(Received for publication, June 21, 1995)
From the
To address the mechanisms through which agonists stimulate actin
polymerization, we examined the roles of monomer sequestering proteins
and free barbed ends on actin polymerization induced by guanosine
5`-3-O-(thio)triphosphate (GTPS) in neutrophils
permeabilized with streptolysin O. Addition of profilin (without
GTP
S) caused a net decrease in F-actin. Thus, merely making
profilin available in the cell was not sufficient to induce actin
polymerization. On the other hand, addition of profilin hardly affected
the polymerization induced by GTP
S, while thymosin
or DNase I decreased this polymerization. These data suggested
that GTP
S induced polymerization by increasing the availability of
barbed ends. In the presence of cytochalasin B, profilin did inhibit
polymerization induced by GTP
S, demonstrating that GTP
S did
not inhibit profilin's monomer sequestering ability.
The
F-actin induced by GTPS was not limited by a time-dependent loss
of G-actin or G-proteins from permeabilized cells since, following
stimulation with suboptimal concentrations of GTP
S, addition of
more GTP
S induced further polymerization. Barbed ends remained
free after F-actin reached plateau since (a) cytochalasin B
caused depolymerization of induced F-actin and (b) profilin
did not depolymerize induced F-actin unless the cells were first
treated with cytochalasin to cap barbed ends. The data indicate that
GTP
S maintains an increased level of F-actin by keeping at least a
few barbed ends available for polymerization.
Neutrophils treated with inflammatory mediators increase their F-actin level by shifting the steady state from G- to F-actin. However, which of the many factors that cause this shift in vitro account for it in vivo are unknown. Possible factors include: (a) a shift in the nucleotide bound to G-actin from ADP to ATP or (b) inhibition of the major monomer sequestering proteins. These factors now seem unlikely (Rosenblatt et al., 1995; Carlier et al., 1993; Redmond et al., 1994; Safer et al., 1990; Cassimeris et al., 1992; Nachmias et al., 1993), so interest is focused on (c) the availability of profilin and (d) the availability of free barbed ends.
Profilin, first identified from its ability to inhibit
actin polymerization (Carlsson et al., 1977), is now also
known to promote polymerization (Pantaloni and Carlier, 1993). When
profilin is added in vitro to a mixture of thymosin
(T
4), (
)G-actin, and F-actin, the
F-actin decreases or increases depending on whether the filament barbed
ends are capped or free (Pantaloni and Carlier, 1993; Carlier and
Pantaloni, 1994). Cells contain T
4, G-actin, and F-actin. Thus, if
in the resting cell some barbed ends are free but profilin is
sequestered, an agonist could induce polymerization merely by releasing
profilin from a sequestered pool. Profilin might well be sequestered in
cells because it is distributed nonhomogeneously in the cell (Buss et al., 1992) and because it binds strongly to inositol
bisphosphate (Lassing and Lindberg, 1985). Furthermore, some agonists
may release profilin bound to inositol bisphosphate, thus freeing it to
promote polymerization (Sohn and Goldschmidt-Clermont, 1994; Machesky
and Pollard, 1993). The hypothesis that profilin release induces actin
polymerization has not been tested.
Free barbed ends are of interest
because in vitro increasing the fraction of filaments with
free (uncapped) barbed ends induces polymerization (Yin and Stossel,
1979; Pollard, 1986). However, in cells, evidence that free barbed ends
regulate the actin steady state is equivocal. On one hand, cells lysed
after stimulation with agonist have an increased number of sites that
nucleate barbed end elongation of exogenous actin (Carson et
al., 1986; Condeelis et al., 1988; Hall et al.,
1989; Hartwig, 1992; Nachmias et al., 1993). This increase in
nucleation sites occurs even when net polymerization is blocked by
cytochalasin, suggesting these sites may be the cause rather than the
effect of polymerization (Hartwig, 1992). ()On the other
hand, most of the filaments in the lysate of resting neutrophils appear
to have free barbed ends, and the increase in number of barbed ends
upon stimulation is matched by an equal increase in pointed ends (Cano et al., 1991; Carson, et al., 1986). Merely doubling
the number of filaments should not shift the critical concentration.
