Characterization of the actin cross-linking properties of the scruin-calmodulin complex from the acrosomal process of Limulus sperm.

During activation of the Limulus sperm acrosomal process, actin filaments undergo a change in twist that is linked with the conversion from a coiled to a straight scruin-actin bundle. Since scruin had not been purified, its identity as an actin-binding protein has not been demonstrated. Using HECAMEG (methyl-6-O-(N-heptylcarbamoyl)-α-D-glucopyranoside) detergent extraction in concert with high calcium, we purified native scruin and identified it as an equimolar complex with calmodulin. I-Calmodulin overlays and calmodulin-Sepharose indicate that scruin binds calmodulin in calcium but not in EGTA. Overlay experiments also map the calmodulin binding site between the putative N- and C-terminal β-propeller domains within residues 425-446. Immunofluorescence microscopy reveals that calmodulin colocalizes with scruin and actin in the coiled bundle. Although scruin binds calmodulin, pelleting assays and electron microscopy show that the scruin cross-links F-actin into bundles independently of calcium. Based on our biochemical and structural studies, we suggest a model to explain how scruin controls a change in twist of actin filaments during the acrosome reaction. We predict that calcium subtly alters scruin conformation through its calmodulin subunit and the conformation change in scruin causes a shift in the relative positions of the scruin-bound actin subunits.

During activation of the Limulus sperm acrosomal process, actin filaments undergo a change in twist that is linked with the conversion from a coiled to a straight scruin-actin bundle. Since scruin had not been purified, its identity as an actin-binding protein has not been demonstrated. Using HECAMEG (methyl-6-O-(N-heptylcarbamoyl)-␣-D-glucopyranoside) detergent extraction in concert with high calcium, we purified native scruin and identified it as an equimolar complex with calmodulin. 125 I-Calmodulin overlays and calmodulin-Sepharose indicate that scruin binds calmodulin in calcium but not in EGTA. Overlay experiments also map the calmodulin binding site between the putative N-and C-terminal ␤-propeller domains within residues 425-446. Immunofluorescence microscopy reveals that calmodulin colocalizes with scruin and actin in the coiled bundle. Although scruin binds calmodulin, pelleting assays and electron microscopy show that the scruin cross-links F-actin into bundles independently of calcium. Based on our biochemical and structural studies, we suggest a model to explain how scruin controls a change in twist of actin filaments during the acrosome reaction. We predict that calcium subtly alters scruin conformation through its calmodulin subunit and the conformation change in scruin causes a shift in the relative positions of the scruin-bound actin subunits.
In many examples of cell motility including: cytokinesis, phagocytosis, exocytosis, chemotaxis, and extension of the lamella, movement or force is generated by either actin-myosin interactions or the reversible assembly of actin filaments (1). Contrary to these examples, extension of the acrosomal process in Limulus sperm may be a movement of an actin spring in which potential energy, stored as a coiled bundle at the base of sperm body, is unleashed at fertilization to uncoil and extrude the bundle through a channel in the nucleus (2,3). During the uncoiling process, the actin bundle untwists by an impressive 60°per 700 nm. This action is accompanied by slippage and a modest (Ϫ0.23°per subunit) untwisting of the actin filaments (4,5). As a result of these events, the bundle forms a 60-m-long membrane extension, which bridges the egg jelly coat to fuse with the egg plasma membrane.
The factors that maintain the coiled state of the bundle or signal its rotation and slippage are unknown, but the target of their action must be scruin, an actin-associated protein in the acrosomal process. Previous studies show that the acrosomal process consists of a 1:1 complex of actin and scruin (M r 102,000) (6). In EM 1 reconstructions, scruin decorates the outside of an actin filament, with each scruin molecule bound to a pair of actin subunits along the actin one-start helix (7). Presumably, actin cross-links are maintained by interactions between scruin proteins on neighboring filaments.
