The Mechanism of Concanavalin a Cap Formation in Leukocytes

J. Cell Set. a6, 57-75 (1977) 57 Printed in Great Britain

THE MECHANISM OF CONCANAVALIN A CAP FORMATION IN LEUKOCYTES

D. F. ALBERTINI*, R. D. BERLIN* AND J. M. OLIVERf Departments of Physiology* and Pathologyf, University of Connecticut Health Center, Farmington, Connecticut 06032, U.S.A.

SUMMARY The process of Concanavalin A (Con A) cap formation on human blood polymorphonuclear leukocytes, monocytes and lymphocytes and rabbit alveolar macrophages has been studied by correlative use of light, fluorescence and electron microscopy. The most important precondition for Con A capping on these cells is the disassembly of cytoplasmic microtubules by colchicine or the glutathione-oxidizing agent, 'diamide'. Incubation of microtubule-depleted leukocytes with fluorescein-conjugated Con A (F-Con A) leads to the aggregation of lectin into a cap which usually occupies a protuberance at one pole of the cell. F-Con A can also be concentrated at a constriction in the cell body. The protuberance is shown to consist of highly plicated membrane subtended by a network of densely packed microfilaments. Additional microfUaments originate from this network and course into individual plications of the protuberance. How- ever, the formation of the protuberance with its organized structure follows the disassembly of microtubules alone and does not require Con A. Thus when cells are treated with colchicine or diamide, then fixed and labelled with F-Con A the typical changes in cell shape that are associated with capping are observed but lectin is distributed homogeneously over the cell surface. Similarly if cells are first capped with low concentrations of unlabelled lectin, then fixed and incubated with F-Con A, fluorescencei s again uniformly distributed over the whole membrane. This indicates that membrane Con A receptors have not been concentrated over the protuberance despite the prior aggregation of microfilaments. By contrast, when precapped cells are labelled with F-Con A before fixation, fluorescence is concentrated through the previously established cap. Thus extensive organization of microfilaments and unlabelled lectin does not inhibit the movement of F-Con A-receptor complexes on unfixed cells. Further, the Con A cap is sufficiently fluid to permit mixing of sequentially formed Con A-receptor complexes. Although the aggregation of microfUaments into a protuberance and the concentration of Con A into the membrane of the protuberance are clearly separable events, microfilaments and Con A-receptor complexes are ultimately found in close association in the cap. This association appears to stabilize the localization of both the surface-bound lectin and the submembranous network of microfilaments. Such stabilization could result from physical interactions between microfilaments and Con A within the protuberance. However, we favour an alternative mechanism in which a region of low membrane fluidity that limits further diffusion is established following microtubule disassembly and is preserved by microfilament-membrane interactions.

INTRODUCTION The concept that intracellular structures such as microtubules and microfilaments play a role in the control of cell surface topography is derived in large part from studies of the movement of Concanavalin A (Con A) on the surfaces of leukocytes. In granulocytes and lymphocytes, disruption of microtubules by colchicine permits Con A-receptor complexes to move from an inherently random distribution into surface 5 8 D.F. Albertini, R. D. Berlin andj. M. Oliver caps (Edelman, Yahara & Wang, 1973; Unanue & Karnovsky, 1974; Oliver, 1976a, b). This suggests a role for microtubules in limiting the lateral movement of Con A. Further, the aggregation of Con A into caps can be prevented by metabolic inhibitors and by agents such as cytochalasin B and local anaesthetics that may impair microfilament function (Ryan, Unanue & Karnovsky, 1974; de Petris, 1975; Schreiner & Unanue, 1976). Thus, Con A cap formation has been inferred to depend on active contractile movements of microfilaments. Despite these findings, there is surprisingly little ultrastructural information relating microtubules or microfilaments to surface events. We recently established that microtubule disassembly and an extreme degree of Con A cap formation are induced by agents such as diazene dicarboxylic acid bis (iV,iV-dimethylamide) 'diamide' that oxidize glutathione (Oliver, Albertini & Berlin, 1976). Application of these agents to a variety of leukocytes has revealed considerable detail of the localization of microfilaments in microtubule-depleted cells and of the movement of surface-bound Con A during capping. Three new findings are presented here. First, Con A caps which usually occupy a protuberance or constriction in the cell, are underlain by a dense network of microfilaments in human peripheral blood polymorphonuclear leukocytes (PMN), monocytes and lymphocytes as well as in rabbit alveolar macrophages. Second, the accumulation of filaments and the formation of a protuberance or constriction is not induced by Con A but follows disassembly of microtubules per se. This accumulation of filaments does not lead to the movement of unoccupied Con A receptors into the protuberance. Thus, microfilaments are unlikely to physically move Con A-receptor complexes into caps even though the eventual site of surface Con A aggregation corresponds to the region of microfilament concentration. Third, when Con A receptors that are distributed diffusely over the surface bind Con A, they can move into preformed caps. Thus, ligand-receptor complexes can move despite prior assembly of microfilaments at a distant site. Furthermore, the capped membrane is sufficiently fluid to allow mixing of sequentially formed ligand- receptor complexes.