Furthermore, it is unclear if these barbed ends are available in the
intact cell or freed only upon lysis and dilution. Indeed, at high cell
concentrations, lysates contain sufficient capping activity to cap all
the barbed ends present in both control and stimulated cells
(Cassimeris et al., 1992; Southwick and DiNubile, 1986). Thus,
it is unclear what fraction of filaments are free in the intact cell
and whether this fraction is altered by stimulation. Attempts to
determine whether free barbed ends stimulate actin polymerization in
intact cells by injection of free barbed ends (small actin filaments)
gives negative or ambiguous results (Sanders and Wang, 1990; Handel et al., 1990).
Also equivocal is evidence that F-actin is regulated by freeing barbed ends that is based on experiments with cytochalasin, a barbed end capper. Cytochalasin inhibits agonist-induced polymerization in neutrophils, suggesting that polymerization occurs primarily at barbed ends (MacLean-Fletcher and Pollard, 1980; White et al., 1983). However, this does not indicate that the freeing of barbed ends is the regulated event; a similar inhibition by cytochalasin would be expected were agonist releasing profilin to facilitate polymerization on existing free ends. Furthermore, cytochalasin has many effects such as stabilizing actin dimers and increasing the rate of ATP hydrolysis (Sampath and Pollard, 1991). Thus, cytochalasin could exert its effects in cells by shifting the nucleotide bound to G-actin from ATP to ADP. So overall, the evidence supporting regulation of F-actin by freeing of barbed ends is weak.
One cannot study how an agonist causes actin polymerization in vitro because after cell lysis both natural agonists and
GTPS cease to stimulate an increase in F-actin. However,
permeabilized neutrophils, like intact cells, double their F-actin
level upon addition of appropriate agonist (Downey et al.,
1989; Therrien and Naccache, 1989; Bengtsson et al., 1990;
Redmond et al., 1994). Neutrophils permeabilized with
streptolysin O (SLO) have large pores, allowing entry of exogenous
proteins up to 120,000 Da (Redmond et al., 1994; Bhakdi et
al., 1993). This permitted us to modulate the cytoplasmic G-actin
by means of exogenous monomer binding proteins. We utilized the
different properties of monomeric actin binding proteins to dissect the
changes that lead to actin polymerization. In particular, we utilized
the unusual properties of G-actin profilin complex, which can
contribute G-actin to the barbed but not pointed end of an actin
filament (Tilney et al., 1983; Pollard and Cooper, 1984; Pring et al., 1992; Pantaloni and Carlier, 1993; Giuliano and
Taylor, 1994). The results put on firmer ground the idea that increase
in F-actin by chemoattractants is mediated through an increase in the
availability of free barbed ends.
Thymosin 4 (T
4) was isolated from calf spleen by the
method of Cassimeris et al.(1992) with minor modifications.
The amount
of G-actin, T4, and profilin in a permeabilized cell is not
constant because endogenous proteins are continually leaving. Analysis
of the G-actin released into the medium (assayed by DNase I inhibition)
indicated that at the time of stimulation with GTP
S (i.e. 2 min) about half of the G-actin pool has left the cell (Redmond et al., 1994). Since T
4 is in rapid equilibrium with the
G-actin, we assumed that half of the T
4 has also left the cell.
Indeed, slightly more than 50% of the profilin released by Triton lysis
was released into the medium after warming SLO-permeabilized cells for
2 min. Profilin released from permeabilized cells or after Triton lysis
was collected on a polyproline column, eluted with urea, and quantified
by comparison to profilin standards on Coomassie Blue-stained SDS gels
(Kaiser et al., 1989).
Thus after 2 min, with about 125
µM T4 and 57.5 µM G-actin remaining in
the cell, about 57 µM T
4 would be bound to G-actin
and the free G-actin concentration would be
0.5 µM (in equilibrium with the free pointed end). We then calculated the
amount of G-actin that would be released from T
4 if the affinity
of F-actin were decreased to 0.1 µM, allowing actin to
polymerize until the free G-actin decreased to 0.1 µM.
