(Received for publication, October 17, 1995; and in revised form, January 8, 1996)
From the
-Thymosins are the currently favored candidates for
maintaining the large actin monomer (G-actin) pool in living cells. To
determine if
-thymosin behaves like a simple G-actin buffering
agent in the complex environment of a cell, we overexpressed thymosin
10 (T
10) in NIH3T3 cells and determined the effect on the
monomer/polymer equilibrium. T
10 is the predominant
-thymosin
isoform in the NIH3T3 cell line, and it is present in approximately
equal molar ratio to profilin and cofilin/actin depolymerizing factor,
two other well characterized actin monomer binding proteins. Clonal
cell lines that overexpressed three times more T
10 had
23-33% more polymerized actin than control cells, and the
filaments appeared thicker after staining with fluorescent phalloidin.
There was no change in total actin, profilin, and cofilin/actin
depolymerizing factor content. The overexpressing cells were more
motile; they spread faster and had higher chemotactic and wound healing
activity. Assuming that there is no compensatory inactivation of the
other classes of monomer binding proteins, our paradoxical observation
can be accounted for quantitatively by a parallel in vitro study (Carlier, M.-F., Didry, D., Erk, I., Lepault, J., Van Troys,
L., Vanderkekove, J., Perelroizen, I., Yin, H. L., Doi, Y., and
Pantaloni, D.,(1996) J. Biol. Chem. 271, 9231-9239).
-Thymosin at levels comparable with that found in the
overexpressing cells binds actin filaments and decreases the critical
concentration (C
) for actin polymerization. This
reduces the monomer buffering ability of
-thymosin, so that above
a certain threshold an incremental increase in thymosin does not lead
to a corresponding increase in G-actin. Furthermore, the decrease in C
reduces the buffering capacity of the other
actin monomer binding proteins. As a consequence, an increase in
-thymosin does not necessarily result in a proportionate increase
in actin monomer content in a complex environment containing other
actin monomer binding proteins. The outcome depends on the level of
-thymosin expression relative to the composition of the other
actin monomer binding protien. Our results suggest that
-thymosins
are not simple actin buffering proteins and that their biphasic action
may have physiological significance.
The actin cytoskeleton of nonmuscle cells responds to
extracellular stimuli through a spatially and temporally regulated
series of polymerization and depolymerization reactions. Monomer
binding proteins, such as -thymosin, ADF(
)/cofilin, and
profilin, buffer actin monomers (G-actin) to prevent them from
polymerizing spontaneously (reviewed in (1) and (2) )). During cell activation, polymerization is initiated by
filament uncapping at the cell cortex, which lowers the critical
concentration for actin
polymerization(1, 3, 4, 5, 6) .
Actin monomer binding proteins amplify the effect of filament uncapping
because they create a reservoir of G-actin that can be desequestered to
supply actin to filament ends. Furthermore, because profilin-actin
complexes facilitate barbed end filament growth (7) and
profilin competes with
-thymosin for actin, profilin can tap into
the
-thymosin-actin pool (7, 8, 9) to
fuel massive polymerization. After polymerization, actin filaments are
transported centripetally(10, 11) and subsequently
depolymerize(12, 13) . The newly released actin
subunits are captured by monomer-binding proteins and recycled toward
the cell front.
Among the monomer-binding proteins identified thus
far, -thymosins appear to best fulfill the criteria for a simple
monomer-buffering protein because no interaction with other proteins
and no regulation of their activity has been
described(14, 15, 16) . Up until now, much of
the focus has been on
-thymosin in neutrophils and platelets that
contain sufficient
-thymosins to sequester the bulk of
G-actin(15, 16) . The contributions of other
monomer-binding proteins are therefore ignored. However, in many other
types of cells,
-thymosin does not predominate, and its
contribution has to be considered in the context of the other
G-actin-binding proteins.
In this paper, we examined the fundamental
assumption that -thymosin behaves like a simple actin
monomer-buffering protein in the intracellular milieu.
-Thymosin
was overexpressed to a moderate level by cDNA mediated transfection,
and the consequences on the polymer/monomer equilibrium were examined.
