isolation of an immunoreactive analogue of brain fodrin that is associated with the cell cortex of...

Cell Motility and the Cytoskeleton 11:303-317 (1988)

Isolation of an lmmunoreactive Analogue of Brain Fodrin That Is Associated With the Cell Cortex of

Dictyostelium Amoebae

Holly Bennett and John Condeelis

Department of Anatomy and Structural Biology, Albert Einstein College of Medicine, Bronx, New York

We have used a polyclonal affinity-purified antibody made against chicken brain fodrin (both 240 and 235 Kd subunits) as a probe to determine if a fodrinlike protein exists in amoebae of Dictyostelium discoideum. In Western blots of whole cells and the isolated cell cortex, polypeptides measuring 220 and 70 Kd are recognized by the fodrin antibodies. In situ localization by indirect immunofluorescence with antifodrin indicates that the immunoreactive polypeptides are cortical. The immunoreactive analogues copatch and cocap with concanavalin A. At the level of resolution of the electron microscope, immunocytochemistry with antifodrin and colloidal gold confirms that the immunoreactive analogues are cortical proteins associated with microfilaments on the cytoplasmic side of the plasma membrane. We have isolated and characterized the 220 Kd protein to determine if it is similar to fodrin and to investigate its relationship to the 70 Kd polypeptide. The 220 Kd protein can be extracted from the cortex in the absence of detergent and isolated by gel filtration and sucrose density gradient sedimentation. The 220 Kd is a rod-shaped protein 118 ? 17.8 nm (N = 37) in length. It has a sedimentation coefficient of 9.3 S and Stokes’ radius of 13 nm and exists as a dimer of approximately 500,000 daltons (Mr). Isolated 220 Kd binds to actin filaments in vitro when assayed by rotary shadowing. Morphological criteria distinguish 220 Kd from Dictyostelium myosin I1 heavy chain (215 Kd) and the filaminlike protein at 240 Kd. The 70 Kd polypeptide appears to be a cleavage fragment of the 220 Kd, since it is found after prolonged storage when formerly only the 220 Kd was present. Furthermore, the 220 and 70 Kd polypeptides exhibit similar one-dimensional peptide maps when treated with TPCK trypsin. On the basis of its physical and immunoreactive characteristics, and location in the cell, the 220 Kd may be a fodrinlike protein.

Key words: spectrin, actin, membrane skeleton, cytoskeleton

INTRODUCTION

The amoeboid stage of Dictyostelium discoideum is typified by actively motile cells that undergo endocyto- sis, patching and capping, chemotaxis, ruffling, exten- sion of pseudopodia, and locomotion on a substrate. Actin-binding protcins in thc cortcx arc bclicvcd to bc responsible for the regulation of these motile events [ 1,9,10,13,14,49].

We have previously isolated the cortex of Dic- tyostelium amoebae [ 11 and found, by morphological

techniques, that it is composed of a network of microfilaments and residual plasma membrane. The microfilaments in the cortex make numerous contacts with the plasma membrane. There is an average of approximately

Received March 28, 1988; accepted August 18, 1988

Holly Bennett is now at the Whitehead Institute for Biomedical Research, Nine Cambridge Center, Cambridge, MA 02 142.

Address reprint requests to John Condeelis, Department of Anatomy and Structural biology, Albert Einstein College of Medicine, Bronx, NY 10461.

0 1988 Alan R. Liss, Inc.

304 Bennett and Condeelis

25,000 contacts per isolated cortex and these are prima- rily of two types: end-on and lateral. Microfilaments that contact the membrane end-on do so at the barbed end; other microfilaments are linked to the membrane by lateral bridges approximately 15 nm in length. The lateral bridges bind along the microfilaments with a periodicity of 36 nm.

Both types of contacts between microfilaments and the membrane are reminiscent of those seen in microvilli of the chicken intestinal brush border. In the brush border, the molecular mechanism for end-on attachments of microfilaments to the membrane if any, is unknown, whereas the lateral attachments are mediated by lateral bridges composed of a 110,000 dalton protein (1 lOK), which binds actin and calmodulin [ 18,24,34].

However, some of the microfilaments in the isolated cortex of Dictyostelium are associated with the plasma membrane by long, thin filaments, suggesting that these links might represent another mechanism for lateral membrane linkage. In fact, another type of well characterized membrane-linkage site for microfilaments in the human erythrocyte, involving spectrin and its associated proteins, exhibits this kind of morphology of membrane attachment [S] . Binding studies have revealed that the molecular mechanism for attachment of actin oligomers to the erythrocyte membrane is via spectrin tetramers [5,37], which are bound to ankyrin [2]. Anky- rin binds spectrin to band 3 an integral membrane protein that functions as an anion channel [3].

Proteins related immunologically and structurally to spectrin have been reported in various cell types as diverse as Acanthamoeba [38], sea urchin coelomocytes [ 171, lymphocytes [27,36,41], and chicken intestinal brush borders 119,201. This suggests that the molecular mechanism for linkage of actin to the membrane first identified in erythrocytes may be a conserved mechanism.

We have explored the possibility that some of the filamentous links between the membrane and microfilaments in the cortex of Dictyostelium amoebae are mediated by a spectrinlike protein. In this study, we prepared an antibody to both subunits of chicken brain fodrin, the nonerythroid form of spectrin [4,19], and with it have identified two polypeptides that are immunoreactive in Dictyostelium amoebae. We have localized the immunoreactive analogues in situ and isolated the larger polypeptide to determine its physical characteristics and to compare it to proteins of the spectrin family.

MATERIALS AND METHODS Preparation of Antichicken Brain Fodrin

Chicken brain fodrin was isolated according to the methods of Glenney et al. [20], which are a modification

of the method of Levine and Willard [27]. We modified the protocol by including phenylmethylsulfonyl fluoride (PMSF) and Trasylol (Mobary, New York, NY) in buffers as inhibitors of proteolysis. We also concentrated the protein by vacuum dialysis (Biomolecular Dynamics, Beaverton, OR) rather than by use of Aquacide. As a final purification step, a known amount of fodrin as determined by the Lowry assay [30] was applied to preparative 4% sodium dodecyl sulfate (SDS) Laemmli gel [26], and the 240K and 235K bands were resolved by gel electrophoresis and cut out of the gel and electroeluted. The 240K/235K polypeptides were dialyzed against phosphate-buffered saline (PBS; 0.9% NaCl, 20 mM KP04), pH 7.0, to remove the SDS before injection into rabbits or conjugation to cyanogen bromide-activated (CNBr-activated) Sepharose 4B (Pharmacia Fine Chemicals, Piscataway , NJ) for affinity purification of the immune serum.

