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1 IUBMB Life, 49: , 2000 Copyright c 2000 IUBMB /00 $ Original Research Article Simultaneous Interaction of Actin With -Actinin and Calponin Olesya O. Panasenko and Nikolai B. Gusev Department of Biochemistry, School of Biology, Moscow State University, Moscow, Russia Summary Interaction of calponin and -actinin with actin was analyzed by means of cosedimentation and electron microscopy. G-actin was polymerized in the presence of calponin, -actinin, or both of these actin-binding proteins (ABPs). The single and bundled actin laments were separated, and the stoichiometry of ABPs and actin in both types of laments was determined. Binding of calponin to the single or bundled actin laments was not dependent on the presence of -actinin and did not displace -actinin from actin. In the presence of calponin, however, less -actinin was bound to the bundled actin laments, and the binding of -actinin was accompanied by a partial decrease in the calponin/actin stoichiometry in the bundles of actin laments. Calponin had no in uence on the binding of - actinin to the single actin laments. The structure of actin bundles formed in the presence of the two ABPs differed from that formed in the presence of either one singly. We conclude that calponin and -actinin can coexist on actin and that nearly each actin monomer can bind one of these ABPs. IUBMB Life, 49: , 2000 Keywords Actin; a -actinin; calponin; cytoskeleton. INTRODUCTION a -Actinin is a dimeric F-actin binding protein (ABP) enriched in Z-disks of skeletal muscle, cytoplasmic and membraneassociated dense bodies of smooth muscle, focal adhesion contacts, stress bers, and the cortical web of nonmuscle cells (1, 2). Calponin, a ubiquitous, predominantly monomeric, ABP (3), is detected both in contractile and cytoskeleton domains of smooth muscle (2, 4) and in stress bers and the cortical web of nonmuscle cells (2, 5). Recent biochemical and structural investigations have revealed that calponin and a -actinin interact with partially overlapping sites on the surface of actin. Indeed, the a -actinin binding Received 10 January 2000; revised 3 February 2000; accepted 10 February Address correspondence to Nikolai B. Gusev. Fax: NBGusev@mail.ru sequences on actin are located within residues (or 87 96) and residues (or ) of actin subdomain 1 (6, 7). The biochemical data indicate that three sequences of actin monomer (residues 1 226, , and ) are implicated in calponin binding (8). Three-dimensional reconstruction of actin laments containing calponin shows that calponin contacts residues of one actin monomer and residues (and 43 48) on the axially neighboring actin monomer (9). The same spatial organization has also been found for the complex of a -actinin with actin (7). Hence, a -actinin and calponin occupy almost the same sites on the surface of actin monomer, and their spatial organization on the actin lament seems to be very similar. In addition, both calponin and a -actinin are located in the same intracellular compartments. Therefore, one would expect these two ABPs to compete with each other for actin binding. However, before the present investigation, the simultaneous interaction of a -actinin and calponin with actin was not analyzed in detail. This paper deals with detailed investigations of the mutual effects of calponin and a -actinin on actin binding and compares the structure of actin bundles formed in the presence of these two ABPs, either singly or combined. Preliminary results of this study were reported at the 28th European muscle congress (10). EXPERIMENTAL PROCEDURES Rabbit skeletal actin and chicken (or duck) gizzard a -actinin and calponin were puri ed by methods described previously (11 13). The purity of the proteins was checked by sodium dodecyl sulphate (SDS) gel electrophoresis (14), and the concentrations of the proteins were determined spectrophotometrically. Molecular masses of actin, calponin, and a -actinin were taken to be 43, 34, and 200 kda, respectively. Two different approaches were used to obtain actin laments containing ABPs. In the rst, polymerization of G-actin proceeded in the presence of various amounts of ABPs. For this purpose ABPs at the desired molar ratio were mixed in Eppendorf tubes with an appropriate amount of buffer. The polymerization 277

