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Latrunculin B or ATP depletion induce cofilin-dependent translocation of actin into
nuclei of mast cells.a
Annmarie Pendleton, Brian Pope+ Alan Weeds+ and Anna Koffer*
Physiology Department, University College London, University Street, London WC1E 6JJ
+MRC Laboratory of Molecular Biology, Hills Road, Cambridge, CB2 2QH, UK
*Address correspondence to Anna Koffer, Physiology Department, University College
London, University Street, London WC1E 6JJ, UK. Tel: +(44) 171 2096094. Fax: +(44) 171
3876368. E-mail: a.koffer@ucl.ac.uk
Running Title:
Nuclear actin in mast cells
a We acknowledge support by a studentship from the Medical Research Council (AMP), and by grants (AK) from the Wellcome Trust and the National Asthma Campaign. The confocal microscope was purchased with funds obtained from the Wellcome Trust. We would like to thank Arnold Pizzey for his generous help with the EPICS Elite flow cytometer.
Copyright 2003 by The American Society for Biochemistry and Molecular Biology, Inc.
JBC Papers in Press. Published on February 3, 2003 as Manuscript M206393200 by guest on M
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SUMMARY
Increasing cellular G-actin, using latrunculin B, in either intact or permeabilised 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
visualised with an actin antibody, indicating its monomeric or a conformationally distinct
state, e.g. cofilin-decorated filaments. Introduction of anti-cofilin into permeabilised cells
inhibited nuclear actin accumulation, implying that an active, cofilin-dependent, import exists
in this system. Nuclear actin was localised outside the ethidium bromide-stained region, in the
extrachromosomal nuclear domain. In permeabilised cells, the appearance of nuclear actin
and cofilin was not significantly affected by increasing [Ca2+] and/or adding GTP-γ-S, 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greatly promoted when ATP was withdrawn.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 actin’s nuclear import.
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INTRODUCTION
We have previously used latrunculin B (LBb) 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 recognisable 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 actin’s regulated nuclear import and export 2. Actin has
been shown to contain two functional leucine-rich nuclear export sequences; their disruption
leads to nuclear actin accumulation and consequently to a decrease in cell proliferation 3.
However, actin itself does not possess any nuclear localisation sequence. The actin-binding
protein, cofilin, contains a classical bipartite SV40-type nuclear localisation sequence 4 and
translocates into nuclei together with actin after a heat shock 4;5 or DMSO 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 stabilised 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 compartmentalisation of
functions or reorganisation of chromatin 2. Recently, a novel protein, EAST, has been
characterised as a component of the nuclear matrix, localised 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
b The Abbreviations used are: ABA, anti - β- actin; CB, chloride buffer; DMSO, dimethylsulfoxide; EB, ethidium bromide; FA, FITC-annexin; LB, latrunculin B; GTP-γ-S, guanosine-5'-O-(3-thiotriphosphate); GB, glutamate buffer; PI, propridium iodide; RPMC, rat peritoneal mast cells; RP, rhodamine-phalloidin; SL-O, Streptolysin-O
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within it. Relevant to our study is the finding that cellular levels of G actin control activation
of a transcription factor, Serum Response Factor, SRF. An increase in G-actin level, induced
by an actin- destabilising drug, latrunculin B, or by manipulation of some actin-regulatory
proteins, suppresses specific SRF 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 MAPK 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 permeabilisation despite leakage of cytosolic proteins. Using the permeabilised 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 permeabilised cells also promoted an
increase of nuclear actin and cofilin. In this case, the nuclear translocation was associated
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EXPERIMENTAL PROCEDURES
Latrunculin B (LB) was from Calbiochem (428020-S), it was dissolved (5 mg/ml) in
dimethylsulfoxide (DMSO) and stored in small aliquots at –20oC. GTP-γ-S and ATP were
obtained from Boehringer Mannheim. Streptolysin-O (SL-O) was from Murex Diagnostics
(CX MR16, distributed by Corgenix, UK). Glass 'Multitest' slides (6 mm diameter wells)
were from ICN Biomedicals (Aurora, Ohio 44202). All other reagents were obtained from the
Sigma Chemical Company (Poole, UK).
