From the Physiology Department, University College London,
University Street, London WC1E 6JJ, United Kingdom and the
Medical Research Council Laboratory of Molecular Biology,
Hills Road, Cambridge, CB2 2QH, United Kingdom
Received for publication, June 27, 2002, and in revised form, December 19, 2002
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ABSTRACT |
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Increasing cellular G-actin, using latrunculin B,
in either intact or permeabilized rat peritoneal mast cells, caused
translocation of both actin and an actin regulatory protein, cofilin,
into the nuclei. The effect was not associated with an increase in the proportion of apoptotic cells. The major part of the nuclear actin was not stained by rhodamine-phalloidin but could be visualized with an actin antibody, indicating its monomeric or a conformationally distinct state, e.g. cofilin-decorated filaments.
Introduction of anti-cofilin into permeabilized cells inhibited nuclear
actin accumulation, implying that an active,
cofilin-dependent, import exists in this system. Nuclear
actin was localized outside the ethidium bromide-stained region, in the
extrachromosomal nuclear domain. In permeabilized cells, the appearance
of nuclear actin and cofilin was not significantly affected by
increasing [Ca2+] and/or adding guanosine
5'-O-(3-thiotriphosphate), but was greatly promoted
when ATP was withdrawn. Similarly, ATP depletion in intact cells also
induced nuclear actin accumulation. In contrast to the effects of
latrunculin B, ATP depletion was associated with an increase in
cortical F-actin. Our results suggest that the presence of actin in the
nucleus may be required for certain stress-induced responses and that
cofilin is essential for the nuclear import of actin.
We have previously used latrunculin B
(LB)1 to explore the
relationship between actin cytoskeleton and secretion in rat peritoneal mast cells (RPMC) (1). During these studies, we have consistently noticed accumulation of actin in the nuclei of LB-treated cells. Nuclear actin was visible even in cells that, as a consequence of the
LB treatment, have lost all other structures recognizable by
phalloidin. Here we address the mechanisms involved in and conditions
necessary for this translocation.
The existence of nuclear actin has long been questioned, but recent
years have brought several lines of strong evidence indicating the
regulated nuclear import and export of actin (2). Actin has been shown
to contain two functional leucine-rich nuclear export sequences,
and their disruption leads to nuclear actin accumulation and
consequently to a decrease in cell proliferation (3). However, actin
itself does not possess any nuclear localization sequence. The
actin-binding protein, cofilin, contains a classical bipartite
SV40-type nuclear localization sequence (4) and translocates into
nuclei together with actin after a heat shock (4, 5) or dimethyl
sulfoxide (Me2SO) treatment (6).
Very little is known about the physiological function of nuclear actin.
Monomeric actin is a well known inhibitor of DNase I (7), and the
actin-DNase I complex is stabilized by cofilin (8), but the relevance
of these interactions is still unknown. Actin may constitute a part of
the nuclear matrix, the non-chromatin fraction of the nucleus, enabling
compartmentalization of functions or reorganization of chromatin (2).
Recently, a novel protein, EAST, has been characterized as a component
of the nuclear matrix, localized exclusively to the extrachromosomal
nuclear domain (9). An increase in the levels of EAST (after a heat
shock or due to its overexpression) led to an expansion of this domain
and to accumulation of actin within it. Relevant to our study is the finding that cellular levels of G-actin control activation of a
transcription factor, serum response factor. An
increase in G-actin level, induced by an
actin-destabilizing drug, latrunculin B, or by manipulation of some
actin regulatory proteins, suppresses specific serum response factor
target genes such as srf and vinculin (10). In another
study, latrunculin B was found to delay nuclear division in yeast;
latrunculin B-induced disruption of actin filaments led to
an incorrect spindle position, which in turn seems to promote stress-activated mitogen-activated protein kinase pathway
(11).
Here we report that latrunculin B-induced disassembly of F-actin in
mast cells promotes an entry of actin and cofilin into the nuclei. The
competence to translocate actin is retained after cell permeabilization
despite leakage of cytosolic proteins. Using the permeabilized cell
system, we have shown that accumulation of nuclear actin is blocked by
addition of an anti-cofilin antibody. Depletion of ATP from either
intact or permeabilized cells also promoted an increase of nuclear
actin and cofilin. In this case, the nuclear translocation was
associated with an increase in F-actin content.
