(Received for publication, July 11, 1995; and in revised form, October 5, 1995)
From the
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.
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 (-0.23° per subunit) untwisting of the actin filaments(4, 5) . As a result of these events, the bundle forms a 60-µm-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 102,000)(6) . In EM (
)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 50 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 cross-links 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.
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 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
,
one half of the actin pellets at low g-forces leaving scruin
in the supernatant (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
) standards, scruin fractionates as a monomer
with an apparent molecular weight of 107,000.
Figure 1: Isolation and calcium treatment of scruin-actin bundles and stoichiometry of scruin-actin-calmodulin complex. A, SDS-PAGE of true discharges extracted with HECAMEG (lane T) show the presence of scruin, actin, and a 17-kDa protein. The supernatant (lane S) of the calcium extract contained mostly scruin and a 17-kDa protein, whereas the pellet (lane P) was enriched in actin. B, HPLC traces of washed HECAMEG true discharges indicate that the ratio of scruin:actin:calmodulin is essentially 1:1:1.
Figure 2: Copurification of calmodulin and scruin by gel filtration and quaternary amine ion exchange chromatography. A, the low speed supernatant was chromatographed through Superdex 200 HR (top panel). SDS-PAGE (bottom panel) of the fractions shows scruin and a 17-kDa polypeptide co-fractionate. B, a pool of scruin-containing fractions was loaded onto a Q-HiTrap column. The chromatograph (top panel) indicates a single peak elutes at at 40% buffer B. SDS-PAGE (bottom panel) shows that scruin co-purifies with a 17-kDa band that was determined by internal protein sequencing to be calmodulin.
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. = ± 7.6, n = 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.
Figure 3:
Distribution of scruin and calmodulin in Limulus sperm. a and b, DIC images of
unactivated sperm show the presence of long flagella and an apical
vesicle. c, phalloidin (green) and -scruin (red) colocalize to the coiled actin bundle at the base of the
nucleus. d, calmodulin (green) primarily colocalizes
with phalloidin (red) in the bundle at the base of the sperm.
Additionally, the calmodulin also is also located in the nuclear region
and at the perimeter of the acrosomal vesicle. Bar =
5.0 µm.
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.
Figure 4:
Scruin binds calmodulin avidly in calcium
based on blot overlays with I-calmodulin. Purified scruin
was electrophoresed through SDS-PAGE gels and either stained with
Coomassie Blue (A) or electroblotted to nitrocellulose
membranes (B and C). The membranes were incubated
with
I-bovine calmodulin in the presence (B) or
absence (C) of calcium. The calmodulin bound only to scruin in
calcium; little or no binding was detected in EGTA. Similar results
were observed with gel overlays (not shown). Myosin I bound calmodulin
in calcium and EGTA as reported previously (data not shown; (24) ). D, competition experiments with scruin and
PSN1, the peptide containing the calmodulin binding site. Calmodulin
binding to scruin was inhibited by nanomolar concentrations of PSN1.
The estimated K
is <50 nM.
Each point is an average of two intensity
values.
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 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 1). 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 C-terminal half of
scruin (454C and 590C, produced by expression in E. coli; (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.
Figure 5: The putative calmodulin binding site. a, the protease-sensitive neck region of scruin contains an amphipathic helix that is similar to the calmodulin binding IQ motif of MYO 2. Bold text delineates conserved residue matches. b, the helical wheel representation of the peptide PSN1 identifies basic and hydrophobic faces, which are characteristic of a calmodulin binding motif.
Figure 6:
Cosedimentation of scruin with actin in
EGTA and calcium. A, various concentrations of scruin were
incubated with 2 µM actin in the presence of EGTA
() and calcium (
). 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).
Figure 7: Reconstitution of scruin actin bundles. Based on electron microscopy, scruin is capable of forming bundles with rabbit skeletal F-actin in the presence of EGTA (a) and calcium (b). These bundles have a similar packing to the Limulus bundles (c) but are not crystalline. In the absence of scruin F-actin does not form bundles, and in the presence of scruin alone no filamentous structures are observed (data not shown). With the addition of exogenous sperm calmodulin at a 3.75-fold molar excess over scruin, there is no apparent difference in the formation of bundles (data not shown). Bar = 100 nm.
The long term goal of our studies is to understand how the actin bundle uncoils during the acrosome reaction to form the 60-µm-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 actin-associated 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 dye-binding
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 actin-binding 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 neighboring 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.
Figure 8: Model for extension of the bundle during sperm activation. 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.