Freeze-fracture study of mast cell secretion.

Abstract

ABS1RACr Within seconds after exposure of rat peritoneal mast cells to polymyxin B, bulges appear on the surface of the cells. Freeze-fracture electron microscopy reveals that each bulge overlies a mast cell granule. In contrast to the even distribution of intramembranous particles in the plasma membrane of unstimulated cells, the intramembranous particles in the stimulated cells are unevenly distributed in the membrane of the bulges with large patches of membrane lacking intramembranous particles. The membranes over the most prominent bulges are entirely free of intramembranous particles, and in some instances there is an increased concentration of intramembranous particles at the margins of the bulges. Perigranule membranes exhibit the same changes in distribution of intramembranous particles. Electron microscopy of thin sections of rapidly fixed, stimulated mast cells shows a peculiar structure of the membrane overlying some bulges; instead of the pentalaminar membranes previously demonstrated, the membrane at these sites of presumptive fusion of perigranule and plasma membrane assumes the form of a single dense lamina with a fine fuzzy coating on either side. It seems possible that membrane fusion and subsequent pore formation proceed in the stimulated mast cell through a stage of flight of intramembranous particles and molecular rearrangement of the other membrane components.It is well established that specific membrane-bound cytoplasmic granules represent the storage form of histamine and heparin in mast cells (1-3). The granules are extruded from the cell under the influence of a variety of agents. Induced granule secretion has been shown to occur by exocytosis (4), in which apposing membranes of granules and cell surface interact to establish an opening (5). Involvement of membranes surrounding other granules produces a series of channels that penetrate well into the cell domain (6, 7).The extent of membrane interaction occurring during secretion would be expected to make the mast cell a particularly favorable object for the investigation of secretory membrane events. We have previously reported on the appearance of normal unstimulated mast cells with the freeze-fracture technique (8). The application of freeze-fracture technique to the study of mast cell secretion provides information complementary to that available from the previously used modes of morphologic analysis (5). MATERIALS AND METHODSPeritoneal cells, including mast cells, were collected from the peritoneal cavities of 2-to 4-month-old male CDF rats obtained from Charles River Breeding Laboratories Inc., Wilmington, Mass., or bred in our departmental animal facilities from CDF stock. In each of the degranulation experiments performed, six to eight rats were used. Six to ten million mast cells were separated from the other peritoneal cells by centrifugation through 35% albumin (9), so that mast cells constituted at least 80% by number of the final cell suspension. Mast cells were then washed once and suspended in balanced salt solution (9) at...

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

“…This absence of IMP clearing at both the plasma membrane and interior granule membranes of quick-frozen mast cells was in marked contrast to previous observations of such areas in membranes of glutaraldehydefixed mast cells (7,21) . To be sure that IMP clearing could be seen in our mast cell preparations, we fixed mast cells with glutaraldehyde after 15-30 s of stimulation with polymyxin B sulfate .…”

“…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 granule, suggesting that the binding sites for these ligands have been cleared away (21) .…”

“…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) .…”

Rescue of SP-B Knockout Mice with a Truncated SP-B Proprotein

“…While the formation of lamellar bodies in SP-B(Ϫ/Ϫ)/hSP-B ⌬c (ϩ/ϩ) mice appears normal, the regulation of lamellar body size is clearly compromised. Accumulation of tissue PC and dramatically enlarged lamellar bodies are similar to findings in the lipid storage disease seen in beige mice, an animal model of Chediak-Higashi syndrome (31). Although the biochemical basis of Chediak-Higashi syndrome is not known, other lipid storage diseases result from an accumulation of substrate in lysosomes.…”

Membrane fusion of secretory vesicles and liposomes Two different types of fusion

“…In earlier studies it was reported that the exocytotic fusion process is characterized by a clearing of membrane particles from the fusion site [67][68][69]. This was interpreted as evidence against protein involvement in exocytotic membrane fusion.…”