Cells
Rat peritoneal mast 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, (piperazine-
N,N[prime]-bis (2-ethanesulfonic acid) pH 7.2 (chloride buffer, CB). For confocal
microscopy, cells in CB were allowed to attach to glass slides for 1 hour at room temperature
(~ 25000 cells/well). Suspended cells were used for flow cytometry.
Permeabilisation and cell treatments
Glass-attached cells were washed with 137 mM K Glutamate, 2 mM MgCl2, 1 mg/ml BSA,
20 mM NaPIPES, pH 6.8 (glutamate buffer, GB) and then exposed for 8 minutes at room
temperature to SL-O at 0.4 IU/ml in GB-3 mM EGTA. After permeabilisation, 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 one hour before permeabilisation and / or to 20 µg LB /ml
GB-100 µM EGTA for 10 minutes after permeabilisation, both at room temperature. After the
pre-treatments, permeabilised cells were further incubated for 20 minutes at 30oC 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
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Ca2+ EGTA buffer system, pH 6.8, calculated using appropriate dissociation constants as
given by Martell and Smith 13. Pre-treatment of permeabilised 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, 6mM deoxyglucose and
10 µM antimycin in CB, were applied at 30ºC for the indicated times.
Staining
Cells were fixed for 20 minutes at room temperature with 3 % paraformaldehyde - 4 %
polyethylene glycol – 3 mM EGTA - GB, washed with 50mM 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 minutes) or
with anti-β-actin (ABA, 1 hour) or with anti-cofilin (1 hour) all in 1 mM EGTA – GB (GBE).
Blocking with 5 % goat serum in GBE (20 minutes) and washing with GBE was performed
before each step of the immunostaining. ABA was an affinity-purified monoclonal antibody
(clone AC-15) from Sigma (used at 1/200 dilution); it recognises an N-terminal epitope.
Using Elisa assay, we have found the AC-15 antibody to be truly β-actin specific. AC-15
recognises β-F-actin equally well as β-G-actin and its binding to actin is unaffected either by
cross-linking or by actin’s interaction 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. Cofilin antibody’s affinity for cofilin was
unaffected by cofilin’s interaction 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 Chemical Company (Poole, UK).
Cy2-streptavidin (1/50, from Amersham Biosciences UK) was the tertiary layer. In double-
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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.
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 Ar/Kr laser (Omnichrome) and a CSU-10 confocal scanning
unit (Yokogawa, Japan). This system utilises 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 x 1018
pixels (Perkin Elmer Life Sciences, Cambridge, UK). Read-out speed was 0.5 MHz and
sensitivity 0.72 electrons/grey 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 100x objective was used; images of equatorial slices that section nuclei are
shown.
Flow cytometric assay for apoptosis
Suspended intact cells were incubated for 2 hours at 37°C with control buffer
(0.25 % DMSO), LB (20µg/ml), or, as a positive control, staurosporine (2µM) +
cyclohexamide (10µg/ml), all in CB. Cells were then washed and incubated with FITC-
annexin V (FA, 1/100, NeXin Research, Netherlands) - propridium iodide (PI, 2µg/ml) –5
mM CaCl2 –CB for 5minutes at RT in the dark. Cell fluorescence was quantified using flow
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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 analysed per condition.
Western Blotting / Immunoblotting
Samples of cell fractions were dissolved in Laemmli sample buffer 15 and analysed 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).
All figures shown are representative of at least three separate experiments.
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RESULTS
Latrunculin B causes translocation of actin into nuclei of intact cells.
Resting rat peritoneal mast cells (RPMC), stained for filamentous actin with rhodamine
phalloidin (RP), exhibited only the prominent rings of cortical actin with no internal
structures visible (Figure 1, top left panel). Staining with a monoclonal anti-β-actin antibody
(ABA) gave somewhat different results: in addition to the cortex, punctate internal foci were
visible together with low levels of perinuclear, sometimes nuclear, staining (Figure 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 per 106 cells (not shown). The close agreement indicates
that most of the actin in mast cells is β-actin. The cortical staining disappeared within 10
minutes of latrunculin B (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 minutes with LB, most cells exhibited nuclear actin, stained
by both RP and ABA.
