Cloning and characterization of cytokeratins 8 and 19 in adult rat striated muscle. Interaction with the dystrophin glycoprotein complex.
ABSTRACT We used degenerate primers for the amino- and carboxyl-terminal ends of the rod domains of intermediate filament proteins in reverse transcriptase-PCR experiments to identify and clone cytokeratins 8 and 19 (K8 and K19) from cardiac muscle of the adult rat. Northern blots showed that K8 has a 2.2-kb transcript and K19 has a 1.9-kb transcript in both adult cardiac and skeletal muscles. Immunolocalization of the cytokeratins in adult cardiac muscle with isoform-specific antibodies for K8 and K19 showed labeling at Z-lines within the muscle fibers and at Z-line and M-line domains at costameres at the sarcolemmal membrane. Dystrophin and K19 could be co-immunoprecipitated and co-purified from extracts of cardiac muscle, suggesting a link between the cytokeratins and the dystrophin-based cytoskeleton at the sarcolemma. Furthermore, transfection experiments indicate that K8 and K19 may associate with dystrophin through a specific interaction with its actin-binding domain. Consistent with this observation, the cytokeratins are disrupted at the sarcolemmal membrane of skeletal muscle of the mdx mouse that lacks dystrophin. Together these results indicate that at least two cytokeratins are expressed in adult striated muscle, where they may contribute to the organization of both the myoplasm and sarcolemma.
- SourceAvailable from: Karla Garcia-Pelagio[show abstract] [hide abstract]
ABSTRACT: We studied the biomechanical properties of the sarcolemma and its links through costameres to the contractile apparatus in single mammalian myofibers of Extensor digitorum longus muscles isolated from wild (WT) and dystrophin-null (mdx) mice. Suction pressures (P) applied through a pipette to the sarcolemma generated a bleb, the height of which increased with increasing P. Larger increases in P broke the connections between the sarcolemma and myofibrils and eventually caused the sarcolemma to burst. We used the values of P at which these changes occurred to estimate the tensions and stiffness of the system and its individual elements. Tensions of the whole system and the sarcolemma, as well as the maximal tension sustained by the costameres, were all significantly lower (1.8-3.3 fold) in muscles of mdx mice compared to WT. Values of P at which separation and bursting occurred, as well as the stiffness of the whole system and of the isolated sarcolemma, were ~2-fold lower in mdx than in WT. Our results indicate that the absence of dystrophin reduces muscle stiffness, increases sarcolemmal deformability, and compromises the mechanical stability of costameres and their connections to nearby myofibrils.Journal of Muscle Research and Cell Motility 03/2011; 31(5-6):323-36. · 1.36 Impact Factor
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ABSTRACT: Dystrophin is an actin binding protein that is thought to stabilize the cardiac and skeletal muscle cell membranes during contraction. Here, we investigated the contributions of each dystrophin domain to actin binding function. Cosedimentation assays and pyrene-actin fluorescence experiments confirmed that a fragment spanning two-thirds of the dystrophin molecule [from N-terminal actin binding domain (ABD) 1 through ABD2] bound actin filaments with high affinity and protected filaments from forced depolymerization, but was less effective in both assays than full-length dystrophin. While a construct encoding the C-terminal third of dystrophin displayed no specific actin binding activity or competition with full-length dystrophin, our data show that it confers an unexpected regulation of actin binding by the N-terminal two-thirds of dystrophin when present in cis. Time-resolved phosphorescence anisotropy experiments demonstrated that the presence of the C-terminal third of dystrophin in cis also influences actin interaction by restricting actin rotational amplitude. We propose that the C-terminal region of dystrophin allosterically stabilizes an optimal actin binding conformation of dystrophin.Journal of Molecular Biology 02/2012; 416(3):414-24. · 3.91 Impact Factor
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ABSTRACT: In skeletal muscle fibers, forces must be transmitted between the plasma membrane and the intracellular contractile lattice, and within this lattice between adjacent myofibrils. Based on their prevalence, biomechanical properties and localization, desmin and keratin intermediate filaments (IFs) are likely to participate in structural connectivity and force transmission. We examined the passive load-bearing response of single fibers from the extensor digitorum longus (EDL) muscles of young (3 months) and aged (10 months) wild-type, desmin-null, K19-null, and desmin/K19 double-null mice. Though fibers are more compliant in all mutant genotypes compared to wild-type, the structural response of each genotype is distinct, suggesting multiple mechanisms by which desmin and keratin influence the biomechanical properties of myofibers. This work provides additional insight into the influences of IFs on structure-function relationships in skeletal muscle. It may also have implications for understanding the progression of desminopathies and other IF-related myopathies.BioMed Research International 01/2012; 2012:704061. · 2.88 Impact Factor