New Zealand white rabbits were bled for preim- mune serum. Rabbits were injected subcutaneously along their dorsal surfaces with 0.5 mg chicken brain fodrin (240K/235K) mixed 1: 1 (vol/vol) with Freund’s complete adjuvent. Four weeks later, the rabbits were given a booster of 0.5 mg of chicken brain fodrin mixed 1: 1 (vol/vol) with Freund’s incomplete adjuvent and were bled 8 days later. Two weeks later, they were given a second booster of 0.3 mg chicken brain fodrin and were bled 1 week later. Antibodies were prepared, affinity purified, and stored according to the methods of Carboni and Condeelis [9]. lmmunofluorescence Procedure

Dictyosteliurn amoebae were fixed in suspension and stained with 20 pg/ml of antifodrin according to the method of Carboni and Condeelis [9]. For localization of antigen during concanavalin A (Con A) capping, cells were challenged with 10 pg/ml fluorescein Con A (F Con A) and 60 pg/ml Con A and fixed at various time points after challenge. The position of antifodrin was detected with a secondary antibody (rhodamine labelled goat antirabbit; Gibco Laboratories, Grand Island, NY) and, in some experiments, with a tertiary antibody (rhodamine labelled rabbit antigoat; Gibco) as well. To decrease nonspecific staining, both secondary and tertiary antibodies were preabosorbed with Dictyostelium amoebae that had been fixed in 3.2% formaldehyde and permeabilized in -20°C acetone for 25 min. Cells were photographed at 100 x under oil immersion. Micro- graphs of control, and experimental cells were taken and printed under identical conditions of exposure. lmmunocytochemistry With Colloidal Gold

Cortices were isolated according to the method of Bennett and Condeelis [ l ] from cells that had been challenged with 70 pg/ml Con A for 4 min. Cortices

Analogue of Fodrin in Dictyostelium Amoebae 305

220K polypeptide to NC paper, whereas the 70K polypeptide transferred efficiently even in the absence of SDS, thereby resulting in a stronger reaction with the antibody on most immunoblots.

NC paper was stained with amido black, and the gel was stained with Coomassie blue to check for transfer of proteins from the gel to the paper. The NC paper was blocked in 5% BSA dissolved in TBS (200 mM Tris, 0.9% NaCl, pH 7.6), pH 8.2 (i.e., 5% BSA/TBS); rinsed twice in TBS, pH 8.2; and incubated in 1 pglml antifodrin in 1% BSA/TBS, pH 8.2, for a minimum of 3 hr with shaking. After washing to remove traces of the primary antibody, the blot was stained with a secondary antibody and the peroxidase reaction (Vectastain Kit) as described by Carboni and Condeelis [9] or with Iz5I-protein A alone.

The protein A (Amersham, Arlington Heights, IL) was iodinated with lZ5I (Amersham) by a chlorimine T reaction according to the method of Hunter and Green- wood [25]. For staining with '251-protein A, the NC paper was washed three times for 5 rnin in solution A (1 mM ethylendiaminetetraacetic acid [EDTA], 0.02% Triton X-100, TBS, 0.02% NaN3), pH 7.5, for 5 min in solution B (2 M urea, 0.01 glycine, 1.0% Triton X-100) and for 5 rnin in solution A. The NC paper was incubated in 1 pCi/ml '251-protein A in 1% BSA/TBS, pH 7.5, for 2 hr, with shaking. The NC paper was washed as described above, rinsed in TBS and H20, and dried. Autoradiography was performed by exposing NC paper to Kodak X-OMAT AR film (XAR-5) with a Dupont Cronex (Quanta 111) intensifying screen.

Solid-state lmmunoassay

A solid-state immunoassay was used to detect proteins that were immunoreactive with antifodrin during various stages of the purification of 220K. NC paper was soaked in TBS, pH 7.5, and 0.02% NaN3 and assembled into a bio-dot apparatus (Biorad, Richmond, CA) and washed with TBS, pH 7.5. Each well had a protein capacity of 5 pg. Aliquots of approximately 10 p1 each were applied to individual wells by gravity flow and then vacuum dried. Samples were blotted onto NC paper in the presence of 0.1% SDS to retain solubility and unmask blocked epitopes. Unused wells were plugged with 3% gelatin. Sample wells were blocked with 250 pl/well of blocking solution (10% BSA/TBS) by gravity flow and vacuum dried. Wells were washed three times with 300 pl of 500 mM NaCl/TBS containing 0.05% Tween, pH 7.5, and rinsed once with 250 pg/well TBS, pH 7.5. Primary antibody at a concentration of 0.06 pg/well in 1% BSA/TBS, pH 7.5, was added by gravity flow, vacuum dried, and washed as described above. The NC paper was removed from the apparatus and blocked in 5% BSA/TBS in 0.02% NaN3, pH 7.5, for

were labeled by the indirect method with antifodrin and colloidal gold conjugated with IgG as described else- where [15] (also, S. Ogihara, J. Carboni, J. Condeelis, submitted for publication). Briefly, cortices were lightly fixed in 0.5% glutaraldehyde in H3-P (1 mM EGTA, 2.5 mM Pipes, 2.5 mM MgS04, 10 mM KCl, 20 mM KP04), pH 7.0, containing 0.02% NaN3, 10 pg/ml chymostatin, 400 U/ml Trasylol, 12 pg/ml phalloidin, and 20 mM Tris HCl, pH 7.0. Cortices were pelleted and resuspended in NaBH4 in H3-P for 10 min; in H3-P containing 1% bovine serum albumin (BSA), 1% fetal calf serum (FLS) for 40 min, and finally in 10 pg/ml primary antibody in H3-P containing 1 % BSA, 1 % FCS, 0.02% NaN3 for 3-5 hr on ice. Cortices were washed extensively in H3-P containing 0.1% BSA. Cortices were resuspended in 1 ml goat antirabbit IgG-gold at a concentration of between 1 and 5 optical density (OD) units at 520 nm and incubated overnight on ice. Cortices were washed extensively, then fixed in 2% glutaraldehyde/PBS at pH 6.0 for 3 rnin on ice, and stained in 1% uranyl acetate at 4°C for 15 min. The cortices were processed for transmission electron microscopy as described by Bennett and Condeelis [ l] . The density of gold particles in control and experimental cortices was measured from micrographs printed at 60,000 by counting the number of gold particles in 0.25-pm2 areas.

