Differential recognition of monomeric and polymeric forms of actin by anti-actin antibodies

MoCfrr/ar Imnmmlog~. Vol. 17. pp. 1219-1230. 0 Pergamon Press Ltd. 1980. Printed in Great

016i-58~/80/i~1-~2~9

$02.00/O

Brrtain

DIFFERENTIAL POLYMERIC

RECOGNITION OF MONOMERIC AND FORMS OF ACTIN BY ANTI-ACTIN ANTIBODIES

MAGALI DOSSETO and CHRIST0 GORIDIS Centre ~Immunologie

INSERM-CNRS

de Marseille-Luminy, Case 906, 13288 Marseille &iex 2, France

(First received 10 October 1979; in revisedform

14 January 1980)

Abstract-The fine specificity of anti-actin sera raised in rabbits against denatured or insolubilized muscle actin was anaiysed by a radioimmunologi~~ assay procedure. Whereas two different labeled derivatives of monomeric actin lacked immunore~tivity, iodinated stabb polymers produced by cross-linking F- or Gactin bound to the antibodies. Labeled oligomers of greater size were more readily bound. An actin polymer prepared by treating G-actin with polyglutaraldehyde was used as tracer in competition experiments to determine the specificity of the antibodies for different forms of actin. When the test was carried out under conditions favorable for polymerization (0.1 MNaCI, 37”C), F-actin readily competed for antibody binding sites. Under the same conditions, dilutions of G-actin also inhibited the binding of labeled antigen, but somewhat higher concentrations were required. However, actin maintained in its G-configuration by performing the assay in low ionic strength buffer or at low temperature was not recognized by the antibodies. By contrast, a large tr~sin-resist~t proteolytic fragment of G-actin which has lost its ability to polymer& was immunoreactive. Apparently, the anti-(muscle-actin) antibodies are directed against antigenic sites available on polymeric or partially degraded actin, but not on G-actin or labeled monomers. Because of their selectivity for certain forms of actin, such antibodies may be useful probes for the conformational state of actin.

INTRODUCTION

Actin has been identified as a major component of essentially all eukaryotic cells (see Pollard & Weihing, 1974, for review). This protein exists characteristically in two profoundly different conformational states: the soluble, monomeric globular or G-actin and the polymerized form, the fibrous or F-actin. The transformation between G- and F- actin has been studied most extensively using purified muscle actin. Actin can be regarded as a polymerization condensation phenomenon: F-actin is formed from actin monomers above a critical concentration which is a function of ionic conditions and temperature (Szent-Gyorgi, 1951; Oosawa et af., 1961; Higashi & Oosawa, 1965; Stone et al., 1970; West, 1970; Murphy, 1971; Lehrer & Kerwar, 1972). In striated muscle, all actin present is in the polymer&d state. In non-muscle cells, however, a considerable proportion of actin is depolymerized, but some is present as actin filaments which can by visualized by anti-actin antibodies using fluorescence (Lazarides & Weber, 1974; Trenchev et al., 1974; Lazarides, 1975; Osborn et al., 1978; Jockusch et al., 1978; Herman 81 Pollard, 1979), peroxidase (Karsenti

et al., 1978) or ferritin (Webster et al., 1978) labeling. These immunocytochemical techniques have been very useful in establishing the presence of filamentous actin in non-muscle cells, in demonstrating the dynamic state of the actincontaining structures in these cells. The antibodies employed in such studies were directed against non-muscle, smooth muscle or striated muscle actin. The amino acid sequences of muscle and cytoplasmic actins from different species are highly conserved (Vandekerckhove & Weber, 1978 a, b and c) and antibodies against actin from one given source recognize the homologous protein in different tissues and species. The poor immunogenicity of actin can be explained by this autoimmune character of antiactin immunisations. Therefore, denatured (Lazarides & Weber, 1974), insolubilized (Groschel-Stewart et al., 1977; Jockusch et al., 1978) or chemically modified (Benyamin et al., 1979) actin has been used as immunogen to increase the immune responsiveness. The antibodies obtained clearly react with actin filaments. However, the fine specificity of antiactin antibodies for different conformational states of actin has not been investigated in detail. Such information is of particular importance since G-and F-actin are known to coexist in non-

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DOSSETO

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muscle cells, the transformation between the polymerized and unpolymerized forms being regulated by the cell (Goldman et al., 1976; Tilney, 1977; Korn, 1978; Lindberg et al., 1978). This report describes the characterization of anti-actin antibodies by analysing their specificity for monomeric and polymeric actin. The results show that of the two physiological forms of actin, F- but not G-actin are recognized by our antibodies. EXPERIMENTAL

