From the Secretory Process Research Program, Department of
Pharmacology, Faculty of Medicine, University of Ottawa, Ottawa,
Ontario K1H 8M5, Canada
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INTRODUCTION |
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.
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MATERIALS AND METHODS |
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
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 |
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.
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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 ( ) 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 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.
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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.
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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.
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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. , actin;
, actin:r-Sc, 400:1; , actin:Sc5-6, 400:1; ,
actin:ScL5-6, 400:1; , actin:Sc1-4,6, 400:1.
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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.
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DISCUSSION |
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).
We are grateful to S. J. Dunn for typing
the manuscript and to the Ottawa Red Cross for supplying platelet-rich
plasma.