After 1 hour treatment of intact cells with 20 µg 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
FITC-annexin (FA) and propridium iodide (PI) and examined using flow cytometry (Table 1).
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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 propridium 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 two hours 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 permeabilised cells
Streptolysin-O (SLO), 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 20 (and references within). Permeabilised mast cells retain
integrity of their cellular architecture and respond to stimulation by calcium and/or GTP-γ-S
12 as well as to the activation of cell surface receptors 21. After permeabilisation, 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 minutes. This is in a 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).
Mast cells, permeabilised under control conditions (8 minutes with streptolysin-O, washed
with GB followed by a 20 minutes exposure to 3 mM EGTA-3 mM ATP-GB) showed either
none or very weak nuclear staining with RP or ABA, respectively (Fig. 2 A, Control).
Cortical staining was clearly visible but occasionally (see Fig. 2 A, top left panel), a few
spontaneously degranulating cells could be seen with filamentous actin around secretory
granules. When intact cells were pre-treated for 1 hour with LB and then permeabilised in its
absence, many cells retained nuclear actin, stained by both RP and ABA (Fig. 2 A, LBÆSL-
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O). It is noteworthy that actin translocation into nuclei occurred also when LB was applied
after the permeabilisation (Fig.2 A, SL-OÆLB); in this case most of the nuclear actin was not
stained by phalloidin. LB treatment both before and after permeabilisation resulted in the
appearance of nuclear actin in all cells (Fig.2 A, LBÆSL-OÆLB), but only a proportion was
recognised by phalloidin (Fig. 2 B). Distinct dot-like nuclear actin structures were apparent,
particularly when the LBÆSL-OÆLB protocol was used (Fig. 2 B), 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). DIC 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. 3 A). The nuclear structures could be visualised with a DNA/RNA stain,
ethidium bromide (EB), and actin was excluded from these EB-stained domains (Fig. 3 B).
Localisation of cofilin in intact and permeabilised 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. 4 A, 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.
4 A, bottom right panel). In parallel, actin presence in the nuclei of LB–treated cells was
revealed by ABA (Fig. 4 A, top panels).
Cofilin entry into nuclei could also be induced in permeabilised cells. Cells, that were treated
with LB after their permeabilisation (protocol SL-OÆLB), exhibited stronger nuclear staining
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with both anti-cofilin (Fig. 4 B, bottom panels) and with ABA (Fig. 4 B, top panels) relative
to control cells.
Effect of anti-cofilin on the nuclear accumulation of actin.
The permeabilised 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 permeabilised 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. 5 A). In the absence of any
antibody (Fig. 5 B) or in the presence of the control antibody, anti-vinculin (Fig. 5 C), 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 of
actin (Fig. 5 D).
Absence of ATP promotes accumulation of actin in the nuclei of permeabilised and
intact cells.
A small proportion of permeabilised cells exhibit nuclear or perinuclear staining with ABA
even under control conditions (Fig. 6 A, top right panel and see also Figs. 2 A, 4 B and 5 A).
This staining increased considerably when cells were maintained in the absence of ATP (Fig.
6 A, 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. 6 A, 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
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reflected by a stronger cortical staining by ABA of cells without ATP (compare the two right
panels in Figs. 6 A and 6 B).
Immunofluorescence with anti-cofilin of cells exposed to EGTA in the presence and absence
of ATP revealed that ATP in permeabilised cells was also crucial for preventing accumulation
of nuclear cofilin (Fig. 6 B, left panels). Calcium (pCa 5) and GTP-γ-S, two agents capable
of promoting secretion from permeabilised mast cells, did not seem to have any significant
effect on cofilin localisation (not shown). A small decrease in nuclear staining was
occasionally apparent in GTP-γ-S-treated cells, but the effect was marginal. Likewise,
nuclear staining of actin with ABA was somewhat reduced in the presence of GTP-γ-S (not
shown). The main determinant of cofilin nuclear localisation 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. 6 B).