LB was from Calbiochem (428020-S), and it was dissolved (5 mg/ml) in Me2SO and stored in small aliquots at Cells--
RPMC were prepared as described previously (12). The
cells were resuspended in a solution containing 137 mM
NaCl, 2.7 mM KCl, 1.0 mM CaCl2, 2 mM MgCl2, 5.6 mM glucose, 1 mg/ml
bovine serum albumin, and 20 mM NaPIPES, pH 7.2 (chloride
buffer, CB). For confocal microscopy, cells in CB were allowed to
attach to glass slides for 1 h at room temperature (~25,000
cells/well). Suspended cells were used for flow cytometry.
Permeabilization and Cell Treatments--
Glass-attached cells
were washed with 137 mM potassium glutamate, 2 mM MgCl2, 1 mg/ml bovine serum albumin, 20 mM NaPIPES, pH 6.8 (glutamate buffer, GB) and then exposed
for 8 min at room temperature to SL-O at 0.4 IU/ml in GB, 3 mM EGTA. After permeabilization, cells were washed with GB
free of soluble components and excess SL-O. Where indicated, cells were
exposed to 20 µg LB/ml CB for 1 h before permeabilization and/or
to 20 µg LB/ml GB, 100 µM EGTA for 10 min after
permeabilization, both at room temperature. After the pretreatments,
permeabilized cells were further incubated for 20 min at 30 °C with
3 mM EGTA, 3 mM ATP, GB (in some experiments, ATP was omitted). LB, if present, was diluted by half to 10 µg/ml. Where indicated, free Ca2+ concentration was buffered by 3 mM Ca2+ EGTA buffer system, pH 6.8, calculated
using appropriate dissociation constants as given by Martell and Smith
(13). Pretreatment of permeabilized cells with antibodies, anti-cofilin
or anti-vinculin (both at 1:10 dilution), was performed before the
addition of latrunculin B. To deplete ATP in intact cells, metabolic
inhibitors, 6 mM deoxyglucose and 10 µM
antimycin in CB, were applied at 30 °C for the indicated times.
Staining--
Cells were fixed for 20 min at room temperature
with 3% paraformaldehyde, 4% polyethylene glycol, 3 mM
EGTA, GB; washed with 50 mM glycine, 100 µM
EGTA, GB; and then exposed to a mild detergent, lysophosphatidylcholine
(80 µg/ml), 100 µM EGTA, GB. Finally, cells were
stained with either 0.3 µM rhodamine phalloidin (RP) (20 min) or with anti- Confocal Microscopy--
Stained cells were observed using an
IX-70 inverted microscope (Olympus), fitted with an UltraVIEW confocal
imaging system (PerkinElmer Life Sciences, Cambridge, UK). This
comprises a dual wavelength argon/krypton laser (Omnichrome) and a
CSU-10 confocal scanning unit (Yokogawa, Japan). This system utilizes
Nipkow discs to allow real-time confocal imaging. Digital images
(mostly equatorial slices) were collected with no pixel binning, using
a cooled 12-bit digital interline UltraPix FKI 1000 camera (G2) with
1008 × 1018 pixels (PerkinElmer Life Sciences). Read-out speed
was 0.5 MHz and sensitivity 0.72 electrons/gray level. Image capture
was controlled by the software package "Ultraview" ("Spatial
Module" configuration). For the detection of Cy2 fluorescence,
excitation was at 488 nm, and emission was collected with a
multiband-pass filter (transmitting between 500 and 540 nm). For the
detection of RP or EB fluorescence, excitation was at 568 nm, and
emission was collected between 580 and 620 nm. Olympus U Plan
Apochromat 100× objective was used; images of equatorial slices that
section nuclei are shown.
Flow Cytometric Assay for Apoptosis--
Suspended intact cells
were incubated for 2 h at 37 °C with control buffer (0.25%
Me2SO), LB (20 µg/ml), or, as a positive control,
staurosporine (2 µM) + cyclohexamide (10 µg/ml), all in
CB. Cells were then washed and incubated with fluorescein
isothiocyanate-annexin V (FA, 1/100, NeXin Research), propidium
iodide (PI, 2 µg/ml), 5 mM CaCl2, CB for 5 min at room temperature in the dark. Cell fluorescence was
quantified using flow cytometry on EPICS Elite flow cytometer (Coulter
Electronics Inc., Hialeah, FL) equipped with an argon-ion laser.
Excitation was at 488 nm, and emission was collected at 525 nm for FA
and at 575 nm for PI. Each sample contained 10,000-15,000 cells.
Histogram was partitioned into four domains according to FA and PI
fluorescence intensity. Duplicate samples of 10,000-15,000 cells were
analyzed per condition.
