1 Department of Anatomy and Structural Biology, Albert Einstein College of
Medicine, 1300 Morris Park Avenue, Bronx, NY 10461, USA
2 Department of Neuroscience and Cell Biology, UMDNJ-Robert Wood Johnson Medical
School, 675 Hoes Lane, Piscataway, NJ 08854, USA
* Author for correspondence (e-mail: vogniew{at}aecom.yu.edu)
Accepted 4 September 2002
![]() |
Summary |
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Tropomyosin, an inhibitor of the Arp2/3 complex and cofilin function, was localized in relation to actin filaments, the Arp2/3 complex, and free barbed ends of actin filaments in MTLn3 cells, which rapidly extend flat lamellipodia following EGF stimulation. All tropomyosin isoforms examined using indirect immunofluorescence were relatively absent from the dynamic leading edge compartment, but did colocalize with actin structures deeper in the lamellipodium and in stress fibers. An in vitro light microscopy assay revealed that tropomyosin protects actin filaments from cofilin severing. The results suggest that tropomyosin-free actin filaments under the membrane can participate in rapid, dynamic processes that depend on interactions between the activities of the Arp2/3 complex and ADF/cofilin that tropomyosin inhibits elsewhere in the cell.
Key words: Actin, Arp2/3 complex, Tropomyosin, Cofilin, Cytoskeleton
![]() |
Introduction |
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The formation of the branched network in the leading edge domain of the
actin cytoskeleton is spatially and temporally regulated using both positive
and negative mechanisms. For example, the Arp2/3 complex is localized in the
leading edge (Bailly et al.,
1999; Svitkina and Borisy,
1999
), where it is activated by membrane bound factors, Rho-family
GTPases and PIP2, which in turn activate cellular WASp/Scar
proteins that bind to the Arp2/3 complex (reviewed in
Borisy and Svitkina, 2000
;
Higgs and Pollard, 2001
;
Small et al., 2002
). In
addition, actin filaments serve as obligate secondary activators of the Arp2/3
complex activity (Ichetovkin et al.,
2002
). Cortactin stabilizes branches
(Uruno et al., 2001
;
Weaver et al., 2001
). Cofilin,
required for the formation of barbed ends at the leading edge
(Zebda et al., 2000
), severs
actin filaments, thereby increasing the number of free barbed ends,
polymerization, and the number of ATP-containing filaments leading to enhanced
Arp2/3 complex activation (Ichetovkin et
al., 2002
). Cofilin also ensures a supply of actin monomers for
polymerization and the turnover of the Arp2/3 complex by severing and
increasing the off-rate of actin monomers from the pointed end (reviewed by
Bamburg, 1999
;
Carlier et al., 1997
;
Hawkins et al., 1993
;
Condeelis, 2001
). Experiments
using the actin drug jasplakinolide showed that protrusion of lamellipodia in
migrating chick fibroblasts is tightly coupled to actin filament disassembly,
suggesting that ongoing actin filament assembly is facilitated by free actin
monomers derived from filament disassembly
(Cramer, 1999
). Tropomodulin,
a protein that together with tropomyosin inhibits association and dissociation
of actin monomers from the pointed end of actin filaments
(Weber et al., 1994
;
Weber et al., 1999
), may also
favor barbed end incorporation of actin monomers.
Some factors negatively regulate formation of the branched actin filament
network. ATP hydrolysis and phosphate dissociation following actin
polymerization promote dissociation of filament branches
(Blanchoin et al., 2000b).
Phosphorylation of cofilin inhibits its severing and depolymerizing activities
(Agnew et al., 1995
;
Blanchoin et al., 2000c
;
Moriyama et al., 1996
;
Ressad et al., 1998
).
Tropomyosin, a protein that binds along the sides of actin filaments, inhibits
nucleation and branch formation by the Arp2/3 complex
(Blanchoin et al., 2001
) and
cofilin-F-actin interaction in vitro
(Bernstein and Bamburg, 1982
;
Nishida et al., 1985
;
Ono and Ono, 2002
). Proteins
that cross-link actin into stable structures, such as
-actinin, fascin
and fimbrin, may also protect actin filaments against branching and
severing.
The molecular models put forward to date to explain the processes that take
place at the leading edge of motile cells primarily depend on in vitro
experiments with the Arp2/3 complex. Identification of multiple domains of the
actin cytoskeleton within cells and how they relate to the Arp2/3 complex
localization and function at the leading edge requires spatial and temporal
analysis in a well-defined cell model. The extent to which cofilin and the
Arp2/3 complex contribute to lamellipodium extension has been studied in MTLn3
cells following EGF stimulation. Barbed ends at the leading edge are generated
by cofilin during EGF stimulation (Zebda
et al., 2000). Cofilin severing is required for both barbed end
generation and protrusion of the leading edge
(Chan et al., 2000
). Also, the
branching activity of the Arp2/3 complex is required for lamellipodium
protrusion during EGF stimulation (Bailly
et al., 2001
).
