(Received for publication, July 24, 1995; and in revised form, November 17, 1995)
From the
Actin exhibits ATPase activity of unknown function that
increases when monomers polymerize into filaments. Differences in the
kinetics of ATP hydrolysis and the release of the hydrolysis products
ADP and inorganic phosphate suggest that phosphate-rich domains exist
in newly polymerized filaments. We examined whether the enrichment of
phosphate on filamentous ADP-actin might modulate the severing activity
of gelsolin, a protein previously shown to bind differently to ATP and
ADP actin monomers. Binding of phosphate, or the phosphate analogs
aluminum fluoride and beryllium fluoride, to actin filaments reduces
their susceptibility to severing by gelsolin. The concentration and pH
dependence of inhibition suggest that HPO binding to actin filaments generates this resistant state. We
also provide evidence for two different binding sites for beryllium
fluoride on actin. Actin has been postulated to contain two P
binding sites. Our data suggest that they are sequentially
occupied following ATP hydrolysis by HPO
which is subsequently titrated to
H
PO
. We speculate that
beryllium fluoride and aluminum fluoride bind to the
HPO
binding site. The cellular
consequences of this model of phosphate release are discussed.
The hydrolysis of ATP during actin polymerization has been
postulated to alter filament
structure(1, 2, 3, 4) , change the
kinetics of monomer binding(5, 6) , and modulate the
association of actin binding
proteins(7, 8, 9) . Biochemical evidence
suggests that ATP hydrolyzes rapidly as monomers add to the end of a
filament, but that subsequent release of hydrolysis products is
slow(10) . In F-actin ()at steady state, ADP is
bound within the nucleotide binding pocket of the actin
monomer(11) , and this nucleotide is trapped until the monomer
dissociates from the filament(5, 12) . However, the
inorganic phosphate (P
) generated dissociates slowly from
the filament without monomer turnover (13) . Therefore, newly
polymerized filaments will be rich in the hydrolysis intermediate
ADP-P
while older filaments will contain predominantly
ADP(14) .
Inorganic phosphate binds to the actin filament at
a low affinity (mM) site (15) postulated to be one of
two sites occupied by P released from ATP upon
hydrolysis(16, 17) . Binding of P
to F-ADP
actin reduces the off-rate of monomers from the fast-exchanging
(barbed) end of filaments(6) . The changes in actin
monomer-filament interactions may be due to alterations in filament
structure. The electron density map of the actin filament alters when
P
is bound, with domain 2 of the actin monomer moving out
from the helical center of the filament and away from domain 1 of the
next monomer in the two start helix(3) . In contrast, this
domain is not readily visualized in F-actin polymerized from ADP
monomers, suggesting its position is variable.
AlF and BeF
(the terms P
,
AlF
, and BeF
indicate that
the exact ionic species is unknown) have effects similar to P
on the actin filament. Although the exact isoform of
AlF
and BeF
is
unclear(18) , these agents reduce the off-rates of monomers,
change the structure of the filament(3, 19) , and
alter the filament's susceptibility to proteolysis(20) .
BeF
binds with higher affinity to F-actin than
does P
, and its association and dissociation rate constants
are much slower, a finding that suggests it may bind to the
high-affinity P
site within the nucleotide binding site of
the monomer(17) .
The ATP hydrolysis that accompanies actin
polymerization has been suggested to enhance the disassembly of
filaments at a time separate from that of assembly(21) . This
hypothesis implies that the rate of ATP hydrolysis and release of
P can act as a clock, with the age of a filament given by
the relative concentrations of ATP, ADP-P
, and ADP
subunits. We report that the actin filament severing protein gelsolin
can differentiate between ADP-P
and ADP-rich filaments,
with filaments in the ADP-P
state being severed by gelsolin
much more slowly. Binding of P
, AlF
,
or BeF
reduces the extent of severing by both
intact gelsolin and its Ca
insensitive N-terminal
domain. These findings suggest that in cells the assembly and
disassembly of filaments could be regulated not only by signal
transduction pathways, but by the energy charge of the cell and the age
of pre-existing actin filaments.