Treatment of intact mast cells with inhibitors of glycolysis (6mM deoxyglucose) and
oxidative phosphorylation (10µM 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 minutes at
30oC, respectively 22. Figure 7 shows that these inhibitors induced translocation of actin into
nuclei. This was already apparent after 20-minute 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.
Permeabilisation did not eliminate the presence of the punctate internal foci stained by ABA.
In fact, they were visible more clearly in permeabilised than in intact cells and this must be
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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. 6 C, right panel; see also Fig. 2 A, 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 permeabilisation
(Fig. 2 A, bottom right panel).
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DISCUSSION
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 destabilise actin filaments by binding to G-actin at the site adjacent to the
nucleotide-binding cleft 23-25. This prevents actin’s re-incorporation 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-destabilising
drugs, bind to the barbed ends of filaments and prevent elongation and shortening from these
ends. Cytochalasins inhibit polymerisation 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 permeabilised, in response to cytochalasin (not shown). It is
remarkable that LB could induce accumulation of nuclear actin in permeabilised 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 minutes, suggesting an
active process rather than passive diffusion, particularly with regard to the large size of actin
(42 K). 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 permeabilised 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 cofilin’s
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dissociation 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 permeabilised 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 localisation sequence (NLS) is on
the opposite side of ADF /cofilin than its actin-binding face 28;31. We speculate that the
antibody binds nearer the NLS sequence, 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 recently reported 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 DMSO treatment.
Nuclear actin was localised 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 permeabilisation (LB Æ SL-O Æ 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
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(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 recognise a specific actin
conformation, present in the nuclei (but not in the cytoplasm) of these cells 33. Again, the
staining did not co-localise with the DNA-specific Hoechst stain.
Latrunculin B did not increase the proportion of apoptotic (FA-positive) cells (Table 1).
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 pre-treatment 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
hours 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 auto-regulatory 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 permeabilised
mast cells but in this case, levels of F-actin in the cortex actually increased (Figs. 6 and 7).
Nuclear actin was not recognisable 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-γ-S was marginal and needs
to be further investigated. The co-localisation of cofilin and actin in the nuclei again suggests
the existence of actin-cofilin complexes that cannot bind phalloidin. ATP-depletion will
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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.
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, polymerisation 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 previously
reported 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
permeabilised 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 permeabilised 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
polymerisation could be induced in permeabilised mast cells by adding GTP-γ-S or
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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. 6 C). Similar ABA-stained foci were seen in cultured fibroblasts.
Observation of fibroblasts after the microinjection of fluorescently labelled 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 polymerised actin.
In conclusion, it can be expected that cofilin-dependent entry of actin into nuclei forms a part
of cellular response to stress in general. Permeabilised mast cells, which retain their capacity
for both nuclear translocation and de novo polymerisation of actin, should provide an
excellent system for further investigation.
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FOOTNOTES:
a ACKNOWLEDGEMENTS
We acknowledge support by a studentship from the Medical Research Council (AMP), and by grants
(AK) from the Wellcome Trust and the National Asthma Campaign. The confocal microscope was
purchased with funds obtained from the Wellcome Trust. We would like to thank Arnold Pizzey for
his generous help with the EPICS Elite flow cytometer.
b The Abbreviations used are:
ABA, anti - β- actin; CB, chloride buffer; DMSO, dimethylsulfoxide; EB, ethidium bromide; FA,
FITC-annexin; LB, latrunculin B; GTP-γ-S, guanosine-5'-O-(3-thiotriphosphate); GB, glutamate
buffer; PI, propridium iodide; RPMC, rat peritoneal mast cells; RP, rhodamine-phalloidin; SL-O,
Streptolysin-O
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FIGURE LEGENDS
Figure 1:
Latrunculin B induces translocation of actin into nuclei of intact mast cells.
Intact mast cells were treated for the indicated times (minutes at 30oC) 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.
Figure 2:
Latrunculin B induces translocation of actin into nuclei of permeabilised mast cells.
