Journal of Neuroscience, Vol 13, 547-558, Copyright © 1993 by Society for Neuroscience
Dystrophin in a membrane skeletal network: localization and comparison to other proteins
GM Dmytrenko, DW Pumplin and RJ Bloch
Department of Neurology, University of Maryland School of Medicine, Baltimore 21201.
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.
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