Dictyostelium discoideum contains asoluble actin-binding protein that caps actin ifaments at their fast growing ends. The purified protein consists of two subunits ...
The EMBO Journal vol.3 no.9 pp.2095-2100, 1984
New actin-binding proteins from Dictyostelium discoideum
M.Schleicher, G.Gerisch1 and G.Isenberg Max Planck Institute for Psychiatry, and 'Max Planck Institute for Biochemistry, 8033 Martinsried/Munich, FRG Communicated by G.Gerisch
Dictyostelium discoideum contains a soluble actin-binding protein that caps actin ifaments at their fast growing ends. The purified protein consists of two subunits with 34 kd and 32 kd apparent mol. wts. Like similar proteins from Acanthamoeba and bovine brain the capping protein from D. discoideum acts in a C2+4-independent manner. It lacks severing activity as indicated by its inability to disrupt the stress fibers and the microfiament network in detergent-extracted cells. Two actin-binding proteins from a plasma membraneenriched fraction were labeled with [1251]actin using a gel overlay technique. One of these proteins, with an apparent mol. wt. of 17 kd in SDS-polyacrylamide gels, has been purified from high-salt extracts, the other protein with an apparent mol. wt. of 31 kd has been purified from Triton X-100 extracted membranes. Monoclonal antibodies were raised against D. discoideum severin, a-actinin, the larger subunit of the capping protein, and the 17-kd membrane-associated protein. Immunoblotting of proteins from whole cell lysates showed that all these actin-binding proteins were present in both growth phase and aggregation-competent cells. Key words: actin-binding proteins/cytoskeleton/cell motility/ Dictyostelium Introduction Dynaamic rearrangements of actin filaments in non-muscle cells are thought to be necessary for cellular motility. Actinbinding proteins regulate these rearrangements of actin in cell-free systems and also in living cells by either interfering with the polymerization and depolymerization equilibrium of actin, or by cross-linking actin filaments to each other or to other cellular components (for reviews, see Craig and Pollard, 1982; Korn, 1982; Weeds, 1982). Proteins of the first type are the capping proteins (Glenney et al., 1981a; Isenberg et al., 1980, 1983; Kilimann and Isenberg, 1982; Maruta and Isenberg, 1983; Southwick and Hartwig, 1982; Wang and Bryan, 1981), actin-fragmenting proteins (Brown et al., 1982; Hasegawa et al., 1980; Hinssen, 1981; Yamamoto et al., 1982) and proteins which stabilize the monomeric form of actin (Blikstad et al., 1980; Carlsson et al., 1977; Harris and Weeds, 1978; Mannherz et al., 1980; Reichstein and Korn, 1979). Examples of the second type are actin cross-linking or bundling proteins, and proteins which connect actin filaments to membranes (Brenner and Korn, 1979; Bretscher and Weber, 1980; Glenney et al., 1981b; Hartwig and Stossel, 1981; Jockusch and Isenberg, 1981). Amoebae of Dictyostelium discoideum are rapidly moving cells which, like granulocytes, change their orientation within seconds upon chemotactic stimulation. During reorientation IRL Press Limited, Oxford, England.
