Identification of Spectrin-like Repeats Required for High Affinity Utrophin-Actin Interaction

Abstract

Most studies aimed at characterizing the utrophinactin interaction have focused on the amino-terminal tandem calponin homology domain. However, we recently reported evidence suggesting that spectrin-like repeats of utrophin also participate in binding to actin. Here we expressed several recombinant fragments encoding the utrophin amino-terminal domain alone or in combination with various numbers of spectrin-like repeats. We further quantitatively characterized the actin binding properties of each recombinant utrophin fragment using a high-speed sedimentation assay. To evaluate the capacity of each protein to stabilize actin filaments, we compared the effect of utrophin recombinant fragments and full-length utrophin on 6-propionyl-2-(N,N-dimethylamino)naphthalene actin depolymerization. Our results suggest that, whereas the amino-terminal domain is essential for primary interaction between utrophin and actin, spectrin-like repeats have additive effects on the affinity and stoichiometry of binding. Our data indicate that the amino-terminal domain and first 10 consecutive spectrin-like repeats recapitulate the actin binding activity of full-length utrophin more faithfully than the amino-terminal domain alone. These findings support the model for lateral association of utrophin along the actin filament and provide the molecular basis for designing the most effective utrophin "mini-genes" for treatment of dystrophinopathies.

Versatile fluorescent probes for actin filaments based on the actin‐binding domain of utrophin

“…Second, the UtrCH probes have no apparent untoward effects on the actin cytoskeleton. This conclusion is based several findings: even excessive GFP-UtrCH does not increase the amount of pelletable F-actin, in keeping with the report showing that UtrCH does not reduce the rate of actin disassembly in vitro [Rybakova and Ervasti, 2005]. In addition, comparison of the wound healing process by both inspection and quantification revealed no differences between fluorescent actin and the UtrCH probes.…”

“…We chose this domain (UtrCH) based on the demonstration that it binds to, without stabilizing, F-actin in in vitro dilution assays [Rybakova and Ervasti, 2005]. Three fusion constructs based on UtrCH were generated: Green fluorescent protein-UtrCH (GFPUtrCH), Red fluorescent protein-UtrCH (RFP-UtrCH) and photoactivatable GFP-UtrCH [PaGFP-Utr-CH; Patterson and Lippincot-Schwartz, 2002].…”

Mouse Regenerating Myofibers Detected as False-Positive Donor Myofibers with Anti-Human Spectrin

“…S3). Mouse utrophin contains a number of spectrin-like repeat domains that allow its binding to actin (Rybakova and Ervasti, 2005) (Supplementary Fig. S3).…”

Absence of keratin 19 in mice causes skeletal myopathy with mitochondrial and sarcolemmal reorganization

“…Dystrophin, the protein missing or mutated in Duchenne and Becker muscular dystrophies, has an N-terminal actin-binding domain that binds F-actin microfilaments with an affinity of 10-50 M Rybakova et al, 1996;Senter et al, 1993;Sutherland-Smith et al, 2003) but its association with actin is enhanced by sequences in its rod domain Warner et al, 2002;Rybakova and Ervasti, 2005). Dystrophin-actin interactions at the sarcolemma are important, because the absence of dystrophin in a murine muscular dystrophy weakens the association of ␥-actin with the Z-domains of costameres (Rybakova et al, 2000), severely altering the organization of costameres (Williams and Bloch, 1999).…”