Our goal in the present study was to relate cellular compartments in which
the Arp2/3 complex and cofilin-mediated actin polymerization occurs to the
localization of tropomyosin. Our hypothesis was that since these activities
are inhibited by tropomyosin, tropomyosin would be excluded from the leading
edge of actively protruding cells. Although the tropomyosin localization has
been reported in numerous cell types (reviewed in
Lin et al., 1997), its
location in relation to the dendritic actin network has not been investigated.
Here we relate the biochemical effects of tropomyosin on the activities of the
Arp2/3 complex and cofilin to the cellular localization of tropomyosins with
respect to the Arp2/3 complex, barbed ends, and F-actin at the leading edge
during rapid lamellipodium extension. We used an optically flat cell (MTLn3)
with well-defined kinetics of EGF-stimulated protrusion that allows cellular
localization of proteins at the leading edge at high resolution. Our results
indicate that tropomyosin plays a regulatory role to confine the activities of
the Arp2/3 complex and cofilin to the leading edge.
![]() |
Materials and Methods |
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Primary antibodies
Previously characterized mouse monoclonal and rabbit polyclonal antibodies
against tropomyosin were generously supplied by colleagues, or purchased. Six
different antibodies were screened that together recognize tropomyosins
expressed by all four tropomyosin genes representing most, if not all, the
isoforms in MTLn3 cells. Of these, two (LC24 and f9d) were selected for
more extensive analysis based on their staining characteristics and ability to
detect a wide range of tropomyosin isoforms.
LC24 is a mouse monoclonal (IgG), provided by Jim Lin (University of Iowa).
It is specific to TM4, a short TM encoded by the TM4 gene
[-TM (Lin et al.,
1985a
; Lin et al.,
1988
)]. It was prepared against human protein, but it also
recognizes mouse protein.
f9d is a rabbit polyclonal, provided by Peter Gunning (University of
Sydney). It recognizes the C-terminal region of
- and ß-TM
isoforms expressing exon 9d (Schevzov et
al., 1997
). In mouse it crossreacts with a number of short and
long TM isoforms:
TM2,
TM3, ßTM1, ßTM6 (all long TM
isoforms), and
TM5a and
TM5b, and homologous short ß-TM
isoforms.
CG3 is a mouse monoclonal (IgM), provided by Jim Lin (University of Iowa).
It cross reacts with the exon 1b-encoded N-terminus of products of the
TM5 gene (-TM), short, non-muscle tropomyosins
(Lin et al., 1985a
;
Lin et al., 1988
;
Vera et al., 2000
). It was
prepared against human immunogens, but also recognizes mouse proteins.
IV15 is a mouse monoclonal (IgM) provided by Fumio Matsumura (Rutgers
University) with a wide specificity [F. Matsumura, personal communication
(Matsumura et al., 1983)].
CGß6 is a mouse monoclonal (IgM) provided by Jim Lin (University of
Iowa) that crossreacts with TM2 and TM3, long -TM isoforms
(Lin et al., 1985a
;
Lin et al., 1988
) that are
also recognized by
f9d and TM311.
TM311, a mouse monoclonal IgG purchased from Sigma, crossreacts with the
N-terminus of long and ß-TM isoforms
(Nicholson-Flynn et al.,
1996
).
Anti-p34 (AE360) is a rabbit IgG generated using a peptide immunogen of a
sequence of the p34 protein in the Arp2/3 complex
(Bailly et al., 2001).
It is unlikely that the lack of binding of anti-TM at the leading edge is a
consequence of non-linear binding of antibody at low concentration of antigen,
since both monoclonal (e.g. CG3) and polyclonal (e.g. f9d) antibodies
gave the same tropomyosin distribution as far as absence of labeling of the
leading edge is concerned, even though they generally exhibit different
binding curves.
