 |
INTRODUCTION |
Profilin is a remarkable actin-binding protein. This small
(15-kDa), ubiquitous, essential protein binds monomeric (G) actin in a
1:1 molar ratio (1). Although the profilin-actin complex is unable to
spontaneously nucleate actin filaments, it can productively associate
with the barbed ends specifically (2, 3). This unique property confers
a dual function to profilin, depending on the capping of barbed ends.
When barbed ends are blocked by capping proteins, profilin sequesters
G-actin. In contrast, in motile regions of the cell where uncapped
barbed ends are actively elongating, the profilin-actin complex can
actively participate in filament growth. The profilin-MgATP-actin
complex (PA)1 can therefore
be considered as an end-specific quasipolymerizable actin monomer.
Recent results (4) show that this property of profilin is used to
enhance the processivity of treadmilling (i.e. steady-state
barbed end assembly)in the presence of actin depolymerizing factor. In
the mechanism that was proposed based on thermodynamic data (5, 6),
F-actin assembly from profilin-actin is possible only because of its
coupling to ATP hydrolysis, as follows. Profilin-actin associates with
the barbed end; the interaction of profilin with actin is weakened once
the actin-bound ATP has been hydrolyzed; profilin then dissociates from
that end, thus promoting the incorporation of one actin subunit in the
filament and regenerating a free barbed end available for further
growth from PA. Profilin is reused at each cycle and works as a
catalyzer of assembly. While polymerization of actin alone proceeds in
a manner uncoupled from ATP hydrolysis (7, 8), these two reactions are
proposed to occur in a compulsory order when actin filaments grow from
profilin-actin units. In this paper, the proposed mechanism of barbed
end growth from profilin-actin is challenged by kinetic experiments.
Understanding the kinetics of filament assembly from profilin-actin in
depth also has implications concerning the structure of the filament.
In the atomic model of the actin filament proposed by Holmes et
al. (9) and refined by Lorenz et al. (10), the profilin
interaction area on actin is exposed at the barbed end, accounting for
the association of profilin-actin to a growing barbed end, but not to a
pointed end. Hence, within Holmes' model, it is anticipated that
profilin can cap the barbed ends. On the other hand, examination of the
actin-actin contacts in the crystals of the profilin-actin complex (11)
shows evidence for a nonhelical ribbon structure of the profilin-actin
complex, in which extensive actin-actin contacts, however different
from those present in the Lorenz et al. atomic model of the
actin filament (10) are involved. It was proposed (12, 13) that barbed
end growth from profilin-actin could involve the transient extension of
profilin-ATP-actin ribbons. The transition from ribbon to filament
would be coupled to ATP hydrolysis and dissociation of profilin.
According to this model, the profilin-actin ribbon would exist in
solution only in the ATP- or ADP-Pi-bound form, and the
orientation of the actin monomer in the filament derived from the
ribbon would be different from the Holmes atomic model.
In the present work, we first determined the rate parameters for barbed
end elongation from profilin-actin using a turbidimetric method. We
next attempted to find conditions under which profilin-actin would
undergo spontaneous self-assembly, with the goal to characterize the
profilin-actin polymer. The EDC-cross-linked profilin-actin complex
appears able to polymerize into filaments, which exhibit the same
helical structure and same thermodynamic stability as native F-actin
filaments. Profilin-actin filaments therefore provide a tool to probe
the structure of the actin filament and the interface of actin with
other actin-binding proteins.
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MATERIALS AND METHODS |
Proteins--
Actin was purified from rabbit skeletal muscle
acetone powder (14) and isolated as CaATP-G-actin through Sephadex
G-200 chromatography (15) in G buffer (5 mM
Tris/Cl
, 0.1 mM CaCl2, 0.2 mM ATP, 1 mM dithiothreitol, 0.01%
NaN3, pH 7.8). MgATP-G-actin (<20 µM) was
prepared by incubation of CaATP-G-actin with 0.2 mM EGTA
and 1 mol eq plus 10 µM excess MgCl2 for 3 min and used immediately afterward. MgATP-G-actin was polymerized by
the addition of 2 mM MgCl2 and 0.1 M KCl. CaATP-G-actin was polymerized by the addition of 0.1 M KCl. Actin was NBD-labeled (16). Spectrin-actin seeds
were prepared from human erythrocytes, and their molar concentration
was determined as described (17) and by titration by gelsolin.
Gelsolin-actin seeds were prepared at a 2 µM
concentration by incubation of 2 µM human plasma gelsolin (a generous gift from Dr. Yukio Doi) with 4.2 µM
CaATP-G-actin in G buffer. Profilin was isolated from bovine spleen by
poly(L-proline) affinity chromatography (18).
S1(A1) and S1(A2)
isoforms of chymotryptic myosin subfragment-1 were resolved by
SP-trisacryl chromatography (19).
Fluorescence and Light Scattering Measurements--
Fluorescence
measurements were carried out at 20 °C using a Spex Fluorolog 2 spectrofluorimeter at the following wavelengths: NBD-actin,
exc 475 nm and
em 530 nm;
rhodamine-phalloidin,
exc 530 nm and
em
575 nm; tryptophan fluorescence,
exc 295 nm and
em 330 nm.
Turbidity Measurements--
Turbidity measurements were
performed at 20 °C at 310 nm using a Cary 1 Varian spectrophotometer
with 1-cm path cuvettes. All solutions were thoroughly filtered and
degassed before the experiment.