BeSO and Al
(SO
)
were purchased from Aldrich. Phalloidin, tetramethylrhodamine
isothiocyanate-coupled phalloidin (TRITC-phalloidin), NaF, nucleotides,
and all other buffers and salts were purchased from Sigma.
F-ADP-actin was polymerized at
concentrations of 15 to 30 µM in solutions containing 150
mM KCl, 20 mM HEPES, pH 7.4, 0.5 mM ADP, 0.2
mM dithiothreitol, 2 mM MgCl, and 0.2
mM CaCl
. F-ADP-P
actins were generated
by polymerization of G-ADP actin in similar solutions containing
various concentrations of KH
PO
and
K
HPO
(varied to give the appropriate pH) as
indicated under ``Results,'' and KCl, with a total
K
concentration equal to 150 mM(15) .
BeF and AlF
were generated by addition of
BeSO
and NaF, or Al
(SO
)
and NaF, at a molar ratio of 1 aluminum or beryllium to 4 NaF, to
a modified polymerization solution containing 100 mM KCl, 50
mM HEPES, pH 7.4, 0.2 mM CaCl
, 2 mM MgCl
, 0.5 mM ADP, and 0.2 mM dithiothreitol. The final BeF
or AlF
concentrations stated in the text were taken from the
concentration of beryllium or aluminum added to the buffer.
Human
plasma gelsolin was purified by elution from DE-52 ion exchange matrix
in 30 mM NaCl, 3 mM CaCl, 25 mM Tris, pH 7.4, as described by Kurokawa et
al.(24) , dialyzed into 75 mM NaCl, 20 mM Tris, pH 7.4, and 0.2 mM EGTA, rapidly frozen in liquid
nitrogen and stored at -80 °C. Gelsolin concentration was
determined from the absorbance at 280 nm using the extinction
coefficient 1.8 ml mg
cm
. The
N-terminal fragment of gelsolin, s1-3, was purified from Escherichia coli as described(25) .
where I is the fluorescence at time t, I
is the fluorescence of
TRITC-phalloidin in the absence of actin, and I
is the fluorescence of the TRITC-phalloidin F-actin mixture.
While the kinetics of gelsolin severing TRITC-phalloidin-bound
F-actin depend on divalent cation concentrations (27) the
severing by gelsolin s1-3 was found to be completely independent
of the presence of divalent cations, as previously
reported(25) . For experiments using intact gelsolin, the
Ca concentration was measured as described previously (27) using Mag Fura 5 (Molecular Probes, Eugene, OR).
Using gelsolin's ability to displace phalloidin from
F-actin and the enhanced fluorescence of TRITC-phalloidin upon binding
to actin as a severing assay(27) , we investigated whether the
binding of P to F-ADP actin alters actin's
susceptibility to severing. Reduction of free Ca
by
P
was accounted for by titration of
[Ca
] in control buffers with EGTA.
Ca
concentrations were measured using the ratiometric
dye Mag Fura 5, as described previously(27) .
Fig. 1demonstrates that binding of P to F-ADP
actin reduces the extent of severing by human plasma gelsolin. F-ADP
actin was incubated overnight in buffers containing increasing
concentrations of P
, diluted in identical buffers
containing TRITC-phalloidin and allowed to come to equilibrium (as
determined from the increased emission of TRITC-phalloidin). Addition
of gelsolin reduced the fluorescence to baseline levels, as previously
reported. Gelsolin displaced all the TRITC-phalloidin from F-ADP actin
within 5 min at all but the lowest Ca
concentration.
In contrast, F-ADP-P
actins showed a P
concentration-dependent resistance to severing by gelsolin within
this time scale. This result suggests that binding of P
to
F-ADP actin generates filaments resistant to gelsolin. However, to
escape complications from the alterations in free Ca
by P
, we continued these studies using bacterially
expressed N-terminal gelsolin fragment s1-3, which is
Ca
insensitive(25) .