Immunofluorescence
Cells were plated on glass-bottom dishes (MatTek Corporation) as previously
described (Bailly et al.,
1998a) and left untreated or stimulated with EGF. They were fixed
with 3.7% formaldehyde in cytoskeletal buffer (5 mM KCl, 137 mM NaCl, 4 mM
NaHCO3, 0.4 mM KH2PO4, 2 mM MgCl2,
5 mM Pipes, 2 mM EGTA, 5.5 mM glucose, pH 6.1
(Small et al., 1978
), at
37°C for 5 minutes. Cells were permeabilized at room temperature for 20
minutes with 0.5% Triton X-100 in cytoskeletal buffer. They were rinsed once
and then incubated in 0.1 M glycine (in cytoskeletal buffer). After five
washes with TBS (20 mM Tris, 154 mM NaCl, pH 8), cells were blocked/stabilized
in TBS with 1% BSA, 1% FCS, and 5 µM phalloidin (Calbiochem-Novabiochem)
for 20 minutes. For F-actin staining, phalloidin was supplemented with 0.5
µM rhodamine phalloidin (Molecular Probes). This was followed by incubation
with primary antibody at room temperature for one hour. Cells were rinsed five
times for five minutes with TBS containing 1% BSA and incubated with secondary
antibody, goat anti-rabbit fluorescein-conjugated IgG (ICN Biomedicals), or
Cy5 conjugated donkey anti-mouse IgG (Jackson ImmunoResearch), for one hour at
room temperature. After five final washes with TBS (containing 1% BSA), cells
were mounted in 50% glycerol in TBS, supplemented with 6 mg/ml N-propyl
gallate and 0.02% sodium azide.
Visualization of free barbed ends
For double labeling of actin nucleation sites (barbed ends) and tropomyosin
(f9d), a previously described protocol was used
(Bailly et al., 1999
;
Chan et al., 1998
). Briefly,
cells were stimulated with EGF for 50 seconds or 3 minutes, immediately
followed by permeabilization with cytoskeletal buffer, containing 0.45 µM
of biotin-labeled G-actin (Cytoskeleton), 1% BSA, and 0.025% saponin. The
distribution of biotin-actin was identified using a Cy5-coupled anti-biotin
antibody (Jackson ImmunoResearch). Tropomyosin was visualized by incubating
cells with antibody
f9d for 1 hour, followed by incubation with a goat
anti-rabbit fluorescein-conjugated IgG (ICN Biomedicals).
Microscopy and fluorescence quantification
All images were taken on an Olympus IX70 microscope using constant settings
with 60x NA 1.4 infinity-corrected optics coupled to a computer-driven
cooled CCD camera using IP lab spectrum software (VayTek). Images were
captured below the saturation level of the camera. For the fluorescence
quantification, all digitized images were linearly converted in NIH Image
(program developed by the National Institute of Health, available on the
internet at
http://rsb.info.nih.gov/nih-image)
and analyzed using different macros. For double-labeling measurements
(Fig. 5) cell perimeters were
traced by fluorescence threshold in the non-tropomyosin channel and also
applied to the tropomyosin channel. The measurements were taken on
lamellipodia, avoiding regions of cells that did not contain lamellipodia. The
software macro automated the collection of pixel intensity in a perimeter of
the cell starting 1.98 µm outside the cell and extending 4.18 µm into
the cell in 0.22 µm steps. Lamellipodia are flat and of uniform thickness,
so there is negligible contribution of the variation of thickness to the
fluorescence intensity (Bailly et al.,
1998a; Bailly et al.,
1998b
; Chan et al.,
1998
; Rotsch et al.,
2001
). This procedure included subtraction of background
fluorescence from the measured cellular fluorescence. The dark current noise
of the camera is below the level of background fluorescence. Any signal
measured above the dark current noise of the camera is in the linear range of
the camera, up to saturation levels. In the conditions used, any signal above
zero in the graph is in the linear range of the camera and above dark current
noise levels. For a description of the system see
http://www.aecom.yu.edu/aif.
|
|
|
|
Tropomyosin extraction experiments
To estimate the percent of the total tropomyosin that was free, not
associated with the actin cytoskeleton, we used two independent experimental
methods. In the first gel-based method, 100 mm plates of confluent MTLn3 cells
were washed with phosphate buffered saline and permeabilized for one to four
minutes with: 138 mM KCl, 10 mM Pipes, pH 7, 0.1 mM ATP, 3 mM EGTA, 4 mM
MgCl2, 0.025% saponin, 0.5% Sigma protease cocktail. To estimate
total tropomyosin, in parallel, cells on plates were washed and lysed in 10 mM
TrisHCl, pH 8.0, 10 mM EGTA, 0.025% saponin, 0.5% of a Sigma protease
cocktail, for 10 minutes on ice to obtain the total cytoskeletal protein, and
removed from the plates by scraping. NaCl was added to the samples to a final
concentration of 0.2-0.5 M, and the samples were heated for 2 minutes at 100%.
Following centrifugation for 10 minutes in a microfuge at 4°C, the
supernatants containing tropomyosin, and other heat-stable proteins, were
lyophilized. The samples were resuspended in the same volume of sample buffer
and analyzed by SDS PAGE on 12% gels stained with Coomassie Blue.