This would allow G-actin bound to T
4 to be released until the
complex was in equilibrium with 0.1 µM G-actin. The amount
of G-actin that would polymerize in the absence of exogenous monomer
binding proteins (
40 µM) was set at 100%.
Since
the concentration of free G-actin in the medium outside the
SLO-permeabilized cells was extremely low, it was unlikely that G-actin
binding proteins in the external medium would increase the rate of
G-actin exit from permeabilized cells. Thus, the effects must be
mediated by the proteins that entered the cell. To estimate the effects
of exogenous monomer binders on polymerization induced by GTPS
stimulation, we assumed the concentration of monomer binder inside the
cell (bound or free) during the polymerization (i.e. between 3
and 5 min) was equal to the concentration added to the medium. While
this is an overestimate, the actual value would be greater than half of
this concentration.
To evaluate the effects of exogenous monomer
binders on the pool of T4-actin present inside the cell and
responding to the change in free G-actin concentration, we assumed that
essentially all of the DNase I, which has an affinity for G-actin of
about 1 nM (Weber et al., 1992), that entered the
cell would bind G-actin. This would decrease the pool of T
4-actin
complex present, lower the concentration of free G-actin, and cause
F-actin depolymerization, as observed (Fig. 1). We assumed that
a fraction of the T
4 that entered the cell would bind G-actin (the
fraction determined by its affinity, which was assumed to be the same
as endogenous T
4, 0.6 µM). This would increase the
amount of T
4-actin complex present and cause depolymerization.
While some of the G-actin released by depolymerization would leave the
cell, some would bind to the monomer binders present; for this
calculation, the contribution of G-actin released by depolymerization
was ignored.
Figure 1:
Concentration dependence of DNase I,
T4, and profilin on basal and GTP
S-induced F-actin levels.
Cells permeabilized with 2
10
IU SLO/cell
were resuspended in IP buffer containing various concentrations of
DNase I (panel A), T
4 (panel B), or profilin (panel C). After incubating for 3 min at room temperature, 100
µM GTP
S was added to half of the samples (filled
diamonds) at each concentration (open squares,
unstimulated samples). 2 min later, all of the samples were fixed and
stained with TRITC-phalloidin as described under ``Experimental
Procedures.'' The data from three (A) or two (B and C) different experiments were pooled by setting as
100% the basal level of saturable TRITC-phalloidin (basal fluorescence
minus the fluorescence in the presence of excess unlabeled phalloidin). Error bars indicate the S.D. (A) or range of values (B and C).
We then calculated for each concentration of monomer
binder in the cell the change in the amount of G-actin that would be
bound to T4 (endogenous and exogenous) if stimulation changed the
critical concentration as described above. This change was expressed as
a percentage of the change occurring in the absence of exogenous
monomer-binding proteins.
On-line formulae not verified for accuracy
where [P] is free profilin,
[PA] is the profilin-G-actin complex, f is the fraction of barbed filament ends with
profilin bound, and f
is the fraction free to
elongate. K
and K`
are the
affinity constants of profilin for free G-actin and barbed filament
ends, respectively. A is then obtained by setting the net
filament elongation rate to zero
On-line formulae not verified for accuracy
where k, k
, and k`
are the rate constants of filament elongation at the barbed and
pointed ends by G-actin, and at the barbed end by the profilin-G-actin
complex respectively; and k
, k
and k`
are the corresponding depolymerization rate constants. This
equation, which yields a cubic equation in A, was conveniently
solved numerically by binary search with an initial range from zero to
the pointed end critical concentration.