Measurement of the Initial Rate of Filament
Elongation--
Initial rates of filament growth were measured using
turbidity or NBD fluorescence. Spectrin-actin seeds were used to
initiate elongation at the barbed end (20), while gelsolin-actin seeds initiated elongation at the pointed end. At time 0, MgATP-G-actin (or
CaATP-G-actin), in the absence or presence of profilin or profilin-actin covalent complex, was supplemented simultaneously with
seeds and salts, and the time course of assembly was recorded. The
actin concentrations were chosen so that the spontaneous nucleation could be neglected.
The rate of barbed end growth J at a given total
concentration of G-actin ([Ao]) and in the presence of
different total amounts [Po] of profilin was monitored
turbidimetrically. Data were analyzed as follows,
|
(Eq. 1)
|
with [Ao] = [A] + [PA].
In Equation 1, [S] is the concentration of spectrin-actin seeds;
[A] and [PA] are the concentrations of free and profilin-bound G-actin; and k+A and
k+PA are the corresponding barbed
end association rate constants. The contribution of the off rates was
neglected in equation 1 because measurements were carried out at actin
concentrations well above the critical concentration. The change in
J as the concentration [Po[ of profilin was
increased reflected the increase in the contribution of PA to barbed
end assembly as G-actin was gradually saturated by profilin. The
concentration of PA was calculated as follows,
|
(Eq. 2)
|
where KPA represents the equilibrium
dissociation constant for profilin-actin.
Measurements of J in absorbance units/s were converted in
µM assembled F-actin/s using a critical concentration
calibration curve as described previously (21), from which the
polymerization of 1 µM F-actin led to an increase of
0.0017 absorbance units at 310 nm (1-cm optical path).
Combining Equations 1 and 2 led to the expression of J as a
function of [Po], [Ao], and
KPA. Analysis of the data within this expression
led to the determinations of k+A
(from the measurement of J in the absence of profilin),
k+PA (from the measurement of
J in the presence of saturating amounts of profilin), and
KPA, by adjustment of the theoretical curve to
the data at different concentrations of profilin.
The rate of barbed end growth was also measured at different
concentrations of G-actin and either in the absence or in the presence
of saturating amounts of profilin (i.e. 1 mol eq of G-actin plus an excess of 5 µM bovine profilin or of 30 µM Arabidopsis thaliana profilin 3 (6)). The
resulting J(c) plots for actin and profilin-actin
were used to derive the values of
k+B for actin or profilin-actin
and of the critical concentrations Cc for
polymerization, as follows.
|
(Eq. 3)
|
All experiments were performed using freshly prepared G-actin in
G buffer containing 1 mM dithiothreitol to be sure that Cys374 was thoroughly reduced, so that all of the actin in
the preparation was able to bind profilin with high affinity. We
reasoned that if a very small proportion of G-actin was oxidized, it
would give rise to spontaneous polymerization at high actin
concentrations even in the presence of an excess of profilin.
Therefore, where appropriate, the spontaneous polymerization measured
in the absence of seeds was subtracted from the data.
Chemical Cross-linking of Actin and Profilin--
The
cross-linking of Glu364 of actin to Lys125 of
bovine profilin (which corresponds to Lys115 of
Acanthamoeba profilin) was performed using EDC as described (22, 23). Briefly, actin was dialyzed at 4 °C versus 2 mM HEPES, 0.2 mM CaCl2 buffer (pH
7.8); profilin was prepared in 5 mM HEPES, 1 mM
dithiothreitol, 0.2 mM CaCl2 buffer (pH 7.8). Samples were brought to 20 °C. Actin (15 µM, 60 ml)
was activated with 2 mM EDC and 2 mM
sulfo-N-hydroxysuccinimide for 20 min at pH 6.5. 20 µM
profilin and 0.2 mM dithiothreitol were then added, the pH
was adjusted to 7.8, and the reaction was allowed to proceed for 30 min
before it was quenched by 10 mM glycine. The yield of
the cross-link was about 15% (Fig. 1, inset,
lane a).
Purification of the Profilin-Actin Covalent Complex--
The
mixture obtained at the end of the cross-linking reaction was first
loaded on a DEAE-cellulose column (DE52; Whatman; 2.5-cm diameter,
10-cm length) equilibrated in DEAE buffer (5 mM HEPES, 0.2 mM CaCl2, 0.2 mM ATP, pH 7.8) (Fig.
1). Free profilin was recovered in the flow-through. Following a wash
step with one column volume of DEAE buffer supplemented with 50 mM NaCl (Fig. 1, inset, lane
b), the mixture of free actin and covalent profilin-actin
complex was eluted with DEAE buffer containing 250 mM NaCl
(Fig. 1, inset, lane c). The eluate
was applied to a poly(L-proline)-agarose column (1.5-cm
diameter, 12-cm length) equilibrated in PLP buffer (10 mM
Tris/Cl
, 0.2 mM CaCl2, 0.2 mM ATP, 0.1 M glycine, 0.1 M KCl,
pH 7.8). Free actin was recovered in the flow-through (Fig. 1,
inset, lane d), and profilin-actin
covalent complex was then eluted with PLP buffer containing 30%
Me2SO, according to the procedure used for isolation of
profilactin complex (24). The eluted complex was rapidly concentrated
to about 30 µM by ultrafiltration over a Diaflo PM30
membrane in a 100-ml Amicon cell, dialyzed against G buffer, and
centrifuged at 400,000 × g for 45 min.