PROCEDURES

Actin pur$cation Actin was prepared from acetone powders of the back muscles of Hyla white rabbits according to the method of Spudich & Watt (1971). The actin purified by two cycles of polymerization and depolymerization was further purified by a third cycle either of polymerizationdepolymerization or gel filtration on a Sephadex column (Adelstein et al., 1963). Gel electrophoretic analysis in the presence of sodium dodecylsulfate revealed one band with an apparent molecular weight of 42,000. Prior to use as an antigen or before labeling, the solution of G-actin was centrifuged for 2 hr at 100,000 g and 4°C to remove aggregates which might have been formed. The formation of F-actin was induced by adding 0.1 M KC1 and 2 mA4 MgCl, to the solution of purified G-actin. The large protease-resistant fragment of G-actin, called actin-core protein, was prepared by tryptic degradation as described (Jacobson & Rosenbusch, 1976). Antisera Rabbits of the same strain used to prepare the actin were injected with the purified protein. Two different published procedures were followed. According to the first method (Lazarides & Weber, 1974; Karsenti et al., 1978; Lazarides. 1976a), the purified actin was denatured in sodium dodecylsulfate, subjected to polyacryelectrophoresis eluted lamide and gel electrophoretically from the gel. On day 1, the animals received subcutaneously 0.8 mg of complete Freund’s protein emulsified in adjuvant and on days 13 and 71, 0.4 mg of protein emulsified in incomplete Freund’s adjuvant. The rabbits were bled at day 78. Six animals (rabbits I-VI) were injected by this

*Buffer G: 10 mM Trls-HCI buffer (pH 7.8). 0.2 mA4 ATP, 0.2 mA4 CaCI,, 0.5 mM 2-mercaptoethanol.

GORIDIS

method. The method of Jockusch et al. (1978) using actin not denatured in sodium dodecylsulfate, was followed exactly as described. Five animals were injected according to this immunization scheme (rabbits VII-XI). Laheled monomeric

antigens

G-actin was labeled by two different methods: iodination with the aid of chloramine-T and derivatization with [ l 2sI] iodosulfanilic acid. The radioactive antigens obtained were tested for their ability to polymerize and to bind to DNAse I. Before being iodinated by the chloramine-T method (Hunter & Greenwood, 1962), G-actin was dialysed overnight against buffer G*, from which 2-mercaptoethanol had been omitted. The dialysed protein was centrifuged for 2 hr at 100,000 g and 4°C to eliminate any actin which might have polymerized. Bound and free iodine were separated by chromatography on a Sephadex G25 column in buffer G. The purity of the iodinated product was checked by electrophoresis on 100,;) polyacrylamide gels in the presence of 0.1% sodium dodecylsulfate. A single peak of radioactivity was seen which comigrated with unlabeled actin (not shown). G-actin was derivatized with [iz51] iodosulfanilic acid (New England Nuclear, Boston, MA, 1 Ci/pmol) following the instructions of the manufacturer. The labeled protein was separated from the reagents by chromatography on a Sephadex G-25 column in buffer G. The two labeled derivatives of G-actin were partially characterized by testing their ability to copolymerize with an excess of unlabeled actin and to bind to DNAse I. The labeled derivatives (3 x IO5 countsjmin of [iz51] iodo-actin and 8 x lo4 counts/min [ 125I] iodosulfophenyl-azo-actin in 0.5 ml) were added to 4 ml of a solution of G-actin in buffer G (1.5 mgiml). Then the mixture was made 0.1 M in KC1 and 2 mM in MgCl,. After 1 hr at 37°C the solution was centrifuged for 2 hr at 100,OO g and 4°C and the radioactivity of supernatant pellet and determined. Whereas 93”/, of the unmodified actin was found in the pellet as shown by protein determination, 927,) of the radioactivity added as I 251-labeled actin remained in the supernatant (Table 1). Under the same conditions, 5 I::, of the [i 251] iodosulfanilic acid derivative was pelleted. Control experiments showed that only negligible amounts of this derivative were recovered in the pellet without the addition of unlabeled carrier. G-actin has been shown to bind specihcally and

Specificity of Anti-actin

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Table 1. Properties of 12s1-labeled G-actin and G-actin derivatized with [izsI] iodosulfanilic acid

[‘251]Iodoactin

[lzsI]I~osuIfophenylazo-actin

Mole ‘251/mo1 actin Copolymerization with unmodified actin (% radioactivity recovered in a pellet of F-actin)

0.8 8

0.25 51

Binding to DNAse-Sepharose (% radioactivity bound which could be eluted with 3 M guanidinium-HCl)

