Slow components of axonal transport: two cytoskeletal networks

We have identified two slowly moving groups of axonally transported proteins in guinea pig retinal ganglion cell axons (4). The slowest group of proteins, designated slow component a (SCa), has a transport rate of 0.25 mm/d and consists of tubulin and neurofilament protein. The other slowly transported group of proteins, designated slow components b (SCb), has a transport rate of 2-3 mm/d and consists of many polypeptides, one of which is actin (4). Our analyses of the transport kinetics of the individual polypeptides of SCa and SCb indicate that (a) the polypeptides of SCa are transported coherently in the optic axons, (b) the polypeptides of SCb are also transported coherently but completely separately from the SCa polypeptides, and (c) the polypeptides of SCa differ completely from those comprising SCb. We relate these results to our general hypothesis that slow axonal transport represents the movements of structural complexes of proteins. Furthermore, it is proposed that SCa corresponds to the microtubule-neurofilament network, and that SCb represents the transport of the microfilament network together with the proteins complexed with microfilaments.     

Differential axonal transport of isotubulins in the motor axons of the rat sciatic nerve

The axonal transport of the diverse isotubulins in the motor axons of the rat sciatic nerve was studied by two-dimensional polyacrylamide gel electrophoresis after intraspinal injection of [35S]methionine. 3 wk after injection, the nerve segments carrying the labeled axonal proteins of the slow components a (SCa) and b (SCb) of axonal transport were homogenized in a cytoskeleton-stabilizing buffer and two distinct fractions, cytoskeletal (pellet, insoluble) and soluble (supernatant), were obtained by centrifugation. About two-thirds of the transported-labeled tubulin moved with SCa, the remainder with SCb. In both waves, tubulin was found to be associated mainly with the cytoskeletal fraction. The same isoforms of tubulin were transported with SCa and SCb; however, the level of a neuron-specific beta-tubulin subcomponent, termed beta', composed of two related isotubulins beta'1 and beta'2, was significantly greater in SCb than in SCa, relative to the other tubulin isoforms. In addition, certain specific isotubulins were unequally distributed between the cytoskeletal and the soluble fractions. In SCa as well as in SCb, alpha'-isotubulins were completely soluble in the motor axons. By contrast, alpha' and beta'2-isotubulins, both posttranslationally modified isoforms, were always recovered in the cytoskeletal fraction and thus may represent isotubulins restricted to microtubule polymers. The different distribution of isotubulins suggests that a recruitment of tubulin isoforms, including specific posttranslational modifications of defined isoforms (such as, at least, phosphorylation of beta' and acetylation of alpha'), might be involved in the assembly of distinct subsets of axonal microtubules displaying differential properties of stability, velocity and perhaps of function.     

Anterograde Components of Axonal Transport in Motor and Sensory Nerves in Experimental 2,5-Hexanedione Neuropathy

Anterograde slow and fast axonal transport was examined in rats intoxicated with 2,5-hexanedione (1 g/kg/week) for 8 weeks. Distribution of radioactivity was measured in 3-mm segments of the sciatic nerve after labelling of proteins with [35S]methionine or [3H]leucine and glycoproteins with [3H]fucose. The axonal transport of the anterograde slow components was examined after 25 (SCa) and 10 days (SCb), in motor and sensory nerves. SCa showed an increased transport velocity in motor (1.25 +/- 0.08 mm/day versus 1.01 +/- 0.05 mm/day) and in sensory nerves (1.21 +/- 0.13 mm/day versus 1.06 +/- 0.07 mm/day). The relative amount of labelled protein in the SCa wave in both fiber systems was also increased. SCb showed unchanged transport velocity in motor as well as in sensory nerves, whereas the amount of label was decreased in the motor system. Anterograde fast transport in motor nerves was examined after intervals of 3 and 5 h, whereas intervals of 2 and 4 h were used for sensory nerves. Velocities and amounts of labelled proteins of the anterograde fast component remained normal. We suggest that the increase in protein transport in SCa reflects axonal regeneration.     