METHODS Cells. Suspensions of human leukocytes containing approximately 80% PMN and 20% mononuclear cells were obtained from buffy coat of freshly drawn heparinized blood as previously described (Oliver, 1976a). Alveolar macrophages were obtained from rabbit lungs by the method of Myrvik, Leake & Fariss (1961). Lectins. Fluorescein isothiocyanate-conjugated Con A (F-Con A) was prepared from Con A (Sigma; 3 x crystalline) as described before (Oliver et al. 1976). In all experiments both F-Con A and unlabelled Con A were purified by affinity chromatography on Sephadex G50 prior to use. Ricinus comrnunis agglutinin (RCA) was purified by affinity chromatography on agarose gel using the method of Nicolson, Blaustein & Etzler (1974). Con A labelling for light microscopy. Cell suspensions (ioe blood leukocytes/ml; 2 x io5 alveolar macrophages/ml) were incubated at 37 °C in phosphate-buffered saline containing 5 mM glucose (PBS) and microtubule-disrupting drugs (io~6 M colchicine, 30-min incubation; io~4 M diamide, 5-min incubation). Con A or F-Con A (15 ftg/ml) was present during a further 5 min of incubation after which cells were fixed with 2% paraformaldehyde (10 min, room temperature), washed and observed by phase-contrast, Nomarski optics or fluorescence using a Zeiss Photomicroscope III. Con A cap formation 59 Con A labelling for electron microscopy. Cell suspensions (10 x io9 peripheral blood leukocytes/ ml; 25 x io6 alveolar macrophages/ml) were incubated with io~° M colchicine or io~3 M diamide and then labelled for 5 min with Con A (100 /fg/ml) as described above. The cells were fixed at room temperature for 30 min in 1 % glutaraldehyde in 01 M cacodylate buffer, pH 74, then washed in buffer and postfixed in 1 % aqueous osmium tetroxide. Specimens were dehydrated through a graded series of ethanols and embedded in Epon. Thin sections were collected on 300-mesh grids, stained with uranyl acetate (Watson, 1958) and a mixture of lead salts (Sato, 1968) and examined in a Philips 300 electron microscope.