88

32

with high affinity to DNAse I (Lazarides & Lindberg, 1974). F-a&n also interacts with DNAse I, but the formation of a stable complex is a much slower process (Hitchcock et al., 1976). Denatured actin does not bind to DNAse I (Lazarides & Lindberg, 1974). We assessed the binding of the labeled actin derivatives to DNAse I using the affinity chromatography procedure described by Lazarides & Lindberg (1974). [1251]Iodoactin (7 x lo5 counts/min) or [125I] iodosulfophenyl-azo-actin (3 x lo* counts~min) in 0.1 ml buffer G were applied to small columns of DNAse-Sepharose (0.7 x 5 cm) which were then washed and eluted as described. Nearly 90% of the actin iodinated by the chloramine-T method was bound and the bound radioactvity was quantitatively recovered in the guanidinium-HCI eluate. In the case of the sulfanilic acid derivative, 48% of the applied radioactivity was fixed, of which 67% could be eluted by 3 h4 guanidinium-HCl. Labeled polymeric antigens G- and F-a&in were cross-linked with the bifunctional reagents polyglutaraldehyde and succinyl tyrosine, respectively. The polymers obtained were theniodinated by thechloramine-T method. Labeled polymers ofdifferent size classes were separated by gel filtration. N-succinyl tyrosine was prepared in our laboratory by M. A. Delaage (to be published in detail elsewhere). F-actin was cross-linked with this bi-functional reagent as follows. First, 6.5 pmol succinyl tyrosine were activated by reacting it with 18 pmol ethyl chloroformate in the presence of 18 lmol t~ethylamine in 0.2 ml dimethylforma~de for 15 min at 0°C. Then, 50 ~1of this reaction mixture were added to 1 ml of a solution of 1.3 mg/ml F-a&in in 10 mM borate buffer (pH 9.5) containing 0.2 mM ATP, 2 mM MgCl, and 0.1 M KCl. After 15 min at 4”C, the pH was adjusted to 11 and the incubation *Saline/phosphate:10 mM sodium phosphate buffer (pH 7.2), 0.15 A4 NaCI.

continued for 1 hr. The reaction mixture was then dialysed against buffer G without 2-mercaptoethanol (24 hr at 4°C) to remove excess reagents and to depolymerize actin polymers not stabilized by covalent bonds. An aliquot of the dialysed solution corresponding to 1.25 nmole of actin monomer was then iodinated with 1 mCi NaI [1251J in the presence of 10 pg chloramine-T. After 2 min at room temperature, the reaction was stopped by addition of 60 ,ug sodium metabisulfite. Then, 0.5 ml of 10 mM Tris-HCl buffer (pH 7.4) containing 2 mg/ml human serum albumin was added and the reaction products fractionated by chromatography on an Ultrogel AcA 44 column equilibrated and developed in 10 mM Tris-HCl buffer (pH 7.4). Stable polymers of G-actin were prepared by reacting it with glutaraldehyde polymerized by exposure to alkaline pH (Monsan et al., 1975). The pH of a commercial solution of 25% glutaraldehyde (Merck, Darmstadt, G.F.R.) was adjusted to 10 with NaOH. Then, 0.1 ml of this solution were added dropwise to 1.5 mg of G-a&in in 1 ml of buffer G. After 3 hr at room temperature, the reaction was stopped by adding 0.5 ml of 1 M lysine hydrochloride (pH 7.8). The reaction mixture was incubated overnight at 4°C and then dialysed against saline/phosphate* buffer to remove excess reagents. The reaction products were analysed by sodium dodecylsulfate-polyacrylamide gel electrophoresis on 6.7% gels. Most of the material did not enter the gel, but a succession of bands with the molecular weights of actin monomers to tetramers were clearly visible (not shown). An aliquot of the dialysed reaction mixture containing 1.25 nmol of actin monomer was iodinated as described above for succinyl tyrosine-treated F-actin. The iodinated actin polymers were freed from unbound iodine and partially separated by chromatography on an Ultrogel AcA 44 column equilibrated and developed in saline/phosphate buffer. A specific activity of 32 $i/nmol monomer was estimated

MAGALI DOSSETO and CHRIST0 GORIDIS

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for the fraction excluded from the gel. The high molecular weight fractions were diluted to 1 x lo6 counts/min in saline/phosphate buffer containing 2 mg/ml human serum albumin. No deterioration of binding to anti-actin sera was noted after 2 lnonths storage at -20°C.

Radioimmunoassays were performed using the double antibody precipitation method. For antibody dilution assays, serial dilutions of the rabbit antisera (50 ~1) were incubated with appropriate amounts of labeled antigen for 1 hr at 37°C in a total volume of 0.15 ml. Except if stated otherwise, the antisera were diluted in saline/phosphate buffer containing 2% fetal calf serum and the final NaCl concentration of the incubation mixture was 0.1 M. Antiserum dilutions greater than l/l00 were done in l/l00 diluted normal rabbit serum to obtain a macroscopic precipitate with the second antibody. Following incubation with the first antibody, sufficient goat anti-(rabbit IgG*) serum (kindly provided by A. Colle) was added to quantitatively precipitate the rabbit immunoglobulin. After 24 hr at 4°C the precipitates were spun down, washed twice with saline/phosphate buffer, transferred to clean tubes and counted. Competition assays were carried out by incubating varying amounts of unlabeled actin or actin derivatives with a dilution of the antiserum giving 40-50% binding of labeled antigen in the absence of competitor. Typically, 1 ng to 100 pg of competitor were added to the diluted serum, followed immediately by labeled antigen and the samples were processed as described for antibody dilution assays. Results are expressed as the ratio of precipitated vs total radioactivity in the sample (B/7’).