SDS-PAGE, Proteolytic Digests, and lmmunoblotting

Samples were run on either 4%, 5 -20%, or 4-25% SDS Laemmli gels [26] according to the method of Matsudaira and Burgess [33]. Gels were scanned with a Joyce Loebel microdensitometer at 550 nm.

The 220 and 70K immunoreactive analogues were compared by one-dimensional peptide maps according to the methods of Cleveland et al. [ l 11, with the following modifications. The 220 and 70K bands were cut out from SDS-PAGE of the Superose 6B column fractions containing these polypeptides and run on 4-25% SDS- PAGE in the presence of 10 p1 of TPCK trypsin at a concentration of 0.14 mg/ml. The gel was turned off for 0, 10, or 30 min before the proteins entered the resolving gel to permit limited digestion of the samples.

For immunoblotting, proteins separated by SDS- PAGE and left unstained were transferred to nitrocellulose (NC) paper (Schleider and Schuell, Inc., Keene, NH) by electrophoresis in electroblot buffer ( I92 mM glycine, 25 mM Tris, 20% methanol, and 0.1% SDS) according to the method of Towbin et al. [50] for 8 hr at 250 milliamps to obtain transfer of the high-molecular- weight proteins.

Immunoblotting of the 220K polypeptide was prob- lematic. Inclusion of SDS in the tank buffer and long transfer times were required for efficient transfer of the


45 min at 37"C, with shaking. The NC paper was rinsed in TBS, incubated in 1% BSA/TBS and 0.02% NaN3, pH 7.5, with 1 pCi/ml '251-protein A for 2 hr, with shaking. It was washed five times for 5 min each in 500 mM NaCl/TBS with 0.05% Tween 20, pH 7.5, rinsed in TBS, then H20, and dried. The amount of '251-protein A bound to the NC paper was determined by gamma counting or by autoradiography. The conditions used in this assay were developed so that the amount of '251-protein A bound to the NC paper was directly proportional to the amount of bound antigen. Autoradiog- raphy was performed as described for immunoblotting. The autorad was scanned with a Joyce Loebel microdensitometer and the density of staining was plotted on paper. The peaks were cut out, weighed, and expressed as arbitrary units. Exposures of the X-ray film used were chosen so that peak weight was directly proportional to the amount of antigen present on the NC paper.

Isolation of Cell Cortices for Extraction of 220K Protein

For use in the isolation of the 220K, cortices were prepared according to the method of Bennett and Con- deelis [ 11, with the following modifications. Five liters of Dictyostelium ameobae (ax-3) were grown to a density of 5-10 x lo6 cells/ml in HL5 media. After washing, the cells were incubated for 10 min at 22°C in 20 mM KCl, 30 mM NaH2P04, 0.7 mM CaC12, 2 mM MgS04, pH 6.2, containing 1 mM PMSF and 1 pgiml chymostatin. Cells were challenged with 50 pg/ml Con A for 4 min, during which time they began to patch but did not cap the Con A. Cells were washed and resuspended in lysis buffer (1 mM ethylene glycol-bis(beta-amino-ethyl ether)N,N',N'-tetraacetic acid [EGTA], 5 mM Tris, 20 mM KP04), pH 7.6, as usual. Just prior to lysis, an inhibitor cocktail was added to achieve the following inhibitor concentrations: 0.02 ml/ml Trasylol, 5 mM phenanthroline, 2 mM NCZ-phenylalanine, 1 mM PMSF, and 10 pg/ml chymostatin. Cells were lysed in 0.2% Triton X-100 in GSA bottles (Sorval), with gentle mixing by hand for 5 sec. The cortices were pelleted and resuspended in H3 buffer (1 mM EGTA, 5 mM PIPES, 5 mM MgS04, 20 mM KCl), pH 7.0

Isolation of the 220K Protein Dictyostelium amoebae have an abundance of pro-

teases [29]. This presented a special problem when isolating the 220K, because it is very sensitive to proteolysis. To prevent proteolysis, it was necessary to include thiol- arid serine-dependent protease inhibitors, to work on ice, and to isolate the protein as quickly as possible.

The 220K protein was extracted from the cortex by incubation for 30 min with 0.2 M NaCl, 10 mM MgATP

in H3 buffer with inhibitor cocktail, 10 pg/ml chymostatin, and 2 mM PMSF. The extract was clarified by centrifugation at 100,000 g-hr, increased in salt concentration to 0.5 M from a 3 M stock, concentrated 23-fold either by vacuum dialysis (Bio-Molecular Dynamics) or with Aquacide 11, and further clarified by centrifugation at 100,000 g-hr. Ammonium sulfate could not be used at this step for concentrating the 220K, because it rendered the protein insoluble in aqueous buffers; concentration by vacuum dialysis or aquacide was more gentle, although some of the protein was lost by proteolysis.

The 100,000 g-hr supernatant of the concentrated extract was injected into a Superose 6B gel filtration column (Pharmacia Fine chemicals; 1.6 x 55 cm) equilibrated in column buffer (20 mM Tris, 0.15 M NaCl, 1 mM EGTA, 1 mM MgATP, 0.1 mM dithio- threitol [DTT], 0.02% NaN3, 2 mM phenylalanine, 1 mM phenathroline, 1 pg/ml chymostatin, 0.01 ml/ml Trasylol), pH 8, at 5°C. The 220K eluted as a peak in fractions 21-24 and was separated from the 70K, which eluted in fractions 26-29.

The peak fractions containing the 220K were pooled and brought to 0.5 M NaCl, concentrated fourfold, and clarified at 100,000 g-hr. The supernatant was further purified by sedimentation on a 5-2070 sucrose density gradient prepared in TKE buffer (0.5 M NaCl, 10 mM Tris, 0.5 mM EDTA, 0.1 mM DTT, 0.02% NaN3, 2 mM phenylalanine, 1 mM phenanthroline, 0.01 ml/ml Trasylol, 10 pg/ml chymostatin), pH 8, at 5°C. A volume of 0.5 ml was loaded on each gradient and spun at 40,000 rpm for 14 hr in an SE 41 rotor (Beckman). Standards were included on identical gradients for calibration. The peak fraction identified by immunoblotting with antifodrin was found to migrate at 9.3 S.

