(Received for publication, October 18, 1995; and in revised form, January 31, 1996)
From the
Thymosin is acknowledged as a major G-actin
binding protein maintaining a pool of unassembled actin in motile
vertebrate cells. We have examined the function of T
in actin assembly in the high range of concentrations (up to 300
µM) at which T
is found in highly motile
blood cells. T
behaves as a simple G-actin
sequestering protein only in a range of low concentrations (<20
µM). As the concentration of T
increases,
its ability to depolymerize F-actin decreases, due to its interaction
with F-actin. The T
-actin can be incorporated, in low
molar ratios, into F-actin, and can be cross-linked in F-actin using
1-ethyl-3-(3-dimethylaminopropyl)carbodiimide. As a result of the
copolymerization of actin and T
-actin complex, the
critical concentration is the sum of free G-actin and
T
-G-actin concentrations at steady state, and the
partial critical concentration of G-actin is decreased by
T
-G-actin complex. The incorporation of
T
-actin in F-actin is associated to a structural
change of the filaments and eventually leads to their twisting around
each other. In conclusion, T
is not a simple passive
actin-sequestering agent, and at high concentrations the ability of
T
-actin to copolymerize with actin reduces the
sequestering activity of G-actin-binding proteins. These results
question the evaluation of the unassembled actin in motile cells. They
account for observations made on living fibroblasts overexpressing
-thymosins.
It is generally thought that to elicit a motile response to
extracellular signals, living cells regulate their content in F-actin,
by spatially controlled changes in the steady state of filament
assembly(1) . Capping proteins and G-actin binding proteins are
major players in this control. In the presence of ATP, capping proteins
block the dynamics at the barbed ends of actin filaments and establish
the high critical concentration of the pointed ends; when barbed ends
are uncapped, the effective critical concentration is close to the
critical concentration of the barbed end. The changes in critical
concentration, however, represent a very small amount of actin in mass
(less than 1 µM), hence by themselves they cannot elicit
any massive assembly of actin. These changes, however, are largely
amplified by G-actin binding proteins which maintain a pool of
unassembled (sequestered) actin, used for site-directed actin assembly.
The concentration of actin in complex with sequestering proteins is
indeed determined by the concentration of free G-actin, i.e. the critical concentration. The concentration of actin in complex
with sequestering proteins is high when barbed ends are capped, and
decreases locally to yield F-actin upon creation and maintenance of
available barbed ends, which is thought to occur upon stimulation. A
major G-actin binding protein is thymosin (T
) discovered in 1991 (2) in platelets
and later found to be ubiquitous in vertebrate cells (see for review, (3) and (4) ). The function of T
as a
simple passive sequestering protein was demonstrated in vitro(5, 6, 7) as well as in
vivo(8, 9, 10, 11) . The
equilibrium dissociation constant for the G-actin-T
1:1 complex lies in the 0.7-2.5 µM range, from
measurements of its sequestering activity (5, 6, 7) as well as from direct binding
studies(12, 13) . However, in the above experiments,
the concentrations of T
used were lower than the
physiological concentrations, especially those found in motile blood
cells (200-500 µM in platelets and
neutrophils(5, 8) ). In the present work, the function
of T
is explored in greater detail in the higher
concentration range found in motile living cells. The role of
T
appears more complex than previously thought,
because actin filaments fail to totally depolymerize in the presence of
high concentrations (100-200 µM) of
T
, due to incorporation of very low amounts of
T
-actin in the filaments. The consequences of this
property of T
on the structure of filaments and on the
regulation of actin assembly in living cells is examined.
[C]Thymosin
was chemically
synthesized on a model 431A peptide synthesizer (Applied Biosystems
Inc., Foster City, CA) with an additional Gly-Cys at the extreme C
terminus. Previous studies have indicated that the nature of the C
terminus of T
is not important for actin
binding(17) . The C-terminal cysteine residue was then labeled
with iodo-[1-
C]acetamide (Amersham) using a
1:1.5 molar ratio of thymosin
to label. The resulting
[
C]thymosin
had a specific
activity of 20,000 cpm/nmol.