Inclusion of profilin in the buffer at the time of
permeabilization also decreased the basal F-actin level (lower
curve in Fig. 1C). The effects of profilin were
similar, although often not as pronounced as those of T4. After
warming for 4 min in 40 µM profilin, the basal F-actin
decreased by 40 ± 10%. The fact that addition of profilin
decreased the F-actin in unstimulated cells indicated that profilin had
entered the cells and was able to sequester G-actin. The fact that the
addition of profilin in the cell caused a decrease, not an increase, in
F-actin ruled out the possibility that merely freeing profilin from a
sequestered pool could account for the increase in cellular F-actin
upon stimulation.
Figure 2:
Time course of polymerization in the
presence of profilin or T4. Cells were permeabilized and
resuspended in buffer with (triangles) or without (squares) 40 µM profilin (panel A) or 40
µM T
4 (panel B). After warming for 2 min,
100 µM GTP
S was added to half of the samples (filled symbols), which were then fixed and stained with
TRITC-phalloidin at various times. In each case, the data are from one
experiment representative of two.
The
freeing of barbed ends, by shifting the affinity of F-actin (for
G-actin), could account for the concentration dependence of both DNase
I and thymosin on the amount of GTP
S-induced
actin polymerization. The data from these experiments were compared
with those predicted from a simple model (see ``Experimental
Procedures'') in which the F-actin increase equalled the amount of
G-actin released from a T
4 complex and polymerizing when the
affinity of the F-actin shifted from 0.5 to 0.1 µM. When
we assumed that the concentration of monomer binder in the cell by the
time of fixation (5 min) equalled the concentration added to the
medium, the predicted increases in F-actin were similar to those
observed (Fig. 3). The fact that the model fits both the DNase I
and T
4 data suggested that stimulation was not selectively
inactivating T
4, since this would have made T
4 a less
effective inhibitor than DNase I. The model did not replicate the
slight increase in the magnitude of the F-actin induced by GTP
S in
the presence of low concentrations of T
4. An increase in the
magnitude of F-actin induced by GTP
S was also seen with profilin
at all concentrations but not with DNase I. This increase was probably
due to polymerization of the profilin and T
4-actin complexes that
arose as a result of the depolymerization of basal F-actin. These
complexes were ignored in the model, as noted under ``Experimental
Procedures,'' but could contribute to the increase in F-actin
after stimulation. However, the DNase I- actin complexes are of too
high affinity to contribute.
Figure 3:
Model of the effects of T4 and DNase
I on the polymerization induced by GTP
S. Data on the amount of
F-actin induced by GTP
S in the presence of different
concentrations of T
4 (filled squares) or DNase I (filled triangles) from Fig. 1, A and B, are compared with predictions from a model of monomer
sequestering activity (open squares) as described under
``Experimental Procedures.''
The hypothesis that GTPS acts by
making free barbed ends available is supported by experiments with
cytochalasin. Cytochalasin B inhibited much of the GTP
S-induced
polymerization. Maximal inhibition was achieved with concentrations of
cytochalasin B between 2 and 10 µM. At these
concentrations, the polymerization induced by 100 µM GTP
S was inhibited by 64 ± 3% (Fig. 4A). Since in intact cells cytochalasin B
completely inhibits the polymerization induced by chemoattractant, it
is not clear why the inhibition in SLO-permeabilized cells was not
complete. In the presence of cytochalasin, the magnitude of the
decrease in F-actin caused by either profilin (Fig. 4B)
or 40 µM T
4 (not shown) was similar before and after
GTP
S stimulation. This ruled out the possibility that the failure
of profilin to decrease the GTP
S-induced F-actin in the absence of
cytochalasin was due to GTP
S inhibiting its sequestering ability.
The presence of cytochalasin B had little effect on basal F-actin level
or on the ability of 40 µM profilin to lower the basal
F-actin levels (Fig. 4B). This is consistent with the
hypothesis that the steady state was already determined primarily by
the pointed ends.
Figure 4:
Effects of cytochalasin on
GTPS-induced F-actin levels. A, dose-response of
cytochalasin B on basal and GTP
S-induced F-actin levels.