Western Blotting/Immunoblotting--
Samples of cell
fractions were dissolved in Laemmli sample buffer (15) and analyzed by
electrophoresis on 10% polyacrylamide vertical slab gels. Proteins
were transferred onto nitrocellulose. The membranes were probed with an
ABA (1/2000) followed by goat anti-mouse antibody (1/2000) as described
(16) and finally developed using ECL reagents (Amersham
Biosciences).
All figures shown are representative of at least three separate experiments.
Latrunculin B Causes Translocation of Actin into Nuclei of Intact
Cells--
Resting RPMC, stained for filamentous actin with RP,
exhibited only the prominent rings of cortical actin with no internal structures visible (Fig. 1, top
left panel). Staining with a monoclonal ABA gave somewhat
different results; in addition to the cortex, punctate internal foci
were visible together with low levels of perinuclear, sometimes
nuclear, staining (Fig. 1, top right panel, and Fig. 7,
top left panel). No staining was observed in the absence of
the primary antibody (not shown). RPMC contain about 2.6 ng total actin
per 106 cells (as determined by densitometry of Coomassie
Blue-stained gels (17); densitometry of Western blots after ABA
staining produced a value of 2.3 ng/106 cells (not shown).
The close agreement indicates that most of the actin in mast cells is
After 1-h treatment of intact cells with 20 µg of LB/ml, RP staining
decreases by 60-70%; secretion from these cells is inhibited by about
40%, and the effect is fully reversible (1). The plasma membrane of
these cells remains intact as indicated by Bodipy-sphingomyelin staining (18). Consequently it is unlikely that nuclear actin is
associated with apoptotic cells. To confirm that actin changes were not
linked to apoptosis, LB-treated cells were stained with FA and PI and
examined using flow cytometry (Table I).
Annexin V marks the early stage of apoptosis (hi FA-low PI), it binds with high affinity to phosphaphatidyl serine, which appears on the
outer leaflet of the plasma membrane during the early stages of
programmed cell death. During the mid-late stages of apoptosis, membrane integrity is lost allowing propidium iodide to associate with
DNA (hi FA-hi PI). Treating cells with staurosporine and cycloheximide,
agents known to induce apoptosis (19), provided a positive control.
After 2 h of LB treatment, there was no increase in the proportion
of early or mid-apoptotic cells.
Latrunculin B Causes Translocation of Actin into Nuclei of
Permeabilized Cells--
SL-O, a bacterial exotoxin, binds to membrane
cholesterol and then oligomerizes to form pores with a diameter up to
30 nm, allowing passage of proteins of molecular weight up to 400 kDa (Ref. 20 and references within). Permeabilized mast cells retain integrity of their cellular architecture and respond to stimulation by
calcium and/or GTP
Mast cells, permeabilized under control conditions (8 min with
streptolysin-O, washed with GB followed by a 20-min exposure to 3 mM EGTA, 3 mM ATP, GB) showed either none or
very weak nuclear staining with RP or ABA, respectively (Fig.
2A, Control).
Cortical staining was clearly visible, but occasionally (see Fig.
2A, top left panel), a few spontaneously
degranulating cells could be seen with filamentous actin around
secretory granules. When intact cells were pretreated for 1 h with
LB and then permeabilized in its absence, many cells retained nuclear
actin, stained by both RP and ABA (Fig. 2A, LB Localization of Cofilin in Intact and Permeabilized Mast Cells;
Effect of Latrunculin B--
Cofilin, a small actin-binding protein,
has previously been reported to enter nuclei together with actin after
a heat shock (4, 5). To establish whether F-actin disassembly induces nuclear entry of cofilin concomitantly with that of actin, the presence
of this protein in control and LB-treated cells was studied using an
affinity-purified anti-cofilin. Intact cells exhibited diffuse staining
throughout the cell with some cells showing low levels of nuclear
staining (Fig. 4A,
bottom left panel). No staining was observed in the absence
of the primary antibody except for the few cells, most probably those
that have degranulated spontaneously (not shown); such cells with
artificial high staining are also visible in Fig. 4. There was a
significant increase in anti-cofilin staining of the nuclei of intact
cells after LB treatment (Fig. 4A, bottom right
panel). In parallel, actin presence in the nuclei of LB-treated
cells was revealed by ABA (Fig. 4A, top
panels).