the rear end of a cell can be turned into a moving front, and vice versa (Gerisch, 1982; Gerisch and Keller, 1981; Swanson and Taylor, 1982). It is supposed that the flexibility of the motile system of D. discoideum cells is based on fast and efficient regulation of actin polymerization and filament crosslinking. A 40-kd actin-binding protein, severin, which fragments actin filaments in a Ca2+-dependent manner has been purified from D. discoideum cells (Brown et al., 1982; Yamamoto et al., 1982). Actin filament cross-linking proteins purified from D. discoideum cells are a 95-kd protein, which in its function resembles non-muscle cx-actinin (Brier et al., 1983; Condeelis and Vahey, 1982; Fechheimer et al., 1982), a 120-kd protein (Condeelis et al., 1982) and a 30-kd protein (Fechheimer and Taylor, 1984). We have found additional actin-binding proteins in D. discoideum using low shear viscometry and labeling of proteins separated by SDS-polyacrylamide gel electrophoresis with iodinated actin in gel overlays. One of these proteins is a capping protein composed of two subunits with apparent mol. wts. of 34 and 32 kd. This protein caps actin filaments at their fast growing ends in a Ca2 + -independent manner. Monoclonal antibodies raised against this capping protein, against severin, ca-actinin and a 17-kd membrane-associated actin binding protein of D. discoideum indicate that these proteins are immunologically distinct. Similarities between the capping protein from D. discoideum and those from Acanthamoeba (Isenberg et al., 1980) and bovine brain (Kilimann and Isenberg, 1982) suggest a common role of these proteins in cells of different origin.
Results
Purification of actin-binding proteins from the cytosol From the soluble fraction of cell homogenates, three actinbinding proteins were purified using low shear viscometry as an assay (Figure 1). Two of these proteins, severin and cxactinin, have already been described (Brown et al., 1982; Fechheimer et al., 1982; Condeelis and Vahey, 1982). The third protein proved to be a capping protein. Severin was recovered in the flow-through fraction of a DEAE-cellulose column. The capping protein was eluted from the column at lower ionic strength than ci-actinin (Figure 2). For its final purification, the capping protein was eluted from a DEAE-cellulose column by a shallow NaCl-gradient (Figure 3A). The peak of the Ca2 +-independent activity coincided with the presence of two polypeptides which were separated by SDS-polyacrylamide gel electrophoresis under reducing conditions (Figure 3B). Their apparent mol. wts. are 34 kd and 32 kd. As revealed by scanning of Coomassie bluestained gels, the polypeptides were present at a 1: 1 ratio. The native protein eluted from a calibrated gel filtration column as a polypeptide with a Stokes radius of -3.5 nm. These results indicate that the capping protein is a heterodimer of - 65 kd mol. wt. 2095
M.Schleicher, G.Gerisch and G.Isenberg D I C T Y O S T E L I U M
D I S C O I D E UM
AX2; aggregation competent cells
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homogenization (Parr bomb)
sediment
+
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l supernatant
Dextran / PEG partitioning
100,000 g supernatant
flowthrough
_
ammoniumsulfate fractionation
I hydroxyapatite I phosphocel -
ammoniumsulfate fractionation
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extraction with 1.5 N KCl
supernatant
30-65X sat.
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DEAE
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cellulose
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I
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activity pocol
hydroxyapatite
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40,000 g
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plasma - membrane enriched fraction
+
cellulose gradients 0-350 mM NaCl
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+
DEAE
DEAE
cellulose hydroxyapatite
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hydroxyapatite gradient: 0-300 mM phosphate
SEVERIN ATNI
I activity pool DEAE - cellulose gradients 50-120 sM NaCl
|
17 KD PROTEIN
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-
l
1
31 KD PROTEIN|
I CAPPING PROTEIN / 34 +32 KD
Flg. 1. Fractionation scheme for actin-binding proteins of D. discoideum. Details of purification of the capping protein are given in Materials and methods. The purification procedure for ca-actinin was essentially the same as described by Condeelis and Vahey (1982). The purification of severin followed the description of Yamamoto et al. (1982), except that gel filtration was replaced by phosphocellulose chromatography.
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Fig. 2. Chromatography of the soluble fraction of cell homogenates on DEAE-cellulose with a 0-350 mM linear gradient of NaCI (x x). Fractions 60-72 contained the capping protein. The peak fractions reduced the viscosity relative to a control sample to < 5%, as measured by low shear viscometry (0 0). In fractions 95-105 the viscosity was raised up to > 900% due to the cross-linking activity of a-actinin (0-*). 5 id of the fractions (fraction size: 12.5 ml) were tested in a total volume of 160 1d in the presence of I mM EGTA.
