Knockdown of dystrophin Dp71 impairs PC12 cells cycle: localization in the spindle and cytokinesis structures implies a role for Dp71 in cell division.

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 ß-dystroglycan, proteins involved in aspects of the cell division cycle; therefore, we compared the distribution of Dp71 with that of lamin B1 and ß-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 ß-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 ß-dystroglycan levels.

Dystrophin Dp71 is critical for stability of the DAPs in the nucleus of PC12 cells.

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.

Neuronal differentiation modulates the dystrophin Dp71d binding to the nuclear matrix.

The function of dystrophin Dp71 in neuronal cells remains unknown. To approach this issue, we have selected the PC12 neuronal cell line. These cells express both a Dp71f cytoplasmic variant and a Dp71d nuclear isoform. In this study, we demonstrated by electron and confocal microscopy analyses of in situ nuclear matrices and Western blotting evaluation of cell extracts that Dp71d associates with the nuclear matrix. Interestingly, this binding is modulated during NGF-induced neuronal differentiation of PC12 cells with a twofold increment in the differentiated cells, compared to control cells. Also, distribution of Dp71d along the periphery of the nuclear matrix observed in the undifferentiated cells is replaced by intense fluorescent foci localized in the center of the nucleoskeletal structure. In summary, we revealed that Dp71d is a dynamic component of nuclear matrix that might participate in the nuclear modeling occurring during neuronal differentiation.