RESULTS Con A cap formation and associated cell shape cfianges: a morphological description. The surface distribution of Con A on leukocytes has been extensively analysed by fluorescence microscopy. The corresponding appearance of Con A-treated cells by phase-contrast microscopy and Nomarski optics has not been emphasized, and examination of the ultrastructure of Con-A-treated leukocytes has not revealed details of filament and membrane organization (Yahara & Edelman, 1975). We show below that combined application of these techniques immediately provides new insights into the process of Con A cap formation. The appearance by light, fluorescence, and electron microscopy of cells incubated with Con A alone is illustrated in Figs. 1 and 2. The cells are spherical (Fig. 1 A) and lectin is distributed homogeneously over the cell surface (Fig. IB). By electron microscopy (Fig. 2) Con A-treated leukocytes show the same irregularly ruffled surface and zone of granule exclusion under the membrane that is typical of untreated cells. Cells exposed to Con A differ from untreated cells by the presence of numerous microtubules radiating from the satellites of the centrioles. As described before (Hoffstein, Soberman, Goldstein & Weissmann, 1976; Oliver et al. 1976) exposure of leukocytes to Con A for 5 min induces a marked increase in the number of centriole- associated microtubules. Most PMN, lymphocytes, monocytes and macrophages treated with either diamide or colchicine followed by F-Con A are capped (Fig. 1 D). By phase-contrast microscopy, a bulge or protuberance is visible at the site of cap formation (Fig. IE). When viewed by Nomarski optics, this structure most frequently appears as a discrete projection of ruffled surface membrane that extends beyond a constriction in the cell body (Fig. 3 A, inset left, Fig. 3B, inset). At the ultrastructural level, cells that were capped with diamide or colchicine and Con A are readily recognized by the presence of a protuberance and by the relatively smooth contour of the remaining non-capped membrane (Fig. 3). The protuberance in the capped monocyte (Fig. 3 A) contains a large number of spherical or elongated vesicles. These may arise both from imaginations of the capped membrane and from internalization of Con A-receptor complexes. The inset (right) in Fig. 3 A shows the typical appearance by fluorescence microscopy of intracellular vesicles containing F-Con A within a cap. An important feature in all leukocytes is the accumulation of electron-dense material within the protuberance. This is emphasized in Fig. 3B, a section through a capped lymphocyte. Cytoplasmic organelles are excluded from this specialized region. 60 D. F. Albertini, R. D. Berlin andj. M. Oliver The protuberance can assume a variety of different shapes. The minimal clearly recognizable shape change is simply the appearance of a small projection of granular cytoplasm beyond a slight constriction in the cell body (Fig. 4A). By contrast, the PMN shown in Fig. 4B displays a tight, elongated constriction with a region of granular cytoplasm on either side of the constriction, while the macrophage in Fig. 4 c has developed a furrow or constriction that divides the cell into 2 comparably sized parts. On cells demonstrating these marked changes in shape, F-Con A is usually not capped at one pole of the cell. Rather, lectin accumulates at the constriction and is absent from the remaining membrane (Fig. 1E, F).

Fig. 1. The shape and distribution of F-Con A on peripheral blood PMN; phase- contrast and fluorescence microscopy. PMN treated with F-Con A (15 /ig/ml) alone maintain a rounded cell shape (A) and a homogeneous surface distribution of lectin (B). Cells treated with diamide followed by F-Con A usually show a protuberance (c) or more rarely a constriction towards the cell centre (E). F-Con A is concentrated over the protuberance (D) or at the constriction point (F). Initial magnification x 1250.

The appearance by electron microscopy of a PMN with a constriction is illustrated in Fig. 5 A. The constriction divides the cell into 2 segments, both containing granules and portions of the nucleus. Membrane ruffling activity is visible in the constricted area and the remaining plasma membrane is relatively smooth. At high magnification (Fig. 5B) a region of electron-dense material is visible at the constriction point. Con A cap formation 61 Straight filaments, 10-0-11-5 nm in diameter, course for several microns through the constricted region of the cell. The continuity of these filaments between the 2 sections of the cell emphasizes that a single cell is being examined. The relationship between cell shape change and Con A capping. The data above establish that the localization of a Con A cap corresponds to the site of formation of a protuberance or constriction point on the cell. However, they do not establish whether Con A cap formation and cell shape changes are independent or simultaneous events. The following experiments provide evidence that the shape changes induced by microtubule disassembly are clearly separable from the capping of Con A.

Fig. 2. The shape and ultrastructure of Con A-treated PMN. The inset illustrates the spherical shape and uniform distribution of granules in a Con A-treated PMN by Nomarski optics. The electron micrograph of a similar Con A-treated PMN shows an irregularly contoured plasma membrane, a cortical zone of granule exclusion and a centriole with associated microtubules (arrows), x 12100; inset, initial magnification x 1250.