Slow Axonal Transport Impairment of Cytoskeletal Proteins in Streptozociti-Induced Diabetic Neuropathy

The impairment of slow axonal transport of cytoskeletal proteins was studied in the sciatic nerves of streptozocin-diabetic rats. [35S]Methionine was unilaterally injected into the fourth lumbar ganglion and spinal cord, to label the sensory and motor axons, respectively, and then the polymerized elements of the cytoskeleton and the corresponding soluble proteins were analyzed separately. In addition, the pellet/supernatant ratio for tubulin and actin was also assessed. Our results indicate that the velocity of slow component a (SCa) of axonal transport, particularly that of neurofilaments, was strongly reduced (by 60%) in sensory axons. At the same time, a decreased pellet/supernatant ratio of tubulin, possibly owing to a depolymerization of stable microtubules, was also observed. The transport of slow component b (SCb) of axonal transport was also impaired, but the extent of this impairment could not be precisely evaluated. In contrast, motor axons showed little or no impairment of both SCa and SCb at the time studied, a result suggesting a delayed development of the neuropathy in motor axons.     

Proteins transported in slow components a and b of axonal transport are distributed differently in the transverse plane of the axon

The distribution of the proteins migrating with the slow components a (SCa) and b (SCb) of axonal transport were studied in cross-sections of axons with electron microscope autoradiography. Radiolabeled amino acids were injected into the hypoglossal nucleus of rabbits and after 15 d, the animals were killed. Hypoglossal nerves were processed either for SDS-polyacrylamide gel electrophoresis fluorography to identify and locate the two components of slow transport, or for quantitative electron microscope autoradiography. Proteins transported in SCa were found to be uniformly distributed within the cross-section of the axon. Labeled SCb proteins were also found throughout the axonal cross-section, but the subaxolemmal region of the axon contained 2.5 times more SCb radioactivity than any comparable area in the remainder of the axon.     

Axotomy accelerates slow component b of axonal transport.

Because the integrity of an axon depends on the supply of proteins synthesized in the cell body, we examined the effect of axotomy on the transport of structural proteins in rat motor axons, and the effect of altered transport on the rate of outgrowth after a subsequent testing axotomy. To examine the axonal transport of structural proteins, we labeled newly synthesized proteins with 35S-methionine 7 days after a "conditioning" lesion of the sciatic nerve, and removed the nerve 7-21 days later for SDS-PAGE. Tubulin, actin, calmodulin, and the 68-kD light neurofilament protein (NF-L) were identified by fluorography and removed for liquid scintillation counting. The fastest moving structural proteins were carried by slow component b (SCb) of axonal transport, which advanced 20% faster in conditioned axons: 4.2 versus 3.5 mm/day (p less than 0.01). NF-L was not accelerated, indicating that the motor for subcomponent a (SCa) of slow axonal transport was unaffected by axotomy. To measure outgrowth distances, the testing lesions was made 7 days after the conditioning lesion, and growth cones were located by the fast transport method 3 or 9 days later. The regression analysis of outgrowth distance on time showed that sprouts elongated 25% faster in conditioned axons: 4.0 versus 3.2 mm/day (p less than 0.001). These accelerated sprouts were formed too far from the spinal cord to contain SCb proteins that were synthesized after axotomy. Because the rate of outgrowth correlated closely with the rate of SCb in outgrowing sprouts (McQuarrie and Jacob, J. Comp. Neurol. 305:139-147, 1991), we conclude that SCb is accelerated throughout the length of the axon by 7 days after axotomy.     

Organization and slow axonal transport of cytoskeletal proteins under normal and regenerating conditions

The organization of the axonal cytoskeleton was investigated by analyzing the solubility and transport profile of the major cytoskeletal proteins in motor axons of the rat sciatic nerve under normal and regenerating conditions. When extracted with the Triton-containing buffer at low temperature, 50% of tubulin and 30% of actin were recovered in the insoluble form resistant to further depolymerizing treatments. Most of this cold-insoluble form was transported in slow component a (SCa), the slower of the two subcomponents of slow axonal transport, whereas the cold-soluble form showed a biphasic distribution between SCa and SCb (slow component b). Changes in slow transport during regeneration were studied by injuring the nerve either prior to (experiment I) or after (experiment II) radioactive labeling. In experiment I where the transport of proteins synthesized in response to injury was examined, selective acceleration of SCb was detected together with an increase in the relative proportion of this component. In experiment II where the response of the preexisting cytoskeleton was examined, a shift from SCa to SCb of the cold-soluble form was observed. The differential distribution and response of the two forms of tubulin and actin suggest that the cold-soluble form may be more directly involved in axonal transport.     

