Proc. Natl. Acad. Sci. USAVol. 73, No. 8, pp. 2823-2827, August 1976Cell Biology

Freeze-fracture study of mast cell secretion(cell membrane/intramembranous particles/polymyxin B/membrane fusion/exocytosis)

EMIL Y. CHI*, DAVID LAGUNOFFt, AND JAMES K. KOEHLERtDepartments of * Pathology and *Biological Structure, University of Washington, Seattle, Wash. 98195

Communicated by Earl P. Benditt, June 8, 1976

ABS1RACr Within seconds after exposure of rat peritonealmast cells to polymyxin B, bulges appear on the surface of thecells. Freeze-fracture electron microscopy reveals that eachbulge overlies a mast cell granule. In contrast to the even dis-tribution of intramembranous particles in the plasma membraneof unstimulated cells, the intramembranous particles in thestimulated cells are unevenly distributed in the membrane ofthe bulges with large patches of membrane lacking intramem-branous particles. The membranes over the most prominentbulges are entirely free of intramembranous particles, and insome instances there is an increased concentration of in-tramembranous particles at the margins of the bulges. Peri-granule membranes exhibit the same changes in distributionof intramembranous particles. Electron microscopy of thinsections of rapidly fixed, stimulated mast cells shows a peculiarstructure of the membrane overlying some bulges; instead ofthe pentalaminar membranes previously demonstrated, themembrane at these sites of presumptive fusion of perigranuleand plasma membrane assumes the form of a single denselamina with a fine fuzzy coating on either side. It seems possiblethat membrane fusion and subsequent pore formation proceedin the stimulated mast cell through a stage of flight of in-tramembranous particles and molecular rearrangement of theother membrane components.

It is well established that specific membrane-bound cytoplasmicgranules represent the storage form of histamine and heparinin mast cells (1-3). The granules are extruded from the cellunder the influence of a variety of agents. Induced granulesecretion has been shown to occur by exocytosis (4), in whichapposing membranes of granules and cell surface interact toestablish an opening (5). Involvement of membranes sur-rounding other granules produces a series of channels thatpenetrate well into the cell domain (6, 7).The extent of membrane interaction occurring during se-

cretion would be expected to make the mast cell a particularlyfavorable object for the investigation of secretory membraneevents. We have previously reported on the appearance ofnormal unstimulated mast cells with the freeze-fracture tech-nique (8). The application of freeze-fracture technique to thestudy of mast cell secretion provides information comple-mentary to that available from the previously used modes ofmorphologic analysis (5).

MATERIALS AND METHODSPeritoneal cells, including mast cells, were collected from theperitoneal cavities of 2- to 4-month-old male CDF rats obtainedfrom Charles River Breeding Laboratories Inc., Wilmington,Mass., or bred in our departmental animal facilities from CDFstock. In each of the degranulation experiments performed, sixto eight rats were used. Six to ten million mast cells were sepa-rated from the other peritoneal cells by centrifugation through35% albumin (9), so that mast cells constituted at least 80% bynumber of the final cell suspension. Mast cells were then washed

once and suspended in balanced salt solution (9) at a concen-tration of 2 X 105 cells per ml. Degranulation was studied incells equilibrated at room temperature (220), 150, or 9°. His-tamine release was induced by the addition of polymyxin Bsulfate to a final concentration of 2 ,ug/ml in balanced salt so-lution. At intervals varying from several seconds to 15 min afteraddition of polymyxin B, 5 ml of 4% glutaraldehyde in 0.1 Mcacodylate buffer, pH 7.4, was added to 5 ml of cell suspen-sion.

Immediately after the addition of 4% glutaraldehyde, cellswere centrifuged at 40, and 2% glutaraldehyde in cacodylatebuffer was added to the pellet. After fixation for 2 hr, the cellswere washed three times with cacodylate buffer and transferredto 20-25% glycerol for 10-14 hr at 4°. The cells were subse-quently processed as described (8). Replicas were examined ina JEOL-100B electron microscope at 60 kV.

For thin section, transmission electron microscopy, cells werefixed in 2% glutaraldehyde for 2 hr, washed three times withbuffer, post-fixed in 1% OS04, and handled as described (7).

