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 mem- brane 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, fluorescence is 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 concen- tration of Con A into the membrane of the protuberance are clearly separable events, micro- filaments 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 micro- filament 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 poly- morphonuclear 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 concen- tration. 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