Localization by Segmental Deletion Analysis and Functional Characterization of a Third Actin-binding Site in Domain 5 of Scinderin*

Monica G. Marcu, Li Zhang, Abdelbaset Elzagallaai, and José-María TrifaróDagger

From the Secretory Process Research Program, Department of Pharmacology, Faculty of Medicine, University of Ottawa, Ottawa, Ontario K1H 8M5, Canada

    ABSTRACT
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Scinderin is a Ca2+-dependent actin filament severing protein present in a variety of secretory cells. Previous work suggests that scinderin-evoked cortical F-actin disassembly is required for secretion because local disassembly of cortical cytoskeleton allows secretory vesicle exocytosis (Vitale, M. L., Rodríguez Del Castillo, A., Tchakarov, L., and Trifaró, J.-M. (1991) J. Cell Biol. 113, 1057-1067). Scinderin has six domains each containing three internal sequence motifs, two actin, and two phosphatidylinositol disphosphate-binding sites in domains 1 and 2. In this paper we report the presence of another actin-binding site at the NH2-terminal of domain 5 (Sc511-518). This site binds actin in a Ca2+-independent manner and a recombinant fragment (Sc5-6 or Sc502-715) containing this site binds to actin-DNase-I-Sepharose 4B beads, co-sediments with actin and is able to nucleate actin assembly. Recombinant ScL5-6, a fusion protein devoid of the actin-binding site (Sc519-715), did not exhibit these properties. Moreover, Sc-ABP3, a peptide constructed with sequence (RLFQVRRNLASIT) identical to Sc511-523 blocked the binding of Sc5-6 to actin. Sc5-6 and Sc-ABP3 also prevented the actin severing activity of recombinant full-length scinderin (r-Sc) and inhibited the potentiation by r-Sc of Ca2+-evoked release of serotonin from permeabilized platelets. On the other hand, ScL5-6 failed to block the effect of r-Sc on platelet serotonin release. Sc1-4,6, a construct devoid of domain 5, was able to sever but unable to nucleate actin, indicating that an actin nucleation site of scinderin was in domain 5. The results suggest that scinderin, in addition to binding actin on sites present in domains 1 and 2, must bind actin on a third site in domain 5 to sever and nucleate actin effectively.

    INTRODUCTION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Trifaró et al. (1), Bader et al. (2), and Ashino et al. (3) first showed the presence of gelsolin-like proteins in chromaffin cells. Bader et al. (2) also showed the presence of a Ca2+-dependent actin-binding protein that was immunologically different from gelsolin. The isolation and characterization of this protein was carried out simultaneously by two independent laboratories (4-6). These publications showed that chromaffin cells contain a Ca2+-dependent actin-severing protein that was distinct from gelsolin, although gelsolin was also present in these cells (2, 5). The names "scinderin" (4, 5) and "adseverin" (6) were given to this protein. Cloning of scinderin cDNA and sequence analysis (7, 8) demonstrated that, similarly to gelsolin, scinderin has six domains with two actin-binding sites in domains 1 and 2 (7, 9, 10). Two Ca2+-binding sites were also described for scinderin (5), and contrary to gelsolin (11-13) but similar to villin (14, 15), scinderin dissociated from actin in the presence of EGTA (4-6). Similar to gelsolin and villin, the actin-severing activity of scinderin resides in its NH2-terminal half (16), precisely domains 1 and 2 (7, 9, 10). The F-actin severing activity of scinderin seems to play a role in secretion (17). Scinderin is present only in secretory cells (5, 18, 19) and in chromaffin cells is highly concentrated together with F-actin in the cortex (17). Cortical F-actin acts as a barrier to the free movement of secretory vesicles to release sites, and only during cell stimulation induced Ca2+ entry, cortical F-actin is disassembled, most probably through the activation of scinderin (17). This will allow the movement of secretory vesicles to release sites on the plasma membrane (17, 20). In this regard, recent experiments have shown that recombinant scinderin potentiates Ca2+-evoked F-actin disassembly and exocytosis in platelets as well as in chromaffin cells (9, 10), effects blocked by PIP21 (9, 10). Two PIP2-binding sites have been described for scinderin, and as with gelsolin, they are present in segments 1 and 2 (7). PIP2 inhibits the actin-severing activity of scinderin, but unexpectedly phosphatidylserine also binds to scinderin and inhibits its actin-severing activity (21, 22). This is a distinct feature of scinderin when compared with gelsolin. Although the actin-severing activity of scinderin is localized in its NH2-terminal half (16), and this binds a molecule of G-actin (20), the COOH-terminal half also binds a molecule of G-actin (16, 20) with formation of a 2:1 scinderin-actin complex (20).

The present experiments were directed to identify and characterize a third actin-binding site that was found to be present in the COOH-terminal half of scinderin. Fusion proteins corresponding to either the full-length or truncations of scinderin were prepared and tested for their interaction with actin. Here we demonstrate that a third binding site for actin is localized in the NH2-terminal half of domain 5 of scinderin. Moreover, experiments with a recombinant truncated scinderin containing this site or a peptide constructed on the basis of this site sequence demonstrated that when this third site is occupied or blocked, the severing activity of intact scinderin is reduced. Similarly, either this peptide or a truncated scinderin fragment containing domain 5 was able to block the increases in Ca2+-evoked release of serotonin from platelets induced by recombinant full-length scinderin, suggesting again that the third actin-binding site is necessary for full interaction of scinderin with actin. Furthermore, experiments with a scinderin construct with deletion of domain 5 indicated that the actin-binding site present in this domain contributes to the actin nucleation activity of scinderin.

    MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Protein Expression and Purification-- The Thioredoxin (TRX) Thio-Fusion System (Invitrogen, San Diego, CA) was used for the expression of fusion proteins. Full-length recombinant scinderin (r-Sc) was prepared as described previously (9, 10), and the different truncations (deletions) of scinderin were obtained by PCR. Primers were designed to include a BamHI restriction site for 5' end and a SalI for 3' end. The obtained PCR products were digested and subcloned in expression vector pTrxFus using the same restriction sites. Recombinant plasmids were then transfected into the host Escherichia coli GI698. Correct subcloning and reading frames of all clones were confirmed by dideoxy sequencing (ABI 373 Automated Sequencer). PCR primers used to construct the fusion proteins were as follows: 5'-ACT GCA GGA TCC ATT GGT TTC AGA TGC CAG TGG-3' forward for Sc3-6, Sc3-4, and Sc3-1/24; 5'-GTC GTG TCG ACA GTC AGC ATC CAG CAG AGC TA-3' reverse for Sc, Sc5-6, ScL5-6, Sc1/25-6, and Sc6; 5'-GTC GTG TCG ACT TGC TCC TTG CCA GGT GTA G-3' reverse for Sc3-1/24; 5'-ACT GCA GGA TCC AGA AGG TCA GGC ACC AGC C-3' forward for Sc5-6; 5'-CTC GTG TCG ACC TGG TGC CTG ACC TTC TTT C-3' reverse for Sc3-4; 5'-ACT GCA GGA TCC AGA GAA AGG AGC AGA GTA CG-3' forward for Sc1/25-6; 5'-ACT GCA GGA TCC AGG CCT GGC TTC GAT CAC CAG-3' forward for Sc1/25-6; 5'-ACT GCA GGA TCC AGC ATC CCA GGC TGA AGA CCA TC-3' forward for Sc6; 5'-ACT TGG TAC CAA TGG CCC AGG GGC TGT AC-3' forward for Sc1-4,6; and 5'-TAG CAG GAT CCA CTG GTG CCT GAC CTT CTT TC-3' reverse for Sc1-4,6. Sc1-4,6 was obtained starting from Sc6 cloned into vector pTrxFus. A PCR product of Sc1-4 was amplified and then subcloned into the vector carrying Sc6 that was cut at the 5' end with KpnI and BamHI. One new amino acid was introduced just in front of the amino end of segment 6 of scinderin. Transformed E. coli were grown in Rich Medium containing 100 µg ampicillin/ml and induced with 100 µg tryptophan/ml. Proteins were obtained by osmotic shockate to a purity of at least 80%, as described previously (9). For the release studies, r-Sc was further purified by actin-DNase-I-Sepharose 4B affinity chromatography as described by Marcu et al. (9). Fusion proteins were dialyzed against deionized water and lyophilized as described previously (9, 10). A schematic representation of the position of the different recombinant fragments in the scinderin molecule is shown in Fig. 1.

Actin Binding Assay-- Actin-DNaseI-Sepharose 4B affinity beads were prepared as described previously (9) and used in binding experiments performed "in bulk." Rabbit skeletal muscle actin (G-150) was purchased from Cytoskeleton (Denver, CO). The buffer used for binding assays was either 1 mM CaCl2, 150 mM KCl, 1 mM dithiothreitol, 1 mM NaATP, 1 mM phenylmethylsulfonyl fluoride, 1 µM leupeptin, and 20 mM Tris-HCl, pH 7.5 (buffer A) or Ca2+-free 10 mM EGTA in the same buffer as above (buffer B). Washings were with either buffer A or B, this time including 600 mM KCl. For the Ca2+-free experiments, actin binding was performed in buffer B. 300 µl of actin-DNaseI-Sepharose 4B beads were incubated at 4 °C for 1 h on a rotary shaker with 50 µg of each fusion protein. Preparations were then centrifuged at 5000 × g for 1 min and washed several times with the respective buffer and centrifuged again to recover the beads. For all conditions, the actin-DNaseI-Sepharose 4B bead sediments and supernatants (buffer B eluates) were analyzed by SDS-PAGE and Western blots.

Actin Cosedimentation Assay-- The ability of fusion proteins to bind actin was also investigated by an actin-cosedimentation assay (23). Monomeric actin (0.5 mg/ml) (Cytoskeleton, Denver, CO) was induced to polymerize in 100 mM KCl, 2 mM MgCl2, 1 mM NaATP, 1 mM dithiothreitol, 10 µM CaCl2, and 10 mM Tris-HCl, pH 7.4, or the same buffer devoid of Ca2+ but containing 10 mM EGTA (Ca2+-free buffer). Aliquots of fusion proteins were mixed with actin and incubated for 90 min at 20 °C. Different ratios between recombinant truncated proteins and actin were used: 0.15:1; 0.35:1; 0.5:1; 0.75:1; 0.95:1; 1:1; and 1.2:1. Filamentous actin was sedimented by centrifugation at 100,000 × g for 60 min; sediments and supernatants were tested for the presence of proteins by SDS-PAGE and Western blots. To determine affinities to actin and Kd values of scinderin constructs, 4 µM actin samples were polymerized in the presence of different molar ratios of constructs to actin. After 90 min of incubation, the mixtures were centrifuged as above. The amounts of constructs and actin in supernatants and pellets were determined by densitometric scanning (Ultrascan XL Bromma Laser densitometer) of Coomassie Blue-stained gels. Scatchard's plots were prepared with the data, and Kd values were calculated from the plots.