The tropomyosin in the heat stable fraction was quantified by densitometry of the stained gels using a Molecular Dynamics model 300A computing densitometer. The tropomyosin region of the gel was identified by immunoblots using specific tropomyosin antibodies and tropomyosin standards. In the absence of permeabilization and lysis buffers there was no staining in the tropomyosin region of the gel. With the permeabilization procedure, 15±4% (n=5) of the total tropomyosin was removed.
In the second method based on immunofluorescence, a monolayer of EGF stimulated cells were fixed with formaldehyde and stained with LC24 as described in `Immunofluorescence'. Another monolayer of EGF-stimulated cells was permeabilized with saponin, fixed with formaldehyde and stained with LC24 as described in `Visualization of barbed ends'. Digital images of cells were taken at 20x magnification and pixel intensities of the cell were measured using the NIH Image software as described in `Microscopy and fluorescence quantification'. Cells extracted with saponin and then fixed with formaldehyde showed an 18% loss of fluorescence intensity presumably reflecting extraction of free tropomyosin not bound to actin filaments.
Protein purification
Actin was purified from rabbit skeletal muscle acetone powder
(Spudich and Watt, 1971).
Recombinant rat TM5a, a short non-muscle TM product of the
-TM
gene, was cloned and expressed in E. coli, and purified as previously
described (Moraczewska et al.,
1999
). Recombinant cofilin was expressed in E. coli and
purified as previously described (Bamburg
et al., 1991
) with modifications as stated in Ichetovkin et al.
(Ichetovkin et al., 2000
).
Light microscopy severing assay
This severing assay has been described in detail and validated previously
(Chan et al., 2000;
Ichetovkin et al., 2002
;
Ichetovkin et al., 2000
).
Briefly, 10 µM recombinant rat TM5a in perfusion buffer (20 mM Pipes pH
7.0, 2 mM MgCl2, 5 mM EGTA, 50 mM KCl, 1 mM ATP, 1 mM
dithiothreitol) was reduced by heating to 58°C for 2 minutes, cooled to
room temperature and kept on ice for the duration of the experiments. Chambers
with pre-bound actin filaments (Ichetovkin
et al., 2002
) were perfused for 45 minutes with either 10 µM
TM5a or perfusion buffer as a control. After 45 minutes, chambers were placed
on a CCD-equipped inverted microscope (Olympus IX70), and unbound TM5a was
washed away with anti-bleaching wash buffer (20 mM Pipes pH 7.0, 2 mM
MgCl2, 5 mM EGTA, 50 mM KCl, 1 mM ATP, 100 mM DTT, 5 mg/ml BSA, 6
mg/ml glucose, 0.2 mg/ml glucose oxidase, 0.036 mg/ml catalase), `0 second'
time point rhodamine-fluorescent images were collected (with the 60x
objective). Chambers were perfused under the microscope with 100 nM
recombinant rat cofilin in 10 mM Tris pH 7.5, 100 mM DTT, 5 mg/ml BSA, 6 mg/ml
glucose, 0.2 mg/ml glucose oxidase, 0.036 mg/ml catalase. After 1 minute of
incubation with cofilin, images of the same field were taken. Images were
quantified using NIH Image by counting number of filaments using automatic
thresholding. An increase in the total number of filaments in the same field
indicated cofilin-severing activity. To avoid counting false breaks in the
filament due to non-continuous labeling, thresholding levels were set to score
only gaps between filaments larger than 0.3-0.4 mm. That led to the
underscoring of small gaps even though they were clearly visible by eye
(nearly all breaks formed after an initial 30 seconds), but resulted in much
more consistent and reproducible quantitative data.
![]() |
Results |
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EGF-stimulated lamellipodium extension in MTLn3 cells depends on actin
polymerization and results in a transient increase in the number of free
barbed and pointed ends of filaments accompanied by the formation of a
branched filament network in a narrow zone at the extreme leading edge
(Bailly et al., 1999;
Chan et al., 2000
). This zone
is enriched in the Arp2/3 complex and cofilin, proteins required for
lamellipodium extension (Bailly et al.,
2001
; Bailly et al.,
1999
; Chan et al.,
2000
; Zebda et al.,
2000
).