Cofilin entry into nuclei could also be induced in permeabilized
cells. Cells, that were treated with LB after their permeabilization (protocol SL-O Effect of Anti-cofilin on the Nuclear Accumulation of
Actin--
The permeabilized cell system, with its capacity to support
nuclear entry of both actin and cofilin, provides a good opportunity to
examine whether cofilin is required for latrunculin B-induced nuclear
accumulation of actin. The 9771 cofilin antibody is entirely specific for cofilin and is able to bind cofilin both free and when
complexed to actin (see Supplemental Material). The antibody was
introduced into permeabilized cells prior to latrunculin B treatment
and anti-vinculin was used as a control. As expected, control cells
exhibited actin cortex and very low levels of nuclear/perinuclear staining (Fig. 5A). In the
absence of any antibody (Fig. 5B) or in the presence of the
control antibody, anti-vinculin (Fig. 5C), LB caused
cortical actin disassembly and an increase in nuclear actin. In the
presence of anti-cofilin, LB induced cortical actin disassembly but
failed to induce nuclear accumulation actin (Fig. 5D).
Absence of ATP Promotes Accumulation of Actin in the Nuclei of
Permeabilized and Intact Cells--
A small proportion of
permeabilized cells exhibit nuclear or perinuclear staining with ABA
even under control conditions (Fig. 6A, top right panel
and see also Figs. 2A, 4B, and 5A).
This staining increased considerably when cells were maintained in the
absence of ATP (Fig. 6A, bottom right panel).
Phalloidin did not recognize this nuclear actin, but RP staining of the
cortex was somewhat increased in the absence of ATP (Fig.
6A, bottom left panel). Flow cytometric assay of
RP fluorescence showed the relative F-actin level of cells maintained
in the absence of ATP to increase by 5-10% relative to those with ATP
(not shown). This is also reflected by a stronger cortical staining by
ABA of cells without ATP (compare the two right panels in
Fig. 6, A and B).
Immunofluorescence with anti-cofilin of cells exposed to EGTA in the
presence and absence of ATP revealed that ATP in permeabilized cells
was also crucial for preventing accumulation of nuclear cofilin (Fig.
6B, left panels). Calcium (pCa 5) and
GTP
Treatment of intact mast cells with inhibitors of glycolysis (6 mM deoxyglucose) and oxidative phosphorylation (10 µM antimycin) causes depletion of ATP; concentrations of
ATP are reduced to <5% and <1% of the original level (~2.3
mM) within 10 and 20 min at 30 °C, respectively (22).
Fig. 7 shows that these inhibitors induced translocation of actin into nuclei. This was already apparent after 20-min treatment and became very prominent with increasing time.
Nuclear actin was not recognized by phalloidin (not shown). Again,
cortical actin staining increased progressively with the time of
metabolic inhibition.
Internal Foci of Monomeric Actin--
Permeabilization did not
eliminate the presence of the punctate internal foci stained by ABA. In
fact, they were visible more clearly in permeabilized than in intact
cells, and this must be due to the leakage of soluble actin and
therefore to a higher signal/noise ratio. At higher magnification,
these foci could be seen throughout the cell, as if delineating
cellular organelles/structures (Fig. 6C, right
panel; see also Fig. 2A, control cells, bottom left panel). Examination of a stack of confocal slices (taken 0.2 µm apart along the z axis) has shown the foci of actin to be present at all planes (not shown). After latrunculin B treatment, these foci are less prominent, especially when LB was applied both
before and after permeabilization (Fig. 2A, bottom
right panel).
This paper demonstrates cofilin-dependent
translocation of actin into nuclei of mast cells exposed to two
different stress-related conditions: 1) latrunculin B-induced
disassembly of cortical F-actin and 2) ATP depletion, associated with
an increase in F-actin.
Latrunculins destabilize actin filaments by binding to G-actin at the
site adjacent to the nucleotide-binding cleft (23-25). This prevents
the re-incorporation of actin into filaments and depletes cellular
F-actin over a period of time; the rate of this process depends on the
rate of filament turnover (26, 27). In contrast, cytochalasins, the
most frequently used actin-destabilizing drugs, bind to the barbed ends
of filaments and prevent elongation and shortening from these ends.
Cytochalasins inhibit polymerization of actin in response to cell
activation but usually do not deplete cellular F-actin. Indeed, we have
not seen any reduction in RP staining of resting mast cells, intact or
permeabilized, in response to cytochalasin (not shown). It is
remarkable that LB could induce accumulation of nuclear actin in
permeabilized cells, indicating that the components required for the
translocation are retained although a large proportion of cytosolic
proteins have leaked out.