Permeabilized cells were resuspended in IP buffer containing various
concentrations of cytochalasin B (CB). After incubating for 2
min at room temperature, 100 µM GTP
S was added to
samples (filled circles) at each cytochalasin B concentration (open circles, unstimulated samples). 2 min later, all samples
were fixed and stained with TRITC-phalloidin as described under
``Experimental Procedures.'' The data from four different
experiments were normalized as in Fig. 1. Error bars indicate the S.D. B, the effect of cytochalasin on
polymerization induced in the presence of profilin. Cells were
incubated in buffer with or without 40 µM profilin and
with or without 2 mM cytochalasin B. After 2 min, 100
µM GTP
S was added, and the samples were fixed and
stained 2 min later. Data plotted are duplicates from a single
experiment representative of two
experiments.
Figure 5:
Time-course of TRITC-phalloidin staining
in SLO-permeabilized cells stimulated with different concentrations of
GTPS. Cells permeabilized with 0.9
10
IU SLO/cell were warmed in IP buffer for 2 min and then incubated
(at time = 0) with 0 (open circles), 0.1 (filled
circles), 1 (open triangles), or 100 µM (filled triangles) GTP
S for various times. All samples
were fixed and stained as described under ``Experimental
Procedures.'' Values were set as percentage of the basal level of
saturable TRITC-staining (basal fluorescence minus the fluorescence in
the presence of excess of unlabeled phalloidin). The data are from a
single experiment representative of four
experiments.
To determine
if the cessation of polymerization at the plateau was due to depletion
of GTPS, we examined whether GTP
S was still available at the
time the plateau level of F-actin was reached. Supernatants of
permeabilized cells that had been incubated 15 min with or without a
suboptimal concentration of GTP
S (300 nM) were
ultrafiltered (cut off 10,000 daltons) to remove proteins such as
G-actin and monomer binding proteins and then tested for their ability
to stimulate polymerization in freshly permeabilized cells. The
supernatant from control cells had no effect on F-actin levels, but the
supernatant from stimulated cells induced polymerization to
approximately the same level as that observed with the original cells
(data not shown). Thus, the termination of net polymerization at
plateau was not limited by depletion of GTP
S.
Figure 6:
Effect of sequential addition of an
increased dose of GTPS on induced F-actin levels. Permeabilized
cells were warmed in IP buffer for 2 min, then incubated (at time
= 0) without (open circles) or with 100 nM GTP
S (open squares). At 4 min, 100 µM GTP
S was added to some samples of resting (filled
circles) and stimulated cells (filled squares). Samples
were fixed at various times. The data from two different experiments
were pooled as described in Fig. 1.
Figure 7:
Time course of change in basal and
GTPS-induced F-actin levels caused by addition of cytochalasin B.
Cells, permeabilized and warmed in IP buffer for 2 min, were incubated
(at time = 0) without (open circles) or with 300 nM GTP
S (open squares). At 3 min, 4 µM cytochalasin B was added on a fraction of both resting (filled
circles) and stimulated (filled squares) cells. Samples
were fixed and stained with TRITC-phalloidin at various times. The data
from 3 different experiments were pooled as described in Fig. 1. Error bars indicate the S.D.
This
conclusion that barbed ends remained free was further supported by the
observation that addition of profilin to stimulated cells did not
decrease the plateau level of F-actin. The TRITC-phalloidin staining
remained unchanged or was increased slightly (Fig. 8A).
Profilin did cause depolymerization of basal F-actin in unstimulated
cells that had been permeabilized for the same period of time,
indicating that prolonged permeabilization did not on its own make
cells insensitive to profilin (Fig. 8B). However, when
profilin was added to cells that had been stimulated with GTPS and
then treated with cytochalasin B to block all barbed ends, it did cause
depolymerization (Fig. 8A). Thus, when the barbed ends
were capped with cytochalasin, profilin did cause depolymerization even
in the presence of GTP
S. This indicated that the differential
effects of profilin on basal and stimulated cells were due to
differences in the availability of free barbed ends and reinforced the
conclusion that GTP
S did not inhibit the G-actin sequestering
ability of profilin.
