Binding of dystrophin's tandem calponin homology domain to F-actin is modulated by actin's structure.

Dystrophin has been shown to be associated in cells with actin bundles. Dys-246, an N-terminal recombinant protein encoding the first 246 residues of dystrophin, includes two calponin-homology (CH) domains, and is similar to a large class of F-actin cross-linking proteins including alpha-actinin, fimbrin, and spectrin. It has been shown that expression or microinjection of amino-terminal fragments of dystrophin or the closely related utrophin resulted in the localization of these protein domains to actin bundles. However, in vitro studies have failed to detect any bundling of actin by either intact dystrophin or Dys-246. We show here that the structure of F-actin can be modulated so that there are two modes of Dys-246 binding, from bundling actin filaments to only binding to single filaments. The changes in F-actin structure that allow Dys-246 to bundle filaments are induced by covalent modification of Cys-374, proteolytic cleavage of F-actin's C-terminus, mutation of yeast actin's N-terminus, and different buffers. The present results suggest that F-actin's structural state can have a large influence on the nature of actin's interaction with other proteins, and these different states need to be considered when conducting in vitro assays.

The dystrophin complex forms a mechanically strong link between the sarcolemma and costameric actin.

The absence of dystrophin complex leads to disorganization of the force-transmitting costameric cytoskeleton and disruption of sarcolemmal membrane integrity in skeletal muscle. However, it has not been determined whether the dystrophin complex can form a mechanically strong bond with any costameric protein. We performed confocal immunofluorescence analysis of isolated sarcolemma that were mechanically peeled from skeletal fibers of mouse hindlimb muscle. A population of gamma-actin filaments was stably associated with sarcolemma isolated from normal muscle and displayed a costameric pattern that precisely overlapped with dystrophin. However, costameric actin was absent from all sarcolemma isolated from dystrophin-deficient mdx mouse muscle even though it was localized to costameres in situ. Vinculin, alpha-actinin, beta-dystroglycan and utrophin were all retained on mdx sarcolemma, indicating that the loss of costameric actin was not due to generalized membrane instability. Our data demonstrate that the dystrophin complex forms a mechanically strong link between the sarcolemma and the costameric cytoskeleton through interaction with gamma-actin filaments. Destabilization of costameric actin filaments may also be an important precursor to the costamere disarray observed in dystrophin-deficient muscle. Finally, these methods will be broadly useful in assessing the mechanical integrity of the membrane cytoskeleton in dystrophic animal models lacking other costameric proteins.

Dystrophin binding to nonmuscle actin.

We purified actin from bovine brain by DNase I affinity chromatography in order to compare the binding of dystrophin to muscle actin with its binding to nonmuscle actin. While both beta- and gamma-nonmuscle actins are expressed in brain, Western blot analysis with isoform-specific antibodies indicated that our purified brain actin was exclusively the gamma-isoform. The recombinant amino-terminal, actin-binding domain of dystrophin bound to muscle and brain actin in a saturable manner (approximately 1 mol/mol actin) with similar Kd values of 13.7+/-3.5 and 10.6+/-3.7 microM, respectively. We further demonstrate that intact dystrophin in the dystrophin-glycoprotein complex bound with equal avidity to muscle and brain F-actin. These data argue that a preferential binding of dystrophin to nonmuscle actin is not the basis for its targeting to the muscle cell plasmalemma but do support the hypothesis that dystrophin is capable of interacting with filamentous actin in nonmuscle tissues.

Dystrophin-glycoprotein complex is monomeric and stabilizes actin filaments in vitro through a lateral association.

