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Interaction of immunoglobulin with actin
Immunologic. Vol Press Ltd. 1979.
16. pp. 881-888. Prmted in Great
Bntam
INTERACTION MARCUS Department
OF IMMUNOGLOBULIN FECHHEIMER,
of Biology,
The Johns
JOHN L. DABS
WITH ACTIN*?and JOHN J. CEBRA
Hopkins University, Charles MD 21218, U.S.A. (Received
and 34th Streets,
Baltimore,
I March 1979)
Abstract-Actin can form specific, direct associations with immunoglobulin resulting in soluble complexes or cross-linked matrices. This interaction can be detected by four in vitro assays using purified components: (1) actin enhances the cytophilic activity of guinea pig IgG2; (2) in solutions of low ionic strength, actin and IgG2 co-precipitate; (3) soluble complexes exist in 0.1 M KC1 as revealed by the displacement of actin from its expected sedimentation pattern in a gradient of sucrose when in the presence of IgGl, IgG2, or IgM; (4) by crossimmunoglobulin (IgGl, IgG2, BGG)$ increases the viscosity of F-actin solutions, presumably linking F-actin filaments. These data suggest that direct interaction of a cytoskeletal protein with a cell surface receptor is possible.
INTRODUCTION
electrophoresis and peptide mapping indicates that the ubiquitous, unexpected component is actin (Jones, 1977; Delovitch et al., 1979). Second, fluorochrome-labelled antisera directed against immunoglobulin and actin have been used to show that actin redistributes to the region of B lymphocytes containing patches or caps of cross-linked immunoglobulin molecules (Bourguignon & Singer, 1977; Gabbiani et al., 1977). Some evidence suggests that thiscapacity to interact with actin is a special property of membrane immunoglobulin not shared by most other membrane proteins (Braun et al., 1978a,b). Third, an increased amount of cell surface immunoglobulin becomes associated with actin when myeloma cells are treated with bivalent antibody against their cell surface immunoglobulin (Flanagan & Koch, 1978). None of the experiments cited above allows one to determine whether the interaction is direct, or is mediated by intervention of ‘linker’ proteins. Thus, we have investigated the interaction of actin with immunoglobulin in vitro, and have demonstrated a direct interaction of these two proteins in four assay systems.
Interest in the interaction of cytoskeletal elements with membrane components has been stimulated by efforts to understand regulation of the distribution and lateral mobility of receptors, the mechanisms by which signals received at the cell surface may be transmitted to the cytoplasm, and the manner in which contractile proteins in the cytoplasm mediate changes in cell shape and motility. Actin-immunoglobulin interaction is one of few specific cytoskeleton membrane interactions which has been component subjected to analysis. Three experimental observations support the hypothesis that actin can interact either directly or indirectly with immunoglobulin both associated with cells and in cell extracts. First, a 45,000 dalton molecular weight protein contaminates immunoprecipitates formed by cell-derived antigens reacting with antisera having a variety of specificities from many species. Analysis by both two dimensional gel
*This work was supported by grants from the National Science Foundation (GB-38798), National Institute of Allergy and Infectious Diseases (AI-09652) and the DuPont Corporation. Two of us (M.F. and J. L. D.) have been supported as pre-doctoral trainees by a training grant from the National Institutes of Health (GM00057). t Some of this work appeared in a preliminary form in Cebra et al. (1977). $ Abbreviations used: G-Actin, globular actin; F-Actin, filamentous actin; BGG, bovine gamma globulin; BSA, bovine serum albumin; PEC, peritoneal exudate cells; DNP, dinitrophenyl; DTT, dithiothreitol; DDAO, di-DNPdiaminooctane; SDS, sodium dodecyl sulfate; ATP, adenosine tri-phosphate; HEPES, N-2hydroxyethylpiperazine-N’-2-ethanesulfonic acid.
MATERIALS AND METHODS Buffers
Phosphate-buffered saline without Ca2 + and MgZC (PBS) (Dulbecco & Vogt, 1954; Trisbuffered KC1 (TK): 0.01 M Tris (Sigma 7-9, Sigma Chemical Co., St. Louis, MO), 0.1 M KCl, pH 7.5; G actin buffer: (adapted from Spudich & Watt, 1971), 2 mM Tris, 0.2 mM ATP (Grade II, 881
L. DAlSS
Sigma). 0.2 mM CaCI,, 0.5 mA4 DTT (Sigma), pH 8.0, were used as indicated in Results. Proteins
Actin was isolated from rabbit skeletal muscle as described by Spudich and Watt ( 197 1). Purity was assessed by polyacrylamide gel electrophoresis in the presence of SDS according to Stephens (1975). The actin was sterilized by filtration and stored in G buffer at 4°C. All guinea pig IgGl, lgG2 and fragments thereof were anti-dinitrophenyl (anti-DNP) antibodies. IgGl and IgG2 anti-DNP antibodies were isolated essentially according to Oliveira et al. (1970). Purity was assessed by gel electrophoresis and by immunoelectrophoresis using anti-guinea pig Ig antisera. Small amounts of IgG2 in the IgGl were removed by passage over a column of goat anti-guinea pig IgG2 pFc’ conjugated to Sepharose 4B (Pharmacia Fine Chemicals, Piscataway, NJ). Guinea pig IgM, isolated from normal serum essentially by the method of Leslie and Cohen (1970), was the generous gift of Mr. William Ohriner. These proteins were stored at -20°C in PBS. Protein concentrations were determined spectrophotometrically using E&, for actin, IgG 1 and lgG2 as 10.9, 15.0 and 14.0, respectively (Rees & Young, 1967; Leslie & Cohen, 1970). Fab, F(ab’),. and the Fc fragments of guinea pig IgG2 anti-DNP antibody were prepared as described by Leslie et cd. (1971), except that the different cleavage products comprising the Fc fragments were not separated. Columns of DNPBSA or goat anti-guinea pig lgG2 pFc’coupled to Sepharose 4B were used to remove minor contaminants in the preparations of Fc and Fab, respectively. Bovine serum albumin (Cohn Fraction V) and bovine gamma globulin (Cohn Fraction II) were from Sigma. Egg albumin (2 x crystallized) was from Nutritional Biochemical Co., Cleveland, OH. All immunoglobulins were column-purified on Sepharose 6B the day before use to remove any aggregated material. Dimers of lgG2 anti-DNP were formed by mixing IgG2 (5 mg/ml) with an equimolar amount of di-DNP-diaminooctane (DDAO), and allowing the mixture to stand for l-2 days. The mixture was then fractionated on a Sepharose 6B column (2.1 x 70 cm) and the dimer fraction was taken for experiments. Proteins were iodinated by the chloramine T method (Klinman & Taylor, 1969) with 1 mCi NalzsI (Amersham Searle, carrier-free
and JOHN
J. C‘EBKA
Arlington Heights, IL), except that actin was kept in G buffer. Unincorporated rriI was removed by desalting on a column of Sephadex G-25 (Pharmacia) followed by extensive dialysis. r 2s1 was counted in a Packard Auto-Gamma Scintillation Spectrometer Model 3002. Co-precipitation
qf’ actin
nith
immunoglohulin
Since actin-immunoglobulin precipitates are labile during repeated washing. an assay which avoids the need for washing was devised using 22Na as a marker for trapped volume (adapted from Tsay & Schlamowitz, 1975). Proteins in G buffer were mixed in final volumes of 90 ~1 in 0.4 ml polypropylene centrifuge tubes (Arthur H. Philadelphia, PA), and 100,000 Thomas, Nuclear, counts/min of Z2Na (New England Boston, MA) in 10 ~1 of G buffer was added to each sample. The mixtures were allowed to stand for 2 hr at room temperature in a sealed chamber. Samples were spun for 2 min in a microfuge (Beckman Instruments, Inc., Spinco Division, Palo Alto, CA), and the pellets were counted for 12sI and 22Na. The small amount of I 251-actin or I2 sl-IgG non-specifically sticking to the tube was determined at each concentration used, and was subtracted from actin-IgG mixtures where appropriate. Mixtures of iz51-actin and cold IgG or cold actin and 12sI-IgG were measures of the and IgG precipitated, respectively. actin Mixtures of “‘I-actin and 1251-lgG measured the amount of total protein in the precipitate. Corrections for spillover between the 22Na and * 2sI channels in the gamma spectrometer were made by determining the fraction of the counts which appeared in the ‘incorrect’ channel when only 12Na or i2 5I was present. We assumed that the counting efficiencies for 22Na and izsl were constant. Real counts were calculated from the observed counts by solving two simultaneous equations. In order to determine the amount of protein in the precipitate, it was necessary to determine both the volume of trapped superthe final concentration of natant, and components in the supernatant. The amount of protein precipitated (2) is equal to the corrected l 2‘I counts/min (I,) minus the product of the trapped volume (V,) times the final protein concentration in the supernatant (P,). z = IR_T/lP/ The value of V, is calculated by dividing the corrected 22Na countslmin by the concentration
