changes in plasma-membrane-associated filaments during endocytosis and exocytosis in...

Cell, Vol. 24, 905-914. June 1981, Copyright @3 1981 by MIT

Changes in Plasma-Membrane-associated Filaments during Endocytosis and Exocytosis in Polymorphonuclear Leukocytes

Janet Boyles* and Dorothy F. Bainton Department of Pathology University of California School of Medicine San Francisco, California 94143

Summary

We examined the filaments associated with the cytoplasmic surface of the plasma membrane in rabbit exudate PMNs during phagocytosis of particles, or during “frustrated phagocytosis” with exocytosis of storage granules. Cells were plated onto yeast particles glued to coverslips with polylysine or onto coverslips coated with sheets of heat-agglutinated IgG. After periods ranging from 1 to 15 min, we disrupted the cells by a jet of salt solution and exposed their inner membranes. These broken cells were fixed immediately and processed for SEM. Whole cells were also prepared for SEM or TEM. At the site of PMN adherence to an opsonized yeast particle, a network of globular centers and thin, branched filaments appears on the cytoplasmic surface of the plasma membrane, while the outstretch- ing lamellipodia contain a mesh of such filaments but no globular centers. Within 1 to 2 minutes, these structures disappear from the invaginating portion of the developing vacuole, and the cell’s storage granules fuse with the barren membrane regions. These activities occur in rapid sequence over the vacuolar membrane after the first contact, until the phagocytosed particle is wholly encircled by a smooth, loose membrane, separated from the cell surface. A comparable filament pattern or complex was seen during “frustrated phagocytosis” on IgG sheets. At times between 1 and 5 min after plating, the cytoplasmic surfaces of these adherent membranes contain denuded central regions and peripheral nets of globular centers with radiating, thin, branched filaments. Granules apparently fuse with the bare areas. Thus we have obtained evidence of filament association with the plasma membrane at sites of adherence (to phagocytosable or nonphagocytosable surfaces) and have traced the subse- quent disappearance of the filaments with degranulation.

Introduction

Neutrophilic polymorphonuclear leukocytes (PMNs), cells specialized for host defense, can adhere to surfaces, locomote by amoeboid movements either at random or in reaction to gradients of chemotactic agents and ingest certain particles. These cells also release stored granules internally into phagocytic vac-

l Present address: Department of Cell Biology. Yale University School of Medicine. New Haven, Connecticut 08510.

uoles, or externally into the surrounding media when the stimulating substance is too large to ingest. Such processes, which are integral to normal PMN function, are common to many cell types and are believed to depend upon contractile elements. In fact, PMNs have been shown to contain 1 OYO actin, 1% myosin and 1% actin-binding protein (Stossel and Pollard, 1973; Boxer and Stossel, 1976). Although the organization of contractile proteins has been clearly delineated in muscle, the arrangements of these proteins in leukocytes remain obscure.

In a previous study (Boyles and Bainton, 19791, using a combination of ultrastructural techniques, we examined the cytoplasmic face of the PMN plasma membrane during adherence and spreading with re- sultant locomotion. Our major finding was the assembly of a subplasmalemmal filament complex on the adherent membrane, with changing patterns that cor- related with specific cellular activities. When nonad- herent PMNs were observed by scanning electron microscopy (SEM), the inner surfaces of their mem- braIN? were free of filaments. Lamellipodia, however, contained a felt of filamentous, finely granular material, as seen by transmission electron microscopy (TEM). After the attachment of the cell to a substrate, a three-dimensional, interlocking network of globular projections and radiating microfilaments was associated with the cytoplasmic surface of the plasma membrane. As the cells began locomotion, this pattern gave way to bundles of anastomosing filaments, within which thicker, beaded filaments appeared. These ultrastructural data complemented the biochemical data of other laboratories (Stossel and Pollard, 1973; Boxer et al., 1974; Senda et al., 1975; Boxer and Richardson, 1976; Boxer and Stossel, 1976) by pro- viding some information on the organization of the PMN’s contractile apparatus during the initiation of amoeboid movements. These same contractile elements have also been implicated in the process of phagocytosis and degranulation (Malawista et al., 1971; Zigmond and Hirsch, 1972; Davies et al., 1973; Reaven and Axline, 1973; Boxer et al., 1974; Korn et al., 1974; Berlin and Oliver, 1978; Bowers, 1980; Hartwig et al., 1980; Stendahl et al., 1980; Valerius et al., 19801-a cellular response to surface contact that is generally thought to be radically different from locomotion.