To investigate which of the two subunits contain an actinbinding site, gels were overlayed with 1251-labeled actin, and bound actin was visualized by autoradiography. During purification (Figure 4A C) strong actin-binding activity remained associated with the 34-kd subunit of the capping protein (Figure 4A' C'). The 32-kd subunit, as well as contaminants that were present in impure fractions, showed little if any actin-binding activity. -
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Purification of actin-binding proteins from a plasma membrane-enriched fraction From the particulate fraction of aggregation-competent cells a plasma membrane-enriched preparation was obtained. This preparation contained at least two actin-binding proteins as monitored during purification by overlay of SDS-polyacrylamide gels with [1251]actin. One of these proteins was recovered in the soluble fraction after high-salt extraction of the membranes. After SDS-polyacrylamide gel electrophoresis and Coomassie blue staining the purified protein showed a single band corresponding to an apparent mol. wt. of 17 kd (Figure 4E). This band was strongly labeled with actin (Figure 4E'). For comparison, partially purified severin was applied to the same gel (Figure 4F,F'). The 40-kd band of the severin was also labeled, but less strongly than that of the 17-kd protein. membrane-enriched fraction remained bound to the particulate material during high-salt extraction but was extracted with Triton X-100, indicating that this protein was associated with the membranes by hydrophobic interactions. After purification the protein exhibited a single Coomassie bluestained band indicating an apparent mol. wt. of 31 kd (Figure 4D). The gel overlay technique revealed strong binding of actin to this band (Figure 4D'). Distinction of actin-binding proteins using specific monoclonal antibodies Monoclonal antibodies were raised at early stages of purification of the actin-binding proteins. The specificity of antibodies was determined by immunoblotting. Some of the obtained antibodies cross-reacted with several proteins including
Actin-binding proteis in Dictyostelium 25
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proteins were incubated with culture supernatants of cloned hybridomas and labeled with [PI]sheep anti-mouse IgG. For SDS-polyacrylamide gel electrophoresis in either 10% gels (left panel) or in 15% gels (right panel) proteins from I x 106 cells/slot were applied . Numbers of the monoclonal antibodies were: mAb 47-19-2 (anti-a-actinin), mAb 42-65-23 (anti-severin), mAb 43442-1 (anti-capping protein), mAb 54-165-12 (anti-17 kd protein).
--18
B 46
48
50
52
FIg. 3. Final purification of the capping protein by elution from DEAE x). cellulose with a shallow 50-120 mM gradient of NaCl (x (A) The viscosity reducing activity was eluted as a sharp peak in fractions 48-50, and was found to be Ca2+-independent as measured by low shear 0) or 200 pM Ca2+ viscometry in the presence of I mM EGTA (0 (0 *0). (B) The activity peak coincided with two polypeptides with apparent mol. wts. of 34 kd and 32 kd. Polypeptides of fractions 42-52 were separated by SDS-polyacrylamide gel electrophoresis in 12% gels and stained with Coomassie blue.
95-
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FIg. 5. Immunoblots of total cellular proteins from aggregation-competent cells (A) or growth phase cells (G). After transfer to nitrocellulose the
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42
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Fig. 4. Actin binding assayed with a gel overlay technique. D. discoideum capping protein at different grades of purity (lanes A-C, A' -C'), purified D. discoideum 31-kd (D,D') and 17-kd (E,E') membraneassociated proteins, and severin (F,F') were electrophoresed in 12% SDSpolyacrylamide gels, labeled with [mI]actin and subjected to autoradiography (A' -F').