Actin-Immunoglobulin
of 22Na. The value of Ps may be calculated algebraically or determined by measuring the protein concentration in the supernatant at the end of the experiment. The values of Z, converted to pg, are the values reported in the text and figures. Sucrose gradients One volume of 1251-actin in G buffer was mixed with two volumes of immunoglobulin in Tris-KC1 to yield final concentrations of 0.2 and 2.0 mg/ml, respectively. The mixture was then made 1 mg/ml in BSA and 0.1 M in KCl. 100 ~1 samples were loaded onto 3.8 ml linear gradients of 5-20’;/, sucrose in Tris-KC1 in 7/16 in. x 2-3/8 in. polyallomer tubes (Beckman Instruments), and overlaid with mineral oil. Centrifugation was performed at 265,000 g for 9 hr at 4°C in an SW-60 rotor. Gradients were fractionated by puncturing the bottoms of the tubes, and the O.lml fractions were counted for lzsI counts/min. To facilitate comparison of results from different gradients, data are expressed as the per cent of the recovered radioactivity present in each fraction. Binding of IgG2 to Fc receptors on guinea pig peritoneal exudate cells. Peritoneal exudate cells (PEC) were obtained by peritoneal lavage of strain 13 guinea pigs 4-5 days after injection of 20 ml sterile mineral oil (Ampak, American Quinine, Plainview, NJ) intraperitoneally. After hypotonic lysis of red blood cells, PEC were washed twice with Hank’s Buffered Saline Solution (Grand Island Biological Co., Grand Island, NY), incubated for 1 hr at 37°C in RPM1 1640 (GIBCO), and resuspended at 2.5 x 10’ cells/ml in RPM1 1640 supplemented with l:/, BSA, 0.04% NaN, and 13 mM HEPES (Sigma), pH 7.4. These cells were greater than 95:/, viable and about 70”/, macrophages. 100 ~1 of PEC were added to monomeric l 251IgG2 in 100 ~1 PBS plus 50 ~1 of G buffer with or without actin. After a 90-minute incubation at 4°C with intermittent shaking, 200 ~1 samples were removed. Cells bearing Fc receptor-bound IgG2 were separated from unbound IgG2 by the method of Segal and Hurwitz (1977). Low shear viscometry Interaction of actin with immunoglobulin was demonstrated by falling ball viscometry in G buffer containing 0.1 M KC1 (Griffith & Pollard, 1978). Actin (120 ~1) in G buffer plus 180 ~1
883
Interaction
immunoglobulin in G buffer containing 0.167 M KC1 were mixed and immediately drawn into 100 ~1 micropipets (Van Lab, VWR Scientific, Baltimore, MD). These were plugged with clay and placed in a 25°C bath. Viscosity was determined by placing each sample on a ramp in a 25°C water bath inclined either 50” or 80” from the horizontal. The time for an 0.64-mm steel ball (Microball Co., Peterborough, NH) to roll through the mixture is directly proportional to absolute viscosity. The fall time was determined in two 4-cm intervals during the descent of a ball in each capillary. Duplicate capillaries were prepared for each sample. Values reported are the means of 4 measurements of which the range was about 109% of the mean.
RESULTS
Effect of actin on the binding of IgG2 to macrophage Fc receptors. We first detected actin-immunoglobulin interaction by testing the effect of actin on the binding of IgG2 to peritoneal macrophages. Increasing concentrations of actin were added to guinea pig PEC and a concentration of 1251IgG2 sufficient to bind 50% of the Fc receptors on the cells. The result was monotonic
Fig. 1. Scatchard plots of the binding of 1251-lgG2 to Fc receptors of peritoneal exudate cells in the presence or absence of actin. Each tube contained IgG2 at the indicated concentration, 4 mg/ml BSA, and 10’ cells/ml in the presence or absence of actin at a final volume of 0.25 ml. (0) IgG2 alone; (0) IgG2 plus 0.4 mg/ml actin. The lines drawn on the figure were derived by linear regression analysis of the data. r, Concentration of cell-associated IgG2 in pg/ml; c, concentration of free IgG2 in pg/ml; n, the number of receptors per macrophage; KaPP, the apparent association constant of ligand for receptor in I/mol.
884
MARCUS
FECHHEIMER,
JOHN
Molar
Ratio
L DAIS ActIn
and JOHN
J. CEBRA
/ Ig GZ 6.0 16.8
8.7 14.3 ActIn+IgG~(meosured)
70’ 65 60 -
[ ActIn] Fig. Z Co-precipitation
performed
of’actin
*
mg /ml
ofactln and IgG2 mixed in different proportions at low ionic strength. in G buffer at pH 8.0. The concentration of IgG2 was 0.5 mg/ml.
enhancement of the binding of lgG2 as the concentration of actin was increased from 25-800 pg/ml. In order to define this more precisely, increasing phenomenon concentrations of 12jI-IgG2 were mixed with PEC in the presence or absence of actin. Scatchard plots of the results of this experiment. shown in Fig. 1, demonstrate that actin increases the apparent number of Fc receptors on the cells increase in by 25”‘, with an accompanying affinity. To determine whether apparent enhancement of IgG2 binding is due to fluid phase complex formation or to a direct effect of actin on the cells, the binding of actin by itself to PEC was measured. In these experiments we did not detect any specific binding of actin to cells (data not shown). The enhancement ofcytophilic activity of IgG2 in the presence of actin can be most easily explained by binding to the cells of complexes formed in the fluid phase. Co-prrcipitation
1npui ppt
and
IgC2
at low
ionic
strength
Preliminary experiments attempting to define a soluble complex in mixtures of actin and IgG2 were performed in G buffer with the hope of maintaining actin in a monomeric state. Analyses of these mixtures in the Model E ultracentrifuge showed that actin and IgG2 formed discrete 4s and 7s peaks, respectively (data not shown). Although no small complexes were apparent, a substantial quantity of material sedimented to the bottom of the cell in only a few minutes. This was never observed during the sedimentation of either component alone. When
Assays
the mixtures were allowed to stand for a few hours. visible precipitates formed. A co-precipitation assay employing 12jIlabelled proteins and 22Na as a trapped volume marker was devised to measure the contribution of each protein to the precipitate. Figure 2 shows the composition of the precipitate when a fixed amount of IgG2 was mixed with increasing concentrations of actin. The similarity of the curves for actin plus IgG2 determined separately or together demonstrates that unlabelled and labelled proteins behave identically. The precipitates contain more than 50”” of each protein present. As the concentration of actin is increased, the quantity of lgG2 precipitated increases and then decreases, suggesting that cross-linking is occurring. The molar ratio of actinjIgG2 at the point of maximal precipitation of IgG2 is 1.8. The interaction is specific since no coprecipitation occurs between actin and BSA, or IgG2 and ovalbumin. Co-precipitation is readily reversible by dilution or by increasing the ionic strength. We observed 95:; inhibition with 0.15 MKCI, and complete inhibition with 0.5 MKCl. Since lgG2 anti-DNP is a basic protein and actin is acidic, it seemed possible that coprecipitation at pH 8.0 at low ionic strength may reflect an electrostatic interaction of components of opposite charge. Figure 3 shows the effect of pH on the amount of IgG2, IgGl and F(ab’)z of lgG2 coprecipitated with actin. Precipitation of IgG2 is enhanced at acid pH reaching 95”, at pH 6.4. IgGl and F(ab’), of IgG2 show no and little precipitation, respectively, even at pH values
~ctia-~mmunoglobul~n
,
<
I
,
Interaction
X85
at pH 7.3 and 8.6, respectively, and this precipitation was totally inhibited by added aetin. The existence of soluble complexes suggests that actin-Ig~o-pre~jp~tation is at least a two-step process. Thus, co-precipitation is not necessarily a direct measure of binding.
