Dystrophin, the protein absent in patients with Duchenne muscular dystrophy (Hoffman et al., 1987), shows structural and functional similarities to cytolinkers, which suggests the hypothesis that dystrophin performs a cytolinker role in muscle. Dystrophin’s large molecular mass of 427 kD, spectrinlike repeats, and ability to bind actin filaments via a tandem CH domain (Way et al., 1992) highlight three similarities with cytolinkers. Although dystrophin lacks a plakin repeat domain, dystrophin–intermediate filament interactions have been documented (Stone et al., 2005; Bhosle et al., 2006). Thus, the ability to link the actin and intermediate filament cytoskeletons to the transmembrane dystroglycan complex (Suzuki et al., 1992) illustrates how dystrophin functions similarly to other cytolinkers. Finally, the muscle membrane fragility associated with the loss of dystrophin (Petrof et al., 1993) parallels the structural deficiencies observed in other cytolinker-deficient tissues, further demonstrating a close relationship between dystrophin and other cytolinkers. Collectively, these data support the hypothesis that dystrophin may function as a cytolinker in skeletal muscle.

Although dystrophin exhibits many characteristics of a cytolinker, a direct dystrophin–MT interaction has not been documented. Dystrophin lacks either a Gas2-related or a glycine/serine/arginine domain, but recent studies indicated that dystrophin at least indirectly influences MT organization or stability (Percival et al., 2007; Ayalon et al., 2008). For instance, the dystrophin-deficient mdx mouse exhibited MT disorganization in skeletal muscle with the costameric MTs most severely affected (Percival et al., 2007). Dystrophin’s enrichment at costameres and the restoration of costameric MT organization through virally mediated expression of a microdystrophin (Percival et al., 2007) indicates that dystrophin is necessary for proper costameric MT organization in skeletal muscle. Moreover, acute knockdown of ankyrin-B, a protein necessary for delivery of dystrophin to the sarcolemma and neuromuscular junction, caused the loss of costameric MTs and aberrant MT organization in a subset of MTs underlying the neuromuscular junction (Ayalon et al., 2008).

Next, we performed a tissue-based MT cosedimentation assay (Fig. 2 A) to determine whether dystrophin cosedimented with MTs. Under conditions that induced MT depolymerization, virtually no muscle protein pelleted (Fig. 2 B). However, numerous proteins pelleted under MT-stabilizing conditions, and this fraction of proteins represents MTs and the MAPs of skeletal muscle (Fig. 2 B). We Western blotted each fraction obtained from the tissue cosedimentation assay performed on wt mice expressing full-length dystrophin or transgenic mdx mice expressing Dp260, microdystrophin (ΔR4-23), or Dp71 (Fig. 2 C, right). Full-length dystrophin, Dp260, and ΔR4-23 all pelleted with MTs, whereas Dp 71 did not (Fig. 2 C, left). By comparing the dystrophin domains present or absent in each construct (Fig. 2 C, right) along with each construct's ability to cosediment with MTs, we suggest that spectrinlike repeat 24 through the first third of the WW domain encodes a novel MT-binding domain.

To test for a direct interaction between dystrophin and MTs, we performed MT cosedimentation using two purified recombinant dystrophin constructs and purified tubulin. The two recombinant constructs used were Dp260, which encodes from spectrinlike repeat 10 through the carboxy terminus of dystrophin, including the proposed MT-binding domain, and DysN_Term-R₁₀ (Rybakova et al., 2006), which encodes the amino-terminal, tandem CH actin-binding domain and spectrinlike repeats 1–10 of the middle rod domain absent from Dp260. A small amount of Dp260 pelleted in the absence of MTs, but substantially more Dp260 shifted to the pellet fraction when MTs were present (Fig. 3 A). After subtracting self-pelleting Dp260, Dp260 displayed a concentration-dependent and saturable cosedimentation with a Dp260/α-β tubulin heterodimer stoichiometry of 1:1.4 and a K_d of 0.66 µM (Fig. 3 C). As predicted, DysN_Term-R₁₀ did not cosediment with MTs up to concentrations approaching 10 µM (Fig. 3, B and C). Next, we assessed how the presence of 1 µM Dp260 affected the tubulin–MT equilibrium in vitro. Dp260 had no significant effect on the fraction of tubulin in the MT fraction when incubated at room temperature (67.3 ± 0.72% vs. 68.6 ± 1.3%). However, the presence of Dp260 significantly increased the fraction of tubulin retained in the MT pellet (33.6 ± 2.9% vs. 42.2 ± 2.0%) when MTs were induced to depolymerize by incubating at 4°C. (Fig. 3, D and E). Collectively, these results demonstrate that dystrophin directly binds and stabilizes MTs from cold-induced depolymerization.