T was oxidized into
Met
-sulfoxide-T
by incubation for 1 h at
room temperature in the presence of 2 M H
O
(6% v/v), immediately followed by lyophilization. Profilin was
purified from bovine spleen as described (18) . Gelsolin was
purified from pig plasma as described(19) . Recombinant CapG
was expressed and purified as described(20) .
Barbed ends were capped by either gelsolin or CapG. Gelsolin was added at a gelsolin:actin ratio of 1:200 to 1:500. When CapG, which is a weaker capping protein, was used, it was added at a constant concentration of 120 nM in all samples, independently of actin concentration. Preliminary assays were run with each batch of CapG used in this work, to verify that the critical concentration of the pointed end was established as soon as at least 80 nM CapG was present in solution. The amount of F-actin and unassembled actin present at steady state was converted into molar concentrations by comparison of the fluorescence readings with a critical concentration curve carried out in parallel using the same actin solution, in the same concentration range as in the samples.
where A represents the critical concentration at the
pointed end. The value of K
was derived from the
slope - A
/(A
+ K
) of the linear decrease in the fluorescence of
pyrenyl-F-actin versus T
.
In the second method,
the rate of filament growth at a given concentration C of
G-actin was measured in the presence of different concentrations of
T
. Since only free G-actin can appreciably participate
in assembly, filament growth was inhibited due to the formation of the
T
-actin complex(6) . Gelsolin-capped filaments
were used as seeds, and the value of C
was chosen low
enough (C
3 µM) for the free G-actin
concentration dependence of the rate of growth at the pointed ends of
actin filaments to vary linearly in the range (O to
C
)(22) . Under these conditions, the fraction of
T
-bound actin,
, was directly proportional to the
percent of inhibition of filament growth:
where V(0), V(T), and V(
) were the elongation
rates measured in the absence or in the presence of a concentration
T
of T
, or at infinite concentration of
T
, respectively. (Note that V(
) theoretically
equals the rate of depolymerization of filaments upon dilution, which
was experimentally verified.) Data were analyzed within the following
equation which describes the hyperbolic binding of T
to G-actin:
The value of K was derived from the slope of
1/(1-
) versus [T
]/
.
Figure 2:
T causes incomplete
F-actin depolymerization at high concentration. A, series of
samples containing F-actin at 3 µM (
) (data from Fig. 1), 5 µM (
), 10 µM (
), 20 µM (
), 120 nM CapG and
T
at the indicated concentrations were incubated
overnight. Data were analyzed and presented as in Fig. 1. The dotted line represents the variation of [A] +
[TA] expected within . Note that the origin of
the ordinate scale is set at 0.5 µM. Inset: samples of 15 µM F-actin containing 120
nM CapG and O (a, a[prime]), 40 (b,
b`), 100 (c, c`), and 250 (d, d`) µM T
were incubated for 16 h and centrifuged at
400,000
g for 30 min at 20 °C. The amount of actin
in the electrophoresed supernatants (a-d) and pellets
resuspended in the initial volume (a`-d`) is shown. 50 µl
of each sample were loaded on the gel. B, comparison of the
effect of T
on F-actin with capped and uncapped barbed
ends. Actin (12.5 µM, 1% pyrenyl labeled) was assembled in
the absence (
) or presence (
) of 120 nM CapG and
T
at the indicated concentrations. The concentration
of F-actin at steady state was derived from pyrene fluorescence
readings following overnight incubation. Each sample containing CapG
was then supplemented with 0.2 mM EGTA and fluorescence was
read again 3 h later (
). C, time course of
repolymerization upon uncapping of barbed ends, at different
concentrations of T
. F-actin (12.5 µM, 1%
pyrenyl labeled) was incubated overnight in the presence of 120 nM CapG and the following concentrations of T
, in
µM, top to bottom curves a-e: 44, 124,
195, 221, 248. At time 0, 0.2 mM EGTA was added to each
sample, and the time course of repolymerization due to the lowering of
critical concentration of G-actin was monitored by pyrene fluorescence.
The emission shutter was periodically opened for 5 s and closed for 30
s.