The covalent complex obtained at this stage was at least 90% pure as
juged by SDS gel electrophoresis and appeared able to self-assemble
reversibly upon the addition of salt. The capacity of covalent
profilin-actin to undergo self-assembly declined with time following
isolation of the complex. About 70% of the covalent profilin-actin
complex that eluted from poly(L-proline)-agarose was
polymerizable on day 1. This proportion declined by 2-fold within 2 weeks. A cycle of polymerization was added as a final step of the
purification of covalent profilin-actin, as follows. The solution of
covalent PA was incubated with 0.2 mM EGTA and 40 µM MgCl2 for 3 min and then overnight with 2 mM MgCl2 and 0.1 M KCl at room
temperature. The solution was centrifuged at 400,000 × g for 45 min. The pellet, which typically contained about
70% of initial PAcov, was resuspended and dialyzed against
G buffer for 24 h, with a brief sonication after 16 h. The
solution was again centrifuged at 400,000 × g for 45 min. No visible pellet was found. The supernatant consisted of pure
covalent profilin-actin (Fig. 1,
inset, lane e). About 2.5 mg of
covalent profilin-actin complex were obtained, corresponding to 7% of
the initially reacted actin. This material was used within a week.
Small amounts of oligomers of high molecular mass, occasionally present
after the cross-link, were eliminated in the purification steps
(compare lanes a and e). Several
independent preparations of covalent profilin-actin complex yielded a
material that behaved in a reproducible fashion in the different
experiments. The concentration of the covalent profilin-actin complex
was determined by the Bradford assay (Bio-Rad reagent) using G-actin as
a standard.

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Fig. 1.
Purification of the EDC-cross-linked
profilin-actin complex. Shown is the elution profile of the
DEAE-cellulose chromatography. Separation of free profilin from the
mixture of actin and covalently cross-linked profilin-actin is shown.
Inset, gel electrophoresis pattern of PAcov at
different stages of purification. Lane a,
reaction mixture before purification; lane b,
pooled fractions 6-12 from the DEAE column; lane
c, pooled fractions 32-36 from the DEAE column;
lane d, flow through from the
poly-L-proline column; lane e,
purified profilin-actin covalent complex eluted from
poly-L-proline and submitted to one cycle of
polymerization; lane f, molecular mass standards
(in kDa).
|
|
Electron Microscopy--
Profilin-actin covalent complex (1.5 µM) in G buffer was first converted into MgATP-bound
PAcov by incubation with EGTA and MgCl2 as
described above for G-actin and was induced to polymerize by the
addition of 2 mM MgCl2 and 0.1 M
KCl at 20 °C. At different times of the polymerization process,
4-µl aliquots of the sample were deposited on air glow-discharged
carbon coated grids. Following adsorption to the grid for 10 s,
the specimens were negatively stained with 2% uranyl acetate and
observed in a CM12 (Philips) electron microscope. Electron micrographs
taken at a 35,000-fold magnification on electron image plates (Eastman
Kodak Co.) were developed for 5 min in full-strength D19 developer
(Kodak). Images were selected according to the quality of their optical
diffraction pattern (no astigmatism, defocus value in the range 0.5-1
µm, presence of the first and sixth layer lines). Images were
digitized with a rotating drum microdensitometer (Optronics P-1000)
using a scan raster of 25 µm. The filaments were straightened and cut to a length corresponding to 4-7 axial repeats and a width slightly larger than the diameter. The axial repeat is typically equal to 36.5 nm. The mean value of the densities surrounding the filaments was
subtracted from the images, which were floated in 512 × 512 arrays. Fourier transforms were calculated. They were characterized by
the presence of three layer lines (ll0, ll1, and ll6), which can be
indexed by the selection rule l =
6n + 13m.
Sedimentation Assay for Binding of S1 to
F-PAcov--
Actin or PAcov complex in G
buffer was freed from ATP by Dowex-1 treatment (25), polymerized at 5 µM, and split into samples supplemented with 0, 4, 6, 8, 10, or 20 µM S1(A1). Samples were centrifuged 15 min later at 400,000 × g for 10 min at
20 °C in the TL 100 Beckman ultracentrifuge. Pellets were
resuspended in the original volume of G buffer. Supernatants and
resuspended pellets were submitted to SDS-polyacrylamide gel
electrophoresis and Coomassie Blue-stained.
ATPase Measurements--
Actomyosin MgATPase activity of myosin
subfragment-1 was measured in the presence of 17 µM
F-actin or F-PAcov and 1 µM
S1(A1) at 20 °C in a buffer containing 5 mM Tris/Cl
, pH 7.8, 0.1 M KCl, 2 mM MgCl2, 1 mM
-32P-labeled ATP. The reaction was started by the
addition of S1(A1). Aliquots of the reaction
mixture were removed from the solution at 30-s intervals; brought into
1 N HCl, 10 mM ammonium molybdate; and
processed for 32Pi extraction as described
(8).
