Freeze-fracture evidence for the presence of cholesterol in particle-free patches of basal disks and the plasma membrane of retinal rod outer segments of mice and frogs

Abstract

The freeze-fracture technique was used to examine the membranes of the photoreceptors of mice and frogs. Particle-free patches were found in the plasma membrane and basal disk membranes of the outer segments of both mice and frogs housed at room temperature, but not in frogs kept in a cold room. These patches were shown not to be artifacts of cryoprotection or fixation, and they persisted when fresh isolated outer segments were frozen by an ultrarapid method. They were also found to persist in mouse rods when retinas were incubated and subsequently fixed at temperatures up to 80 degrees C. Cholesterol was implicated as a significant component of the patches by the observation that, in the outer segments, pits, induced by treatment with the sterol-specific polyene antibiotic filipin, were present in and confined to the particle-free patches. That these lesions are not inherently limited to particle-free membrane areas was evident in the apical plasma membrane of the photoreceptor inner segments, where particles and pits were intermixed. Treatment with saponin, a surface-active agent which specifically complexes cholesterol, resulted in the disappearance of the particle-free patches. Patches were found in basal disks of both mouse and frog rods but not in older disks nearer the pigment epithelium, which indicates that changes occur in the composition of disk membranes and/or in the molecular ordering of their protein and lipid components during the early phase of their transit from the base towards the apex of the outer segment.