Immunofluorescence on monolayer cells was done following the method described by Lazarides (19766). Mouse 3T3 and human Chang liver cells showed a very similar staining pattern. Immunoglobulin fractions of anti-actin or control sera were used at concentrations of 1 mg/ml protein. Protein was determined by the method of Lowry rt al. ( 195 1) with bovine serum albumin as standard or, if applicable, by measuring the

* IgG: Immunoglobulin

G.

absorbance at 290 nm (E = 0.63 mg- 1 x cm ’ for actin solutions (Houk & Ue, 1974). Polyacrylamide gel electrophoresis in the presence of sodium dodecylsulfate was performed in vertical slab gels according to Laemmli (1970). Routinely, the gels contained 10% polyacrylamide. Gels containing radioactive samples were frozen at -8O”C, sliced and counted.

RESULTS When assayed by indirect fluorescence on cultured cells grown on glass cover slips, immunoglobulin fractions from 10 out of 11 immunized rabbits gave staining patterns very similar to those found in the literature (Lazarides & Weber, 1974; Lazarides, 1975; Lazarides, 1976b), and the fluorescence was completely abolished by absorption with purified F-actin, thus indicating the production of specific antibodies regardless of the immunization protocol employed (results not shown). Staining intensities were the same whether a human or a mouse cell line was used. In order to investigate the reactivity of our antibodies with soluble actin, we tested them against monomeric labeled antigens. None of the sera or immunoglobulin fractions. which had given positive results by immunofluorescence, bound significant amounts of G-actin iodinated by the chloramine-T method. Although the labeled derivative appeared to have conserved the overall conformation of G-actin as judged from its binding to DNAse I. its inability to polymerize indicated more subtle changes due to derivatizing an essential tyrosine residue or due to exposure to the reaction mixture (Table 1). These changes, then, could have abolished the antigenicity t 251-labeled G-actin. of the Alternatively, G-actin could be intrinsically devoid of immunoreactivity. These possibilities were investigated further by preparing a different labeled derivative of G-actin. Diazotized sulfanilic acid can be coupled to accessible histidine, lysine or tyrosine residues under very mild conditions (Berg & Hirsch, 1975). The [12sI] iodosulfanilic acid derivative of G-actin thus obtained, which contains an average of only 0.25 mol 1251/mol actin, differed from its analogue prepared by the chloramine-T method in that around 507; of it copolymerized with an excess of cold G-actin (Table 1). Nevertheless, the labeled conjugate did also not bind to our antisera. The results

Specificity

of Anti-actin

obtained with G-actin labeled by two different methods suggested that the lack of immunoreactivity of the derivatives was due to their being in the monomeric state rather than to changes introduced by the labeling. Labeled derivatives of F-actin cannot be used as tracers in a radioimmunoassay, because of the high dilutions in which labeled antigens have to be employed and which preclude the formation of F-actin even under conditions favorable for polymerization. We therefore synthesized stable polymers of actin which could be iodinated and used as radioactive antigens. First, a novel bifunctional reagent, succinyl tyrosine, was used to cross-link F-actin. Succinyl tyrosine presents the advantage of introducing additional tyrosine residues susceptible to iodination by chloramineT. Cross-linking of F-actin with succinyl tyrosine produced stable high molecular weight polymers since most of the product eluted in the void volume of an Ultrogel AcA 44 column after exhaustive dialysis against a low ionic strength buffer. This treatment should have depolymerized all F-actin not stabilized by

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covalent bonds (Fig. 1). Trace amounts of the iodinated polymers excluded from the Ultrogel column were incubated with increasing dilutions of immune or preimmune serum. The antibody dilution curves clearly show that an anti-actin serum, which lacked reactivity towards the monomeric actin derivatives, bound the iodinated conjugates (Fig. 2). Preimmune serum complexed only insignificant amounts of this labeled antigen. Although most of the iodinateti actin derivatized with succinyl tyrosine eluted in the void volume of the Ultrogel column (fractions 14 and 15), subsequent fractions still contained appreciable radioactivity, nearly all of which was trichloroacetic acid precipitable. They were separately diluted to contain approximately the same concentration of lz51 as the diluted fractions 14 and 15. As shown in Fig. 3, a l/l0 dilution of the antiserum bound more than 60% of the radioactivity of fractions 14 and 15, but only 34% of fraction 17 and less than 10% of the following fractions, indicating that higher weight polymers molecular were better recognized by the antibodies.

3 I

4 .