Based on sequence analysis, limited proteolysis, and EM image reconstructions, scruin is organized into two 40-kDa domains connected by a highly helical protease-sensitive neck (7)(8)(9). Each domain consists of a six-fold repeat of ϳ50 amino acids that based on the work of Bork and Doolittle (10), is predicted to fold into a four stranded ␤-sheet motif (9). This motif typifies a protein superfamily, which includes galactose oxidase (10), several open reading frames in the genome of pox viruses (11), a mouse placental transcript, MIPP (12), and kelch, the Drosophila gene that is important for nutrient transport during oogenesis (13). Although there is some understanding of the structural organization of scruin, its regulation and biochemical properties are not understood because the protein has not been purified in a native, soluble state.
To identify the mechanism that causes the dynamic conformational changes in the acrosomal process during the acrosome reaction, we must first purify scruin and characterize its actin binding properties. In this report, we describe the isolation of scruin and report its association with calmodulin. Furthermore, we show that the scruin-calmodulin complex crosslinks F-actin into bundles but, surprisingly, the cross-linking activity is independent of calcium. Our results suggests that scruin is always bound to actin filaments, and we hypothesize that during the acrosome reaction the conformational changes in the actin filament and acrosomal process may be caused by a subtle conformation change in scruin.

EXPERIMENTAL PROCEDURES
Materials-Pepstatin, leupeptin, aprotinin, benzamidine, and ATP were purchased from Sigma. HECAMEG and calcium ionophore A23187 were purchased from Calbiochem. 125 I-Calmodulin was purchased from DuPont NEN. Artificial sea water was purchased from Tropic Marin. Protein assays were performed with the Bio-Rad assay reagent.
Purification of the True Discharge-The true discharge was isolated using a protocol modified from Schmid et al., (6). Twenty ml of sperm, * This work was supported by National Institutes of Health Grants CA55621 and GM52703 (to P. M.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ To whom all correspondence should be addressed: Whitehead Institute, Nine Cambridge Center, Cambridge, MA 02145. collected from 30 -40 adult male horseshoe crabs, was divided into six aliquots, layered on 33 ml of a 1:1 mixture of artificial sea water and 50 mM calcium chloride, and then gently mixed. Sperm were activated by adding 100 l of 19 mM A23187 (in dimethyl sulfoxide) to each tube and incubated in the dark for 30 min. To prevent proteolysis, a mixture of protease inhibitors were added to each step (final concentrations: 0.1 mM phenylmethylsulfonyl fluoride, 0.1 mM benzamidine, 0.1 mM pepstatin A, 1.0 KIU/ml aprotinin, and 0.2 g/ml leupeptin). The activated sperm were sheared three times through a 21-gauge needle and centrifuged twice at 2420 ϫ g for 10 min to remove the sperm heads. The supernatant was then centrifuged at 43,140 ϫ g for 15 min to pellet the acrosomal bundles. Each pellet was resuspended in 5 ml of scruin buffer A (10 mM Tris, pH 8.0, 1 mM dithiothreitol, 1 mM CaCl 2 , 100 mM NaCl, 0.01% NaN 3 ) with 19 mM HECAMEG for 15 min on ice. The volume was diluted to 30 ml with scruin buffer A and centrifuged at 43,140 ϫ g for 15 min. The pellets were pooled into 1 ml of buffer A and pelleted at 16,000 ϫ g in a microcentrifuge for 15 min. To quantify the stoichiometry of scruin and calmodulin by HPLC, this washing step was repeated 5 times to reduce the protein contaminants. In a typical preparation, 5.2 mg of true discharge were obtained as determined by the Bio-Rad assay. The purity of the true discharge preparations was assessed by SDS-PAGE.