Viscometry Analysis-- Apparent viscosity was measured at low shear rates by the falling ball technique as described by MacLean-Fletcher and Pollard (24). G-actin was induced to polymerize (4 mg/ml) for 1 h at 22 °C, and then it was diluted to 1 mg/ml (0.22 µM) in the following buffer: 100 mM KCl, 2 mM MgCl2, 5 mM EGTA, 0.005% NaN3, 10 µM free Ca2+ (4.6 mM CaCl2) 1 mM NaATP, 1 mM phenylmethylsulfonyl fluoride, 1 mg leupeptin/ml, 40 mM PIPES, pH 6.8. Actin alone or mixed (at 0 °C) with fusion proteins in different ratios was drawn into 100-µl glass capillaries, sealed with plasticine at one end, and held horizontally at room temperature for 2 h before determination of the apparent viscosity. One stainless steel ball was used per capillary; one measurement per tube and three measurements per condition were done. Capillary tubes were placed at an angle of 45 °, and the time for the ball to fall between two points was recorded. Apparent viscosity was calculated from a calibration curve obtained by measuring ball falling times through glycerol solutions (from 0-100%) of known viscosity, and the results were expressed in centipoises.

Actin Nucleation Assay-- 6 µM G-actin containing 5% pyrene-iodoacetamide-labeled actin (Cytoskeleton, Denver, CO) were dissolved in low salt buffer (0.2 mM NaATP, 0.5 mM dithiothreitol, 0.2 mM CaCl2, and 5 mM Tris-HCl, pH 8.0) and left on ice for 30 min to allow complete depolymerization of small oligomers that might form during defrosting. The solution was then centrifuged at 100,000 × g for 1 h at 4 °C, and the actin present in the supernatant was polymerized alone or in the presence of r-Sc or recombinants Sc1-4,6, Sc5-6, or ScL5-6 (the molar ratio of actin to any of the recombinants was 400:1) by addition of <FR><NU>1</NU><DE>50</DE></FR>th volume of polymerization inducer buffer (2.5 M KCl, 100 mM MgCl2 and 50 mM NaATP). Actin polymerization was monitored for 120 min at 22 °C by measuring the change in fluorescence, using a 10-nm bandwidth and excitation and emission wavelengths of 365 and 407 nm, respectively (Perkin-Elmer LS5 Fluorometer).

Gel Electrophoresis and Immunoblotting-- Monodimensional electrophoresis (SDS-PAGE) was performed according to Doucet and Trifaró (25). Gels were either stained with Coomassie Blue or transferred to nitrocellulose membranes. Immunolabeling was performed with a monoclonal antibody against thioredoxin (Invitrogen, CA) in 1:5000 dilution. A scinderin polyclonal antibody previously raised in our laboratory against full-length scinderin (5) did not recognize most of the fusion proteins corresponding to small truncations of scinderin except Sc3-6, Sc3-4, and Sc1-4,6. The second antibody was alkaline phosphatase-conjugated goat anti-mouse IgG (Bio-Rad), and it was used in a dilution of 1:3000. Color was developed by treatment of membranes with a mixture of p-nitro-blue-tetrazolium chloride and 5-bromo-4-chloride-3-indolylphosphate toluidine salt. Coomassie Blue-stained protein bands were densitometrically scanned with an Ultrascan XL Bromma Laser Densitometer.

Preparation of Scinderin-derived Peptides-- Peptides with a sequence corresponding to the 5' end of scinderin 5-th domain (RLFQVRRNLASIT) and with scramble sequence (AVNIRLRFSTLQR) were prepared by solid phase peptide synthesis (26). Sequences were checked by Edman chemistry and peptide purity determined by mass spectroscopy (27). No homology for scramble sequence was found in the GenBankTM data.