Sedimentation analysts in gr~d~e~rs of sucrose was employed to demonstrate soluble compiexes of actin and IgG in 0.1 M KCI. The existence of such complexes was suggested by results of Fc receptor titrations and the co-pr~c~pita~io~ assay. Figure 4 shows sucrose gradient profiles of Fig. 3. Effect of pW on co”~r~ip~tati~~ of actin with IgG 1) radioIabeII~d actin in the presence or absence of lgG2 and F(ab’f, offgG2. (IL?)I&l; (0) 1gG2; {A, F(ab’), of IgG2. Mixtures contained 0.5 mg/ml of each protein IgG2. IgG-2 causes a displacement into more present. Since ionic strength affects co-pr~~~itation, the rapidly sedimenting protein of about 10% of the couducti~t~ of each G buffer solution was adjusted to 160 actin with a c~~~ornitant diminution ofthe peak pmho with NaCf after titration to the proper pH. of monomeric actin, IgGI, IgG2, dimeric JgG2 below their isoelectric points. Since an and IgM were all effective in causing actin appreciably fraction of the actin precipitated in displacem~~~t. The shape of the gradient profiles tubes containing actin alone at pW 5.8, the suggest that actin may dissociate from IgG sedimentation. The actin itself increased precipitation of F(ab’), and IgGt at during this PI-I may be due to a different sort of polymerized if higher concentrations (2 rng~rn1~ interaction. These results demonstrate that co- were used, and the F-a&n induced a down~eld precipitation is not governed simply by the net displacement of IgG2. The foilowing speciality charges of the interacting proteins, and that it is controls were performed: BSA did not induce a down~eld djsplacement of actin; lgC2 did not apparently F~-mediated. The preserve of sohtbie complexes in this induce a dow~~eld djsplacement of BSA; an system is suggested both by the immuno- excess of u~iab~Il~d BSA did not inhibit the precipitation-like nature of the curve in Fig. 1, displacement of 12sf-actin by IgG2, and by the fact that precipitation of I ~~~~1~~~~~~~~ of actin-immlmo~1~hlllin itself was blocked by the presence of actin. In one interaction by ~is~~rn~tr~ experiment, 23 and 34% of the IgGl precipitated Viscometry was employed to study act~n-immunogiobuli~ i~teractj~n because potentially interacting components do not have to be separated in the process of aI~~Iyzing a mixture. PreIimi~ary experiments using au ~stwald-type viscometer showed that IgG 1, IgGZand BGG all increased the specific viscosity of actin solutions by 400/,, while BSA had no effect (data not shown). Repeated measurement of the same sample resulted in a decay of the viscosity toward values observed for actin alone indicating djsruption of the interaction by high shear forces. Thus, the method of low shear falling ball vi~~metry described by Gr~f~tb and Pollard (1978) was employed in subsequent experiments. luring such measnreme~ts, the Froctlon Number solution itself remains static and each sample is Fig. 4. 3i~din~ of actin to immuno~obuli~ on sucrose only subjected to a single evaIuation. gradients, (1) 1251-actin alone; (0) i2s1-actin plus XgGZ. mixtures of actin and immunoglobui~~ were Samples of 0. t ml contained 2 mg/ml XgG2and 0.2 mg]ml of ‘“%actin. held for 24 hours at 25°C before the viscosity was
886
MARCUS
\ ii
I
301 / I /
/
/ /
FECHHEIMER.
/
JOHN
I I
Fig. 5. Interaction of actin with IgGl, IgG2, Fab and Fc of lgG2, and BSA measured by falling ball viscometry. (0) IgG 1; (0) IgG2; ( x ) Fab of IgG2; (+) Fc of IgG2: (0) BSA. The concentration of actin was 0.73 mgiml. Since fall times for all immunoglobulin solutions an; BSA at the highest concentration used were less than 0.5 set/cm, the viscosity of mixtures are significantly greater than the sum of the viscosities of the components.
since preliminary experiments determined indicated that 20-30 hours were required for stable values to be attained. Figure 5 shows the concentration dependence of enhancement of actin viscosity by IgG2, IgGl, Fab and Fc of IgG2, and BSA. IgGl and IgG2 increase the viscosity equally well, possibly by cross-linking actin filaments. Fab, Fc and BSA induce modest increases in viscosity. At higher concentrations (20 @4), the fragments of immunoglobulin were slightly more effective than BSA at enhancing actin viscosity (data not shown). These other proteins and fragments may act by nonspecifically stabilizing actin, since the viscosity of actin alone was frequently observed to decrease
I
I 140-
100 -
.67 Concentration
I.33
, 2.0
of Added
2.67 IqG2
3.33 LpM)
Fig. 6. Interaction of actin with monomeric or dimeric IgG2 measured by falling ball viscometry. (0) Monomeric lgG2; (0) dimeric IgG2. The concentration of actin was 0.63 mg/ml. the unit of concentration on the abcissa is moles Fe/l, so that the monomer and dimer may be directly compared on a weight basis.
L. DAK?
and JOHN
J. CEBRA
from its peak value by 20-go”,, after 20-30 hours of incubation. Dimers of IgG2 prepared with DDAO are up to four times more effective than monomeric IgG2 at enhancing actin viscosity as shoK;n in Fig. 6. It is not clear whether this reflects a higher avidity of the dimeric IgG2 for actin. or a higher efficiency of cross-linking. The break in the concentration curves between 0.3 and 1.5 ,uM was observed reproducibly for IgGl, IgG2 and IgG2 dimer, suggesting that more than one type of interaction may be occurring.