Purification of Soluble Scruin-Scruin was dissociated from actin by gently shaking the isolated bundles in 1 ml of 1 M calcium for 1 h at room temperature, and then centrifuging the extract for 15 min in a microcentrifuge (16,000 ϫ g) to remove most of the actin filaments. The low speed supernatant containing primarily scruin, calmodulin, actin, and some minor contaminants (3.7 mg of total protein) was successively filtered through 0.8-, 0.45-, and 0.2-m filters. In the later stages of the project, nucleic acids in the extract were removed by filtration through a 0.8-m filter and then a Qiagen Tip 5. The filtrate was loaded onto a Superdex 200 HR FPLC gel filtration column pre-equilibrated and run in scruin buffer A supplemented with inhibitors. The fractions that contained scruin were pooled (1.1 mg of total protein), loaded onto a Q-HiTrap (Pharmacia Biotech Inc.) ion exchange column with scruin buffer A, and eluted with a 0 -1 M NaCl gradient in buffer A. Scruin protein eluted in 0.4 M NaCl. The scruin-containing fractions (300 g of total protein) were either shell-frozen in liquid nitrogen and stored as a lyophilate or concentrated by Ultrafree-MC centrifugal membrane filters (Millipore) and used immediately.
Identification of Calmodulin-The stoichiometry of the scruin-actincalmodulin complex was determined by size exclusion chromatography through a TSK 300SWLX column (Toyo Soda) under denaturing conditions (6 M guanidine HCl). The absorbance was measured at 230 nm. The molar ratio was calculated from the integrated peak areas. The 17-kDa polypeptide was identified as calmodulin by protein sequence obtained by mass spectrometry or chemical sequencing. First, the scruin-calmodulin complex was dissociated in 6 M guanidine HCl and separated by size exclusion chromatography. The purified calmodulin was cleaved with cyanogen bromide in 70% formic acid for 12 h as described previously (Matsudaira, 1992). Following incubation the sample was diluted in water and dried under vacuum in a fume hood. The fragments were purified by reverse phase chromatography on the HPLC. The fractions that were found to contain a single polypeptide by mass spectrometry (LASERMAT, Finnigan MAT) were N-terminally sequenced by gas phase sequencing on a model 2090E sequencer (Beckman Instruments Inc.) equipped for the identification of phenythiohydantoin amino acids. In later experiments, Limulus sperm calmodulin was purified from sperm with a phenyl-Sepharose column on the FPLC using standard methods (14). The purified protein was confirmed to be calmodulin by calcium-dependent mobility shift on SDS-PAGE and sequence by mass spectrometry using a PerSeptive Biosystems Voyager Elite time of flight mass spectrometer.
Identification of the Calmodulin Binding Site-125 I-Calmodulin gel overlays were performed as described previously (15) with minor modifications (16). 125 I-Calmodulin nitrocellulose blot overlays were performed as described (17) using 0.05% Tween 20 and 30 mg/ml bovine serum albumin as the blocking agents. For scruin, we found that either method worked, but the nitrocellulose blot overlays had a higher signal to noise ratio. In contrast, the myosin I positive control only worked with the gel overlay method. The percent binding was quantitated using NIH Image software and is represented as the ratio of the signal intensity and the total signal in calcium and EGTA ϫ 100. A value of zero indicates the signal was not above the background levels. Competition assays with scruin and the peptide PSN1 (described below) were performed in calcium only.