Figure 1:
T is a simple G-actin
sequestering protein and fully depolymerizes actin at low
concentration. F-actin (3 µM, 1% pyrenyl labeled)
containing 120 nM CapG was incubated overnight in the presence
of the indicated amounts of T
. The amount of
unassembled actin formed at steady state, [A] +
[TA], was derived from pyrene fluorescence measurements (see
``Materials and Methods'') and varied linearly with
T
according to , until all F-actin
(2.45 µM) was depolymerized. The slope of the line was
consistent with a value of 2 ± 0.2 µM for K
, using a value of 0.5 µM for the critical concentration at the pointed
end.
In the
second method, unlabeled bovine spleen T was used.
T
present in the pellets of F-actin was assayed by
HPLC of the perchloric extract of the resuspended pellets, and
comparison of the peak areas of the absorbance elution diagram at 220
nm with a calibration curve using standards in the range 0-2 nmol
of T
. The proportion of contaminant T
trapped in the pellet was estimated as in the first method.
When barbed ends are capped, A is the critical
concentration at the pointed ends of filaments, which is higher than at
the barbed ends, hence T
sequesters G-actin more
efficiently. This situation is the most favorable, for economy of
material, to measure the affinity of T
for G-actin. Fig. 1shows that upon addition of increasing amounts of
T
to a solution of 3 µM F-actin capped by
gelsolin, the concentration of unassembled actin (A + TA)
increased linearly with total
-thymosin until all actin was
depolymerized. A value of 2 ± 0.2 µM was derived
for
from the slope of the plot, in good agreement with
previous data (5, 6, 7) obtained in the same
range of actin and T
concentrations.
It was found appropriate to verify that the ATP had
not been extensively hydrolyzed during the 16-h incubation period, due
to the steady-state ATPase of F-actin, and that even the most
concentrated F-actin samples could be truly considered as being at
steady state in the presence of ATP. G-actin (20 µM) was
equilibrated in G-buffer in which the 200 µM ATP were
traced by [-
P]ATP, and polymerized by
addition of 2 mM MgCl
and 0.1 M KCl as
described under ``Materials and Methods.'' The solution was
then split into 3 samples containing 0, 50, and 200 µM T
. After 16 h incubation, the amounts of
hydrolyzed ATP were 29, 28.8, and 31.8 µM, respectively,
in the three samples. Hence free nucleotide in the medium then
consisted of about 10 µM ADP and 190 µM ATP.
These numbers testify that the measurements made after 16 h truly
reflect a situation in which F-actin is at steady-state in ATP, hence
free G-actin is ATP-G-actin at the critical concentration, and
T
binds ATP-G-actin as described by .
Thus far the conclusion that T fails to totally
depolymerize F-actin at high concentration relies on fluorescence
measurements of pyrenyl actin. To verify that the measurements truly
reflect equilibrium values of F-actin, G-actin, and
T
-G-actin, the following controls were carried out. 1)
Identical fluorescence readings were obtained 8 h later, indicating
that measurements reflected a stable situation; 2) identical results
were obtained with actin solutions containing different fractions of
pyrene-labeled actin; 3) sedimentation of the samples of F-actin
containing different concentrations of T
and SDS-gel
electrophoresis of the pellet and supernatant (Fig. 2A,
inset) established the validity of the interpretation of
fluorescence measurements in terms of F-actin and unassembled actin; 4)
sedimentation velocity of T
at different
concentrations up to 250 µM showed that T
was a monomeric 5-kDa protein in the whole range of
concentrations investigated. Finally quantitatively identical results
showing incomplete depolymerization of F-actin were obtained with
chemically synthesized T
, which eliminates the
possibility that a minor contaminant present in the preparation of
T
from spleen would be responsible for the incomplete
depolymerization of F-actin at high T
.