Figure 1:
Gelsolin severs F-ADP-P actin less effectively than it does F-ADP actin. 40 µM G-ADP actin was polymerized overnight in either F-ADP buffer, pH
7.4, or F-ADP buffer containing various concentrations of
P
, pH 7.4, as described under ``Materials and
Methods.'' The increase in fluorescence of 1 µM TRITC-phalloidin in F-ADP and F-ADP-P
buffers was
measured upon addition of 400 nM F-actin and found to be
identical for both ADP and ADP-P
F-actins (data not shown).
Addition of 200 nM human plasma gelsolin displaced all the
TRITC-phalloidin within 5 min from F-ADP actin (open circles)
at all but the lowest Ca
concentration. Addition of
200 nM gelsolin to F-ADP-P
actins (closed
circles, P
concentration indicated to the right of the
data point) consistently displaced less of the TRITC-phalloidin than
that observed for F-ADP at equivalent Ca
concentrations. Free Ca
was determined as
described under ``Materials and
Methods.''
Figure 2:
F-ADP-P actin is resistant to
severing by gelsolin s1-3. ADP actin was polymerized overnight at
30 µM in the presence of either 150 mM KCl or 50
mM KH
PO
, pH 6.8, 100 mM KCl;
with 10 mM HEPES, 2 mM MgCl2, 0.5 mM ADP;
0.2 mM dithiothreitol. F-actin was diluted to 300 nM in the above buffer containing 1 µM TRITC-phalloidin.
Subsequent addition of 180 nM gelsolin s1-3 displaced
all the phalloidin from F-ADP actin (open circles) as
indicated by the complete loss of enhanced fluorescence, yet displaced
only 40% of that bound to F-ADP-P
actin (closed
circles). Addition of gelsolin s1-3 at 360 nM (closed squares) or 540 nM (closed
triangles) did not increase the amount of TRITC-phalloidin
displaced on this time scale.
Gelsolin interacts with the phosphates of
phosphoinositide lipids(28) , and with ATP(29) .
However, it appears unlikely that the effect of P is on
gelsolin rather than actin. Increasing concentrations of gelsolin
s1-3 do not lead to an increased loss of F-actin (Fig. 2),
consistent with decreased substrate availability. Also, incubation of
gelsolin s1-3 in 50 mM P
buffer before
addition to TRITC-phalloidin-bound F-ADP actin led to complete severing
which could not be distinguished from controls (data not shown).
A
previous study demonstrated a binding site for the
HPO
ion (15) on F-ADP
actin, based on the apparently tighter binding at pH 6.8 compared with
pH 8.0, where the HPO
ion is more
prevalent. We used a similar approach to define the specific phosphate
ion involved in generating gelsolin-resistant actin by determining the
phosphate concentration and pH dependence of inhibition of severing by
gelsolin s1-3. Consistent with the results using human plasma
gelsolin, generation of F-actin resistant to severing by gelsolin
s1-3 requires tens of millimolar P
at pH 6.8 (Fig. 3). In contrast, at pH 8.0, 50% inhibition is detected at
12 mM P
, and 25 mM maximally reduces
severing. These results suggest that it is the binding of
HPO
, not
H
PO
, which generates the
severing-resistant state. However, we cannot exclude a more complex
mechanism by which the binding of both ions generates the severing
resistant state.
Figure 3:
Concentration and pH dependence of the
inhibitory effect of P on the severing of F-actin by
gelsolin s1-3. G-ADP actin was polymerized in buffers that
differed in their P
concentration and pH. KCl was varied
such that the total K
concentration was 150
mM. 400 nM actin was diluted into buffers containing
1 µM TRITC-phalloidin and the increase in fluorescence
upon binding was measured. The increase in fluorescence was similar for
all samples and was normalized to 1.0 as described under
``Materials and Methods.'' 200 nM gelsolin
s1-3 was added and the loss of fluorescence followed over time.
The concentration dependence is altered, with lower concentrations
showing inhibitory activity at higher pH. Time is in
seconds.
Binding of P to F-ADP actin, as
indicated in Fig. 3, causes a dramatic reduction in the extent
of severing, when assayed over a relatively short period of time.
