Alterations in the morphology of rabbit skeletal muscle plasma membrane during membrane isolation

This study describes changes in morphology of plasmalemma from fast skeletal muscle in the course of tissue disruption and isolation. We find that conditions used to solubilize muscle contractile elements, in the isolation of plasmalemma, including the use of 0.6 M KCl or 0.4 M LiBr in the cold (0-4 degrees C), lead to altered plasmalemma morphology. The intramembrane particles, as revealed by freeze-fracture electron microscopy, become aggregated, leaving large domains devoid of particles. The square arrays in the P face and the complementary "pits" in the E face also become aggregated, sometimes forming sizeable aggregates of square arrays. Thin-section electron microscopy using tannic acid enhancement reveals plasma membrane associated components, on both cytoplasmic and extracellular faces, are largely reduced by the salt treatment. Pyrophosphate and magnesium at lower concentrations, sometimes used instead of high salt, also resulted in particle aggregation, although less pronounced than with concentrated salt solutions. The plasma membrane-associated proteins on both plasma membrane surfaces were likewise decreased by this treatment. Pyrophosphate treatment also separated the basal lamina from the plasma membrane. Incubation of muscle in isoosmotic sucrose does not alter the morphology of the plasmalemma with regard to particle aggregation, diminution of membrane associated components, or separation of the basal lamina. Our observations suggest that membrane-associated protein and/or cytoskeleton constrains the mobility of components in the plane of the membrane and that removal of this constraint leads to aggregation of intramembrane particles.