Differential Axonal Transport of Soluble and Insoluble τ in the Rat Sciatic Nerve

Abstract: Axonal transport of microtubule-associated protein τ was studied in the motor fibers of the rat sciatic nerve 1–4 weeks after labeling of the spinal cord with [³⁵S]methionine. As 60–70% of low molecular weight τ in this system was found to be insoluble in 1% Triton-containing buffer, labeled proteins in 6-mm consecutive nerve segments were first separated into Triton-soluble and insoluble fractions. Two-dimensional gel electrophoresis and immunoblotting with anti-tau antibody confirmed the presence of τ among labeled, transported proteins in both fractions. Isoform composition of labeled τ was similar to that of bulk axonal τ, the most acidic species with apparent molecular mass of 66 kDa being the major component. Transport profiles obtained by measuring radioactivities associated with this major isoform showed that soluble and insoluble τ were transported at different rates. Insoluble τ, which contained the majority of τ-associated radioactivity, was transported at 1.7 mm/day in slow component a (SCa), whereas soluble τ was transported faster, at 3 mm/day, corresponding to the rate of slow component b (SCb). Cotransport of insoluble τ with insoluble tubulin in SCa suggests its association with stable microtubules.     

Axotomy accelerates slow component b of axonal transport

Because the integrity of an axon depends on the supply of proteins synthesized in the cell body, we examined the effect of axotomy on the transport of structural proteins in rat motor axons, and the effect of altered transport on the rate of outgrowth after a subsequent testing axotomy. To examine the axonal transport of structural proteins, we labeled newly synthesized proteins with ³⁵ S-methiomine 7 days after a “conditioning” lesion of the sciatic nerve, and removed the nerve 7–21 days later for SDS-PAGE. Tubulin, actin, calmodulin, and the 68-kD light neurofilament protein (NF-L) were identified by fluorography and removed for liquid scintillation counting. The fastest moving structural proteins were carried by slow component b (SCb) of axonal transport, which advanced 20% faster in conditioned axons: 4.2 versus 3.5 mm/day (p < 0.01). NF-L was not accelerated, indicating that the motor for subcomponent a (SCa) of slow axonal transport was unaffected by axotomy. To measure outgrowth distances, the testing lesion was made 7 days after the conditioning lesion, and growth cones were located by the fast transport method 3 or 9 days later. The regression analysis of outgrowth distance on time showed that sprouts elongated 25% faster in conditioned axons: 4.0 versus 3.2 mm/day (p < 0.001). These accelerated sprouts were formed too far from the spinal cord to contain SCb proteins that were synthesized after axotomy. Because the rate of outgrowth correlates closely with the rate of SCb in outgrowing sprouts (McQuarrie and Jacob, J. Comp. Neurol. 305:139–147, 1991), we conclude that SCb is accelerated throughout the length of the axon by 7 days after axotomy.     

Synthesis, axonal transport, and turnover of the high molecular weight microtubule-associated protein MAP 1A in mouse retinal ganglion cells: tubulin and MAP 1A display distinct transport kinetics

Microtubule-associated proteins (MAPs) in neurons establish functional associations with microtubules, sometimes at considerable distances from their site of synthesis. In this study we identified MAP 1A in mouse retinal ganglion cells and characterized for the first time its in vivo dynamics in relation to axonally transported tubulin. A soluble 340-kD polypeptide was strongly radiolabeled in ganglion cells after intravitreal injection of [35S]methionine or [3H]proline. This polypeptide was identified as MAP 1A on the basis of its co-migration on SDS gels with MAP 1A from brain microtubules; its co-assembly with microtubules in the presence of taxol or during cycles of assembly-disassembly; and its cross-reaction with well-characterized antibodies against MAP 1A in immunoblotting and immunoprecipitation assays. Glial cells of the optic nerve synthesized considerably less MAP 1A than neurons. The axoplasmic transport of MAP 1A differed from that of tubulin. Using two separate methods, we observed that MAP 1A advanced along optic axons at a rate of 1.0-1.2 mm/d, a rate typical of the Group IV (SCb) phase of transport, while tubulin moved 0.1-0.2 mm/d, a group V (SCa) transport rate. At least 13% of the newly synthesized MAP 1A entering optic axons was incorporated uniformly along axons into stationary axonal structures. The half-residence time of stationary MAP 1A in axons (55-60 d) was 4.6 times longer than that of MAP 1A moving in Group IV, indicating that at least 44% of the total MAP 1A in axons is stationary. These results demonstrate that cytoskeletal proteins that become functionally associated with each other in axons may be delivered to these sites at different transport rates. Stable associations between axonal constituents moving at different velocities could develop when these elements leave the transport vector and incorporate into the stationary cytoskeleton.     

