Résumé :
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Communication n° 494 Introduction. Dystrophin is an essential component in the assembly and maintenance of the dystrophin-associated protein complex (DAPC), which includes members of the dystroglycan, syntrophin, sarcoglycan and dystrobrevin protein families. Distinctive complexes have been described in the cell membrane of different tissues and cultured cells. Dystrophin Dp71 is the main DMD gene product in non-muscle tissues; however, its function remains to be elucidated. Recently, we showed that alternative splicing suffered by Dp71 at exons 70-74 and 78 regulates its cytoplasmic or nuclear localization. The nuclear presence of Dp71 opens new avenues towards the elucidation of its cellular function. In this work, we report the identification and characterization of a novel dystrophin associated complex present in the nuclei, which contains dystrophin Dp71 as a key component. Objectives a) To identify nuclear proteins that associate with Dp71. b) To analyze the subnuclear localization of Dp71 Methods and Results. We found, by confocal microscopy studies and cell fractionation analysis, the presence of Dp71, b sarcoglycan, b-dystroglycan, g2-syntrophin, a1- and b- dystrobrevin, and nNOS in the nuclei of HeLa cells. Furthermore, we demonstrated by coimmunoprecipitation experiments that most of these proteins form a complex in the nuclear compartment. As an attempt to define the role of this nuclear complex, we analyzed its possible association with the nuclear matrix. We observed the presence of Dp71, b-dystroglycan, nNOS, b-sarcoglycan, a1-dystrobrevin and b-dystrobrevin, in the nuclear matrix protein fractions and in situ nuclear matrix preparations from HeLa cells; moreover, we found that Dp71, b-dystroglycan and b-dystrobrevin coimmunoprecipitated with the nuclear matrix proteins lamin and actin Conclusions. Our findings suggest the presence of a Dp71-DAPC in the nuclei of HeLa cells; in addition, the association of members of this protein complex with the nuclear matrix may indicate their function as scaffolding proteins involved in nuclear architecture.
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