RESULTSMast cells equilibrated at either 150 or 22° and then exposedto 2 iig/ml of polymyxin B for several seconds before additionof glutaraldehyde showed essentially the same alterations.Numerous low bulges (Fig. 1) appeared on the surfaces of mostcells. The formation of the surface bulges was associated withthe disappearance of virtually all the surface ridges charac-teristic of the unstimulated mast cell (8). More prominent bulges(Figs. 2 and 4) were also frequently observed. Unlike, the moreor less even distribution of intramembranous particles in theplasma membrane of the normal mast cell, patches free of theparticles were prevalent in the membrane of the bulges (Figs.1 and 2). The membranes of the most prominent (Fig. 4) bulgeswere completely free of intramembranous particles. In manyinstances, although not invariably, it appeared that there wasan increased concentration of intramembranous particles at thebases of bulges that were partially or completely depleted ofthe particles (Figs. 5 and 6). Similar change in distribution ofintramembranous particles in perigranule membranes was alsoevident (Fig. 6). Study of transmission electron micrographsof cells fixed seconds after addition of polymyxin B revealedincipient pores, pentalaminar fusions of perigranule membraneand plasma membrane (5), and membrane structures we havenot previously observed. The latter covered well-developedbulges and consisted of a central dense line with very finestriations forming a fuzzy coat on either side of the line (Fig.3). These structures were relatively rare.

Mast cells fixed 20 sec, 1 min, or 15 min after the additionof polymyxin B at 220 had essentially the same appearance. Thebulges had largely receded, and the cells resumed their, moreor less, regular surface contours (Fig. 7). Within the cell thelarge channels previously observed by transmission microscopywere also evident in the freeze-fracture preparation (Fig. 7).

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t To whom correspondence should be addressed: Department of Pa-thology SM-30, University of Washington, Seattle, Wash. 98195.

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FIGS. 1-3. Fig. 1. Freeze-fractured mast cell exposed to 2 ,gg/ml ofpolymykin B at 15° for 2-3 sec before addition of glutaraldehyde. Numerouslow bulges cover the surface of this cell. Surface ridges are sparse. The cleavage plane is largely through the plasma membrane, exposing theP face. [The nomenclature used for labeling fracture faces is that proposed by Branton et al. (10).] In a few sites, the E face of the membraneof an underlying granule is seen. Both plasma membrane and perigranule membrane in the bulges exhibit regions free of intramembranousparticles (arrows). X23,500. Fig. 2. Freeze-fractured mast cell exposed to 2 ,gg/ml of polymyxin B at 22° for 2-3 sec before addition of glutaral-dehyde. A single bulge is present, rising from the plasma membrane close to a cross-fractured surface ridge. The cleavage plane is through theplasma membrane, revealing a substantial portion of the P face of the membrane over the bulge devoid of intramembranous particles. To theleft of the base of the bulge there appears to be a somewhat increased density of intramembranous particles. X25,500. Fig. 3. Thin section ofa mast cell exposed to 2 vg/ml of polymyxin B at 15° for 2-3 sec before addition of glutaraldehyde. The section passes through two bulges atthe surface of the cell. The membrane covering the bulge on the right has the form of a single central dense lamina covered on both sides by afuzzy layer. A similar region is evident in a portion of the membrane covering the other bulge. X23,500.

The intramembranous particles appeared to be evenly dis-tributed in the channel-lining membrane (Fig. 10), and obviouspores were present (Fig. 8).

At 90, even by 15 min the secretory process had not devel-oped beyond a few low bulges with some diminution of in-tramembranous particles in the overlying plasma membrane(Fig. 9).

DISCUSSION

The mast cell is a secretory cell; the ultrastructural featuresassociated with its secretory activity have been studied in anumber of laboratories. A variety of secretory stimuli have beenused: polymyxin B (4, 5), compound 48/80 (11-13), toluidineblue (14), ATP (15), the antigen-antibody reaction (16), chy-motrypsin (17), and the calcium ionophore A23187 (18). Mastcell secretion induced by these noncytotoxic agents occurs bya common process of channel formation with consequent ex-ternalization of the granules. Pentalaminar fusions and pre-sumptive early pores have both been found (5) and interpretedas possible antecedents to channel formation.

In a study of mucocyst extrusion from Tetrahymena (19),the application of freeze-fracture has revealed preformed sites

for secretion which take the form of rosettes or necklaces ofintramembranous particles in the plasma membrane. Mem-brane fusion during the extrusion process was proposed to in-volve association of such sites with complementary rosettes inthe mucocyst membrane. Similar circular arrangements ofintramembranous particles have been demonstrated in mam-malian cells in relation to pinocytosis (20). As yet, no suchgrouping of intramembranous particles has been found asso-ciated with exocytosis in mammalian cells (21-24). In ourprevious reported study of unstimulated mast cells, no orga-nized arrays suggestive of predetermined loci for secretion werefound (8); and in the present study of cells stimulated withpolymyxin B no structured arrays of intramembranous particleswere found at sites of incipient membrane fusion.The earliest ultrastructural event we have observed is the

bulging of the plasma membrane overlying peripheral granules.Increased prominence of a bulge is associated with loss of in-tramembranous particles from the overlying plasma membraneand the underlying perigranule membrane, with the collectionof intramembranous particles at the margin of the bulge. Twodistinct membrane structures have been observed in thin sec-tions of bulging membranes: fusion of perigranule and plasmamembranes to form a pentalaminar membrane and an unusual

