The function of dystrophin Dp71 in neuronal cells remains to be established. Previously, we revealed the involvement of this protein in both nerve growth factor (NGF)-induced neuronal differentiation and cell adhesion by isolation and characterization of PC12 neuronal cells with depleted levels of Dp71. In this work, a novel phenotype of Dp71-knockdown cells was characterized, which is their delayed growth rate. Cell cycle analyses revealed an altered behavior of Dp71-depleted cells, which consists of a delay in G0/G1 transition and an increase in apoptosis during nocodazole-induced mitotic arrest. Dp71 associates with lamin B1 and β-dystroglycan, proteins involved in aspects of the cell division cycle; therefore, we compared the distribution of Dp71 with that of lamin B1 and β-dystroglycan in PC12 cells at mitosis and cytokinesis by means of immunofluorescence and confocal microscopy analysis. All of these three proteins exhibited a similar immunostaining pattern, localized at mitotic spindle, cleavage furrow, and midbody. It is noteworthy that a drastic decreased staining in mitotic spindle, cleavage furrow, and midbody was observed for both lamin B1 and β-dystroglycan in Dp71-depleted cells. Furthermore, we demonstrated the interaction of Dp71 with lamin B1 in PC12 cells by immunoprecipitation and pull-down assays, and importantly, we revealed that knockdown of Dp71 expression caused a marked reduction in lamin B1 levels and altered localization of the nuclear envelope protein emerin. Our data indicate that Dp71 is a component of the mitotic spindle and cytokinesis multi-protein apparatuses that might modulate the cell division cycle by affecting lamin B1 and β-dystroglycan levels.

We have adopted the PC12 cell line as in vitro cell model for studying Dp71 function in neuronal cells. These cells express a cytoplasmic (Dp71f) and a nuclear (Dp71d) isoform of Dp71 as well as various dystrophin-associated proteins (DAPs). In this study, we revealed by confocal microscopy analysis and Western blotting evaluation of cell fractions the presence of different DAPs (beta-dystroglycan, beta-dystrobrevin, epsilon-sarcoglycan and gamma1-syntrophin) in the nucleus of PC12 cells. Furthermore, we established by immunoprecipitation assays that Dp71d and the DAPs form a dystrophin-associated protein complex (DAPC) in the nucleus. Interestingly, depletion of Dp71 by antisense treatment (antisense-Dp71 cells) provoked a drastic reduction of nuclear DAPs, which indicates that Dp71d is critical for DAPs stability within the nucleus. Although Up71, the utrophin gene product homologous to Dp71, exhibited increased expression in the antisense-Dp71 cells, its scarce nuclear levels makes unlikely that could compensate for Dp71 nuclear deficiency.

Even though the Duchenne muscular dystrophy (DMD) gene product Dystrophin Dp71d is involved in various key cellular processes through its role as a scaffold for structural and signalling proteins at the plasma membrane as well as the nuclear envelope, its subcellular trafficking is poorly understood. Here we map the nuclear import and export signals of Dp71d by truncation and point mutant analysis, showing for the first time that Dp71d shuttles between the nucleus and cytoplasm mediated by the conventional nuclear transporters, importin (IMP) α/β and the exportin CRM1. Binding was confirmed in cells using pull-downs, while in vitro binding assays showed direct, high affinity (apparent dissociation coefficient of c. 0.25nM) binding of Dp71d to IMPα/β. Interestingly, treatment of cells with the microtubule depolymerizing reagent nocodazole or the dynein inhibitor EHNA both decreased Dp71d nuclear localization, implying that Dp71d nuclear import may be facilitated by microtubules and the motor protein dynein. The role of Dp71d in the nucleus appears to relate in part to interaction with the nuclear envelope protein emerin, and maintenance of the integrity of the nuclear architecture. The clear implication is that Dp71d's previously unrecognised nuclear transport properties likely contribute to various, important physiological roles.