The response to LB was very fast; nuclear actin appeared within 10 min,
suggesting an active process rather than passive diffusion, particularly with regard to the large size of actin (43 kDa). Initially, this actin was not accessible to phalloidin; RP staining of
nuclei was delayed relative to that by ABA (Fig. 1). This indicates that actin enters as a monomer or that it forms oligomers complexed with cofilin. Cofilin binding to the filaments changes their
conformation (twist), which prevents RP binding (28). Indeed, we have
observed cofilin translocation into nuclei in both intact and
permeabilized cells after LB treatment (Fig. 4). At the later stages of
LB treatment, RP staining of nuclei became progressively more apparent
although the major part of nuclear actin was still not accessible to RP (Fig. 1). Since not all actin translocated to nuclei will be complexed with LB, it means that after the dissociation of cofilin from actin
monomers the LB-free actin could form RP-accessible filaments. This
dissociation from cofilin may be promoted by the presence of LIM kinase
(LIMK) in the nucleus, which would phosphorylate and therefore
inactivate cofilin (29, 30).
Cofilin antibody inhibited accumulation of nuclear actin in LB-treated
permeabilized cells (Fig. 5). The antibody is entirely specific for
cofilin and able to bind cofilin both free and complexed to actin
(Supplemental Material). The nuclear localization sequence is on the
opposite side of ADF/cofilin than its actin-binding face (28, 31). We
speculate that the antibody binds nearer the nuclear localization
sequence and prevents access to it and therefore nuclear
translocation. Our results support the existence of an active,
cofilin-dependent nuclear import of actin in this system.
Cofilin binds away from the LB-binding site, similar to but distinct
from the gelsolin-binding site (31). This is consistent with
cofilin-dependent increase in nuclear translocation of
actin after LB treatment; more actin monomer is available to the
cofilin to bind to and subsequently to translocate into the nuclei.
Accumulation of actin in the nuclei of latrunculin B-treated maize root
cells was reported recently (32). The authors suggest that this may be
due to the changed conformation of G-actin after latrunculin binding
that may interfere with the nuclear export of G-actin. This is an
interesting possibility, but it would not explain the appearance of
nuclear actin under other conditions such as ATP depletion, heat shock,
or Me2SO treatment.
Nuclear actin was localized outside the region of high DNA/RNA density
(Fig. 3), similar to the reported presence of actin (after a heat shock
or after overexpression of EAST protein) in the extrachromosomal
nuclear domain (9). Nuclear actin in mast cells was often present in
the form of distinct dots, apparent on all confocal slices throughout
the nuclei. This suggests a formation of either vertical actin rods or
very concentrated actin grains. The protocol, including LB treatment
both before and after the permeabilization (LB Latrunculin B did not increase the proportion of apoptotic
(FA-positive) cells (Table I). Nuclear accumulation of actin seems to
be controlled differently from the induction of apoptosis. For
example in hepatocytes, inhibitors of protein synthesis were found to
induce both apoptosis and an increase in nuclear G-actin staining, but
only apoptosis could be prevented by a pretreatment with caspase
inhibitors (34). It seems that accumulation of nuclear actin forms a
part of a general response to stress that is not causally related to
apoptosis, but it may precede it. We have, however, not detected any
increase in FA-positive cells even after 6 h of LB treatment (not
shown). Stress-induced sequestration of actin into the nucleus may be
important for preventing formation of inappropriate cytosolic
structures. An increase in monomeric actin level activates
autoregulatory feedback mechanisms at both the transcriptional and the
post-transcriptional level, which lead to a decreased synthesis of
actin and some actin-binding proteins (35-37). Specific serum response
factor target genes are suppressed (10) and nuclear division delayed
(11). Our results imply that some of these responses may involve
regulatory mechanisms dependent on the presence of actin in the nucleus.
Nuclear entry of actin could also be induced by ATP depletion of both
intact or permeabilized mast cells, but in this case, levels of F-actin
in the cortex actually increased (Figs. 6 and 7). Nuclear actin was not
recognizable by phalloidin. As confirmed by immunostaining with cofilin
antibody, ATP depletion has also caused cofilin to enter the nuclei of
mast cells, and this was independent of calcium (not shown). The effect
of GTP Increase in F-actin level has been previously reported in ischemic
proximal tubule kidney cells; this was coincident with a collapse of
microvilli and appearance of F-actin in the perinuclear region (43,
44). This rather paradoxical observation (in vitro, polymerization of actin is promoted by ATP) may be due to the presence
of a range of cytosolic actin-regulatory proteins (45) as well as to
the rigor state of the actomyosin in the cortex. We have reported
previously that myosin II-based contraction of the membrane
cytoskeleton is a prerequisite for its disassembly; the latter is
induced by addition of ATP together with calcium to permeabilized mast
cells (46). Addition of ATP alone causes a small but reproducible
decrease in cortical F-actin level (5-10% decrease in RP
fluorescence), while withdrawal of ATP causes an F-actin increase.
