Fluorescent localization of membrane sites in glycerinated chicken skeletal muscle fibers and the relationship of these sites to the protein composition of the Z disc

Abstract

Didansyl derivatives of amino acids and N-phenyl-1-naphthylamine were used to localize membrane hydrophobic sites in glycerol-extracted chicken skeletal muscle fibers. Epifluorescence microscopy revealed that such sites coincide with the distribution of mitochondria, the transverse tubular (T) system and the sarcoplasmic reticulum (SR). They are specifically associated with myofibril Z lines and occasionally extend from one Z plane to the next longitudinally along the muscle fiber. The hydrophobic probes interact noncovalently with the Z lines, and their induced fluorescence can be eliminated by exposure of the myofibrils to ionic detergents, nonionic detergents, or phospholipase C, before or after addition of the hydrophobic label. Extraction of glycerinated fibers with 0.6 M KI removes the majority of sarcomeric actin and myosin and leaves a scaffold of longitudinally interconnected Z planes. Membrane fluorescence remains tightly associated with these Z planes and with the remnant mitochondria. Shearing of such scaffolds results in the cleavage of the longitudinal connections and the production of large sheets of interconnected, close-packed Z discs in a honeycomb-like array. Comparison of the localization of two Z disc proteins, desmin and alpha-actinin, with that of the membrane material reveals that alpha-actinin is localized in the interior of each myofibril Z disc whereas both desmin and the membrane material surround each disc. Thus, glycerination and KI extraction of muscle fibers leaves remnants of T system and SR membranes tightly associated with the Z disc honeycomb lattice. Because the Z discs are connected at their peripheries through the T system appear to the plasma membrane, desmin and this membrane structure appear to be connected throughout the whole Z plane up to and including the plasma membrane. The congruent localization of desmin and the T system strongly suggests that this molecule mediates the adhesion of this membrane system around each Z disc.