Protein Expression of Scruin Fragments-The calmodulin binding site in scruin was mapped using either expressed scruin fragments or synthetic peptides to specific regions in scruin as described below. The 454C and 590C domains of scruin were expressed in Escherichia coli and purified as described previously (9). Additionally, three constructs, GST1, GST2, and GST3, consisting of residues 390 -429, 430 -469, and 470 -509 of scruin fused to the GST domain, respectively, were engineered by polymerase chain reaction using standard methods. Briefly, an in-frame BamHI site was introduced 5Ј to the first amino acid codon and a TAG stop/EcoRI site after the last codon. The resulting polymerase chain reaction products were cloned into the BamHI-EcoRI sites of pGEX-2T (Pharmacia) and the fidelity of the final expression constructs confirmed by sequencing. GST1 and GST3 were transformed into DH5␣ and expressed upon addition of isopropyl-1-thio-␤-D-galactopyranoside. However, because the GST2 fusion construct did not express in E. coli under a number of expression conditions, a synthetic peptide, PSN1 (CGAAKKVQRRWRRYIEQKSITKRM), was synthesized. The N-terminal sequence CG was included to aid in generating a sequence-specific polyclonal antibody. Two other synthesized peptides to a sequence near the protease-sensitive neck region of scruin and a sequence in ␤-scruin (R34, CKAKPQPGSKPTSVK; R35, CTTRSGSRKTQKTLK, respectively) were also used as controls (18).
Actin Cosedimentation Assays-Cosedimentation assays with purified scruin (0 -4 M), sperm calmodulin (15 M), and rabbit skeletal actin (2 M) were performed in the presence of 1 mM EGTA or 1 mM calcium as described previously (16). Briefly, samples were incubated for either 4°C overnight or for 1 h at room temperature and were centrifuged for 30 min in a bench-top ultracentrifuge (TL100, Beckman Instruments Inc., Fullerton, CA) at 75,000 rpm at 4°C. Pellets and supernatants from the centrifugation were adjusted to equal volumes and analyzed by SDS-PAGE, and electron microscopy. To determine whether the ability of scruin to bind and bundle F-actin was affected by calmodulin, exogenous sperm calmodulin was added to scruin at 3.75fold molar excess.
Reconstitution of Scruin-Actin Bundles-Scruin-actin bundles were formed by incubating scruin and actin, with or without exogenous sperm calmodulin at a molar ratio of at a 2:1:3.75, respectively, for 1 h at room temperature prior to absorbing the samples onto carbon films. The samples were negatively stained with 1.0% uranyl acetate and examined in a Phillips 410 electron microscope at an accelerating voltage of 80 kV. In some instances calmodulin was added to the samples at 0 -5-fold molar excess of scruin.
Immunofluorescence Microscopy-A rabbit polyclonal antiserum (R213.5) was generated against scruin; the specificity to crude sperm extracts has been described elsewhere (18). The mouse monoclonal anti-calmodulin antiserum was purchased from Upstate Biotechnology, Inc. (Lake Placid, NY). To reduce the nonspecific background of the anti-scruin antibody for immunofluorescence, the antibody was affinitypurified against scruin blotted to nitrocellulose membrane (19). Sperm were fixed and stained as described elsewhere (18). Coverslips were examined in a Zeiss Axioskop microscope using differential interference contrast optics and a 100ϫ/NA 1.3 Plan Neofluar objective configured with a Bio-Rad MRC600 Confocal ion laser. The ratio of the fluorescent intensity in the bundle and the entire sperm was quantified using the standard confocal image analysis software. For reproduction, the images were transferred to Adobe Photoshop and Illustrator (Adobe Systems Inc., Mountain View, CA) for cropping, contrast/brightness adjustment, placement of each image panel, and annotation.

RESULTS
Treatment of true discharges, isolated in Triton X-100, with 1 M calcium disassociated scruin from actin. However, after a few hours, the soluble scruin precipitated from solution (not shown). Subsequent experiments determined that long term solubility depended on the removal of the Triton X-100 by dialysis at high ionic strength (0.45 M NaCl). Other treatments such as denaturing agents (8 M urea) and low pH (0.2 M glycine, pH 4.2) were found to also disassociate the scruin actin bundles, but less than half of the scruin remained active, as judged by high speed sedimentation with rabbit skeletal actin (100,000 ϫ g for 30 min: not shown). Based on these findings, we replaced Triton X-100 with the non ionic detergent HECAMEG. HECAMEG is easier to remove by gel filtration or dialysis (critical micelle concentration 19.5 mM) and does not absorb at 280 nm. After demembranation of the actin bundle with HECAMEG and extraction of scruin with 1 M CaCl 2 , one half of the actin pellets at low g-forces leaving scruin in the superna-tant (Fig. 1A). The minor protein contaminants, DNA, and HECAMEG in the calcium extract were removed by gel filtration chromatography through Superdex HR 200 and ion exchange (Fig. 2). Based on relative molecular mass (M r ) standards, scruin fractionates as a monomer with an apparent molecular weight of 107,000.