actin, others labeled only a single band after SDS-polyacrylamide gel electrophoresis. Monospecifc antibodies have been obtained against severin, a-actinin, and the 17-kd membraneassociated protein. Highly specific antibodies have been obtained against the 34-kd subunit of the capping protein, whereas none of the antibodies produced by hybridomas of
two different mice reacted with the 32-kd subunit of this protein. During development from the growth phase stage to aggregation competence, D. discoideum acquires full chemotactic sensitivity to cyclic AMP and the capability of converting into long, bipolar cells. To investigate whether these changes are correlated with the up or down regulation of specific actin-binding proteins, monoclonal antibodies were applied to blots of crude homogenates from growth phase or aggregation-competent cells. No differences in labeling of severin and a-actinin were recognized when proteins of the two developmental stages were compared. The capping protein and the 17-kd membrane-associated protein were also present in both stages, but their quantity seems to be higher in the aggregation-competent stage (Figure 5). The specificity of the antibodies indicates that each of the four actin-binding proteins depicted in Figure 5 contains unique regions which are not shared by any of the other actinbinding proteins or by actin. This result does not exclude the existence of sequences that are common to two or more of these proteins. Cross-reactivities of certain antibodies against actin-binding proteins will be presented elsewhere. Properties of the capping protein Ca2+ -independent inhibition of actin polymerization. The purified capping protein inhibited actin polymerization in a concentration-dependent manner in the presence of 0.2 mM Ca2+ or 1 mM EGTA (Figure 6). 50% inhibition was obtained in the presence of Ca2+ at 2.1 Ag of capping protein/ml, and in the presence of EGTA at 2.6 pg/rml. The difference is considered to be too small to account for a major role of Ca2+ in regulating the activity of the capping protein. Absence of severing activity from the capping protein. To prove that the D. discoideum capping protein lacks severing activity, cytoskeletons were prepared from astrocytes by extraction with Triton X-100 and incubated with buffer, with D. discoideum severin, and with the capping protein (Figure 7). After fixation and staining of the F-actin, control cytoskeletons and those treated with the capping protein were indistinguishable. In cytoskeletons which were incubated with 2097
M.Schleicher, G.Gerisch and G.lsenberg
severin the microfilament network and the stress fibers were disrupted. Binding of the capping protein to the fast growing ends of actin filaments. Fragmented actin filaments decorated with myosin subfragment 1 were used to nucleate the polymerization of monomeric actin. The capping protein inhibited the growth of filaments only at their barbed ends (Figure 8A). As 100
0.25
0.50 0.75 100 CAPPING PROTEIN, jig
1.25
1.50
Flg. 6. Effect of the capping protein on actin polymerization in the presence or absence of Ca2+. Capping protein was added, as indicated on the abscissa, to a solution of G-actin (0.5 mg/ml) and polymerization was started at either 200,uM Ca2 (e *) or I mM EGTA (O O) in a total volume of 160 id. After 10 min of incubation at 25°C the viscosity was measured by low shear viscometry and expressed as a percentage of controls lacking capping protein. Data are means from triplicates with standard errors as indicated.
A
B
expected, control samples to which no capping protein was added grew bidirectionally with a preferred growth at their barbed ends (Figure 8B). Discussion The fractionation of soluble proteins of D. discoideum cells led to the purification of a new actin-binding protein, which proved to cap actin filaments at their fast growing ends. This capping protein is the fifth soluble actin-binding protein known to be present in D. discoideum cells. Three of the other proteins, the cr-actinin (Condeelis and Vahey, 1982; Fechheimer et al., 1982), a 120-kd protein (Condeelis et al., 1982) and a 30-kd protein (Fechheimer and Taylor, 1984) are cross-linkers of actin filaments, the fourth protein, severin, is an actin filament fragmenting protein (Brown et al., 1982). Membrane-associated actin-binding proteins are of particular interest because they may anchor the cytoskeleton to membranes. We have overlayed SDS-polyacrylamide gels with radiolabeled actin for identification of actin-binding proteins in extracts of a plasma-membrane enriched fraction. Two proteins strongly bound the actin: a 17-kd protein extracted at high ionic strength, and a 31-kd protein extracted
with detergent. It remains to be proven whether or not the 17-kd protein is a peripheral membrane protein, i.e., a protein that is directly associated with membranes. This protein might be only bound to actin that forms a network of microfilaments beneath the plasma membrane, and might be extracted when this network is destroyed. Identification of actin-binding proteins by the gel overlay technique has to be done with caution. Weak binding is of no significance because actin tends to interact unspecifically with many proteins. No conclusion can be drawn from negative results because denaturation by SDS or separation of
C
I;g. 7. Effect of severin and the capping protein on cytoskeletons from Triton X-100 extracted astrocytes. The cytoskeletons were incubated for 5 min with buffer (A) or with 50 pg per ml of purified capping protein (C), or for 2 min with 50 pg per ml of D. discoideum severin (B). After incubation the preparations were fixed with glutaraldehyde and stained with rhodamine-conjugated phalloidin. Magnification: 360 x; the bar represents 50 pm.