The fact
that T fails to totally depolymerize F-actin in a
range of high concentrations is a first indication that it can bind to
F-actin as well as to G-actin, albeit with a lower affinity. Because
other actin-binding proteins like ADF/cofilin (26, 27) have been shown to bind preferentially either
F- or G-actin depending on pH, the depolymerization of F-actin (20
µM) by increasing amounts of T
was
measured at pH 6.5 and 7.8 in parallel. Practically identical curves
[TA] = f([T
]) showing
incomplete depolymerization were obtained at the two pH values (data
not shown). On the other hand, the failure of T
to
totally depolymerize F-actin at high concentration was only observed at
physiological ionic strength (0.1 M KCl). In a polymerization
buffer containing only 2 mM MgCl
, addition of
increasing amounts of T
to 20 µM F-actin
led to eventual complete depolymerization, and a linear curve
[TA] = f([T
]) was
obtained (like in Fig. 1), consistent with a value of K
of 0.8 µM, in agreement with previous determinations
at low ionic strength(12) .
The incomplete depolymerization
of F-actin at high concentration of T was also
observed when barbed ends were uncapped. Fig. 2B shows
the amount of F-actin observed at steady-state upon addition of
increasing amounts of T
to 12 µM F-actin,
with barbed ends either capped by CapG, or uncapped, in two parallel
series of samples. When 0.2 mM EGTA was added to the samples
containing barbed end-capped F-actin, the dissociation of CapG (28) led to the partial repolymerization of actin filaments due
to the shift in critical concentration caused by uncapping. The amount
of F-actin reached at the end of this relaxation process was the same
as the one measured in the uncapped samples, confirming that
measurements shown in Fig. 2A reflect an equilibrium
situation.
The data show that the extent of repolymerization upon
uncapping of barbed ends, which is the difference between the two
curves, first increases with the total concentration of
T, and reaches a finite limit of
3 µM at high concentration of T
. The time courses of
actin desequestration/repolymerization upon uncapping by EGTA,
displayed in Fig. 2C, show that the repolymerization
process gets slower at higher concentrations of T
.
This result is very surprising for the following reason. Upon uncapping
of barbed ends due to EGTA-induced dissociation of CapG, the initial
rate of repolymerization is expected to be the following:
where kistherateconstantforadditionofG-actintotheuncappedendsatconcentration [F],
C
istheconcentrationoffreeG-actinattimeofuncapping, whichisequaltothecriticalconcentrationofthepointedends, and C
isthecriticalconcentrationforactinassemblyatthebarbedends, whichisreacheduponcompletionoftheuncapping-linkedrepolymerizationprocess. Accordingto,
theinitialrateofrepolymerizationshouldbethesameatallconcentrationsofT
. OnlytheextentofrepolymerizationshouldvaryinproportionwithT
, reflectingthedifferenceintheamountofT
-actincomplex (TA), whenbarbedendsarecappedoruncapped, asfollows.
Figure 3:
T causes the lowering of
the critical concentration. a, F-actin (1% pyrenyl labeled)
was assembled at 10 µM in the presence of 120 nM CapG and either in the absence (
) or presence (
) of 50
µM T
. Profilin was added to the samples
at the indicated concentration. Samples were incubated overnight before
fluorescence was measured. The linear decrease in fluorescence
represents the decrease in F-actin due to the formation of
profilin-actin complex. The less steep slope observed in the presence
of 50 µM T
implies a 3.8-fold lower value
of A
in the presence of 50 µM T
. B, the concentration of free G-actin
at steady state, or partial critical concentration of G-actin,
[A], was derived from the measurements of unassembled actin,
[A] + [TA], shown in Fig. 2a (
,
) using .
which leads to a quadratic equation in [A], the solution which is:
The change in [A] versus [T] can therefore be derived from the
measurements of [A
] at different values of
[T
]. Fig. 3B shows that [A]
calculated according to decreases cooperatively upon
increasing [T
]. It is interesting to observe that
50 µM T
, a value of [A] of 0.15
µM was derived from the data shown in Fig. 2A, that
is a 3.3-fold decrease in critical concentration, in good agreement
with the 3.8-fold decrease found in the experiment shown (Fig. 3A) carried out also at 50 µM T
, and in which profilin was used to derive the
value of [A]. Hence two independent methods agree
quantitatively to demonstrate that the partial critical concentration
of G-actin decreases as larger amounts of TA complex are formed at
steady-state. This result accounts for: 1) the limited extent of
repolymerization upon uncapping and 2) the slowing down in the
repolymerization process upon uncapping observed in Fig. 2C.