The native molecular weight of the dystrophin-glycoprotein complex and its effect on actin depolymerization and polymerization were examined. First, we determined that the native molecular weight of purified dystrophin-glycoprotein complex is only large enough (Mr 1,200,000) to contain one copy of each protein in the complex, including dystrophin. Using different approaches, we also demonstrated that dystrophin-glycoprotein complex significantly protected a fraction of actin filaments from disassembly, while individual recombinant actin binding fragments of dystrophin or calpain-digested dystrophin-glycoprotein complex had no effect on F-actin depolymerization. The protective effect of dystrophin-glycoprotein complex on F-actin depolymerization saturated at a dystrophin:actin molar ratio of 0.04, corresponding to 1 dystrophin/25 actin monomers, which is highly consistent with the 1:24 stoichiometry of dystrophin-glycoprotein complex binding to F-actin previously measured at equilibrium. However, dystrophin-glycoprotein complex did not bind G-actin or alter the kinetics or extent of actin polymerization. This excluded the possibility that dystrophin-glycoprotein complex inhibited actin depolymerization by capping the ends of actin filaments. It therefore appears that actin binding domains separated on the dystrophin molecule from each other by almost 1,200 amino acids act in concert to protect F-actin from depolymerization. Our data suggest that dystrophin stabilizes F-actin in vitro by binding alongside an actin filament and bridging actin monomers in a manner analogous to the actin side binding protein tropomyosin. It is noteworthy that we did not find any effect of skeletal muscle tropomyosin on dystrophin-glycoprotein complex binding to F-actin. This indicates that dystrophin-glycoprotein complex and tropomyosin may simultaneously bind the same actin filament and identifies another feature that distinguishes dystrophin from the other proteins in the actin-cross-linking superfamily.

A new model for the interaction of dystrophin with F-actin.

The F-actin binding and cross-linking properties of skeletal muscle dystrophin-glycoprotein complex were examined using high and low speed cosedimentation assays, microcapillary falling ball viscometry, and electron microscopy. Dystrophin-glycoprotein complex binding to F-actin saturated near 0.042 +/- 0.005 mol/ mol, which corresponds to one dystrophin per 24 actin monomers. Dystrophin-glycoprotein complex bound to F-actin with an average apparent Kd for dystrophin of 0.5 microM. These results demonstrate that native, full-length dystrophin in the glycoprotein complex binds F-actin with some properties similar to those measured for several members of the actin cross-linking super-family of proteins. However, we failed to observe dystrophin-glycoprotein complex-induced cross-linking of F-actin by three different methods, each positively controlled with alpha-actinin. Furthermore, high speed cosedimentation analysis of dystrophin-glycoprotein complex digested with calpain revealed a novel F-actin binding site located near the middle of the dystrophin rod domain. Recombinant dystrophin fragments corresponding to the novel actin binding site and the first 246 amino acids of dystrophin both bound F-actin but with significantly lower affinity and higher capacity than was observed with purified dystrophin-glycoprotein complex. Finally, dystrophin-glycoprotein complex was observed to significantly slow the depolymerization of F-actin, Suggesting that dystrophin may lie along side an actin filament through interaction with multiple actin monomers. These data suggest that although dystrophin is most closely related to the actin cross-linking superfamily based on sequence homology, dystrophin binds F-actin in a manner more analogous to actin side-binding proteins.

Nonnuclear DNA binding proteins in striated muscle.

The mechanism by which striated muscle internalizes plasmid DNA is unknown. A survey of nonnuclear membrane-associated DNA binding proteins from both skeletal and cardiac muscle revealed several sarcoplasmic reticulum restricted DNA binding species. 22P-DNA overlay and DNA-cellulose chromatography were used to identify membrane-associated DNA binding proteins that may mediate the uptake and expression of plasmid DNA by striated muscle. A total membrane vesicle fraction prepared from rabbit skeletal muscle contained 95-, 60-, and 28-kDa proteins that bound double-strand DNA specifically and with high affinity. The DNA binding proteins appear to originate from the sarcoplasmic reticulum because of their absence in purified sarcolemma vesicles and codistribution with several sarcoplasmic reticulum markers after subcellular fractionation. Several distinguishing biochemical features as well as cross-reactivity with triadin-specific antibodies indicated that the 95- and 60-kDa DNA binding proteins are triadin or proteolytic fragments of triadin, respectively. The role of these sarcoplasmic reticulum proteins in the transport of plasmid DNA is discussed.