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FIGS. 4-6. Fig. 4. Freeze-fractured mast cell exposed to 2 pg/ml ofpolymyxin B at 150 for 2-3 sec before addition of glutaraldehyde. Thebulges are particularly prominent in this preparation, and the P faces of the membrane are also entirely free of intramembranous particles.In a small region ofplasma membrane separating two bulges, there appears to be an increased density of the particles. X20,000. Fig. 5. Freeze-fractured mast cell exposed to 2 pg/ml ofpolymyxin B at 150 for 2-3 sec before addition of glutaraldehyde. Two small bulges are present in theplasma membrane. The one on the right shows some depletion of intramembranous particles in the P face with an accumulation of the particlesat the base of the bulge (arrow). X25,000. Fig. 6. Freeze-fractured mast cell exposed to 2 ug/ml of polymyxin B at 150 for 2-3 sec before additionof glutaraldehyde. This cleavage plane reveals the E face ofthe plasma membrane covering several bulges. Two portions ofperigranule membraneremain adherent to the plasma membrane, and their P faces also show loss of intramembranous particles. X20,000.

membrane form with a central dark layer and associated fuzzymaterial on either side. The relationship of the loss of in-tramembranous particles to the two membrane structures hasnot yet been determined. The increased number of in-tramembranous particles at the periphery of the bulges supportsthe interpretation that the intramembranous particles move outof the bulges. The redistribution of intramembranous particlesis apparently transitory, since a normal distribution of in-tramembranous particles is observed in the membrane of fullyformed channels. Associated with the formation of bulges thereis a diminution in surface ridges, leading us to suggest that theincrement in membrane required to cover the bulges is re-cruited from the ridges. More extensive data from scanningelectron microscopy are needed to substantiate this proposal.

Since secretion of mast cell granules at 370 is very rapid (25),reduction of the temperature was used to slow the process and

thereby to examine the early structural changes better. Thisstrategy introduces the possibility of perturbing cell membraneswith the attendant consequence of significant distortion ofmembrane structure. Wunderlich et al. (26), for instance, havedescribed the induction of regions free from intramembranousparticles of plasma membrane by incubating cells at low tem-peratures. The prominence of bulges free from intramembra-nous particles at 150 and 220 and the failure of bulges to de-velop fully at 90 lead us to reject the hypothesis that bulgeformation and intramembranous particle flight are relatedsimply to low temperature. Furthermore, mast cells kept at 150without exposure to polymyxin B do not develop bulges orparticle-free regions. The particle-poor bulges we have observedare also distinct from the smooth blisters described on the sur-face of lymphocytes treated with dimethylsulfoxide withoutprior aldehyde fixation (27).

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FIGS. 7-10. Fig. 7. Freeze-fractured mast cell exposed to 2 .ug/ml of polymyxin B at 220 for 15 min before addition of glutaraldehyde. Severallarge channels formed by fusion of multiple granules are evident. No bulges are present at this time. X11,800. Fig. 8. Freeze-fractured mastcell exposed to 2 jsg/ml of polymyxin B at 220 for 20 sec before addition of glutaraldehyde. There is a large pore connecting a channel to the outsideof the cell (arrow). X17,600. Fig. 9. Freeze-fractured mast cell exposed to 2 Mg/ml of polymyxin B at 90 for 15 min. Cross fractures of surfaceridges and microvilli are seen. The bulges are poorly developed, and only a few regions are partially depleted of intramembranous particles X17,600.Fig. 10. Freeze-fractured mast cell exposed to 2 ug/ml of polymyxin B at 220 for 2-3 sec before addition of glutaraldehyde. The cleavage planeexposes the P face of a moderately large channel. The intramembranous particles appear essentially evenly distributed in the membrane liningthe channel. X18,600.

We have relatively little basis for speculation on the mech-anism of formation of the bulges. They could conceivably resultfrom an active contractile process within the cell forcing theperigranule membranes against the plasma membrane; alter-

natively, the bulges could be formed by osmotic swelling of thegranules within their membranes following a change in thepermeability characteristics of the perigranule membrane.While the bulges and change in distribution of inframem-

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branous particles are consistent observations, we do not yetknow if these events are essential to the exocytotic process; nor

have we been able to obtain any electron micrographs oftransitions between particle-free bulges and frank pores. It isnonetheless tempting to propose that a local movement of in-tramembranous particles from the membranes, passive or ac-

tive, permits localized rearrangements of membrane moleculesthat generate the formation of discrete pores through which thecontents of the granule may be discharged. Ahkong et al. (28)have previously suggested that chemically induced fusion oferythrocytes occurs in regions of perturbed lipid bilayer thatare denuded of intramembranous particles.

This investigation was supported by National Institutes of HealthGrants HL-03174, GM-13543, and GM-16598.

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