2 278 PANASENKO AND GUSEV was started by adding a solution of G-actin. The nal probes contained 10 mm imidazole/hcl (ph 7.5), 2 mm MgCl 2, 150 mm KCl, 0.2 mm ATP, and 10 mm b -mercaptoethanol (buffer A). The nal concentration of actin in the incubation mixture was usually 19 l M, and the concentrations of a -actinin and calponin were varied between 0 and 10 or 0 and 20 l M, respectively. The polymerization of actin was allowed to proceed for 25 min at room temperature, followed by 40 min at 4 ± C. In the second method, G-actin was rst polymerized by the addition of MgCl 2 to a nal concentration of 2 mm; subsequently, F-actin was added to the incubation mixture containing one or both ABPs in the buffer A and incubated as described earlier. Interaction of actin with the ABPs was determined by twostep centrifugation. The incubation mixture containing actin and ABP was at rst subjected to low-speed centrifugation (60 min, 7,000g), and the pellet, which contained the actin bundles, was separated from the supernatant containing single actin laments. Single laments were then sedimented by high-speed centrifugation (60 min, 130,000g). The protein composition of the low- and high-speed supernatants and pellets was determined by quantitative SDS-gel electrophoresis. Under the conditions used, the areas of the protein peaks were linearly related to the quantity of the protein loaded on the gel. The molar ratio of ABP/actin in the low- and high-speed pellets was plotted against the concentration of free ABP remaining in the supernatant. The apparent binding constant and the maximal binding of ABP at saturation were determined by tting the titration curves as rectangular hyperbola. For visualization of ABP-containing actin laments, G-actin (19 l M) was polymerized in the presence of calponin ( nal concentration 15 l M), a -actinin ( nal concentration 6.5 l M), or a mixture of both ABPs. Polymerization was started by adding G-actin to the incubation mixture containing ABPs and the desired quantity of buffer. After polymerization, the samples were diluted vefold in buffer A, loaded onto Formvar-carbon coated grids (3.05 mm, 200 mesh), stained with 2% uranyl acetate (Merck), and observed under a JEM 100CE (Tokyo) electron microscope. RESULTS Polymerization of G-actin in the mixture containing increasing quantities of calponin or a -actinin is accompanied by enrichment of the pellet formed by single or bundled actin laments with these two ABPs. Under the conditions used, not more than 5 7% of isolated calponin, isolated a -actinin or of the mixture of two ABPs was precipitated both at low- and high-speed centrifugation. Therefore, we concluded that enrichment of the actin pellet with ABPs re ects interaction of the actin with the two proteins analyzed. In preliminary experiments we found that the actin was completely saturated by calponin if the molar ratio for calponin/gactin in the initial mixture was close to 0.8. Also, under the conditions used, the actin was saturated by a -actinin when the molar ratio for a -actinin/g-actin in the incubation mixture was These results agree with the data reported in the literature (15, 16). In the following experiments G-actin was polymerized in the presence of saturating amounts of one ABP and various amounts of the second ABP (Fig. 1). When G-actin was mixed with saturating quantities of a -actinin in the absence of calponin, the molar ratio a -actinin/actin in the bundles was close to (Fig. 1A), whereas the same ratio for single actin laments was (Fig. 1B). Addition of increasing quantities of calponin resulted in a very small displacement of a -actinin from its complex with actin. The molar ratio for a -actinin/actin in the bundles decreased from the range to (Fig. 1A); for the single laments the ratio remained practically unchanged: (Fig. 1B). When we repeated the same experiment in reverse order polymerizing G-actin in the presence of saturating quantities of calponin and various amounts of a -actinin (Fig. 1C, D) the actin bundles contained 0.50 mol of calponin per mole of actin in the absence of a -actinin, whereas the calponin/actin molar ratio on the single laments was close to Including a -actinin induced a 30 35% displacement of calponin from the bundled actin laments (Fig. 1C) but had only a very small effect on the amount of calponin bound to the single actin laments (Fig. 1D). Qualitatively similar results were obtained if F-actin was mixed with one or both ABPs. However, in this case a -actinin induced a very small (<10%) displacement of calponin from the bundled actin laments. Overall, the data presented mean that calponin practically does not compete with a -actinin either on single or bundled actin laments. At the same time, a -actinin induces 35% displacement of calponin from the bundled actin laments but has no any effect on calponin bound to the single actin laments. Continuing our investigation, we polymerized G-actin either in the presence of different quantities of the rst ABP or in the mixture containing different quantities of the rst ABP and saturating quantities of the second ABP. The protein composition of the bundled actin laments formed in the course of these experiments was determined by quantitative SDS-gel electrophoresis. Titration curves of free actin or of actin containing a -actinin as titrated with calponin were practically indistinguishable (Fig. 2A). This means there was no hindrance to the binding of calponin with the complex of actin and a -actinin. However, the binding of a -actinin to free actin was different from the binding to actin containing calponin (Fig. 2B) possibly because of the dif culty of incorporating rather large molecules of a - actinin into tightly packed complexes of F-actin and calponin (see below). To analyze the structure of complexes of actin with two ABP, we used electron microscopy. Under the conditions used, calponin induces formation of thin actin bundles with tight parallel alignment of actin laments (Fig. 3B). At the same time, a -actinin induces formation of thick actin bundles (Fig. 3C). Actin laments are widely separated from each other, and the space between laments is readily accessible to uranyl acetate (Fig. 3C). Sometimes cross-bridges connecting actin laments

3 a -ACTININ AND CALPONIN BINDING TO ACTIN 279 Figure 1. Interaction of actin with calponin and a -actinin. (A, B) G-actin (19 l M) was polymerized in the presence of a xed concentration of a -actinin (6.5 l M) and various concentrations of calponin. (C, D) G-actin (19 l M) was polymerized in the presence of a xed concentration of calponin (15 l M) and various concentrations of a -actinin. The molar ratio for ABP/actin in the pellets obtained after low-speed (A, C) and high-speed (B, D) centrifugation is plotted against the concentration of free ABP remaining in the supernatant. Figure 2. Interaction of two ABPs with isolated actin (open symbols) or with actin containing saturating quantities of another ABP (closed symbols). (A) Isolated G-actin (19 l M) or G-actin in the presence of 6.5 l M of a -actinin was polymerized in the presence of various amounts of calponin. (B) Isolated G-actin (19 l M) or G-actin in the presence of calponin (15 l M) was polymerized in the presence of various amounts of a -actinin. The molar ratio for ABP/actin in the low-speed pellet is plotted against the concentration of free ABP.

4 280 PANASENKO AND GUSEV Figure 3. Electron microscopy of isolated F-actin (A), F-actin containing calponin (B), F-actin containing a -actinin (C), and F-actin containing both ABPs (D). The scale bar of 100 nm applies to all four images. and a -actinin molecules can be seen on the micrographs (Fig. 3C) (16). Actin bundles obtained in the presence of both ABPs were of intermediate width (Fig. 3D). The actin laments were packed less tightly than in the complex with calponin, and the space between laments was less accessible to uranyl acetate than in the complex formed by actin and a -actinin. Thus the ndings by electron microscopy agree with the biochemical results and show that calponin and a -actinin may coexist on actin laments. DISCUSSION As mentioned earlier, calponin and a -actinin occupy overlapping sites on actin monomers and their spatial organization on actin laments is very similar (6 9). In addition, both calponin and a -actinin contain one or two calponin homology domains (CH domains) that, at least in the case of a -actinin, are directly involved in the binding of these ABPs to actin (7, 17). Therefore, we would expect mutual exclusion of these ABPs from actin laments. Instead, however, both calponin and a -actinin are colocalized in adherens junctions, dense bodies, and contractile apparatus of different cells (2, 4, 5). To solve this apparent contradiction, we performed a detailed analysis of the interactions of these two ABP with actin. We found that the presence of a -actinin does not affect the interaction of calponin with actin