2098
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Actin-binding proteins in Dictyostelium
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Fig. 8. Blockage of the growth of actin filaments by the D. discoideum capping protein. F-actin from rabbit skeletal muscle was labeled with subfragment-l from chicken gizzard myosin. The decorated fragments were
used as seeds for polymerization of monomeric actin in the presence of capping protein (A) or in its absence (B). Magnification: 74 200 x; the bar represents 0.1 p.
subunits may destroy the actin-binding activity of a protein. Furthermore, the overlay technique works only with proteins that can bind to monomeric actin. Despite these shortcomings the technique provides a basis for focussing further work on proteins that strongly bind actin in gels. The properties of the 17-kd and 3 1-kd membrane-associated proteins discovered by the overlay technique will be presented elsewhere. In the present paper emphasis has been placed on the capping protein of D. discoideum. This protein resembles the capping proteins from Acanthamoeba and bovine brain in two respects: (i) it caps the fast growing ends of actin
flaments in a Ca2 +-independent manner, and (ii) it is a heterodimer with a native mol. wt. of 65 kd. The apparent mol. wts. of the subunits are 34/32 kd for the D. discoideum protein, 36/31 kd for the bovine brain protein (Kilimann and Isenberg, 1982), and 31/28 kd for the capping protein of Acanthamoeba (Isenberg et al., 1980). It remains to be investigated whether these similarities are also reflected in homologies of amino acid sequences and by common in vivo functions. The larger polypeptide of the capping protein of D. discoideum appears to be the actin-binding subunit. The smaller subunit might play a regulatory role, and it would be of interest to know whether the factors controlling the capping activity in amoebae are the same as in mammals. -
Materials and methods Proteins Rabbit skeletal muscle actin was prepared according to Spudich and Watt (1971) and gel filtered on Sephadex G150 (Mac Lean-Fletcher and Pollard, 1980). Myosin subfragment-l from chicken gizzard was kindly provided by Dr. A. Sobieszek, Institute for Molecular Biology, Salzburg, Austria. The
95-kd cross-linking protein (a-actinin) was prepared from aggregationcompetent D. discoideum cells essentially as described (Condeelis and Vahey, 1982). The preparation of severin (Yamamoto et al., 1982) was slightly modified. The gel filtration as a last purification step was replaced by a cationexchange column. The sample was loaded onto a phosphocellulose resin (P-11, 2.5 x 8 cm, equilibrated in TEDA-buffer) and severin was eluted with a linear salt gradient (2 x 150 ml, 0-250 mM NaCl) between 4.6 and 6.0 mS. Protein concentrations were determined according to Lowry et al. (1951) using BSA as standard. Purijication of the Dictyostelium capping protein D. discoideum strain AX2-214 was grown axenically in fermenters (usually 3 x 10 1) at 23°C (Claviez et al., 1982) and starved in 17 mM Soerensen phosphate buffer, pH 6.0. The development to aggregation competence was enhanced by pulses of 20 nM cAMP applied every 6-7 min for 6-8 h (Gerisch et al., 1975; Stadler et al., 1982). After cooling to 4°C the cells were harvested with a continuous flow centrifuge at 2000 g, washed twice with Soerensen buffer, suspended in homogenization buffer (2 ml/g of packed cells), and opened with a Parr bomb (5 min/400 p.s.i.). The homogenate was centrifuged for 