Arrest of membrane fusion events in mast cells by quick-freezing.

Abstract

We have used quick-freezing and freeze-fracture to study early stages of exocytosis in rat peritoneal mast cells. Mast cells briefly stimulated with 48/80 (a synthetic polycation and well-known histamine-releasing agent) at 22°C displayed single, narrow-necked pores (some as small as 0.05 pm in diameter) joining single granules with the plasma membrane . Pores that had become as large as 0.1 lm in diameter were clearly etchable and thus represented aqueous channels connecting the granule interior with the extracellular space. Granules exhibiting pores usually did not have wide areas of contact with the plasma membrane, and clearings of intramembrane particles, seen in chemically fixed mast cells undergoing exocytosis, were not present on either plasma or granule membranes. Fusion of interior granules later in the secretory process also appeared to involve pores; granules were often joined by one pore or a group of 2-4 pores . Also found were groups of extremely small, etchable pores on granule membranes that may represent the earliest aqueous communication between fusing granules .Ultrastructural studies have shown that histamine secretion by mast cells, in response to either antigens or synthetic polycations, is mediated by a rapid, compound exocytosis (I, 3, 15, 16, 18, 19, 28). Early in the secretory process, single granules fuse with the plasma membrane, whereas at later stages membranes of adjacent granules in the cell interior fuse in succession to produce deep invaginations in the cell surface (1,19,28). These morphological observations correlate well with chemical measurements of release showing that 40-80% of the total cell histamine is secreted within 2 min (3, 28). There is good evidence that secretion is initiated by a rise in intracellular calcium activity (9,10,16,17,22), and for this reason the mast cell represents an important model of calcium-mediated stimulus-secretion coupling (8,12).This rapid exocytotic response of the mast cell has proven extremely useful for studying membrane fusion because numerous fusion events occur within a short period ; electron microscopy studies have already demonstrated what appear to be preliminary stages in this process. In thin sections of stimulated mast cells, granule membranes often contact the plasma membrane at sites where the intervening cytoplasm has been expressed (7,21), forming what Palade and Bruns (25) termed a "pentalaminar figure ." The occurrence of these structures in many types of secretory cells during exocytosis has suggested that they are a preliminary stage in membrane fusion (2,24,29,30) . In mast cells these contacts can be extensive and are often seen where the granule bulges against the plasma membrane. In freeze-fracture, these bulges frequently have plasma membrane domes that have been cleared of intramembrane particles (IMP) (7, 21). Furthermore, Lawson et al. (21) have shown in thin sections that a variety of ferritin-conjugated surface ligands will not bind to the plasma membrane where it contacts an underlying secretory...

Plasma-membrane diversity in a highly polarized cell.

“…These two macrodomains also contain microdomains. The band of membrane fronting the tip of the nucleus is less perturbable by PXB than the surrounding membrane (14,30) and susceptible to clearing of sterol/filipin complexes and intramembrane particles when glycerinated as a standard freeze-fracture cryoprotectant procedure (12,14,33) (34,35). Even more obvious are certain intramembrane-particle and lipid microdomains in the sperm tail (Fig.…”

Lipidic intramembranous particles

“…It was assumed that regions free of intramembrane particles or at least relatively large exposed bilayer regions were necessary for actual fusion to occur. However, using fast-freezing methods [98,99], no particle clearance could be detected indicating that only a very small area of the lipid bilayer of both fusing membranes is necessary for fusion.…”

A quantitative model for membrane fusion based on low-energy intermediates

“…In exocytosis, electron microscopy shows that the plasma membrane bends inward toward the granule membrane to establish local and intimate membrane contact. Viewed from the outside of the cell, the projection of membrane appears as a ''dimple'' (7,8). When cells expressing the fusion protein of influenza virus, hemagglutinin, are fused to red blood cells, both morphological (2) and functional (9) evidence indicates that the membranes bend toward each other to establish local contact.…”