To examine these events in this study, we permitted the cells to phagocytose yeast particles or to attempt to ingest sheets of heat-agglutinated IgG too large to be internalized. After periods ranging from 1 to 30 min, the upper surface of the plasma membrane and most of the cellular content were sheared away with a jet of buffered salt solution, and the adherent membranes were fixed and examined by high-resolution SEM. This revealed changes in the organization of filaments associated with the plasma membrane during phagocytosis-from the first extension of a la-

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mellipodium, through internalization, and finally, to fusion of the vacuolar membrane and PMN storage granules.

Results

Phagocytosis of Yeast Particles PMNs were plated onto coverslips coated with opsonized yeast held in place by polylysine. When examined by phase-contrast microscopy, the bond between the yeast and the polylysine is found to be fragile; and yeast particles not contacted by cells float free from the coverslips within a minute or two after the addition of exudate or salt solutions. Settling onto this layer of yeast, cells rapidly devour the particles they touch and stretch forth lamellipodia to engulf other, neigh- boring particles. Within 1 minute after contact, before the yeast is completely internalized, PMN granules (seen by phase microscopy) meet the forming phagosome and fuse with it in a series of “flashes,” as originally described by Hirsch and Cohn (1960) and Hirsch (1962). After 3 to 4 min of contact, the yeast particle can usually be observed drifting in the cytoplasm, as the cell commences to move over the coverslip. (These same events can be viewed by both TEM and SEM.)

In conventional thin sections (Figure la). a thick zone of homogeneous cytoplasm, lacking cellular organelles, can be seen at the foci of particle-membrane contact. Lamellipodia originate from this zone,

which is indistinguishable from the one created when the cell abuts upon a carbon surface. Just as PMNs produce layers of lamellipodia as they spread on carbon or glass surfaces, so do waves of lamellipodia advancing from behind points of contiguity with yeast particles progressively engulf the particles (Figure 1 b).

Phagocytosis can be followed from the inside of the cell by disrupting it and using SEM. This technique allows soluble cytoplasmic proteins to escape, and provides a surface view of a substantial portion of the plasma membrane and the formed elements of the cortical cytoplasm that are tightly associated with it. Where the cell was successfully opened soon after adherence to a yeast particle and the upper lamelli- podial membrane was ripped away, images like Figure 1 c were obtained. These membranes were bordered by a feltwork of thin, short, irregularly branched filaments identical to the feltwork seen in the lamellipodia of PMNs adhering to other surfaces. Behind this was a complex of composite globular projections with similar radiating filaments. This complex is also identical to the one found on the subplasmalemmal surface of the plasma membrane of PMNs adhering to a nonphagocytosable surface, glass or carbon (Boyles and Bainton, 1979).

Surveyed in thin section (Figure 1 d), the later stage of engulfment preceding closure of the phagocytic vacuole was characterized by the disappearance of the homogeneous cytoplasmic area from the top (old-