DISCUSSION
Evidence from four in vitro assay systems demonstrates a direct and specific interaction between actin and immunoglobulin. Since all of the interacting proteins were highly purified, the fraction of molecules interacting is larger than may be attributed to a possible contaminant. This extensive participation is most dramatically illustrated by the fact that 30 and 95”” of the IgG2 is co-precipitated with actin at pH 8.0 and 6.4, respectively. Interaction with actin seems to be a general property of immunoglobulin, since guinea pig IgGl, IgG2 and IgM, and bovine IgG have all been shown to be competent. The interaction is limited, and hence specific, since substitution of unrelated proteins for either actin or immunoglobulin in each of the assay systems resulted in lack of reactivity. Actin and immunoglobulin are competent to form soluble complexes in buffers of both low and physiologic ionic strength. The presence of complexes at low ionic strength may be inferred both from the fact that the amount of IgG2 coprecipitating with actin goes down with increasing actin concentration, and that actin apparently increases the solubility of IgGl in solutions of low salt concentration. Complexes of actin and immunoglobulin at physiologic ionic strength were demonstrated directly in sucrose gradient experiments, and indirectly by Fc receptor titrations and viscometry. Both filamentous and non-filamentous forms of actin were found to be capable of interaction with immunoglobulin. The fact that co-precipitation is salt labile, while binding of actin and immunoglobulin is not, suggests that the salt inhibits a step subsequent to simple binding. A second piece of evidence which distinguishes binding from
Actin-lmmunoglobulin
precipitation is that although both lgG1 and IgG2 bind actin, only IgG2 co-precipitates. This qualitative distinction between the two isotypes of guinea pig IgG may reflect differences either in hydrodynamic behaviour, such as in their segmental flexibility (Cebra ef al., 1977) or in stable structural features of the molecules. It is interesting to note that actinimmunoglobulin precipitation is similar to euglobulin precipitation in its salt lability and concentration dependence. Although it is not clear that the two processes are mediated by similar molecular interactions, one may predict that small amounts of actin released into serum during clot retraction would modulate the amount of IgG in the euglobulin fraction of that serum. The binding of actin to monomeric immunoglobulin appears to be of low affinity, since the appearance of sucrose gradient profiles suggests that the actin which is displaced downfield may have dissociated from complexes with immunoglobulin. Viscometry experiments show that dimeric IgG2 interacts more efficiently with actin than does monomer. It is tempting to speculate from this result that the free and reversible interaction of actin with monomeric immunoglobulin in cells may take on a more concrete nature upon cross-linking of the immunoglobulin by exogenous ligands. Efforts to demonstrate that actinimmunoglobulin interaction is mediated by a specific domain of the immunoglobulin molecule have not been entirely successful. The low effectiveness of both Fab and Fc in the enhancement of actin viscosity may be attributed to damage to the actin binding site during proteolytic cleavage, ability to bind but not cross-link actin filaments, or a genuine absence of the actin binding site. Further, the failure of F(ab’), to form a co-precipitate with actin cannot be construed to indicate Fc-specificity of the interaction, since lack of co-precipitation does not demonstrate lack of binding. Immune precipitates formed with DNP-BSA and F(ab’), of IgG2 anti-DNP in the presence of actin consistently failed to bind actin. Although similar immune precipitates formed with the parent molecule sometimes bound actin to an appreciable extent, this association was not consistently observed (data not shown). Nonetheless, the bulk of available evidence indicates that the ubiquitous unexpected component is indeed actin (Jones, 1977; Delovitch et al., 1979). As noted above, the
Interaction
887
lability of actin-Ig coprecipitation in low salt buffers to salt, dilution or high pH may reflect the inhibition of an interaction secondary to the association of actin with immunoglobulin. Consequently, our results suggest no simple means for elimination of actin contamination of specific immune precipitates formed in whole cell extracts. We find that ‘pre-clearing’ with an irrelevant antisera is usually effective in reducing contamination of immune precipitates by actin. There is no evidence for the existence of direct actin-immunoglobulin interaction in cells. The appearance of actin in immunoprecipitates of antigens could result from cell surface association after cell lysis. Experiments which demonstrate that actin co-caps with immunoglobulin, and that cross-linking cell surface immunoglobulin results in increased association with actin suggest at least an indirect association. The co-capping experiments must be interpreted with caution since it is not clear that a large fraction of the cellular actin is present and available for staining by antibody after fixation. The possibility that the shape changes which accompany capping are responsible for the observed staining patterns must also be considered. Models of actin-immunoglobulin interaction may be divided into those in which association occurs on the cytoplasmic face of the membrane, on the outside of the cell, or in the plane of the membrane. These models are not mutually exclusive, and are all compatible with either direct or indirect association of actin with immunoglobulin. Evidence that actin may be present on the surface of B lymphocytes (Owen et al., 1978) lends plausibility to the notion that actinimmunoglobulin interaction may occur on the outside of the cell. In such an interaction, both actin and/or immunoglobulin could be either endogenously or exogenously derived. A possible function of such an interaction is that the actin acts as a co-receptor to stabilize the interaction of immunoglobulin with protein or lipid receptors in the membrane. Actin-immunoglobulin interaction in the plane of the membrane or on the cytoplasmic face of the membrane could function in membrane -+ cytoplasm or cytoplasm + membrane communication. Differentiation between these alternatives will be possible only if precise information becomes available concerning the distribution of these proteins in the membrane and the existence of a linker molecule.
X88
MAKC’US
IEC‘HHEIMER,
JOHN
The present experiments demonstrate that a direct interaction of a cytoskeletal protein with a cell surface receptor is possible. In the future, it will be of interest to determine whether cytoskeleton-receptor interactions occur in cells, whether they are direct or mediated by linker proteins, and whether these interactions are associated with any specific functions in the cell. Ackrzo~~~lrdgrmmts~We thank Drs. S. MacLean and T. Pollard for instruction in low shear viscometry and for their gift of 0.64-mm diameter stainless steel balls. We thank Dr. V. Pigiet for use of his facilities for analytical and preparative ultracentrifugation.
REFERENCES Bourguignon L. Y. W. &Singer S. J. (1977) Transmembrane interactions and the mechanism of capping of surface receptors by their specific ligands. Proc. natn. Acad. Sci. U.S.A. 74, 5031-5035. Braun J., Fujiwara K., Pollard T. D. & Unanue E. R. (1978~) Two distinct mechanisms for redistribution of lymphocyte surface macromolecules. I. Relationship to cytoplasmic myosin. J. C&l Biol. 78, 409-418. Braun J.. Fuiiwara K.. Pollard T. D. & Unanue E. R. (19786) Two distinct mechanisms for redistribution of lymphocyte surface macromolecules. II. Contrasting effects of local anesthetics and a calcium ionophore. J. CeN Biol. 78, 419426. Cebra .I. J., Brunhouse R., Cordle C.. Daiss J.. Fechheimer M., Richard0 M., Thunberg A. & Wolfe P. B. (1977) Isotypes of guinea pig antibodies: Restricted expression and bases for interactions with other molecules. In Progress in Immunology I11 (Edited by Mandel T. E., Cheers C., Hosking C. S., McKenzie I. F. C. & Nossal G. J. V.) pp. 269-277. Elsevier/North Holland, New York. Delovitch T. L.. Fegelman A., Barber B. H. & Frelinger J. A. (1979) Immunochemical characterizatton of the Ly X.2 murine lymphocyte alloantigen: Possible relationship to actin. J. Immun. 122, 326-333. Dulbecco R. & Vogt M. (1954) Plaque formation and isolation of pure lines with poliomyelitis viruses. J. r’.up. Med. 99, 167-182.