Indeed, because c
decreases from 0.5 µM to less than 0.1 µM (Fig. 3B) while
C
cannot decrease by more than 0.1
µM, the value of
[TA] is lower than
expected () and the value of V () decreases
upon increasing
([TA])T
Figure 4:
Evidence for low affinity binding of
T to F-actin. Samples of F-actin (20 µM)
capped by gelsolin (0.05 µM) containing the indicated
amounts of T
were sedimented at 400,000
g and the amount of bound T
per F-actin subunit was
determined by HPLC after correction for T
trapped in
the pellet (see ``Materials and Methods''). Inset,
EDC cross-linking of T
to F-actin and G-actin. Samples
of F-actin (15 µM) capped by gelsolin and containing
either 200 µM (+) or 0(-)
C-T
were cross-linked for 1 h by
EDC-sulfo-NHS as described under ``Materials and Methods.''
The samples were centrifuged and the pellets were resuspended in a
volume of buffer 2.5-fold smaller than the original volume. 25 µl
of supernatants (sup.) and 15 µl of the resuspended
pellets (pel.) were submitted to SDS-polyacrylamide gel
electrophoresis (top panel) followed by autoradiography of the
gel (bottom panel).
Chemical cross-linking of T to F-actin
displayed in Fig. 4, inset, confirmed that at high
concentration T
bound to F-actin. Approximately
5-10% of F-actin could be covalently cross-linked to T
at 200 µM
C-T
, leading
to a 47-kDa
C-labeled polypeptide migrating at the same
position as the covalent T
-G-actin complex. The mass
amount of T
-actin cross-linked polypeptide was only
about 3-4-fold lower than the mass amount of the
T
-G-actin cross-linked polypeptide found in the
supernatant of the sedimented cross-linked mixture, which rules out the
possibility of a significant contamination of covalent
T
-actin by covalent T
-G-actin trapped
in the pellet. On the other hand, an artifact might arise if the
covalently cross-linked T
-G-actin complex aggregated
and sedimented together with F-actin. To test this possibility, G-actin
(15 µM) was supplemented with 200
µMT
, followed by 1 mM MgCl
and 0.1 M KCl, and the mixture was immediately submitted
to cross-linking. No polymerization of G-actin could occur during
cross-linking due to the high amount of T
. The sample
was centrifuged at 400,000
g. Although no pellet could
be seen by the eye, any putative sedimented material was carefully
resuspended. No covalent actin-T
adduct was observed
in gel electrophoresis of the resuspended material. Therefore the
cross-linking experiments also demonstrate weak binding of T
to F-actin. The Tricine-SDS gel patterns of the cyanogen bromide
digests of the covalent
C-T
-G-actin and
C-T
-F-actin complexes were identical,
which provided an indication that the contact points between
T
and either G- or F-actin were identical.
Figure 5:
Destabilization of the filament structure
by T and intertwining of actin filaments. Panels
a-d, negatively stained samples of F-actin at steady state in the
presence of O (a), 22 µM (b), 100
µM (c), and 250 µM (d)
thymosin
. Actin was polymerized in the presence of
0.1 M KCl and 1 mM MgCl
added to G
buffer, and gelsolin in a 1:300 ratio to actin. Total actin
concentration was 2.5 µM in a, 7 µM in b, 9 µM in c and d. Arrows indicate the separations between the two strands of the
long pitch helix. Panels e and f, freeze dried and
shadowed samples of F-actin standard (e) and F-actin in the
presence of 250 µM T
(f). The circled arrow in the upper left corner indicates the
direction of shadowing. Black arrows point to the separations
between the two strands of the filament.
In a first assay, the initial rate of filament
depolymerization upon 20-fold dilution in F-buffer was measured in the
presence of increasing amounts of T. With both capped
and uncapped barbed ends, the rate of depolymerization was unaffected
by T
up to 200 µM.