Figure 1. PMNs Plated onto a Layer of Yeast Particles

(a) In this oriented thin section, the cell has contacted the serum-coated carbon surface (CS). over which it is spreading, as well as a floating yeast particle (Y). in the early stages of being phagocytosed. Storage granules and islands of partially extracted glycogen are visible in the cytoplasm. A denser, homogeneous zone of cytoplasm (double-headed arrows) prevents these organelles from closely approaching the membrane at sites of cell-carbon and cell-yeast contact. The cytoplasm of both the lamellipodia touching the yeast and carbon surface (L) and of those reaching out from the region above the contact sites (L’) is continuous with this subplasmalemmal band of organelle exclusion. Specimen was fixed for 30 min at 37°C in 1.5% glutaraldehyde in 0.1 M sodium cacodylate buffer containing 1% sucrose, kept oyernight at 4°C. osmicated for 1 hr in 1% OsOn in 0.1 M Na4P04-NaHP04 buffer containing 4% sucrose, stained en bloc with uranyl acetate, dehydrated and embedded in Spurr’s resin. 9000x. (b) In this scanning electron micrograph, the advancing ruffle at the mouth of the phagocytic vacuole comprises two layers of lamellipodia-one in various stages of adherence (L) and the other extending from above the contact sites (L’) with a yeast particle(Y). Specimen was fixed for 1 hr at 37°C in a 1 :l mixture of SBS and 4% glutaraldehyde in 20 mM Na2P04-NaHP04 buffer, osmicated for 1 hour in 1% 0~0, in 0.1 M N&P04- NaHP04 buffer containing 4% sucrose, treated with uranyl acetate, dehydrated, critical-point-dried and coated with 5 to 7 nm platinum-carbon. 9000x. (c) The PMN in this high-magnification view has been ruptured and the cytoplasmic-membrane surface at the edge of a forming phagosome is exposed. The rough exterior of the opsonized particle (Y) can be seen in the upper left corner, beyond the jagged edge of the torn phagocytic- vacuole membrane. Here, at the tip of the developing vacuole, an irregular granular filamentous felt (double-headed arrows) covers the exposed membrane surface. Behind this band of felt lies a network of globular centers (circles) composed of varying numbers of smaller subunits and interconnected by thin-branched filaments. Specimen was prepared as described in b, except for the omission of osmication from the fixation protocol. 28.000~. (d) Oriented thin section of a cell that has almost finished engulfing a yeast particle (Y) attached by polylysine to the carbon surface (CS). No cellular organelles inhabit the subplasmalemmal cytoplasm near the closing “lips” of the phagocytic vacuole (double-headed arrows). Above this region, however, granules draw very near to the vacuolar membrane (arrows). The greater membrane-scalloping here than that at the mouth of the vacuole, and the dark, undissolved granule content clinging to the cell wall of the yeast particle, indicate that degranulation (d) has already occurred. Specimen was prepared as described in a. 11,000x. (e) This scanning electron micrograph depicts the cytoplasmic-membrane surface of a ruptured PMN that has partially phagocytosed a yeast particle and has extensively spread over the glass substrate. A network of branched filaments and globular centers (circles), the filament complex, covers the surface of the glass-adherent portion of the membrane in the lower half of the micrograph. At the top, the membrane overlies the yeast particle, about half of which has been ingested. A filamentous network displaying globular centers (circles) decorates the rim of the phagocytic vacuole (double-headed arrows). The “older.” central part of the vacuole is blanketed by a sizeable field of barren membrane. Granules (g) are fusing with this filament-free stretch of membrane, making it IOOSS and baggy at sites of degranulation (d). Specimen was prepared as described in c. 17.000x.

Changes in Plasma-Membrane-associated Filaments 907

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est portion) of the vacuole, affording granules a close approach to this part of the membrane as they contin- ued to be excluded from the advancing or closing “lips” of the phagosome. Where the granule and phagosomal membranes had fused, the phagosomal membrane was loose or baggy and no longer firmly encased the particle.

Viewed by SEM after cell rupture, the phagosomal membrane of yeast particles partially ingested by PMNs had a peripheral zone of filaments (Figure le) and an adjacent zone of round, adherent granules, at times clearly fused with the vacuolar membrane. The central part of the phagosomal membrane appeared slack and flabby. Although the site of granule fusion did not seem filamentous, neither was it completely smooth (possibly because of the roughness of the yeast-covered surface directly below).

In later samples (adherent for 4 min or longer), neither free yeast particles outside the cell nor phagocytic vacuoles were often encountered. Evidently, they were washed away by the deluge of the buffer jet. Those vacuoles that managed to escape the flood were enclosed in lax membranes with some filaments remaining at their bases (Figure 2a). Occasionally, a completely smooth phagosome sat atop filaments cov- ering the glass-adherent membrane beneath (Figure 2b). Many of the plasma membranes adherent to the

coverslip, though lacking phagosomes, had round holes or raised, circular ridges in their filamentous webs; presumably these were remnants left behind by the ripping away of the phagosomes by the buffer jet either before or after closure, but anteceding their natural separation.

The inclusion or omission of OsO., from the fixation protocol had no apparent effect on the filaments associated with the phagocytic vacuole, nor did this factor observably increase the size and number of filament-free areas. When osmication was omitted, however, the membrane overlying the yeast particle, particularly at the borders of both barren and filamentous areas, became rough, making the presence or absence of filaments difficult to ascertain. This effect probably resulted from collapse of the membrane onto the rough surface of the yeast particle below.

