Localization of sarcoglycan, neuronal nitric oxide synthase, β‐dystroglycan, and dystrophin molecules in normal skeletal myofiber: Triple immunogold labeling electron microscopy

Abstract

In order to investigate the mode of existence of the sarcoglycan complex, neuronal nitric oxide synthase (nNOS), beta-dystroglycan, and dystrophin in the normal skeletal myofiber, we examined the ultrastructural localization and mutual spatial relationship of nNOS, beta-dystroglycan, dystrophin, and the individual components of the sarcoglycan complex by using triple immunogold labeling electron microscopy. Each molecule of alpha-, beta-, gamma- and delta-sarcoglycans is located intracellularly or extracellularly near the muscle plasma membrane mostly in accordance with the sarcoglycan antigenic sites against which the antibodies were generated. The association of different two and/or three sarcoglycan molecules out of alpha-, beta-, gamma- and delta-sarcoglycan molecules was frequently observed. Each molecule of nNOS, beta-dystroglycan, and dystrophin was ultrastructurally noted along the cell surface of normal skeletal myofibers. Moreover, the close relation of a sarcoglycan molecule with beta-dystroglycan and dystrophin, and the association of nNOS with dystrophin were also confirmed ultrastructurally. Thus, this study demonstrated that the constituting molecules of the sarcoglycan complex, nNOS, beta-dystroglycan, and dystrophin existed in the form of a cluster at the normal muscle plasma membrane. The association of nNOS with dystrophin and its associated glycoproteins may form a macromolecular signaling complex at the muscle plasma membrane.

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).…”

δ-Sarcoglycan-deficient muscular dystrophy: from discovery to therapeutic approaches

“…The DGC confers structural stability to the sarcolemma, and dissociation of the complex by loss of one of its components renders the muscle more susceptible to contraction-induced damage. Dissociation of the DGC complex is also thought to have widespread implications for a number of signalling processes, as it is closely associated with a diverse range of molecules, such as neuronal nitric oxide synthase (nNOS) [34-36] and members of the integrin family of signalling proteins [37-39]. …”

“…Displacement of nNOS from the sarcolemmal membrane has been noted in patients with DMD and more recently in LGMD patients with SG mutations and animal models of sarcoglycanopathy [35,120]. nNOS is associated with dystrophin at the sarcolemmal membrane [34] and generates NO, a molecule with a pivotal role in modulating blood flow [121], through a calcium-dependent process. It is thought that disruption of nNOS renders the muscle more susceptible to focal ischaemia and damage by superoxides [121,122].…”

Biophysical Stimulation for Engineering Functional Skeletal Muscle

“…12 Strain has also been shown to increase the nNOS expression in skeletal muscle, which associates with the cytodystrophin complex and increases the release of nitric oxide (NO). 34,35 This chain of events increased the apparent Young's Modulus of the cells through higher production of talin and vinculin 36 (Fig. 1) and could lead to a positive feedback loop for amplification of the strain signal.…”