20

25

30

35

FRACTIONS Fig. 1. Chromatography of the reaction products obtained by iodinating cross-linked actin on an Ultrogel AcA 44 column. (i) F-actin was reacted with succinyl tyrosine and iodinated as described in the Experimental Procedures section. The iodination mixture was applied to the column (90 x 1.5 cm) equilibrated with 1OmM Tris-HC1 buffer (pH 7.4). The radioactivity of the effluent was monitored by a Nardeux E 500 radiometer, and is given in arbitrary units. Fractions of 2.8 ml were collected. The elution profile is given by the dotted line. The small molecular weight peak represents free iodine and iodinated ohgomers of succinyl tyrosine. (ii) G-actin was reacted with polyglutaraldehyde and iodinated as described in the Experimental Procedures section. The iodination mixture was applied to the same column equilibrated and developed with saline/phosphate buffer (solid line). The small molecular weight peak represents free iodine. In a separate run, the elution volume ofblue dextran and ofproteins ofknown molecular weight was determined (1 = blue dextran, 2 = catalase dimer, 3 = transferrin, 4 L chymotrypsinogen). From this experiment, the following approximate molecular weights could be assigned to the radioactive fractions: fraction (17): 150,000; fraction (18): 120,000; fraction (19): 95,000.

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1 I w’

1

’ -3 x)

I

l

’ -5 10

DILUTIONS Fig. 2. Serum dilution curve with iodinated succinyl tyrosine derivative of F-actin. Labeled antigen (45,000 counts/min) eluted in the void volume of the Ultrogel AcA 44 column was incubated with the indicated dilutions of preimmune serum (A) or anti-actin serum (rabbit VII) (0). Results are presented as the ratio of precipitated vs total radioactivity (B/7)

0.6

‘2

10

’

I

103

I

I

d

DILUTIONS Fig. 3. Immunoreactivity of iodinated succinyl tyrosinelinked actin polymers of various size classes. Aliquots (45,000 counts/minf of the high molecular weight fractions eluted from the Ultrogel column were incubated with dilutions of anti-actin serum (rabbit VII). The elution profile is shown in Fig. 2. (0) fractions 14 + 15; (0) fraction 16; (W) fraction 17; (0) fraction 18; (A) fraction 19.

To investigate further the apparent specificity of our anti-muscle actin antibodies, we prepared soluble polymers by reacting G-actin with glutaraldehyde. In contrast to Lehrer (1972), we had no difficulties in preparing glutaraldehydelinked polymers from G-actin using a somewhat different technique. Actin was not reacted directly with the commercial solution of glutaraldehyde, but with polyglutaraldehyde prepared by exposing the glutaraldehyde solution to alkaline pH (Monsan et al., 1975). When analysed by gel electrophoresis on 6”,, polyacrylamide gels in the presence of sodium dodecylsulfate, most of the cross-linked actin did not enter gel, but a succession of bands corresponding to different oligomers were also observed (results not shown). The actin polymers were iodinated by chloramine-T and chromatographed on an Ultrogel AcA 44 column (Fig. 1). The high molecular weight fractions were separately diluted and their immunoreactivity determined by the double antibody precipitation assay. As in the case of the succinyl tyrosinelinked actin, binding by the antiserum was highest for the excluded fraction and declined progressively thereafter (Fig. 4). From calibration of the column with proteins of known molecular weights, a molecular weight of around 120,000 could be assigned to the iodinated of fraction 18, the smallest components molecular weight fraction showing significant binding to the antiserum. Whereas the maximum binding of the

Specificity

of Anti-actin

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DILUTIONS

Fig. 4. Immunoreactivity of iodinated polyglutaraldehydelinked actin polymers of various size classes. Aliquots (45,000 counts/min) of the high molecular weight fractions eluted from the Ultrogel column were incubated with dilutions of anti-actin serum (rabbit VII). The elution profile is given in Fig. 5. (m) fraction 15; (0) fraction 16; (0) fraction 17; (0) fraction 18; (+) fraction 19, (A) fraction 20.

excluded peaks by low dilutions of the antiserum were not very different for the succinyl tyrosineand the polyglutaraldehyde-linked polymers, the higher affinity of the antiserum for the glutaraldehyde derivative is clearly revealed by the slope of the dilution curves, 50% of the labeled antigen being bound by l/l 30 or l/20 diluted serum for glutaraldehydeor succinyl tyrosine-treated actin, respectively. Therefore, the iodinated, high molecular weight polymers prepared with polyglutaraldehyde (fraction 15 of Figs. 1 and 4) were used for competition assays. We first tested the binding of this antigen to different immune sera, which had all given negative results when assayed with *251-labeled G-actin. All sera reacted with the labeled glutaraldehyde derivative (Fig. 5). A low level of binding was also observed with preimmune serum, suggesting that antibodies recognizing polyglutaraldehyde-linked actin were present before immunization; this is consistent with recent observations on the natural occurrence of antibodies to cytoskeletal proteins (Karsenti et al., 1977). However, at the dilution of the immune sera used for competition assays (l/ 1000),little binding by preimmune serum was observed. In order to investigate the reactivity of the