However, even at 50 mM P
, pH 8.0, there is some
small amount of fluorescence loss on this time scale. To address
whether the inhibition observed was actually a reduction in the
kinetics of severing, we continued the analysis over a longer time
scale. As illustrated in Fig. 4, gelsolin will sever ADP-P
F-actin, but requires thousands of seconds to do so. It should
also be noted that the loss of fluorescence is not well described by a
single exponential function.
Figure 4:
Severing of ADP-P actin by
gelsolin s1-3 goes to completion, but with much slower kinetics.
Severing of 400 nM F-ADP (closed circles) or
F-ADP-P
actin (open circles; 50 mM P
, pH 8.0, F-buffer) by 200 nM gelsolin
s1-3 was followed over a longer time scale. Complete loss of
fluorescence was defined as that lost by F-ADP actin in the presence of
excess gelsolin s1-3. Time is in
seconds.
Figure 5:
Low
concentrations of AlF and BeF
generate F-actin resistant to severing. In the top
panel, G-ADP actin was polymerized in the presence of various
concentrations of BeSO
and NaF to give the concentration of
BeF
indicated in the panel. F-actin was diluted to
a final concentration of 300 nM in buffer containing 1
µM TRITC-phalloidin and a concentration of BeF
equal to that of the polymerization buffer. The
TRITC-phalloidin was allowed to bind for 5 min. 300 nM gelsolin s1-3 was added at 0 s. The final fluorescence after
addition of gelsolin s1-3 to F-ADP actin was 68 to 69
fluorescence units and was equal to the fluorescence of 1 µM TRITC-phalloidin in the absence of F-actin. The bottom panel illustrates a similar experiment, except that G-ADP-actin was
polymerized and analyzed in various concentrations of
AlF
, generated as described under ``Materials
and Methods.'' The concentration of AlF
is
indicated to the right of the relevant curve. Time is in
seconds.
Binding of BeF to F-ADP actin reduces the
fluorescence of pyrene iodoacetamide-labeled F-ADP actin(17) .
Incubation of 10 µM F-PIA-ADP actin with either 10 or 100
µM BeF
depresses the pyrene fluorescence with
a second order rate constant of approximately 20 M
s
(Fig. 6A). The extent and kinetics of
fluorescence depression are similar to those previously
reported(17) . In contrast, 1 mM BeF
caused an increase in fluorescence of F-PIA actin, not a
decrease, with a rate constant of approximately 1 M
s
. This increase in
the fluorescence of F-PIA ADP actin upon exposure to BeF
concentrations greater than 100 µM was quite
apparent in an independent experiment examining equilibrium effects (Fig. 6B). These results provide further evidence for
two BeF
binding sites on F-ADP actin.
Figure 6:
The
effects of BeF on the fluorescence of F-PIA-ADP
actin indicate the presence of two binding sites for this molecule on
F-ADP actin. In panel A, the kinetics of the fluorescence
change of 10 µM F-PIA-ADP actin were measured upon
addition of BeF
concentrations of 0.01 (open
circles), 0.1 (closed circles), and 1 mM (open squares). Only every 10th data point is plotted for
clarity. The line indicates the change in fluorescence fit to
a second order rate constant of 20 M
s
for 10 and 100 µM BeF
, and 1 M
s
for 1 mM BeF
.
In a separate experiment (panel B) the fluorescence change of
10 µM F-ADP-PIA after exposure to various concentrations
of BeF
for 24 h is
illustrated.
Inhibition of
severing by AlF requires higher concentrations than
BeF
. As illustrated in Fig. 5B, 100
µM AlF
has little effect on the rate of
severing, while 0.5 mM shows a significant reduction. In
contrast to BeF
, AlF
did not show a reduction
in inhibitory activity within the concentration range tested. The
concentration dependence of AlF
action is consistent with
previous reports comparing the ability of AlF
and BeF
to recreate the ADP-P
like state on
F-actin(17) .