Low-Angle Rotary Shadowing Protein samples were rotary shadowed according to

the method of Tyler and Branton [12,51]. Briefly, the samples were dialyzed against 1 M ammonium acetate, pH 7.0; mixed with 40% gylcerol; and sprayed onto freshly cleaved mica. The samples were dried under vacuum and shadowed at 4" with platinum in a Balzers freeze-fracture apparatus. Measurements of filaments were made from representative prints at a final magnifi- cation of x 90,000.

For shadowing with F-actin, we follbwed the method of Glenney et al. [21], with the following changes. Fractions from the sucrose density gradient containing the 220K protein were dialyzed and concentrated twofold against low-salt buffcr (10 mM Pipcs, 50 mM KCl, 1 mM EGTA), pH 6.8, containing the following inhibitors: 2 mM phenylalanine, 1 mM phenanthroline, 10 pg/ml chymostatin, and 0.01 ml/ml Trasyol. The solution was then clarified at 100,000 g-hr and


dialyzed against 50% glycerol in low-salt buffer, pH 6.8. The 220K protein at a concentration of 0.012 mg/ml (0.024 pM) following clarification was mixed with 0.12 mg/ml(2.9 pM) G-actin from rabbit skeletal muscle [48] and 1 mM MgS04 and then incubated for 10 min at room temperature and then for 1 hr on ice. Phalloidin at a concentration of 25 pM was added to stabilize the actin filaments. After an overnight incubation at 4"C, the mixture was fixed in 1 % glutaraldehyde for 1 hr on ice and then sprayed and shadowed as described above.

Falling Ball Viscometry

Viscometry was performed according to MacLean- Fletcher and Pollard [32] at an angle of 30". Proteins were dialyzed into 20 mM KCl; 5 mM Pipes, pH 7.0; 1 mM EGTA; 1 mM MgS04. The 220K protein, at final concentrations of 0, 0.022, 0.044, and 0.066 mgiml, was incubated at 25°C for 1 hr with F-action, which was at a final concentration of 1.4 mg/ml.

Negative Staining

The 220K was dialyzed into a low-salt buffer (20 mM KCl, 2 mM MgS04, 5 mM Pipes, 2 mM EGTA, pH 7.0) and then clarified at 100,000 g-hr. The 220K at a concentration of 0.11 mg/ml was mixed with 0.12 mg/ml G-actin and 1 mM MgS04 and allowed to copolymerize, or the 220K was mixed with prepolymer- ized F-actin [48]. Controls included F-actin alone and 220K alone. Samples were placed on formvar-coated grids and were stained with 2% uranyl acetate, pH 4.

RESULTS Specificity of the Antibody and the lmrnunoreactive Analogues of Fodrin in Dictyostelium Amoebae

The specificity of the polyclonal affinity-purified antibody that was prepared against chicken brain fodrin is shown in Figure 1. Both the 240K and the 235K chains of fodrin stained with the antibody (Fig. lb). Only polypeptides migrating at 240K and 235K were stained in western blots of whole-brain homogenates (Fig. lc).

Antifodrin cross reacts with a 70K polypeptide in whole cell homogenates of Dictyostelium amoebae(Fig. 2b). However, whole-cell homogenates were also found to contain a 220K polypeptide that stained with antifodrin when heavier loads were applied to 4% gels (Fig. 3e). The antibody showed a moderate to weak reaction with the 220K polypeptide compared to its reaction with chicken brain fodrin. This 220K polypeptide was not Dictyostelium myosin 11, since purified Dictyostelium myosin heavy chain did not stain with antifodrin (Fig. 1 d). Furthermore, antibodies absorbed with chicken brain fodrin failed to stain western blots of lysates of D.

:

a b C d Fig. 1 . 4-20% SDS-PAGE and immunoblots stained with antifodrin. a: Standards stained with Coomassie blue are (from top): rabbit brain fodrin (240,000 and 235,000 daltons), Diciyosteliurn myosin heavy chain (215,000 daltons), rabbit skeletal muscle myosin heavy chain (200,000 daltons), phosphorylase b (92,500 daltons), transfernin (78,000 daltons), bovine serum albumin (67,000 daltons), actin (42,000 daltons), and concanavalin A (27,000 daltons). Each subsequent pair of lanes shows Coomassie blue (left) and immunoblots stained with antifodrin (right). b: Purified chicken brain fodrin. c: Hornogenate of chicken brain. d: Dictyosteliurn myosin.

discoideum (not shown). The size of the polypeptide that cross reacted with antifodrin was estimated to be 220K based on comparison with human erythrocyte spectrin (fig. 3a-c).

Using this antibody in a solid-state immunoassay it was estimated that the total immunoreactive material (220K and 70K) present in the whole cell is only 0.1% of the total cell protein. This is probably an underestimate, because the standard curve was based on the binding of antifodrin to brain fodrin.

The cortex isolated from Dictyostelium amoebae as described in Materials and Methods also contained the immunoreactive analogues of fodrin (Fig. 2c). Western blots demonstrated that both 220K and 70K were stained with antifodrin at approximately the same intensity in isolated cortices (Fig. 2c), unlike western blots of whole cells (Fig. 2b), suggesting that 220K is enriched in the isolated cortex.

Quantification by the solid-state immunoassay of the amount of immunoreactive analogue in Dictyostelium amoebae u p 1 1 lysis and in the subsequent cortical and soluble fractions demonstrated that 5 1 % of the 220/70K was recovered with the cortex, whereas only 16% was recovered with the soluble fraction (Table I). The miss- ing 33% was degraded to small peptides that were not


a C d Fig. 2. 4-20% SDS-PAGE and immunoblots stained with antifodrin. a: Standards stained with Coomassie blue are (from top): Dictyosieliurn myosin heavy chain (215,000 daltons), rabbit skeletal muscle myosin heavy chain (200,000 daltons), phosphorylase b (92,500 daltons), transferrin (78,000 daltons), bovine serum albumin (67,000 daltons), actin (42,000 daltons), and concanavalin A (27,000 daltons). Each subsequent pair of lanes shows Coomassie blue (left) and immunoblots stained with antifodrin (right). b: Whole cell homogenate of Dicfyostelium amoebae. c: Cortices isolated from

c-

a b c d e

Fig. 3. Top of 4% SDS-PAGE and immunoblot stained with antifodrin. First four wells (left to right) are stained with Coomassie blue. Fifth well is an immunoblot stained with antifodrin. a: Human erythrocyte spectrin (240,000 and 220,000 daltons). b: Purified Dictyostelium 220K. c: Dicfyostelium 220K and human erythrocyte spectrin. d: Whole-cell homogenate of Dicfyosteliurn amoebae. e: Autoradiogram of western blot of whole cell homogenate of Dicfyosie- liurn amoebae after staining with antifodrin. Arrow points to a band stained with antifodrin measuring 220,000 daltons.

retained on the nitrocellulose paper and, therefore, were not detected in the assay.