1

1

lOJ

18

J

DILUTIONS Fig. 5. Reactivity of sera from rabbits immunized according with two different protocols iodinated to polyglutaraldehyde-linked actin polymer. Labeled antigen (45,000 counts/min) was incubated with dilutions of sera from rabbits immunized with skeletal muscle actin as described by Lazarides (1976~) (0) or by Jockusch et al. (1978) (0). The radioactivity bound by preimmune serum is represented by the closed triangles. The sera of the remaining nine immunized rabbits were tested at a single dilution (l/IO); eight of them bound significantly more radioactivity than the preimmune serum.

antisera towards underivatized actin, experiments were carried out in which unlabeled Gand F-actin were used as competitors for binding of iodinated, polyglutaraldehyde-linked actin polymers to an anti-actin serum. For this study, a well-characterized serum (rabbit VII) with high affinity for the iodinated polymer was selected. As expected, the unlabeled synthetic polymer was an effective competitor, around 1 pg being required for half-displacement (Fig. 6A). The displacement curve obtained for F-actin was not very different indicating similar affinity of the antibodies for the two actin polymers. In Fig. 6B, displacement of labeled antigen by F-actin is compared with the competition obtained when dilutions of G-actin were added to the test. About twice as much G- as F-actin had to be added to attain half-maximum displacement. This result can be interpreted in two ways: either antibodies were present recognizing native, monomeric actin but with a somewhat lower affinity or else actin polymers were formed when dilutions of G-actin in low ionic strength buffer were added to the incubation mixture. In fact, the conditions of the incubation with the first

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0.2..

Fig. 6. Competition between polyglutaraldehyde-treated actin, F-actin or G-actin and iodinated actin polymer. (A) Dilutions of either unlabeled polyglutaraldehyde-treated actin (0) or F-actin (0) in saline/phosphate buffer were incubated with iodinated polyglutaraldehyde-linked actin (45,000 counts/min) and anti-actin serum (l/IO3 diluted). Final concentration of NaCl 0.1 M. (B) Dilutions of F-actin in polymerization buffer (10 mMTris-HCl, pH 7.8,0.2 mM ATP, 0.2 mMCaCI,. 0.5 mM2-mercaptoethanol, 0.1 M KCI) (0) or of G-actin in buffer G (Cl) were incubated with iodinated polymer (45,000 countsmin) and anti-actin serum (l/lo3 diluted). The final concentration of NaCl was 0.1 M in all samples. The B/T values in the absence of added competitor were obtained by adding buffer only. (C) Dilutions of G-actin in buffer G were incubated (i) for 1 hr at 37°C with iodinated conjugate (45,000 counts/mm) which had been dialysed against 10 mMTris-HCl (pH 7.8), 0.2 mM ATP, 0.2 mM CaCl, and antiserum diluted I/lo3 in the same buffer plus 2% fetal calf serum (o), (ii) for 1hr at 11 “C with iodinated conjugate (45,000 countsjmin) in saline/phosphate buffer and antiserum diluted 1/lo0 in the same buffer plus 2 9’, fetal calf serum (A). Prior to use, the serum diluted in low ionic strength buffer was centrifuged at 100,000 g for 1 hr to remove immunoglobulin aggregates which might have been formed by reducing the salt concentration. A visible precipitate was not obtained. All results are presented as the ratio of precipitated vs total radioactivity in the sample (B/T).

Specificity

antibody (1 hr at 37°C in a final concentration of 0.1 M NaCl) strongly favor the formation of F-actin (Szent-Gyorgi, 19.51). To test these possibilities, double antibody precipitation assays with G-actin as competitor were carried out in the presence of low ionic strength or at low temperature, conditions unfavorable for the Gz$F transformation. Dilutions of G-actin were incubated with labeled antigen and anti-actin serum in 10 mM Tris-HCl buffer (pH 7.8) containing 0.2 mMCaC1, and 0.2 mM ATP. Under these conditions, binding of the tracer was not reduced: a l/l000 dilution of the antiserum complexed 48% of the radioactivity in the above buffer and 42% in 0.1 M NaCl-10 mM sodium phosphate (pH 7.2). However, the apparent immunoreactivity of G-actin was abolished if the test was carried out in the low ionic strength buffer (Fig. 6~); no displacement of the labeled antigen was observed over the range of actin concentrations studied (6.7 x 1O-s-6.7 x 1O-2 g/l). Lowering the temperature has been shown to delay considerably the polymerization of monomeric actin even in the presence of salt concentrations which favor formation of F-actin (Kasai’, 1969). When competition experiments with G-actin were carried out in 0.1 A4 NaCl for 1 hr at 11°C instead of 37”C, the labeled polymer was only marginally displaced (Fig. 6C). The displacement seen under these conditions is in accord with the results of Kasai’ (1969), who showed that a small amount of Gactin polymerized during 1 hr at 11°C. Since at 11 “C binding of the labeled antigen was lowered considerably, the antiserum was diluted less (l/100) to give between 45 and 50% binding. These results clearly demonstrate that of the two physiological forms of actin, only fibrous actin was recognized by our antiserum. However, a polymeric state of actin was not the sole prerequisite for its immunoreactivity, since a large proteolytic fragment, which has lost the ability to polymerize, was also bound by the antiserum. Native G-actin can be cleaved by trypsin into a large fragment of around 34,000 daltons, called which is resistant to further actin-core, proteolytic attack (Jacobson & Rosenbusch, 1976). This large proteolytic fragment was iodinated by chloramine-T and tested for its binding to anti-actin serum. As shown in Fig. 7A, the labeled core protein was complexed by the antiserum. The immunoreactivity of iodinated actin-core protein cannot be explained by the

