Dystrophin in a membrane skeletal network: localization and comparison to other proteins

Abstract

We studied the location, relative abundance, and stability of dystrophin in clusters of ACh receptors (AChRs) isolated from primary cultures of neonatal rat myotubes. Although variable amounts of dystrophin were found at receptor clusters, dystrophin was always associated with organized, receptor-rich domains (AChR domains). Dystrophin was occasionally seen in focal contact domains, but never in clathrin-coated domains. Dystrophin was also present in a diffuse, punctate distribution in regions of myotube membrane that did not contain AChR clusters. Immunogold labeling at the ultrastructural level localized dystrophin in a spectrin-rich filamentous network closely applied to the cytoplasmic surface of the cell membrane at AChR domains. Dystrophin was not associated with overlying actin filaments. Semiquantitative immunofluorescence studies indicated that dystrophin was present in relatively small amounts in these preparations, with only one molecule of dystrophin for every approximately 5 AChR, 43 kDa and 58 kDa molecules, and for every approximately 20-35 beta-spectrin molecules. Clusters were disrupted, but the total amount of dystrophin was not significantly reduced, when myotubes were incubated with sodium azide or in Ca(2+)-free medium, and when isolated AChR clusters were extracted at low ionic strength, at high pH, or in 6 M urea. These treatments extract other peripheral membrane proteins from AChR clusters. Labeling for dystrophin was completely eliminated when clusters were incubated with chymotrypsin, however. Thus, dystrophin forms part of a membrane skeleton at AChR clusters, but it is more difficult to remove than other proteins in the network. This suggests that dystrophin attaches to cluster membrane in a unique way.

Developmental studies of dystrophin and other cytoskeletal proteins in cultured muscle cells

“…In our cultured muscle cells, granular distribution of dystrophin was observed just underneath the plasma membrane and no clear lattice-like pattern was detected. These ultrastructural appearances of dystrophin are consistent with those in the regions of AChR clusters in cultured rat myotubes (Dmytrenko et al, 1993). Therefore, in living cells dystrophin may have a folded or condensed configuration as integral mem- brane protein-like spectrin in the erythrocyte membrane (Ursitti et al, 1991).…”

“…It is well known that the formation of innervation during development gives dramatic changes in the differentiation and maturation of muscle fibers and also motor neurones themselves. Recently it has been reported that dystrophin and DRP are densely concentrated in neuromuscular junctions (Huard et al, 1992;Khurana et al, 1991;Love et al, 1991;Pons et al, 1991;Shimizu et al, 1989) and they are closely associated with acetylcholine receptors (Chang et al, 1989;Dmytrenko et al, 1993;Jasmin et al, 1990) and integral membrane glycoprotein (Campbell and Kahl, 1989;Ervasti et al, 1990;Ervasti and Campbell, 1991). Our present studies show that dystrophin and DRP are concentrated in some nerve terminals with AChR clusters, which are supposed to be neuromuscular junctions, in cultured innervated human muscle cells similar to adult muscle fibers.…”

Disorganization of dystrophin costameric lattice in Becker muscular dystrophy

“…7,11 At the other end or N-terminal site, dystrophin may interact directly or indirectly with other cytoskeletal proteins, including ␣-actinin, F-actin, talin, vinculin, and integrin. 5,8,12,15,20 In DMD, it has been postulated that the absence of dystrophin causes a disruption of the dystrophin-glycoprotein complex which, in turn, triggers the cascade of events leading to muscle cell degeneration and death. 4,18 We have previously described that dystrophin colocalizes with vinculin at the muscle cell surface.…”

Biomechanics of the sarcolemma and costameres in single skeletal muscle fibers from normal and dystrophin-null mice

“…Dystrophin is a 427 kDa cytoskeletal protein enriched at costameres that is thought to connect the cytoskeleton to integral proteins of the plasma membrane (Porter et al 1992; Ervasti and Campbell 1991, 1993; Williams and Bloch 1999a; Rybakova et al 2000; Ursitti et al 2004; Bhosle et al 2006; Ervasti 2007; Stone et al 2007), and through the membrane, to the basal lamina (Ohlendieck et al 1991; Dmytrenko et al 1993; Winder 1997; Campbell and Stull 2003; Beedle et al 2007). Dystrophin and the proteins with which it associates at the plasma membrane, termed the dystrophin–glycoprotein complex, have been linked to different forms of muscular dystrophy.…”