L. DAISS and JOHN
J. C’EBK,.‘!
Flanagan J. & Koch G. L. t,. (I Y7X) C‘ross-linked \urt;rce lg attaches to actin. .YNII~I.~~. Lo& 273. 27X-2x I. Gabbrani G.. Chappomcr C’.. Lumbe A. & Vassal11 P. (107;) Actin and tubulin co-cap with surface rmmunoglobultn~ in mouse B lymphocytes. ,!v’cr/rrrc,.L,,,rt/. 269. hY7-6YX Griffith L. M. & Pollard T. D. (lY7X) Evtdcnce 1’01 actm filament-microtubule interaction mcdiatcd h\ mitt-otuhulc associated proterns. J. (‘c/i Broi. 78, Y5X-Yhj Jones P. P. (1977) Analysrs of H-2 and la molecule\ by two dimensional gel electrophoresis. ./. “\,I, l-lc,/ 146, 3261-1279. Klinman N. R. & Taylor R. B t 1969) General methods forthe study of cells and serum during the immune response: The response to dinitrophenyl in mice. C‘lirr. c’.\,r. /~r/,rw,~. 4.473-487. Leslie R. G. Q. & Cohen S. (19701 C‘hemical properties 01 guinea-pig immunoglobulins ;,,G, ;‘,G. and ;,M H,oc/~c~,rl. J. 120, 787-795. Leslie R. G. Q., Melamed M. D. & Cohen S. ( lY7l ) The products from papain and pepsin hydrolyses ofguinea-ptg immunoglobulins ;,,G and ;,,G. Bioc,/,ejn. J. 121. X19-X37. Oliveira B.. Osler A. G.. Siraganian R. P & Sandhcr-g A L. (1970) The biologic activiticr of guinea pig antrbodics. I Separation of ;‘, and ;‘l nnmunoglobulin~ and thcit participation in allergic reactions of the tmmediate type. .I Iw7tmm. 104, 32&328. Owen M. J.. Auger J.. Barber B. H.. Ldwat-ds A J.. Wal\h F. S. & Crumpton M. J. t lY781 Actin may he present on the lymphocyte surface. Proc,. wm. .Awd ,SC1. I ..S. I. 75. 44844488. Rees M. K. & Young M. (lY67) Studies on the rsolation and molecular properties of homogeneous globular actrn .I hiol. C‘hcw. 242, 4449-445X. Segal D. M. & Hurwrtz E. (lY77) Binding of altin~ry ct-asslinked oligomers of IgG to cells hearing Ic rcccptors. .I I~?Zmun. 118, 1338-1347 Spudich J. A. & Watt S. I 1971) The regulatron 01 trnhbrt skeletal muscle contraction. I. Biochemtcal studies of the interaction of the tropomyostn-troponin cornpIe\ with actin and the protcolytic tragments of myosin. .I /JIM/ Chcm. 246, 486&4X73, Stephens R. E. (1975) Hugh-resolutton preparative SI>Spolyacrylamide gel electrophoresis: Fluorescent visuals/ation and electrophoretic elution-concentration ofpt-otetn bands. Anrrl~t. Bioc~/wrn.65, 369%37’). Tsay D. D. & Schlamowitz M. (1975) Comparison of the binding affinities of rabbit IgG fractions to the rabbit fetal yolk sac membrane: llse of”Na to facilrtate quantitatinn of rZ51-IgG binding .I. Immir,r. I IS, Y3Y-943.
16. pp. 881-888. Prmted in Great
Bntam
INTERACTION MARCUS Department
OF IMMUNOGLOBULIN FECHHEIMER,
of Biology,
The Johns
JOHN L. DABS
WITH ACTIN*?and JOHN J. CEBRA
Hopkins University, Charles MD 21218, U.S.A. (Received
and 34th Streets,
Baltimore,
I March 1979)
Abstract-Actin can form specific, direct associations with immunoglobulin resulting in soluble complexes or cross-linked matrices. This interaction can be detected by four in vitro assays using purified components: (1) actin enhances the cytophilic activity of guinea pig IgG2; (2) in solutions of low ionic strength, actin and IgG2 co-precipitate; (3) soluble complexes exist in 0.1 M KC1 as revealed by the displacement of actin from its expected sedimentation pattern in a gradient of sucrose when in the presence of IgGl, IgG2, or IgM; (4) by crossimmunoglobulin (IgGl, IgG2, BGG)$ increases the viscosity of F-actin solutions, presumably linking F-actin filaments. These data suggest that direct interaction of a cytoskeletal protein with a cell surface receptor is possible.
INTRODUCTION
electrophoresis and peptide mapping indicates that the ubiquitous, unexpected component is actin (Jones, 1977; Delovitch et al., 1979). Second, fluorochrome-labelled antisera directed against immunoglobulin and actin have been used to show that actin redistributes to the region of B lymphocytes containing patches or caps of cross-linked immunoglobulin molecules (Bourguignon & Singer, 1977; Gabbiani et al., 1977). Some evidence suggests that thiscapacity to interact with actin is a special property of membrane immunoglobulin not shared by most other membrane proteins (Braun et al., 1978a,b). Third, an increased amount of cell surface immunoglobulin becomes associated with actin when myeloma cells are treated with bivalent antibody against their cell surface immunoglobulin (Flanagan & Koch, 1978). None of the experiments cited above allows one to determine whether the interaction is direct, or is mediated by intervention of ‘linker’ proteins. Thus, we have investigated the interaction of actin with immunoglobulin in vitro, and have demonstrated a direct interaction of these two proteins in four assay systems.
Interest in the interaction of cytoskeletal elements with membrane components has been stimulated by efforts to understand regulation of the distribution and lateral mobility of receptors, the mechanisms by which signals received at the cell surface may be transmitted to the cytoplasm, and the manner in which contractile proteins in the cytoplasm mediate changes in cell shape and motility. Actin-immunoglobulin interaction is one of few specific cytoskeleton membrane interactions which has been component subjected to analysis. Three experimental observations support the hypothesis that actin can interact either directly or indirectly with immunoglobulin both associated with cells and in cell extracts. First, a 45,000 dalton molecular weight protein contaminates immunoprecipitates formed by cell-derived antigens reacting with antisera having a variety of specificities from many species. Analysis by both two dimensional gel
*This work was supported by grants from the National Science Foundation (GB-38798), National Institute of Allergy and Infectious Diseases (AI-09652) and the DuPont Corporation. Two of us (M.F. and J. L. D.) have been supported as pre-doctoral trainees by a training grant from the National Institutes of Health (GM00057). t Some of this work appeared in a preliminary form in Cebra et al. (1977). $ Abbreviations used: G-Actin, globular actin; F-Actin, filamentous actin; BGG, bovine gamma globulin; BSA, bovine serum albumin; PEC, peritoneal exudate cells; DNP, dinitrophenyl; DTT, dithiothreitol; DDAO, di-DNPdiaminooctane; SDS, sodium dodecyl sulfate; ATP, adenosine tri-phosphate; HEPES, N-2hydroxyethylpiperazine-N’-2-ethanesulfonic acid.