Figure 8:
Time course of change in basal and
GTPS-induced F-actin levels caused by sequential addition of
cytochalasin B and profilin. Cells were permeabilized and warmed in IP
buffer for 2 min, then incubated (at time = 0) without (panel B, open squares) or with 300 nM GTP
S (panel A, open squares). At 4 min, 4
µM cytochalasin B was added to a fraction of resting or
stimulated cells (B, open triangles and A, open circles, respectively). At 6 min, 35 µM profilin was added to half of the cells for each condition: in A, control (filled squares) or cytochalasin B-treated (filled circles) stimulated cells; in B, control (filled squares) or cytochalasin B-treated (filled
triangles) resting cells. Samples were fixed and stained at
various times. The data are from a single experiment representative of
two experiments.
Figure 9:
Time course of change in basal and
GTPS-induced F-actin levels caused by addition of DNase. Cells
were permeabilized and then incubated without (basal, open
circles) or with 100 µM GTP
S (open
squares). After 2.25 min, 25 µM DNase was added to
the cell suspension of resting (filled circles) and stimulated (filled squares) cells. Samples were fixed and stained at
various times. The data from three different experiments were pooled as
described in Fig. 1.
To determine if this slight increase in rate was
due to barbed-end depolymerization, we compared the rate of
depolymerization induced by DNase I in the presence and absence of
cytochalasin, which blocks barbed end depolymerization. The presence of
cytochalasin did not detectably decrease the rate of depolymerization
over that seen with DNase alone (data not shown). Nor did the presence
of cytochalasin decrease the rate of depolymerization observed when (a) the DNase concentration was increased to 100 µM to insure that the G-actin concentration was below the critical
concentration of the barbed end or (b) the DNase was added
soon after GTPS when the F-actin level was still rising.
Figure 10:
Time course and dose-response of
nucleation sites in SLO cells stimulated with GTPS. A,
time course. Cells were permeabilized, warmed in IP buffer for 2 min
and then stimulated with 100 µM GTP
S (at time
= 0). At various times, 100 µl of cells were withdrawn and
mixed with 900 µl of an assay buffer that contained 1
µM pyrenyl G-actin. The initial rate of increase in
pyrenyl G-actin polymerization (pyrenyl fluorescence) was plotted as a
function of stimulation time before lysis. The data from five different
experiments were pooled by setting as 100% the rate of pyrenyl G-actin
polymerization induced by unstimulated permeabilized cells. Error
bars indicate the S.D. B, dose response.
SLO-permeabilized cells were stimulated with various concentrations of
GTP
S. 3 min later, the cells were lysed, and the initial rate of
pyrenyl G-actin polymerization was measured as described above. The
data from four different experiments were pooled as described
above.
It is important to note that unstimulated cells also nucleated barbed-end elongation after lysis. The presence of 2 µM cytochalasin reduced the rate of polymerization of 1.5 µM pyrenyl-actin more than 80%, suggesting that after lysis, greater than 20% of the filaments had free barbed ends (Korn et al., 1987). However, as noted above, the effects of adding cytochalasin and profilin to unstimulated cells suggested that most barbed ends were capped. Thus, sites that nucleate barbed end polymerization after lysis may not have been free in the permeabilized cell before lysis.
Figure 11:
Dependence of apparent critical
concentration on the fraction of barbed ends that are uncapped in the
presence of profilin. Calculated apparent actin critical concentrations
(free [G-actin] at which no net elongation or
depolymerization occurs) in µM at 0 (open
squares), 5 (open circles), and 30 µM (open triangles) total profilin are shown as a function
of the percentage of the barbed filament ends that are free (uncapped). Profilin binding to free G-actin and to the barbed
filament ends was assumed to be at equilibrium, after Pantaloni and
Carlier(1993), with K values of 0.5 and 7
µM, respectively. The on and off rate constants of both
G-actin and profilin-G-actin at the barbed ends were 10
µM
s
and 1
s
, and for G-actin at the pointed ends 0.4
µM
s
and 0.2
s
.