Although RP and ABA cortical staining of cells without ATP was stronger
than that of cells with ATP, no such increase was visible for cortical
staining with anti-cofilin. In most cells, cofilin concentration is
much smaller than that of actin (47). In ATP-depleted mast cells, actin
moved into the nuclei accompanied by cofilin, but actin enrichment at
the cortex seems to be cofilin-independent.
Finally, where does the nuclear actin come from? This question is
pertinent considering that nuclear translocation of actin occurs in
permeabilized cells that have lost ~60% of their total actin by
leakage. We have previously postulated the existence of an actin
monomer pool that is bound to intracellular structures. This premise
was based on the fact that de novo actin polymerization could be induced in permeabilized mast cells by adding GTP In conclusion, it can be expected that cofilin-dependent
entry of actin into nuclei forms a part of cellular response to stress in general. Permeabilized mast cells, which retain their capacity for
both nuclear translocation and de novo polymerization of
actin, should provide an excellent system for further investigation.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
20 °C.
GTP
S and ATP were obtained from Roche Molecular Biochemicals.
Streptolysin-O (SL-O) was from Murex Diagnostics (CX MR16, distributed
by Corgenix). Glass "Multitest" slides (6-mm diameter
wells) were from ICN Biomedicals (Aurora, OH). All other reagents were
obtained from the Sigma (Poole, UK).
-actin (ABA, 1 h) or with anti-cofilin (1 h)
all in 1 mM EGTA, GB (GBE). Blocking with 5% goat serum in
GBE (20 min) and washing with GBE were performed before each step of
the immunostaining. ABA was an affinity-purified monoclonal antibody
(clone AC-15) from Sigma (used at 1/200 dilution), and it recognizes an
N-terminal epitope. Using enzyme-linked immunosorbent assay, we have
found the AC-15 antibody to be truly
-actin-specific. AC-15
recognizes
-F-actin equally well as
-G-actin, and its binding to
actin is unaffected either by cross-linking or by the interaction of
actin with cofilin. Anti-human cofilin antibody ("9771") was raised
in rabbits against complexes of recombinant human cofilin (14) and
rabbit skeletal muscle. The complexes were prepared from F-actin mixed
(~1:1) with cofilin at pH 8.0 and centrifuged to remove residual
F-actin. The cofilin antibodies were then affinity purified on
Sepharose-cofilin and used at 1/50 dilution. The affinity of cofilin
antibody for cofilin was unaffected by the interaction of cofilin with
actin. The antibody binds equally well to cofilin and cofilin-actin
complexes. Results of our antibody tests are available as Supplemental
Material. The secondary antibodies (both at 1/50 dilution in GBE) were
goat-anti-mouse IgG-biotin and goat-anti-rabbit-IgG-biotin,
respectively, both from Sigma. Cy2-streptavidin (1/50, from Amersham
Biosciences) was the tertiary layer. In double-label
experiments, RP or ethidium bromide (EB, 0.5 µM) was
added with the secondary antibody. This is particularly important for
ABA + RP staining, since phalloidin was found to interfere with the ABA
staining, while ABA did not seem to affect the staining with phalloidin.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-actin. The cortical staining disappeared within 10 min of LB
treatment (Fig. 1); ABA-stained cells showed stronger diffuse staining
throughout the cell due to the increase in the monomer concentration,
and in some cells, intense nuclear staining was already apparent. The
proportion of cells with nuclear actin increased with the time of
treatment and this was also true for RP-stained cells, although with a
discernible delay. After 60 min with LB, most cells exhibited nuclear
actin, stained by both RP and ABA.
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Fig. 1.
Latrunculin B induces translocation of actin
into nuclei of intact mast cells. Intact mast cells were treated
for the indicated times (minutes at 30 °C) with LB (20 µg/ml),
fixed, and stained with RP or ABA. Bar = 10 µm. Note
that nuclear actin appears at earlier times with antibody
staining.
Proportion of apoptotic cells after LB treatment
S (12) as well as to the activation of cell
surface receptors (21). After permeabilization, cytosolic components
gradually leak out of the cells. Using ABA and Western blotting, we
have found that about 60% of cellular actin leaks out within 15 min.
This is in good agreement with our previous determination of
actin leakage by densitometry of Coommassie Blue-stained gels
(17). The leakage was increased to ~95% in the presence of LB,
and no such increase was seen in the presence of cytochalasin (not shown).