AlF and BeF
do not
appear to affect gelsolin directly. Incubation of gelsolin s1-3
in maximally inhibitory concentrations of AlF
,
BeF
, or P
followed by dilution into buffer
lacking these molecules had no effect on the kinetics and extent of
severing (data not shown). This result is not due to rapid dissociation
of the ion from gelsolin. Gelsolin s1-3 severed F-ADP actin
incubated for 5 min in the presence of 1 mM AlF
,
with kinetics similar to that observed in the absence of AlF
(Fig. 7). In contrast, overnight incubation of F-ADP actin
in 1 mM AlF
strongly reduced the susceptibility to
severing by gelsolin s1-3. This result, and the fact that
increasing gelsolin s1-3 concentration does not increase the
amount of ADP-P
actin severed (Fig. 1) strongly
suggest that the target of these agents is the actin filament, not
gelsolin.
Figure 7:
Incubation of F-ADP actin in 1 mM AlF does not rapidly lead to the generation
of severing resistant F-actin. The severing of F-ADP actin in two
control experiments (open symbols) is compared with that of
G-ADP actin polymerized overnight in 1 mM AlF
and assayed in 1 mM AlF
(closed triangles) and with that of F-ADP actin
incubated in 1 mM AlF
for 5 min and then
assayed in 1 mM AlF
(closed
squares). The F-actin and gelsolin s1-3 concentrations were
300 nM each. Time is in seconds.
A significant fraction of cellular ATP metabolism is associated with polymerization of actin(30) , yet the function of actin's polymerization-stimulated ATPase is unclear. ATP hydrolysis is not necessary for polymerization, since ADP-G actin will polymerize(10) . Several potential roles for ATP hydrolysis have been suggested, including regulating the structure of the actin filament(1, 2, 3, 4, 31) , regulating interactions with actin binding proteins(7, 8, 32, 33) , and regulating the dynamics of monomer and filament interaction(6, 34) , effects which are not necessarily mutually exclusive.
While the exact role of ATP hydrolysis in actin
dynamics remains unclear, there is some agreement on the mechanistic
details. Association of G-ATP actin monomer with a filament end is
rapidly followed by hydrolysis of the ATP(35) . However, the
release of hydrolysis products is relatively slow, with ADP released
upon monomer dissociation from the filament and the P generated from hydrolysis released at a rate approximately 2
orders of magnitude slower than ATP hydrolysis(13) . Rapid
polymerization or elongation of actin filaments would produce domains
within individual filaments preferentially enriched in ATP,
ADP-P
, and ADP. Under cellular conditions ATP hydrolysis by
actin is postulated to be a vectorial, not stoichastic,
process(36) . Therefore, domains enriched in ADP-P
or ADP would be large and continuous, rather than dispersed
throughout the filament length.
Several protein families have been
proposed to regulate or enhance the disassembly of actin filaments in
cells. The work described here and elsewhere suggests that members of
both the gelsolin and the cofilin/destrin families of severing proteins
cannot sever ADP-P-rich F-actin(37) . Furthermore,
the presence of P
on the actin filament dramatically
reduces monomer dissociation(6, 38) , preventing the
disassembly of actin filaments by the monomer sequestering proteins
found in cells. We speculate that in cells the activation of actin
filament disassembly by filament severing and monomer sequestering
proteins is regulated in part by the relative proportions of ADP and
ADP-P
bound to the filament. These species, in turn, depend
on the age of the actin filament and the energy charge of the cell.
The binding to F-actin of BeF, AlF
, and to a
lesser degree P
increases the stability of the actin
filament, raising the denaturation temperature from 65 °C to as
high as 82 °C(4) . Similarly, 10 mM P
reduces the susceptibility of filaments to denaturation by SDS or
KI(39) . One mechanism by which the multiple effects of
creating P
-rich domains on actin filaments can be explained
is by the alteration of actin filament structure(1) . Binding
of P
, BeF
, or AlF
alters the
position of subdomain 2 of the actin monomer in the actin
filament(3, 19) . In the ADP-P
state this
domain interacts strongly with the next monomer along the long pitch
helix in the filament. In the absence of bound P
, this
domain either has multiple orientations, or is fixed in a position that
interacts weakly if at all with the next monomer(3) .