In Situ Localization of the lmmunoreactive Analogues of Fodrin

Indirect immunofluorescence was used to determine the location of the fodrin immunoanalogues in Dictyostelium amoebae. No staining occurred in the absence of antifodrin (Fig. 4, row a) or when the cell was stained with antifodrin that had been preabsorbed with

f h i Diciyosieliurn ameobae. d: 100,000 g-hr supernatant of membranes extracted with 0.2 M NaCl and 10 mM MgATP, pH 7.0. e: Concentrated soluble extract loaded onto superose 6B gel filtration column. f: Fraction 22 from the gel filtration column. g: Fraction 26 from the gel filtration column (arrowhead indicates 70K). h: Gradient load prepared from fractions 20-23, from the gel filtration column, concentrated and clarified at 100,000 g-hr. i: Fraction 20 from sucrose density gradient.

chicken brain fodrin (Fig., 4, row b). In the presence of antifodrin, staining occurred at the periphery of the cell (Fig. 4, rows c,d) and was sometimes punctate in this area (Fig. 4, row c)., Antifodrin stained pharopodia (Fig. 4, row d, arrow) but not the uropod found at the opposite end of the cell. The immunoanalogues of fodrin copatch- ed (Fig. 5 , rows a, b) and cocapped with Con A (Fig. 5 , rows c 4 ) . After capping was complete, the fodrin immunoanalogues moved to other regions of the cell and into pseudopodia opposite the cap (Fig. 5 , lane f) .

To determine the location of polypeptides within the cortex that are immunoreactive with antifodrin, isolated cortices were stained with antifodrin and goat antirabbit IgG-colloidal gold for viewing in the electron microscope (Fig. 6b). A control preparation, shown in Figure 6a, was stained with goat antirabbit IgG-colloidal gold in the absence of antifodrin. A smail amount of nonspecific staining is observed in such preparations (9.8 gold particledpm’), but morphometry indicates that the density of label in such control preparations (N = 6 fields) is about one-third that found in experimental preparations (27 gold particlesipm’) (N = 6 fields). Figure 6 b d shows experimental preparations stained with antifodrin and IgG-colloidal gold. Filamentous structures that are associated with microfilaments in the cortex and on the cell membrane are labeled with gold.

Analogue of Fodrin in Llictyosteliurn Amoebae 309

TABLE I. Distribution of Irnmunoreactive Analogue of Chicken Brain Fodrin in Dictyosteliurn Amoebae

Fraction Amount of antigen

(arbitrary units) Recovery (%)

Cell lysate 357 ? 23.0 100 Supernatant (soluble) 51.6 2 5.6 16 2 1.6 Pellet (cortex) 182 ? 17.9 51 2 5.0

Fig. 4. Antifodrin staining of Dicryosteliurn amoebae. Phase (left) and antifodrin (right). a: Antifodrin omitted. b: Antifodrin preabsorbed with chicken brain fodrin. c,d: Antifodrin staining. Bar = 10 pm.

The gold label was always found on the cytoplasmic side of the membrane, which indicates that immunofluorescence associated with the cell periphery (Figs. 4, 5 ) was not due to labeling of cell surface components.

Isolation of the 220K Protein

The cross reaction of 220K and 70K polypeptides with antifodrin and their association with cortical microfilaments in situ suggest that they might be related to the fodrin (nonerythroid spectrin) family of actin binding

Fig. 5 . Antifodrin staining of Dictyosteliurn amoebae after challenge with Con A. The immunoanalogue of fodrin copatches and cocaps with Con A. Phase (left), fluorescein-labeled Con A (middle), and indirect labeling with antifodrin (right). Rows show various stages of Con A capping. (a, b, c): Copatching. d, e: Cocapping. f: Distribu- tion after capping when Con A caps are being shed and/or internalized. Bar = 10 pm.

proteins. To investigate this further, we isolated the 220K protein to determine if it is similar to fodrin and investigate its relationship to the 70K polypeptide.

The isolated cortex (Fig. 2c) contains both the 220 and 70K polypeptides. To extract the 220K, the isolated


Fig. 6. Electron micrographs of thin sections of isolated cortices. a: Control. No primary antibody present, but incubated with IgG gold. b: Cortex stained with antifodrin and goat antirabbit IgG-gold. Circles

indicate the position of colloidal gold. Bar = 200 nm. c, d: Examples of filaments associated with the membrane that are stained with antifodrin and IgG-gold. Bar = 200 nrn.

cortex was used as starting material because, as dis- cussed above, the 220K appears to be enriched in this preparation when compared to the whole cell. The antifodrin antibody was used as a probe to follow the isolation of the 220K protein. The 220K protein was extracted from the cortex by incubation with 0.2 M NaCl and 10 mM MgATP. Myosin is a contaminant of the extract when 220K is extracted from the cortex under these conditions [ 13,391. Although 220K could also be extracted with 0.6 M NaCl, thereby minimizing myosin

contamination at this step, we chose the former conditions because they gave more efficient extraction of 220K than that with 0.6 M NaCl. In addition, when 0.6 M NaCl was used, there was a significant actin contamination, which was more difficult to resolve in the subsequent purification steps. After extraction of the cortices, the extract was clarified by centrifugation at 100,000 g-hr, which sedimented some of the contami- nating actin and myosin. The soluble extract (S,) is shown in Figure 2, lane d.


loor

c 3

-0.10 t ? +

-0.08 5

10 I5 20 25 30 35 40 45 50 55 60 FRACTION NUMBER

Fig. 7. Gel Filtration on superose 6B. Solid line, percent transmit- tance. Dashed line, amount of antifodrin bound from fractions dotblotted onto nitrocellulose paper as determined by solid-state immunoassay. See Figure 2, lanes f , g.

