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

Abstract

We studied the developmental changes of localization of dystrophin and other cytoskeletal proteins, especially actin, spectrin and dystrophin related protein (DRP) using immunocytochemistry and quick-freezing and deep-etching (QF-DE) method. In developmental studies of mouse and human muscle cultures, some myoblasts had positive-reactions to spectrin, DRP, and F-actin, but not dystrophin. In aneurally cultured myotubes, dystrophin, DRP, and spectrin were localized diffusely in the cytoplasm and later in discontinuous patterns on the plasma membrane, when myotubes became mature. Spectrin and DRP had more positive reactions in immature myotubes, compared with those of dystrophin. In some areas of myotubes, dystrophin/spectrin and spectrin/actin were localized reciprocally. In innervated cultured human muscle cells, dystrophin and DRP were localized in neuro-muscular junctions, which were co-localized with clusters of acetylcholine receptors. By using the QF-DE method, dystrophin was localized just underneath the plasma membrane, and closely linked to actin-like filaments (8-10 nm in diameter), most of which were decorated with myosin subfragment 1. In actin-poor regions, spectrin was detected as well-organized filamentous structures in highly interconnected networks with various diameters. DRP was distributed irregularly with granular appearance inside the cytoplasm and also under the plasma membrane in immature mouse myotubes. Our present studies show that dystrophin, spectrin, and DRP are localized differently at the developmental stages of myotubes. These results suggest that dystrophin, spectrin, and DRP are organized independently in developing myotubes and these cytoskeletal proteins might play different functions in the preservation of plasma membrane stability in developing myotubes.

Dystrophin isoforms Dp71 and Dp427 have distinct roles in myogenic cells

“…Although Dp427 contains an N-terminal actin binding domain, it has not been reported to localize along the length of actin filaments, and is restricted to the surface membrane and adhesion zones. 5,10,16,19,24,35,37,42,46,57,61,62,64,69 To confirm that the dystrophin we detected in the SFLS was Dp71 and not Dp427, we also included, in these experiments, an antibody against the rod domain of Dp427 (Dys 2) which would not cross-react with Dp71. As demonstrated in Figure 5e , localization of Dp427 to the SFLS was not observed with this antibody, confirming that the SFLS localization was specific for Dp71.…”

Of Membrane Stability and Mosaics: The Spectrin Cytoskeleton

“…In mammalian systems, the switching of {3 spectrin isoforms occurs later in development. The overall profile is similar, with {3II spectrin diminishing until it is absent at birth, and {3I12 spectrin increasing from day E12 onward (246,534). Thus the {3II spectrin subunit can be thought of as the "fetal" {3 spectrin isoform during myogenesis and the {3I12 subunit is the "adult" {3 spectrin isoform.…”

Immunohistological intensity measurements as a tool to assess sarcolemma‐associated protein expression

“…We chose β‐spectrin as our internal control to account for differences in the integrity of the fibres. We have previously shown that spectrin is an ideal marker of sarcolemmal integrity as it is not a protein of the dystrophin complex [25] and is not affected by dystrophin deficiency, except on necrotic and regenerating fibres [26]. All measurements were normalized with their corresponding serial section labelled for β‐spectrin.…”