Identification of Calmodulin-At each stage of the purification, a 17-kDa polypeptide consistently cofractionated with scruin ( Figs. 1 and 2). Although the N terminus was blocked to chemical sequencing, we obtained sequence from two fragments generated by cyanogen bromide cleavage (MKDTD-SEEEI and MIREADIDGDGQVNYEEFVTM). Data base searches with these two sequences showed an exact match with calmodulin from Drosophila. Additionally, this 17-kDa protein shows the characteristic calcium-dependent mobility shift on SDS-PAGE gels and is recognized by a monoclonal antibody to calmodulin (not shown).
The presence of calmodulin was also confirmed by immunofluorescence microscopy (Fig. 3). In unactivated sperm cells, calmodulin is localized to the base of the nucleus as a ring of fluorescence, which colocalizes with the rhodamine phalloidin staining pattern of F-actin or immunostaining of scruin. Quantitation of the fluorescence showed that 71% (S.D. ϭ Ϯ 7.6, n ϭ 7) of the calmodulin was found to localize to the bundle at the base with a less apparent staining in the nucleus and the perimeter of the acrosomal vesicle. No calmodulin staining was observed within the flagellar region of the sperm or the interior of the acrosomal vesicle.
To determine the stoichiometry of the scruin, actin, and calmodulin in the acrosomal process, we quantified the peak areas of samples separated by gel exclusion chromatography in denaturing conditions. Based on the integrated peak areas and the known molecular masses for the proteins, the scruin:actin: calmodulin molar ratio was 1.00:1.15:0.97 (Fig. 1B). Although scruin and calmodulin were always seen to co-purify, the molar ratio of the two proteins was sometimes 1:0.5, depending on the individual preparation. This variability in the ratio of scruin to calmodulin after purification either is due to the extraction conditions or is merely a consequence of an equilibrium for binding and subsequent dissociation of calmodulin during purification.
Calmodulin Binding Site-To confirm that calmodulin is a scruin subunit, we tested binding of exogenous bovine calmodulin to scruin. By two criteria: 125 I-calmodulin blot and gel overlays (Fig. 4) and calmodulin-Sepharose chromatography (not shown), calmodulin binds scruin in calcium but not in EGTA.
Although scruin is predicted to be mainly ␤-sheet, secondary structure analysis predicted that the neck region (Fig. 5a) between the N-and C-terminal domains of scruin is highly helical and amphipathic. A comparison of this region with the calmodulin binding IQ motif of MYO2 highlights the similar pattern of basic and hydrophobic residues. A helical wheel representation of a portion of the neck region clearly shows a basic and a hydrophobic face of the helix ( Fig. 5b; Refs. 20 and 21). Because calmodulin binds to the basic face of an amphipathic helix, we examined calmodulin binding to various synthetic peptides, proteolytic fragments, and GST fusions that shows that scruin co-purifies with a 17-kDa band that was determined by internal protein sequencing to be calmodulin. spanned the neck region. Two GST fusion constructs, GST1 and GST3, contain sequences that border outside of the predicted calmodulin binding site. These fusion constructs did not bind the radiolabeled calmodulin (Table I). The third GST fusion (GST2) which contains this region did not express in E. coli; however, a synthetic peptide, PSN1, to the region 425-446 did bind calmodulin in EGTA and calcium. The peptide at a concentration of 50 nM also inhibited calmodulin binding to intact scruin by 61% (Fig. 4D). In other experiments, the Cterminal half of scruin (454C and 590C, produced by expression in E. coli; Ref. 9), a tryptic digestion of scruin, or natural breakdown products of scruin did not bind to the radiolabeled calmodulin in the presence or absence of calcium (not shown). These proteolytic sites have been previously mapped to the protease-sensitive neck region of scruin (9), suggesting that the calmodulin binding site is not in the C-or N-terminal halves of scruin.
