RESUMO
The temperature-sensitive mutant tsp53val135 accumulates in the cytoplasm of cells kept at the non-permissive temperature (39 degrees C), but is rapidly transported into the cell nucleus at the permissive temperature (30 degrees C). tsp53 thus may serve as a model for analysing cellular parameters influencing the subcellular location of p53. Here we provide evidence that retention of tsp53 in the cytoplasm at the non-permissive temperature is due to cytoskeletal anchorage of the p53 protein. Two sublines of C6 rat glioma cells differing in their expression of the intermediate filament protein vimentin (vimentin expressing or vimentin negative cells) were stably transfected with a vector encoding tsp53. Whereas cells of vimentin expressing C6 subclones retained tsp53 in the cytoplasm at the non-permissive temperature, cells of vimentin negative subclones exclusively harbored the tsp53 within their nuclei. Intermediate filament deficient cells that had been reconstituted with a full length vimentin protein again showed a cytoplasmic localization of tsp53, whereas in cells expressing a C-terminally truncated (tail-less) vimentin tsp53 localized to the nucleus. We conclude that cytoplasmic sequestration of tsp53 requires an intact intermediate filament system.
Assuntos
Compartimento Celular , Citoplasma/metabolismo , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Vimentina/metabolismo , Actinas/metabolismo , Animais , Transporte Biológico , Compartimento Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Citocalasina B/farmacologia , Citoesqueleto/metabolismo , Citoesqueleto/ultraestrutura , Imunofluorescência , Glioma , Camundongos , Mutação , Ratos , Tubulina (Proteína)/metabolismo , Células Tumorais Cultivadas , Vimentina/ultraestrutura , Vimblastina/farmacologiaAssuntos
Adesão Celular/fisiologia , Fibrina , Hidroxibutiratos , Polímeros , Próteses e Implantes , Ácido 3-Hidroxibutírico , Animais , Divisão Celular/fisiologia , Fibroblastos/citologia , Humanos , Teste de Materiais , Camundongos , Microscopia de Força Atômica , Peso Molecular , Propriedades de SuperfícieRESUMO
Whole mount electron microscopy of extracted cells combined with immunogold labeling techniques can be used to characterize the cytoskeletal architecture of cultured cells. As shown with subclones of the C6 rat glioma cell line, heavy metal shadowing was suitable for getting basic information concerning the arrangement of the various filament types within the networks. Pure carbon shadowing combined with immunogold double labeling proved to be optimal to identify linkages between filaments, to localize filament associated proteins and to follow the arrangement of filaments in dense arrays such as lamellipodiae and cell margins. Thin connecting filaments which interact with actin as well as with vimentin filaments and can be labeled with antibodies to the intermediate filament associated protein plectin may play a major role in the structural organization of the cytoskeleton of these cells.
Assuntos
Proteínas do Citoesqueleto/análise , Citoesqueleto/química , Citoesqueleto/ultraestrutura , Glioma/ultraestrutura , Imuno-Histoquímica , Microscopia Eletrônica , Actinas/análise , Animais , Células Clonais , Glioma/química , Glioma/metabolismo , Proteínas de Filamentos Intermediários/análise , Filamentos Intermediários/química , Filamentos Intermediários/ultraestrutura , Plectina , Ratos , Células Tumorais Cultivadas , Vimentina/análiseRESUMO
Intrauterine and neonatal manifestations of congenital long QT syndrome are associated with a high cardiac risk, particularly when atrioventricular block and excessive QT prolongation (> 600 ms(1/2)) are present. In a female newborn with these features, treatment with propranolol and mexiletine led to complete reduction of arrhythmia that was maintained 1.5 years later. High throughput genetic analysis found a sodium channel gene (LQT3) mutation. Disappearance of the 2:1 atrioventricular block and QTc shortening (from 740 ms(1/2) to 480 ms(1/2)), however, was achieved when mexiletine was added to propranolol. This effect was considered to be possibly genotype related. Early onset forms of long QT syndrome may benefit from advanced genotyping.