A model for slow axonal transport and its application to neurofilamentous neuropathies

A model for slow axonal transport is developed in which the essential features are reversible binding of cytoskeletal elements and of soluble cytosolic proteins to each other and to motile elements such as actin microfilaments. Computer simulation of the equations of the model demonstrate that the model can account for many of the features of the SCa and SCb waves observed in pulse experiments. The model also provides a unified explanation for the increase and decrease of neurofilament transport rates observed in various toxicant-induced neuropathies.     

Clathrin is axonally transported as part of slow component b: the microfilament complex

During axonal transport, membranes travel down axons at a rapid rate, whereas the cytoskeletal elements travel in either of two slow components, SCa (with tubulin and neurofilament protein) and SCb (with actin). Clathrin, the highly ordered, structural coat protein of coated vesicles, has recently been shown to be able to interact in vitro with cytoskeletal proteins in addition to membranes. The present study examines whether clathrin travels preferentially with the membrane elements or the cytoskeletal elements when it is axonally transported. Guinea pig visual system was labeled with tritiated amino acids. Radioactive SDS-polyacrylamide gel electrophoresis profiles from the major components of transport were coelectrophoresed with clathrin. Only SCb had a band comigrating with clathrin. In addition, radioactive clathrin was purified from guinea pig brain containing only radioactive SCb polypeptides. Kinetic analysis of the putative clathrin band in SCb revealed that it travels entirely within the SCb wave. Thus we conclude that clathrin travels preferentially with the cytoskeletal proteins making up SCb, rather than with the membranes and membrane-associated proteins in the fast component.     

Nerve-specific enolase and creatine phosphokinase in axonal transport: soluble proteins and the axoplasmic matrix

The axonal transport of two soluble enzymes of intermediary metabolism was evaluated: the nerve-specific form of the glycolytic enzyme enolase (NSE) and the brain isozyme of creatine phosphokinase (CPK). Previously, little was known about the intracellular movements of the soluble proteins of the cell. Although the soluble enzymes of glycolysis and other pathways of intermediary metabolism have been thought to be freely diffusing in the cytosol, many are required in the axonal extremities of the neuron and must be transported to the sites of utilization. Comigration of purified enzymes with radioactive polypeptides associated with specific rate components of axonal transport in two-dimensional gel electrophoresis indicates that both NSE and CPK move in the axon solely as part of the group of proteins known as slow component b (SCb) at a rate of 2 mm/day. Peptide mapping following limited proteolysis confirmed identification of NSE and CPK in SCb. Materials associated with SCb have been shown to move coherently along the axon and to behave as a discrete cellular structure, the axoplasmic matrix. Association of two soluble enzymes, NSE and CPK, with the SCb complex of proteins requires a reevaluation of the assumption that these and other soluble proteins of the axon are freely diffusible.     

Increased slow transport in axons of regenerating newt limbs after a nerve conditioning lesion

