biallelic missense and frameshift pathogenic variants in the gene encoding human nucleoporin NUP214 causing acute febrile encephalopathy. Clinical symptoms include neurodevelopmental regression, seizures, myoclonic jerks, progressive microcephaly, and cerebellar atrophy. NUP214 and NUP88 protein levels were reduced in primary skin fibroblasts derived from affected individuals, while the total number and density of nuclear pore complexes remained normal. Nuclear transport assays exhibited defects in the classical protein import and mRNA export pathways in affected cells. Direct surface imaging of fibroblast nuclei by scanning electron microscopy revealed a large increase in the presence of central particles (known as "plugs") in the nuclear pore channels of affected cells. This observation suggests that large transport cargoes may be delayed in passage through the nuclear pore channel, affecting its selective barrier function. Exposure of fibroblasts from affected individuals to heat shock resulted in a marked delay in their stress response, followed by a surge in apoptotic cell death. This suggests a mechanistic link between decreased cell survival in cell culture and severe fever-induced brain damage in affected individuals. Our study provides evidence by direct imaging at the single nuclear pore level of functional changes linked to a human disease.
Asunto(s)
Encefalopatía Aguda Febril/etiología , Fibroblastos/patología , Mutación del Sistema de Lectura , Canales Iónicos/fisiología , Mutación Missense , Proteínas de Complejo Poro Nuclear/genética , Poro Nuclear/patología , Transporte Activo de Núcleo Celular , Encefalopatía Aguda Febril/metabolismo , Encefalopatía Aguda Febril/patología , Apoptosis , Núcleo Celular/genética , Núcleo Celular/metabolismo , Proliferación Celular , Células Cultivadas , Niño , Preescolar , Femenino , Fibroblastos/metabolismo , Humanos , Lactante , Masculino , Poro Nuclear/genética , Poro Nuclear/metabolismo , Proteínas de Complejo Poro Nuclear/química , Proteínas de Complejo Poro Nuclear/metabolismo , Linaje , Conformación ProteicaRESUMEN
Field emission scanning electron microscopy (FESEM) is a well-established technique for acquiring three-dimensional surface images of nuclear pore complexes (NPCs). We present an optimized protocol for the exposure of mammalian cell nuclei and direct surface imaging of nuclear envelopes by FESEM, allowing for a detailed morphological comparison of individual NPCs, without the need for averaging techniques. This provides a unique high resolution tool for studying the effects of cellular stress, specific genetic manipulations and inherited diseases on the ultrastructure of human NPCs.