Ankyrin-B Interactions with Spectrin and Dynactin-4 Are Required for Dystrophin-based Protection of Skeletal Muscle from Exercise Injury

Abstract

Costameres are cellular sites of mechanotransduction in heart and skeletal muscle where dystrophin and its membrane-spanning partner dystroglycan distribute intracellular contractile forces into the surrounding extracellular matrix. Resolution of a functional costamere interactome is still limited but likely to be critical for understanding forms of muscular dystrophy and cardiomyopathy. Dystrophin binds a set of membrane-associated proteins (the dystrophin-glycoprotein complex) as well as γ-actin and microtubules and also is required to align sarcolemmal microtubules with costameres. Ankyrin-B binds to dystrophin, dynactin-4, and microtubules and is required for sarcolemmal association of these proteins as well as dystroglycan. We report here that ankyrin-B interactions with β2 spectrin and dynactin-4 are required for localization of dystrophin, dystroglycan, and microtubules at costameres as well as protection of muscle from exercise-induced injury. Knockdown of dynactin-4 in adult mouse skeletal muscle phenocopied depletion of ankyrin-B and resulted in loss of sarcolemmal dystrophin, dystroglycan, and microtubules. Moreover, mutations of ankyrin-B and of dynactin-4 that selectively impaired binary interactions between these proteins resulted in loss of their costamere-localizing activity and increased muscle fiber fragility as a result of loss of costamere-associated dystrophin and dystroglycan. In addition, costamere-association of dynactin-4 did not require dystrophin but did depend on β2 spectrin and ankyrin-B, whereas costamere association of ankyrin-B required β2 spectrin. Together, these results are consistent with a functional hierarchy beginning with β2 spectrin recruitment of ankyrin-B to costameres. Ankyrin-B then interacts with dynactin-4 and dystrophin, whereas dynactin-4 collaborates with dystrophin in coordinating costamere-aligned microtubules.

Annexin B9 binds to βH-spectrin and is required for multivesicular body function in Drosophila

“…However, spectrin is now emerging as a significant modulator of trafficking processes. Spectrin binding is an essential step for Rab7-stimulated dynein activation during transport to lysosomes (Johansson et al, 2007) and for the localization of dynactin to costameres in muscle (Ayalon et al, 2011), suggesting that spectrin is an integral part of the dyneindynactin system. Furthermore, expression of mutant b-spectrin isoforms in the fly leads to dynein-dynactin-based axonal transport defects (Lorenzo et al, 2010).…”

Obscure functions: the location–function relationship of obscurins

“…Obscurin polypeptides interact with ankyrin-B to provide a molecular bridge between membrane and cytoskeletal proteins, thereby promoting the assembly of essential protein complexes in skeletal muscle (Randazzo et al 2013). At costameres, ankyrin-B complexes with dynactin-4 and β 2 -spectrin, both of which are necessary for the proper localization of dystrophin, a key cytoskeletal protein (Ayalon et al 2008(Ayalon et al , 2011. In the absence of giant obscurins, ankyrin-B is absent from costameres, which results in mislocalized dystrophin and alterations in the organization of costamere-associated microtubules (Randazzo et al 2013).…”

Obscurin is required for ankyrinB-dependent dystrophin localization and sarcolemma integrity

“…These authors proposed a hierarchical model, in which the organization of a functional DGC at the costamere level starts with the recruitment of ankB by the cytoskeletal protein 2-spectrin. AnkB in turn, interacts with both dystrophin and dynactin-4 to coordinate the correct delivery of dystrophin and -dystroglycan through the subsarcolemma microtubule cytoskeleton (Ayalon et al, 2011).…”

“…More recently, two muscle-specific ankyrin isoforms, ankB and ankG, have been shown to have a key role in the organization of dystrophin and -dystroglycan at costameres and at neuromuscular junctions in skeletal muscles fibers (Ayalon et al, 2008(Ayalon et al, , 2011. Dystrophin and -dystroglycan participate in the assembly of the dystrophin-glycoprotein complex (DGC), containing integral membrane components (sarcospan, -, -, -, and -sarcoglycans, and -dystroglycan) and peripheral membrane proteins (-dystroglycan, 1/1-syntrophins, and dystrophin; Crosbie et al, 1997;Ervasti, 2003).…”