In order to determine the general localization of tropomyosin in MTLn3
cells we used a broad spectrum of antibodies that recognize tropomyosin
isoforms encoded by all four tropomyosin genes (, ß,
,
), and all tropomyosin types expressed in the cell (see Materials and
Methods). The tropomyosin isoforms expressed in MTLn3 cells were evaluated
using immunoblots of total cell extracts from unstimulated cells
(Fig. 1). Antibody
f9d
crossreacted with at least three major bands and additional minor bands, both
long and short tropomyosins, some of which were identified using the TM311 and
CGß6 antibodies. These represent tropomyosins TM1/TM6 (not separated in
this blot), TM2, TM3 and possibly small amounts of short isoforms. Two short
isoforms encoded by the
-TM gene, TM5a and TM5b, were not
detected using another antibody specific for these isoforms. The CG3 antibody
crossreacted with one major band that consists of up to 11 isoforms of the
-TM gene (TM5nm tropomyosins). The antibody LC24 crossreacted
with a single band indicating that TM4, the only non-muscle isoform encoded by
the
-TM gene, is expressed well in MTLn3 cells. In summary,
the major tropomyosin isoforms expressed in MTLn3 cells are the long isoforms
TM1/TM6, TM2, TM3 and the short isoforms TM4 and one or more TM5nm.
|
The distribution of tropomyosin and F-actin during lamellipodium
extension
Previous studies using these antibodies have shown the presence of multiple
microfilament compartments within cells
(Lin et al., 1985a;
Lin et al., 1985b
;
Lin et al., 1988
;
Nicholson-Flynn et al., 1996
;
Percival et al., 2000
).
However, from these studies it is unclear whether tropomyosin is present in
the leading edge of protruding lamellipodia [defined as the first 0.9 µm
next to the membrane (Chan et al.,
2000
)]. This is the case either because the cell types studied had
no distinct, protruding leading edge, or the history of the cell protrusion
was not followed, or the staining was not related to the cell edge by phase
contrast images or double labeling with markers for the leading edge. We
examined cells stained with
f9d, LC24, CG3, CGß6, TM311, and IV15
antibodies, which recognize tropomyosins encoded by all four tropomyosin
genes, as discussed above. The results using all the antibodies were the same
in that none stained the leading edge of protruding lamellipodia, although
they stained other actin-containing structures to differing extents. Since the
tropomyosin isoforms recognized by TM311 and CGß6 were also recognized by
f9d, we did not proceed with a detailed cellular analysis using these
antibodies. Illustrative results using the antibodies
f9d, LC24, CG3
and IV15 are shown in Figs
2,3,4.
The antibodies LC24 and
f9d were selected for more extensive analysis
because they stained actin structures well, including the base of lamellipodia
close to the leading edge. All other antibodies mainly stained structures
toward the middle of the cell, such as stress fibers. In addition,
f9d
and LC24 recognized bands in immunoblots of MTLn3 cell extracts that are
encoded by different tropomyosin genes
(Fig. 1). The results of this
analysis are shown in Fig.
5.
MTLn3 cells double-labeled for F-actin (rhodamine-phalloidin, Fig. 2A,E,I) and tropomyosin (LC24, Fig. 2B,F,J) showed bright F-actin staining at the leading edge before (Fig. 2A-D,M,N) and after (Fig. 2E-L,O,P) EGF stimulation, reflecting the newly polymerized F-actin network adjacent to the plasma membrane that is generated during lamellipodium extension. However, tropomyosin was absent from this dynamic leading edge compartment, although there was substantial overlap of F-actin and tropomyosin staining in the rest of the cell (overlays, Fig. 2C,G,K,M-P), especially in stress fibers and the diffuse non-stress fiber actin network in the base of the lamellipodium (indicated by an asterisk). Higher magnification images of the boxed regions of the cells in Fig. 2M,O show that in both unstimulated cells (Fig. 2N) and EGF-stimulated cells (Fig. 2P) tropomyosin (red) was absent from the dynamic leading edge compartment whereas F-actin (green) was present up to the cell membrane, and thickens after stimulation. However, both tropomyosin and F-actin were present just beyond the dynamic leading edge compartment at the base of lamellipodia.
To determine the approximate ratio of actin to tropomyosin at the leading
edge, we quantified the fluorescence intensity for F-actin and tropomyosin as
a percentage of the total fluorescence for each fluorophor in different
regions of stimulated cells (Table
1). The greatest fraction of both actin and tropomyosin, and the
highest actin-to-tropomyosin ratio, was in the stress fibers. If we assume
actin filaments in stress fibers are saturated with tropomyosin, then by
comparison of the fluorescence intensities, only 25% of the filamentous
actin at the leading edge [defined as the first 0.9 µm next to the membrane
(Chan et al., 2000
)] have
tropomyosin bound. If the actin filaments in stress fibers are not saturated
with tropomyosin, then 25% is an overestimate. In the base of lamellipodia
(0.9-1.8 µm from the membrane), there is sufficient tropomyosin to saturate
70% of the filamentous actin.
|
These calculations are based on the following measurements. In MTLn3 cells
the total actin is about 153 mM (Edmonds
et al., 1998), only about half of it being filamentous
(Edmonds et al., 1996
).
