Freeze-fracture identification of sterol-digitonin complexes in cell and liposome membranes.

Abstract

Digitonin in freeze-fracture investigations of model lipid vesicles and tissues was used in an attempt to understand the organization of cholesterol within cell membranes. Cholesterol suspensions or multilamellar liposomes composed of phosphatidylcholine with and without cholesterol were exposed to digitonin. Freeze-fracture replicas of those multilamellar liposomes containing cholesterol displayed either 50-60 nm wide intramembrane corrugations or extramembrane tubular complexes. Comparable intramembrane hemitubular scallops and extracellular free tubular complexes were observed in thin sections. Exposure of guinea pig sperm, human erythrocytes (whole and ghosts), and intact Swiss mouse tissues (skin, liver, adrenal gland, epididymis) to digitonin produced the same types of intra- and extramembrane complexes or furrows as were formed in liposomes. The plasma membrane of guinea pig serum tail had 2 unfurrowed regions: the annulus and the zipper. Incubating erythrocyte membranes with digitonin resulted in rapid displacement of cholesterol, accompanied by intramembrane particle clustering and membrane faceting, a feature which was not seen in the intact epithelia studied. In freeze-fractured epithelia, plasma membranes, lysosomes, and some vesicular organelles commonly furrowed, but mitochondrial membranes and nuclear envelopes were generally spared, correlating well with their known cholesterol content. Plasma membrane corrugations approached but did not impinge on either gap or tight junctions, or on coated vesicles. Freeze-fracture of membranes exposed to digitonin reveals distinctive cholesterol-digitonin structural complexes, distinguishes cholesterol-rich and -poor organelle membranes and demonstrates membrane domains rich or poor in cholesterol.