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An experimental approach is described that enables the analysis of interactions between exogenous surface ligands and components of the cytoplasm in neutrophil leukocytes . Neutrophils treated with the nonionic detergent Lubrol FIX, under controlled conditions, yield intact detergent-insoluble ghosts . Morphological analysis of neutrophil ghosts shows that they retain the original dimensions of the cell and consist almost entirely of a peripheral filamentous network, representing the submembranous cortical web, concentric to nuclear remnants . All intracellular membrane-bounded organelles, plasma membrane, and background cytoplasmic electron density are absent .Biochemical analysis of the ghosts shows that <10% of enzyme markers for the soluble and granule fractions remain, and that >90% of total cell phospholipid is removed during detergent extraction . The major proteins remaining in the ghosts comigrate, on polyacrylamide gels in the presence of SDS, with chicken gizzard actin, myosin, filamin, and a 110-kdalton protein .Patches and caps induced on neutrophils with either fluorescein isothiocyanate-concanavalin A or ferritin-concanavalin A retain their original location and morphology on ghosts after lysis, as determined by both fluorescence and electron microscopy . In similar experiments, but using '25 1-labeled lectins, 37% of total cell bound concanavalin A (Con A) and 25% succinylated Con A remain attached to the ghosts .A major "51-labeled membrane glycoprotein (80 kdaltons) is associated with ghosts prepared from intact neutrophils iodinated in the presence of exogenous lactoperoxidase . Further ' 25 1-labeled membrane glycoproteins (217, 170, and 147 kdaltons) become associated with ghosts prepared from iodinated cells treated before lysis with Con A, but not with succinylated Con A.These data taken together suggest that linkages exist in neutrophils between proteins exposed on the outer surface of the plasma membrane and the peripheral filamentous network independent of the presence of lipid bilayer. The implications of these findings for surface motile phenomena will be discussed .Since the wide acceptance of a membrane structure based on the fluid mosaic model proposed by Singer and Nicolson (35), it has become apparent that the lateral mobility of some membrane proteins may be constrained or influenced by their interaction, through the lipid bilayer, with components of the cytoplasm (14). This property of certain membrane proteins can be inferred from their behavior in the presence of multivalent ligands (34) and from lateral diffusion data obtained in photobleaching experiments (30,31,36) . In some cases these phenomena can be enhanced or inhibited by treatment of cells with agents that interfere with microtubule or microfilament THE JOURNAL OF CELL BIOLOGY " VOLUME 90 SEPTEMBER 1981 743-754 © The Rockefeller University Press " 0021-9525/81/09/0743/12 $1 .00 integrity (13,14), thus leading to the predominant view that these structures are somehow involved either directly or indirectly in...

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The free calcium ion concentration, [Ca2+]i, in the cytoplasmic matrix of quin2-loaded neutrophil leucocytes increases rapidly after addition of concanavalin A. This increase is effectively abolished by a short (3 min) preincubation with 10 nM-TPA (12-O-tetradecanoylphorbol 13-acetate). TPA also inhibits a [Ca2+]i rise of similar magnitude induced by low concentrations (10 nM) of calcium ionophore A23187, suggesting that phorbol ester does not interfere with a physiological influx mechanism. To investigate the effects of TPA further, cells were depleted of Ca2+ during quin2 loading and then re-equilibrated with normal extracellular [Ca2+]. The return to a stable [Ca2+]i value was preceded by a transient overshoot in [Ca2+]i, implying delayed activation of an efflux mechanism by rising [Ca2+]i. TPA abolished the transient, suggesting preactivation by TPA of the efflux mechanism before Ca2+ influx. TPA also stimulates net Ca2+ efflux from neutrophils and neutrophil cytoplasts. These observations are consistent with the thesis that TPA stimulates a Ca2+-efflux mechanism in these cells.