MATERIALS AND METHODS Buffers
Phosphate-buffered saline without Ca2 + and MgZC (PBS) (Dulbecco & Vogt, 1954; Trisbuffered KC1 (TK): 0.01 M Tris (Sigma 7-9, Sigma Chemical Co., St. Louis, MO), 0.1 M KCl, pH 7.5; G actin buffer: (adapted from Spudich & Watt, 1971), 2 mM Tris, 0.2 mM ATP (Grade II, 881
L. DAlSS
Sigma). 0.2 mM CaCI,, 0.5 mA4 DTT (Sigma), pH 8.0, were used as indicated in Results. Proteins
Actin was isolated from rabbit skeletal muscle as described by Spudich and Watt ( 197 1). Purity was assessed by polyacrylamide gel electrophoresis in the presence of SDS according to Stephens (1975). The actin was sterilized by filtration and stored in G buffer at 4°C. All guinea pig IgGl, lgG2 and fragments thereof were anti-dinitrophenyl (anti-DNP) antibodies. IgGl and IgG2 anti-DNP antibodies were isolated essentially according to Oliveira et al. (1970). Purity was assessed by gel electrophoresis and by immunoelectrophoresis using anti-guinea pig Ig antisera. Small amounts of IgG2 in the IgGl were removed by passage over a column of goat anti-guinea pig IgG2 pFc’ conjugated to Sepharose 4B (Pharmacia Fine Chemicals, Piscataway, NJ). Guinea pig IgM, isolated from normal serum essentially by the method of Leslie and Cohen (1970), was the generous gift of Mr. William Ohriner. These proteins were stored at -20°C in PBS. Protein concentrations were determined spectrophotometrically using E&, for actin, IgG 1 and lgG2 as 10.9, 15.0 and 14.0, respectively (Rees & Young, 1967; Leslie & Cohen, 1970). Fab, F(ab’),. and the Fc fragments of guinea pig IgG2 anti-DNP antibody were prepared as described by Leslie et cd. (1971), except that the different cleavage products comprising the Fc fragments were not separated. Columns of DNPBSA or goat anti-guinea pig lgG2 pFc’coupled to Sepharose 4B were used to remove minor contaminants in the preparations of Fc and Fab, respectively. Bovine serum albumin (Cohn Fraction V) and bovine gamma globulin (Cohn Fraction II) were from Sigma. Egg albumin (2 x crystallized) was from Nutritional Biochemical Co., Cleveland, OH. All immunoglobulins were column-purified on Sepharose 6B the day before use to remove any aggregated material. Dimers of lgG2 anti-DNP were formed by mixing IgG2 (5 mg/ml) with an equimolar amount of di-DNP-diaminooctane (DDAO), and allowing the mixture to stand for l-2 days. The mixture was then fractionated on a Sepharose 6B column (2.1 x 70 cm) and the dimer fraction was taken for experiments. Proteins were iodinated by the chloramine T method (Klinman & Taylor, 1969) with 1 mCi NalzsI (Amersham Searle, carrier-free
and JOHN
J. C‘EBKA
Arlington Heights, IL), except that actin was kept in G buffer. Unincorporated rriI was removed by desalting on a column of Sephadex G-25 (Pharmacia) followed by extensive dialysis. r 2s1 was counted in a Packard Auto-Gamma Scintillation Spectrometer Model 3002. Co-precipitation
qf’ actin
nith
immunoglohulin
Since actin-immunoglobulin precipitates are labile during repeated washing. an assay which avoids the need for washing was devised using 22Na as a marker for trapped volume (adapted from Tsay & Schlamowitz, 1975). Proteins in G buffer were mixed in final volumes of 90 ~1 in 0.4 ml polypropylene centrifuge tubes (Arthur H. Philadelphia, PA), and 100,000 Thomas, Nuclear, counts/min of Z2Na (New England Boston, MA) in 10 ~1 of G buffer was added to each sample. The mixtures were allowed to stand for 2 hr at room temperature in a sealed chamber. Samples were spun for 2 min in a microfuge (Beckman Instruments, Inc., Spinco Division, Palo Alto, CA), and the pellets were counted for 12sI and 22Na. The small amount of I 251-actin or I2 sl-IgG non-specifically sticking to the tube was determined at each concentration used, and was subtracted from actin-IgG mixtures where appropriate. Mixtures of iz51-actin and cold IgG or cold actin and 12sI-IgG were measures of the and IgG precipitated, respectively. actin Mixtures of “‘I-actin and 1251-lgG measured the amount of total protein in the precipitate. Corrections for spillover between the 22Na and * 2sI channels in the gamma spectrometer were made by determining the fraction of the counts which appeared in the ‘incorrect’ channel when only 12Na or i2 5I was present. We assumed that the counting efficiencies for 22Na and izsl were constant. Real counts were calculated from the observed counts by solving two simultaneous equations. In order to determine the amount of protein in the precipitate, it was necessary to determine both the volume of trapped superthe final concentration of natant, and components in the supernatant. The amount of protein precipitated (2) is equal to the corrected l 2‘I counts/min (I,) minus the product of the trapped volume (V,) times the final protein concentration in the supernatant (P,). z = IR_T/lP/ The value of V, is calculated by dividing the corrected 22Na countslmin by the concentration
Actin-Immunoglobulin
of 22Na. The value of Ps may be calculated algebraically or determined by measuring the protein concentration in the supernatant at the end of the experiment. The values of Z, converted to pg, are the values reported in the text and figures. Sucrose gradients One volume of 1251-actin in G buffer was mixed with two volumes of immunoglobulin in Tris-KC1 to yield final concentrations of 0.2 and 2.0 mg/ml, respectively. The mixture was then made 1 mg/ml in BSA and 0.1 M in KCl. 100 ~1 samples were loaded onto 3.8 ml linear gradients of 5-20’;/, sucrose in Tris-KC1 in 7/16 in. x 2-3/8 in. polyallomer tubes (Beckman Instruments), and overlaid with mineral oil. Centrifugation was performed at 265,000 g for 9 hr at 4°C in an SW-60 rotor. Gradients were fractionated by puncturing the bottoms of the tubes, and the O.lml fractions were counted for lzsI counts/min. To facilitate comparison of results from different gradients, data are expressed as the per cent of the recovered radioactivity present in each fraction. Binding of IgG2 to Fc receptors on guinea pig peritoneal exudate cells. Peritoneal exudate cells (PEC) were obtained by peritoneal lavage of strain 13 guinea pigs 4-5 days after injection of 20 ml sterile mineral oil (Ampak, American Quinine, Plainview, NJ) intraperitoneally. After hypotonic lysis of red blood cells, PEC were washed twice with Hank’s Buffered Saline Solution (Grand Island Biological Co., Grand Island, NY), incubated for 1 hr at 37°C in RPM1 1640 (GIBCO), and resuspended at 2.5 x 10’ cells/ml in RPM1 1640 supplemented with l:/, BSA, 0.04% NaN, and 13 mM HEPES (Sigma), pH 7.4. These cells were greater than 95:/, viable and about 70”/, macrophages. 100 ~1 of PEC were added to monomeric l 251IgG2 in 100 ~1 PBS plus 50 ~1 of G buffer with or without actin. After a 90-minute incubation at 4°C with intermittent shaking, 200 ~1 samples were removed. Cells bearing Fc receptor-bound IgG2 were separated from unbound IgG2 by the method of Segal and Hurwitz (1977). Low shear viscometry Interaction of actin with immunoglobulin was demonstrated by falling ball viscometry in G buffer containing 0.1 M KC1 (Griffith & Pollard, 1978). Actin (120 ~1) in G buffer plus 180 ~1
883
Interaction
immunoglobulin in G buffer containing 0.167 M KC1 were mixed and immediately drawn into 100 ~1 micropipets (Van Lab, VWR Scientific, Baltimore, MD). These were plugged with clay and placed in a 25°C bath. Viscosity was determined by placing each sample on a ramp in a 25°C water bath inclined either 50” or 80” from the horizontal. The time for an 0.64-mm steel ball (Microball Co., Peterborough, NH) to roll through the mixture is directly proportional to absolute viscosity. The fall time was determined in two 4-cm intervals during the descent of a ball in each capillary. Duplicate capillaries were prepared for each sample. Values reported are the means of 4 measurements of which the range was about 109% of the mean.
RESULTS
Effect of actin on the binding of IgG2 to macrophage Fc receptors. We first detected actin-immunoglobulin interaction by testing the effect of actin on the binding of IgG2 to peritoneal macrophages. Increasing concentrations of actin were added to guinea pig PEC and a concentration of 1251IgG2 sufficient to bind 50% of the Fc receptors on the cells. The result was monotonic
Fig. 1. Scatchard plots of the binding of 1251-lgG2 to Fc receptors of peritoneal exudate cells in the presence or absence of actin. Each tube contained IgG2 at the indicated concentration, 4 mg/ml BSA, and 10’ cells/ml in the presence or absence of actin at a final volume of 0.25 ml. (0) IgG2 alone; (0) IgG2 plus 0.4 mg/ml actin. The lines drawn on the figure were derived by linear regression analysis of the data. r, Concentration of cell-associated IgG2 in pg/ml; c, concentration of free IgG2 in pg/ml; n, the number of receptors per macrophage; KaPP, the apparent association constant of ligand for receptor in I/mol.