laments (Fig. 2), and binding of calponin does not induce signi cant dissociation of a -actinin from either bundled or single actin laments (Fig.1A, B). Interaction of a -actinin with actin leads to formation of ladderlike structures (16), in which widely separated actin laments are cross-linked by a -actinin forming rungs of the ladder (Fig. 4A). A rather small molecule of calponin may easily penetrate the space between laments that are cross-linked by a -actinin and thus occupy sites on actin (Fig. 4B). Therefore, interaction of calponin with actin that contains saturating quantities of a -actinin is not very different from the interaction with isolated actin (Fig. 2A), and detachment of a -actinin is not a prerequisite for the binding of calponin (Fig. 1A). In contrast, less a -actinin was bound to actin containing saturating quantities of calponin than to the isolated actin (Fig. 2B);

5 a -ACTININ AND CALPONIN BINDING TO ACTIN 281 Figure 4. Hypothetical scheme of calponin and a -actinin binding inside of actin bundles. (A) Polymerization of G-actin in the presence of excess a -actinin leads to formation of ladderlike structures with widely separated actin laments. (B) Polymerization of G-actin in the mixture containing saturating quantities of a -actinin and various quantities of calponin is not accompanied by repacking of a ladderlike structure. Calponin easily penetrates this network and binds to actin laments without displacing a -actinin. (C) Polymerization of G-actin in the presence of excess calponin leads to formation of tightly packed parallel bundles of actin. (D) Polymerization of G-actin in the mixture containing saturating quantities of calponin and various quantities of a -actinin results in formation of a network with an intermediate structure in which actin laments are pushed slightly apart by a -actinin and a portion of calponin dissociates from the bundled laments. moreover, under certain conditions, a -actinin induced partial displacement of calponin from the bundled actin but not from the single actin laments (Fig. 1C). Interaction of actin with calponin results in formation of tight bundles in which parallel actin laments are very close to each other (Fig. 3B). This type of packing can be caused by the presence of at least two closely separated actin-binding sites in the structure of calponin (18). The tight packing of actin laments in the complex with calponin (Fig. 4C) induces steric hindrances for the binding of a -actinin. Nevertheless, a -actinin seems to be able to push apart actin laments cross-linked by calponin (Fig. 4D), a process accompanied by partial dissociation of calponin and formation of a looser network of actin laments. Thus, calponin and a -actinin can coexist on the bundled actin laments. The three-dimensional reconstructions of the complexes of actin with calponin or a -actinin indicate that both ABPs interact with (or at least contact) two adjacent actin monomers (7, 9). If these reconstructions re ect the real interaction of actin with two ABPs, then the maximal stoichiometry of binding of both calponin and a -actinin cannot be more than 0.5 mol of ABP per mole of actin. However, the sum of calponin and a - actinin bound to the bundled or single actin laments is generally > 0.55 mol/mol and in some cases is 0.7 (Fig. 1B). This apparent contradiction may result from a nonspeci c interaction between calponin and a -actinin. However, we and others (19) have not detected any direct interaction between isolated calponin and a -actinin. Although we cannot completely exclude the possibility that after binding to actin the calponin and a -actinin start to interact with each other, such an interaction seems rather improbable for explaining the unusually high stoichiometry of ABPs bound to actin. Thus, although both ABPs contact two monomers of actin at the same time, only one of these monomers is directly involved in the binding of ABP. The next molecule of actin may interact with another APB molecule or the same ABP. This means that actin laments may accommodate a rather large number of ABPs. While this manuscript was in preparation, the paper of Leinweber et al. (19) was published. Our data mainly agree with the results of that publication. However, in contrast to Leinweber et al. (19), we found that under certain conditions a -actinin can partially displace calponin. 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