Because misregulation of other MAPs can alter tubulin expression and MT stability (Harada et al., 1994; Takahashi et al., 2003), we investigated how the loss of dystrophin affects the regulation of tubulin expression and the tubulin–MT equilibrium in skeletal muscle fibers. Tubulin levels in wt and mdx skeletal muscle extracts were examined by quantitative Western blot analysis. With mAb B512, we observed no difference in α-tubulin expression between wt and mdx skeletal muscle extracts (Fig. 4, A and B), which was consistent with what we (Prins et al., 2008) and others (Barton et al., 2002) reported previously. However, mAb DM1A showed an ~2.5-fold increase in α-tubulin expression in mdx skeletal muscle (Fig. 4, A and B). Because levels of α- and β-tubulin are coregulated (Gonzalez-Garay and Cabral, 1995), we investigated β-tubulin levels to determine whether α-tubulin is up-regulated in mdx skeletal muscle. β-Tubulin expression was elevated 2.5-fold in mdx skeletal muscle (Fig. 4, A and B), suggesting that expression of both α- and β-tubulin is increased in mdx skeletal muscle. Thus, we conclude that mAb DM1A is able to recognize a population of α-tubulin not detected by mAb B512. To examine MT stability in mdx skeletal muscle, we analyzed levels of tyrosinated α-tubulin, a marker of dynamic MTs (Gundersen et al., 1984, 1987), and acetylated α-tubulin, a marker of long-lived MTs (Bulinski and Gundersen, 1991). The levels of tyrosinated α-tubulin were increased ~2.5-fold in mdx extracts (Fig. 4, A and B), whereas the levels of acetylated α-tubulin were not (Fig. 4, A and B). The loss of dystrophin's MT-stabilizing ability may explain why acetylated α-tubulin was not more abundant in mdx skeletal muscle extracts, but alterations in the tubulin–MT equilibrium could also explain the lack of more stable MTs. Therefore, we examined the tubulin–MT equilibrium in wt and mdx skeletal muscles and found that the loss of dystrophin did not affect the equilibrium (Fig. 4, C and D). Collectively, these results show that tubulins are misregulated in dystrophin-deficient skeletal muscle without affecting the tubulin–MT equilibrium. The loss of dystrophin's MT-stabilizing ability likely explains why there are not more stabilized MTs even in the presence of more tubulin dimer in dystrophin-deficient skeletal muscle.

An indirect link between dystrophin and MTs mediated by ankyrin-B was recently shown to be important for proper trafficking of dystrophin and β-dystroglycan to the sarcolemma (Ayalon et al., 2008). However, costameric MTs are disorganized in mdx skeletal muscle even in the presence of properly localized ankyrin-B (Ayalon et al., 2008). Because the MT- and ankyrin-B–binding domains of dystrophin do not overlap (Fig. 5), our results and previous results suggest that dystrophin interacts with MTs in vivo through two distinct mechanisms. We propose that ankyrin-B delivers dystrophin to the sarcolemma dependent on MTs and that dystrophin and ankyrin-B collaborate to stabilize and organize MTs in skeletal muscle.

As with other cytolinkers, the ability to bind multiple components of the filamentous cytoskeleton likely allows dystrophin to protect the sarcolemma from mechanically induced damage. One highly truncated microdystrophin construct (ΔR4-23) is very effective in restoring function in the dystrophin-deficient mdx mouse (Harper et al., 2002). Interestingly, the ΔR4-23 microdystrophin contains all sequences required for interaction with the three cytoskeletal filament systems: the amino-terminal tandem CH domain, which binds actin (Way et al., 1992) and cytokeratin filaments (Stone et al., 2005), the spectrinlike repeat 3 and the cysteine-rich regions, which are necessary for synemin intermediate filament binding (Bhosle et al., 2006), and the MT-binding domain. In contrast, Dp260 lacks the cytokeratin filament–binding domain and portions of the synemin- and actin-binding domains, which likely alters the binding affinities to both actin and synemin filaments and may explain why transgenic overexpression of Dp260 only partially alleviates the mdx phenotype (Warner et al., 2002). Because Dp116 harbors only the MT-binding domain and one of two synemin-binding sequences, an inability to bind cytokeratin filaments and actin filaments likely explains why transgenic overexpression of Dp116 fails to rescue the mdx phenotype (Judge et al., 2006). Finally, the mild muscle phenotypes of γ_cyto-actin (Sonnemann et al., 2006) or keratin 19 knockout mice (Stone et al., 2007) may be explained by dystrophin's linkage with the remaining components of the cortical cytoskeleton. Collectively, these results support the hypothesis that dystrophin must bind all three components of the cellular cytoskeleton to function properly in skeletal muscle.