Peripheral blood leukocytes and alveolar macrophages were preincubated with or without diamide or colchicine. The leukocytes were then either fixed with 2 % paraformaldehyde to prevent further redistribution of Con A-receptors, and subsequently labelled with F-Con A, or labelled and then fixed. Cell shape was examined by phase-contrast microscopy and the distribution of F-Con A determined by D. F. Albertini, R. D. Berlin and J. M. Oliver Con A cap formation 63 fluorescence microscopy using the same cell suspensions. The results obtained are given in Table 1. Cells exposed to buffer alone are mostly round (Table 1, lines 1,7)- Exposure of such cells to F-Con A either before or after fixation causes no marked effects; the cells remain round and Con A receptors show a relatively uniform surface distribution (Table 1, lines 2, 3, 8, 9). By contrast, exposure of cells to diamide or colchicine leads to the formation of either a constriction or a distinct protuberance identical to that on Con A-treated leukocytes on about 60% of cells (Table i, lines 4, 10). When these microtubule-depleted cells are fixed and then labelled with F-Con A, they maintain the same range of cell shapes. However, lectin is bound uniformly over the cell surface (Table 1, lines 5, 11; Fig. 6A, B). Thus, the shape changes induced by microtubule disrupting agents can occur independently of the movement of Con A-receptor complexes into caps. This observation was confirmed by electron microscopy. The cells in Fig. 6 c were treated with diamide but not Con A. A marked polarization of membrane activity is apparent. A band of electron-dense material is present underneath the folded membrane. This morphology is very similar to the morphology of Con A-capped cells (Fig. 3 A, B; see also Fig. 8 A). Centrioles were oriented at random with respect to the protuberance, suggesting that no precise spatial relationship exists between the protuberance and cytoplasmic structures. The data in Table 1, lines 6, 12 show that F-Con A labelling of diamide- or colchicine-treated cells prior to fixation not only leads to accumulation of fluorescence in the cap but also leads to an increase in the proportion of cells showing a protuberance. To further establish the separation between the formation of a protuberance and the movement of Con A into caps, we examined the distribution of F-Con A under conditions in which most cells had previously established protuberances rather than a range of shape changes. The strategy was to incubate leukocytes with colchicine or diamide followed by 5 /tg/ml unlabelled lectin (either Con A or a lectin of different specificity such as the Ricinus communis agglutinin (RCA)). This concentration of lectin was sufficient to induce the distinct protuberance characteristic of capped cells without occupying all of the Con A receptors when pretreatment was with Con A. (Control experiments under the same conditions with 5 /tg/ml F-Con A or F-RCA showed capped fluorescence.) The cells were then labelled with F-Con A (15 /

Fig. 3. Light- and electron-microscopic views of leukocytes capped by incubation with diamide and Con A. The insets (left) show the typical appearance of Con A caps on monocytes (A) and lymphocytes (B) by Nomarski optics. Both cells have a prominent protuberance of ruffled membrane. The fluorescence micrograph (A, inset right) shows intracellular vesicles containing F-Con A within the capped protuberance of a monocyte. The electron micrograph of a monocyte (A) reveals the presence of elongated vesicles within the prominent protuberance. In the lymphocyte (B) the membrane of the Con A-capped protuberance is extensively plicated. Note the increased cytoplasmic density beneath the cap and the smooth contour of non-capped regions of the plasma membrane, A, X 11300; B, X 12100. Insets, initial magnification x 1250. 5-2 % D. F. Albertini, R. D. Berlin and J. M. Oliver Fig. 7. When macrophages or PMN were incubated with Con A or RCA alone at 5 /Jg/ml and then labelled with F-Con A, the cells remained round and fluorescence was distributed uniformly over the surface (Fig. 7A). When cells pretreated with diamide or colchicine and unlabelled lectins were fixed and then labelled with F-Con A at 37 °C, or labelled without fixation at 4 °C, most cells showed a protuberance. Nevertheless, F-Con A was distributed over the entire surface (Table 2, lines 2, 4, 6, 8; Fig. 7B). Treatment with diamide or colchicine and unlabelled lectin followed by incubation with F-Con A at 37 °C did not increase the proportion of cells with a protuberance. However, F-Con A was now capped (Table 2, lines 1, 3, 5, 7; Fig. 7c).

Fig. 4. Nomarski views of leukocytes exposed to diamide and Con A. A, PMN showing small projection of granular cytoplasm; B, tightly constricted PMN with granular cytoplasm on either side of the constriction; c, an alveolar macrophage with a deep central furrow. Initial magnification of A, B, X 1250; c, x 500.