In a second
experiment, the initial rate of filament growth was measured in the
presence of 3 µM G-actin and increasing concentrations of
T. The inhibition of filament growth was complete at
saturation by T
, i.e. eventually filaments
depolymerized when the concentration of free G-actin fell below the
critical concentration. The data (Fig. 6A) analyzed as
described under ``Materials and Methods'' were perfectly
consistent with binding of T
to G-actin with an
equilibrium dissociation constant of 1.6 µM, a value
identical to the one derived from steady-state measurements of the
sequestering activity of this protein. Therefore free
T
, in a large range of concentrations (0-100
µM) does not appear to influence the kinetic parameters
for filament growth, but simply acts as a G-actin sequestering protein.
The T
-actin complex, in the concentration range from 0
to 3 µM, does not appreciably affect the kinetic
parameters for filament growth. On the other hand, different results
were obtained when the inhibition of filament growth by T
was assayed at higher concentrations (6.5 µM, 10.5
µM, 14 µM) of G-actin. Using the value of 1.6
µM derived above for the equilibrium dissociation constant
of the TA complex, the values of [TA] and [A] were
calculated for each pair of total concentrations of G-actin
(C
) and of T
(T
) (see e.g.). Since the incorporation of TA in filaments is
extremely low, the process of filament growth was fed essentially by
addition of free G-actin to filament pointed ends. Accordingly, the
rate of elongation J varied linearly with the concentration of
free G-actin, but the plots obtained at 10.5 µM and 14
µM total G-actin did not superimpose, in the region
0-3 µM free G-actin, with the regular J(c)
plot obtained in the absence of T
(Fig. 6B). The linear J(c) plots
obtained at about 90% saturation of G-actin by T
, i.e. in a range of concentrations of free G-actin of 0-2
µM (i.e. in the presence of 20-100
µM T
), were characterized by a higher
slope than the control J(c) curve carried out in the absence
of T
, a lower critical concentration (defined as the
concentration of free G-actin at which the rate of filament growth is
zero), and the same value of the ordinate intercept. At the somewhat
lower value of C
of 6.5 µM, data clearly
showed a gradual shift from the coincidence with the standard J(c) at concentrations of TA
3 µM, toward
coincidence with the plots obtained at A
= 10.5
µM and 14 µM and high T
concentrations, as the saturation of G-actin by T
increased. In other words, at high concentrations T
is less efficient to inhibit filament growth than expected from
the extent of inhibition at low concentration of T
, a
result essentially in agreement with previous observations(6) .
These data demonstrate that in the presence of large concentrations of
TA complex, the partial critical concentration of actin is lower, and
this decrease in critical concentration is mediated by an increase in
the rate constant k
for association of
G-actin to filament ends, while the dissociation rate constant k
seems practically unchanged. This kinetic
piece of data agrees with and further expands upon the data of
incomplete depolymerization of actin filaments in the presence of high
concentrations of TA reported in Fig. 2, A and B, the slow rate of repolymerization upon uncapping shown in Fig. 2C, and the steady state measurements of the
decrease in critical concentration shown in Fig. 3.
Figure 6:
Effect of T and TA
complex on the kinetic parameters for pointed end filament growth. The
initial rate of elongation onto pointed ends was assayed as described
under ``Materials and Methods.'' A, inhibition of
filament growth by T
at low actin concentration (3.2
µM). The inset shows the analysis of the data
using (see ``Materials and Methods''). B, effect of high concentrations of TA on the rate of filament
growth. The rate of filament growth was measured at different
concentrations of free G-actin either in the absence of T
(
), or in the presence of T
at three
different total concentrations of G-actin (
, 6.5
µM;
, 10.5 µM;
, 14
µM). Note that at the two higher concentrations of
G-actin, the rates are measured in a range of T
concentrations of 20-100 µM, in which
90-99% of G-actin consists of TA complex. That is [TA]
= constant = 9 ± 1 and 13 ± 1.5
µM, respectively. The concentrations of free G-actin were
calculated using . The curve obtained at 6.5 µM total G-actin (
) exhibits a change in regime between 3 and
5.5 µM TA during which the kinetic parameters change from
those of the control curve to those operating at high
TA.