Characterization of Actin Cross-linking Activity-Because scruin is the only actin-associated protein in the acrosomal process, we tested it for actin binding activity using a cosedimentation assay and electron microscopy. Scruin binds actin independently of calcium. In calcium and EGTA, the majority of the scruin is found associated with actin in the pellet (Fig. 6). There is no difference between binding in EGTA and calcium. Furthermore, the amount of actin found in the pellet is invariant suggesting that scruin does not alter the F-to G-actin equilibrium. In the absence of actin, scruin remains in the supernatant in high g-force sedimentation assays (not shown). Since exogenously added calmodulin may affect the binding affinity of scruin for actin, cosedimentation assays were also performed with a 3.75-fold molar ratio of exogenously Limulus calmodulin (not shown). Examination of the samples by electron microscope before centrifugation shows that actin bundles form in the presence and absence of calcium (Fig. 7). The results from sedimentation assays and electron microscopy show that the presence or absence of excess calmodulin does not detectably affect actin binding activity of scruin.

DISCUSSION
The long term goal of our studies is to understand how the actin bundle uncoils during the acrosome reaction to form the 60-m-long acrosomal process. We have made major strides toward this goal through the development of methods to separate and purify native scruin from actin. Although there are several three-dimensional views of the filaments in the acrosomal process (7,8,22), this study provides the first biochemical information about the protein components of the acrosomal process. Two tricks enabled us to purify scruin as a soluble and native protein and characterize it as an actin cross-linking protein. First, because calcium disrupts the acrosomal process (2), we were able to extract scruin as a native protein without using denaturants. Second, we modified the demembranation step by replacing the detergent Triton X-100 with HECAMEG. This eliminated the aggregation of the purified scruin and improved the overall yield of protein. With the purified scruin, we first confirmed that scruin is an actin cross-linking protein. Previously the only evidence for actin binding was the observation that scruin is the only actinassociated protein in the acrosomal process and that EM reconstructions reveal the outside of the actin filament is decorated in a 1:1 stoichiometry by a two-domain protein, approximately the mass of scruin. Although these are powerful arguments that scruin is the actin cross-linking protein in the acrosomal process, they do not substitute for direct evidence. Thus, it was critical to show that scruin binds actin filaments and reconstitutes actin filaments into bundles. In the reconstituted bundles, the approximate equimolar ratio of scruin and actin is maintained but the reconstituted actin bundles do not display the characteristic banding pattern that is indicative of the crystalline organization of F-actin and scruin in the acrosomal process. This difference in apparent organization between the in vitro and in vivo assembled bundles could easily be attributed to the assembly conditions. For example, the slow assembly of the actin bundle during spermatogenesis may be more favorable to the formation of a crystalline bundle, while the relatively rapid assembly of the bundle in vitro may cause the disorder.
One novel and important finding reported in this paper is that scruin is complexed with a single calmodulin molecule. The identification of calmodulin and its binding site in the neck region provides a more detailed model of scruin structure. In EM reconstructions, a neck region connects two large domains each of which binds a separate actin subunit on the same filament. Based on secondary structure predictions and similarity with galactose oxidase, the protein sequence of scruin is organized as two large ␤-sheet domains, which are separated by a highly helical region. The domain organization suggested by the sequence is in exact correspondence with the EM reconstructions; thus, we propose that the helical region of sequence is the neck region. Within this helical region is the calmodulin binding sequence (residues 425-446) of PNS1. Thus, calmodulin bound to the neck would be flanked by a pair of actin-binding domains. We had not detected calmodulin previously because of its low molecular weight and relatively poor dyebinding capacity (6,8,23). Because the previous EM reconstructions were of acrosomes that may have been partially depleted of calmodulin (prepared in the absence of calcium), we are checking the new preparations to see if the neck region is thicker from the presence of calmodulin.