Platelet Permeabilization, Labeling of Serotonin Store, and Serotonin Release-- Platelet-rich plasma was obtained from the Ottawa Red Cross and centrifuged at 200 × g for 15 min to eliminate red blood cells. The supernatant was centrifuged at 800 × g for 15 min to obtain a platelet sediment. This sediment was resuspended in Ca2+-free Locke's solution (154 mM NaCl, 2.6 mM KCl, 2.14 mM K2HP04, 0.85 mM KH2P04, 1.2 mM MgC12, 10 mM glucose, and 2.0 mM EGTA, pH 7.2), and the platelet concentration was adjusted to 7.5 × 108/ml. Platelets were then incubated at 37 °C for 90 min with 0.6 nmol [3H]5-hydroxytriptamine (serotonin, [3H]5-HT)/ml (specific activity = 25.4 Ci/mmol, DuPont) (28), and [3H]5-HT-labeled platelets were washed by incubation with six changes of 10 ml of Ca2+-free Locke's solution over a 60-min period. [3H]5-HT-labeled platelets were permeabilized by 5 min of treatment with 15 µM digitonin in K+-glutamate buffer (160 mM K+-glutamate, 12.5 mM MgC12, 2.5 mM EGTA, 2.5 mM EDTA, 5 mM ATP, 20 mM HEPES, pH 7.4) (29). Platelets were then centrifuged at 900 × g for 2 min (4 °C) and resuspended in K+-glutamate buffer. Ca2+ concentrations (pCa values) were calculated as described previously (30). Samples (100 µl) containing 7.5 × 107 permeabilized platelets in K+-glutamate buffer were stimulated with 10 µM Ca2+ for 45 s in the absence or the presence of different recombinant scinderins, a scinderin-derived actin-binding peptide, or both. Release experiments were carried out as described previously (9). [3H]5-HT output was expressed as a percentage of total content after subtraction of values for spontaneous (absence of Ca2+) release. A minimum of eight samples/condition were measured, and the means ± S.E. were plotted.

    RESULTS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Recombinant Scinderins: Expression and Purification-- Different truncations of scinderin cDNA obtained by PCR were subcloned into expression vector pTrxFus using the restriction sites as indicated under "Materials and Methods." Recombinant vectors were introduced into E. coli GI698 to express the corresponding fusion proteins. Lysates obtained from tryptophan-induced cultures showed in SDS-PAGE a new band that corresponded to each truncated scinderin (see Fig. 2). The molecular masses of these TRX fusion proteins were as follows: Sc1-4,6, 79 kDa; Sc3-6, 64 kDa; Sc3-4, 43 kDa; Sc3-1/24, 34 kDa; Sc5-6, 41 kDa; ScL5-6, 40.6 kDa; Sc1/25-6, 35 kDa; and Sc6, 27 kDa. A schematic representation of the position of these scinderin fragments in the scinderin molecule is shown in Fig. 1. Some fusion proteins (Sc1-4,6 and Sc3-4 to ScL5-6) were extremely well expressed amounting to more than 50% among total E. coli proteins (Fig. 2, A and C). Because the scinderin polyclonal antibody did not recognize several scinderin fragments (only Sc1-4,6, Sc3-6, and Sc3-4 were recognized by the antibody), most Western blots (Fig. 2B) were stained with a mouse TRX monoclonal antibody (Invitrogen, CA). However, when dealing with r-Sc (Sc1-6) and Sc1-4,6 blots were stained with a scinderin polyclonal antibody (Fig. 2D). Enterokinase does not efficiently cut TRX-Sc fusion proteins, and because scinderin is very sensitive and undergoes fast proteolysis at room temperature, all experiments were carried on with TRX-Sc fusion proteins as previously shown (9, 10). TRX does not interfere with the function of scinderin (9, 10) and has no effect in in vitro tests when compared with TRX-Sc (data not shown).


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Fig. 1.   Schematic representation of scinderin domains (Sc1-6), including full-length and fragments obtained by different deletions. The number of the first and last amino acid of every recombinant protein is indicated. The amino acid sequences and position of three scinderin-derived actin-binding peptides (Sc-ABP1, Sc-ABP2, and Sc-ABP3) are also shown.


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Fig. 2.   Expression of different truncated scinderins in E. coli GI698. Bacteria were transfected with recombinant pTrxFus, grown, and induced with tryptophan. Some truncated proteins (Sc1-4,6 and Sc3-4 to Sc1/25-6) were better expressed than others. A and C, aliquots of bacterial lysates (30 µg of protein) were subjected to SDS-PAGE followed by staining with Coomassie Blue. The Western blot corresponding to A is depicted in B; TRX fusion proteins were identified with a monoclonal antibody against thioredoxin (Invitrogen, CA). In D r-Sc (Sc1-6) and Sc1-4,6 were detected with an antibody against scinderin. The position of the recombinant fragments within the scinderin molecule is depicted in Fig. 1.