Actomyosin Motility Assay--
Actin or PAcov was
polymerized overnight at 1 µM in the presence of 0.05 M KCl, 2 mM MgCl2, and 1 µM tetramethyl rhodamine-phalloidin. In vitro
motility assays were conducted using whole myosin tethered to
monoclonal antibodies (anti LMM 5C3-2) immobilized onto a
nitrocellulose-coated glass coverslip, as described (26). The
rhodamine-phalloidin filaments were diluted to 3-5 nM in
motility buffer (25 mM imidazole, 25 mM KCl, 4 mM MgCl2, 5 mM 2-mercaptoethanol,
0.2 mM CaCl2, 7.5 mM ATP, 0.1%
methylcellulose, pH 7.6) supplemented with oxygen scavengers.
Observations were made on a Zeiss microscope using a 100× Plan
Apochromat objective equipped with epifluorescence optics. Images of
moving filaments were recorded with a SVHS video recorder.
 |
RESULTS |
Filament Assembly at the Barbed Ends from Profilin-Actin Requires
Mg2+ and Not Ca2+ as Divalent Metal Ion Tightly
Bound to G-actin--
Since profilin binds derivatized actin very
poorly (27), the conventional method using the increase in fluorescence
of N-pyrenyl-carboxyamidomethyl- or NBD-labeled actin as a
probe of polymerization is inadequate in its presence. Turbidimetry,
which has proven useful to monitor actin polymerization in the case of
ADF/cofilin (21), was used. The initial rate of barbed end assembly
from spectrin-actin seeds was measured at a given G-actin concentration
and in the presence of increasing amounts of profilin. Fig.
2a shows that when filament barbed ends elongated from CaATP-G-actin subunits, profilin inhibited the growth in a concentration-dependent fashion. Total
inhibition of growth was observed at saturation by profilin. Data,
analyzed as described under "Materials and Methods," indicate that
profilin binds CaATP-G-actin with an equilibrium dissociation constant of 1.2 ± 0.2 µM and that the profilin-CaATP-actin
complex does not participate in barbed end assembly. Preassembled
F-actin (5 µM) was also used to measure barbed end growth
in the presence of 5 µM CaATP-G-actin, either in the
absence or presence of 20 µM profilin. Turbidity
increased in the absence of profilin, demonstrating active barbed end
growth, but decreased when profilin was added together with G-actin.
70% depolymerization of the F-actin seeds was observed within 5 min.
In conclusion, in the presence of CaATP-actin, profilin acts as a pure
G-actin-sequestering protein. In particular, it does not cap the barbed
ends.

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Fig. 2.
Polymerization properties of profilin-actin
complex. a, profilin-MgATP-actin, but not
profilin-CaATP-actin, participates in filament growth at the barbed
ends. Barbed end growth off spectrin-actin seeds was initiated by
adding 2.8 nM spectrin-actin seeds, 2 mM
MgCl2, and 0.1 M KCl to a solution containing
3.5 µM MgATP-G-actin and profilin at the indicated
concentrations in G buffer (closed circles). The
same experiment was carried out with CaATP-actin at 5.1 µM in the presence of 4.6 nM seeds
(open circles) and at 7.5 µM
(open squares) in the presence of 3.7 nM seeds. Turbidity at 310 nM was monitored to
determine the initial rate of elongation. The rate of elongation in the
absence of profilin was normalized to 1. Solid
lines were calculated using Equations 1 and 2 with
KPA = 0.1 µM and
k+PA = 0.6 k+A for MgATP-actin, and
KPA = 1.2 µM and
k+PA = 0 for CaATP-actin. The
inset represents typical raw data for 3.5 µM
MgATP-actin in the presence of 0 and 6 µM profilin.
b, kinetic parameters for filament barbed end assembly from
profilin-MgATP-actin. The initial rate of barbed end elongation was
monitored turbidimetrically at the indicated concentrations of
MgATP-actin in the presence (open circles) or
absence (closed circles) of a saturating amount
of bovine profilin, 2.8 nM spectrin-actin seeds, 2 mM MgCl2, and 0.1 M KCl.
Inset, the J(c) plot deviates from
linearity at high profilin-actin concentration. Open
circles, bovine profilin-actin; closed
diamonds, Arabidopsis profilin-actin. Values of
the control without seeds (closed triangles) have
been subtracted from the data shown.
|
|
In contrast, when barbed ends elongated from MgATP-G-actin subunits,
profilin caused only a partial (40%) inhibition of growth. The data
(Fig. 2a) are quantitatively consistent with the view that
profilin binds MgATP-G-actin with an equilibrium dissociation constant
of 0.1 µM, and the profilin-MgATP-actin complex
associates productively with the barbed ends of actin filaments. At
saturation by profilin, filaments grew from profilin-actin units
exclusively, at a rate 40% lower than from MgG-actin alone. No further
inhibition was observed at concentration of profilin as high as 100 µM, again indicating no detectable capping of the barbed
ends by profilin.
In conclusion, in the presence of CaATP-actin, profilin acts as a pure
G-actin-sequestering protein. In contrast, the complex of profilin with
MgATP-actin participates in barbed end assembly. The different
behaviors of profilin-Ca-actin and profilin-Mg-actin are in agreement
with the conclusion of previous works (5, 6) that the effective
participation of profilin-actin in barbed end growth required ATP
hydrolysis to be coupled to polymerization, a condition that was
fulfilled for profilin-Mg-actin but not for profilin-Ca-actin. The
hydrolysis of ATP during polymerization of profilin-Mg-actin was
measured at a high concentration (46 µM
Mg-[
-32P]ATP-G-actin 1:1 complex plus 60 µM profilin). A short sonication was applied immediately
after the addition of salt to enhance the rate of polymerization by
fragmentation. Filament assembly showed a short lag followed by a rapid
increase in light scattering (complete in less than 1 min). ATP was
hydrolyzed in a manner that showed no detectable uncoupling from
polymerization. However, a definite proof that elongation is coupled to
hydrolysis requires the demonstration that the rate of elongation be
kinetically limited at high concentration by the rate of ATP hydrolysis.