The results with cells fixed prior to F-Con A labelling indicate that unoccupied receptors can remain uniformly distributed over the cell surface despite the presence of a distinct protuberance. The results with cells fixed after F-Con A labelling demon- strate that a second wave of movement of F-Con A-receptor complexes over the surface and into a preformed cap can occur at 37 °C. Moreover, the second wave can spread throughout the membrane in the region of the preformed cap. Thus, although Con A- receptor complexes are concentrated in the cap they are clearly capable of moving within the confines of the capped membrane. Ultrastructure of the Con A cap. Further ultrastructural analysis of the capped membrane and underlying cytoplasm established that the region of granule exclusion is composed primarily of microfilaments. The cells in Figs. 8 and 9 were capped by incubation with diamide and Con A. A monocyte sectioned through the longitudinal axis of the cap is illustrated in Fig. 8 A. The capped membrane is extensively folded and a band of electron-dense material underlies the entire cap. Granules are excluded from the region above the dense band but elongated membrane vesicles most likely containing internalized Con A are prominent below this region. At higher magnification (Fig. 9) it is apparent that the electron-dense band is composed primarily of tightly packed microfilaments. At the margins of the cap a gradual thinning of the Con A cap formation

Fig. 5. Ulrrastructure of a constricted PMN. The cell was exposed to diamide and Con A. The cytoplasm and nucleus of this PMN are partitioned by a deep furrow or constriction (A). Electron-dense material is prominent at the constriction point. At higher magnification (B) intermediate (io-nm) filaments (arrows) course between the 2 sections of the cell. Cytoplasmic granules (g) are trapped in surface plications in the region of the constriction, A, X 9200; B, X 40700. 66 D. F. Albertini, R. D. Berlin andj. M. Oliver dense band occurs and individual microfilaments, 6-8 nm in diameter, can be resolved. Other microfilaments emerge from the dense band and course into the plications of the capped membrane. It is apparent from the inset to Fig. 9 that the latter microfilaments can extend the entire length of these membrane processes. Periodic attach- ments are visible between the filaments and the membrane. A transverse section

Table 1. Separation of cell shape changes from Con A capping

B. Rabbit alveolar :macrophages 7 PBS 97 2 1 — — 8 PBS, F-Con A 97 3 0 85 15 9 PBS, Fix, F-Con A 98 2 0 100 0 10 Colchicine 37 31 32 •— — 11 Colchicine, Fix, F-Con A 37 28 35 100 0 12 Colchicine, F-Con A 21 15 64 15 85 Cells were pretreated with drugs then exposed to F-Con A for 5 min either before or after fixation. The cells were washed, mounted on slides and the distribution of F-Con A and the shape of cells were determined respectively by fluorescence and phase-contrast microscopy. At least 100 cells per slide were scored for both cell shape and distribution of Con A. • Constricted cells include those with limited polarization in shape (as in Fig. 6 B) through those with a pronounced indentation separating the cell into 2 equal or unequal regions of granular cytoplasm (as in Fig. 5). Cells with protuberances are those where a single bulge of cytoplasm and ruffled membrane projects from one pole of the cell (Fig. 3). •f The distribution of F-Con A is classified as uniform when fluorescence occupies the whole membrane either in a homogeneous distribution or in surface patches. Con A is capped when fluorescence is concentrated at a constriction point or over a protuberance. A few cells also showed a polar accumulation of fluorescence not associated with an obvious change in cell shape. Hence the proportion of capped cells is somewhat larger than the proportion of cells showing a shape change when examined by phase-contrast. through the cap of a lymphocyte is shown in Fig. 8B. This section is approximately perpendicular to the section in Fig. 8 A. A nearly complete ring of microfilaments is visible. In addition, thicker (IO-O-II-5 nm in diameter) rod-like filaments are present within the circle of microfilaments. These fibres are structurally similar to the myosin thick filaments that were observed in human blood platelets by Niederman & Pollard (1975)- Con A cap formation

Fig. 6. Cell shape changes induced by diamide. The fluorescence micrographs illustrate PMN that were treated with diamide, fixed and subsequently labelled with F-Con A. The cells show either a distinct protuberance (A) or a moderate shape change (B). However, F-Con A is bound uniformly over the surface of both cells. The slight increase in labelling intensity in A reflects the increased amount of membrane in a protuberance (see c). The electron-microscopic profiles of PMN exposed to diamide alone (c) establish the confinement of surface plications at one pole of the cell. Electron-dense material is concentrated under this specialized region. A centriole depleted of microtubules is present in the lower cell (co). A, B initial magnification X 1250; C, X 15400. D. F. Albertini, R. D. Berlin and J. M. Oliver