Binding of Recombinant Scinderin Fragments to Actin-- The ability of scinderin fragments to bind actin was tested using two different assays. (a) The first assay was binding to actin-DNaseI-Sepharose 4B beads. In Ca2+ containing buffer (buffer A), only two Sc-derived proteins, Sc3-6 and Sc5-6, were found to bind actin (Fig. 3A). Moreover, once bound in the presence of 1 mM Ca2+, Sc3-6 and Sc5-6 could not be eluted by a EGTA buffer (buffer B). As a matter of fact, both Sc3-6 and Sc5-6 were found to bind to actin equally well in the presence or absence (10 mM EGTA) of 1 mM Ca2+ (Fig. 3C). Due to the fact that the Sc3-6 and Sc5-6 could not be eluted with EGTA containing buffer, all gel wells were loaded with supernatants obtained after the Sepharose 4B beads were boiled in SDS-PAGE sample buffer. Therefore, the presence of actin and DNase I was evident in all Coomassie Blue-stained gels (Fig. 3, A and B). A deletion of 16 amino acids (502-518) from the beginning of domain 5 produced ScL5-6, a fragment corresponding to amino acids 519-715 of scinderin, which did not bind actin. This suggests the presence of an actin-binding site at NH2-terminal of domain 5. Because it has been shown that domain 4 of gelsolin contains an actin-binding site (36), a set of experiments tested Sc3-4 and Sc5-6 for binding under different conditions (Fig. 3B). These two constructs did not bind to either Sepharose 4B or Sepharose 4B-DNase I beads (Fig. 3B, lanes 1-4). When tested for binding to Sepharose 4B-DNase I-actin beads, only Sc5-6 was found to bind to the beads (Fig. 3B, lane 6). (b) The second assay was co-sedimentation of recombinant truncated scinderins with actin. Here again, with the exception of Sc3-6 (data not shown) and Sc5-6, all recombinant scinderin fragments failed to co-sediment with actin under these experimental conditions (Fig. 4A). This, together with the observation that ScL5-6 did not co-sediment with actin, whereas Sc5-6 did, suggests the presence of a third actin-binding site at the 5' end of domain 5 (Sc5) of scinderin. To further demonstrate the presence of a third actin-binding site a 13-amino acid peptide (Sc-ABP3) with the sequence RLFQVRRNLASIT was constructed. This peptide is 6 amino acids longer than the possible actin-binding site (RLFQVRR). The length of the peptide was chosen just to be sure that the difference in binding to actin between Sc5-6 and ScL5-6 was not due to a possible disruption of a binding site whose length remains, at this point, to be elucidated. The scinderin-derived peptide (Sc511-523 or Sc-ABP3) but not a scrambled 13-amino acid peptide (AVNIRLRFSTLQR; no sequence homology in the EBI Data Bank), when present in a ratio to actin of 10:1, reduced by 49% (as shown by densitometry measurements) the binding of either Sc5-6 or Sc3-6 (data not shown) to actin when tested in the actin co-sedimentation assay (Fig. 4B). The binding of Sc5-6 to actin in the presence of the scramble peptide was 101% of that observed in its absence. The deletion of 16 amino acids that produced ScL5-6 was enough to suppress the binding to actin. Consequently, the actin-binding site should be localized at the very beginning of domain 5 of scinderin. This is a stretch of 13 amino acids starting at amino acid 511 and ending at amino acid 523. 


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Fig. 3.   Binding of recombinant scinderin fragments to actin-DNaseI-Sepharose 4B beads. A, truncated scinderins were tested for binding to actin in the presence of 1 mM Ca2+ as indicated under "Materials and Methods." Because two of the bound proteins (Sc3-6 and Sc5-6) could not be eluted with either buffer A or B (Ca2+-free 10 mM EGTA), at the end of incubations, the Sepharose 4B beads carrying the protein complexes were boiled with the SDS sample buffer, and the supernatants were loaded onto the gel. Therefore, actin (*) and DNase I (triangle ) are also visible in the Coomassie Blue-stained gel. Only recombinants Sc3-6 and Sc5-6 were found to be bound to actin under these conditions. B, because domain 4 of gelsolin has been shown to contain an actin-binding site (36), an additional experiment was carried out to further analyze Sc3-4 lack of binding to actin using Sc5-6 as a positive control. In the absence of actin and the presence of either Sepharose 4B (lanes 1 and 2) or Sepharose 4B-DNase I (lanes 3 and 4), both Sc3-4 and Sc5-6 did not bind to the beads, and they were recovered in the supernatants (lanes 1'-4'). When Sepharose 4B-DNase I-actin beads were present in the medium (lanes 5 and 6), Sc3-4 did not bind, and it was recovered in the supernatant (lane 5'). On the other hand, Sc5-6 was found to be bound to the beads (lane 6). Actin and DNase I bands are indicated by * and triangle  respectively. C, Western blot of an experiment performed to test the binding of Sc3-6 and Sc5-6 in either 1 mM Ca2+ or Ca2+-free 10 mM EGTA. Both proteins bind well under these two conditions. Samples were prepared as above.


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Fig. 4.   A, cosedimentation of various recombinant scinderin fragments with actin. The fusion proteins were mixed with actin and incubated in the presence of 10 µM CaCl2 as described under "Materials and Methods." Preparations were centrifuged at 100,000 × g for 60 min; sediments were washed, resuspended in the SDS sample buffer, and then loaded onto the gel. The asterisk indicates the position of actin. Sc3-6 (data not shown) and Sc5-6 were the only two contructs that cosedimented with actin. (An additional experiment was performed in Ca2+-free 10 mM EGTA, with the same result (data not shown).) B, inhibition of Sc3-6 cosedimentation with actin in the presence of a scinderin-derived actin-binding peptide (Sc-ABP3). Sc-ABP3 (lane 3) and a control scramble peptide (lane 4) were incubated together with Sc5-6 and actin in molar ratios of 100:1:10 and then centrifuged. Pellets (P) and supernatants (S) thus obtained were subjected to SDS-PAGE. Sc-ABP3 (lane 3) reduced by 49% the binding of Sc5-6 to actin when compared with control-scramble peptide (lane 4). Densitometric analysis of the gel shown in B resulted in areas of 0.98 and 1.05 arbitrary units for the actin bands in lanes 3 and 4, respectively, whereas the areas of Sc5-6 in the same lanes were 0.058 and 0.030 arbitrary units, respectively. Lanes 1 and 1' represent actin polymerized alone; lanes 2 and 2' actin polymerized in the presence of Sc5-6.