The present results demonstrate that the function of
T in the regulation of actin assembly is more complex
than previously thought. We confirm that in a range of low
concentrations (<20 µM), typical of the physiological
concentration of
thymosins in many cells, T
acts
as a simple G-actin binding protein. The 1:1 thymosin-actin complex,
which may accumulate at steady state up to
4 µM when
barbed ends are capped, does not participate in actin assembly and does
not affect the kinetic parameters of filament growth. On the other
hand, in a range of higher concentrations (20-250
µM), T
appears to also interact with
F-actin with a very low affinity (K
5-10 mM). The weak incorporation of T
into filaments creates defects in the structure of the polymer.
These defects can be described in terms of local points of
destabilization of actin-actin contacts perpendicular to the filament
axis, which results in local separation of the two strands of the
long-pitch helix, thus enhancing the incidental ``lateral
slipping'' feature noticed by U. Aebi on standard
filaments(29) . Therefore our results indicate that
T
binding to G-actin inhibits polymerization by
interfering with the formation of lateral actin-actin bonds along the
short pitch helix, rather than with the formation of longitudinal
bonds. At high filament concentration, the destabilized, partially
unravelled filaments tend to self-associate and twist around each other
to yield thick rope-like structures. The effect of T
on the F-actin structure may be compared to the effect of
intercalating drugs on DNA structure.
The main consequence of the
incorporation of T in F-actin is the decrease in the
partial critical concentration of G-actin. The
T
-G-actin complex cannot be considered as a good
polymerizing actin monomer since filaments containing on average 5% or
less T
-actin subunits exhibit a destabilized
structure. These unstable filaments therefore are maintained at steady
state by exchanging subunits at their ends with a pool of monomers at a
high critical concentration. The total critical concentration of
monomeric actin is the sum of free G-actin and
T
-G-actin at steady state. The partial critical
concentration of T
-actin monomer is 2 orders of
magnitude higher than the partial critical concentration of G-actin
itself under these conditions, while the copolymer consists of less
than 5% T
-actin subunits. These figures illustrate the
fact that although T
-actin is a weak polymerizing
species, its contribution to filament assembly, via a high partial
critical concentration, helps to diminish the partial critical
concentration of G-actin. Therefore, in addition to its G-actin
sequestering function, T
also possesses the power to
control the steady state of actin assembly, like capping proteins and
profilin do(18) , but in contrast to profilin, it can do it at
both ends. The decrease in partial critical concentration of G-actin
occurs smoothly over a range of high concentrations of T
(only a 3-4-fold decrease was observed at 50 µM T
).
The following consequences can be derived
from our results: the property of T to decrease the
concentration of G-actin at steady state causes a self-limitation of
the G-actin sequestering function of T
, but also
promotes a decrease in the amount of G-actin sequestered by other
G-actin binding proteins like ADF/cofilin and, when barbed ends are
capped, of profilin (as illustrated in Fig. 3A). The
present in vitro results and their analysis provide an
explanation of in vivo observations of actin dynamics in cells
overexpressing T
(a variant of T
),
which show evidence for a paradoxical decrease in the amount of
unassembled actin in overexpressing cells as compared to control cells
(see accompanying article, (33) ). Examination of the measured
cellular amounts of T
and other G-actin binding
proteins leads to the conclusion that a putative 2-fold decrease in
critical concentration linked to the 3-fold overexpression of
T
is enough to account for the observed lower amount
of unassembled actin. Under these conditions, the moderate increase in
T
-actin complex in overexpressing cells cannot fully
compensate the actual decrease in the pool of actin sequestered by
other G-actin binding proteins which is linked to the decrease in
steady state concentration of G-actin. It should be emphasized here
that, within our model, the change in the amount of unassembled actin
linked to overexpression of
-thymosin can lead to very different
situations depending on the concentration of other G-actin binding
proteins in cells. Let us assume that a living cell contains
T
and other ``bona fide'' G-actin binding
proteins that do not affect the critical concentration. For simplicity,
all these non-T
G-actin binding proteins will be
collectively considered as a single species called ``GBP.''