15 min at 12 000 g and the resulting pellet was used for further purification of plasma membranes (Figure 1). The 12 000 g supernatant was centrifuged at 100 000 g for 3 h, the clear, yellow supernatant was adjusted to pH 7.5 and loaded onto a DEAE-column (DE-52; 5 x 40 cm; equilibrated with DEAE-buffer). After washing the column with at least three column volumes, bound proteins were eluted with a linear salt gradient (2 x 1000 ml; 0-350 mM NaCl). The resulting fractions were tested by low shear viscometry and a strong viscosity decreasing activity was eluted at a conductivity range between 5.0- 6.8 mS. Solid ammonium sulfate (30% saturation) was added to the activity pool, the precipitate removed by centrifugation (12 000 g/l15 min) and the ammonium sulfate concentration raised to 65 % saturation. The resulting pellet was dissolved in a minimum amount of G150-buffer and loaded onto a gel filtration column (G150; 2.7 x 114 cm) without further dialysis. Activity containing fractions were pooled, dialysed against 10 mM KPO4 (pH 7.2) and loaded onto a hydroxyapatite column (2.5 x 10 cm). Viscosity decreasing activity was eluted by a linear gradient of 0-300 mM KPO4 (2 x 200 ml) at a phosphate concentration of 100130 mM KPO4. The last purification step was performed with a shallow gradient on a second DEAE-column (DE-53; 1.5 x 5 cm; equilibrated in TEDAbuffer; 2 x 100 ml; 50-120 mM NaCl). The yield of purified protein was between 100 and 200 Ag/l00 g packed cells. Membrane preparation The sediment obtained by a 12 000 g spin of the crude homogenate was washed twice with Soerensen buffer and then partitioned in a two-phase system of dextran 500/polyethylene glycol mol. wt. 6000 essentially as described (Stadler et al., 1982). The plasma membranes were washed twice in TEDA-buffer and extracted with TEDA-buffer containing 1.5 M KCI. The high-salt extract was centrifuged for 30 min at 40 000 g and the resulting pellet was treated with TEDA-buffer containing 0.5% Triton X-100 (w/v). SDS-polyacrylamide gel electrophoresis Using the buffer system of Laemmli (1970) SDS-PAGE was performed on minislab gels (110 x 83 x 0.5 mm, Matsudaira and Burgess, 1978) with a minigel apparatus obtained from IDEA-Scientific, Corvallis, OR 97339, USA.
Gel overlay with iodinated actin Rabbit skeletal muscle actin was iodinated by the method of Bolton and Hunter (1973) as previously described (Snabes et al., 1981b). The overlay procedure was performed essentially as described (Van Eldik and Burgess, 1983; Snabes et al., 1981a, 1983) except that the overlay buffer was composed of 50 mM Tris-HCl, pH 7.6, 0.2% (w/v) BSA, 0.25% (w/v) gelatin, 0.2 M NaCl, 0.05% NaN3. Viscometry Low shear viscometry was carried out after 10 min of incubation at 25°C in a falling ball viscometer (Mac Lean-Fletcher and Pollard, 1980). The reaction mixture (160 Al) contained usually 0.5 mg/ml rabbit skeletal muscle actin and polymerization was started by addition of buffered MgCl2 (final concentration: 2 mM MgCl2, 10 mM imidazole, pH 7.5, 1 mM EGTA or 0.2 mM
CaClo). Electron- and fluorescence microscopy Protein samples were negatively stained with 0.75% uranyl formate, pH 4.25, on parloidon-coated, glow-discharged and cytochrome c-pretreated electron microscope grids. Actin filaments were decorated with S-1 and the effect of Dictyostelium capping protein was checked as previously described (Kilimann and Isenberg, 1982). Triton X-100 cell models were prepared from astrocytes as described by
Isenberg et al. (1983).
