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

Gai Ayalon,Janell D Hostettler,Jan Hoffman,Krishnakumar Kizhatil,Jonathan Q Davis,Vann Bennett

doi:10.1074/jbc.m110.187831

Gai Ayalon, Janell D Hostettler + Show 4 more

Open Access

https://doi.org/10.1074/jbc.m110.187831

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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.

Highlights

Potential payoff includes understanding and modifying disease processes
Knockdown of ankyrin-B to undetectable levels by immunofluorescence results within 4 days in loss of sarcolemmal dystrophin, dystroglycan, dynactin-4, and a subpopulation of microtubules aligned with costameres (Fig. 1D) [11]
We found that depletion of ␤2 spectrin results within 5 days in loss of sarcolemmal ankyrin-B, dynactin-4, dystrophin, ␤-dystroglycan, as well as costamere-associated microtubules (Fig. 4C)

Summary

EXPERIMENTAL PROCEDURES

TA muscles were harvested for fiber isolation, processing, and imaging 4 days after ankyrin-B and dynactin-4 siRNA and rescue transfections and 5 days after ␤2 spectrin siRNA transfections. Fibers were washed twice in PBS and incubated 2 h at 4° C in secondary antibody in 3% BSA, 0.05% saponin in PBS. Fibers were washed twice in PBS and incubated with secondary antibody in 3% BSA, 0.2% Tween in PBS for 2 h at 4° C. Eluted dynactin-4 was adjusted to 0.1 mM zinc sulfate and immediately incubated with glutathione-Sepharose beads for immobilization and washed and equilibrated in binding assay buffer (see below). Ankyrin-B-Zu5-DD WT and with the 1320 DD/AA mutation were first isolated on nickel-Sepharose beads followed by an amylose affinity column (New England Biolabs) washed with wash buffer (20 mM Tris, pH 7.4, 0.2 mM NaCl, 1 mM DTT, 0.5 mM EGTA) eluted with the addition of 10 mM maltose. Modeling the location of ankyrinB-DD1320 on the UNC5b structure was based on the alignment of ankyrin-B and UNC5b [20]

RESULTS

DISCUSSION

Davis and Vann Bennett