A) Glass attached mast cells were exposed for 1 hour at room temperature to control buffer
(Control, SL-OÆLB) or to 20 µg /ml LB (LBÆSL-O, LBÆSL-OÆLB). Cells were then
permeabilised with SL-O and treated for 10 minutes at room temperature with either GB-100
µM EGTA (Control, LBÆSL-O) or 20 µg /ml LB in GB-100 µM EGTA (SL-OÆLB,
LBÆSL-OÆLB). Finally, EGTA and ATP were added, both to 3 mM final concentration,
and cells were further incubated for 20 minutes at 30oC. LB, when present, was diluted by
half to 10µg/ml. Cells were fixed and stained with either RP or with ABA. B) Cells were
treated as above for the LBÆSL-OÆLB protocol and double-stained with RP and ABA. Note
that a large proportion of nuclear actin is not accessible to phalloidin. Bars 10 µm.
Figure 3:
Nuclear actin is localised into areas of low DNA/RNA density.
Mast cells were exposed for 1 hour to LB (20 µg /ml), permeabilised and treated for 10
minutes with LB (20 µg /ml) in GB-100 µM EGTA (protocol LBÆSL-OÆLB). Cells were
then further incubated for 20 minutes at 30oC with 3 mM ATP, 3 mM EGTA, 10 µg/ml LB in
GB, fixed and (A) stained with RP or (B) double-stained with 0.5 µM ethidium bromide (EB)
and ABA. Bar 5 µm. Nucleus is clearly visible in the DIC image; RP staining is concentric
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with the structure seen in the middle of the nucleus. The intense EB staining does not co-
localise with actin.
Figure 4:
Latrunculin B induces translocation of cofilin into nuclei of both intact and
permeabilised mast cells.
(A) Intact cells were treated with LB (20 µg/ml) for 60 minutes, fixed and stained with either
affinity-purified anti-cofilin or ABA. (B) SL-O-permeabilised cells were treated 10 minutes
with 20 µg/ml LB - 100 µM EGTA - GB and then for a further 20 minutes with 10 µg/ml LB -
3 mM ATP - 3 mM EGTA – GB, fixed and stained as above. Bar 10 µm.
Figure 5:
Effect of anti-cofilin on translocation of actin into nuclei of permeabilised cells
Permeabilised 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 minutes at room
temperature, LB was added (20 µg /ml final concentration) to all samples except the control,
diluting the antibodies by half. After further 10 minutes, EGTA and ATP were added, both to
3 mM final concentration, and cells were further incubated for 20 minutes at 30oC. LB and
the antibodies, when present, were diluted by half and a quarter, respectively. Cells were then
fixed and stained with ABA.
Figure 6:
(A and B) Absence of ATP promotes nuclear translocation of both actin and cofilin.
Permeabilised mast cells were exposed for 20 minutes at 30ºC to 3 mM EGTA-GB in the
presence or absence of 3mM ATP, fixed and double labeled with RP and ABA. The few
bright-staining cells are the contaminating neutrophils. (B) As for (A) but cells were stained
with anti-cofilin and ABA. Bar 10 µm. (C) Foci of actin remain in mast cells after
permeabilisation. Cells were exposed to EGTA-ATP-GB, fixed and stained as in (A).
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Nuclear actin and the foci of actin in the cytoplasmic space are both recognized by ABA but
not by phalloidin. Bar, 5 µm.
Figure 7:
ATP depletion induces translocation of actin into the nuclei of intact mast cells.
Intact cells were treated with metabolic inhibitors (6mM deoxyglucose and 10 µM antimycin
in CB) for the indicated times (minutes) at 30ºC. Cells were stained with ABA after fixation.
Bar 10 µm.
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TABLES
healthy cells
(%)
early apoptotic
(%)
mid–late
apoptotic (%)
broken cells
(%)
Fluorescence
Intensity
low FA – low PI Hi FA – low PI hi FA – hi PI low FA – hi PI
Control 80.7 5.5 10.2 3.6
LB 90.2 3.8 5.1 0.9
Stauro + CHI 58.6 22.6 18.0 .8
Table 1.