Table 2. Cell shape and distribution of F-Con A on precapped cells

% cells % cells with a pro- capped with Line Treatment tuberance F-Con A A. Human peripheral blood leukocytes 'X Diamide, Con A, F-Con A 67 92 M Diamide, Con A, Fix, F-Con A 65 o Diamide, RCA, F-Con A 61 89 Diamide, RCA, Fix, F-Con A 64 o

B. Rabbit alveolar macrophages Colchicine, Con A, F-Con A $$, I* Colchicine, Con A, Fix, F-Con A $$ f Colchicine, RCA, F-Con A $f if Colchicine, RCA, Fix, F-Con A 69 o Cells were pretreated with drugs then exposed to unlabelled lectin for 5 min, washed once, resuspended in PBS containing F-Con A and incubated for a further 5 min. Fixation was either before or after the second incubation with lectin. The cells were washed, wet-mounted on glass slides, and 100 cells scored in each slide, for the presence or absence of distinct protuberance (by phase-contrast microscopy) and for concentration of F-Con A into caps (by fluorescence microscopy). The somewhat greater percentage of cells with Con A caps than with protuberances is due in part to the fact that some cells that appear capped show a constricted cell shape and in part to the development of Con A caps on a small proportion of cells in which no shape change is apparent by phase-contrast microscopy.

Fig. 7. The mobility of F-Con A on macrophages prelabelled with RCA. The macrophage in A was exposed to RCA (5 /ig/ml) followed by F-Con A (15 /ig/m\). A uniform distribution of fluorescence is apparent. Macrophages treated with colchicine and RCA, fixed and then labelled with F-Con A showed a prominent protuberance corresponding to the site of RCA capping (B). However, fluorescence due to F-Con A is distributed over the whole membrane. The apparent increased fluorescence observed in the protuberance is probably due to the extensive folding of membrane in this region. Macrophages treated with colchicine and RCA and then labelled with F-Con A at 37 °C showed fluorescence concentrated into a cap (c). Initial magnification x 500. 69

B Fig. 8. Ultrastructure of the Con A cap. A, section through the longitudinal axis of a capped monocyte. A band of electron-dense material is located immediately beneath the cap. Elongated vesicles {ev) extend from the cap towards the cell centre. Note the presence of centrioles (co) lacking microtubules to the left. B, a transversely sectioned profile through the capped region of a lymphocyte, showing a central ring of microfuament (mf) profiles. Dense rod-like structures (arrows) resembling myosin filaments (/) are present within the microfilament ring, A, X 12 300; B, X 55800. 70 D. F. Albertini, R. D. Berlin and J. M. Oliver

DISCUSSION Since the first descriptions of Con A cap formation induced by colchicine in mouse splenic lymphocytes (Edelman et al. 1973) and SV40 virus-transformed fibroblasts (Ukena, Borysenko, Karnovsky & Berlin, 1974) a great deal of research interest has focused on the mechanisms that can restrain the movement of Con A-receptor complexes or permit their movement into caps. The results of pharmacological analyses in this and other laboratories have implicated microtubules in the restriction of Con A movement and microfilaments in the process of movement of Con A into caps in leukocytes (Oliver, 19760,6; Nicolson, 1976; Schreiner & Unanue, 1976). In support of microtubule involvement, we have shown by electron microscopy that a homogeneous surface distribution of Con A is correlated with the presence of cytoplasmic microtubules in PMN and that capping occurs when microtubules have been disrupted (Oliver et al. 1976). A role for microfilaments in Con A capping was suggested from the analysis by Albertini & Anderson (1975) of the spontaneous formation of Con A caps in rabbit ovarian granulosa cells. In these cells, patches of surface-bound Con A aggregate into a central cap that is bordered by an underlying ring of microfilaments. Similarly, microfilaments are present beneath immunoglobulin G caps that develop spontaneously in lymphocytes (de Petris, 1975). Nevertheless, no general agreement about the mechanism(s) by which cytoplasmic filaments and microtubules control the topographical distribution of Con A has yet been reached. The data presented here provide additional information that may be of value in establishing a generally acceptable model for Con A cap formation. The following points are of major importance: (1) Disassembly of microtubules produces a range of shape changes and redistributions of microfilaments in PMN, lymphocytes, monocytes and alveolar macrophages. These changes may be identical to those associated with cap formation, but (2) the microfilament redistributions and shape changes can occur in the absence of binding of Con A. (3) Microfilament redistribution can occur without redistribution of the Con A receptors. (4) When microtubule-depleted cells are treated with Con A, microfilaments and Con A-receptor complexes are closely associated within a cap or, less frequently, at a constriction point in the cell. (5) Con-A receptor complexes can move into pre-established caps, even though numerous microfilaments are already extensively organized at a distance from the complexes. These data are strengthened by their similarity in the diverse cell types studied: PMN, lymphocytes, monocytes and alveolar macrophages. However, they are not entirely consistent with the current views of Con A cap formation. For example