Kinetic Parameters for Barbed End Assembly from Profilin-Actin
Complex--
The rate constants for profilin-MgATP-actin association
to and dissociation from the barbed ends can be derived from the
analysis of the dependence of the rate of filament growth on
profilin-actin concentration (J(c) plot; Ref. 8).
The J(c) plots for Mg-actin and profilin-Mg-actin
(Fig. 2b) were derived from growth rate measurements using
spectrin-actin seeds. The values found for the association rate
constants were 4.9 ± 0.5 µM
1
s 1 for Mg-actin, in agreement with Pollard and Mooseker
(28), and 3.5 ± 0.4 µM
1
s
1 for profilin-Mg-actin. The rate constants for
MgATP-actin and profilin-MgATP-actin dissociation from the barbed ends
were derived from the ordinate intercepts of the plots. Values of
0.6 ± 0.2 s
1 and 1 ± 0.3 s
1
were found for actin and profilin-actin, respectively. In other words,
when barbed ends are actively elongating from profilin-actin as well as
from G-actin subunits, terminal subunits dissociate at a rate 1 order
of magnitude lower than the rate of dissociation of ADP-F-actin (8 s
1). In conclusion, the present kinetic data rule out a
simple model for barbed end growth from profilin-actin, according to
which actin incorporation and profilin release from the barbed end
would be tightly coupled to Pi release, as described in
Fig. 7 (Scheme I). In this scheme, the dissociation rate
constant measured in a regime of growth is the dissociation rate
constant of ADP-actin, kD. The resulting
J(c) plot for profilin-actin would then be a
straight line with an ordinate intercept equal to
kD, and the critical concentration (abscissa
intercept) would be 1 order of magnitude higher than the experimentally
observed value of 0.3 µM.
In a range of high concentrations of profilin-actin, the
J(c) plot deviated from linearity and curved
downward (Fig. 2b, inset), indicating that the
association of profilin-actin units to barbed ends was rate-limited by
another reaction. We propose this reaction to be ATP hydrolysis, since
this feature appears to be characteristic of the barbed end growth from
profilin-actin and was not observed when the elongation from G-actin
was measured. Quantitatively identical data were obtained with bovine
profilin and A. thaliana profilin 3 (compare open
circles and closed diamonds in Fig.
2b, inset).
Profilin Is Not Detectably Incorporated in Rapidly Growing
Filaments--
When observed in electron microscopy, filaments
assembled from profilin-actin units displayed a structure strictly
identical to those assembled from unliganded actin. No profilin was
detected in the pellets of sedimented filaments. Efforts were made to
detect nonhelical ribbon-like extensions of filaments that might be
transiently formed at the barbed ends in regimes of rapid growth. At
very high G-actin concentration, ATP hydrolysis is known to be more largely uncoupled from F-actin assembly (7, 29), hence profilin might
remain transiently bound to F-ATP-actin stretches, possibly in a
ribbon-like structure (12). Specimens undergoing rapid assembly at high
concentrations of profilin-actin were rapidly observed in the initial
stages of the reaction. No structural difference was seen between the
core and the end-proximal regions of the filaments assembled from
profilin-actin. The possibility that the ATP-bound profilin-actin
ribbon at the tip of the growing filament was too small to be
detectable by EM cannot be discarded. Finally,
[BeF3
,H2O], a structural
analog of Pi that binds to F-ADP-actin filaments and
reconstitutes the intermediate state in ATP hydrolysis on F-actin (30),
was used to examine whether profilin would be able to bind to
F-ADP-P*-actin. F-ADP-BeF3-actin was incubated overnight in
the presence of different concentrations of profilin. SDS-polyacrylamide gel electrophoresis of the pellets of the sedimented samples showed no profilin bound to F-ADP-BeF3-actin.
In conclusion, all assays failed to detect the incorporation of
profilin into F-actin. To increase the probability of formation of a pure profilin-actin polymer and to identify the nature of the
actin-actin contacts that can be formed when profilin remains bound to
actin, a purification of the covalently cross-linked profilin-actin
complex was elaborated (see "Materials and Methods").
Self-assembly of PAcov--
The covalent
profilin-actin complex was assayed for its nucleotide binding
properties. ATP was found bound to the complex following gel filtration
on Sephadex G-25 (PD10; Amersham Pharmacia Biotech) in G buffer
containing no ATP. To assess whether profilin retained the property to
enhance the rate of nucleotide exchange in the PAcov
complex,
-ATP was mixed with either noncovalent profilin-ATP-actin
or PAcov-ATP 1:1 complex (Ca-ATP-actin, in G buffer, with
20 µM free Ca2+) in the stopped flow, and the
increase in fluorescence of
-ATP associated with the exchange of
-ATP for bound ATP was monitored. Comparison of the nucleotide
exchange kinetics on the noncovalent and covalent complexes, shown in
Fig. 3a, demonstrates that, in the presence of 20 µM free Ca2+ ions, the
rate constants of nucleotide exchange were 0.085 s
1 and
0.025 s
1 for the noncovalent and covalent complex,
respectively, while a rate constant of 0.0006 s
1 was
measured for nucleotide exchange on unliganded actin under the same
conditions. Nucleotide exchange therefore was accelerated 150- and
45-fold on noncovalent and covalent profilin-actin, respectively. The
fact that the covalent profilin-actin complex is purified by
poly-L-proline chromatography also indicates that the
poly-L-proline binding property of profilin (and of
profilin-actin) is not altered in the covalent complex. In conclusion,
the covalent complex is biochemically very similar to the noncovalent
complex.

