Participation of calmodulin in immunoglobulin capping

When mouse B lymphocytes are incubated with antibodies against their surface immunoglobulin (Ig), patching and capping occur in a process that involves the action of the actomyosin cytoskeleton and the mobilization of cell calcium. Calmodulin (CAM) plays a central role in the Ca ++ regulation of many cellular structures and processes, including the cytoskeleton and membrane-bound enzymes, and therefore was investigated for its role in capping. CaM was isolated from mouse lymphocytes by affinity chromatography on Fluphenazine-Sepharose. Lymphocyte CaM co-migrates with calf brain CaM on SDS polyacrylamide gels, where its rt is Ca++-dependent. It stimulates the activity of the CaM-dependent cyclic AMP phosphodiesterase (PDE) of bovine heart. Several phenothiazine and thioxanthene compounds as well as the drugs W7, WS, and R24571 inhibit CaM in in vitro enzyme assays with ID~o's of from 1 /~M to >1 mM. These were tested for their effects on capping of Ig and were found to inhibit capping in dose-dependent fashions with ID~o'S that corresponded to their anti-CaM potencies. The drugs also disrupted preformed caps and were all reversible. CaM was localized in lymphocytes by staining with a highly fluorescent trifluoperazine derivative (TFP*) produced by photo-oxidation. TFP* staining was diffuse in untreated lymphocytes but stained under caps and in uropods in cells capped with anti-lg antibodies. Staining of cells with antibodies against calf brain and rat testis calmodulin gave similar staining patterns. Staining of patched cells with either antibodies or TFP* showed patched distributions of CaM, but submembranous CaM "patches" did not map one-on-one with respect to Ig patches. These observations suggest that calmodulin participates in the latter stages of ligandinduced Ig redistribution probably by regulating the interaction of the cytoskeleton with the membrane. The capping of surface immunoglobulin on B lymphocytes is a dramatic example of surface receptor redistribution mediated by components of the underlying cytoskeleton (5, 21, 32, 53, 57). The arrangement and regulation of the cell's actomyosinbased motility apparatus and the means by which surface receptors are connected to this machinery through the lipid bilayer are two of the central issues in understanding patching and capping. Cross-linking of receptors by multivalent ligands is required to initiate the energy-independent patching of receptors that must occur before the energy-dependent, cytoskeleton-mediated capping process gathers the patched receptors to one pole of the celi (40, 57, 58, 59). It is during the patching phase of receptor movement that initial connections to the cytoskeleton are made (3, 5, 14, 18, 20, 71). The cell's motility machinery then coordinates the movement of receptor aggregates. These two physiologically and structurally distinct steps may be regulated in similar or dissimilar ways. One known regulatory species whose local distribution is altered during cross-linking and patching is the calcium ion. Cross-linking of surface immunoglobulin (Ig) by anti-Ig antibodies induces movement of protein-associated Ca ++ to lipid regions of the membrane, as reported by the fluorescent probe chlorotetracycline (32). 45Ca+÷ efflux from intact lymphocytes occurs subsequent to cross-linking (7). Studies of the effects of free-fatty acids on membrane structure ana capping have led to the description of membrane domains and implicate changes in local Ca ++ distribution as occuring concomitantly with the cross-linking of immunoglobulin receptors (32, 33). THE Iournau or CeLt BIOLOGY • VOLUME 95 DecemBer 1982 771-780 © The Rockefeller University Press • 0021-9S25/82/12/0771/10 $1.00 7 7 1 Calcium plays a regulatory role in the capping of receptors as well as in their patching. In the presence of external calcium, the calcium ionophore A23187 is a potent inhibitor of capping (but not patching) and will even disperse preformed caps in an energy dependent process (6, 53). Cis-unsaturated free fatty acids will inhibit capping (but not patching) and can induce changes in the arrangement of the cytoskeleton--these effects are antagonized by high concentrations of extracellular calcium (29, 32). A variety of tertiary amine anesthetics and tranquilizers act as capping inhibitors and in some cells have been reported to dissociate microfilaments from the cell membrane (2, 6, 42, 48, 51, 72). Their anti-capping affects are also antagonized by high extracellular calcium. These observations indicate a role for calcium in maintaining the connection of the cytoskeleton to the membrane. The regulation by calcium of microtubule polymerization, actomyosin-based contraction and cell motility is well documented (11, 23, 24, 26, 28, 41, 43, 62) and must also be considered in the context of capping. The common theme of calcium control unites these observations and recent studies suggest that the protein calmodulin may provide the key to understanding the molecular basis of this calcium control. Calmodulin (CAM) is a ubiquitous calcium-binding protein that has been found to regulate a variety of cellular processes in eukaryotes; many of these processes are directly relevant to capping (10, 11). Calmodulin regulates the Ca++/Mg ÷+ ATPases of cell membranes and thereby the internal ionic states of cells (34, 44). It regulates the polymerization of microtubules in vitro and is associated with microtubules in vivo (13, 41, 68). Actomyosin in smooth and skeletal muscles is regulated via the calmodulin dependence of myosin light chain kinase (23, 24, 28, 62). The shapes and ligand binding properties of platelets are altered by calmodulin inhibitors (31). Immunocytochemical localization of calmodulin shows it to be associated with stress fibers or microfilament bundles and the mitotic spindles of fibroblasts as well as with postsynaptic densities of nerve membranes (13, 25, 63, 68, 70). Finally, several of the drugs that are known to inhibit capping have been shown to be potent specific inhibitors of calmodulin in in vitro enzyme systems (22, 34, 36, 56, 60, 61, 64, 66, 67). These observations prompted us to investigate directly whether calmodulin plays a role in lymphocyte capping. In this paper we identify and characterize a calmodulin protein from murine splenic lymphocytes. The preferential distribution of calmodulin under immunoglobulin caps is mapped with anti-calmodulin antibodies and a fluorescent calmodulin-binding drug. The dose-dependent inhibition of capping by a variety of calmodulin-specific inhibitors is then demonstrated. Finally, these observations are all interpreted along with earlier observations in terms of how calmodulin may regulate capping of surface immunoglobulin and other