The 220K was separated from the 70K polypeptide by gel filtration on Superose 6B (Fig. 7). Using the solid-state immunoassay , immunoreactive material was detected in several peaks. Western blot analysis demonstrated that the 220K polypeptide eluted as a peak in fractions 21-24 (Fig. 2, lane f ) whereas the 70K polypeptide eluted in fractions 26-29 (Fig. 2, lane 8).

Fractions containing the 220K protein in the leading immunoreactive peak were pooled, brought to 0.5 M NaCl, concentrated fourfold, and clarified at 100,000 g-hr. The supernatant (Fig. 2, lane h) was further purified by sedimentation on a 5-20% sucrose density gradient under conditions of high salt, with protease inhibitors present. The 220K protein was distributed in the gradient fractions as shown in Figure 8. The peak fraction (Fig. 2, lane i) contained the 220K protein, which stained with antifodrin.

The major contaminants in the preparation of 220K were actin and myosin (Dictyostelium myosin 11). Actin contamination was minimized by extracting 220K from isolated membranes with 0.2 M NaCl and 10 mM MgATP as described above. Myosin contamination was effectively minimized by eluting the gel filtration column with 0.15 M NaCl, conditions under which most of the myosin was collected in the void volume. We used rotary shadowing, by which myosin and 220K are readily distinguished, to determine the degree of myosin contamination after gel filtration. Rotary shadowing of the leading peak fractions containing the 220K indicated 8-1 1 % myosin contamination. This myosin contaiiiiiia- tion was removed by sucrose gradient centrifugation in 0.5 M NaCl, conditions under which myosin (S2*, w = 6) [49] and 220K (S20, w = 9.3) were separated because of their different sedimentation coefficients.

80 1 v) L t

60

40

n It

2oi 0 4 8 12 16 2 0 24 28 32

FRACTION NUMBER

Fig. 8. Sucrose gradient centrifugation of 220K protein. The position of 220K in the gradient fractions was determined by SDS-PAGE and quantified by densitometry of Coomassie blue-stained gels. Fractions were collected from the bottom of the tube. See Figure 2, lanes h. i.

Relationship Between 220K and 70K

Storage of the 220K protein in TKE buffer following sucrose density centrifugation for several days re- sulted in the appearance of a 70K polypeptide (Fig. 9A), suggesting a precursor-product relationship between 220K and 70K. This was investigated further by one- dimensional peptide mapping (Fig. 9B). Limited digestion of 220K with trypsin yielded polypeptides at 70K, 57K, 55K, and several lower-molecular-weight species. These polypeptides derived from 220K comigrated with the 70K polypeptide that was produced in vivo and the polypeptides generated by trypsin digestion of 70K.

Physical Properties of the 220K Protein

The physical properties of the protein were determined as shown in Figures 10-12 and summarized in Table 11. These results indicate that the 220K protein is a dimer of approximately 500,000 daltons (M,) under the conditions tested. I Rotary shadowing of the 220K protein in high-salt buffer demonstrated that the 220K is a rod-shaped protein 118 * 17.8 nm (N = 37) in length and 16.1 -+ 3.0 nm (N = 1 1 ) in width (Fig. 12) with a

'The native molecular weight was calculated using the formula M = ([6n x N(a) (s)]/[l - (v) (p)], where M = native M,, N = Avogadro's number, S = sedimentation coefficient, a = Stokes' radius, v = partial specific volume = cm7/gm [the value for spectrin was used 1451, v = 0.7351, and p = density of buffer = gndcm'.


20

16

12

8-

4 -

A B

-

-

-

- I

l6 r

I

I -

I

I

I

a b c d a b c

Fig. 9. A: 4-20% SDS-PAGE of 220K after prolonged storage. a: Standards stained with Coomassie blue are (from top): rabbit skeletal muscle myosin (200,000 daltons), P-galactosidase (1 16,250 daltons), phosphorylase B (92,500 daltons), bovine serum albumin (66,200 daltons), ovalbumin (45,000 daltons). b: Fraction 20 from sucrose density gradient containing Dictyostelium 220K. c: Fraction 20 from sucrose density gradient after storage for several days on ice. The stored fraction contains Dictyostelium 220K and 70K breakdown product (arrow). B: One-dimensional peptide map of a partial trypsin digest of 220K and 70K. Bars show molecular weight markers (from top): 200,000 daltons, 92,500 daltons 66,200 daltons, 45,000 daltons, 31,000 daltons, 21,500 daltons, 14,400 daltons. a: 220K band sliced out of SDS-PAGE of fraction 21 from Superose 6B column and rerun on SDS-PAGE. b: 70K band sliced out of SDS PAGE of fraction 27 from Superose 6B column, electroeluted and rerun on SDS-PAGE. c: Same as a following digestion with trypsin. d: Same as b following digestion with trypsin.

mass:length ratio of 4,237 daltonshm. A few longer rods measuring 260 IT 48 nm (N = 4) in length and 15.0 2 9.7 nm (N = 4) in width were also observed in the same preparation (Fig. 12C), suggesting that the protein may form larger oligomers. The width of the 220K may average about four times its actual width because of the deposition of platinum at 4" based on calibration with F-actin.

Actin Binding Properties of the 220K Protein

The binding of 220K to F-actin could not be quantitated by sedimentation experiments, since 220K would sometimes aggregate in low-salt buffers making interpretation of the assay difficult. To examine the binding of 220K to F-actin, 220K that had been clarilied in low-salt buffer to remove aggregates and traces of myosin, was mixed with actin in low-salt buffer and examined by low-angle rotary shadowing as described in Materials and Methods. In the absence of 220K, the actin

3

r4 0- In

0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 Kav 10

FRACTION NUMBER 11 Fig. 10. Determination of Stokes' radii of immunoreactive proteins from Dictyosfelium. Stokes' radius was determined by fractionation on a Superose 6B gel filtration column equilibrated in 0.15 M NaC1, 20mM Tris, 2 mM EGTA, 0.02% NaN3, pH 8.0, at 5°C. A: 220,000 dalton protein (13.5 nm). B: 70,000 dalton protein (1 1.4 nm). C: Fibrinogen (10.7 nm). D: Thyroglobulin (8.5 nm). E: Catalase (5.2 nm). F: Cytochrome C (1.7 nm).