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Fig. 3.
Functional properties of the
PAcov complex. a, enhancement of the rate of
nucleotide exchange. Noncovalent profilin-actin complex (obtained by
mixing 2 µM ATP-G-actin 1:1 complex with 5 µM bovine profilin) or covalent profilin-actin-ATP 1:1
complex (2 µM) was mixed with 10 µM -ATP
in the stopped flow. The final concentrations were 1 µM
profilin-actin, 5 µM -ATP, and 20 µM
free Ca2+. The increase in fluorescence due to nucleotide
exchange was monitored. Top curve, noncovalent
profilin-actin complex (kobs = 0.084 s 1); bottom curve, covalent
profilin-actin (kobs = .025 s 1).
Note that the extent of fluorescence change is the same in both
samples, indicating that 100% of the covalent complex is active for
nucleotide exchange. b, effect of covalently cross-linked
profilin-actin on F-actin assembly. MgATP-actin (15 µM)
was polymerized by the addition of 2 mM MgCl2
and 0.1 M KCl in the absence (actin) or in the
presence (actin + PAcov) of 1.5 µM
PAcov. Polymerization was monitored turbidimetrically.
Curve PAcov is the curve observed for polymerization of
PAcov alone at 1.5 µM (the final turbidity level was
0.063 absorbance units). c, spontaneous assembly of
profilin-actin covalent complex. Spontaneous aggregation of
Mg-PAcov following the addition of 2 mM
MgCl2 and 0.1 M KCl at concentrations (in
µM) indicated on the curves is monitored by
turbidimetry. The assembly of 8 µM Ca-PAcov
was initiated by adding 0.1 M KCl. At time 70 min
(arrow) the solution was sheared by pipetting. d,
critical concentration plot for self-assembly of the covalent
profilin-actin complex. PAcov was polymerized at 8 µM in F buffer for 8 h at room temperature and
diluted in the same buffer to the indicated concentrations. Samples
were kept at room temperature for 18 h before being centrifuged
for 45 min at 400,000 × g. Closed
circles represent the concentration of PAcov in
the supernatant measured by the Bio-Rad assay. Open
circles are the calculated amounts of polymerized
PAcov derived from the difference between the
concentrations of total PAcov and of PAcov in
the supernatant.
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PAcov was able to self-assemble upon the addition of salt.
The time courses of spontaneous polymerization of actin alone, PAcov alone, and mixed actin and PAcov were
compared (Fig. 3b). The turbidity curve observed when actin
and PAcov are mixed together could not be described as the
sum of the time courses recorded for actin alone and PAcov
alone, which suggests that PAcov can copolymerize with
F-actin. The effect of PAcov on the rates of filament
growth at the barbed and pointed ends confirmed this conclusion.
PAcov had no effect on the rate of growth at the pointed ends and very slightly slowed down the rate of G-actin assembly onto
spectrin-actin seeds monitored by the change in fluorescence of
NBD-actin, in agreement with the data shown in Fig. 3b.
Notably, no high affinity capping of barbed ends by PAcov
was observed. Altogether, these results indicate that PAcov
can copolymerize with F-actin.
The kinetics of spontaneous polymerization of pure PAcov at
different concentrations was examined by turbidimetry. The
polymerization curves shown in Fig. 3c consisted in a lag
phase followed by an exponential increase, suggestive of a
nucleation-growth process similar to the polymerization of actin
itself. The lag phase was much more pronounced for
CaATP-PAcov than for MgATP-PAcov. This feature
again is strongly reminiscent of the slower nucleation of filaments
from CaATP-actin than from MgATP-actin (31). Shearing of the polymers
by pipetting accelerated the polymerization process of
PAcov, consistent with the view that fragmentation of the
PAcov polymers increases the number of elongation sites, as
observed for F-actin filaments. The extent of turbidity change at the
end of the polymerization process increased linearly with the
concentration of PAcov in the polymerizing sample. Samples
of PAcov polymerized at different concentrations in the
range 1.5-10 µM were sedimented at 400,000 × g for 45 min when the turbidity plateau was reached. The
concentration of unassembled PAcov present in the
supernatants was 0.2 µM for all samples. From these data,
the specific increase in turbidity per µM assembled
PAcov was found to be 0.048 cm
1 at 310 nm, a
value 28-fold higher than the one (0.0017 ± 0.0002) measured for
F-actin (21), indicating that the size of the PAcov polymer
that scattered light was much larger than the size of the actin filament.
A conventional critical concentration plot was derived from
measurements of the amounts of unassembled PAcov present in
the supernatants of sedimented samples prepared by serial dilutions of
a preassembled solution of 8 µM PAcov
followed by 18-h incubation at room temperature. Data shown in Fig.
3d demonstrate that PAcov polymerized with a
critical concentration of 0.2 µM.