Proportion of apoptotic cells after LB treatment
Suspended intact cells were incubated for for 2 hours at 37°C with control buffer
(0.25 % DMSO-CB), LB (20µg/ml), or, as a positive control, staurosporine (2µM) +
cyclohexamide (10µg/ml). Cells were then washed and incubated with FITC-annexin V
(1:100, FA) - propridium iodide (2µg/ml, PI) –5 mM CaCl2 –CB for 5minutes at room
temperarure in the dark. Cell fluorescence was quantified using flow cytometry. Histogram
was partitioned into four domains according to FA and PI fluorescence intensity. Duplicate
samples of 10,000 – 15,000 cells were analysed per condition. Results are representative of 3
independent experiments. No increase in % of apoptotic cells is apparent after 2 hours of
treatment with LB and the same was true for longer treatments (4 and 6 hours, not shown).
The top row explains distribution of cells during different stages of apoptosis: early stages
exhibit high annexin V but low propridium iodide staining, while in later stages staining by
both markers is intense.
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Supplemental Material Re: M2:06393 Latrunculin B or ATP depletion induce cofilin-dependent translocation of actin into nuclei of mast cells. Authors: Annmarie Pendleton, Brian Pope Alan Weeds and Anna Koffer. Testing anti-actin and anti-cofilin antibodies We have used the Elisa technique to check for actin binding preferences of the AC-15 antibody used in our studies. We actually screened a panel of 5 monoclonal antibodies (Sigma AC-15 and AC-74, Amersham N350, NeoMarkers Ab-5 and one prepared in house, “2G2”), together with 4 rabbit affinity purified polyclonal antibodies. The latter include the antibody “9785-11”, an anti-actin antibody, raised against actin-cofilin complexes and affinity purified, which was used in the Western blot shown in Fig. 3. All antibodies were tested against both rabbit skeletal α-actin and human platelet β-actin. The actins were used both in F (filamentous) and G (monomeric) states. In each state they were then tested for any effects of x-linker on the efficiencies of antibody binding to actin before the x-linker was then used to stabilise interactions with cofilin. We have also looked for an effect of the cofilin antibody on the cofilin-actin interaction (antibody “9771”, prepared by B. Pope as described in Materials and Methods). We demonstrate the absence of inhibition of cofilin interaction with actin by the cofilin antibody. The antibody binds equally well to cofilin and cofilin-actin complexes. A blot to demonstrate the specificity of the cofilin antibody is also included. Method: Antigen preparations were of α (rabbit skeletal) or β (human platelet) actin either as G or F, x-linked or free. Recombinant human cofilin was prepared as described in reference (1). Complexes of cofilin/actin were prepared by mixing cofilin with F-actin at ratios of 2:1 at pH 6.0 to maximise the interactions with filaments and thus decorate filaments with equimolar ratios of cofilin (1). These were then diluted into a pH 6.0 buffer containing the cross linker. Similarly prepared filaments were adjusted to pH 8.0 and left for 20 minutes to depolymerise the filaments and generate cofilin-G-actin complexes (2). These too were then x-linked. (1) Uncoupling actin filament fragmentation by cofilin from increased subunit turnover. B. J. Pope, S. M. Gonsior, S. Yeoh, A. McGough & A. G. Weeds. J. Mol. Biol. 298, 649-661 (2000) (2) Determining the Differences in Actin Binding by Human ADF and Cofilin S. Yeoh, B. Pope, H. G. Mannherz and A. Weeds. J. Mol. Biol. 315, 911-925 (2002) Dynatech Immulon plates were coated with 2µg of antigen and coupled to 1° antibody using dilutions of 1:100, 200, 400, 800 and 1600. Wells with zero antigen, zero 1° or zero 2° antibody were run as controls. 2° antibodies were HRPO conjugated and the colour indicator developed with TMB (3,3',5,5'-tetramethylbenzidine) in 100mM sodium acetate. Plates were read at 450nm.
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Results: actin antibodies. Fig. 1. ELISA results for actin antibody screen on G v F-actin, with and without cofilin. Final absorbance from ELISA assays on Sigma AC-15 (green), Sigma AC-74 (black) and NeoMarkers Ab-5 (blue) monoclonal actin antibodies. Results with G-actin antigen (open circles), F-actin (open triangles), G-actin-cofilin complexes (closed circles) and F-actin-cofilin complexes (closed triangles).