Fig. 9. Anatomy of the electron-dense band subtending a surface cap. At high magnification a dense filamentous band is resolved beneath the capped membrane. This band extends to the margin of the cap (upper left) and is characterized by the presence of thin (60-80 ran) actin-like microfilaments (arrows, mf) and occasional rod-like fibrous elements (probably myosin filaments,/). The inset shows that microfilaments course from the electron-dense band into the membrane processes. These microfilaments are visible in both longitudinal and cross-sectional profiles (small arrows). Lateral extensions of the microfilaments terminate on the adjacent plasma membrane (large arrows), x 70400; inset x 75000. Con A cap formation 71 72 D. F. Albertini, R. D. Berlin andj. M. Oliver Edelman (1976) considers that Con A-receptor complexes are physically attached to microfllaments. Capping occurs by cross-linking between adjacent complexes with simultaneous aggregation of associated microfllaments. However, we show that filament aggregation can precede movement of Con A-receptor complexes. In addition the apparent mixing of sequentially added lectin-receptor complexes in a cap provides evidence against extensive cross-linking during Con A cap formation. Schreiner & Unanue (1976) envisage a microfilament-directed movement of unlabelled membrane away from a lattice of cross-linked Con A-receptor complexes which thus accumulate passively into a cap at the trailing end of the cell. In contrast our data show that microfilaments subtend the cap and not its opposite pole. Other investigators (Bretscher, 1976; Harris, 1976) have proposed that capping results from inclusion of cross-linked ligand-receptor complexes in a continuous directed flow of membrane lipid or of the whole membrane towards a region where the major membrane components are internalized. The cross-linked complexes cannot be internalized and so accumulate to form a cap. These hypotheses do not explain the apparent involvement of microtubules and microfilaments in the capping process, nor are they consistent with the rapid internalization of vesicles that are enriched for ligand-receptor complexes at the sites of both Con A (Albertini & Anderson, 1975; see also Fig. 3 A) and immunoglobulin G (Unanue & Karnovsky, 1973; Raff & de Petris, 1973) cap formation. In our view, the most important condition leading to Con A cap formation in leukocytes is the disassembly of microtubules which leads to the reorganization of microfilaments into a protuberance. The ultrastructure of the protuberance is similar to that of the brush border of intestinal epithelium (Mooseker & Tilney, 1975). Thus, in analogy to the terminal web, a dense filamentous network underlies the region of membrane ruffling. This dense band of microfilaments does not appear to connect directly with the membrane. Rather, individual microfilaments emerge from the filamentous band and course into the individual plications of the protuberance. These filaments extend towards the tip of the plications. Further, they appear to attach to the membrane via lateral cross-bridges. The plications thus are analogous to the microvilli of the brush border. Myosin-like filaments, previously shown to occur in leukocytes by Stossel & Pollard (1973) and Senda et al. (1975) are present within the microfilament core. In transverse sections through caps, these filaments were prominent within the microfilament band. Thus, the most likely 3-dimensional arrangement of the protuberance is a central array of myosin filaments that attach to a cone of microfilaments. The microfilaments present in membrane plications originate from the outer surface of the filamentous cone. Why microtubule disassembly results in activation or reorganization of microfilaments and other filamentous elements is unknown. One possibility is that microtubule-microfilament interactions normally inhibit microfilament contraction or assembly. In this case, microtubule disassembly would permit microfilament poly- merization. However, mechanisms leading to the concentration of microfilaments at one region of the cell are not obvious and no direct evidence for inhibition of microfilament assembly or function by microtubules is available. Another possibility is that Con A cap formation 73 microtubule disassembly leads to formation of a protuberance by permitting the development of a regional imbalance of forces at the membrane wherein intracellular hydrostatic pressure exceeds the counter force derived from membrane surface tension. The mechanism leading to such an imbalance is not denned. However, evidence that microtubules may control the