SL-O). It is noteworthy that actin translocation into nuclei
occurred also when LB was applied after the permeabilization (Fig.
2A, SL-O
LB); in this case most of the
nuclear actin was not stained by phalloidin. LB treatment both before
and after permeabilization resulted in the appearance of nuclear actin
in all cells (Fig. 2A, LB
SL-O
LB), but
only a proportion was recognized by phalloidin (Fig. 2B).
Distinct dot-like nuclear actin structures were apparent, particularly
when the LB
SL-O
LB protocol was used (Fig. 2B), and
they could be seen on all Z axis confocal slices throughout
the nuclei (taken 0.2 µm apart along the z axis; not
shown). Differential interference contrast microscopy showed
that these dot structures lie close to and within the nuclear envelope,
concentrically with the structure seen in the middle of the nucleus,
presumably a nucleolus (Fig.
3A). The nuclear structures
could be visualized with a DNA/RNA stain, EB, and actin was excluded
from these EB-stained domains (Fig. 3B).
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Fig. 2.
Latrunculin B induces translocation of actin
into nuclei of permeabilized mast cells. A,
glass-attached mast cells were exposed for 1 h at room temperature
to control buffer (Control, SL-O LB) or to 20 µg/ml LB (LB
SL-O, LB
SL-O
LB).
Cells were then permeabilized with SL-O and treated for 10 min at room
temperature with either GB, 100 µM EGTA
(Control, LB
SL-O) or 20 µg/ml LB in GB,
100 µM EGTA (SL-O
LB, LB
SL-O
LB). Finally, EGTA and ATP were added, both to 3 mM final concentration, and cells were further incubated
for 20 min at 30 °C. LB, when present, was diluted by half to 10 µg/ml. Cells were fixed and stained with either RP or with ABA.
B, cells were treated as above for the LB
SL-O
LB
protocol and double-stained with RP and ABA. Note that a large
proportion of nuclear actin is not accessible to phalloidin.
Bars = 10 µm.
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Fig. 3.
Nuclear actin is localized into areas of low
DNA/RNA density. Mast cells were exposed for 1 h to LB
(20 µg/ml), permeabilized, and treated for 10 min with LB (20 µg/ml) in GB, 100 µM EGTA (protocol LB SL-O
LB). Cells were then further incubated for 20 min at 30 °C with 3 mM ATP, 3 mM EGTA, 10 µg/ml LB in GB, fixed,
and stained with RP (A) or double-stained with 0.5 µM EB and ABA (B). Bar = 5 µm. The nucleus is clearly visible in the differential interference
contrast image; RP staining is concentric with the structure
seen in the middle of the nucleus. The intense EB staining does not
co-localize with actin.
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Fig. 4.
Latrunculin B induces translocation of
cofilin into nuclei of both intact and permeabilized mast cells.
A, intact cells were treated with LB (20 µg/ml) for 60 min, fixed, and stained with either affinity-purified anti-cofilin or
ABA. B, SL-O-permeabilized cells were treated 10 min with 20 µg/ml LB, 100 µM EGTA, GB and then for a further 20 min
with 10 µg/ml LB, 3 mM ATP, 3 mM EGTA, GB,
fixed, and stained as above. Bar = 10 µm.
LB), exhibited stronger nuclear staining with both
anti-cofilin (Fig. 4B, bottom panels) and with
ABA (Fig. 4B, top panels) relative to control cells.
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Fig. 5.
Effect of anti-cofilin on translocation of
actin into nuclei of permeabilized cells. Permeabilized mast cells
were exposed to GB, 100 µM EGTA with no added antibody
(A, B) or with anti-vinculin (C, 1:10)
or with anti-cofilin (D, 1:10). After 10 min at room
temperature, LB was added (20 µg/ml final concentration) to all
samples except the control, diluting the antibodies by half. After a
further 10 min, EGTA and ATP were added, both to 3 mM final
concentration, and cells were further incubated for 20 min at 30 °C.
LB and the antibodies, when present, were diluted by half and a
quarter, respectively. Cells were then fixed and stained with
ABA.
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Fig. 6.
A and B, absence of ATP
promotes nuclear translocation of both actin and cofilin. Permeabilized
mast cells were exposed for 20 min at 30 °C to 3 mM
EGTA, GB in the presence or absence of 3 mM ATP, fixed, and
double labeled with RP and ABA. The few bright-staining cells are the
contaminating neutrophils. B, same as described for
A, but cells were stained with anti-cofilin and ABA.