of Anti-actin

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A 0.3

0.2.

s

0.1

DILUTIONS

I

0

lo

t

lo3

I

8

I

lo”

ng PROTEIN ADDED

Fig. 7. Immunoreactivity of actin-core protein. (A) The large trypsin-resistant fragment obtained by tryptic degradation of G-actin was iodinated by the chloramine-T method and the reaction products fractionated on a Sephadex G 25 column. The radioactivity in the excluded peak corn&rated with unlabeled actin core-protein on sodium dodecylsulfate-polyacrylamide gels. This material (45,000 counts/min) was incubated with dilutions of anti-actin (rabbit VII, l) or preimmune (A) serum. (B) Unlabeled actin-core protein was incubated with iodinated polyglutaraldehyde-treated actin polymer (45,000 counts/min) and antiserum (dilution 1/103) in saline/phosphate buffer.

presence of polymers or aggregates in our preparation, since less than 3% of lz51-labeled actin-core protein eluted from Ultrogel columns corresponding to a molecular weight greater than 40,O~ daltons. The seemingly lower affinity of the antiserum for this antigen as compared with the glutaraldehyde-treated polymer was confirmed by competition assays (Fig. 7B). Actin-core protein competed with iodinated polyglutaraldehyde-treated actin for binding to the antiserum but the amounts of protein were about 10 times higher than the amounts of F-actin needed to compete at the 50% level. Since complete displacement was attained at high concentrations of actin-core protein, its recognition does not seem to implicate an

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DOSSETO and CHRIST0

antibody species different from the one reacting with F- or polyglutaraldehyde-linked actin. DISCUSSION

In non-muscle cells a considerable portion of the cellular actin is in the monomeric state (see Tilney, 1977 and Korn, 1978 for reviews), and it has been reported that cells fixed by consecutive treatments with formaldehyde and acetone, the most frequently employed method, retain 90% of their actin content (Herman & Pollard, 1978). Nevertheless, published pictures of immunofluorescent staining with anti-actin antibodies generally show distinct cytoskeletal structures, whereas the monomeric actin present should result in diffuse cytoplasmic staining. This apparent contradiction is readily explained by our observation that anti-actin sera react only with F- but not with G-actin. It remains to be seen, however, to which extent our results apply also to antibodies raised against actin from other sources or following other immunization protocols. The fact that 10 of 11 sera from rabbits injected with skeletal muscle actin according to two different immunization l 2SI-labeled polyglutaralprotocols bound dehyde conjugates of purified actin but no i* 51labeled G-actin indicates that preference for actin polymers may be a general property of antibodies raised against actin from striated muscle. Similarly, recent evidence indicates that human smooth-muscle antibodies react better with F- than with G-actin (Andersen et al., 1979). However, Trenchev & Holborow (1976) reported than an antiserum prepared against skeletal muscle actin reacted with G-actin. Their conclusion was based on the inhibition of actin polymerization by addition of immune serum as determined by viscosity measurements and not on radioimmunological analysis. Anti-actin antibodies have been clearly shown to bind to actin coupled to Sepharose (Herman & Pollard, 1979; Jockusch et al., 1978; Lessard et al., 1979) to polyacrylamide-agarose beads (Karsenti et al., 1978) or to plastic tubes (Kurki, 1978). It is, however, not clear whether the actin bound is in its G- or F-configuration. In fact, the conditions employed by some authors (Herman & Pollard, 1979; Jockusch et al., 1978; Kurki, 1978) favor polymerization. While this manuscript was in brief characterization of preparation, a acidantibodies raised against performic oxidized actin by enzyme-linked immunoassay has appeared (Benyamin et al., 1979). Based on a comparison of antigen binding at two different

