Sarcomeric binding pattern of exogenously added intact caldesmon and its C-terminal 20-kDa fragment in skinned fibers of skeletal muscle

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 signi ficantly altering the actin attachment of force producing, strong binding c ross-bridges (Brenner et al., 1991; Kraft et al., 1995a). However, the sarc omeric distribution and the specific binding of externally added caldesmon to the myofilaments of skeletal muscle fibers was not known. It was e.g., u nclear whether caldesmon binds along actin in a manner similar to tropomyos in or whether it also binds to myosin. In this study, we determined the bin ding pattern of exogenously added intact caldesmon and its C-terminal 20-kD a fragment, respectively, in MgATP-relaxed rabbit skeletal muscle fibers us ing electron (EM) and confocal fluorescence microscopy (CFM). EM showed tha t similar to what has been demonstrated earlier for smooth muscle thin fila ments (Lehman et al., 1989), intact caldesmon binds periodically every 38 n m along the thin filaments. CFM revealed that rhodamine-labeled intact cald esmon 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 unl abeled, both when equilibrated at normal and long sarcomere lengths. The wi dth of the unlabeled region seems to depend on ionic strength. The 20-kDa C -terminal caldesmon fragment binds in essentially the same pattern as intac t caldesmon. This indicates that the high fluorescence intensity in the ove rlap region seen with intact caldesmon does not depend on caldesmon binding to myosin. X-ray diffraction was used to monitor the effects on filament l attice. Intact caldesmon at > 0.3 mg/ml induced disorder in the myofilament lattice. No such disordering was observed, however, when fibers were equil ibrated with up to 0.8 mg/ml of the 20-kDa caldesmon fragment.