We have previously shown that a nerve conditioning lesion (CL) made 2 weeks prior to amputation results in an earlier onset of limb regeneration in newts. Studies in fish and mammals demonstrate that when a CL precedes a nerve testing lesion, slow component b (SCb) of axonal transport is increased compared to axons that had not received a CL. We wanted to know whether the earlier initiation of limb regeneration after a CL was associated with an increase in SCb transport. The transport of [35S]methionine labeled SCb proteins was measured by using SDS-PAGE, fluorography, and scintillation counting. The rate of transport and quantity of SCb proteins was determined at 7, 14, 21, and 28 days after injection of [35S]methionine into the motor columns of normal; single lesioned (i.e., transection axotomy, amputation axotomy, or sham CL followed by amputation); and double-lesioned limb axons (i.e., nerve transection CL followed 2 weeks later by amputation axotomy). The rate of SCb transport in axons of unamputated newt limbs was 0.19 mm/day. There was an increase in the amount of labeled SCb proteins transported in axons regenerating as the result of a single lesion but no acceleration in the rate of SCb transport, which was 0.21 mm/day in axons that received a sham CL followed by limb amputation. The rate of SCb transport doubled (0.40 mm/day) and the amount of labeled SCb proteins being transported was increased when amputation was preceded by a CL. This study demonstrates that the earlier onset of limb regrowth, seen when amputation follows a CL, is associated with an increased transport of SCb proteins. This suggests that limb regeneration is, in part, regulated by axonal regrowth. We propose that the blastema requires a minimum quantity of innervation before progressing to the next stage of limb regeneration, and that the transport of SCb proteins determines when that quantity will be available.     

Selective Alterations in Presynaptic Cytomatrix Protein Organization Induced by Calcium and Other Divalent Cations That Modulate Exocytosis

Rises in intracellular calcium cause several events of physiological significance, including the regulated release of neuronal transmitters. In this study, the effects of divalent cations on the structural organization of cytomatrix in presynaptic terminals was examined. [35S]Methionine-radiolabeled guinea pig retinal ganglion cell cytomatrix proteins were axonally transported [in slow component b (SCb) of axonal transport] to the neuron terminals in the superior colliculus. When the peak of radiolabeled cytomatrix proteins reached the terminals, synaptosomes containing the radiolabeled cytomatrix proteins were prepared. Approximately 40% of each SCb protein was soluble after hypoosmotic lysis of the radiolabeled synaptosomes in the presence of divalent cation chelators. Lysis of synaptosomes in the presence of calcium ions over a range of concentrations, however, caused a dramatic decrease in solubility of the presynaptic SCb proteins. The cytoplasmic effects may result from a calcium-dependent condensation of cytoplasm around presynaptic terminal membrane systems. There are two major presynaptic SCb proteins (at 60 and 35 kDa), that exhibited exceptional behavior: they remained as soluble in the presence of calcium as under control conditions, suggesting that they were relatively unaffected by the mechanism causing the decrease in SCb protein solubility. Also examined were the effects of other alkaline earth and transition metal divalent cations on the presynaptic SCb proteins.     

Two 68-kDa Proteins in Slow Axonal Transport Belong to the 70-kDa Heat Shock Protein Family and the Annexin Family

The major 68-kDa protein found selectively in the faster of the two subcomponents of slow axonal transport [group IV or slow component b (SCb)] in the rat sciatic nerve has been characterized. It was found to contain two distinct classes of proteins, S1 and S2, both of which have isoelectric points of 5.7, but differ in their solubility in the presence of calcium. The S1 protein, which contributes up to 70% of the 68-kDa component, was soluble in the presence or absence of calcium, whereas the S2 protein was bound to the cytoskeleton in a calcium-dependent manner. Further characterization of the two proteins by peptide mapping and immunological methods revealed that the S1 protein belonged to a family of proteins related to the 70-kDa heat shock protein, whereas the S2 protein was identical to 68-kDa calelectrin (annexin VI). Selective occurrence in SCb of these proteins with potential abilities to regulate protein-protein or protein-membrane interactions suggests that they may play important roles in the control of cytoskeletal organization in the axon, because SCb contains mainly cytoskeletal proteins in a more dynamic form compared with the slowest rate component, slow component a, which is enriched in the stably polymerized form of these proteins.     

Microtubule gelation-contraction in vitro and its relationship to component a of slow axonal transport

Microtubule proteins, isolated by cycles of assembly, will undergo ATP-dependent gelation-contraction in vitro. A particulate component is present in these preparations, which is required for the gelation-contraction of microtubules assembled from purified tubulin. These particulates contain tubulin, neurofilament, spectrin, MAP2, and other as yet unidentified proteins. The particulates have a microtubule-stimulated ATPase that may be unique and is the likely motor for microtubule gelation-contraction. The basic structural unit of these particulates appears to be a crescent-shaped, or hemispherical, granule about 20 nm in diameter. The particles move along microtubule walls at a rate of about 1 micron. When compared to known physiological phenomena, microtubule gelation-contraction has striking similarities to component a of slow axonal transport (SCa), but displays no relationship to slow component b or to fast transport. On the basis of their similarities in composition, solubility, and rate of movement, we have proposed that the particulates responsible for microtubule gelation-contraction are the insoluble protein complexes, which have been suggested to be the transported component of SCa. We have termed these structures "slow component a particulates" or "SCAPs." It is probable that similar motile protein complexes exist in cells other than neurons, and we propose the term "dynasome" to describe such structures in general.     