Previous overexpression studies (17, 18, 19) demonstrate that swamping the
cell with a large excess of
-thymosin causes extensive actin
disassembly, but the cortical filaments are surprisingly resistant and
the relation between
-thymosin dose and depolymerization response
is not analyzed quantitatively. We find that a 3-fold increase in the
predominant
-thymosin isoform (T
10) in NIH3T3 cells enhanced
cell spreading, chemotaxis, and wound healing. It decreased the G-actin
pool, contrary to the expectation for an exclusively buffering
function. The paradoxical result may be explained by our recent in
vitro studies, which show that thymosin
4 (T
4), an
isoform that is functionally identical to T
10(14) , binds
actin filaments with low affinity and decreases the C
(47) . This reduces the impact of
-thymosin overexpression and has repercussions for all the other
monomer-binding proteins in the intracellular milieu as well.
For some experiments, NIH3T3 cells were starved in DMEM/0.2% serum for 24 h and in a completely defined 1:1 DMEM/Ham's F-12 medium supplemented with 20 mM Hepes, pH 7.4, 0.5 mg/ml bovine serum albumin, 1 µg/ml insulin, and 1 µg/ml transferrin (Sigma) (Q-medium) for an additional 18 h.
For
-thymosin detection, cells were trypsinized from the monolayers
and washed in phosphate-buffered saline. They were resuspended in a
buffer containing 100 mM Tris-HCl, pH 7.4, 4 mM MgCl
, 1.8 mM CaCl
, 50 mM KCl, 2 mM phenylmethylsulfonyl fluoride, and 2 µg/ml
each of aprotinin, leupeptin, and pepstatin and sonicated with a
microtip probe for 30 s at 4 °C. An aliquot was removed for protein
quantitation by the micro-BCA method (Pierce), and the remainder was
centrifuged at 14,000 rpm for 10 min in an Eppendorf microfuge. The
supernatant was boiled in SDS gel sample buffer and electrophoresed in
5-20% gradient SDS-polyacrylamide gels. Western blotting was
performed as described previously(17) , using glutaraldehyde to
cross-link
-thymosin in the acrylamide gel prior to
electrophoretic transfer to enhance retention on the nitrocellulose
membrane. Greater than 99% of
-thymosin was recovered in the
supernatant.
For detection of proteins other than -thymosin,
cells were lysed in cold RIPA buffer (50 mM NaCl, 0.1% SDS, 1%
Nonidet P-40, 2 mM EDTA, 2 mM EGTA, 50 mM Hepes, pH 7.4) containing the protease inhibitor mixture.
Polypeptides in SDS gels were transferred to nitrocellulose without
glutaraldehyde fixation (28) .
Immunoreactive bands were
visualized with the ECL detection system (Amersham Corp.). The
intensity of the stained bands was determined by scanning with a
Molecular Dynamics 300A computing densitometer. Serial dilutions of the
cell lysates were analyzed, and samples within the linear range were
compared with actin or actin-binding protein standards to estimate
their content. Purified recombinant rat T4 and T
10
concentrations were determined by amino acid analyses. Bovine spleen
profilin, rabbit muscle actin, human plasma gelsolin, recombinant
chicken ADF, and chicken cofilin (gifts of J. Bamburg) were prepared by
standard methods, and their protein concentrations were determined by
the micro-BCA method.
Fluorescence images were acquired with a cooled CCD camera
(Photometrics, Tuscon, AZ) on an Axiovert 135 microscope (Carl Zeiss
Instruments, Thornwood, NY) using 63 or 100
oil immersion
Plan-Neofluar objectives.
Figure 1:
Analysis of T10
overexpression. A, Western blot. T
10 antibody was used to
probe NIH3T3 cells transfected with Ctrl or T
10 expression vector.
15 µg of cell extracts were analyzed. B, Western blot.
T
4 antibody was used to probe purified T
4 standards, CV1 and
NIH3T3 cells. Lanes 1-3, 7, 14, 28 ng of T
4; lanes 4 and 5, 1.8 and 3.8 µg of CV1 extract; lanes 6 and 7, 15 µg of Ctrl and TK9 extracts.
The streak between lanes 5 and 6 was due to gel
loading problems and should be ignored. C, analytical HPLC.
Equal amount of lysate samples were analyzed. The arrow indicates the peak that is not T
4. The TK9 tracing was more
compressed relative to the others. D, Northern blot. Right
panel, T
10 cDNA probe was used to probe U937, TK9, and Ctrl
RNA. Left panel, T
4 probe. 10 µg of total RNA was
analyzed per lane.
