DEMONSTRATION OF THE OUTER SURFACE OF FREEZE-ETCHED RED BLOOD CELL MEMBRANES

Abstract

Freeze-etching promises to be a valuable technique for the ultrastructural study of cells and subcellular components. The tissue specimen is cleaved while frozen by liquid nitrogen, and the exposed faces are replicated by platinum-carbon shadowing while under high vacuum. Thus, this technique eliminates the artifacts of thin-section microscopy produced by fixation, dehydration, embedding, and heavy metal staining. Through the use of this technique, a new feature of cell membrane ultrastructure has been revealed. Cleavage of cell membranes results in the appearance of regularly spaced globular units approximately 85 A in diameter on the exposed membrane face (1, 2). The chemical composition and possible functional properties of the globular units are as yet unknown, and there is also some controversy as to the anatomical location of these structures within the membrane. This controversy is based on a fundamental disagreement as to which face of the membrane is exposed by the cleavage process. Moor and M/ihlethaler (1) and others (3, 4) have suggested that membranes are cleaved along planes which expose either their true outer or cytoplasmic surfaces. According to this view, the 85-A particles are located on the outside surface of the cell membrane; some are also present on the cytoplasmic side of the membrane. It has even been suggested that the particles extend completely through the thickness of the membrane (5). However, studies by Branton (2, 6) question the above interpretations. An analysis of the appearance of membranes in freeze-etched preparations and freeze-cleaving experiments with lipid bilayers has led Branton to conclude that the freeze-fracture process actually splits membranes along a plane within the membrane itself rather than cleaving along its outer or cytoplasmic surface. If this is the case, then the globular particles seen on freeze-cleaved membrane faces would be located somewhere within the interior of the membrane rather than directly on the surface. The usefulness of the freeze-etching technique in the study of membranes clearly requires that the controversy over the location of the cleavage plane in membranes be resolved. One solution would be to label the outer or inner surface of tile membrane with molecules of known structure which can be identified in the freeze-etch prepara-