Slow axonal transport mechanisms move neurofilaments relentlessly in mouse optic axons

Pulse-labeling studies of slow axonal transport in many kinds of axons (spinal motor, sensory ganglion, oculomotor, hypoglossal, and olfactory) have led to the inference that axonal transport mechanisms move neurofilaments (NFs) unidirectionally as a single continuous kinetic population with a diversity of individual transport rates. One study in mouse optic axons (Nixon, R. A., and K. B. Logvinenko. 1986. J. Cell Biol. 102:647-659) has given rise to the different suggestion that a significant and distinct population of NFs may be entirely stationary within axons. In mouse optic axons, there are relatively few NFs and the NF proteins are more lightly labeled than other slowly transported slow component b (SCb) proteins (which, however, move faster than the NFs); thus, in mouse optic axons, the radiolabel of some of these faster-moving SCb proteins may confuse NF protein analyses that use one dimensional (1-D) SDS-PAGE, which separates proteins by size only. To test this possibility, we used a 2-mm "window" (at 3-5 mm from the posterior of the eye) to compare NF kinetics obtained by 1-D SDS-PAGE and by the higher resolution two-dimensional (2-D) isoelectric focusing/SDS-PAGE, which separates proteins both by their net charge and by their size. We found that 1-D SDS-PAGE is insufficient for definitive NF kinetics in the mouse optic system. By contrast, 2-D SDS-PAGE provides essentially pure NF kinetics, and these indicate that in the NF-poor mouse optic axons, most NFs advance as they do in other, NF-rich axons. In mice, greater than 97% of the radiolabeled NFs were distributed in a unimodal wave that moved at a continuum of rates, between 3.0 and 0.3 mm/d, and less than 0.1% of the NF population traveled at the very slowest rates of less than 0.005 mm/d. These results are inconsistent with the proposal (Nixon and Logvinenko, 1986) that 32% of the transported NFs remain within optic axons in an entirely stationary state. As has been found in other axons, the axonal transport system of mouse optic axons moves NFs and other cytoskeletal elements relentlessly from the cell body to the axon tip.     

Axoplasmic transport in the crayfish nerve cord. The role of fibrillar constituents of neurons

(a) Axoplasmic transport of tritium-labeled proteins in crayfish nerve cord was confirmed at a slow rate of 1 mm/day. A second proteinaceous component which moves at a rate of 10 mm/day was also detected. Radioautography and biochemical analysis indicate that proteins migrating at these velocities have a perikaryal origin and move caudad within axons as sharply defined peaks. (b) Evidence is presented for the blockage of the slow and the fast movement of proteins by intraganglionic injection of the anti-mitotic agent vinblastine sulfate (0.1 mM). (c) Electron microscope observations of vinblastine-treated ganglia revealed a reduction in the number of axonal microtubules and the formation of intracellular aggregates presumably composed of microtubular protein. (d) These findings would be compatible with the involvement of microtubules in both slow and fast axoplasmic transport. However, the block induced by vinblastine was detected in regions of the cord (up to 10 mm away from the injection site) where the number and morphology of microtubules appeared unaltered. In addition, axons showing effects of vinblastine occasionally contained mitochondria with remarkably dense and thickened membranes. (e) In association with the surfaces of axonal microtubules are lateral filamentous elements (40–80 A in diameter) which also showed vinblastine-induced alterations. Our observations indicate that such filiform structures, associated with microtubules, may be a necessary component in the transport mechanism(s).     

The cytoskeletal system of nucleated erythrocytes. II. presence of a high molecular weight calmodulin-binding protein

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 cytoskeletons were found to contain a calcium-dependent calmodulin-binding protein, CBP, by two independent techniques: (a) 125I-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.