microviscosity of the leukocyte membrane and hence its physical properties was recently obtained (Berlin, 1975). One con- sequence of their disassembly could be reduction in surface tension within a limited domain of the membrane. Microfilaments might then be recruited to prevent the spread of such domains and to permit resorption of the protuberance. The variable shape of cells treated with diamide and colchicine alone may reflect such dynamic changes in membrane organization. Subsequent Con A treatment increases the proportion of cells with an extreme protuberance, perhaps by inhibiting the relaxation of microfilaments. Consistent with this model, Trinkaus (1973) and Ramsey (1972) have indicated that in certain cell types, including PMN, movement appears to involve a rapid extension of a bleb or lamellipodium from the surface followed by flow of cytoplasm into the bleb rather than a 'sol-gel' transformation as in the extension of pseudopods by amoebae. Dipasquale (1975) has shown that bleb formation can be inhibited in fibroblasts by hypertonic solutions of sorbitol, suggesting that the extension depended on excess turgor pressure. We emphasize that no directionality can be assigned to the protuberance in our cells since all experiments were performed in suspension. The absence of a consistent relationship between the protuberance and the centrioles suggests that protuberances in microtubule-depleted cells may develop at any point on the membrane. In the presence of Con A, lectin-receptor complexes accumulate at regions of microfilament density from which they are subsequently removed by internalization. Con A-receptor complexes can move within the confines of a cap, and can be recruited from distances considerably removed from the cap. Thus, cap formation is unlikely to occur by extensive cross-linking of adjacent receptors into a functionally frozen aggregate even though limited cross-linking is not excluded. What factors, then, lead to the final disposition of Con A-receptor complexes and microfilaments in the cap ? One possibility is that although movement of Con A-receptor complexes is independent of their interaction with filaments, interactions between microfilaments and membrane components may ultimately occur in the cap. Such interactions could limit lateral diffusion of Con A-receptor complexes without influencing the mobility of unoccupied receptors. Such microfilament-receptor interactions would clearly have to be sufficiently dynamic to permit movement of a second wave of Con A-receptor complexes through a preformed cap. No marked differences in microfilament organization between cells simply treated with colchicine or diamide and cells treated in addition with Con A that might support this hypothesis have been observed. Thus, we favour an alternative explanation that extends the suggestion above that the chemical or physical properties of the membrane of a protuberance may differ from those of the bulk membrane. We propose that cells treated with Con A alone maintain a uniform membrane composition in which either no preferred environment for Con A-receptor complexes exists or in which a series of 74 D. F. Albertini, R. D. Berlin andj. M. Oliver preferred domains promoted by microtubule assembly exist over the whole membrane. The result is a homogeneous distribution of F-Con A at the level of the fluorescence microscope. Microtubule-depleted cells may develop a region of decreased fluidity (increased microviscosity) in the protuberance. This modified membrane may limit the diffusion of Con A-receptor complexes (but not free receptors). By this model, microfilaments do not attach to Con A-receptor complexes. Rather, they may poten- tiate capping by helping to balance the forces created by regional alterations in the physical properties of the capped membrane. Experiments to test for physical associations between actin filaments and membranes by fluorescence resonance energy transfer techniques developed in this laboratory (Becker, Oliver & Berlin, 1975) are in progress. We are also studying the microviscosity of capped and non-capped regions of leukocyte membranes by fluorescence polarization techniques (Berlin, 1975; Fernandez & Berlin, 1976). These data may provide direct evidence for or against these 2 potential mechanisms for Con A cap formation.

We thank Ms Geraldine Crean and Christine Pearson for their excellent technical assistance. Supported in part by grants ES-onc6, CA-18564 and GM-22621 from the National Institutes of Health and by grant BC-179 from the American Cancer Society.

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(Received 16 December 1976)