Affinity of the Sc5-6 Site for Actin-- Actin co-sedimentation experiments were also performed at different construct:actin molar ratios to determine affinity constants (Kd). The experiments were carried out either in the presence or the absence (10 mM EGTA) of Ca2+ to determine whether or not Ca2+ would affect the affinity for actin. In these experiments, Sc3-4 was also included to rule out the possibility that the absence of its binding to actin described above was due to the fact that only one concentration of Sc3-4 and only one ratio to actin was tested. Again, the experiments clearly demonstrated that only Sc5-6 was bound to actin at all ratios tested (Fig. 5, bottom). Scatchard analysis of the data (Fig. 5, top) revealed a Kd for Sc5-6 of 0.30 µM when tested in the presence of Ca2+ and a Kd of 0.33 µM in Ca2+-free medium; thus, indicating that Sc5-6 binds actin with equal affinity either in the presence or the absence of Ca2+.


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Fig. 5.   Determination of dissociation constants (Kd) by co-sedimentation of actin with either Sc3-4 of Sc5-6. Construct Sc3-4 or Sc5-6 was co-sedimented as indicated under "Materials and Methods" after incubation in the presence of 10 µM Ca2+ at the following construct. The construct actin molar ratios were 0.15:1 (lane 2), 0.35:1 (lane 3), 0.5:1 (lane 4), 0.75:1 (lane 5), 0.95:1 (lane 6), and 1.2:1 (lane 7). Preparations were centrifuged at 100,000 × g for 60 min, and the content of pellets (P) and supernatants (S) were separated by SDS-PAGE. The Coomassie Blue-stained gels are shown in the bottom panel. Lane 1 represents actin alone. Only Sc5-6 was found to co-sediment with actin. Protein bands were scanned, and ratios between free and bound Sc5-6 and between bound Sc5-6 and actin were determined and then plotted (Scatchard method) (A). The Sc5-6 Kd obtained from this analysis was 0.30 µM. In a similar experiment, this time performed in Ca2+-free 10 mM EGTA, the Kd obtained was 0.33 µM.

Severing Activity of Recombinant Full-length Scinderin and Its Fragments-- The severing activity of recombinant scinderins was determined by measuring F-actin gel low shear apparent viscosity using a falling ball viscometer. Previous experiments from our laboratory have shown that unlike full-length scinderin, Sc3-6 does not increase the secretory response of chromaffin cells (10). Measurement of viscosity of actin gels in the presence of recombinant Sc, Sc3-6, Sc5-6, Sc1-4,6, and Sc-ABP3 showed that Sc3-6 (data not shown) and Sc5-6 do not seem to have any effect on viscosity of actin gels (Fig. 6A). However, and as expected, r-Sc was very effective in decreasing viscosity (Fig. 6A). Mixtures of r-Sc with actin in the presence of either Sc5-6 or Sc-ABP3 in different molar ratios were tested in the presence of 10 µM Ca2+. Under these conditions r-Sc alone decreases the viscosity of actin gels (Fig. 6B). However, in the presence of either Sc5-6 or Sc3-6 (data not shown), r-Sc ability to decrease viscosity of actin gels was reduced (Fig. 6B). Moreover, when r-Sc was tested in the presence of Sc-ABP3, a similar inhibition of r-Sc effect on actin gels was observed (Fig. 6B). In view of these results, which indicate that the binding to actin by either domain 5 or Sc-ABP3 (a peptide with the binding sequence) interferes either with the binding or activity of r-Sc, Sc1-4,6, a construct with a deletion of domain 5, was tested. Sc1-4,6 was as effective as r-Sc in reducing the apparent viscosity of actin, and in this case, Sc-ABP3 was ineffective in blocking the actin severing activity of the construct (Fig. 6C). Moreover, when r-Sc and Sc1-4,6 were combined, there was a summation of their actin severing activities (Fig. 6C).


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Fig. 6.   Effect of r-Sc, Sc1-4,6 and Sc5-6 scinderins and peptide Sc-ABP3 on the apparent viscosity of F-actin. A, effect of various molar ratios of r-Sc or Sc5-6 to actin. G-actin was induced to polymerize for 1 h at 22 °C, and then it was diluted to 0.22 µM in PIPES-based buffer, mixed with various concentrations of either r-Sc or Sc5-6 on ice, then drawn into 100-µl glass capillaries, sealed at one end, and held horizontally at room temperature for 2 h, and measurement of apparent viscosity was performed as indicated under "Materials and Methods." B, effect of Sc5-6 and Sc-ABP3 on the fall in apparent viscosity of actin produced by r-Sc. The molar ratios used in the test are specified in the figure. C, effect of Sc1-4,6 on the apparent viscosity of actin.

Nucleation-- Actin polymerization was evaluated by measuring the increase in fluorescence of pyrene-labeled actin as indicated under "Materials and Methods." In the presence of either r-Sc or Sc5-6 and after a lag period of approximately 10 and 15 s, respectively, the fluorescence intensity increased exponentially reaching a maximum at 110 s (Fig. 7). In the absence of r-Sc or Sc5-6, actin polymerized slowly reaching after 300 s 33% and after 120 min 30% of the fluorescence intensity reached in the presence of either recombinant scinderin, thus suggesting that spontaneous nucleation was very slow. ScL5-6 and Sc1-4,6 did not nucleate actin assembly (Fig. 7). The results indicate that Sc5-6, even when tested at a small molar ratio to actin (1:400) is a very powerful inducer of actin polymerization and that this activity is localized at the NH2-terminal end of domain 5 (Sc502-518).


