Reorganization of Membrane Cholesterol at anEarly Stage of Exocytosis (Degranulation) in Rat Mast Cells: Inferred from the Ditribution of Filipin-Cholesterol Comlexes

Abstract

Using filipin, a sterol-specific polyene antibiotic, we examined the redistribution of cholesterol molecules present at an early stage of degranu-lation (release of heparin-protein complexes) in rat peritoneal mast cells with freeze-fracture electron microscopy. Isolated mast cells first were stimulated to undergo degranulation with compound 48/80 (1 μg/ml) for 3 sec, then were treated with a glutaraldehyde solution containing filipin (50 μg/ml) for 30 min. Freeze-fractures of the plasma membrane of these cells showed small depressions which lacked filipin-cholesterol complexes. These are the first recognizable membrane alterations and are suggested to correspond to the initial sites of fusion between the plasma and granule membranes. Subsequent-ly, these depressions became bulges. Filipin-cholesterol complexes are absent from the portion of the bulges in close contact with the plasma membrane, probably because the bulges have not yet fused with the underlying granule membrane. These complexes also were absent from the periphery of the two apposed membranes at exocytotic sites during fusion. Even after fusion was complete, no complexes appeared in the circumferential zone where the plasma membrane and the secretory granule membrane had fused. Our results suggest that membrane cholesterol is reorganized at the fusion sites between the plasma and granule membranes during mast cell secretion, and that this alteration in the organization of cholesterol in the plasma membrane represents an initial event during mast cell secretion. A hypothetical model for the sequential changes in the plasma membrane during degranulation is proposed.