The Cytoskeletal System of Nucleated Presence of a High Molecular Weight Protein Erythrocytes. II. Calmodulin-binding

Calmodulin was detected in dogfish erythrocyte lysates by means of phosphodiesterase activation. Anucleate dogfish erythrocyte cytoskeletons bound calmodulin. Binding of calmodulin was calcium-dependent, concentration-dependent, and saturable. Cytoskeletons consisted of a marginal band of microtubules containing primarily tubulin, and trans-marginal band material containing actin and spectrinlike proteins. Dogfish erythrocyte ghosts and cytosekeletons were found to contain a calcium-dependent calmodulin-binding protein, CBP, by two independent techniques: (a) 12Sl-calmodulin binding to cytoskeletal proteins separated by SDS PAGE, and (b) in situ azidocalmodulin binding in whole anucleate ghosts and cytoskeletons. CBP, with an apparent molecular weight of 245,000, co-migrated with the upper band of human and dogfish erythrocyte spectrin. CBP was present in anucleate ghosts devoid of marginal bands and absent from isolated marginal bands. CBP therefore appears to be localized in the trans-marginal band material and not in the marginal band. Similarities between CBP and high molecular weight calmodulin-binding proteins from mammalian species are discussed. Generation and maintenance of correct cytoskeletal organization is a subject of primary interest in cells such as erythrocytes (3) in which specific mechanical properties and asymmetric morphology are vital to cell function. Evidence obtained in recent years implicates calcium ion in the control of such organization as well as many other cellular phenomena. Assembly of both microtubules (29, 36) and microfflaments (18, 38) has been shown to be calcium-mediated in vitro, and localized intracellular calcium levels are believed to regulate formation of at least some microtubule systems in vivo (21, 28). Calmodulin is a known mediator in the control of numerous cellular enzymes by calcium (12, 25) and its interaction with microtubule-associated proteins (MAPs) has been suggested as the mechanism by which calcium causes microtubule disassembly (7). To elucidate the possible role of calcium and calmodulin in the formation and function of the erythrocyte cytoskeleton, we have examined noumammalian erythrocytes for ealmodulin content and for the presence of calmodulin-binding proteins associated with the cytoskeleton. The noumammalian erythrocyte was chosen for this study because it is a relatively simple nucleated cell type with characteristic flattened elliptical morphology, and is obtainable in pure populations. Its cytoskeletal system contains components of universal interest, including a marginal band of microtubules (3, 14), a spectrin-containing network of trans-marginal band material (TBM; reference 15), and intermediate filaments attached to the nucleus (37). The cytoskeleton of nonmammalian erythrocytes resembles those of nucleated nonerythroid cells more closely than does the mammalian erythrocyte cytoskeleton and therefore may serve as a more appropriate model system. The dogfish erythrocyte is well suited for this study because it is available in quantity and, due to its relatively large size (15), provides a cytoskeleton readily visualized by phase-contrast microscopy. MATERIALS AND METHODS Diisopropyl fluorophosphate (DFP), EGTA, HEPES, PIPES, p-tosyl ar~nine methyl ester (TAMe), and lactoperoxidase were obtained from the Sigma Chemical Co., St. Louis, MO. Nat~SI, 2 Ci/mmol, was a product of Amersham Cozp. (Arlington Heishts, IL). Standard molecular weight markers were obtained from THE JOURNAL OF CELL BIOLOGY • VOLUME 95 OCTOBER 1982 278-284 278 © The Rockefeller U niversity Press • 0021-9525/82/10/0278/07 $1.00 on A ril 6, 2017 D ow nladed fom Published October 1, 1982