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Fig. 7.   Fluorescence monitoring of actin nucleation. Actin alone (6 µM) and in combination with r-Sc, Sc5-6, ScL5-6 or Sc1-4,6 at the indicated molar ratios was polymerized in the presence of 0.2 mM Ca2+. All samples contained 5% pyrene-iodoacetamide-labeled actin. The fluorescence intensity was measured at 407 nm (emission) after excitation at 365 nm. bullet , actin; open circle , actin:r-Sc, 400:1; black-down-triangle , actin:Sc5-6, 400:1; down-triangle, actin:ScL5-6, 400:1; black-square, actin:Sc1-4,6, 400:1.

Effect of Sc5-6, ScL5-6, and Sc1-4,6 on the Increase in Serotonin Release from Platelets Produced by r-Sc-- The inhibitory effects of both Sc5-6 and Sc-ABP3 on the actin severing activity of full-length scinderin suggest that either the third acting-binding site of scinderin is also required for full severing activity or when occupied by the peptide does not allow the proper binding of full-length scinderin to actin. Therefore, the ability of r-Sc to potentiate Ca2+-evoked release of serotonin in the presence of Sc5-6, ScL5-6, Sc1-4,6 or Sc-ABP3 was tested. Digitonin-permeabilized platelets showed increased release of serotonin in response to 10 µM Ca2+ (Fig. 8, A, B, and C). This secretory response is potentiated in the presence of either 0.1 µM r-Sc or Sc1-4,6 (Fig. 8, A and C). On the other hand, the serotonin release response evoked by 10 µM Ca2+ was unchanged by the presence in the medium of 0.1 µM Sc5-6, 0.1 µM ScL5-6 (Fig. 8A), or 10 µM Sc-ABP3 (Fig. 8B). However, when both r-Sc (0.1 µM) and Sc5-6 (0.1 µM) were present together in the incubation medium, the potentiating effect of r-Sc on Ca2+-induced release of serotonin was blocked by Sc5-6 (Fig. 8A). Similarly, the potentiating effect of r-Sc on serotonin release was blocked in the presence of Sc-ABP3 (Fig. 8B). On the other hand, ScL5-6 failed to block the potentiating effect or r-Sc on Ca2+-evoked serotonin release (Fig. 8A). Sc1-4,6 not only failed to block the effect of r-Sc on release, but having an effect of its own, showed a summation effect when added together (Fig. 8C). Moreover, as expected, Sc-ABP3 failed to decrease the potentiation of Ca2+-evoked release produced by Sc1-4,6 (Fig. 8C). Thus the inhibitory effects of Sc5-6 and Sc-ABP3 and the lack of effect of ScL5-6 on r-Sc responses suggests again that the third actin-binding site of scinderin may be required for its full activity or alternatively be necessary for the proper binding of scinderin to actin.


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Fig. 8.   Effect of recombinant Sc5-6, ScL5-6, Sc1-4,6, and peptide Sc-ABP3 on the potentiation by r-Sc of Ca2+-evoked serotonin release from permeabilized platelets. A, samples containing each 7.5 × 107 permeabilized platelets were incubated in 100 µl of K+-glutamate buffer, pH 7.4, for 45 s with 10 µM Ca2+ alone or in the presence of 0.1 µM r-Sc, 0.1 µM Sc5-6, 0.1 µM ScL5-6, or combinations of these fragments in a molar ratio (1:1). B, platelet samples were incubated and stimulated with Ca2+ as in A in the presence of 0.1 µM r-Sc, 10 µM peptide Sc-ABP3 (RLFQVRRNLASIT), or both. C, samples containing platelets were incubated and stimulated with Ca2+ as in A in the presence of 0.1 µM r-Sc, 0.1 µM Sc1-4,6, 10 µM Sc-ABP3, or combinations of these peptides. [3H]5-HT release was measured in A, B, and C as described under "Materials and Methods." The bars (means ± S.E.; n = 8) represent [3H]5-HT outputs after subtraction of spontaneous release.

    DISCUSSION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Scinderin is a Ca2+-dependent actin-severing protein of 715 amino acids that shares 63 and 53% homology, respectively, with gelsolin and villin (7). Scinderin shares six internal repeats of short sequence motifs (A, B, and C) with gelsolin and villin (7, 31). After alignment of motifs A, B, and C, these proteins reveal six (Sc1-6) domains (31). Previous work has demonstrated the presence and functional properties of two actin-binding sequences in domains 1 and 2 of scinderin (7, 9, 10). The high homology of these two actin-binding sites in these proteins (7, 31) would suggest that the type of interaction described for domains 1 and 2 of gelsolin with actin (32) might be similar for scinderin and villin. However, the binding of domain 1 of gelsolin to actin seems to be Ca2+-independent (33, 34), whereas in the case of scinderin, this binding requires the presence of Ca2+ (20). Therefore, because of this property, scinderin resembles villin rather than gelsolin.