884
MARCUS
FECHHEIMER,
JOHN
Molar
Ratio
L DAIS ActIn
and JOHN
J. CEBRA
/ Ig GZ 6.0 16.8
8.7 14.3 ActIn+IgG~(meosured)
70’ 65 60 -
[ ActIn] Fig. Z Co-precipitation
performed
of’actin
*
mg /ml
ofactln and IgG2 mixed in different proportions at low ionic strength. in G buffer at pH 8.0. The concentration of IgG2 was 0.5 mg/ml.
enhancement of the binding of lgG2 as the concentration of actin was increased from 25-800 pg/ml. In order to define this more precisely, increasing phenomenon concentrations of 12jI-IgG2 were mixed with PEC in the presence or absence of actin. Scatchard plots of the results of this experiment. shown in Fig. 1, demonstrate that actin increases the apparent number of Fc receptors on the cells increase in by 25”‘, with an accompanying affinity. To determine whether apparent enhancement of IgG2 binding is due to fluid phase complex formation or to a direct effect of actin on the cells, the binding of actin by itself to PEC was measured. In these experiments we did not detect any specific binding of actin to cells (data not shown). The enhancement ofcytophilic activity of IgG2 in the presence of actin can be most easily explained by binding to the cells of complexes formed in the fluid phase. Co-prrcipitation
1npui ppt
and
IgC2
at low
ionic
strength
Preliminary experiments attempting to define a soluble complex in mixtures of actin and IgG2 were performed in G buffer with the hope of maintaining actin in a monomeric state. Analyses of these mixtures in the Model E ultracentrifuge showed that actin and IgG2 formed discrete 4s and 7s peaks, respectively (data not shown). Although no small complexes were apparent, a substantial quantity of material sedimented to the bottom of the cell in only a few minutes. This was never observed during the sedimentation of either component alone. When
Assays
the mixtures were allowed to stand for a few hours. visible precipitates formed. A co-precipitation assay employing 12jIlabelled proteins and 22Na as a trapped volume marker was devised to measure the contribution of each protein to the precipitate. Figure 2 shows the composition of the precipitate when a fixed amount of IgG2 was mixed with increasing concentrations of actin. The similarity of the curves for actin plus IgG2 determined separately or together demonstrates that unlabelled and labelled proteins behave identically. The precipitates contain more than 50”” of each protein present. As the concentration of actin is increased, the quantity of lgG2 precipitated increases and then decreases, suggesting that cross-linking is occurring. The molar ratio of actinjIgG2 at the point of maximal precipitation of IgG2 is 1.8. The interaction is specific since no coprecipitation occurs between actin and BSA, or IgG2 and ovalbumin. Co-precipitation is readily reversible by dilution or by increasing the ionic strength. We observed 95:; inhibition with 0.15 MKCI, and complete inhibition with 0.5 MKCl. Since lgG2 anti-DNP is a basic protein and actin is acidic, it seemed possible that coprecipitation at pH 8.0 at low ionic strength may reflect an electrostatic interaction of components of opposite charge. Figure 3 shows the effect of pH on the amount of IgG2, IgGl and F(ab’)z of lgG2 coprecipitated with actin. Precipitation of IgG2 is enhanced at acid pH reaching 95”, at pH 6.4. IgGl and F(ab’), of IgG2 show no and little precipitation, respectively, even at pH values
~ctia-~mmunoglobul~n
,
<
I
,
Interaction
X85
at pH 7.3 and 8.6, respectively, and this precipitation was totally inhibited by added aetin. The existence of soluble complexes suggests that actin-Ig~o-pre~jp~tation is at least a two-step process. Thus, co-precipitation is not necessarily a direct measure of binding.
Sedimentation analysts in gr~d~e~rs of sucrose was employed to demonstrate soluble compiexes of actin and IgG in 0.1 M KCI. The existence of such complexes was suggested by results of Fc receptor titrations and the co-pr~c~pita~io~ assay. Figure 4 shows sucrose gradient profiles of Fig. 3. Effect of pW on co”~r~ip~tati~~ of actin with IgG 1) radioIabeII~d actin in the presence or absence of lgG2 and F(ab’f, offgG2. (IL?)I&l; (0) 1gG2; {A, F(ab’), of IgG2. Mixtures contained 0.5 mg/ml of each protein IgG2. IgG-2 causes a displacement into more present. Since ionic strength affects co-pr~~~itation, the rapidly sedimenting protein of about 10% of the couducti~t~ of each G buffer solution was adjusted to 160 actin with a c~~~ornitant diminution ofthe peak pmho with NaCf after titration to the proper pH. of monomeric actin, IgGI, IgG2, dimeric JgG2 below their isoelectric points. Since an and IgM were all effective in causing actin appreciably fraction of the actin precipitated in displacem~~~t. The shape of the gradient profiles tubes containing actin alone at pW 5.8, the suggest that actin may dissociate from IgG sedimentation. The actin itself increased precipitation of F(ab’), and IgGt at during this PI-I may be due to a different sort of polymerized if higher concentrations (2 rng~rn1~ interaction. These results demonstrate that co- were used, and the F-a&n induced a down~eld precipitation is not governed simply by the net displacement of IgG2. The foilowing speciality charges of the interacting proteins, and that it is controls were performed: BSA did not induce a down~eld djsplacement of actin; lgC2 did not apparently F~-mediated. The preserve of sohtbie complexes in this induce a dow~~eld djsplacement of BSA; an system is suggested both by the immuno- excess of u~iab~Il~d BSA did not inhibit the precipitation-like nature of the curve in Fig. 1, displacement of 12sf-actin by IgG2, and by the fact that precipitation of I ~~~~1~~~~~~~~ of actin-immlmo~1~hlllin itself was blocked by the presence of actin. In one interaction by ~is~~rn~tr~ experiment, 23 and 34% of the IgGl precipitated Viscometry was employed to study act~n-immunogiobuli~ i~teractj~n because potentially interacting components do not have to be separated in the process of aI~~Iyzing a mixture. PreIimi~ary experiments using au ~stwald-type viscometer showed that IgG 1, IgGZand BGG all increased the specific viscosity of actin solutions by 400/,, while BSA had no effect (data not shown). Repeated measurement of the same sample resulted in a decay of the viscosity toward values observed for actin alone indicating djsruption of the interaction by high shear forces. Thus, the method of low shear falling ball vi~~metry described by Gr~f~tb and Pollard (1978) was employed in subsequent experiments. luring such measnreme~ts, the Froctlon Number solution itself remains static and each sample is Fig. 4. 3i~din~ of actin to immuno~obuli~ on sucrose only subjected to a single evaIuation. gradients, (1) 1251-actin alone; (0) i2s1-actin plus XgGZ. mixtures of actin and immunoglobui~~ were Samples of 0. t ml contained 2 mg/ml XgG2and 0.2 mg]ml of ‘“%actin. held for 24 hours at 25°C before the viscosity was
886
MARCUS
\ ii
I
301 / I /
/
/ /
FECHHEIMER.