Tropomyosin is 1.5-2% of the total protein, or
25 mM [in chicken embryo
fibroblasts and human blood platelets (Lin
et al., 1985a
) (S.E.H.-D., unpublished)], more than sufficient to
saturate the filamentous actin with the usual 1 tropomyosin to 6 or 7 actin
subunits in F-actin. When unstimulated MTLn3 cells growing in monolayers were
permeabilized upon brief, mild, detergent treatment as in the experiments to
assay barbed end labeling (see Materials and Methods), 15±4%
(n=5) of the tropomyosin was extracted, based on quantitative
analysis of tropomyosin using SDS-PAGE of supernatants and pellets. Similarly,
analysis of immunofluorescence of cells stained for tropomyosin using the LC24
antibody showed an 18% loss of fluorescence intensity after saponin treatment
of a monolayer of EGF stimulated cells (see Materials and Methods), presumably
reflecting extraction of free tropomyosin not bound to actin. Assuming the
extractable tropomyosin is unbound, there should be sufficient free
tropomyosin (2.5-7.5 mM) to bind to F-actin with a Kd of
0.1 mM.
Tropomyosin is not associated with polymerizing filaments at the
leading edge
The dynamic actin filament network at the leading edge was more
specifically visualized by polymerizing biotin-labeled actin monomer into the
barbed ends of growing actin filaments
(Bailly et al., 1999;
Chan et al., 1998
). As
previously reported (Bailly et al.,
1999
), unstimulated cells only had weak barbedend staining at the
leading edge (Fig. 3A), but
cells EGF-stimulated for 50 seconds or 3 minutes showed an increase in the
incorporation of actin monomer into filaments at the leading edge
(Fig. 3F,K,P). The tropomyosin
antibody
f9d stained actin stress fibers as well as more diffuse
F-actin networks at the base of lamellipodia. However,
f9d did not
label the dynamic leading edge compartment
(Fig. 3B,G,L), which is
enriched with barbed ends before and after EGF stimulation (see overlay in
Fig. 3C,H,M). The CG3 antibody
predominantly labeled perinuclear stress fibers but not the diffuse F-actin
network at the base of lamellipodia [(Fig.
3Q) (Percival et al.,
2000
)] and did not colocalize with newly formed actin filaments at
the leading edge (Fig. 3R). The
difference in the staining patterns of the
f9d and CG3 antibodies with
regard to the diffuse F-actin network at the base of lamellipodia is similar
to that found in NIH 3T3 cells (Percival
et al., 2000
), suggesting these antibodies recognize different
populations of microfilaments in MTLn3 cells as well.
Fig. 3D,I,N,S show higher
magnification images of the boxed areas in
Fig. 3C,H,M,R. The tropomyosin
staining (red) does not extend to the edge of the cell that is enriched in
barbed ends of growing actin filaments (green).
Another probe for the dynamic actin filaments at the leading edge is the
presence of the Arp2/3 complex. An antibody to the Arp2/3 complex, anti-p34,
labeled the leading edge of the cells, especially after EGF stimulation
(Fig. 4A,E,I)
(Bailly et al., 1999). The
tropomyosin antibody LC24 labeled stress fibers and actin networks within the
cells, but did not label the leading edge at any time
(Fig. 4B,F,J), as seen in the
overlays (Fig. 4C,G,K). Similar
results were obtained with antibody IV15
(Fig. 4N,O), which stained
stress fibers as well as the base of lamellipodia, but not the leading edge
compartment.
The localization of tropomyosin with respect to F-actin, free barbed ends
and the Arp2/3 complex at the leading edge of lamellipodia is quantified in
Fig. 5. The tropomyosin
antibodies used in this analysis were LC24 and f9d, which recognize
many different tropomyosin isoforms, as discussed earlier. As described in the
Materials and Methods, all measurements were in the linear range and above the
noise level of the CCD camera. In unstimulated cells, tropomyosin was
minimally associated with actin-containing structures close to the membrane,
with the bulk of the labeling occurring much deeper in the cell as well as in
stress fibers (Fig. 5A,D,G).
Upon EGF stimulation, F-actin (phalloidin-actin), barbed end labeling and the
Arp2/3 complex increased and peaked within 0.5 µm of the leading edge, but
tropomyosin did not (Fig.
5B,C,E,F,H,I,J,K). Labeling for barbed ends and the Apr2/3 complex
was maximal at 50 seconds of EGF stimulation, as previously reported
[(Fig. 5D,E,F,G,H,I)
(Bailly et al., 1999
)]. The
maximal labeling for F-actin, however, did not occur until 3 minutes of EGF
stimulation (Fig. 5A,B,C,J).
Tropomyosin remained minimal, possibly decreasing in amount at the edge
(Fig. 5B,C,K). The localization
of tropomyosin at the leading edge did not change upon EGF stimulation
(Fig. 5K). This leading edge
region is the same region of lamellipodia that contains branched actin
filaments after EGF stimulation (Bailly et
al., 1999
).