“Frustrated Phagocytosis” of Heat-agglutinated W Films or sheets of antibody elicit a phagocytic response from PMNs-that is, the cells attempt to ingest them, and, as if in frustration, degranulate against the surface too large to engulf (Henson, 1971). Such films, prepared on glass coverslips, fulfilled our re- quirements for high-resolution SEM of degranulation better than the round, rough yeast particles, since the

Figure 2. Exposed Cytoplasmic Membrane Surfaces of Two PMNs, in the Later Stages of Phagocytosis when Ruptured and Fixed

(a) With the yeast particle internalized, filaments (D mainly appear at the lower edge of the phagocytic vacuole (PV) (where the vacuole joins the plasmalemma). blending with the filamentous network on the glass-adherent area of the membrane. Granules(g) continue to fuse with the widened expanse of filament-free vacuolar membrane. Specimen was prepared as described in Figure lc. 20,000x. (b) When this cell was ruptured, the phagocytic vacuole (PV) was nearly ready to separate from the plasma membrane. Note the smoothness of the loose-fitting vacuolar membrane overlying the ingested particle. Compare this membrane to the glass-adherent membrane with its thick coat of filaments (0. Specimen was prepared as described in Figure 1 b. 20.000X.


sheets have flat, stable surfaces. Thin films of heat-agglutinated IgG are transparent,

so that the cells can be followed easily by phase- contrast microscopy. Cells settling on the surface spread rapidly, becoming flatter than they do on glass surfaces. Degranulation, observed as “flashes,” can occur anywhere on the bottom cell surface, leaving its hallmark-motionless, dark, undissolved granules surrounded by white halos. Although degranulations may occur upon initial contact of PMNs and antibody film, they are frequently not detected in the spread cell until 5 min after contact, and then continue for another 5 to 10 min. Not all cells degranulate in this system: those that do so are stationary, unstirring except for their cytoplasmic contents, which con- stantly seethe or churn. The percentage of cells degranulating on the antibody film varies from area to area of the same coverslip-and also from experiment to experiment. Particularly in regions where no cells are degranulating, a few cells can be found moving over the surface. But more often at these sites, the cells sit and rock their contents slowly from side to side, almost as if they were trying (unsuccessfully) to

depart first in one direction, then in another, but were tightly stuck in place.

Specimens of exposed adherent membranes were obtained l-30 min after plating. In samples collected at 6 min, the cytoplasmic surface of cells manifested a complex of globular centers with radiating branched filaments- a pattern that we had previously observed on cells adherent to glass for less than 3-5 min only (Boyles and Bainton, 1979). This network of filaments was irregular in thickness. Areas of bare membrane were frequent (Fig 3). Out of the filament- poor membrane, bubbles of membrane protruded in some samples (Figure 4). These appear to be sites of extracellular degranulation-that is, fusion of the granule membrane with the plasma membrane (Fig- ures 4b and 4~). Similar patterns were seen at earlier time intervals, but the degranulation sites tended to be minute and the network of filaments less devel- oped. While a few membranes had granules associated with the remaining filaments at the borders of these areas, obvious states of granule-plasma membrane fusion could be distinguished only in essentially filament-free zones. Where sites of degranulation

Figure 3. A PMN Layered for 6 min onto a Sheet of Heat-agglutinated IgG (a Surface That Elicited a Phagocytic Response), Ruptured and Examined by SEM

A thick complex of globular projections with radiating microfilaments (circles) coats the membrane. A denuded zone of cytoplasmic membrane surface is present. Specimen was prepared asdescribed in Figure 1 b. 15.000X.

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Figure 4. Another PMN in Contact with Heat-agglutinated IgG for 6 min

(a) A network of slender, branched filaments radiating from irregularly shaped globular projections appears only at the periphery of this me The relatively filament-free membrane demonstrates several sites of granule fusion or degranuiation Cd). 5225 X. (b and c) in these enlargements of the same specimen, note that the membrane surface seems patchy-either smooth or pebbled w projections, apparently associated with the few remaining filaments (9. Biebs of smooth membrane mark the sites of degranulation residual filaments on this membrane are thin and smooth. At the periphery of the barren membrane, several large, irregular centers (circi scant, radiating filaments. are evident. Specimen was prepared as described in Figure 1 b. 38.400X.

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were present, the membrane surface resembled a mosaic of smooth and pebbled islets (Figures 4b and 4~). Most often, granule fusion seemed to take place in smooth areas, for a ring of smooth membrane commonly surrounded these sites. The membranes of these fused granules were either perfectly smooth or dotted, with small projections slightly larger than those on the plasmalemma. Rarely, a filament or two ran from a granule to the plasma membrane. The few short filaments left in the area were generally associated with the pebbled surface. In fact, filaments and “pebbles” were so intimately linked that in many instances it was difficult to pinpoint where a filament ended. Filaments in denuded zones were single and short, or were arranged in small isolated patches of mesh.