GORIDIS

temperatures in the presence of 0.1 M KCl, these authors conclude that G- and F-actin are both recognized by their anti-actin antibodies. However, physico-chemical data (Oosawa et al., 1961) supported by theoretical considerations (Oosawa & Kasai’, 1962) indicate that, in the presence of 0.1 M KCl, actin is not in its G-configuration even below the critical concentration for polymerization. In particular, Rich & Estes (1976) have shown that at subcritical concentrations in 0.1 M KCl, actin assumes a conformation resembling that of a F-artin monomer, although viscosity measurements give no indication of polymer formation. Considering the important conformational changes known to occur in actin (Higashi & Oosawa, 1965; Stone et al., 1970; West, 1970; Murphy 1971; Lehrer & Kerwar, 1972), a difference in immunoreactivity between G- and F-actin is not surprising. However, the antibodies have been prepared not against polymeric actin, but against actin denatured by sodium dodecylsulfate or adsorbed onto alum. We interpret this to mean that in denatured and alum-precipitated actin, determinants are available which are not accessible in G-actin. One could argue that a polyvalent antibody molecule shows higher affinity for a polymer due to binding at multiple sites. We deem this rather unlikely, since the antibodies bound the protease-resistant actin-core protein which has lost its ability to polymerize. Hence, actin-core may share antigenic determinants with F-actin which are masked in the intact monomer. The puzzling fact remains that the antiserum bound both a synthetic polymer prepared from G-actin and native F-actin with approximately the same affinity. Antigenic sites not available on native G-actin may be unmasked upon treating monomeric actin by polyglutaraldehyde, although direct evidence for that is lacking. Iodinated synthetic oligomers of greater size were more readily bound. This can be explained by assuming that not all of the monomers are in the antigenic configuration and that antigenic determinants are distributed along the polymers in a statistical manner. Hence, the probability of containing at least one antigenic monomer increases with increasing length of the oligomers. The presence of one antigenic site in the molecule would then be sufficient for antibody binding and precipitation by the second antibody. Interestingly, a similar preference of an antitubulin antibody for polymeric tubulin has been reported (Van de Water & Olmsted, 1978).

Specificity of Anti-actin

This specificity of antibodies directed against cytoskeletal proteins for the polymers may be related to the autoimmune character of such immun~ations. Of the two physiolo~c~ forms of actin, F-actin may be localized exclusively within cells, whereas monomeric actin could escape into the circulation and/or be accessible at the cell surface (Owen et al., 1978). Hence, only G-actin may be recognized as a ‘self’ molecule. Then, the immunogenicity of actin may not depend on its being denatured but on the exposure of antigenic sites not available on G-actin. Alternatively, antibodies to G-actin may be produced but they could be complexed by circuIating or surface-expose G-actin. In any case, studies of the precise specificity of antibodies directed against autologous molecules and of the immunogenicity of highly conserved proteins may lead to new insights into the problem of immunological tolerance. The radioimmunolo~cal method described using iodinated polyglutaraldehyde-linked actin polymers as labeled antigen is suitable for the quantitation of anti-actin antibodies. In principle, this assay can detect all immunoglobulin classes of different species provided that a second antibody of the proper specificity is used. By contrast, the use of protein A-linked assays (Lessard et al., 1979) is limited by the restricted specificity of this reagent (Goding, 1978). It is interesting to note that actin iodinated by chloramine-T, which contained no more than 1 mol lzsI per mol actin, had lost its ability to polymerize. In fact, as shown by Elzinga & Collins (1973) modification of a single tyrosine residue is sufficient to inhibit actin polymerization. We have shown that anti-muscle actin antibodies did not bind G-actin under conditions preventing polymerization. F-actin, however, competed for binding of labeled antigen. The use of au immunoassay for determining actin has been advocated (Lessard er ai., 1979). Our results indicate the need for caution in interpreting such assays as long as the forms of actin which bind to and compete for the antibody have not been determined. Since our antibodies differentiate between F- and G-actin, they may be useful as sensitive probes of actin conformation. Concentrations of F-actin as low as 0.6 pg/ml produced half-displacement of labeled antigen (Fig. 6B), a sensitivity which cannot be attained by physico-chemical methods. The selectivity of the antisera most probably reflects the exposure of antigenic determinants on F-actin which are

1229

masked in the monomeric form. This implies that small oligomers of F-actin which are difficult to detect using current methodology are recognized by the antibodies. In accord with this conclusion, synthetic iodinated oligomers with a molecular weight of actin tetramers were clearly bound by the antiserum. These data are currently being extended to determine the affinity of the antibodies for actin oligomers of various size ranges. Small strands of F-actin may play an important role as nucleation centers for actin polymerization in various in vivo and in vitro systems. For example, it has been suggested (Cohen et af., 197X) that red cell ghosts possess sites for actin polymerization nucleation consisting of small amounts of fragments of Factin, but evidence for this is lacking. The study of this and similar questions may be facilitated by the use of antisera selectively recognizing Factin. Acknowledgements-We thank M. A. Delaage for continuing interest in this work and for preparing succinyl tyrosine, F. M. Kourilsky for carefully reading manuscript and E. Baechler and M. Hirsch for help with cell culture and immunofluorescence techniques.

his the the the

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