Tropomyosin protects actin filaments from severing by cofilin
Previous biochemical results indicate that tropomyosin can inhibit the
binding of cofilin to F-actin and cofilin-induced depolymerization
(Bernstein and Bamburg, 1982;
Nagaoka et al., 1995
;
Nishida et al., 1985
;
Ono and Ono, 2002
). Since
depolymerization and severing by cofilin are separable activities
(Pope et al., 2000
) and
cofilin can sever actin filaments at concentrations 100-fold lower than that
required for depolymerization (Ichetovkin
et al., 2002
), we tested the ability of tropomyosin to inhibit
severing by cofilin using a light microscopy assay that directly measures the
severing activity of cofilin (Fig.
6). Actin filaments were immobilized on nitrocellulose and
addition of cofilin severed the actin filaments
(Fig. 6, top panels).
Pre-incubation of the actin filaments with TM5a, a short non-muscle isoform,
inhibited cofilin's severing activity (Fig.
6, bottom panels).
|
![]() |
Discussion |
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We base our conclusion on negative immunofluorescence at the leading edge
of protruding lamellipodia. One possible explanation for the absence of
tropomyosin at the leading edge is that the tropomyosin epitopes are blocked
in this region, which is unlikely, since none of the six antibodies we tested,
that together detect all tropomyosin isoforms in MTLn3 cells, stained the
leading edge, while they all stained actin structures elsewhere in the cell.
It is improbable that the different epitopes recognized by the six antibodies
would all be buried at the leading edge, but not in other cytoskeletal
domains. The tropomyosin antibodies stained MTLn3 cells with similar intensity
before and after EGF stimulation, suggesting that epitope availability does
not correlate with protrusion or motility in this case. In addition, MTLn3
cells are continuously motile even before stimulation with EGF
(Shestakova et al., 1999).
This is unlike the case reported by Hegmann et al., where a correlation was
found between motility and epitope availability for a monoclonal tropomyosin
antibody (Hegmann et al.,
1988
).
The spatial segregation of tropomyosin isoforms in cells is well
established (Gunning et al.,
1998; Lin et al.,
1988
; Lin et al.,
1997
; Percival et al.,
2000
). Tropomyosin has long been known to be preferentially
localized in regions of stable actin filaments in cells
(Lazarides, 1976
), but until
now its distribution was not related to either actin function or other actin
binding proteins in the actin-rich lamellipodium. Previous studies
(Lin et al., 1988
;
Warren et al., 1985
) showed
the presence of tropomyosin in peripheral ruffles in normal chicken embryonic
fibroblasts and transformed rat kidney cells. However, ruffles are
structurally distinct from protruding lamellipodia observed in EGF stimulated
MTLn3 cells. Protruding lamellipodia are planar with well-defined leading
edges while ruffles are regions of membrane that fold back on themselves, are
not planar and not necessarily regions of outward extension but often
retraction structures involved in endocytosis
(Bailly et al., 1998b
). Since
MTLn3 cells cease to form ruffles during EGF stimulation and lamellipodia
extension (Rotsch et al.,
2001
), the present study only focuses on whether tropomyosin is
present in rapidly extending lamellipodia and their leading edges.
Fig. 7 is a model to
illustrate three types of actin compartments defined by tropomyosin in
protruding MTLn3 cells. The main body of the cell is rich in actin-containing
structures, stress fibers and other long actin filaments containing
tropomyosin that protects them from cofilin and other actin severing proteins,
and prevents branch formation nucleated by the Arp2/3 complex. The
lamellipodium of the cell is divided into two regions, the leading edge and
base (Chan et al., 2000). In
the base, the chronologically older region of the lamellipodium, there are
long, unbranched actin filaments that contain tropomyosin, protected from
cofilin and the Arp2/3 complex. Although cofilin and the Arp2/3 complex are
distributed throughout the cell, a dynamic branched network of actin filaments
forms only at the leading edge immediately under the plasma membrane (<1
µm) where tropomyosin is absent. Since the cofilin concentration is highest
at the leading edge of carcinoma cells
(Chan et al., 2000
), cofilin
mediated severing and depolymerization would occur at the leading edge and not
at the base of lamellipodia, as has been proposed
(Small et al., 2002
).