Fig. 1 I . Determination of S20,w of 220K from Dictyostelium. S20,w was determined by sedimentation on a 5-20% sucrose gradient in 0.5 M NaCI, 10 mM Tris, 0.5 mM EDTA, 0.1 mM DTT, 0.02% NaN3, pH 8.0, at 5°C. A: Thyroglobulin (19.3 S ) . B: Catalase (1 1.3 S). C: Dictyosfelium 220,000 dalton proteins (9.3 S). D: Bovine serum albumin (4.3 S). E: Cytochrome C (1.9 S).

filaments were smooth and devoid of surface projections (Fig. 13a,b). When the 220K protein was mixed with F-actin, one end of the rod-shaped 220K protein was observed to bind to the side of an actin filament (Fig. 13c,d). However, cross linking of actin filaments by 220K dimers was riot observed.

Based on the concentration of 220K and actin present, the expected frequency of binding was one 220K dimer per 120.8 monomers (302 nm) if 100% of the 220K present was bound to actin. The observed


investigated further by low-shear falling ball viscometry . The inverse velocity (vel-' = sedcm) of 1.4 mg/ml F-actin was decreased slightly from 4.9 k 0.47 sec/cm (N = 9) in the absence of 220K to 4.1 2 1.0 sec/cm (N = 9) in the presence of 220K at concentrations of 220K between 0.022 and 0.066 mg/ml.

F-actin and mixtures of F-actin and 220K were viewed by electron microscopy after negative staining with uranyl acetate. Under the conditions described in Materials and Methods, mixtures of F-actin and 220K were indistinguishable from F-actin alone (not shown), suggesting that 220K did not cross link or aggregate actin filaments. Furthermore, in our hands, the 220K protein could not be visualized following negative staining with uranyl acetate.

Fig. 12. Low-angle rotary shadowing of Dicfyostelium 220K protein. Samples in 0.6 M ammonium acetate, pH 7.0, and 40% glycerol were sprayed on freshly cleaved mica and rotary shadowed at an angle of 4". a: 220K protein dimer. Bar = 100 nm. b: Panel of six 220K dimers. Bar = 100 nm. c: Panel of three 220K oligomers (possibly tetramers, since the length of each is twice that of the dimer). Bar = 200 nm.

frequency of binding of 220K to actin filaments was one 220K dimer per 127.4 monomers (318.5 nm) (N = 6 fields containing 35 actin filaments).

Cross-linking of actin filaments by 220K was

DISCUSSION Antifodrin Stains a Unique High-Molecular-Weight Protein in Dicfyostelium Amoebae

We have isolated from the cortex of Dictyosteliurn amoebae a 220K protein that is an immunoreactive analogue of chicken brain fodrin. The 220K is a unique high-molecular-weight protein whose properties distinguish it from other high-molecular-weight actin binding proteins of Dictyostelium amoebae.

Reines and Clarke [39] observed that Dictyosteliurn amoebae have many polypeptides whose mobilities by SDS-PAGE are similar to myosin heavy chain (M, 215K) but do not cross react with monoclonal antibodies prepared to Dictyosteliurn myosin. Our results suggest that one of these is the 220K protein. We could not use migration by SDS-PAGE to distinguish 220K from myosin, because 220K and myosin heavy chain were not well resolved on standard SDS-PAGE. However, we were able to use antifodrin as a specific probe for the 220K because Dictyostelium myosin I1 heavy chain does not cross-react with the antifodrin used in these studies. Furthermore, myosin and the immunoreactive analogue of fodrin have different locations in the cell. Myosin has a diffuse distribution in unstimulated randomly moving amoebae [9], whereas the immunoreactive analogue of fodrin has a cortical distribution. Under the conditions employed for the isolation of 220K, we could separate and recognize myosin from 220K based on its different hydrodynamic properties and morphology.

Another high-molecular-weight actin-binding protein found in Dictyosteliurn amoebae is the 240K protein. Unlike 220K, the 240K protein resembles smooth muscle filamin in morphology, actin-binding activity, and immunoreactivity with antibodies prepared to filamin from chicken gizzard [23]. The 240K protein can also be distinguished from the 220K by its molecular weight on SDS-PAGE and lack of cross reactivity with antifodrin.


TABLE 11. Comparison of Physical Properties of 220K Protein From Dictyostelium Amoebae With Other High-Molecular-Weight Actin-Binding Proteins

Human Pig Pig Chicken Cell and Dictyostelium Erythrocyte brain brain gizzard Dictyostelium protein 220K spectrin fodrin MAP 2 filamin 240K

Stokes’ 13 12 24 15 12 8.6 radius

s,,,., 9.3 8.7- 10.2 I I 4.4 10 12 Native 500K 460K 1,090K 266K 498K 434K

molecular weight

subunits Number of Dimer Dimer Tetramer Monomer Dimer Dimer

Subunit 220K 240Kl220K 240Ki235K 270K 250K 240K Contour 1 I8 100 200 147 160 140

length (nm)

(daltonsinm) Massilength 4,237 4,600 5,450 1,810 3,113 3,100

References 45.51.52 4.21 6,53 52,54 23

Furthermore, antifilamin from chicken gizzard does not cross react with the 220K [23].

220K and 70K Exhibit a Precursor-Product Relationship

Both the 220K and the 70K polypeptides show cross reactivity with antifodrin. In immunoblots of homogenates of whole cells, where there is a plethora of proteases [29], the 70K is the predominant immunoreactive species, suggesting that 70K is derived from 220K by the action of endogenous proteases. This was con- firmed during the isolation of 220K, when the 70K appeared in samples that previously contained only the 220K. One-dimensional peptide maps of trypsin digests of 220 and 70K further suggest that the 70K is a cleavage fragment of the 220K.

Like the 220K, both fodrin and spectrin are proteins that are susceptible to proteolysis [7,46]. Trypsin proteolysis of fodrin yields a band at approximately 70,000 Daltons [7], which may be related to the 70K immunoreactive polypeptide in Dictyostelium amoebae. Fodrin is also a substrate for calpain I, a calcium- activated thiol protease. The action of calpain on fodrin in the presence of CaCI2 produces a major 70K polypeptide [46]. It has been suggested that brief increases in the level of free, intracellular calcium mediate the degrada- tion of fodrin and that this acts to modify the cytoskeleton, distribution of cell surface receptors, and cell shape [3 I ,46,47].