Cells ripped open more than 10 min after plating

still appeared as described above, except that many showed evidence of previous intense degranulation, exhibiting large pockets of smooth membrane balloon- ing up from the foci of adherence. In general, such pockets (or bubbles) had been partially torn away (along with the cell contents) by the shearing stream of buffer. Micrographs with still-intact pockets, as in Figure 5, were rarely obtainable. Finally, after more than 30 min, the number of filaments at the cellular periphery tended to diminish.

Discussion

Phagocytosis is a local response of a segment of the cell’s surface to signals generated by specific particle-plasma membrane interactions. Griffin and co- workers (Griffin and Silverstein, 1974; Griffin et al.,

Figure 5. PMN Broken 30 min after Having Been Plated on Heat-agglutinated IgG

This PMN has at its center a large collapsed bubble (B) left from repeated degranulations. The immediately surrounding membrane appears firmly attached to the surface, but barren. Only at the cell’s extreme periphery are filaments (1) and globular centers found. The buffer jet has torn the cell’s upper membrane surface 9JMS) to the side, exposing the cell’s multilobed nucleus(n). many granules (g) and many of the thicker filaments (tf) that run from the nuclear area to the adherent surface. Fixed as described in Figure 1 b. 10,500x.

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1975, 1976) have proposed a model of ligand-medi- ated phagocytosis, wherein ligand-receptor interaction sends forth a signal, initiating a regional aggre- gation of contractile proteins, which leads to pseudo- podial extension in the area of the attached particle and the possibility of additional ligand-receptor interaction. This process may be repeated until the plasma membranes meet and fuse to form a completed phagocytic vacuole. Recently, it has been reported that, in addition to actin, the pseudopodia of phagocytosing macrophages (Stendahl et al., 1980) and PMNs (Val- erius et al., 1980) contain concentrates of actin-binding protein and myosin. In an attempt to explain the interaction of these ingredients, Stossel and cowork- ers (Hartwig et al., 1980; Stendahl et al., 1980) pos- tulated a dynamic model of specific contractile and accessory protein interactions designated “the rigidity shear hypothesis.” To date, however, there have been no ultrastructural data to support or refute any model. As a step towards acquiring such data, we have documented the changing organization of the cortical filament system during phagocytosis.

The initial contact of a particle with the PMN is frequently via lamellipodia, since PMNs crawl towards foreign bodies along gradients of chemotactic factors (Zigmond, 1978). These lamellipodia are filled with a feltwork of fine filaments and granular material (Boyles and Bainton, 19791, which is reorganized into a complex of composite globular centers with radiating, branched filaments when the lamellipodia membrane adheres to a particle during phagocytosis. These events are identical to those that occur during adhesion to a nonphagocytosable substance. However, the cell does not retract from the site of its attachment during phagocytosis, as it does after spreading over a surface that permits locomotion. Rather, during phagocytosis, the filament complex disappears as the particle moves into the cytoplasm and the cell’s granules fuse with the exposed vacuolar membrane. This series of activities continues in sequence across the membrane of the vacuole as more and more of the particle is engulfed. Ultimately, the baggy membrane that forms the endocytic vacuole breaks free from the surface membrane and completes the internalization of the particle. The observations made during “frustrated phagocytosis” were the same as those made during yeast-particle engulfment. Moreover, the presence of essentially filament-free, or nude, membrane surfaces at sites of degranulation was clearer in ex- amples of “frustrated phagocytosis.”

Although the technique of opening cells allows a new view of the cytoplasmic surface of the plasma membrane, it is well to remember that the structures we have documented are those firmly adherent to the membrane. Lost are the majority of the cell’s contents and architecture, and possibly also elements that are important for the processes of phagocytosis and degranulation. Despite these limitations, the results we