For the studies described here we have chosen to
stimulate polymerization with GTPS. GTP
S presumably acts, at
least in part, through the pertussis toxin-sensitive trimeric G-protein
through which chemoattractants act, although it may also activate
downstream small G-proteins (Redmond et al., 1994).
Stimulation with GTP
S has the advantages that (a) it
induces a large and stable polymerization of F-actin and (b)
the response is not limited by receptor dynamics, e.g. phosphorylation and internalization, or by GTP hydrolysis. Thus
the magnitude of the F-actin increase may closely reflect the
properties of actin steady state.
While the cytoplasm of a permeabilized cell is changing over time as soluble components diffuse out, it is possible to systematically investigate the effects of exogenous factors within a limited time window. Because the experiments are very rapid, one can alter the composition of the cytoplasm without the secondary effects that can result from chronic alterations, for example, following transfection of intact cells.
A 5-fold decrease in the critical concentration caused
by freeing barbed ends could account for the greater inhibition of
basal than stimulated F-actin by DNase I and T4. The fact that
both DNase and T
4 were equally well fit by a simple model suggests
that the properties of T
4 were not modified, even transiently, by
stimulation with GTP
S. Furthermore, in the presence of
cytochalasin, both profilin and T
4 caused similar decreases in
F-actin in the presence or absence of GTP
S, indicating that
neither protein was inhibited by GTP
S. This extends previous
studies that had shown that there was no stable modification of T
4
following stimulation (Safer et al., 1990; Cassimeris et
al., 1992; Nachmias et al., 1993), but had left open the
possibility that it might be transiently modified at the time of
stimulation. Local modification of T
4 or profilin is not ruled out
by the studies presented here.
The fraction of filaments that have
free barbed ends before and after polymerization in the permeabilized
cell is not known. In the resting cell, the fraction appears to be low
as profilin acts as an effective monomer-sequestering protein. However,
the fact that profilin did not decrease resting F-actin levels as well
as T4 may indicate that in the resting cell some barbed ends are
free. Indeed, in some experiments cytochalasin did decrease the basal
F-actin slightly, and profilin had a slightly greater effect on basal
F-actin when cytochalasin was present (see Fig. 4b).
Although cell lysates made after stimulation showed a large increase in
the number of free barbed ends, in the permeabilized cell, it was not
possible to detect the presence of free barbed ends by studies of the
rate of depolymerization. The 2-fold increase in rate of
depolymerization induced by DNase I in stimulated versus control cells may result from a 2-fold increase in filament number
associated with the increase in F-actin, i.e. 2 times more
pointed ends (Cano et al., 1991). Furthermore, the presence of
cytochalasin did not detectably slow the DNase-induced depolymerization
of stimulated F-actin.
One can imagine various factors including the
presence of cross-linking proteins that might limit our ability to
detect barbed ends from the kinetics of depolymerization. We therefore
also examine the rate of polymerization induced by phalloidin. Addition
of phalloidin to permeabilized cells stimulated actin polymerization in
both resting and stimulated cells. The phalloidin
presumably acts by inhibiting depolymerization while allowing addition
of monomer at either filament end. However, the rate of polymerization
was no greater in stimulated than in control cells, even when the cells
were stimulated with suboptimal concentrations of GTP
S to insure
that a reservoir of G-actin remained. Nor could we detect an effect of
cytochalasin on this rate, even though in vitro cytochalasin
effectively inhibited polymerization at the barbed end in the presence
of phalloidin.
The discrepancy between the data showing that free
barbed ends determine the stimulated F-actin steady state and the
inability to actually measure free barbed ends in stimulated
permeabilized cells is reconciled by a calculation showing that in the
presence of profilin, a very small fraction of filaments with free
barbed ends can shift the critical concentration. Thus, the number
available at any time may be well below current detection limits. These
studies shed no light on the mechanism through which barbed ends become
available upon GTPS stimulation. They may arise from uncapping,
cutting of filaments or de novo nucleation of new filaments.
These mechanisms remain to be defined.