The pool of unassembled actin consists of T
-actin and
GBP-actin complexes. The concentration of unpolymerized actin in cells
at different total concentrations of T
and GBP is
described in a three-dimensional plot shown in Fig. 7. Iso-GBP
lines outline the effect of overexpression of T
at
different concentrations of GBP. At low concentration of GBP, the
increase in T
-actin concentration predominates over
the decrease in GBP-actin concentration, and a net increase in
unassembled actin is linked to overexpression of T
. In
the intermediate range of GBP concentration, the two effects roughly
compensate each other, and very little change in unassembled actin is
observed upon overexpression of T
. At high
concentration of GBP, the decrease in GBP-actin complex predominates
over the increase in T
-actin, resulting in a net
decrease in unassembled actin upon overexpression of
T
. Our in vitro results therefore allow
understanding of the discrepancies reported by different groups
concerning the effects of T
overexpression on the
level of actin assembly, in terms of differences in concentrations of
GBPs in different cell types. It will be interesting to challenge this
interpretation by detailed measurements of the amounts of GBPs in
different cell types.
Figure 7:
Overexpression of T lead
to an increase or a decrease in the concentration of unassembled actin,
depending on the cellular amount of G-actin binding proteins (GBP) other than T
. The concentration of
unassembled actin was calculated as the sum of [TA] and
[GBP-A] complexes. It was assumed that the values of
[A] were 0.5 µM in the absence of T
and varied with T
as described in the legend to Fig. 3. The values of the equilibrium dissociation constants for
TA and GBP-A complexes were assumed to be 1.6 µM and 0.5
µM, respectively. The concentration of unassembled actin
was calculated as follows.
Bold lines represent the change in concentration of
unpolymerized actin upon increasing T, at the
following GBP concentrations: 0 µM, 5, 15, 25, and 40
µM (front to back).
The present in vitro data also
provide an explanation for the unexplained slower rate of propulsion of Listeria in Xenopus egg extracts supplemented with
F-actin together with high amounts of T ( Fig. 6in (30) ). According to the proposed model for
actin-based Listeria movement, the rate of actin assembly is
controlled by the difference in critical concentrations between the
bulk cytoplasm (where capping proteins acts to establish the high
critical concentration of pointed ends) and the bacterium surface
(where anchored uncapped barbed ends, characterized by a low critical
concentration, are actively growing). If the critical concentration in
the bulk cytoplasm is decreased by large amounts of T
,
then the rate of assembly at the bacterium surface is expected to
decrease (such as observed here in Fig. 2C).
Our
work raises questions concerning the actual amount of actin sequestered
by T in resting platelets and neutrophils and the
physiological significance of the ``in vitro physiological ionic conditions.'' Clearly according to the
present in vitro data, very little actin (
10
µM) would be sequestered by T
in resting
platelets or neutrophils, while in vivo data clearly indicate
that at least 100 µM actin would be unpolymerized in these
cells, profilin (estimated at 50 µM in platelets) and
T
(estimated at 400 µM in platelets)
being the major actin sequestering agents. Therefore some cytoplasm
component, or macromolecular crowding, has to be thought of, which
would limit the effects of T
at the high concentration
that we observed in vitro. This component could either
stabilize F-actin (as tropomyosin would do) and consequently prevent
the incorporation of T
-actin in filaments, or it could
screen the effects of ionic strength, thereby favoring the sequestering
activity of T
over its interaction with F-actin.
Nonetheless, the intrinsic properties of T
illustrated
here have to be considered to some extent in the in vivo situation. It may be worth noting that complete agreement has not
been reached among different groups concerning the actual value of the
T
content of motile blood
cells(5, 11) . In addition, the dilution of cytoplasm
that takes place in the preparation of cellular extracts without
fixation causes depolymerization of a part of the F-actin
pool(31, 32) , which may lead to a somewhat
overestimated concentration of unassembled actin.
From a structural
point of view, the fact that T-actin is able, although
weakly, to copolymerize with actin, accounts for the difficulties
encountered in the crystallization of the T
-actin
monomer in salt-containing solutions. The observation that
T
-actin incorporates into filaments only in high ionic
strength (0.1 M KCl) assembly buffers indicates that either
electrostatic bonds in the actin-T
interface have to
be weakened, or hydrophobic bonds have to be strengthened, to allow
incorporation of T
-actin in the filament. More
detailed studies of the structure of T
-actin complex
will challenge these expectations.