have presented are in general agreement with numer- ous studies of phagocytosis in PMNs and other cell types carried out with thin-section techniques (Reaven and Axline, 1973; Korn et al., 1974; Griffin et al., 1976; Klock and Bainton, 1976; Moore et al., 1978; Berlin and Oliver, 1978). A zone of dense cytoplasm, which in some cases can be seen to contain filaments presumed to be contractile elements, has been found at the points of particle adherence and within lamellipodia. In PMNs, this zone rapidly disappears from the forming vacuole, and granules then closely approach the phagosomal membrane and fuse with it. Recent photographs of immunofluorescent localization of actin (Berlin and Oliver, 19781, myosin and actin-binding protein (Stendahl et al., 1980) in macrophages and PMNs (Valerius et al., 1980) have shown each of these substances concentrated in the cortical-cytoplasm-enveloping particles and in much smaller con- centrations in the pits of forming vacuoles. The filament patterns we have shown in the same areas of the phagocytic vacuoles of PMNs may, at least in part, represent the organization of these proteins during phagocytosis. The loss of filaments and globular centers from the internalizing vacuole is also consistent with the low concentration of contractile elements at this site in the immunofluorescent studies and with the loss of the dense “fuzz” from this area in thin-sec- tioned material (Figure Id). It is therefore probably a real phenomenon, and not due to a loss of filaments in the shearing buffer jet. The similarity of the filament clearing, both during endocytosis of yeast particles and during exocytosis on flat IgG sheets, also adds to the argument that this is not a shearing artifact, particularly as cells on surfaces that support locomotion rather than phagocytosis do not have such filament- free regions. This filament loss seems to precede degranulation. It should also be noted that among cells plated onto IgG can be found individual cells that have areas of clearing (Figure 3) with no evidence of degranulation.

Since phagocytosis in PMNs occurs by engulfment of a particle through successive waves of lamellipodia, it must instigate considerable remodeling of the membrane and associated filaments. In this regard, one interesting phenomenon we noted was that lamellipodia themselves are extremely difficult to open by the buffer jet we used. As in the case of glass-adherent cells, they tend to be lost with the upper cell surface and contents of the cell, or to remain intact and unopened at the edge of the phagosome. These pro- pensities hint at an association of the filaments with both the upper and lower membranes of the lamellipodium. Upon membrane contact with a surface and the development of a filament complex with globular centers, however, the situation changes: the upper membrane is now easily ripped away, and the filament complex always persists with the particle-adherent membrane. The external constriction at the base of


the phagosome or root of the lamellipodium, then, represents the point where the translamellipodial feltwork of filaments is transforming into a filament complex, separable from the upper membrane surface. These changes in the lower lamellipodia and their filament anchorage or crosslinking sites during adherence may allow the remodeling necessary for particle engulfment.

Soon after the filament complex is formed it disappears. In the case of “frustrated phagocytosis,” the complex appears to be truly disassembled (through a loss of cohesion between its components, breakage of some element or even depolymerization of these elements). Any relocation of the filaments by being pulled to the periphery is not obvious, for the peripheral mesh is no thicker. Nor can large, myosin-like filaments or thin filaments aggregated into bundles usually be seen. The lack of aggregated or bundled filaments as well as beaded myosin-like filaments (seen in the retracting filament network of motile cells) during degranulation is not absolute. A few mobile cells on IgG films may also eventually degranulate, with areas of degranulation surrounded by aggregated filaments.

With degranulation, the tight phagocytic-vacuole membrane becomes flaccid and sack-like with the added membrane from successive granule fusions. Although the mechanism of vacuole closure is un- known, the presence of a lax membrane, with sup- porting filaments only at the base of the vacuole, prior to separation from the surface suggests that vacuolar sealing may be attributable to the eventual tearing and resealing of a narrow communication to the surface. Such a mechanism was recently proposed in a study of Acanthamoeba (Bowers, 1980). The lack of vacuolar closure sometimes found in phagocytosing PMNs, along with the persistence of a small canal to the surface (Jacques and Bainton, 19781, can then be explained by postulating that forces sufficient to tear the canal have not yet been encountered. A separation system necessitating some special “fusability” at the tips of the meeting lamellipodia would predict the fusion of two cells engulfing the same particle when their lamellipodia tips chanced to touch, but two PMNs attempting to internalize the same particle never fuse. Those filaments present at the mouth of the vacuole before the vacuole’s separation do not appear to be a contracting neck or “purse string.” Their frequent connection of the vacuole’s surface to the cell’s surface seems opposite to what would be expected of filaments forming a tightening “purse string.” Rather, these filaments appear to be the last of the series of filaments that have first organized on, and then been lost from, the internalizing phagocytic vacuole as more of the yeast particle has been internalized. If they, too, were lost, the narrow neck of the phagosome membrane could be easily torn. Rapid sealing of the break would complete the process.