2099
M.Schelcher, G.Gerisch and G.Jsenberg Production of monoclonal antibodies BALB/c mice were immunized with partially purified actin-binding proteins, the spleen cells fused with NS-I or NSO/u myeloma cells using polyethylene glycol mol. wt. 4000 (Polysciences, Warrington, PA). Hybridoma supernatants were screened by solid-phase radioimmunoassay, and the cells cloned in microtiter plates by examining small droplets under the microscope and adding medium plus macrophages to droplets containing single cells. Monoclonal antibodies 42-65-23 and 43-442-1 against severin and capping protein, respectively, were obtained by injecting alternatively protein without adjuvant or with 100-200 pl of Bordetella pertussis antigen (Seruminstitut Bern, Switzerland). Three i.p. injections were given to each mouse at intervals of 1 week, and boosts without adjuvant at 7 or 11 weeks after the last injection. For antibody 54-165-12 against the 17-kd protein the schedule was similar, except that the mouse received antigen treated with 1% SDS, and injections were given alternatively with 100 1d Alugel S (Serva, Heidelberg), 100 p1 B. pertussis antigen, or with no adjuvant. At 4 weeks after the last injection two boosts were given at subsequent days, the first with Alugel S, the second without adjuvant. For antibody 47-19-2 against a-actinin the mouse received a single i.p. injection of protein with Freund complete adjuvant, and 5 weeks later a boost with incomplete adjuvant. The clones were grown up in RPMI 1640 medium containing 10% fetal calf serum, and culture supernatants were used for immunoblotting. For immunoblots, cells of AX2 were harvested either during growth or as aggregation-competent cells at 6 h of starvation in 17 mM Soerensen phosphate buffer, pH 6.0. Whole cells were lysed in sample buffer containing 2% SDS (Laenmli, 1970), the proteins were subjected to SDS-polyacrylamide gel electrophoresis and blotted onto nitrocellulose filters (BA85, Schleicher and Schfill). The blots were labeled according to Towbin et al. (1979) with minor modifications (Stadler et al., 1982), using hybridoma supernatants and iodinated sheep anti-mouse IgG, adjusted to 1 x 10' c.p.m./ml. Buffers Homogenization buffer: 25 mM Tris-HCI, pH 7.5; 1 mM dithiothreitol (DTT); 2 mM EGTA; 0.2 mM ATP; 5 mM benzamidine; 0.5 mM phenylmethyl sulfonylfluoride (PMSF); 0.02Gb (w/v) NaN3, 30% (w/v) sucrose. DEAE-buffer: 10 mM Tris-HCI, pH 7.5; 1 mM DTT; 1 mM EGTA; 1 mM benzamidine; 0.5 mM PMSF; 0.02% NaN3. TEDA-buffer: 10 mM Tris-HCI, pH 7.5; 1 mM DTT; 1 mM EGTA; 0.02% NaN3. G150-buffer: TEDA-buffer containing 0.2 M NaCl. Reagents DEAE-cellulose (DE-52, DE-53) and phosphocellulose (P-11) were purchased from Whatman, hydroxyapatite from BioRad, Sepharose G150 from Pharmacia, Bolton/Hunter Reagent from NEN. All chemicals were of analytical grade.
Acknowledgements We thank Dr. A.Williams, Oxford, for sending us the NSO/u myeloma line of Dr. C.Milstein, Dr. E.Rieske for cultures of astrocytes, Dr. Th.Wieland for a gift of rhodamine-coupled phalloidin, and Dr. A.Sobieszek for the gift of myosin S-1. We are grateful to Daniela Rieger and Barbara Fichtner for their excellent assistance in monoclonal antibody production and to Gertrud Wagle for cell culture. The work was supported by the grant IS 25/4-1 from the Deutsche Forschungsgemeinschaft to G.Isenberg and G.Gerisch.
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