MEMBRANE STEROL HETEROGENEITY - FREEZE-FRACTURE DETECTION WITH SAPONINS AND FILIPIN

Although alterations in cholesterol content are known to influence the permeability, fluidity, and enzymatic activity of cell membranes, little information is available about the planar distribution of cholesterol in individual membranes. Using the saponins, tomatin and digitonin, and the polyene antibiotic, filipin, we demonstrated regions containing cholesterol-reagent aggregated in artificial membranes as well as membranes of whole cells and solid tissues. Ultrastructural and quantitative studies revealed that both the saponins and filipin bound free cholesterol, 7-dehydrocholesterol, cholestanol, desmonsterol, and lanosterol (but not epicholesterol) in multilamellar liposomes (phosphatidylcholine:sterol, 2:1 mole ratio). Whereas the saponins formed tubular complexes (50-60 nm in diameter), filipin produced hemispherical bulges (15-25 nm wide). Comparable structures appeared when suspensions of 3-β-hydroxysterols were treated with the saponins and filipin, indicating that the sterol itself is the membrane constituent which determines the configuration of the aggregates. In mammalian tissues (liver, epidermis) and cell suspensions (sperm, leukocytes), both the saponins and filipin plicated plasma membranes and Golgi complex-derive secretory vesicles, but generally spared the nuclear envelope and mitochondria. Both the saponins and filipin revealed consistent heterogenous patterns of aggregate density in sperm and epithelial plasmalemmae, but not in leukocyte membranes. However because filipin:sterol aggregates are discrete and countable, gradations in apparent sterol content were more readily detected with filipin than with the saponins. In the sperm plasm membrane, complexes were profuse in the acrosomal cap of the head and in the tail's principal piece, sparse in the postacrosomal segment, and nearly absent in the annulus and zipper. In epithelia, desmosomes, gap junctions, and tight-junction strands were devoid of complexes. We conclude that both saponins and filipin: 1) form distinctive complexes with free 3-β-hydroxysterols, and 2) exhibit individual, reproductible patterns of membrane-protuberance heterogeneity in both sperm and epithelial tissues and thereby provide a novel cytochemical tool for freeze-fracture.