While there seems to be little doubt that we are viewing the assembly and reorganization of contractile elements on the cytoplasmic surface of the plasma membrane during phagocytosis and degranulation, a precise and complete comprehension of these events must await direct identification of the components and regulators of this cortical-filament complex.

Experimental Procedures

Collection of Cells Exudates containing 95% neutrophils were produced and harvested according to the method of Hirsch (1956). New Zealand White rabbits were intraperitoneally injected with 150 ml 0.1% shellfish glycogen (Schwartz/Mann) in normal saline, and the peritoneum was drained 4 hr later. The cells were used within 4 hr. (Washed cells were used immediately after washing.)

Phagocytosis of Yeast Particles Dried baker’s yeast (50 mg) was killed by boiling in phosphate- buffered Krebs’ solution (PBK) for 30 min and then washed 3 times with saline. The washed yeast was incubated in 5 ml fresh rabbit serum at 37’C for 30 min and then washed 3 times with distilled water. Subsequently, the particles were applied as a slurry to coverslips cleaned with acid and wetted with a thin layer of poly-L-lysine (Sigma; molecular weight 200,000 (1 m&ml). Excess yeast particles were gently washed off. The coverslips were used immediately, thoroughly drained but not dried.

“Frustrated Phagocytosis” Human IgG (Hyland) was diluted 1 :19 with PBK or Hank’s balanced salt solution (HBSS) (6 mg/ml protein) and spread in a thin layer over a coverslip. The IgG was agglutinated by heating in a moist chamber for 1 hr at 63°C. The coverslips were rinsed and incubated in rabbit serum at 37’C for 30 min before use.

Disruption of Cells and Preparation of Samples Cells in exudate of HBSS were allowed to settle on the prepared coverslips. After periods ranging from 1 to 30 min, we ruptured the cells with a jet of standard breaking solution (SBS) (150 mM KCI. 2 mM MgCI?, 20 mM Na2P04-NaHP04 buffer [pH 7.4u. delivered from a syringe with a small-gauge needle. The coverslips were directly dropped into glutaraldehyde fixative at 37°C. These samples and other cell layers that had not been disturbed were processed as previously described (Boyles and Bainton, 1979). Samples initially placed in glutaraldehyde solution (1 :l SBS and 4% glutaraldehyde [Electron Microscope Sciences] in 20 mM Na2P04-NaHP04 [pH 7.41 for SEM; or 1.5% glutaraldehyde plus 1% sucrose in 0.1 M sodium cacodylate buffer [pH 7.41 at 37°C for TEM) were allowed to cool to room temperature and held overnight or for up to 3 days. Samples for thin sectioning were held at 4°C overnight. Then, without a buffer rinse, they were postosmicated for 1 hr in 1% 0~0, in 100 mM Na2P04-NaHPOI (pH 7.4) plus 4% sucrose: rinsed in Michaelis buffer

(pH 6.0) plus 7% sucrose; and submerged for 1 hr in 0.5% uranyl acetate in the same buffer plus 3% sucrose. Uranyl acetate was used not only to enhance the membranes in thin sections but also to remove the glycogen, which otherwise in SEM samples coats the exposed internal surfaces of PMNs and the glass or film supports they rest upon. Osmication was omitted in some samples of yeast- particle phagocytosis to test its effects. Ethanol dehydration preceded embedding in either Araldite or Spurr’s resins, or critical-point drying in either CO2 or Freon at 40°C.

Afler drying, the coverslips of SEM specimens were mounted on aluminum stubs with conductive paint and were coated that same day. (Samples must be coated on the same day or the exposed filaments tend to be indistinct, as if partially dissolved or covered by a thin layer of some substance. After they have been coated, however, specimens remain stable for months.) Platinum-carbon (5-7 nm) (as monitored by the shade of grey produced on white cards, a shade

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previously calibrated by the increase in thickness of known structures) was evaporated onto the samples in a tilting Omnirotor (Denton Vacuum Inc.). The source of evaporation was platinum wire wound between two sharpened carbon rods and melted to form a droplet.

Acknowledgments

This work was supported by grants from the NIH (AM) and from the Cancer Coordinating Fund of the University of California.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 USC. Section 1734 solely to indicate this fact.

Received September 15, 1980; revised February 2. 1981

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changes in plasma-membrane-associated filaments during endocytosis and exocytosis in...

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