Our present evidence shows both calcium-dependent binding of calmodulin to intact scruin and calcium-independent binding to the PNS1 scruin neck peptide. We speculate that the flanking large domains in scruin partially inhibit the binding of calmodulin in the absence of calcium. In either case the immunofluorescence localization of calmodulin within the membrane-limited acrosomal process suggests the possibility that the local concentration of calmodulin could be high. A high calmodulin concentration would ensure a calmodulin-scruin complex is maintained in unactivated sperm.
The presence of a calmodulin subunit immediately suggests that actin binding by scruin is calcium regulated. Normally, in many enzymatic complexes, calmodulin is a regulatory subunit, which binds or dissociates from a target catalytic subunit and thus acts as a calcium-dependent switch to activate or inactivate the enzyme. However, our biochemical studies suggest a different type of regulatory mechanism, because scruin binds actin independently of calcium. We conclude that calmodulin does not regulate actin binding activity in an on-off fashion. This finding eliminates a simple on-off binding event as a mechanism for inducing the conformation changes in the actin filament. Instead, our results suggest that scruin is bound to actin before as well as after the acrosome reaction. Although the coiled bundle of unactivated sperm has not been studied, our speculation is supported by a related structure, the supercoiled false discharge, which has been shown to contain scruin (8). Based on our studies, we propose a model that calmodulin may instead control the twist of an actin filament or bundle by regulating the conformation of scruin. We envision a mechanism in which calmodulin acts as a wedge between the actinbinding domains (Fig. 8). In this position, changes in the conformation of calmodulin could alter the relative positions of the actin binding domains. The conformation change in scruin is then transmitted to the underlying actin subunits which allows the actin filaments to untwist by 0.23°between subunits (4, 5).  The local change in twist is multiplied along the length of the filament causing a large change in the filament. The change in filament twist breaks scruin-scruin cross-links between neigh-boring filaments which allows the filaments to slip as the bundle uncoils into the straight acrosomal process. The caveat to this model is that we do not yet appreciate how scruin-scruin interactions allow for the formation of bundled filaments, since under the present conditions purified scruin appears to be monomeric. It is possible that scruin interactions with itself are much weaker than actin-scruin or scruin-calmodulin interactions. Alternatively, the scruin-scruin binding site may not be exposed until scruin binds actin.  A cross-section diagram (viewed from the tip) of a scruin-bound actin filament in the acrosomal process before and after calcium binding. A pair of actin subunits is associated with a scruin calmodulin complex that is not drawn to scale, and the angular untwisting is exaggerated to show this subtle change in structure. Upon sperm activation, calcium ions bind to calmodulin, which initiates a series of conformation changes. First, in calcium, calmodulin binds scruin more tightly, which then induces a conformation change in scruin. Consequently, scruin binds more tightly to actin, which causes a subtle rearrangement in the pair of actin subunits. This rearrangement allows the unbending and extension of the actin bundle.
FIG. 6. Cosedimentation of scruin with actin in EGTA and calcium. A, various concentrations of scruin were incubated with 2 M actin in the presence of EGTA (Ⅺ) and calcium (q). SDS-PAGE samples of supernatants and pellets show that in calcium and EGTA scruin binds avidly. B, quantitation of the pelleting assays shows the slightly higher affinity for actin in calcium than in EGTA. However, there is no apparent difference in actin binding in the presence of exogenous sperm calmodulin (data not shown). In the absence of actin, all scruin remained in the supernatant (data not shown).