Co-localization of Dystrophin and β-Dystroglycan Demonstrated in En Face View by Double Immunogold Labeling of Freeze-fractured Skeletal Muscle

Abstract

SUMMARY An absence of dystrophin causes Duchenne muscular dystrophy, but the precise mechanism underlying necrosis of the muscle cells is still unclear. Dystrophin and ␤ -dystroglycan are components of a complex of at least nine proteins, the dystrophin-glycoprotein complex (DGC), that links the membrane cytoskeleton to extracellular elements in skeletal and cardiac muscle. Biochemical studies indicate that dystrophin is bound to other components of the DGC via ␤ -dystroglycan, which suggests that the distribution of these two proteins should be almost identical. In this study, therefore, we examined the spatial relationship between dystrophin and ␤ -dystroglycan with a range of different imaging techniques to investigate the extent of the predicted co-localization. We used (a) double immunogold fracture-label, a freeze-fracture cytochemical technique that allows high-resolution face-on views of labeled membrane components in thin sections and in platinumcarbon replicas, (b) double immunogold labeling of cryosections and (c) confocal microscopy. Both dystrophin and ␤ -dystroglycan were found over the entire fiber surface and, when labeled singly, the nearest neighbor spacing of labeling sites for the two proteins was indistinguishable. With double labeling, very close co-localization could be demonstrated. The results support the conclusion that dystrophin and ␤ -dystroglycan directly interact at the muscle plasma membrane. (J Histochem Cytochem 46:945-953, 1998)

β-Dystroglycan Restoration and Pathology Progression in the Dystrophic mdx Mouse: Outcome and Implication of a Clinically Oriented Study with a Novel Oral Dasatinib Formulation

“…A likely hypothesis for the scarce overall efficacy of DAS in mdx mice, also in a better formulation and over a long-term, could be that, despite the increase of β-DG levels in skeletal muscle fibers, the protein may be not sufficiently expressed at the sarcolemma. In fact, considering the direct interactions of dystrophin and β-DG at the membrane level in normal muscle fibers [ 13 , 33 ], the stability of β-DG at sarcolemma remains weak or incorrectly localized in dystrophin-lacking fibers, in turn limiting DAS effects at the functional and structural level. In this view, further insights will be gained by means of dedicated immunofluorescence and molecular biology experiments.…”

Fukutin-Related Protein Resides in the Golgi Cisternae of Skeletal Muscle Fibres and Forms Disulfide-Linked Homodimers via an N-Terminal Interaction

“…In immunoelectron microscopy, monoclonal mouse anti-MG160 Golgi complex antibody (AE-6) (ab58826) (Abcam, UK), monoclonal mouse anti-ß-dystroglycan (ß-dys) antibody, (7D11) (sc-33701) (Santa Cruz Biotechnology, USA), and mouse anti- Protein Disulfide Isomerase (PDI) antibody (RL90) (ab2792) (Abcam, UK) served as Golgi [32], sarcolemma [39], [40] and endoplasmic reticulum (ER) markers [41], respectively. The secondary antibodies used were gold conjugated goat F(ab)2 anti-rabbit IgG (5 nm) and goat anti- mouse IgG (10 nm) (British Bio Cell International, UK).…”

Plasma membrane cytoskeleton of muscle: a fine structural analysis

“…Double labeling experiments using antibodies to the C-termini of both molecules show that this is indeed the case (Fig. 3b) and careful measurement of the distance and orientation of one gold probe with respect to the other shows that the two epitopes must be separated by Ͻ15 nm (Cullen et al, 1998).…”

“…Immunogold labeling of ␤-dystroglycan shows that the gold conjugate lies on the membrane or extremely close to it, on either side of the membrane (Fig. 3a) (Cullen et al, 1998). The primary antibody that was used was raised against 15 of the last 16 amino acids at the C-terminus of ␤-dystroglycan, i.e., at the cytoplasmic end of the molecule.…”