T10 overexpression and the predominance
of endogenous T
10 over T
4 were confirmed by analytical HPLC (Fig. 1C). CV1, which contains abundant T
4 and
T
10(17) , was used as a standard for identifying the
thymosin isoforms in the HPLC profiles. Two major peaks that coincided
with purified T
4 and T
10 standards were present at a 1.7:1
ratio. Ctrl NIH3T3 cells had a small T
10 peak and no peak
coinciding with the T
4 standard. The neighboring peak (indicated
by an arrow), which overlapped slightly with the tail end of
the bona fide T
4 peak, did not shift the mobility of
actin on nondenaturing gels (data not shown) and was unlikely to be
T
4. TK4 and TK9 had progressively more T
10 than Ctrl cells.
The extent of overexpression, based on the integrated area under the
peaks, were 1.6- and 2.7-fold, respectively (Table 1). These
values were similar to Western blot estimates.
The abundance of
T10 relative to T
4 in Ctrl cells and the overexpression of
T
10 in TK cells were also evident at the mRNA level. In Northern
blots, the T
10-specific probe hybridized strongly with Ctrl cells
and more strongly with the T
10-transfected clone TK9 (Fig. 1D). Only one positive band was detected in the
overexpressing cells, because the T
10 overexpression construct was
approximately the same size as the T
10 mRNA. In contrast, the
T
4-specific probe gave minimal signal for either clone, even
though it hybridized strongly to U937, a mouse macrophage cell line.
These results demonstrated that the T
10 and T
4 probes were
specific for each mRNA species and T
10 was the dominant isoform in
the NIH3T3 line used in these studies.
where S is the concentration of
G-actin-binding proteins S
, and K
is the equilibrium dissociation constant for the binding of S
to G-actin.
T10 and actin accounted for
0.06 ± 0.007 and 2.8 ± 0.1% (average and range) of total
cell proteins in Ctrl cells, respectively (Table 2). Based on the
protein content per cell and cell diameter (Table 3), the
cytoplasmic concentrations of T
10 and actin in Ctrl cells were 8.8
and 44.9 µM, respectively. The unpolymerized actin pool,
estimated from the amount of actin that was Triton X-100 soluble after
a 10-min centrifugation at 14,000 rpm in an Eppendorf microfuge, was
46.6 ± 5.2% (mean ± S.E., n = 5) of the
total actin. Because actin filaments that contain fewer than 50
monomers and are not cross-linked to the cytoskeleton will not pellet
under these conditions(34) , the figure for unpolymerized actin
is likely to be overestimated somewhat. Thus, the upper limit for
unpolymerized actin was 20.9 µM.
The amount of
T10-G-actin complex [TA] can be calculated as
follows:
where [T] is the T
10
concentration, K
is the equilibrium
dissociation constant for the TA complex. Using values of 0.6
µM for K
(14, 32) and 0.5 µM for C
(when barbed ends of actin filaments are capped
as would be expected in ``resting'' cells), 8.8 µM T
10 would bind 4 µM actin monomers (Table 2). Assuming that the other actin monomer-binding proteins
were maximally active and that each bound a single actin in a mutually
exclusive manner(8) , the total actin sequestered would be 15.6
µM, and [A
] would be 16.1
µM. The latter value was in reasonable agreement with the
experimentally determined upper limit of 20.9 µM.
TK9
that overexpressed T10 2.9-fold had a cytoplasmic T
10
concentration of 25.5 µM. Western blotting showed the
concentrations of actin, and the other actin-binding protein were not
altered significantly in TK cells. For example, TK9 had 97.8 ±
0.7% (mean ± S.E., n = 5) and 102.8 ±
0.5% (n = 5) of the actin and profilin, respectively,
of Ctrl cells. Assuming that there is no change in C
or K
in TK cells, [TA]
would increase to 11.6 µM, and
[A
] would be 23.7 µM (compared with 16.1 µM for Ctrl). This represented a
47.2% increase in [A
], which would be at
the expense of polymerized actin.