The
structural difference between ADP-actin and ADP-P-actin can
account for the reduced dissociation of actin monomers from the
filament and the reduced ability of severing molecules to disrupt the
filament. Monomer dissociation requires the coordinate disruption of
multiple contacts with other monomers in the filament. Increasing the
number of these contacts reduces the probability of dissociation.
Furthermore, gelsolin has been suggested to sever actin filaments by
interrupting the actin-actin contacts along the long-pitch helix of the
filament(40) . Enhancing the strength of these contacts through
the increased interaction of subdomain 2 of one monomer with subdomain
1 of the next (3) should make it more difficult to sever the
filament.
The binding of P and BeF
to F-ADP
actin has been
characterized(3, 15, 16, 17, 20) .
Direct measurements demonstrate a binding site for
H
PO
with an affinity of
several millimolar(15) . Similarly, BeF
(exact
stoichiometry unknown) has been reported to bind F-ADP actin with a K
of 30 µM(17) . These two
molecules compete with each other for binding to F-ADP
actin(17) . However, these results do not preclude potential
lower affinity binding sites for these molecules. The binding of
H
PO
to a site on F-ADP actin
is consistent with its effects on the off-rate of monomers (6, 15) and the inhibition of small molecular weight
severing proteins like actophorin(37) . However, the pH
sensitivity and concentration dependence of severing inhibition
reported here is more consistent with the binding of
HPO
to F-ADP actin. Furthermore, the
presence of two binding sites for P
-like molecules on the
actin filament is supported by the bimodal effects of BeF
on the fluorescence of F-PIA-ADP actin.
The data presented
here and in previously published work provide evidence of binding sites
for both HPO
and
HPO
, or their BeF
equivalents, on the actin filament. These two sites would be
sequentially filled in the release of P
generated by ATP
hydrolysis upon actin polymerization. In such a reaction
HPO
is released upon ATP hydrolysis,
where it remains bound in the nucleotide pocket until titrated to
H
PO
, which is slowly released
from the actin filament. Consistent with both previous and current
speculations, BeF
was hypothesized to bind the P
binding site first occupied upon nucleotide
hydrolysis(18) . Resistance of F-actin to severing by gelsolin
correlates both with saturation of the high affinity BeF
site and with binding to a low-affinity, at physiological pH,
P
site. In the context of actin turnover in the cell, newly
polymerized actin filaments would first become sensitive to severing by
gelsolin once HPO
is titrated to
H
PO
, but degradation by
cofilin-like molecules and by monomer sequestering proteins would
require H
PO
dissociation.
The hypothesis that F-actin has binding sites for both
HPO and
H
PO
, which fill sequentially
upon ATP hydrolysis during polymerization and which modulate the
association and activities of different actin binding proteins has
implications for the turnover of actin filaments in cells. Newly
polymerized actin filaments, rich in HPO
would be resistant to severing and depolymerization. Titration of
HPO
to
H
PO
would allow severing by
gelsolin-like molecules, but this shortened filament would be
relatively resistant to disassembly by cofilin-like molecules and to
depolymerization. This gelsolin capped filament would then have some
time, based on the dissociation rate of
H
PO
, to move to a new site in
the cell, where it could uncap or incorporate into new cytoskeletal
structures. If the filament is not stabilized, either by further
elongation with accompanying ATP hydrolysis, or by association of other
stabilizing factors such as tropomyosin, the time-dependent loss of
H
PO
increases the probability
of further fragmentation by cofilin-like molecules and depolymerization
by monomer dissociation.
This model adds a temporal component to the
many previous models that considered actin turnover in the context of
the spatial organization of the cell. Several studies of actin turnover
in cells suggest that actin filament depolymerization is not a
spatially uniform process(41, 42) , but the mechanism
for locally altering depolymerization is undefined. Consideration of
the effects of nucleotide hydrolysis by actin on filament severing and
depolymerization suggests that the dynamics of filament turnover in
cells are defined by the ATPase rate of actin and the titration and
release of P, as well as the regulated action of actin
binding proteins.