Dystrophin Dp71 is Critical for Stability of the DAPs in the Nucleus of PC12 Cells

Abstract

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.

Dystrophin Dp71: The Smallest but Multifunctional Product of the Duchenne Muscular Dystrophy Gene

“…These two proteins have been involved in aspects of the cell division cycle [128,129]. It is noteworthy that these researchers observed drastically decreased staining for both lamin B1 and β-dystroglycan in the mitotic spindle-cleavage furrow and in the midbody of Dp71-depleted cells, as well as a marked reduction in their levels in total extracts [60,127]. In light of this data, we postulate that Dp71 is a component of the mitotic spindle and cytokinesis multiprotein apparatuses that might modulate the cell-division cycle by binding to lamin B1 and β-dystroglycan and conferring proper localization and stability upon them (Fig.…”

“…It was found by cell fractionation and immunoprecipitation of nuclear extracts that Dp71 concurs in the nucleus with sarcoglycans, β-dystroglycan, syntrophins, and dystrobrevins, and that all these are assembled into a nuclear DAPC in HeLa [59], C2C12 [54], and PC12 [60] cell lines. Selective nuclear import of proteins involves the recognition of a conventional nuclear localizing signal (NLS), generally short stretches of basic amino acids either alone (monopartite) or separated by a linker region of 10-12 amino acids (bipartite), located in the cargo protein, by a cytoplasmic receptor that consists of importins α and β. Proteins carrying a classical or bipartite NLS are bound by importin α, which serves as adaptor for importin β−NLS binding.…”

“…Interestingly, researchers in the laboratory of Dr. Cisneros have recently provided experimental evidence linking Dp71 with the cell division cycle [60]. It was observed that Dp71-knockdown PC12 cells display a marked delay in cell growth with no alteration in cell death, suggesting a failure in the cell division cycle.…”

Expression of Dystrophins and the Dystrophin-Associated-Protein Complex by Pituicytes in Culture

“…Several isoform of DAPC have been described and may account for the multiple additional bands we saw on Western blots. In exemple, if the major band of b-DG corresponds to the 43 kDa isoform, a band at 65 kDa has been described by others [39]. Recently several isoform of e-SG have been found in the brain in addition to the major form [40].…”

Identification of Dp71 Isoforms Expressed in PC12 Cells: Subcellular Localization and Colocalization with β-Dystroglycan and α1-Syntrophin

“…These results suggest that exons 71-74 play an important role in the cytoplasmic localization of Dp71a and Dp71e isoforms because Dp71c and Dp71ec, which lack these exons, do not show cytoplasmic localization. Most likely, each Dp71 isoform forms a specific complex with the membrane, cytoplasm, or nuclear proteins because Dp71-DAP complexes have been described in the end-feet and cell membranes of retina Müller glial cells (Fort et al 2008) and in the cytoplasm and nuclei of several cell line models (Fuentes-Mera et al 2006;Romo-Yáñez et al 2007;González-Ramírez et al 2008;Villarreal-Silva et al 2010).…”