The present experiments describe a third actin-binding site in scinderin. This site is localized to the NH2-terminal half of segment 5 of scinderin because construct Sc5-6 but not ScL5-6 was able to bind actin. The first 5 amino acids in the sequence (RLFQVRRNL) of this site (scinderin 511-519) are the same as those present in positions 161-165 of the second actin-binding site (RLFQVKGRR) of gelsolin segment 2. The site (amino acids 511-519) present in Sc5-6 is able to bind actin monomers, similar to gelsolin S4-6 (35). However, the actin-binding site in this part of gelsolin has been localized to segment 4 (36). The results from different types of experiments (Figs. 3-5) presented here clearly indicate that segment 4 of scinderin has no actin binding properties. Furthermore, and opposite to what has been described for gelsolin S4-6 and similar to gelsolin S-2 (35), the binding of actin to Sc5-6 is Ca2+-independent, because the present experiments show that Sc5-6 was bound to actin DNase-I-Sepharose 4B beads or co-sedimented with actin quite effectively in the presence of 10 mM EGTA. Moreover, the affinity of Sc5-6 for actin was similar in Ca2+ or Ca2+-free solutions as indicated by similar Kd values (0.30 and 0.33 µM) obtained by Scatchard plot analysis of the data obtained in these two buffer solutions. These Kd values are within the range of those published for gelsolin domains S4 (36) and S2-3 (37). We have demonstrated that intact native scinderin interacts with actin in a Ca2+-dependent manner and is eluted from actin Sepharose 4B with EGTA buffers (4, 7). Therefore, it is quite possible that the third actin-binding site of scinderin is hidden and that it is only exposed upon binding of Ca2+ and/or actin to other scinderin sites with the consequent changes in the configuration of the protein. The binding of actin to Sc3-6 or Sc5-6 observed in a Ca2+-free environment could be explained by the fact that these fusion proteins are perhaps not folded as the native one, and therefore, the third actin-binding site is permanently exposed. Alternatively, either one-third or two-thirds of the NH2-terminal part of the scinderin is missing in Sc3-6 and Sc5-6, respectively, and this might produce a more "open" configuration in these fusion proteins, permitting the access and binding of actin to the third site in a Ca2+-free environment. The Sc5-6 fragment not only binds monomers of actin but also, similar to gelsolin S4-6 (35), is capable of nucleating actin assembly. In this case Sc5-6 was as effective as r-Sc. However, ScL5-6 and Sc1-4,6 were ineffective in promoting nucleation, suggesting again the presence of an actin-binding site with actin nucleation properties at the NH2-terminal half of domain 5 of scinderin.

More about the third actin-binding site of scinderin was learned from the viscometry and platelet serotonin release experiments described here. We have previously demonstrated that recombinant full-length scinderin potentiates Ca2+-evoked release of serotonin from permeabilized platelets, an effect blocked by PIP2 or peptides with sequences corresponding to the two actin-binding sites present in domains 1 and 2 of scinderin (9). Opposite to r-Sc and Sc1-4,6, Sc5-6 did not increase Ca2+-evoked release of serotonin from platelets. Moreover, in the presence of Sc5-6 or Sc-ABP3, r-Sc was completely ineffective, but it was quite effective when added with ScL5-6 in potentiating Ca2+-evoked serotonin release. Furthermore, Sc1-4,6, in addition to increase Ca2+-evoked release of serotonin, was able to show a summation of effects when combined with r-Sc. All this suggests that the third actin-binding site of scinderin should be occupied by actin for scinderin to display full activity. This idea gained support when the severing activity of scinderin was evaluated by viscometry of actin gels. The decrease in viscosity produced by r-Sc was completely blocked in the presence of either Sc5-6 or peptide Sc-ABP3 (with amino acid sequence corresponding to the third actin-binding site). The inhibitory effect of Sc-ABP3 was observed at micromolar concentrations. These concentrations were necessary because the peptide binds to actin (the scinderin substrate), an abundant cellular protein.

In summary, the experiments presented here demonstrate the presence of the third actin-binding site, which needs to be occupied by actin to position scinderin in such a way as to allow its full severing activity. The experiments do not discard the possibility that in addition to the two actin-binding sites present at the NH2-terminal half of scinderin, other actin-binding sites may also be present, because a systematic study of the NH2-terminal half of gelsolin recently published indicated the presence of an additional actin-binding sequence at the COOH-terminal of segment 2 of gelsolin (38).

    ACKNOWLEDGEMENTS

We are grateful to S. J. Dunn for typing the manuscript and to the Ottawa Red Cross for supplying platelet-rich plasma.

    FOOTNOTES

* This work was supported by a grant from the Ontario Heart and Stroke Foundation (to J.-M. T.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger To whom correspondence should be addressed: Dept. of Pharmacology, University of Ottawa, 451 Smyth Rd., Ottawa, ON K1H 8M5, Canada.

1 The abbreviations used are: PIP2, phosphatidylinositol disphosphate; r-Sc, recombinant full-length scinderin; TRX. thioredoxin; PCR, polymerase chain reaction; PAGE, polyacrylamide gel electrophoresis; PIPES, 1,4-piperazinediethanesulfonic acid; 5-HT, 5-hydroxytriptamine.

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Abstract
Introduction
Materials & Methods
Results
Discussion
References

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