Figure 2:
Effect of T10 overexpression on actin
filament organization. NIH3T3 cell lines transfected with Ctrl or
T
10 vectors were fixed, permeabilized, and stained with rhodamine
phalloidin. A and C, Ctrl and TK9 cells maintained in
DMEM/10% FCS. B and D, Ctrl and TK9 cells that were
serum-deprived. Bar, 20 µm.
This was confirmed directly from the rate of spreading after reseeding. Within 0.5 h of plating, TK cells were more spread than Ctrl cells (Fig. 3), and the cell margins were highly stained with phalloidin. Cell spreading was quantitated by tallying the number of adherent cells that did not have a round shape. TK9 had almost twice as many spread cells as Ctrl at 0.7, 1, and 1.5 h after plating (Fig. 4B). After 2 h, TK and Ctrl had comparable numbers of spread cells. TK4 spread at a rate intermediate between that of TK9 and Ctrl. Direct quantitation of phalloidin binding showed that at zero time, TK9 cells in suspension had 1.19 ± 0.05 times (n = 6; p < 0.05) more polymerized actin than Ctrl. One hour after plating, TK had 1.25 ± 0.06 times (n = 6; p < 0.05) more polymerized actin than Ctrl (Fig. 4A). Therefore, TK cells in suspension had more polymerized actin than Ctrl, and the difference was maintained during spreading.
Figure 3:
Effect of T10 overexpression on cell
spreading. Cells were trypsinized, resuspended in DMEM/10% FCS, and
plated on coverslips at 37 °C. Coverslips were fixed at intervals
and labeled with rhodamine-phalloidin after permeabilization. A, C, E, G, and I, Ctrl
cells 0.5, 1, 1.5, 2, and 5 h after plating. B, D, F, H, and J, TK9 cells after similar
intervals. Bar, 60 µm.
Figure 4:
Effect of T10 overexpression on cell
spreading and actin polymerization. A, quantitation of
phalloidin binding to cells during spreading. The error bars are S.E. (n = 6). B, percentage of cell
spread as a function of time. Cells that had irregular outlines were
scored and expressed as a percentage of total cells. 100-120
cells from each cell line were counted per time point. The results
shown were from one experiment. Similar results were obtained with two
other experiments.
Likewise, motility as measured by the rate of migration of cells into monolayer wounds was also significantly higher in TK than Ctrl. The extent of increase was not as high as in chemotaxis.
The actin monomer pool in cells is an important component of
the actin machinery(1, 9) , and -thymosins are
the currently favored candidates for maintaining actin in the reserve
pool(2, 35) . In this paper, we find that T
10
does not behave like a simple G-actin buffering agent in vivo.
Raising T
10 concentration to a modest extent in NIH3T3 fibroblasts
unexpectedly promotes filament assembly. Consistent with an increase in
F-actin, the cells are more motile in a variety of actin-based
activity, including spreading, chemotaxis, and wound healing. This
phenotype is observed with several overexpressing cell lines,
confirming that it is a direct consequence of overexpression and not
due to clonal variations or artefacts of DNA integration. Enhancement
of actin polymerization is contrary to what is expected for an increase
in monomer buffering capacity and suggests that additional factors are
involved. A discrepancy between
-thymosin level and actin monomer
pool size has also been reported by another group in a preliminary
form(35, 36) .
Overexpression of profilin by cDNA-mediated
transfection in tissue culture cells also increases F-actin
content(38) . However, unlike -thymosin overexpression,
this is the result of a preferential increase in filaments in the
dynamic cell cortex accompanied by disassembly of stress fibers. We do
not observe a redistribution of actin filaments between these two
compartments in the
-thymosin overexpressing cells, implicating a
different mechanism for raising F-actin concentration.
The
-thymosin overexpression phenotype is also superficially similar
to that of CapG (22) and gelsolin (39) overexpression.
These proteins are filament barbed end capping proteins that are
regulated by Ca
and
polyphosphoinositides(40) . In addition, gelsolin also severs
actin filaments and binds monomers(41) . When they are
overexpressed using the same vector backbone and in the same parent
NIH3T3 cell line as employed in the present study, the cells are more
active in chemotaxis and wound healing. However, there is no increase
in actin filament content and cell spreading. Instead, increased
motility is associated with enhanced polyphosphoinositide signaling.
Parallel studies with TK cells did not show an increase in
polyphosphoinositide metabolism, (
)again suggesting that
although the motile phenotypes are superficially similar, different
mechanisms are involved in increasing cell motility. In the case of
-thymosin overexpression, an increase in F-actin content can best
explain the phenotypic changes.