Properties of 220K Protein

Hydrodynamic measurements indicate that the 220K is a dimer in buffers containing either 0.15 or 0.5

M NaCl. However, one4imensional SDS-PAGE under a variety of conditions failed to resolve the 220K polypeptide into more than one band indicating that the two subunits are of identical molecular weight.

The 220K is associated with microfilaments in vitro and in situ. Rotary-shadowed images of mixtures of 220K and F-actin indicate that one end of the 220K dimer can interact with the side of a microfilament. These images are consistent with results obtained when cortices of Dictyostelium amoebae are stained by immunocytochemistry with colloidal gold, which indicates that the immunoreactive analogue of fodrin is associated with microfilaments in situ. However, in our hands, 220K does not cross link actin filaments in vitro. Both falling ball viscometry and negative staining with many1 acetate performed under a variety of conditions failed to detect cross linking of actin filaments in the presence of 220K.

Immunofluoresence of Dictyostelium ameobae and immunocytochemistry with colloidal gold of the cortical preparations containing plasma membrane indicate that the immunoreactive analogue of fodrin is a cortical protein. The coisolation of 220K with the cortical preparations containing plasma membrane and the extraction of 220K from those cortices further suggest that the 220K is a cortical protein.

220K Protein May Be a Member of the Fodrin Family of Proteins

The 220K protein shares immunoreactive epitopes with fodrin. However, this is not proof that the 220K is another fodrin. For example, pig brain microtubule associated protein (MAP) 2 (270,000 Mr), which belongs

Analogue of Fodrin in Bctyostelium Amoebae 315

Fig. 13. Rotary shadowing of mixtures of rabbit skeletal muscle F-actin and Dicfyosfelium 220K protein. Samples, in 25 mM KCI, 5 mM Pipes, 0.5 mM MgS04, 0.5% glutaraldehyde, pH 6.8, and 50% glycerol were sprayed onto freshly cleaved mica and rotary shadowed at an angle of 4". a,b: Rabbit skeletal muscle F-actin in the absence of 220K. c,d: Rabbit skeletal muscle F-actin and Dicfyosfelium 220K protein. Vertical filaments are F-actin. Arrow indicates where one end of a 220K protein has bound to the side of an actin filament. Bar = 100 nm.

to the class of high-molecular-weight MAPS also cross reacts with antispectrin [16]. Likc spcctrin, MAP 2 is a filamentous protein that binds to actin [22,43,44]. How- ever, it is distinct from spectrin in its ability to bind to microtubules, in its lack of ankyrin binding 1161, and in its physical properties (Table 11).

We do not believe that the 220K is a MAP 2-like protein. Microtubules have been observed in a distinctive pattern in Dictyostelium amoebae when stained with antitubulin [42]. However, the pattern of staining that we observe when Dictyostelium amoebae are stained with antifodrin suggests that the immunoreactive analogue of fodrin is not associated with microtubules in situ. Fur- thermore, the morphology and hydrodynamic properties of 220K are distinct from those of MAP 2 when both are measured under similar conditions [6,53].

The 220K protein shares properties with the fodrin family of proteins. The 220K protein has a Stokes' radius and sedimentation coefficient that are similar to those of erythrocyte spectrin dimer [45] (Table 11). When the 220K is rotary shadowed, it has a morphology similar to that of spectrin. It is a rod-shaped molecule of similar size, with a maslength ratio of 4,237 daltonshm compared to 4,600 daltonshm for erythrocyte spectrin, suggesting the presence of two subunits in close side to side contact.

Like fodrin in lymphocytes [28,36], the fodrin immunoanalogues in Dictyostelium cocap with Con A. Furthermore, 220K/70K may be associated with microfilaments that are bound to the cell membrane, because they are found in the cortex in association with microfilaments that are close to the plasma membrane by immunocytochemistry at the level of resolution of the electron microscope.

However, there are differences between the 220K protein and fodrin. As was mentioned above, the two subunits of 220K dimer do not exhibit different mobilities upon SDS-PAGE. This result is reminiscent of the 260K protein from Acanthamoeba, which is also an immunoreactive analogue of the heterodimer spectrin but is not resolved as a heterodimer upon SDS-PAGE [38].

Although erythrocyte spectrin can be obtained in physiological buffers as a dimer, fodrin favors the tetra- meric configuration and will not remain as a dimer unless treated with NaBr [4]. Thus it appears that, whereas the 220K dimer can bind to microfilaments in situ, as a dimer,


it may not be able to cross link microfilaments, because it does not form a tetramer as readily as some members of the fodrin family and thus lacks the two binding sites required to cross link actin filaments.

Finally, 220K tends to aggregate at low ionic strength, which made sedimentation assays designed to investigate the binding of 220K to F-actin uninterpret- able, whereas fodrin is reported to remain soluble under these conditions [20]. More sophisticated approaches such as fluorescence energy transfer may be required to analyze the interaction between 220K and F-actin.

Members of the fodrin family are commonly thought of as membrane-associated proteins. Immuno- reactive analogues of fodrin found in other cell types have been localized by immunofluorescence to the membrane [7,27,28,36,40,41], but, so far, only spectrin and fodrin have been found to bind ankyrin, the protein, first identified in the human erythrocyte, that links spectrin to the anion channel band 3 [2-41. The ankyrin binding domain of spectrin has been mapped to the 220K subunit [35], which suggests that a change in the 220K subunit of spectrin may determine to what extent a molecule will function in membrane linkage. Whether a membrane binding site exists on the 220K of Dictyostelium and whether, like fodrin and spectrin, it is specific for ankyrin are questions that remain to be answered.

ACKNOWLEDGMENTS

We thank Dr. J.R. Glenney, Jr., for his kind gift of antifodrin, which we used in preliminary experiments. We also thank Dr. S. Ogihara for his preparation of IgG-colloidal gold. We thank Dr. J. Carboni, Dr. P. Detmers, Dr. S. Ogihara, Dr. R. Hock, A. Hall, A. Bresnick, and S. Glenn for helpful discussions. Various stages of this work were presented at the 24th, 25th, and 26th annual meetings of the American Society for Cell Biology. This work was submitted by Holly Bennett in partial fulfillment of the requirements for the PhD in the Sue Golding Graduate Division of Medical Sciences, Albert Einstein College of Medicine. This work was supported by grants from the NIH, a training grant (CA09475), and the Hirschl Trust.

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isolation of an immunoreactive analogue of brain fodrin that is associated with the cell cortex of...

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