/
JOHN
I I
Fig. 5. Interaction of actin with IgGl, IgG2, Fab and Fc of lgG2, and BSA measured by falling ball viscometry. (0) IgG 1; (0) IgG2; ( x ) Fab of IgG2; (+) Fc of IgG2: (0) BSA. The concentration of actin was 0.73 mgiml. Since fall times for all immunoglobulin solutions an; BSA at the highest concentration used were less than 0.5 set/cm, the viscosity of mixtures are significantly greater than the sum of the viscosities of the components.
since preliminary experiments determined indicated that 20-30 hours were required for stable values to be attained. Figure 5 shows the concentration dependence of enhancement of actin viscosity by IgG2, IgGl, Fab and Fc of IgG2, and BSA. IgGl and IgG2 increase the viscosity equally well, possibly by cross-linking actin filaments. Fab, Fc and BSA induce modest increases in viscosity. At higher concentrations (20 @4), the fragments of immunoglobulin were slightly more effective than BSA at enhancing actin viscosity (data not shown). These other proteins and fragments may act by nonspecifically stabilizing actin, since the viscosity of actin alone was frequently observed to decrease
I
I 140-
100 -
.67 Concentration
I.33
, 2.0
of Added
2.67 IqG2
3.33 LpM)
Fig. 6. Interaction of actin with monomeric or dimeric IgG2 measured by falling ball viscometry. (0) Monomeric lgG2; (0) dimeric IgG2. The concentration of actin was 0.63 mg/ml. the unit of concentration on the abcissa is moles Fe/l, so that the monomer and dimer may be directly compared on a weight basis.
L. DAK?
and JOHN
J. CEBRA
from its peak value by 20-go”,, after 20-30 hours of incubation. Dimers of IgG2 prepared with DDAO are up to four times more effective than monomeric IgG2 at enhancing actin viscosity as shoK;n in Fig. 6. It is not clear whether this reflects a higher avidity of the dimeric IgG2 for actin. or a higher efficiency of cross-linking. The break in the concentration curves between 0.3 and 1.5 ,uM was observed reproducibly for IgGl, IgG2 and IgG2 dimer, suggesting that more than one type of interaction may be occurring.
DISCUSSION
Evidence from four in vitro assay systems demonstrates a direct and specific interaction between actin and immunoglobulin. Since all of the interacting proteins were highly purified, the fraction of molecules interacting is larger than may be attributed to a possible contaminant. This extensive participation is most dramatically illustrated by the fact that 30 and 95”” of the IgG2 is co-precipitated with actin at pH 8.0 and 6.4, respectively. Interaction with actin seems to be a general property of immunoglobulin, since guinea pig IgGl, IgG2 and IgM, and bovine IgG have all been shown to be competent. The interaction is limited, and hence specific, since substitution of unrelated proteins for either actin or immunoglobulin in each of the assay systems resulted in lack of reactivity. Actin and immunoglobulin are competent to form soluble complexes in buffers of both low and physiologic ionic strength. The presence of complexes at low ionic strength may be inferred both from the fact that the amount of IgG2 coprecipitating with actin goes down with increasing actin concentration, and that actin apparently increases the solubility of IgGl in solutions of low salt concentration. Complexes of actin and immunoglobulin at physiologic ionic strength were demonstrated directly in sucrose gradient experiments, and indirectly by Fc receptor titrations and viscometry. Both filamentous and non-filamentous forms of actin were found to be capable of interaction with immunoglobulin. The fact that co-precipitation is salt labile, while binding of actin and immunoglobulin is not, suggests that the salt inhibits a step subsequent to simple binding. A second piece of evidence which distinguishes binding from
Actin-lmmunoglobulin
precipitation is that although both lgG1 and IgG2 bind actin, only IgG2 co-precipitates. This qualitative distinction between the two isotypes of guinea pig IgG may reflect differences either in hydrodynamic behaviour, such as in their segmental flexibility (Cebra ef al., 1977) or in stable structural features of the molecules. It is interesting to note that actinimmunoglobulin precipitation is similar to euglobulin precipitation in its salt lability and concentration dependence. Although it is not clear that the two processes are mediated by similar molecular interactions, one may predict that small amounts of actin released into serum during clot retraction would modulate the amount of IgG in the euglobulin fraction of that serum. The binding of actin to monomeric immunoglobulin appears to be of low affinity, since the appearance of sucrose gradient profiles suggests that the actin which is displaced downfield may have dissociated from complexes with immunoglobulin. Viscometry experiments show that dimeric IgG2 interacts more efficiently with actin than does monomer. It is tempting to speculate from this result that the free and reversible interaction of actin with monomeric immunoglobulin in cells may take on a more concrete nature upon cross-linking of the immunoglobulin by exogenous ligands. Efforts to demonstrate that actinimmunoglobulin interaction is mediated by a specific domain of the immunoglobulin molecule have not been entirely successful. The low effectiveness of both Fab and Fc in the enhancement of actin viscosity may be attributed to damage to the actin binding site during proteolytic cleavage, ability to bind but not cross-link actin filaments, or a genuine absence of the actin binding site. Further, the failure of F(ab’), to form a co-precipitate with actin cannot be construed to indicate Fc-specificity of the interaction, since lack of co-precipitation does not demonstrate lack of binding. Immune precipitates formed with DNP-BSA and F(ab’), of IgG2 anti-DNP in the presence of actin consistently failed to bind actin. Although similar immune precipitates formed with the parent molecule sometimes bound actin to an appreciable extent, this association was not consistently observed (data not shown). Nonetheless, the bulk of available evidence indicates that the ubiquitous unexpected component is indeed actin (Jones, 1977; Delovitch et al., 1979). As noted above, the
Interaction
887
lability of actin-Ig coprecipitation in low salt buffers to salt, dilution or high pH may reflect the inhibition of an interaction secondary to the association of actin with immunoglobulin. Consequently, our results suggest no simple means for elimination of actin contamination of specific immune precipitates formed in whole cell extracts. We find that ‘pre-clearing’ with an irrelevant antisera is usually effective in reducing contamination of immune precipitates by actin. There is no evidence for the existence of direct actin-immunoglobulin interaction in cells. The appearance of actin in immunoprecipitates of antigens could result from cell surface association after cell lysis. Experiments which demonstrate that actin co-caps with immunoglobulin, and that cross-linking cell surface immunoglobulin results in increased association with actin suggest at least an indirect association. The co-capping experiments must be interpreted with caution since it is not clear that a large fraction of the cellular actin is present and available for staining by antibody after fixation. The possibility that the shape changes which accompany capping are responsible for the observed staining patterns must also be considered. Models of actin-immunoglobulin interaction may be divided into those in which association occurs on the cytoplasmic face of the membrane, on the outside of the cell, or in the plane of the membrane. These models are not mutually exclusive, and are all compatible with either direct or indirect association of actin with immunoglobulin. Evidence that actin may be present on the surface of B lymphocytes (Owen et al., 1978) lends plausibility to the notion that actinimmunoglobulin interaction may occur on the outside of the cell. In such an interaction, both actin and/or immunoglobulin could be either endogenously or exogenously derived. A possible function of such an interaction is that the actin acts as a co-receptor to stabilize the interaction of immunoglobulin with protein or lipid receptors in the membrane. Actin-immunoglobulin interaction in the plane of the membrane or on the cytoplasmic face of the membrane could function in membrane -+ cytoplasm or cytoplasm + membrane communication. Differentiation between these alternatives will be possible only if precise information becomes available concerning the distribution of these proteins in the membrane and the existence of a linker molecule.
X88
MAKC’US
IEC‘HHEIMER,
JOHN
The present experiments demonstrate that a direct interaction of a cytoskeletal protein with a cell surface receptor is possible. In the future, it will be of interest to determine whether cytoskeleton-receptor interactions occur in cells, whether they are direct or mediated by linker proteins, and whether these interactions are associated with any specific functions in the cell. Ackrzo~~~lrdgrmmts~We thank Drs. S. MacLean and T. Pollard for instruction in low shear viscometry and for their gift of 0.64-mm diameter stainless steel balls. We thank Dr. V. Pigiet for use of his facilities for analytical and preparative ultracentrifugation.
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L. DAISS and JOHN
J. C’EBK,.‘!
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