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Intact caldesmon and particularly the actin-binding C-terminal fragment (20-kDa) of caldesmon have been shown in skeletal muscle fibers to selectively displace low affinity, weakly bound cross-bridges from actin without significantly altering the actin attachment of force producing, strong binding cross-bridges (Brenner et al., 1991; Kraft et al., 1995a). However, the sarcomeric distribution and the specific binding of externally added caldesmon to the myofilaments of skeletal muscle fibers was not known. It was e.g., unclear whether caldesmon binds along actin in a manner similar to tropomyosin or whether it also binds to myosin. In this study, we determined the binding pattern of exogenously added intact caldesmon and its C-terminal 20-kDa fragment, respectively, in MgATP-relaxed rabbit skeletal muscle fibers using electron (EM) and confocal fluorescence microscopy (CFM). EM showed that similar to what has been demonstrated earlier for smooth muscle thin filaments (Lehman et al., 1989), intact caldesmon binds periodically every 38 nm along the thin filaments. CFM revealed that rhodamine-labeled intact caldesmon and the 20-kDa caldesmon fragment bind along nearly the entire length of the thin filaments. A portion of the I-band near the Z-line appears unlabeled, both when equilibrated at normal and long sarcomere lengths. The width of the unlabeled region seems to depend on ionic strength. The 20-kDa C-terminal caldesmon fragment binds in essentially the same pattern as intact caldesmon. This indicates that the high fluorescence intensity in the overlap region seen with intact caldesmon does not depend on caldesmon binding to myosin. X-ray diffraction was used to monitor the effects of filament lattice. Intact caldesmon at0.3 mg/ml induced disorder in the myofilament lattice. No such disordering was observed, however, when fibers were equilibrated with up to 0.8 mg/ml of the 20-kDa caldesmon fragment. |
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