Bar = 10 µm. C, foci of actin remain in
mast cells after permeabilization. Cells were exposed to
EGTA/ATP/GB, fixed, and stained as described in the legend to
A. Nuclear actin and the foci of actin in the cytoplasmic
space are both recognized by ABA but not by phalloidin.
Bar = 5 µm.
S, two agents capable of promoting secretion from permeabilized
mast cells, did not seem to have any significant effect on cofilin localization (not shown). A small decrease in nuclear staining was
occasionally apparent in GTP
S-treated cells, but the effect was
marginal. Likewise, nuclear staining of actin with ABA was somewhat
reduced in the presence of GTP
S (not shown). The main determinant of
cofilin nuclear localization was absence of ATP, and the same was true
for actin. Excepting the nuclei, the staining with anti-cofilin was
dispersed throughout the cell, and no increase in the cortical staining
was apparent in the absence of ATP, unlike that with ABA (Fig.
6B).
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Fig. 7.
ATP depletion induces translocation of actin
into the nuclei of intact mast cells. Intact cells were treated
with metabolic inhibitors (6 mM deoxyglucose and 10 µM antimycin in CB) for the indicated times (minutes) at
30 °C. Cells were stained with ABA after fixation.
Bar = 10 µm.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
SL-O
LB),
resulted in the sharpest images of such dots. This could be a
consequence of an aggregation of nuclear actin under these conditions
together with leakage of any non-aggregated actin (thus increasing the
signal to noise ratio). The existence of nuclear actin in dot-like
structures was previously revealed in differentiated myogenic cells by
immunofluorescence with an actin antibody, 2G2. This antibody (but not
phalloidin) seems to recognize a specific actin conformation, present
in the nuclei (but not in the cytoplasm) of these cells (33). Again, the staining did not co-localize with the DNA-specific Hoechst stain.
S was marginal and needs to be further investigated. The
co-localization of cofilin and actin in the nuclei again suggests the
existence of actin-cofilin complexes that cannot bind phalloidin. ATP
depletion will promote dephosphorylated, and therefore active state, of
cofilin (38). It has been shown that cofilin is capable of nuclear
translocation in this dephosphorylated state (39, 40). Moreover, ATP
depletion will also increase levels of ADP-G-actin, which binds to
cofilin more strongly than ATP-G-actin (41, 42), promoting its nuclear accumulation. In cultured neurones, ATP depletion caused formation of
cytoplasmic actin-ADF/cofilin rods, and this was associated with an
increase in the level of dephosphorylated ADF/cofilin (38). No rods,
however, were visible in the nuclei of these ATP-depleted neurons.
S or V14RhoA, a constitutively active mutant of Rho (48, 49). Immunostaining with ABA has indeed revealed foci of monomeric actin that seemed to be
associated with internal cellular organelles/structures (Fig.
6C). Similar ABA-stained foci were seen in cultured
fibroblasts. Observation of fibroblasts after the microinjection of
fluorescently labeled actin revealed that these foci form near the
leading edge and move centripetally toward the nucleus (50). Such actin
foci could be the source/storage sites for both nuclear and newly
polymerized actin.
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ACKNOWLEDGEMENT |
---|
We thank Arnold Pizzey for his generous help with the EPICS Elite flow cytometer.
![]() |
FOOTNOTES |
---|
* This work was supported by a studentship from the Medical Research Council (to A. P.) and by grants (to A. K.) from the Wellcome Trust and the National Asthma Campaign. The confocal microscope was purchased with funds obtained from the Wellcome Trust.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The on-line version of this article (available at
http://www.jbc.org) contains additional "Results" and
supplemental Figs. 1-3.
§ To whom correspondence should be addressed: Physiology Dept., University College London, University St., London WC1E 6JJ, UK. Tel.: 44-171-2096094; Fax: 44-171-3876368; E-mail: a.koffer@ucl.ac.uk.
Published, JBC Papers in Press, February 3, 2003, DOI 10.1074/jbc.M206393200
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ABBREVIATIONS |
---|
The abbreviations used are:
LB, latrunculin B;
ABA, anti--actin;
CB, chloride buffer;
EB, ethidium bromide;
FA, fluorescein isothiocyanate-annexin;
GTP
S, guanosine
5'-O-(3-thiotriphosphate);
GB, glutamate buffer;
PI, propidium iodide;
RPMC, rat peritoneal mast cells;
RP, rhodamine-phalloidin;
SL-O, streptolysin-O;
PIPES, piperazine-N,N'-bis(2-ethanesulfonic acid).
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