The ATP bound to actin in the PAcov complex was hydrolyzed
during the polymerization of PAcov. When 7.7 µM PAcov was polymerized in the presence of
-32P-labeled ATP, no 32P was found in the
pellet of the sedimented material at the end of the polymerization
process. 75 µM Pi were found in the
supernatant after 16 h, indicating that PAcov
filaments turn over.
In conclusion, the polymerization of PAcov shares
mechanistic similarities with the polymerization of F-actin.
PAcov Polymerizes into Helical Filaments--
The
structure of the PAcov polymer was examined by electron
microscopy. Observation of negatively stained specimens of assembled PAcov at steady state showed a homogeneous population of
bundled filaments. The ends of the bundles often appeared blunt,
suggesting that the filaments that composed the bundle grew together in
a synchronous fashion. The kinetics of formation of these bundles was
examined in electron microscopy and turbidity simultaneously, at a
concentration of PAcov low enough (1.5 µM)
for the different steps of nucleation and growth to be clearly
time-resolved. Electron micrographs of the polymerizing sample at
different times of the polymerization process shown in Fig.
4 indicate that during the lag time only
short individual filaments were formed, which gradually interacted with
each other in bundles. Essentially, bundles of ~4-6 filaments were
visible on the grid at the end of the lag phase (t = 6 min) when the change in turbidity was less than 1% of the total
change. Bundles became thicker with time. The Fourier transforms of the
PAcov filaments displayed clear layer lines at spacings of
36 and 6 nm, corresponding to the first and sixth layer lines of
F-actin. These results indicate that PAcov polymerizes into
helical filaments that have the same helical periodicities as F-actin.
Fourier transforms of the bundles observed at later stages of assembly
show an identical pattern. Bundling most likely results from the change
in charge of filaments due to profilin, which is a basic protein.
Native filaments contain repulsive charges that maintain a distance
between each polymer in solution. Binding of ligands can abolish the
repulsion between filaments and favor their mutual interactions (32).
The poly-L-proline binding site exposed at the surface of
profilin may also mediate hydrophobic contacts between the
PAcov filaments and favor the formation of bundles.
Accordingly, bundling was less extensive at low ionic strength.

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Fig. 4.
Electron microscopy morphology of filaments
assembled from profilin-actin covalent complex. Top
panel, electron micrograph of a control Mg-F-actin filament
polymerized in the presence of 1 mM MgCl2 and
0.1 M KCl. Inset, diffraction pattern. The first
and sixth layer lines are indicated by an arrowhead.
Bottom panel, PAcov filaments
assembled from Mg-PAcov under the same ionic conditions as
above. a, initial stage of polymerization kinetics of 1.5 µM PAcov as recorded by turbidimetry.
Sampling times for EM are indicated by arrows.
b-g, electron micrographs of representative specimens
observed on the grid at the following times: t = 0 (b); t = 1 min (c);
t = 4 min (d); t = 6 min
(e); t = 8 min (f);
t = 12 h (stationary state) (g). The
Fourier transforms of the individual PAcov filaments and of
the bundles are shown in insets to panels
c and f.
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To understand whether the bundling resulted from the EDC/NHS and/or
dimethyl sulfoxide treatment of actin, in a control experiment the
uncross-linked actin recovered from the poly-L-proline
affinity column at the end of the PAcov preparation (see
"Materials and Methods") was supplemented with 30%
Me2SO and processed like the covalent complex. Turbidity
measurements showed that this G-actin material polymerized at the same
rate and to the same extent as standard actin and did not form bundles
(data not shown).
Binding of phalloidin to the PAcov filaments was monitored
by the increase in fluorescence of tetramethylrhodamine-phalloidin (33,
34). The time courses for binding 1 mol eq of
tetramethylrhodamine-phalloidin to 1.6 µM either F-actin
or F-PAcov were superimposable (Fig. 5), indicating that the binding site of
phalloidin is very similar in the two structures.

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Fig. 5.
Polymers of profilin-actin covalent complex
and actin filaments interact with phalloidin in a similar fashion.
The time course of interaction of 1.6 µM F-actin
(closed circles) or 1.6 µM
PAcov filaments (open circles) with 1 mol eq of TMR-phalloidin (1.6 µM) was followed by
rhodamine-phalloidin fluorescence. F-actin or PAcov polymer
was added to TMR-phalloidin at time indicated by the
arrow.
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Interaction of the Myosin Head with the Profilin-Actin
Filament--
Several assays were carried out to understand how the
cross-link of profilin to actin affects the actin-myosin interface and its functional properties. The binding of myosin subfragment-1 to
standard filaments and PAcov filaments in the absence of
ATP was examined in parallel in a sedimentation assay. Data shown in
Fig. 6 show that
S1(A1) binds tightly to PAcov
filaments; however, the stoichiometry was lower than the 1:1 value
confirmed here for S1 binding to F-actin. A maximum of
0.4-0.5 S1 was bound per PAcov subunit.

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Fig. 6.