Because
the amount of unpolymerized actin (A) is also
dictated by the K
of the binding protein-actin
complex and the C
, these variables will be
considered. The K
of the regulatory protein for
actin can be altered by the nucleotide bound state of the actin monomer
as well as the activation state of the binding protein. Among the
proteins examined,
-thymosin is particularly sensitive to the
nucleotide content of actin(4) , but
-thymosin itself is
not known to be regulated directly by other effectors. Its K
for ATP-actin is 50-fold less than ADP-actin, so
a shift from ATP-actin to ADP-actin in TK cells will render T
10
inactive. However, there is no evidence for a large pool of actin
clamped in the ADP-bound form in cells(43) . Furthermore,
ADP-actin will increase the C
and cannot explain
the increase in F-actin in TK cells. On the other hand, inactivation of
other monomer-binding proteins by increasing their K
for actin is possible, because each of the proteins studied
(except for
-thymosin) can be regulated by physiologically
relevant signals or post-translational modifications. These include pH (44, 45) and phosphorylation for cofilin and
ADF(26) , Ca
and phospholipids for
gelsolin(41, 46) , and phospholipids for
profilin(20) . However, because each class of proteins is only
present at approximately equimolar ratio to the endogenous
-thymosin and a portion of it is probably already in the inactive
state (e.g. 38% of the ADF in NIH3T3 are phosphorylated and
therefore inactive(26) ), more than one class of actin
monomer-binding proteins will have to be inactivated simultaneously.
This possibility is beyond the scope of the present paper and cannot be
addressed definitively. A major problem is that it is difficult to
directly ascertain the intracellular activation states of these
proteins because each is regulated by multiple signals and can bind
both G- and F-actin. For example, although phosphorylation of ADF and
cofilin can be assessed by two-dimensional gel electrophoresis (26) , the activity of the unphosphorylated proteins is not
known, because they are further regulated by pH and can bind actin
filaments as well.
The possibility that -thymosin
overexpression reduces the C
is supported by our
recent in vitro data with T
4(47) . We find that
although T
4 behaves like a simple actin sequestering protein at
low concentration (<20 µM), it fails to depolymerize
actin as efficiently as expected at higher concentrations.
Sedimentation and chemical cross-linking show that T
4 binds actin
filaments with low affinity and substoichiometric binding induces a
dose-dependent increase in C
. Electron microscopy
shows that the filaments are twisted around each other into bundles.
This activity of
-thymosin was not noticed previously because
relatively low concentrations of
-thymosin were used.
Lowering
the C mitigates the impact of T
10
overexpression and reduces the effectiveness of the other
monomer-binding proteins as well. Assuming from the in vitro data (47) that 26 µM T
10 decreases the C
from 0.5 to 0.2 µM, total
sequestered actin will drop from 23.2 to 14.3 µM (Table 2). The unpolymerized actin pool in TK9 will be 9.9%
smaller than Ctrl, in spite of a 3-fold increase in T
10. Thus, our in vitro observation that raising intracellular
-thymosin
decreases C
can, in theory, account for our in
vivo results quantitatively. Likewise, T
4 overexpression in
another NIH3T3 cell line also does not increase the actin monomer
pool(35, 36) . However, unlike our results, the actin
monomer pool is not decreased, possibly because the cell line used has
a different monomer binding profile compared with our cells.
Nonetheless, the important conclusion is that an increase in
-thymosin does not necessarily result in a proportionate increase
in actin monomer content in a complex environment containing different
types of actin monomer-binding proteins.
The actin stress fibers in
TK cells appeared thicker, suggesting that they may be bundled. The
finding that even TK cells in suspension had more F-actin than Ctrl is
consistent with the possibility that the C is
decreased. Cells in suspension do not have extrinsic cues generated by
substrate attachment, favoring a mechanism that alters an intrinsic
property of the actin machinery itself. Although our preliminary
immunofluorescence studies did not show colocalization of T
10 with
phalloidin-stained actin filaments, (
)low affinity binding
of thymosin to actin filaments is a viable mechanism because
-thymosin binds F-actin substoichiometrically and therefore may
not be present in sufficient quantity to be detected.