|
The mechanism by which newly formed filaments of the leading edge are
tropomyosin-poor remains to be established. Since the Arp2/3 complex and
G-actin are rapidly recruited to the leading edge of lamellipodia upon
stimulation, we would anticipate that tropomyosin would be too, if it could
bind to the actin filaments there and if there are not dramatically different
rates of diffusion. As mentioned in the Results section, there appears to be
sufficient free tropomyosin (15-18%) in the cell to bind to F-actin. The low
actin-to-tropomyosin ratio at the leading edge after stimulated protrusion
(Table 1) during the peak
period of cofilin and Arp2/3 complex activity over several minutes
(Bailly et al., 1999;
Chan et al., 2000
) suggests
that something may prevent recruitment of tropomyosin to the leading edge
filaments. Competitive inhibitors or a more direct regulation of binding of
tropomyosin to newly formed actin filaments may exclude tropomyosin from the
actin network
The demonstration that tropomyosin inhibits actin filament severing by
cofilin is consistent with an antagonistic interaction between cofilin and
tropomyosin that might confine the severing activity of cofilin to the leading
edge actin filaments that are tropomyosin-free. Elevated concentrations of
cofilin at the leading edge after EGF stimulation may inhibit binding of
tropomyosin to F-actin. During stimulated protrusion of MTLn3 cells, cofilin
is strongly recruited to the leading edge with 8% of the cell's 6-25 µM
cofilin accumulating there at times of peak barbed end generation
(Chan et al., 2000). Cofilin
severs actin filaments at the leading edge, aiding in the nucleation of
polymerization (Chan et al.,
2000
; Zebda et al.,
2000
). The high cofilin concentration relative to tropomyosin at
the leading edge should be sufficient to competitively inhibit binding of
tropomyosin to F-actin, possibly through changing the helical twist of the
filament. Therefore, the timing of cofilin recruitment and activation at the
leading edge by 50 seconds after stimulation
(Chan et al., 2000
) may
prevent tropomyosin recruitment there. In the rest of the cell, much of the
cofilin is likely to be inactivated by Lim kinase and less favorable pH
(reviewed by Bamburg, 1999
).
The higher levels of tropomyosin would be able to protect actin filaments from
severing by competing with any remaining active cofilin.
It is more difficult to evaluate the possible effect of the Arp2/3 complex
on the distribution of tropomyosin at the leading edge. The Arp2/3 complex is
approximately 2 µM in crawling cells
(Bailly et al., 1999) and 3% is
found in the leading edge in MTLn3 cells during the first 3 minutes of
stimulated protrusion. There may be sufficient free tropomyosin to inhibit
Arp2/3 complex nucleation and branching, based on in vitro experiments
(Blanchoin et al., 2001
), but
few actin filaments have bound tropomyosin. However, little is known about the
mechanism of Arp2/3 complex inhibition by tropomyosin. Also, only a small
amount of this Arp2/3 complex will be active in the leading edge at any time.
The complex moves away from the membrane quickly during protrusion
(Bailly et al., 1999
) as the
Arp2/3 complex disassociates from the side of F-actin and WASp under the
regulation of rapid nucleotide hydrolysis
(Blanchoin et al., 2000b
;
Dayel et al., 2001
).
Nevertheless, any active Arp2/3 complex that does diffuse into a region where
actin filaments have tropomyosin bound, such as the base of the lamellipodium,
should be inhibited from nucleation and branching.
Recent work is consistent with synergy between the Arp2/3 complex and
cofilin in the formation of a dynamic, branched actin filament network at the
leading edge (Ichetovkin et al.,
2002). The Arp2/3 complex-mediated branch formation in vitro
preferentially occurs from the sides of newly grown filaments containing
ATP-actin. Since the dynamic leading edge compartment contains branched,
rapidly polymerizing actin networks, it is likely that filaments in this
region are enriched in ATP-actin near the fast growing ends (ATP caps). This
would bias the Arp2/3 complex-generated branched filaments to grow in one
direction, the direction of lamellipodium extension. Thus, it appears that the
Arp2/3 complex binds to and initiates branching of newly polymerized actin
filaments at the leading edge, while tropomyosin protects older, ADP-actin
containing filaments away from the leading edge from the binding and
activation of the Arp2/3 complex.
The leading edge of cells may also be the site of other functions inhibited
by tropomyosin. Tang and Ostap reported that myosin 1b, a myosin whose motile
function is inhibited by tropomyosin, is localized in the tropomyosin-poor,
actin-rich cortex of NRK cells that would be expected to contain the Arp2/3
complex (Tang and Ostap,
2001).
The results presented here, as well as previously published work, may explain how cofilin severing and the Arp2/3 complex-nucleated branching result in new filament ends and filament branching only at the leading edge even though both proteins are present throughout the cytoplasm. Only filaments not saturated with tropomyosin are severed upon activation of cofilin or serve as substrates for branch formation. Therefore, even if cofilin and the Arp2/3 complex were globally activated, they would only act on filaments free of tropomyosin, such as those at the leading edge.
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Acknowledgments |
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