15001000500000.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
1°Ab dilution
A45
0
The binding of the NeoMarkers Ab-5 antibody was maximal to G-actin but the signal fell 67% from the G to F actin result. Likewise, cofilin binding to G-actin interfered with the actin antibody binding. These results for Ab-5 serve to demonstrate the effectiveness of the assay in determining the reactivity of AC-15 with F v G actin +/- cofilin.
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Conclusions: 1) The Sigma AC-15 actin antibody was the most sensitive of those tested. 2) AC-15 recognises β-F-actin equally well with β-G-actin. 3) Cross linking does not impair AC-15 antibody binding to actin. Total A450 values from a full set of A450 values obtained with Ab dilutions 1:100 right through to 1:1600 for G-actin antigen were compared to a similar set from cross linked antigen. There was 0% effect on G-actin cross linking and only 5% on x-linking F-actin. 4) Cofilin bound and cross linked to actin, does not seriously affect actin antibody binding.
(Total A450 values from a full set of A450 values obtained with Ab dilutions 1:100 right through to 1:1600 for G- or F-actin antigen in x-linking medium were compared to a similar set from cofilin complexed and cross linked to the respective actins. There was a 9% decrease on the signal with G-actin complexes and 10% with F-actin complexes. This is most certainly within the experimental limits as for AC-74 antibodies the same type of assay gave 14% and 2% respectively, whereas for NeoMarkers Ab-5 there were 21% (cofilin on G-) and 78% (cofilin on F-) effects). 5) The Sigma AC-15 is truly specific for β-actin.
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Results: cofilin antibody. Fig. 2. ELISA results for cofilin-9771 and Rat1-ADF antibodies. Final absorbance from ELISA assays on rabbit 9771 cofilin antibody (black) and Rat1 ADF antibody (green) either with G-actin (filled circles) or without actin (open squares).
200015001000500000.0
0.1
0.2
0.3
0.4
0.5
1° antibody dilution
Ab
sorb
anc
e (
450n
m)
Binding of actin to human cofilin had no effect on the binding of the rabbit affinity purified 9771 cofilin antibody. Thus, the affinity of 9771 antibody for cofilin is not impaired by actin binding to cofilin. A similar analysis of a rat polyclonal antibody for ADF showed weaker binding in the presence of actin. Together these results suggest that there is no inhibition of cofilin interactions with actin by the 9771 antibody.
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Fig. 3. Western blot with cofilin-9771 antibody. Lysate, obtained from RBL-2H3 mast cell line (~ 100 000 cells per lane), was analysed by SDS-PAGE and stained with Coomassie Blue. Parallel samples were transferred to nitrocellulose and immunoblotted using affinity purified antibodies against actin (9785-11, used as a reference) and cofilin (9771).
Conclusions: The 9771 antibody is absolutely specific for cofilin. This means that the inhibitory effect of this antibody on the actin translocation to the nucleus, is mediated through cofilin. Our results also show that the cofilin antibody does not interfere with actin-cofilin interaction. Thus the knockout effect of the 9771 antibody on nuclear translocation of actin is outside of cofilin's depolymerising action on actin filaments. Evidently, the antibody binds to a different part of the cofilin to that involved in actin binding. From modelling studies (see McGough, et al., 1997, J. Cell Biol. 138, 771-781 and references therein), actin filament binding involves one face of the cofilin molecule with the opposite face free of actin and exposed into solution. Although site directed mutagenesis of residues on this face do not affect actin binding, they do affect cell viability (Lappalainen et al, (1997) Embo Journal 16: 5520-5530.). This “non-actin binding region”, however, contains the nuclear localisation sequence and antibody binding here would clearly affect the ability of cofilin to be actively translocated to the nucleus. We presume the antibody 9771 masks the NLS on cofilin and therefore blocks its ability to translocate actin into nucleus.
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Coomassieactin 9785-11
cofilin 9771
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Annmarie Pendleton, Brian Pope, Alan Weeds and Anna Koffernuclei of mast cells
Latrunculin or ATP depletion induce cofilin-dependent translocation of actin into
published online February 3, 2003J. Biol. Chem.
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