Myosin subfragment-1 binding to the
profilin-actin filament in rigor. F-actin (top
panel) or F-PAcov (bottom
panel) was incubated at a concentration of 5 µM in physiological ionic strength buffer containing no
ATP, in the presence of S1(A1) at the indicated
concentrations. Samples were centrifuged at 400,000 × g. The supernatants and resuspended pellets were submitted
to SDS-polyacrylamide gel electrophoresis. In the F-actin + S1 samples (top panel), the volume of
loaded pellets corresponds to a 2 times higher amount of the initial
actin plus S1 solution than the supernatants. The positions
of S1 heavy chain, actin, and PAcov are
indicated. Scanning of the gel patterns (not shown) showed that in the
F-actin plus S1 sample, S1 increases linearly
in the supernatant above a total concentration of 5 µM,
consistent with 1:1 binding ratio of S1 to F-actin;
conversely, in the PAcov plus S1 sample,
S1 increases linearly in the supernatant above a total
concentration of 2 µM S1, consistent with a
maximum binding ratio of 0.4 S1 per PAcov
subunit.
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In the electron microscope, filaments assembled from PAcov
and partially decorated (as above) by S1 failed to display
the conventional arrowhead decoration by S1. The myosin
heads were attached irregularly to the PAcov filament with
randomly distributed orientations. No diffraction pattern could be
derived from the images (data not shown).
In an ATP-free low ionic strength buffer,
S1(A1) is known to induce the rapid formation
of G-actin-S1 oligomers, followed by their slower
condensation into arrowhead-decorated F-actin-S1 filaments.
These two reactions can be monitored by light scattering (19, 35).
Under those ionic conditions, no oligomer nor any assembly process
could be detected within 1 h when S1(A1)
at concentrations up to 20 µM was added to
PAcov-ATP 1:1 complex (8 µM) in ATP-free G
buffer. In the control experiment carried out with 8 µM
ATP-G-actin 1:1 complex and 12 µM
S1(A1), the polymerization was complete in 5 min.
On the other hand, the MgATPase of S1(A1) was
clearly enhanced by PAcov filaments. The rate of ATP
hydrolysis, under different ionic conditions (presence
versus absence of 0.1 M KCl), was 2.6-fold lower
than the ATPase rate measured in the presence of the same amount of
F-actin (17 µM) in a parallel sample.
The sliding movements of actin and PAcov filaments over
myosin-coated glass surfaces were assayed in parallel. While actin filaments moved at a speed of 6.6 ± 1.2 µm/min, the
profilin-actin filaments remained immobile and firmly attached to the
myosin-coated surface. Filaments containing 50% actin and 50%
PAcov moved almost as well as standard actin filaments with
an average speed of 3.9 ± 1.0 µm/min and displayed a
discontinuous, somewhat stick-slip motion. Filaments containing
10% PAcov moved at the same speed and displayed the same
regular motile behavior as standard filaments.
Overall, these data suggest, but do not prove, that the
actin-S1 interface remains functional in the profilin-actin
filament, but some steric hindrance due to the presence of profilin
inhibits the movement of the myosin head, which is necessary for its
translocation along actin filaments. Similarly, the fact that
S1 fails to induce oligomer and F-acto-S1
assembly from PAcov indicates that S1 cannot interact with 2 PAcov molecules as it does with
G-actin.
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DISCUSSION |
Under physiological ionic conditions (MgATP-actin, 2 mM MgCl2, 0.1 M KCl), filaments
actively elongate from profilin-actin units, at a rate that is only
30-40% lower than from G-actin subunits. These results are in
agreement with an earlier report (36), which used
Acanthamoeba profilins and the Limulus acrosomal
processes as seeds; however, we find no evidence for a weak capping
activity of profilin even at physiologically high concentrations.
Profilin is not incorporated into the filaments, even in the ATP- or
ADP-Pi-bound state, at variance with the expectations from
a ribbon-to-helix model.
Kinetic data show that filament growth from noncovalent profilin-actin,
although mechanistically coupled to ATP hydrolysis, is not kinetically
coupled to Pi release. Evidence for this conclusion is
provided by the low critical concentration for barbed end assembly from
profilin-actin and extrapolation of the J(c) plot
to a low value of the dissociation rate constant
k
, which is inconsistent with the presence of
rapidly dissociating ADP subunits at the barbed ends in a regime of
growth. In the proposed alternative scheme (Fig.
7), growth from profilin-actin and ATP
hydrolysis are tightly coupled, and a stabilizing ADP-Pi
cap (37) prevents the occurrence of rapidly dissociating ADP subunits
at the ends of elongating barbed ends. The downward curvature of the
plot at high profilin-actin concentration (with both bovine and plant profilins) is due, within this scheme, to ATP hydrolysis, which kinetically limits profilin release and subsequent filament growth. The
rate of ATP hydrolysis would be about 80 s
1 under
physiological ionic conditions and would be consistent with the value
measured here for the association rate constant (4.9 µM
1·s
1). If a higher value
of the association rate constant is used, e.g. 10 µM
1·s
1 (38), then the
corresponding ATPase rate would be 163 s
1.
J(c) measurements carried out at lower ionic
strength (1 mM MgCl2, no KCl) yielded a limit
value of J(c) of 25 ± 5 s
1
(data not shown). This value is in reasonable agreement with results
showing that the rate of hydrolysis accompanying filament elongation
reached a limit of 13 s
1 in the presence of 1 mM MgCl2 (29).

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Fig. 7.
Possible mechanisms for filament barbed end
assembly from profilin-actin complex. The experimental
J(c) plots corresponding to barbed end growth
from G-actin (thin line) and profilin-actin
(thick line) are drawn in comparison with the
theoretical J(c) plot expected for profilin-actin
within Scheme I (dashed
line). Scheme II accounts for the
experimental J(c) plot for profilin-actin.
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