Association of Aciculin with Dystrophin and Utrophin

Abstract

Aciculin is a recently identified 60-kDa cytoskeletal protein, highly homologous to the glycolytic enzyme phosphoglucomutase type 1, (Belkin, A. M., Klimanskaya, I. V., Lukashev, M. E., Lilley, K., Critchley, D., and Koteliansky, V. E. (1994) J. Cell Sci. 107, 159-173). Aciculin expression in skeletal muscle is developmentally regulated, and this protein is particularly enriched at cell-matrix adherens junctions of muscle cells (Belkin, A. M., and Burridge, K. (1994) J. Cell Sci. 107, 1993-2003). The purpose of our study was to identify cytoskeletal protein(s) interacting with aciculin in various cell types. Using immunoprecipitation from cell lysates of metabolically labeled differentiating C2C12 muscle cells with anti-aciculin-specific antibodies, we detected a high molecular weight band (M(r) approximately 400,000), consistently coprecipitating with aciculin. We showed that this 400 kDa band comigrated with dystrophin and immunoblotted with anti-dystrophin antibodies. The association between aciculin and dystrophin in C2C12 cells was shown to resist Triton X-100 extraction and the majority of the complex could be extracted only in the presence of ionic detergents. In the reverse immunoprecipitation experiments, aciculin was detected in the precipitates with different anti-dystrophin antibodies. Immunodepletion experiments with lysates of metabolically labeled C2C12 myotubes showed that aciculin is a major dystrophin-associated protein in cultured skeletal muscle cells. Double immunostaining of differentiating and mature C2C12 myotubes with antibodies against aciculin and dystrophin revealed precise colocalization of these two cytoskeletal proteins throughout the process of myodifferentiation in culture. In skeletal muscle tissue, both proteins are concentrated at the sarcolemma and at myotendinous junctions. In contrast, utrophin, an autosomal homologue of dystrophin, was not codistributed with aciculin in muscle cell cultures and in skeletal muscle tissues. Analytical gel filtration experiments with purified aciculin and dystrophin showed interaction of these proteins in vitro, indicating that their association in skeletal muscle is due to direct binding. Whereas dystrophin was shown to be a major aciculin-associated protein in skeletal muscle, immunoblotting of anti-aciculin immunoprecipitates with antibodies against utrophin showed that aciculin is associated with utrophin in cultured A7r5 smooth muscle cells and REF52 fibroblasts. Immunodepletion experiments performed with lysates of metabolically labeled A7r5 cells demonstrated that aciculin is a major utrophin-binding protein in this cell type. Taken together, our data show that aciculin is a novel dystrophin- and utrophin-binding protein. Association of aciculin with dystrophin (utrophin) in various cell types might provide an additional cytoskeletal-matrix transmembrane link at sites where actin filaments terminate at the plasma membrane.

Specific and Potent RNA Interference in Terminally Differentiated Myotubes

“…However, many disease genes are expressed only in terminally differentiated cells and tissues. For example, components of the dystrophin-glycoprotein complex are required for normal muscle function and prevention of Duchenne muscular dystrophy (38) but are expressed only upon differentiation of myoblasts into myotubes (39). To determine whether RNAi can be used for sequence-specific gene silencing in differentiated mammalian cells, we induced the differentiation of C2C12 myoblasts into skeletal muscle myotubes in culture.…”

Aciculin interacts with filamin C and Xin and is essential for myofibril assembly, remodeling and maintenance

“…Aciculin has primarily been investigated as an adhesion protein and cytoskeletal component of cell-matrix and cell-cell contacts in muscle and non-muscle cells Belkin and Burridge, 1994;Belkin and Burridge, 1995a;Belkin and Burridge, 1995b;Belkin and Smalheiser, 1996). Correspondingly, the previously reported interaction partners of aciculin were dystrophin and its non-muscle homolog utrophin (Belkin and Burridge, 1995a;Belkin and Burridge, 1995b;Belkin and Smalheiser, 1996).…”

“…In striated muscle, it mainly localizes to the intercalated discs of the heart, and to MTJs and costameres of skeletal muscle Belkin and Burridge, 1994;Belkin and Burridge, 1995a;Belkin and Burridge, 1995b;Koteliansky et al, 1989). Because of its distribution and the subsequent identification of dystrophin as a binding partner (Belkin and Burridge, 1995a;Moiseeva et al, 1996), research on aciculin originally focused on its association with cell-cell and cell-matrix contacts.…”

“…In striated muscle, it mainly localizes to the intercalated discs of the heart, and to MTJs and costameres of skeletal muscle Belkin and Burridge, 1994;Belkin and Burridge, 1995a;Belkin and Burridge, 1995b;Koteliansky et al, 1989). Because of its distribution and the subsequent identification of dystrophin as a binding partner (Belkin and Burridge, 1995a;Moiseeva et al, 1996), research on aciculin originally focused on its association with cell-cell and cell-matrix contacts. Given that it is upregulated during muscle cell differentiation, aciculin might also function in muscle development and adaptation (Belkin and Burridge, 1994;Belkin and Burridge, 1995b), for example, following chronic muscle use or disuse (Rezvani et al, 1996).…”

Archvillin, a muscle-specific isoform of supervillin, is an early expressed component of the costameric membrane skeleton

“…The polarized localization of EGFP-tagged archvillin from a construct lacking archvillin 5′-and 3′-UTR sequences indicated that these sequences were not required for protein targeting during early myogenesis. This distribution presaged the appearance of dystrophin at myotube tips because dystrophin is not expressed at appreciable levels until 10-11 days after the induction of differentiation (Belkin and Burridge, 1995;Kobayashi et al, 1995).…”