Membrane sterol heterogeneity. Freeze-fracture detection with saponins and filipin.

Abstract

Although alterations in cholesterol content 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, regions containing cholesterol-reagent aggregates in artificial membranes as well as in membranes of whole cells and solid tissues were demonstrated. Ultrastructural and quantitative studies revealed that the saponins and filipin bound free cholesterol, 7-dehydrocholesterol, cholestanol, desmosterol 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-.beta.-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), the saponins and filipin plicated plasma membranes and Golgi complex-derived secretory vesicles, but generally spared the nuclear envelope and mitochondria. The saponins and filipin revealed consistent heterogeneous patterns of aggregate density in sperm and epithelial plasmalemmae, but not in leukocyte membranes. 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 plasma 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. Saponins and filipin form distinctive complexes with free 3-.beta.-hydroxysterols, and exhibit individual, reproducible patterns of membrane-protuberance heterogeneity in sperm and epithelial tissues and thereby provide a novel cytochemical tool for freeze-fracture.